MICROBIOLOGY

Introduction to Bacteriology
- Industrial Microbiology – use of microorganisms in the production of various
products
DEFINITION AND SCOPE OF MICROBIOLOGY - Biotechnology – commercial use of microorganisms
- Genetic Engineering – use of recommended DNA technology to expand
MICROBIOLOGY
biochemical factories
 The study of microorganisms (unicellular and multicellular organisms)
- Microbial Ecology – relationships between microorganism and their
 It is a specialized branch of biology that deals with microorganisms.
environment
 Microorganisms – minute living things that are usually too small to be seen with
 Benefits of Microbes to Humans
the unaided eye
- Primary decomposers (bacteria are considered as the world’s greatest
 Branches
recyclers)
 Taxonomic
- Food products
 Functional
- Antibiotic production (Penicillium notatum)
 TAXONOMIC APPROACH (see page 76)
- Microbial antagonism (normal flora of the body prevents the invasion of
 Archaea
pathogens)
 Groups of Archaea
- Synthesis of chemicals
 Methanogens – produce methane as a waste product from
- Insect pest control (Bacillus thuringiensis produces natural pesticides)
respiration
- Bioremediation
 Extreme halophiles – organisms able to tolerate extreme salty
- Recombinant DNA technology, gene therapy and genetic engineering
environment
(bacteria can be manipulated to produce enzymes and proteins they
 Extreme thermophiles – organisms able to tolerate extremely hot,
normally would not produce)
sulfurous environment
 Fungi
 Types of Fungi MICROBIAL EVOLUTION
 Mushroom – multicellular organisms that are similar to plants;
incapable of photosynthesis  All living things arise from simple matter.
 Molds – multicellular organisms that create visible masses of  PHYLOGENY – evolutionary relationship between microorganisms;
hyphae (MYCELIA – long filaments that are intertwined, composed interconnectedness
of hyphae)  PHYLOGENIC TREE OF LIFE – establishes the evolution of organisms
 Yeast
 FUNCTIONAL APPROACH MICROBIAL ECOLOGY
- Medical or Clinical Microbiology – microorganisms associated with diseases
- Immunology – immunity  BIOFILM – some microorganisms do not act as individual entity; they exist as a
- Public Microbiology and Epidemiology – when and where diseases occur and community (complex community)
how they are transmitted; prevalence and incidence of diseases  BIOREMEDIATION – use of microbes to remove toxic materials in the environment
- Food Microbiology – beneficial and detrimental effects of microorganisms in (decontamination)
food and food processing  Pseudomonas spp. degrade oil into simple components (in controlled
- Agriculture Microbiology – effects of microorganisms to agricultural products conditions)

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  Strain – genetic variability within species
 Biovars – biochemical or physiologic differences
BACTERIAL TAXONOMY  Morphovars – morphologic differences
 Serovars – differences in antigenic properties
 TAXONOMY – arrangement or catalogue of related organisms into logical groups
 Rules of Nomenclature
 Identification, naming and classification of organisms
 There is only one correct name for an organism.
 IMPORTANCE
 Names that cause error and confusion shall be rejected.
 Establish criteria in the identification
 All names shall be Latinized, regardless of origin.
 Arrange related organisms into groups
 Microorganisms named according to shape (Ex. Staphylococcus
 Provide information on how they evolve
aureus – ‘coccus’ [spherical]; ‘staphylo’ [clusters])
 Provide orderly basis for identification and placing related organisms into
 The first letter of the genus is capitalized. The species is written in
groups or various categories
lowercase letters.
 PHYLOGENY
 Scientific names are underlined or italicized when printed or written.
 Taxon or Taxa – groups of related organisms
 Origin of the Nomenclature of Microorganisms
 Lower level taxa are more similar than the higher level taxa
 Genera named after INDIVIDUALS (Ex. Escherichia coli – Theodore
 Members of specific level taxa are more similar compared to members of
Escherich)
different specific level taxa in some hierarchical level
 Genera named after MICROBE SHAPE (Ex. Streptococcus – spherical in
 Arrangement of organisms depends on their ancestors or origins
chains)
 Phylogenetic Classification System – classification reflects genetic
 Genera named after an ATTRIBUTE OF THE MICROBE
similarity and evolutionary relatedness
 Phenetic Classification System – classification is based on the
convenient observable characteristics BACTERIAL CYTOLOGY
 7 HIERARCHICAL CATEGORIES
 Kingdom (most complex taxa) Glycocalyx
 Phylum  Sugar coat of the BACTERIA; outer layer of the cell wall
 Class  Slime layer – thin, unorganized and loosely attached to the cell wall
 Order  Capsule – thick, organized and closely attached to the cell wall
 Family  External viscous, gelatinous layer of the cell wall composed of either polymers of
 Genus polysaccharides, peptides or both and some phosphates
 Species (most definitive division)  Synthesized intracellularly and is brought out of the cell by an ISOPRENOID LIPID
 BINOMIAL NOMENCLATURE CARRIER
 Nomenclature – naming of microorganisms  Made up of negative charges, which facilitates protection from phagocytosis
 International Code of Nomenclature of Bacteria  White blood cells are also made up of negative charges (like repels like)
 Two name system developed by Carolus Linnaeus  It has a POLYIONIC NATURE, which facilitates attraction and storage of nutrients.
 Two level taxa: Genus and Species  BACTERIAL IDENTIFCATION
 Genus  Moist and glistening colonies
 Comprised of different species with important common features  Negative staining
 Classified based on similarities  Transmission electron microscopy
 Bergey’s Manual of Determinative Bacteriology (David Bergey) –  Antigenic properties
first manual or system of bacterial classification  Quellung Test (Capsular swelling test)
 Species  FUNCTIONS
 Collection of strains  Protects the cell

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  Desiccation – the glycocalyx prevents the cell from dehydration  Types of Movement (either a clockwise or a counterclockwise movement,
because the slime layer and capsule have high water content depending on the cell’s generation of energy)
 Toxic Materials  Run or Swim – counterclockwise rotation; one movement
 Phagocytosis  Tumble – clockwise rotation; periodic abrupt random movement changes
 Virulence Factor  Flagellar receptors
 Pathogen – produced structure that allows the bacterial cell to invade  Flagella moves towards an environment it has sensed; compare or sense the
or evade the immune system and possibly cause disease chemical environment (temporal sensing)
 Adherence of bacteria to cells or mucosal surfaces  Absence of gradients – counterclockwise movement
 Resist phagocytosis  Presence of attractant gradient – changes motility; moves up the gradient;
 Resistance to bactericidal action clockwise movement
 Biofilms  Attractant – chemotactic signal that allows the movement of the flagella
 Protects the cells underneath  Chemotaxis – movement towards a source of nutrients
 Facilitate communication among microorganisms found  Phototaxis – movement towards a source of light
within the biofilm  Aerotaxis – movement towards a source of oxygen
 Survival of the cells by attaching to various surfaces in its  Phase variation – alter the expressed antigenic type of flagella that they produce,
natural environment making detection and identification difficult
 Concentration of nutrients
 Bacterial identification Axial Filament (Endoflagellum)
 Several fibrils that arise between the cell wall and the cell membrane
Flagella  Rotation (rotary movement)
 It arises at the cell membrane, which facilitates movement of bacteria.  Vibration movement; Serpent – like or corkscrew motility – permits boring
 It is long (10 to 20 µm in length) and thin (20 nm in diameter). movement, and is effectively seen in body fluids
 It is a thread – like or whip – like filamentous appendage.  Motility by undulation
 Flagellin – protein found in flagella  Seen in spirochetes (ex. Treponema pallidum)
 Flagella are seen in gram – negative bacteria and some gram – positive bacteria
(ex. Listeria spp. and Enterococcus spp.) Fimbriae (Pili)
 H antigen – flagellar antigen
 Fimbrillin – acts as a scaffolding or skeleton; has adhesions (adhesive molecules)
 H – Haunch (German, meaning breath)
attached
 Parts of the Flagella
 Neisseria gonorrhoeae attaches itself to the urogenital epithelium by means of
 Filament – outermost region of the flagella, which contains the flagellin
the fimbriae
 Hook – distinct protein sleeve where the flagellar filament arises; it attaches the
 Forms
filament to the basal body
 Fimbriae
 Basal Body – complex rings connected by a rod – shaped structure; the
 Non – flagellar hairlike appendages (poles are evenly distributed over
movement of the bacteria results from the rotation of the basal body
the entire cell surface)
 Number and Arrangement
 Few to several hundred per cell
 Atrichous – no flagella
 Functions in biofilm formation
 Monotrichous – single polar flagella
 Facilitates adherence to epithelial surfaces in the body
 Lophotrichous – 2 or more flagella at one pole
 Amphitrichous – single flagella found at both ends or poles of the cell
 Pili
 Amphilophotrichous – 2 or more flagella (tuft) on both poles of the cell
 Longer than the fimbriae and only one or two per cell
 Peritrichous – flagella arises from all over the bacterial cell

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  Functions in conjugation  Periplasmic space – between the cell wall and the cell membrane,
 Conjugation – pili facilitates in the transfer of DNA from the donor where the peptidoglycan layer is located; contains digestive enzymes
cell to another and transport proteins (speed up the entry of nutrients into the cell)
 Involved in cell motility  Lipopolysaccharide component
 Grappling hook model (extension – retraction movement) of  Lipid A – ‘endotoxin’ component of the gram negative wall; upon
twitching motility cell death of the bacteria, the endotoxin is released causing
 Gliding motility (in water content) inflammatory reactions, leading to fever shock
 O polysaccharide – function as antigens; aids in distinguishing
Bacterial Cell Wall gram negative species
 Porins – proteins in the outer membranes of the cell wall, which permits
 Confers rigidity to the bacteria – peptidoglycan layer (murein layer)
the passage of nutrients
 All bacteria have cell walls except Mycoplasma and Ureaplasma.
 Spheroplast – partial destruction of gram – negative cell wall
 Murein layer
 Acid – fast bacteria
 Composed of chains of disaccharides cross – linked by peptide bridges
 Found in Mycobacterium, Nocardia and Corynebacterium species
 CHO backbone – alternating N – acetylglucosamine and N –
 Acid Fast Stain
acetylmuramic acid
 Heat Method (Steam Method) – steam drives the stain to the cell
 Short tetrapeptides – identical short chains of D and L amino acids
wall
 The cell envelope is composed of the 2 barriers.
 Cold Method – utilizes detergent (Tergitol)
 Cell wall – outer barrier
 Atypical cell walls
 Cell membrane – cytoplasmic membrane; inner barrier
 Found in Mycoplasma species
 Exoskeleton
 Cell membrane is composed of lipids (STEROLS)
 Maintains the concentration between the cell and the environment
 Damaged cell wall – PROTOPLAST
  concentration of solutes inside the cell
 Found in Proteus species
  concentration of solutes in the environment
 Cells that underwent incomplete destruction by lysosomes, causing a
 Cells expand in order to accommodate the fluid entering the cell
cell with less wall but intact plasma membrane
 Virulence factors (toxins) are synthesized and stored in the cell wall.
 Capable of carrying on metabolism
 Some antimicrobial agents inhibit cell wall synthesis
 L – form – Lister Institute
 Penicillin – disrupts the normal cell wall production by producing daughter
 Functions of the Cell Wall
cells with dysfunctional cell wall
 Structural rigidity
 Gram – staining – routine staining procedure
 Maintains intracellular water (osmotic) pressure
 Gram positive bacteria
 Maintains association of the wall with cell membrane
 Composed of several layers of peptidoglycan and takes up and traps
 Point of anchorage of the flagella
crystal violet
 Virulence factor
 Composed of TEICHOIC ACID that maintains cell viability by
 Site of antibiotic action
maintaining cell wall charge and permeability
 Differentiates bacteria
 Lipoteichoic acid – spans the entire peptidoglycan layer
 Wall teichoic acid – linked to the peptidoglycan layer
 Gram negative bacteria
 Composed of one or two layers of peptidoglycan and easily
decolorized

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 Endospores  Simple diffusion – from an area of higher concentration to an
area of lower concentration
 Certain types of bacteria are able to produce endospores
 Facilitated diffusion – via carriers or channels
 Bacillus spp. – produce spores in aerobic environment
 Osmosis – from an area of lower concentration to an area of
 Clostridium spp. – produce spores in anaerobic environment
higher concentration
 Spherical or oval in shape; dormant; inactive in taking up nutrients; resting stage
 Active processes
 Location – central, terminal or subterminal
 Active transport – use of ATP
 May or may not swell
 Group translocation – exclusively for prokaryotes; substances
 Sportulation or sporogenesis – formation of spores;/ due to nutritional deprivation
are chemically altered during transport across the cell
 Spores are resistant to destruction
membrane
 Contributors to its resistance
 Site of antibiotic action
 Diplicolinic acid
 The cytoplasm is an amorphous gel that contains water, enzymes, ions, subcellular
 Calcium ions – rigidity of the spores
organelles and granules
 During germination, the endospore coat (calcium ions and diplicolinic acid)
 Nucleoid
are broken down  entry of water  metabolism ensues
 Single and continuous double – stranded DNA, which carries the genetic
information that encodes for cell structures and functions; not surrounded by a
Cytoplasm nuclear envelope
 Cytoplasmic membrane  Extrachromosomal DNA
 Fluid mosaic model – phospholipid bilayers move away to allow substances to  Plasmids – double stranded DNA inherited by the progeny cells; covalently
enter the cell and attaches once again closed circles of dsDNA
 Movement of molecules through the phospholipid bilayer is dependent on  Autonomous replication
molecular size  Genetic information that codes for:
 Basic structure  Bacterial virulence
 Phospholipid bilayer – 30% to 60% of its weight  Antimicrobial resistance
 Glycoproteins (production of toxins) and glycolipids (protect and  Virulence – related adhesins
lubricate the cell)  Toxin production
 No sterol content  Tolerance to toxic materials
 Functions  Resistance to heavy metal ions
 Selective barrier  Synthesis of enzymes
 Enzymes and bacterial toxin synthesis and secretion  Transposons – sequences of DNA that jump from one location to another
 Respiration and oxidative phosphorylation  Ribosomes
 Biosynthesis  Prokaryotes – 70 s
 Energy generation – insulating barrier  Svedberg units (s) – indirect measure of the size of ribosomes; express
 Cell membrane proteins involved in: the amount of ribosomes;/ function of the weight, size and the shape of
 Electron transport the bacteria
 Lipid biosynthesis  Total cellular RNA – 80% rRNA; 20% tRNA and mRNA
 Synthesis of the cell wall constituents  Bacterial RNA
 DNA replication  30s – 16s RNA
 Active transport of materials into the cytoplasm  50s – 23s and 5s RNA
 Passive processes  Functions for protein synthesis and the site of antimicrobial action

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  Some antibacterial agents attack the ribosomes, thus, inhibiting the production  Microbes to microbes
of proteins (ex. Streptomycin, Gentamycin)  Microbes with other organisms
 Enzymes  Microbes with the non – living environment
 Anabolic enzymes – simpler to more complex substances  Humans and microorganisms interact at many levels
 Catabolic enzymes – breakdown of complex to simpler substances
 Inclusions and Granules – function as food reserves
Symbiotic Relationships
 Polysaccharide inclusions – chains of monosaccharides
 Glycogen and starch  Symbiosis – living together or close association of two dissimilar organisms
 Stained by iodine Type Description Example
 Lipid inclusions - Neither symbiont is
 Appear in various species of Mycobacterium, Bacillus, Azotobacter and Neutralism affected by the
Spirillum relationship
 Stained by fat – soluble dyes - Beneficial to one symbiont
- No consequence to the Indigenous (normal)
 Metachromatic dyes or Volutin granules Commensalism
other organism (neither microflora
 Reddish pink (when stained by methylene blue)
harmed nor benefited)
 Composed of inorganic phosphate  converts to ATP for energy
- Normal GIT flora
 Corynebacterium, Yersinia and Mycobacterium
(obtains nutrients
 Sulfur granules
- Beneficial to both from host; produces
 Energy reserves for ‘sulfur bacteria’ Mutualism
symbionts vitamin K)
 Oxidation of sulfur an sulfur-containing compounds - Bacterial
 Carboxysomes antagonism
 Ribulose – 1,5 – diphosphate carboxylase - Beneficial to one symbiont
Opportunistic
 For carbon fixation (carbon as a sole source of energy) Parasitism - Detrimental to other
organisms
 Nitrifying bacteria, Cyanobacteria and Thiobacillus symbiont
 Gas vacuoles
 Hollow cavities covered by proteins SIDE NOTES
 Responsible for the buoyancy of bacteria – position itself on a depth  Bacterial antagonism – normal flora of the host protects it from pathogenic
where it can freely obtain light and oxygen bacterial invasion
 Magnetosomes  Synergism
 Composed of iron oxide – act as a magnet  Synergistic infections – team up of two or more microorganisms to produce a
 Magnetospirillium magnetotacticum disease that neither could cause by itself
 Mix infections – infections caused by multiple bacteria (polymicrobic)
HOST – BACTERIA INTERACTION  Vincent’s Disease (Trench Mouth or Acute Necrotizing Ulcerative
Gingivitis) – caused by Fusobacterium, Actinomyces, Prevotella, and
Ecology spirochetes
 Bacterial Vaginosis – caused by Mobilunous spp. and Gardnerella
 Systematic study of interrelationships that exist between organisms and their
vaginalis
environment and other living organisms and its non – living environment
 Microbial ecology
 Numerous interrelationships between microorganisms and the world around
them

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 BIOSAFETY MICROBIAL RESERVOIR AND TRANSMISSION
 Biosafety level – degree of the disease (severity) that the agent may confer to the Reservoir
individual in contact with it
 Sites where microorganisms thrive and survive until it is transferred to a susceptible
a. Level 1
host
 Healthy individuals; unknown effect to immunocompromised individuals
 Living Reservoir
 Used for laboratory teaching
 Humans – has infectious diseases; carriers
 Passive carriers – carry pathogens without ever having had the
b. Level 2
disease; no symptoms of illness
 Being sought in clinical specimens, commonly isolated in association
 Convalescent carriers – harbor pathogens and transmit the
with disease
pathogen while in a period of recovery
c. Level 3
 Active carrier – completely recover from the disease but continue
 Handling specimens known to contain viruses
to harbor the pathogen (ex. Salmonella typhi)
 Not in routine clinical tests
 Animals
 Causes zoonotic infections
 Direct Transmission – animal bites
d. Level 4
 Indirect Transmission – blood meal from the animal reservoir;
 Bioterrorism – use of microorganisms to impose fear or harm to a
consumption of infected animal products
population
 Insects – bridge the transmission of reservoir and the host
 Non – living Reservoir
Biological Safety Cabinets
 Environment – air, soil and dust
 Inoculating hoods; working under inoculating hoods minimizes the spread of  Food, water and milk – vehicles of transmission via contamination
aerosols  Fomites
 High Efficiency Particulate Air (HEPA)
 Filters that air that may be containing particles (sterilizes the air) Modes of Transmission
 > 0.3 m particles are trapped in the pores of the filter
 Acquisition of microbial agents by the human host
a. Class I
 Principal Modes of Transmission
 Utilizes a vacuum (negative pressure) that pulls the air towards it
 Contact
 HEPA filters and UV light
 Airborne
b. Class II
 Droplet
 Laminar flow biosafety cabinet – air flows in laminar shapes
 Vehicular
 Sterilizes the air inside and the air coming out
 Vector – borne
 Variable sash opening
c. Class III
 Affords the most protection Microorganism Colonization Surface
 Equipped with glove compartments that confer minimum exposure to  Colonization – initial step of encounter; persistent survival of the microorganism on a
biological specimen surface of the human body
 UV light and HEPA filters  Persistent survival – despite efforts to remove the microorganism, the
microorganism remains
 Colonization is dictated by the host’s defenses

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  Inducible defenses – defense is triggered in response to the presence of  Mortality rate – the number of deaths; ratio of the number of deaths during a
the microbial agent (ex. Phagocytosis, Inflammation) specified time per a specified population
 Specific defenses – defenses produced that is specifically directed  Morbidity rate – the number of incidence; the number of individuals affected
towards a particular microorganism (ex. Antibodies) by an infectious diseases during a specified time per a specifically defined
population
 Incidence rate – the number of new cases of a disease in a defined
INTERACTIONS: HOST, PATHOGEN, ENVIRONMENT population in a specific time period
 Prevalence rate
 Three factors play a role in infectious diseases (Koch’s postulates)
 FREQUENCY OF OCCURRENCE OF INFECTIOUS DISEASE
 Pathogen
 Sporadic infections – infectious diseases that occur occasionally within a
 Virulence – degree of pathogenicity (ability to cause disease) of the
population in a given geographic area
organism
 It may be due to (1) neglect in the immunization or (2) sanitation.
 Portal of Entry – gain access to the susceptible host’s body
 Endemic infections – constantly present infectious disease within a population
 Infectious Dose – the number of organisms to be able to cause a
of a particular geographic area
disease
 Factors affecting the rate of occurrence
 Host – considers the following: health status, nutritional status and the
 Conditions of the environment
susceptibility of the host
 Susceptibility of the population
 Environment – considers the appropriate reservoir
 Behavioral factors
 Physical factors – weather, climate or seasons, temperature and
 Number of immune people within a population
geographical location
 Virulence of the pathogen
 Sanitation and hygiene – adequate waste disposal
 Presence of a reservoir
 Availability of potable water
 Epidemic infections – outbreaks: greater than usual number of cases usually
 CHAIN OF INFECTION
occurring within a relatively short period of time
- Pathogen
 Pandemic infections – wide geographic area affecting exceptionally large
- Reservoir
population; diseases that are occurring in epidemic proportions in many
- Portal of Exit
countries simultaneously
- Transmission
 NOSOCOMIAL INFECTIONS
- Portal of Entry
 Hospital – acquired infections; infectious diseases that erupt within fourteen
- Susceptible Host
days of hospital discharge
 Caused by both Gram – positive cocci and Gram – negative bacilli
Epidemiology  Staphylococcus aureus
 The study of diseases, causes, transmission and its source and the factors that  Enterococcus spp.
determine the frequency, distribution and determinants of disease in human  Coagulase – negative Staphylococcus spp.
population.  Pseudomonas aeruginosa
 Communicable disease – infectious diseases that can be transmitted from one host  Enterobacter spp.
to another  Klebsiella spp.
 Contagious disease – communicable diseases that are easily transmitted from one  Infections that develop
person to another  Urinary tract infections
 Zoonotic (Zoonoses) – infections or disease from animal sources transmitted to a  Surgical wound infections
human host  Lower respiratory tract infections
 RATIOS AND RATES  Septicemia

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  Susceptibility to nosocomial infections  Termination – the two DNA helices separate
 Patients who have undergone surgeries  Genetic Recombination
 Burn patients  Natural mechanism of DNA transfer from one microorganism to another
 Diabetic and cancer patients  Vertical Gene Transfer – ‘mutation’; permanent alteration of the genetic
 Extreme ages – pediatric and geriatric patients material in a cell; basis of the production of strains; all future generations
 Patients who are taking in immunosuppressive drugs (patients derived from the parent cell are called MUTANTS
undergoing graft transplants in order to prevent the recipient body from  Horizontal Gene Transfer
attacking the donor organ)  Transformation
 Patients who take in steroids  Uptake of free DNA fragments from the environment and the
 Insertion catheters (hemodialysis patients) expression of the genetic information in the recipient cell
 COMMUNITY ACQUIRED INFECTIONS – infections acquired outside of the health  Competence – refers to the ability of the recipient cell to take up
care facility; the disease must be present upon the patient’s arrival in the health extracellular DNA from the environment (only competent cells can
care facility be transformed)
 Steps:
 Lysis of bacterial cells release DNA fragments composed of 10 –
MICROBIAL GENETICS
20 genes
 Genomics – the study of organism’s genome  DNA is taken up by a competent cell by the competence
 Bacterial DNA factor (located at the poles of the bacteria where DNA uptake
 1/3 of the cell’s total volume; 1.5 nm long; 500x the length of the bacteria is common)
(supercoiled or tightly packed) - Requires enzymes: (1) cell wall degradation enzymes; (2)
 Chromosome – intracellular source of genetic information; it is a single, circular cell membrane transport proteins; and (3) DNA binding
molecule of DNA that is haploid (single set of genetic information); it occurs proteins
singly per cell; localized within the cytosol (nucleoid)  Incorporation occurs when the DNA enters the cell
 Genome – complete set of genes in an organism; varies by species  Internalization; the single DNA recombines with the homologous
 Plasmids regions of the bacterial chromosomes (if compatible)
 Stable extrachromosomal DNA  Transformation; the recipient cell is transformed; confers
 It contains 2% of the total genetic information characteristics to the recipient bacteria
 Types of plasmids  May occur in a crowded or confluent environment; may occur in
 F Plasmids – allows the transfer of genetic materials from donor to environment with more than a single bacterial cell
recipient cell through a recombination process  Transformation may decrease the virulence of the recipient cell, as
 R Plasmids – confers protective functions or resistance of microorganisms the uptake of genes are non – specific (bacteria may take up
to antimicrobial agents defective genes)
 DNA Replication  Conjugation
 Binary fission – two daughter cells are produced and are genetically identical  Two live bacterial cells come together and the donor cell directly
to the parent cell transfers DNA to the recipient cell
 Three stages  Conjugation pilus (ex. E. coli)
 Initiation – DNA unwinds and strands separate  Cell – to – cell contact
 Oric – area where initiation starts  F+ - donor cell
 Elongation – DNA pairing splits; enzymes are synthesized and a new  F- - recipient cell
polynucleotide strand of DNA for each of the two new template is  F factor (F) – plasmid which contains about 100 genes which
produced encodes for plasmid production

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  OriT – region where the plasmid is replicated  Precautionary measures:
 High frequency of recombination (Hfr) strains – cells exhibiting the  PPE
ability to donate chromosomal genes; the F factor has attached to  Personal grooming
the chromosomes  Sterilization
 Transduction  Disinfection
 Requires a virus to carry chromosomal DNA fragments from a donor  Handwashing
to a recipient cell (bacteriophage)  Proper disposal of needles and sharps
 Methods b. Surgical Asepsis
 Lytic cycle (Generalized Transduction) – virulent phages; lyse or  Includes practices used to render and keep objects and areas sterile
destroy the chromosome of the donor cell; penetrate the  Usually practiced in surgery rooms and delivery rooms
recipient cells and replicate within and lyse in order to be  ‘The Sterile Technique’
released  Laboratory Techniques of Infection Control
 Lysogenic cycle (Specialized Transduction) – temperate  Definition of Terms
phages; phages invade the host but do not directly cause lysis;  Sterilization – process by which there is the complete destruction of living
incorporate into the cell as prophages; only specific gene is organisms
transferred, thus a specific trait is expressed by the bacteria  Disinfection – destruction of vegetative pathogens, however, spores are
 Virulent phage – phages the are capable of causing infection and not eliminated
the destruction and death of bacterial cell  Disinfectant – agent used to carry out disinfection; chemical in nature
 Temperate phage – phage DNA is incorporated into a bacteria’s  Antisepsis – prevention of infection by inhibiting or arresting the growth
DNA and is replicated with it and multiplication of pathogens
 Conditions of genetic recombination  Removal of transient microorganisms
 The donor DNA must be ready for transfer  Reduction in the normal flora
 The donor DNA is transferred to the recipient cell.  Asceptic – absence of infectious agents
 The donor DNA is taken up successfully by the recipient cell and must be  Microbicidal – involves the killing of microorganisms
in a stable state in the recipient.  Bactericidal – bacteria
 Fungicidal – fungi
 Virocidal – virus
INFECTION CONTROL  Microbiostasis – inhibition of growth and multiplication of microorganisms
 Degerming – removal of microbes from a limited area (applied in
 Methods or activities that are taken to prevent infections from occurring the health venipuncture)
care settings  Sanitation – treatment intended to lower microbial count in eating and
 Break the chain of infection drinking utensils; facilitate safe public health levels
 Eliminate and contain reservoir  Thermal Death Point – lowest temperature to kill a cell within 10 minutes
 Interrupt the transmission of pathogens  Thermal Death Time – minimal length of time to kill microorganisms in a
 Protect persons from pathogens suspension within a given temperature
 Asepsis or Asceptic Technique  Radical Reduction – time to kill 90% of the population at a given
a. Medical Asepsis temperature
 Includes all precautionary measures necessary to prevent direct transfer  Plasmolysis – shrinkage of the cell’s cytoplasm caused by the osmotic loss
of pathogens and indirect transfer of pathogens through instruments or of water; growth of the cell is inhibited as the plasma membrane pulls
air, equipment and any other inanimate objects away from the ell wall
 ‘The Clean Technique’

10 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Osmotic Lysis – rupturing of the bacterial cell caused by excessive water days with incubation in between
intake due to weak or damaged cell wall heating
 Factors affecting microbial growth DRY HEAT
Burning contaminants to Sterilize inoculating loops at
 Size of bacterial population – the more bacteria, the longer the kill time Direct Flaming
ashes 1870°C
(time for the complete eradication of microorganisms)
Incineration Burning to ashes 1800°C - 6500°C
 Exposure time – specific time of contact with microorganism to effectively
Hot Air Sterilizer Oxidation 150°C to 180°C for 2 to 4 hours
eradicate the microbes Removes microbes by passage of
 Temperature, concentration and pH at which these agents work best Separation of bacteria from
FILTRATION liquid or gas through a screen –
suspending liquid
should be achieved like material
 Protective features of microorganisms COLD
 Spores produced by Bacillus and Clostridium species Refrigeration Bacteriostatic effects
 Porrins seen in Pseudomonas species Decreased chemical
Preservation of microbial culture;
Deep Freezing reactions and possible
 Mycolic acid in the cell walls of Mycobacterium species -50°C to -95°C
changes in protein
 Interaction of the microorganism with the environment
Water removed by increase
 Dirty environment – the presence of substances in the dirt and Lyophilization Dehydration
vacuum at low temperatures
organic substances protect microorganisms Alteration of molecular
 Presence of lipids and fats makes the penetration of chemical agents Preservation of color, flavor and
HIGH PRESSURE structure of proteins and
nutrient values
difficult carbohydrates
 General mechanism of actions of infection control Removing of water from
DESSICATION Disruption of metabolism
 Alteration of microbial permeability microbes; bacteriostatic
 Damage to protein and nucleic acids OSMOTIC PRESSURE Plasmolysis Loss of water from microbial cells
RADIATION
 Damage to the cell wall
Dislodges electron; formation of
Ionizing Destruction of DNA free radicals and highly active
Microbial Control chemicals
260 nm; formation of thymine
Non – ionizing Damage to DNA
dimers
METHOD MOA COMMENTS CHEMICAL METHODS
PHYSICAL METHODS PHENOL
MOIST HEAT Disruption of plasma
Used in throat lozenges and
Heat or boiling water to produce steam; penetrates more efficiently; moist water is a better Phenol (Carbolic acid) membrane and the
sprays
conductor of heat than air. Uses temperature which ranges from 60°C to 135°C. denaturation of enzymes
Boiling or Flowing Steam Denaturation of proteins Kills vegetative pathogens Disruption of plasma Derivatives of phenol that are
Steam under pressure; 10 – 15 Phenolics membrane and the reactive with organic materials
Autoclaving Denaturation of proteins
minutes, 15 psi; 121°C denaturation of enzymes (Ex. O – phenylphenol  Lysol)
Kills vegetative cells, but not Derivatives of phenol that
Boiling Water Denaturation of proteins resistant microorganisms; 100°C contain two phenolic groups
for 30 minutes Disruption of plasma connected by bridges (Ex.
Bisphenols
Heat treatment for milk that kills membrane Triclosan – inhibits the enzymes
PASTUERIZATION Denaturation of proteins pathogens and non – pathogens; needed for the synthesis of fatty
kills Mycobacterium tuberculosis acids)
Sterilize heat sensitive products; Disruption of plasma Biocidal against most vegetative
BIGUANDES
TYNDALLIZATION Denaturation of proteins kills vegetative organisms; 100°C membrane bacteria (Ex. Chlorhexidine)
for 30 minutes in 3 successive HALOGENS

11 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Inhibits protein function and Effective antiseptic (tincture or Ozone Oxidation Chlorination
Iodine
strong oxidizing agent iodophor) Hydrogen Peroxide Oxidation Disinfection
May act alone or as components Benzoyl Peroxide Oxidation Treating wound infection
of inorganic and organic Peracetic acid Oxidation Sporicides; sterilant
compounds
- Calcium hypochlorite
Notes
(Chloride of lime) – disinfect
 Pasteurization
dairy equipment
- Sodium hypochlorite (Chlorox)  Forms of Pasteurization
– household disinfectant  Low Temperature Pasteurization – Classical Holding Method; Batch
Strong oxidizing agent with
- Sodium dichloroisocyanurate Method; Low Temperature – Long Time Method (63°C for 30 minutes)
Chlorine hypochlorous acid; alters
(Chlor – Floc) – contains  Flash Pasteurization – High Temperature – Short Time Method (71.6°C for 15
cellular components
flocculates suspended in water seconds)
- Chlorine dioxide – kills
 Ultrahigh Temperature Pasteurization - 140°C within less than 1 second
endospores and anthrax
 Autoclaving
bacterium
- Chloramine (Chlorine and   pressure =  temperature
ammonia) – disinfectants,  Limitations of Autoclaving
antiseptics and sanitizing  Prions cannot be eliminated (eliminated by heating at 132°C for 4.5 hours)
agents  Melts instruments
Protein denaturation and Kills bacteria and fungi but not  Dulls sharp instruments
ALCOHOL
lipid dissolution endospores
 Chemical breakdown
HEAVY METALS
 Oily substances cannot be treated
Mercury (Mercuric Denaturation of enzymes and
Bacteriostatic
chloride) other essential proteins
Denaturation of enzymes and
Copper (Copper sulfate)
other essential proteins
Destroy green algae MICROBIAL GROWTH AND REPRODUCTION
Zinc (Zinc chloride; zinc Denaturation of enzymes and Mouthwashes (Zinc chloride)
oxide) other essential proteins Antifungal agent (Zinc oxide) Bacterial Physiology
Denaturation of enzymes and Agent against gonorrheal  Nutritional Requirements
Silver (Silver nitrate)
other essential proteins ophthalmic neonatorum
 Physical Requirements
SURFACE ACTIVE AGENTS
 Temperature – degree at which the organism exhibit growth
Mechanical removal of
Soap Emulsification; degerming agent Temperature Optimum
microbes through scrubbing Description
Range Temperature
Enzyme inactivation or Negatively – charged particles
Acid Anionic Detergents
disruption react with plasma membrane Psychrophiles Cold loving bacteria 0°C to 20°C 15°C
Enzyme inhibition, protein
Quarternary Ammonium Bactericidal and bacteriostatic
denaturation, disruption of
Compounds agents
plasma membrane Organisms that cause
ORGANIC ACIDS Metabolic inhibition Food and cosmetics diseases in humans;
Protein denaturation Mesophiles 10°C to 45°C 35°C
moderate – temperature
(formation of crosslinks with growing microbes
ALDEHYDES Antimicrobials
organic functional groups in
proteins)
GASEOUS STERILANTS Protein denaturation Sterilizing agent
Thermophiles Heat loving bacteria 40°C to 70°C 60°C
PEROXYGENS

12 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Oxygen – reflects that mechanism for satisfying requirement needs
Heat resistant
 Obligate aerobes – organisms that require an absolute requirement
microorganisms; Can
tolerate very high for oxygen; oxygen functions as the final electron acceptor for
Hyperthermophiles 75°C to 113°C 80°C cellular energy
temperatures; Can
withstand destruction by  Obligate anaerobes – oxygen is toxic or fatal for the organism;
heat below 113°C growth is inhibited by oxygen tension of 10-5
 Facultative aerobes – organisms capable of growth under both
aerobic and anaerobic environment
 pH
 Microaerophilic microbes – organisms that grow best at lower
 Acidophiles – grow in an acidic medium (pH 1 to 5.5)
oxygen tension; increased oxygen tension is a source of inhibition of
 Neutrophiles – grow in a neutral medium (pH 6 to 8)
growth
 Alkalinophiles – grow in an alkaline medium (pH >8)
 Aerotolerant anaerobes – anaerobes which are not killed by their
 Hydrostatic and Osmotic Pressure and Ionic Strength
exposure to oxygen
 Barophiles – organisms that require high hydrostatic pressure; can
 Carbon Dioxide – Capnophiles (requires increased carbon dioxide
tolerate pressure greater than 16,000 psi
concentration at 5 to 10%)
 Osmophiles – organisms that require high osmotic pressure in order
 Inorganic Ions
to survive
 Carbon – essential for life; chemical structure of all living things
 Halophile – requires high levels of salt; can survive in salt water
 Nitrogen – required for the synthesis of enzymes, cellular proteins,
(marine) environment
nucleic acids; nitrate and ammonia
 Halotolerant – organisms that can tolerate high levels of salt
 Phosphorus – required for the synthesis of nucleotides (DNA and
 Chemical Requirements
RNA), energy storage molecule (ATP) and for the maintenance of
 Energy Source
the cell membrane (phospholipids)
 Chemotrophs – microbes which release energy from organic
 Sulfur – essential for the protein bonds (folding of the proteins)
materials by oxidation
 Trace Elements – needed as cofactors (iron, copper, molybdenum,
 Fermentation – anaerobic process
zinc)
 Respiration – aerobic process
 Organic Nutrients
 Photosynthesis – maintain environmental or ecological balance
 Organotrophs – requires carbohydrates, amino acids, vitamins and
(concentration of oxygen and carbon dioxide)
nucleic acids for growth
 Carbon Source
 Saccarotrophs – sugar – loving bacteria
 Autotrophs or Lithotrophs
 Auxotrophs – organisms that grown in an environment
 Uses carbon dioxides as the sole source of carbon; organisms
supplemented by a particular growth factor that is not required by a
that synthesize carbon skeletons (carbon ready for binding with
wild strain
other elements)
 Growth Factors – promotes growth of organisms, either in vivo or in vitro
 Requires water, carbon dioxide and inorganic salts for growth
 Classification
- Photolithotrophs – derive energy from light Bacterial Division
- Chemolithotrophs – energy derived from oxidation of  Bacterial Growth – increase in the bacterial number; regulated by the nutrition
inorganic substances and molecules obtained by the bacteria from the environment
- Organotrophs – organotrophic microbes; heterotrophic  Binary Fission – normal means of asexual reproduction in prokaryotic cells where
microbes; bacteria that are unable to use carbon dioxide cell divides into two cells whilst containing genetic consistency
as the sole source of carbon; still requires carbon dioxide in  Elongation of the bacteria – enlargement of the cell wall, cell membrane and
its organic form (ex. Glucose) the overall volume

13 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Replication of chromosomes  Phases
 Separation of the newly formed chromosomes; formation of the septa (septal  Lag phase
invagination)  No immediate increase in cell number
 Septum forms in the middle of the cell and deepens and the cell wall  Period of adaptation
separates  Increase in the metabolic activity of the cell
 Generation Time  Synthesis of new enzymes, cofactors and metabolic intermediates
 The time until the microorganism is able to complete cell division and double  No cell division takes place
its population  Old depleted ATP, cofactors and ribosomes
 Time at which specific symptoms of a disease appear  Different medium from where it is initially placed
 Period of recovery
𝐺𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 = 𝑁𝑜2𝑛  Log phase
 Cell growth and cell division at a maximal rate
 Bacterial cells are sensitive to adverse conditions
𝑙𝑜𝑔𝑁 − log 𝑁𝑜  Influenced by:
𝑛 =
𝑙𝑜𝑔2  Temperature
 Where  Carbon source
 N – final count  Nutrition
 No – initial count  Oxygen tension
 n – generation time  Bacterial division continues only when the conditions are optimum
 Active cell division increases the bacterial population
𝑡  Increase in the number of bacterial cells causes an increase in the
𝐺= utilization of nutrients leading to the accumulation of metabolic waste
𝑁 products
  in the density of the culture =  bacterial population =  in the oxygen
 Where
supply
 t – number of hours or minutes of exponential growth
 Difficulty of the oxygen to penetrate the colony
 N – number of generations
 Mass on top of the colonies has a ready access to oxygen while
 Varies among species
other bacterial colonies have no access
 Pseudomonas – 14 minutes
 Cell growth is at a maximal rate and is influenced by the genetic potential
 Staphylococcus aureus – 30 minutes
of the bacteria; nature of the medium and environmental conditions
 Mycobacterium tuberculosis – 15 to 24 hours
 Balanced growth – cell constituents are manufactured at a constant
 Treponema pallidum – 33 hours
rate
 Incubation Period – time from the entry of the pathogen in the body until the first
 Unbalanced growth – rates of synthesis of cellular components vary
symptom appears
relative to one another
 Shift up – microorganism is transferred to a nutritionally poor to a
Bacterial Growth (Batch Culture) nutritionally rich medium; construction of ribosomes occur (to
 Batch Culture – pure culture is introduced in a closed culture vessel enhance the capacity of the organism to synthesize proteins)
 Closed culture vessel  Shift down – microorganism is transferred to a nutritionally rich to
 Single batch of medium – no fresh medium is introduced a nutritionally poor medium; lag is observed (biosynthesis of
  nutrient concentration;  waste concentration unavailable nutrients)
 Stationary phase – phase of equilibrium

14 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Population of living cells = population of dying cells and (2) Turbidostats (turbidity of the culture is held constant by manipulating the
 Population growth ceases rate at which the medium is fed)
 Exhaustion of nutrients  Measurement
 Cells undergo processes for self – preservation  Direct Measurement
 Production of endospores through the process of sporulation  Plate Counts – live bacterial cell count
(completed within 8 hours)  Use of the colony counter
 Bacteria respond to oxygen availability  Viable cell count – measure the colony formed
 Physiologic and genetic expression  Consumes time (16 to 24 hours)
- Produces chaperone proteins (proteins that enable  Each live bacterium grows and divides to produce a single colony
microorganisms to prevent protein denaturation and repair  Serial dilutions are performed –the original inoculum is diluted several
denatured proteins) times
- Enhance the peptidoglycan cross – linkages  Pour plates – uses a vacant petri dish; the colonies grow in and
- Production of DNA binding proteins from starved cells on solidified medium
 Accumulation of metabolic waste products  Spread plates – bacteria are inoculated on a plate containing
 Death phase – Logarithmic decline phase a solid medium and the colonies grow on solidified surface
 Rate of cell division stops completely  Filtration – Membrane Filtration Technique
 Theories  Bacteria are retained on the surface of membrane filter and then
 Viable but Non – culturable (VBNC) transferred to a culture medium to grow and subsequently counted
 Organisms are in the stationary phase without morphological  Applied frequently to detect and enumerate coliform bacteria
changes (fecal pollution of food and water)  bacterial water analysis
 Genetic response to starving  Filter – small, thin porous membrane (<0.3 mm)
 When introduced in a medium where physiologic requirements  The Most Probable Number (MPN) method
are met, cell division resumes  The statistical estimating technique is based on the fact that the
 Programmed Cell Death greater number of bacteria in a sample, the more dilution is needed
 A fraction of the population is programmed to die in order for to reduce the density
the nutrients to leak out and to be used for continuous growth  Used when microbes being counted will not grow in a solid media
 Biphasic Growth  Also used when the growth of bacteria in a liquid differential
 Biphasic – two phases of the growth curve medium is used to identify the microbe
 Seen in bacteria capable of utilizing two different carbon sources  Direct Microscopic Count
 Phases  Microbes in a measured volume of a bacterial suspension are
 Initial Growth Burst – first source of carbon is utilized and exhausted by the counted with the use of a specially designed slide
microorganism  Breed Count Method
 Stationary Phase – synthesis of enzymes and transport mechanisms are  Petroff – Hausser Cell Counter
initiated; proper physiologic conditions are met  Electronic Cell Counter
 Second Phase of Exponential Growth – utilization of the second source of  Indirect Measurement
carbon  Turbidity
  bacteria in a liquid medium =  turbidity of the medium
Bacterial Growth (Continuous Culture System)  Metabolic Activity
 The amount of a certain metabolic product is in direct proportion to
 Culture systems that can maintain a microbial population in exponential growth by
the number of bacteria present (carbon dioxide or acid)
two methods: (1) Chemostats (a population can be kept in the exponential growth
phase indefinitely by draining off the spent medium and adding fresh medium);

15 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Dry Weight  Anaerobic Jar
 Used for filamentous bacteria and molds  Oxygen is removed from the system through reaction with hydrogen
 Fungus is removed from the growth medium, filtered to remove ions (which is externally added by GasPacks), leading to the
extraneous material and dried in a desiccator formation of water
 Palladium catalyst – catalyst that facilitates the reaction between
Incubation Techniques hydrogen and oxygen to form water
 Methylene blue – indicator of oxygen tension
 Grow the microorganisms outside the body and be able to provide the necessary
factors needed for them to grow and thrive outside the body.
Interpretation of Bacterial Culture
 Temperature
 Colony – groups of bacterial cells on a solid medium, which is visible to the naked
 For incubators and water baths
eye and shows various patterns
 Quality control – prevention of wide fluctuations in temperature; no more  Nutrient availability
than + or – 1o to 2oC  Hardness of medium
 Humidity  Bacterial chemotaxis
 Amount of moisture in the air  Nutrient diffusion
 To exhibit maximal growth, a humidity of 70% and greater must be present  Presence of liquid on the medium
 Differences in growth – gradients of oxygen, nutrients and toxic products in the
 pH
colony
 Degree of acidity or alkalinity of the medium
 Interpretation is conducted after 24 hours to 48 hours of incubation
 Optimal pH for growth – pH 6.5 to 7.5  Colony Interpretation
 Bacteria often produce acid as metabolic by – products, which may be  Amount of growth
toxic to microorganisms  None
 Deterioration of growth of microorganisms are exhibited during the  Scanty
stationary phase of bacterial growth (wherein the number of living cells  Few
are equal to the number of dead cells)  Moderate
 Chemical buffers, such as peptones, amino acids and phosphate salts,  Heavy
neutralize the acidic environment.  Appearance of colony – done on isolated colonies
 Size of the colony – diameter of the colonies is determined
 Oxygen
 Pinpoint
 Oxygen is used for energy production  Small
 Concentration of oxygen in the medium  Medium
 Highest concentration on the top of the medium  Large
 Lowest concentration on the bottom of the medium  Form of the colony
 20 -21%  15%  10%  5%  Punctiform
 Effect of oxygen  Circular
 Inactivation of proteins (enzymes)  Irregular
 Sulfhydryl bonds are oxidized and other sensitive groups resent  Filamentous
in enzymes  Rhizoid
 Toxic effects to anaerobes, wherein they are destroyed and  Elevation of the colony
oxidized by oxygen  Flat
 Raised

16 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Convex  Butyric acid – Clostridium species
 Pulvinate  Changes in differential medium
 Umbonate  Differentiation of bacteria through the addition of dyes, pH indicators and
 Margin of the colony other ingredients to the medium and the manifestation of the reaction in
 Entire the differential medium
 Undulate  Mannitol Salt Agar (MSA)
 Robate  Contains 7.5% salt  SELECTIVE to halophilic microorganisms
 Filamentous  Alcohol mannitol – fermentative ability (production of gas and
 Rhizoid acids)
 Color – some bacteria have chromogenic properties or ability, which  Appearance
allows them to produce pigments. It may be diffusible or non – diffusible.  Uninoculated – slight peach in color
 Manner of reporting – amount of growth, changes in the agar, size, elevation,  Fermented – yellow (pH driven change in color by
form, color and margins fermentation)
 Reaction with the medium used  MacConkey Agar (MAC)
 Hemolysis on blood agar medium  Differential to LACTOSE FERMENTERS
 Patterns of hemolysis  Incorporated with crystal violet (dye) to prevent the growth of Gram
 Alpha hemolysis – partial lysis of red blood cells; partial clearing – positive microorganisms
under and surrounding the colonies, producing a greenish  Lactose fermenters form purple colonies (the acids produced by the
coloration of the medium due to the reduction of hemoglobin to bacteria will precipitate the dye causing the colonies to take up the
methemoglobin in the medium. dye)
 Alpha prime hemolysis – a wide area of hemolysis; small zone of  Non – lactose fermenters produce colorless colonies.
complete hemolysis (beta) surrounded by an area of partial lysis  Eosin Methylene Blue (EMB)
(alpha).  Selective to Gram – negative bacteria; differential to lactose
 Beta hemolysis – zone of complete clearing under and the fermenters
surrounding medium of the colony.  Fermentation produces the characteristic green metallic sheen,
 Gamma hemolysis – no hemolysis; the microorganism is incapable which highly suggestive of Escherichia coli but not diagnostic.
of hemolysis  Odor – production of a characteristic odor
 Pigment production  Pseudomonas aeruginosa – grape juice odor
 Biochemical Reactions  Proteus species – burnt chocolate odor
 Carbohydrate fermentation  Eikenella corrodens – bleach – like odor
 Fermentation – it is an energy yielding process, where organic molecules  Alkaligenes faecalis – freshly cut apple odor
serve as both electron donors and acceptors. Carbohydrates undergo  Clostridium species – fecal, putrid odor
glycolysis, yielding characteristic by – products.  Production of acid
 By – products of fermentation – carbon dioxide and water  Triple Sugar Iron Agar (TSIA)
 Bacteria produce a specific, characteristic by – product, which may be  Enteric bacteria identification
useful for identification  Constituents of TSIA
 Lactic acid – Lactobacillus, Streptococcus  Contains three different carbohydrates – lactose, sucrose and
 Alcohol – Zymomonas, Saccharomyces cerivisae glucose
 2, 3 – butanediol – Enterobacter, Bacillus, Serratia - Lactose and sucrose are 10x greater in concentration than
 Formic acid – Escherichia species, Enterobacter species, Proteus glucose
species, Salmonella species

17 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Phenol red – pH indicator; turns yellow in the presence of acid  Inadequate culture method
(pH <6.8)  Faulty collection and transport
- In an uninoculated state, it is buffered at pH 7.4 and is  Bacteria are sensitive to changes in the environment; it must be in a
color red. comfortable environment
- Red in an alkaline condition (pH of 7.4)  Obtaining a specimen
- Yellow in an acidic condition (pH of 6.8)  Obtained before administration of antimicrobial agents
 Beef extract, peptone, yeast extract and Proteus peptone –  One must ask if the patient has been taking in antimicrobial agents
sources of nutrients for non – fastidious organisms.  Obtained where suspected organism is likely to be found
 Iron – in the form of ferrous sulfate or sodium thiosulfate  Considerations
- Assess the ability of the organism to produce hydrogen  Stage of the disease
sulfide  Quantity
 Structure of TSIA  Transport or delivery – conditions at which the bacteria is exposed to; when
 Slant and deep – two chambers of reaction the specimen has arrived in the laboratory, it must still have viable cells in the
- Slant – aerobic reaction; exposed to an oxygen source sample
- Deep – anaerobic reaction; oxygen cannot penetrate the  Clinical information – nature and source of the specimen; information of the
chamber patients
 Length of the slant must be equal to the length of the deep  Rejection of specimen
- Must be 1 cm in length to prevent of the entry of oxygen in  Label does not match the information of the requisition slip
the deep  Transportation errors
 Interpretation of TSIA reaction  Improper temperature
 pH indicator changes (A for acidic; K for alkaline)  Improper medium
- Glucose fermentation  K/A reaction; due to the low  Quantity of the specimen is insufficient for testing
concentration of glucose in the medium.  Leaking of the specimen
 Gas production – cracks within the medium or pulling away of  Harm in the quantity of the specimen
the medium from the bottom of the tube  Harm to the individuals handling the specimen  BIOHAZARDOUS
 Hydrogen sulfide production – black precipitate formation of  Transport time exceeds 2 hours post – collection and is not in a preservative
blackening of the medium  Normal flora overgrows the pathogen
 Starch hydrolysis  Bacteria is exposed to environmental conditions
 Basic biochemical reaction that detects the ability of the organism to  Specimen is received in a fixative
degrade starch by the action of hydrolases  Specimen is obtained from a site known to have anaerobes as part of the
 By – products of starch hydrolysis – glucose, dextrin and maltose normal flora
 Carbohydrate by – products are subjected to fermentation  The specimen is dried up (ex. swabs)
 Information of questionable value
 Other considerations
 Stage of disease as to when a specimen should be collected
SPECIMEN COLLECTION  Enteric pathogens  acute stage
 CSF  soon after the onset of the disease than when the acute symptoms
 Laboratory results are limited to the quality of the specimen and its condition
 Criteria of a good specimen for bacteriologic study appear
 Representative of the disease process  Negative results DOES NOT indicated the absence of the bacteria.
 Ensure complete and accurate examination  Examination of the specimen
 Failure to isolate the causative organism  Gross Examination

18 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Examination of the specimen as a whole  Indicator tests
 Benefits  Directly detect the presence of specific resistance mechanism in a bacterial
 Assess if the quantity is sufficient for testing isolate
 Status of the specimen is checked  Special methods which detect complex antimicrobial organism interaction
 Cloudy or bloody  CONVENTIONAL TESTING METHODS
 Areas with blood or mucus should be located and sampled  General considerations
 To know the part of the specimen to be samples  Inoculum preparation
 Direct Microscopic Examination  Use of pure culture
 Assess the quantity and quality of the specimen  4 to 5 colonies are inoculated in a broth medium or sterile
 Early indications and information regarding what may be wrong with the 0.85% N SS and incubated  turbidity indicated good growth (3
patient to 5 hours)
 Work – up of the specimen can be guided  Standardized inoculum
 Comparison of turbidity of organism suspension with a turbidity
standard (0.5 MacFarland standard)

ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST)

 Preparation of 0.5 MacFarland standard
 Medical intervention to eradicate the infecting pathogen - 1.175% (w/v) BaCl 2 (1.175 g to 18.825 mL of water; 0.048 M)
 Antimicrobial agents (AMA) – substance that either kill or inhibit microorganisms - 1% (v/v) H2SO4 (1 mL to 99 mL of water; 0.36 N)
 Isolation of the organism  Characterization of the organism  Spectrophotometry – 625 nm, with an absorbance of 0.08 to
 Identify the correct species of the organism 0.10
 Determine the antimicrobial susceptibility profile  Contains 1.5 x 108 CFU/mL
 Describes the bacteria’s susceptibility or resistance patterns to particular  Selection of antimicrobial agents – determine if the AMA is appropriate
antimicrobial agents against a microorganism  Antimicrobial Battery or Panel
 Resistant – withstand or tolerate the antimicrobial agent  Group of different AMA that are chosen to appropriately test
 Susceptible – completely killed or inhibited by the antimicrobial against a microorganism, based on CLSI
agent  Criteria
 AST Methods  Organism identification group – Gram – positive or Gram – negative
 Determine the possible AMA that may be used without harming the host  Establishes what organism group is susceptible or resistant against an
 Procedures use to produce antimicrobial susceptibility profiles and detect AMA
resistant that may be used therapeutically  Some AMA are developed to a particular species of bacteria
 Goals  AST method – some microorganisms do not respond to a certain method
 Bacterial etiology  Site of infection
 Resistance to AMA  Activity of AMA are also dependent on the site of infection (ex.
 Potential choices for therapy Nitrofurantoin  effective in the urogenital area)
 Directly measure the activity of one or more antimicrobial agents against a  Availability of the AMA in the formulatory
bacteria  brining the AMA and reacted with one bacterial isolate in one
environment Agar Disk Diffusion Method
 Methods Kirby – Bauer Technique
 Conventional methods – broth dilution, agar diffusion and agar
 Uses a standardized antibiotic – impregnated filter paper disks (disks are soaked in
dilution
AMA of specific, known concentration)
 Commercial susceptibility testing

19 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Most common technique of antimicrobial susceptibility testing  Incubation time – proper incubation time is at 16 to 18 hours;  time – false
 Antimicrobial agents impregnated onto a filter paper disk is placed on the surface resistance;  time – false susceptibility
of Mueller – Hinton agar, which is the standard agar for AST of non – fastidious  Size of plate
organisms  Depth of agar medium – proper depth is at 4 mm
 The antimicrobial agent readily diffuses once in contact with the agar medium,  Too thin - < 4 mm  wider ZOGI
establishing a concentration gradient on the surface of the agar  Too thick - >4 mm  smaller ZOGI
 Gradient -  concentration of the AMA near the disk and decreases in  Spacing of AMA disks
concentration as it moves farther away from the disk such that it will not be  Potency of the AMA disks
effectively killing or inhibiting the bacteria  Composition of the medium – some AMA are affected by the cation content
 Considerations in inoculation of the bacteria of the medium
 Uses a cotton swab – not too wet nor not too dry  Standardization
 Inoculate the entire space of the agar twice by rotating 60°, creating a lawn  Optimizes bacterial growth conditions
of bacterial growth  Optimize conditions for maintaining antimicrobial integrity and activity
 Allow the agar to absorb the broth for 3 to 5 minutes, but must not exceed 15  Maintain reproducibility and consistency of results
minutes
 Considerations in the application of the antimicrobial disk – prevent the
Antimicrobial Agents
overlapping of zone of growth inhibition
 90 mm petri dish may contain a maximum of 6 AMA disks  Mechanism of action of antimicrobial agents
 140 mm petri dish may contain a maximum of 12 AMA disks  Potential antimicrobial agents target different pathways or structure
 A distance of 10 – 15 mm from the edge of the agar must be considered; and  Antimicrobial agents may target the following:
a distance of 24 mm from center of two disks must also be considered  Bacterial cell wall
 The MHA is incubated for 16 to 18 hours  Bacterial cell membrane
 Other considerations (for fastidious organisms)  Protein synthesis
 S. pneumoniae – Mueller – Hinton Sheep Blood  DNA and RNA synthesis
 Haemophilus species – Haemophilus Test Medium Antimicrobial Agent Mode of Action Effect Comments
 N. gonorrhoeae – GC agar INHIBITORS OF CELL WALL SYNTHESIS
 Interpretation of results - It is the largest group of
antimicrobial agents.
 Interpretative Categories
- Common mechanism of
 Resistant – AMA tested is not appropriate for therapy Binds enzymes bacterial resistance
 Intermediate – potential utility of the AMA to body sites where it may be involved in the through the production of
concentrated synthesis of the Halted cell wall beta – lactamases by the
Beta – lactams
 Susceptible – AMA may be an appropriate choice for therapy; bacterial cell wall (penicillin synthesis bacteria
resistance is absent binding proteins - PBP is a transpeptidase
 Technical Factors Influencing the Size of the Zone of Growth Inhibition [PBP]) responsible for the transfer
of peptides necessary to
 Inoculum density
complete the crosslinking of
 Too light – wider; false susceptibility proteins in the cell wall
 Too heavy – smaller; false resistance - Interferes with the PBP
 Timing of disk application – after more than 15 minutes, bacterial cells Binds to enzymes, leading to the
Cessation of cell
increase, causing a smaller ZOGI and a false resistance Glycopeptides precursors of cell defective incorporation of
wall synthesis
 Temperature of incubation – false susceptibility when the temperature is below wall synthesis precursors into the growing
or above the optimal temperature cell wall

20 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 - Examples include - Bacteria that are
vancomycin and tetracycline – resistant are
bacitracin. susceptible to Tigecycline.
INHIBITORS OF CELL MEMBRANE FUNCTION INHIBITORS OF DNA AND RNA SYNTHESIS
- Daptomycin is a - Derivative of nalidixic acid
Disrupts the cell Bind and interfere Prevent DNA
Inhibits cell macromolecule that is - Broad spectrum
Daptomycin wall of Gram – Fluoroquinolones with DNA gyrase supercoiling and
membrane function unable to penetrate Gram antimicrobial agent
positive bacteria enzymes replication
– negative cell membrane - Examples include oxacillin
- Effective against Gram – - Activation of the rug
Direct interaction
negative bacteria through low redox potential
Polymyxin B between the DNA
Leakage of - Used as a last resort of Metronidazole - Potent versus anaerobic
and the
Disrupts bacterial macromolecules treatment (due to its Gram – negative bacteria
activated drug
cell membrane and ions from the toxicity) and amoeba
cytoplasm - Effectiveness varies with the Binds the enzyme
Colistin Prevents the - One of the primary drugs
molecular make – up of the DNA –
Rifampin bacteria to produce for the treatment of
bacterial cell membrane dependent RNA
RNA tuberculosis
INHIBITORS OF PROTEIN SYNTHESIS polymerase
Interrupts protein INHIBITORS OF OTHER METABOLLIC PROCESSES
- Used in combination with
synthesis complex, Inhibits the - Active against Gram –
cell wall inhibitors in order
Binds to 30s reading of mRNA enzyme Disrupts folic acid positive and Gram –
Aminoglycosides to facilitate the entry of the Sulfonamides
ribosomal unit code and dihydropteroate pathway negative bacteria, except
aminoglycosides and
ribosomal – mRNA synthase Pseudomonas aeruginosa
attack the ribosomes
complex - Frequently combined with
Inhibits
Microlide – Disruption of the Disrupts folic acid sulfonamides
Binding to 50s Trimethoprim dihydrofolate
Lincosamine – growing peptide pathway (sulfamethoxazole –
ribosomal unit reductase
Streptogramin (MLS) chain trimethoprim [SXT])
- Chemical derivatives Several targets
related to erythromycin involved in the
Binding to 50S and other macrolides Nitrofurantoin bacterial protein
Ketolides
ribosomal unit - Effective against and DNA
microorganisms resistant to synthesis
macrolides  Mechanisms of antibiotic resistance
Inhibits the  Biologic resistance –changes that result in the organism being less susceptible
addition of new
Binding to 50s to a particular antimicrobial agent that has been previously observed
Chloramphenicol amino acids to the
ribosomal unit  Clinical resistance – observed when a susceptibility is lost that the drug is no
growing peptide
chain longer for clinical use
- Effective against both  Environmentally mediated resistance
Gram – positive and Gram  pH
Incoming tRNa – negative bacteria  Some antimicrobial agents (i.e. erythromycin, glycosides) are less
Binding to the 30s
Tetracycline cannot bind to the - Inhibits or kills intracellular effective in low pH and cannot maximize its potency
ribosomal unit
ribosome bacteria, such as Rickettsia,  Tetracycline has decreased potency in the presence of increased
Chlamydia and Rickettsia –
pH
like organisms
 Anaerobic atmosphere
Binding to the 30s - Synthetic derivatives of
Glycylgycines  Cation concentration
ribosomal unit tetracycline (Tigecycline)
 Presence of magnesium and calcium

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  Aminoglycosides are inhibited by cation concentration
 Pseudomonas aeruginosa is a Gram – negative bacilli with a
negatively charged cell wall. Aminoglycoside is positively
charged; the presence of cations will compete with the
negative charges in the bacteria, leading to the decreased
binding sites for the drug.
 Thymidine concentration
 Enterococcus species utilize thymine and its precursors to
counteract sulfonamides and trimethoprim,
 Microorganism – mediated antimicrobial resistance
 Intrinsic resistance – ‘inherent resistance’
 Conferred by the microorganism which results from the normal
genetic, structural and physiologic state of the microorganism
 The resistance is a product of the genetic expression of the
microorganism.
 It is known, predictable and established.
 Acquired resistance
 Antibiotic resistance exhibited by the microorganism results from the
altered cellular physiologic and structure caused by the changes in
the microorganism’s genetic make – up.
 Due to mutations in the genetic make – up of the microorganisms.

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 MEDICAL MICROBIOLOGY

GRAM – POSITIVE COCCI

 Constitutive – production of the enzyme is continuous
 Genes that code for β – lactamase is conferred by plasmids
Genus Staphylococcus  Toxins
Staphylococcus aureus  Exotoxin – composed of proteins and are released in the environment; it is
cytotoxic (poisonous to different cells) and neurotoxic (acts on the cells of
 General Morphology the CNS)
 Gram positive cocci in clusters  Enterotoxin – can be an exotoxin; active in the gastrointestinal tract of the
 Facultative anaerobes host
 Non – motile, non – sporadic, non – encapsulated  Hemolysins
 Catalase and coagulase positive  Alpha – hemolysin – demonstrates lethal effects on wide variety of
 Halotolerant – able to grow at high concentrations of salt (> 10% NaCl) cells; lyses both red blood cells and white blood cells; induces skin
 Resistant to heat, solar radiation, desiccation and drying cell death (dermonecrotic); potent neurotoxin
 Habitat and Transmission  Beta – hemolysin (Hot – cold hemolysin) – acts on the sphingomyelin
 Human skin and nose, dust, sputum samples (sphingomyelinase); causes lysis of red blood cells at 37°C (activity is
 Transmitted via contaminated hands enhanced at cold temperatures)
 Virulence Factors  Delta – hemolysin – activates adenylcyclase (surfactant), resulting to
 Enzymes cAMP production, trigerring diarrhea
 Catalase – degradation of hydrogen peroxide  Panton – Valentine Leukocidin (PVL) – direct toxic effects on white blood
 Coagulase – formation of fibrin clots that may function as covering of the cells (causes degranulation of the cytoplasm of white blood cells)  cell
bacterial cell wall (antigenic disguise) swelling and lysis by oxidative burst (creates holes on the while blood cells,
 Bound Coagulase leading to the influx of water)
 Free Coagulase  Exfoliative Toxin (Exfoliatin or Epidermolytic Toxin) – dissolution of the skin
 DNAse (Deoxyribonuclease) – liquefies mucoid material at the site of cells in the epidermis; dissolution of the cytoplasm of the epidermis
infection, thus, spread of material occurs when the cell bursts  Toxic Shock Syndrome Toxin (Pyrogenic exotoxin C or Staphylococcal
 Hyaluronidase (Spreading factor) – hydrolyzes hyaluronic acid found in enterotoxin F) – commonly found in post-surgical wound infections,
human cells, thus, neutralizing the mucopolysaccharides in the cells causing osteomyelitis; produces lethal effects on the myocardium, skeletal
 Lipase – hydrolyzes the lipids on the skin, causing invasion to the skin and muscles, kidney tissues and liver, leading to multi – organ failure
the subcutaneous layers  Enterotoxin A to E – heat stable molecules (deactivation at 130°C for 30
 Staphylokinase (Fibrinolysin) – dissolves the fibrin clots causing the spread minutes) that cause contamination of the food; induces signs and
of the organism to the contiguous spaces in the body symptoms by the ingestion of preformed toxin present in the food
 Β – lactamase – neutralizes the β – lactam ring in the β – lactam  Structures
antimicrobial agents  Glycocalyx and Slime Layer
 Types  Protects the cells from phagocytosis
 Inducible – produces the enzyme when the bacteria is exposed
to the antimicrobial agent

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  Promotes adherence of the organism to other bacterial cells and  Sheets of the epidermis burst out, resulting to a skin that resembles
prosthetic devices skin in boiling water
 Capsules are classified into serotypes  Commonly affects neonates, infants and children less than 5 years old
 Antigenic determinants that are reacted with antibiotics  Toxic Shock Syndrome
 S. aureus – antibody 1 to 8  Multiple organ involvement
- Serotype 8 – strains that cause toxic shock syndrome  Due to the presence of super antigen in the bloodstream
- Serotype 5 – confers or exhibits resistance to penicillin and  Sudden onset of fever, chills, vomiting, diarrhea, hypotension, severe red
oxacillin rash which progresses to desquamation
 Protein A – Binds to the Fc region of antibodies – will not produce  Hypotension – oxygen is not very well distributed in the body,
chemotactic factors to signal white blood cells; inhibits phagocytosis; especially in the brain, heart and kidneys
complement will not be activated  Epidemic in menstruation women using tampons
 Cell Wall Constituents – NAM and NAG; teichoic acid  Staphylococcal Food Poisoning
 Adherence to mucosal surfaces of the body  Primary means of bacterial intoxication  due to the presence of
 Deactivates complement; inhibits chemotaxis of inflammatory cells enterotoxin from contaminated food
 Inhibits phagocytosis  Signs and symptoms – nausea, diarrhea, abdominal cramping, loss of
 Pathogenesis of Staphylococcus aureus appetite, vomiting, fever, difficulty of breathing, headache and weakness
 Cutaneous Lesions – initial symptom is fever  Self – limiting infection and does not progress to fatal disease
 Folliculitis (‘pimple’, ‘sty’) – inflammation of the hair follicles; the base of
the hair follicles is red, swollen and pus – filled Disease Virulence Factor
 Furuncles (‘boils’) – large, painful, nodular extensions of folliculitis; spread Scalded Skin Syndrome Exfoliative Toxin
of the infection to the subcutaneous lesions Toxic Shock Syndrome Toxin
Toxic Shock Syndrome
 Carbuncles – occurs when several furuncles coalesce; occurs in areas Delta - hemolysin
where there is thick skin Enterotoxin
 Deep – seated infections Staphylococcal Food Poisoning
Delta – hemolysin
 Osteomyelitis – infection of the bone cavity due to the hematogenous Hyaluronidase
spread of the organism to the bones Lipase
 Endocarditis Cutaneous Lesions
Staphylokinase
 Pneumonia Alpha – hemolysin
 Arthritis
 Bacteremia  Antimicrobial Therapy against Staphylococcus aureus
 Scalded Skin Syndrome (Ritter’s Disease)  Penicillinase – strains that have the enzyme penicillinase exhibit resistance to
 Interepithelial splitting of the skin within the stratum granulosm of the penicillin and other penicillin – related drugs
epidermis  Vancomycin – used for organisms that exhibit resistance to the first group of
 Sloughing off of the epidermis antibiotics; supporting antibiotics include aminoglycosides, β - lactams and
 Layers of the keratinized squamous epithelial cells separate from each quinolones
other of from the underlying tissues  β – lactams – used for organisms that are resistant to naticillin, methicillin and
 Signs and Symptoms oxacillin due to the presence of mecA gene (responsible for coding the
 Reddening and wrinkling of the skin, typically begins on the mouth enzyme penicillinase)
 Sloughing off of the epidermis  Vancomycin – resistant S. aureus – conferred by the gene vacA that confers
 Blisters that contain fluids but lacks white blood cells and bacteria resistance to tetracycline, vancomycin, erythromycin and aminoglycosides

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 Coagulase – Negative Staphylococcus Species  Differential – determines the organism’s ability to ferment
mannitol
Staphylococcus epidermidis - Phenol red – indicator (medium changes from red 
yellow)
 General Characteristics - (+) – yellow medium with yellow colonies – S. aureus
 Normal flora (microbiota) of the skin  Vogel – Johnson Medium (Modified MSA)
 50% to 80% of infections caused by the coagulase – negative Staphylococcus  Medium is incorporated with tellurite, which replaces the salt
species are caused by this organism content of the medium
 Coagulase, mannitol and DNAse negative  Produces yellowish discoloration on the medium with black
 NOVOBIOCIN SUSCEPTIBLE colonies
 Endocarditis – inflammation of the heart valves (native and prosthetic heart valves)  Trehalose – Mannitol – Phosphate Agar – for coagulase – negative
 Biofilm formation – extracellular slime substance that promotes adherence to Staphylococcus species
prosthetic devices  Determines the ability of the organism to ferment carbohydrate alcohols,
 Occurs only when: such as mannitol and trehalose, indicated by the decrease in pH
 The device is contaminated  (+) – purple to yellow – S. saprophyticus
 The skin is not properly disinfected  Also used for the determination of the phosphatase activity of the
 Renders resistance to the organism (antibiotic barrier) organism – a filter paper is impregnated with 0.01% phenolphthalein
 Treated by the removal and replacement of the artificial valve disphosphate  colonies from the medium is introduced into the filter
paper with 1N ammonium hydroxide
 (+) – pink color in the filter paper – S. epidermidis
Staphylococcus saprophyticus  Coagulase Test
 Formation of fibrin clots (Fibrinogen  Fibrin)
 General Characteristics
 Used in order to differentiate pathogenic organisms of Staphylococcus from
 Putrid odor (Sapro – putrid)
non – pathogenic organisms
 NOVOBIOCIN RESISTANT
 Methods
 Urinary Tract Infection
 Slide Method – detects bound coagulase
 Young healthy, sexually active women
 Bound coagulase is found on the cell wall of the organism
 Second cause of cystitis (apart from Escherichia coli)
 Directly reacts with the fibrinogen to form fibrin clots
 Uroepithelial tropism – the organism prefers to infect the cells of the
 Tube Method – detects free coagulase
genitourinary tract
 Free coagulase is secreted extracellularly by the organism
 Needs to react first with coagulase – reacting factor (globulin
Laboratory Diagnosis of Staphylococcus Species plasma factor) to create staphylothrombin to form fibrin clots from
 Gram Staining – Gram positive cocci fibrinogen
 Culture  Uses rabbit plasma in EDTA (most ideal) or human plasma (gives parallel results
 Blood Agar Medium – β – hemolysis (S. aureus); γ – hemolysis (S. epidermidis) with rabbit plasma)
 Mannitol Salt Agar – Yellow medium with yellow colonies (S. aureus)  Some strains of Staphylococcus aureus have delayed formation of fibrin clots.
 Mannitol Fermentation  Incubation is extended to 16 to 18 hours at room temperature
 MSA  Prolonged incubation at 35°C to 37°C will enhance the activity of
 Selective – due to high salt content, favors the growth of fibrinolysin (yielding false negative results)
halotolerant organisms  Microorganisms grown on salt medium tends to exhibit autoagglutination,
yielding false positive results.

25 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  (+) – visible white clots – S. aureus, S. lugdunensis, S. schleiferi  Lysostaphin – a protease that breaks the glycine peptide linkages in the cell
 Methods wall; cleaves the glycine bridge in the peptidoglycan layers, making the cell
 Latex Agglutination – utilizes latex beads coated with plasma, where susceptible osmotic lysis
fibrinogen is bound  Resistant – Micrococcus
 Passive Hemagglutination – sheep red blood cells are sensitized with  Susceptible – Staphylococcus
fibrinogen  Utilizes
 StaphASE – dehydrated plasma is placed un a pouch; plasma is  0.2 mL of Remel Lysostaphin
rehydrated with NSS and a suspension of bacteria is added to the plasma;  10 µg of Lysostaphin
solidifying of the plasma within 1 minute (Staphylococcus)
 DNAse Test
Genus Streptococcus
 Thermostable endonuclease test
 Toluidine Blue or Methyl Green – incorporation of one of the two dyes in a  General Characteristics
nutrient medium  Gram positive cocci in chains or in pairs
 3 mm deep holes are bored into a nutrient medium  Non- motile, non – spore forming
 DNAse is stable and is subjected to heat  Facultative anaerobe
 The suspension of the organism is boiled and poured into the holes  Some are fastidious organisms
 The medium is incubated at 35°C to 37° C (overnight)  Capnophilic
 DNAse hydrolyzes the dye incorporated in the medium, if the organism is  Catalase – negative
present  Homofermentative
 (+) – rose pink (toluidine blue); colorless (methyl green) – S. aureus  Classification of Streptococci
 Sugar Fermentation Test – ability of the organism to utilize carbohydrates as a  Academic or Bergey’s Classification
source of energy  Classification based on the temperature for growth
 Pyogenic group – do not grow at 10°C and 45°C (S. pyogenes)
 Viridans group – do not grow at 10°C but grows at 45°C (S. mutans)
MAN MAL SUC TRE  Enterococcus group – grow both at 10°C and 45°C (Enterococcus
S. aureus + + + + faecalis)
S. epidermidis - + + -  Lactic group – grow at 10°C but do not grow at 45°C (S. lactis)
S. saprophyticus + + W +  Smith’s and Brown’s Classification
 Classification based on hemolytic patterns observed in BAM
 Modified Oxidase Test  α – hemolytic Streptococci
 Detects the enzyme cytochrome oxidase that is secreted by the organism  a – hemolysis – induced by a – hemolysin
 Substrate – tetrmethyl – p – phenylene diamine dihydrochloride  Partial lysis of red blood cells; partial clearing under and
 (+) – purple color – Micrococcus species (Staphylococcus species – negative) surrounding the colony, producing a greenish coloration,
 Antimicrobial Susceptibility Testing indicating the reduction of hemoglobin to methemoglobin
 Bacitracin – antimicrobial agent that inhibits cell wall synthesis  Viridans Streptococci (S. pneumoniae)
 Resistant – Staphylococcus  Β – hemolytic Streptococci
 Susceptible – Micrococcus  Β – hemolysis – zone of complete clearing under and the
 Furazolidone (100 µg of furazolidone) surrounding medium of the colony
 Resistant – Micrococcus  Streptococcus haemolyticus’ group
 Susceptible – Staphylococcus  S. pyogenes; S. agalactiae
 γ – hemolytic Streptococci

26 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  γ – hemolysis – no hemolysis  Via respiratory droplets and cutaneous lesions
 Indifferent Streptococci – Enterococcus species  Virulence Factors
 a’ – hemolytic Streptococci  M Protein
 a’ – hemolysis – inner zone of partial hemolysis surrounded by  Major virulence factor of S. pyogenes; determines the appearance of the
an outer zone of complete hemolysis colonies on BAM
 S. epidemius  Acid, heat – stable substance; present on the outer surface of the cell wall
 Rebecca Lancefield’s Classification (Lancefield Grouping System)  May resist heat and acid agents
 Classification based on the cell surface carbohydrate antigens  Hair – like structure (fibrillar protein) from the cell wall
 Polysaccharides – human group A, B, C, F and G Streptococci  Originates from the cell membrane and extends to the cell wall outside
 Group A – S. pyogenes (rhamnose – N – acetylglucosamine) the capsule of the organism
 Group B – S. agalactiae (rhamnose – N – glucosamine  Counterpart of the protein A in S. aureus – attaches to the Fc region of the
polysaccharide) IgG, thus, inhibiting phagocytosis by white blood cells and prevents the
 Group C – S. dysagalactiae (rhamnose – N – β – activation of the complement cascade
acetylgalactosamine)  Immunogenic – induces antibody production; can recognize the same
 Group F – S. intermedius, S. anginosus, S. milleri, S. constellatus protein and can counter the protein on the second encounter with the
(glucopyrranosyl – N – acetylgalactosamine) organism out of the first invasion
 Lipoteichoic acid – group D Streptococci and Enterococcus  Cross reacts with human tissue, causing non – suppurative infection
 Group D – S. faecium, E. faecalis (glycerol teichoic acid with D – (rheumatic fever)
alanine and glucose)  Capsule – encapsulated cells are more virulent since it prevents phagocytosis
 Erythrogenic Toxin (Streptococcal pyrogenic exotoxin)
Group A Streptococci  Carried by lysogenized S. pyogenes strains (strains that carry
bacteriophages)
Streptococcus pyogenes  Responsible for the red rash in patients infected with the organism
 Seen in patients with Streptococcus Toxic Shock – like syndrome
 Characteristics
 Types
 Gram – positive cocci in chains  Exotoxin A – responsible for Streptococcus toxic shock syndrome
 Non – spore forming, non – motile, encapsulated and scarlet fever
 Facultative anaerobe
 Exotoxin B – no clear function in disease process
 Capnophilic; fastidious  Exotoxin C – also responsible for Streptococcus toxic shock
 Mesophile
syndrome
 Β – hemolytic  Hemolysins
 Colonies on BAM – discoid in shape; appearance is due to the M protein  Streptolysin O (SLO)
content produced by the organism
 Oxygen labile toxin – the presence of oxygen inhibits or inactivates
 Matte colonies –  M protein content the activity of the hemolysin
 Glossy colonies –  M protein content
 Immunogenic – induces antibody production
 Homofermentative  Anti – streptolysin O
 Susceptible to bacitracin (one method to differentiate from group B  Produces subsurface hemolysis on BAM
Streptococci)
 Seen under the colonies, induced through Streak and Stab
 Habitat and Transmission Technique
 Human throat and the skin (but NOT a normal flora of the upper respiratory
 Oxygen cannot penetrate the subsurface of the agar
tract and the skin)

27 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Streptolysin S (SLS) the bacteria, white blood cells (blisters develop honey – colored
 Oxygen stable hemolysin – presence of oxygen does not inactivate crust attached firmly to the skin, causing intense itching)
the hemolysin  Erysipelas – when impetigo spreads to the lymph nodes
 Non – immunogenic  Reddening of the face and upper extremities (distinct red margin)
 Streptokinase  Signs and symptoms
 Counterpart of Staphylokinase  Swollen lymph nodes with pain
 Dissolves fibrin clots, allowing the spread of the organism through  Fever and chills
contiguous spaces  Leukocytosis
 Streptodornase – similar to DNAse  Reddening of the skin are caused by strains that produce the
 Hyaluronidase – hydrolyzes hyaluronic acid in cells erythrogenic toxin
 Leukocidins – induce degranulation of white blood cells, thus, inhibit  May progress to rheumatic heart disease and acute
phagocytosis glomerulonephritis
 Diphosphopyridine nucleotidase – enzyme released by the microorganism  Necrotizing fasciitis
responsible in killing white blood cells  Flesh – eating bacteria
 G – protein – prevents phagocytosis  Digests the fascia and the muscles of the body
 F – protein – fibronectin – binding protein; considered as an adhesion factor,  Causes intense pain and swelling at the site of infection
facilitating the attachment to the pharyngeal epithelium  Seen in the following:
 Pathogenesis of Streptococcus agalactiae  Needle stick injury
 Pharyngitis – ‘strep throat’, ‘tonsillitis’  Abortion
 Back of the pharynx – red and swollen lymph nodes and pus containing  Surgery
abscesses covering the tonsils (characteristic gray and white exudates)   risk in acquiring the infection – cancer, diabetes and chicken pox
 Self – limiting – treatment is sought when it progresses to chronic stages  Treatment involves the complete removal of the site involved;
 25% of those who acquire the disease remain as carriers administration of penicillin and tyndamycin (intravenous)
 Signs and symptoms  Scarlet Fever – ‘Scarlatina’
 Red, swollen lymph nodes  Follows pharyngitis; formation of diffused red rash on the face
 Purulent abscesses  Butterfly rash (cheeks); strawberry tongue
 Fever  Begins on the face and spreads throughout the body; after 1 week, the
 Body malaise rash disappears and the skin sloughs off
 Organism will progress to the larynx (laryngitis) and bronchus (bronchitis)  Strains that produces pyrogenic or erythrogenic toxin A, B and C induces
when untreated scarlet fever
 Laryngitis – hoarseness of the voice  Puerperal fever/Sepsis – ‘Childbirth fever’
 Bronchitis  Affects the uterus, uterine tube and reproductive organs of the mother
 Mucous production – defense mechanism of the body that  The organism is introduced in the vaginal area through contaminated
contains the organism hands
 Coughing – reflex mechanism  Nonsuppurative Infections or Diseases
 Shortness of breath – tension in the lungs is inhibited  Rheumatic Fever
 Common in cold seasons and in elementary and middle school children  Sequelae of pharyngitis
 Skin Lesions  Occurs in untreated strep throat in patients aged 5 to 15 years old
 Impetigo – ‘pyoderma’  Inflammation of the joints, heart, CNS and the subcutaneous tissues
 Inflammation of the epidermis – small flatten red patches seen on  M protein – rheumatogenic; demonstrates tropism to valves and
the skin of the upper limbs; oozing pus – filled vesicles; blisters harbor muscles of the heart

28 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Autoimmune disease – immune defenses are directed towards the  Pathogenesis of Streptococcus agalactiae
itself since antibodies cross react with the muscles of the heart  Neonatal meningitis and sepsis – infections occur during the perinatal and
 Acute Glomerulonephritis neonatal periods  may cause fulminant sepsis, meningitis and respiratory
 Sequelae to strep throat or skin infections distress syndrome during the first month of life
 M protein – nephrotogenic (antigen – antibody complex deposited  Pneumonia (nosocomial infection)
on the kidneys signals inflammatory cells to migrate to the kidneys)  Colonization of the vagina – asymptomatic
 Signs and symptoms  Laboratory Diagnosis
 Edema of the limbs  Gram – staining – Gram – positive cocci in chains
 Hypertension  Culture on BAM
 Urine – smoky appearance  Smaller zone of hemolysis than S. pyogenes
- Hematuria  Colonies are larger
- Hemoglobinuria  Incubation with anaerobic conditions
- Proteinuria  Bacitracin – SXT Susceptibility
 Laboratory Diagnosis  CAMP Test – Christie, Atkins, Munch – Petersen
 Gram – staining – Gram – positive in chains  Production of synergistic hemolysis on BAM
 Culture on BAM  Priniciple – group B Streptococcus produce CAMP factor, which is a
 Wide zone of β – hemolysis – 2 to 4 times larger than the colony diffusible protein – like compound that interacts with the β – hemolysis
 Matte colonies –  M protein content produced by Staphylococcus aureus causing an enhanced, synergistic
 Glossy colonies –  M protein content hemolysis
 Antimicrobial Susceptibility Test  Uses Β – hemolytic strains of Staphylococcus aureus
 Bacitracin Susceptibility  Streaked perpendicularly to colonies of Streptococcus agalactiae
 Presumptive identification of group A Streptococci  (+) – arrowhead hemolysis at the junction where the two colonies meet
 0.04 µg of Bacitracin  Hippurate Hydrolysis
 Taxo A – impregnated in a filter paper disk  Assess the ability of the organism to produce hippuricase
 Differentiates group A and group B Streptococci  Hippuricase hydrolyzes hippurate to benzoate and glycine
- Suscpetible – Group A  Benzoate Test
- Resistant – Group B  Addition of 10% ferric chloride
 Sulfamethoxazole Trimethoprim Test  (+) – precipitation persists for 10 minutes if benzoate is present
 1. 25 µg of Trimethoprim; 23.75 µg of Sulfamethoxazole  Glycine Test
 Susceptible – Group C, F and G  Addition of ninhydrin (strong oxidizing agent, which deaminates glycine,
 Resistant – Group A and B producing ammonia and carbon dioxide)
 (+) – deep purple coloration
Group B Streptococci  PYRase Test
 In conjugation with other tests – presumptive identification of group A
Streptococcus agalactiae Streptococci
 Susbtrate – L – pyrrolidonyl – β – naphthylamide
 Characteristics (refer to S. pyogenes)  Reacted in a broth medium at 35°C to 37°C
 Habitat and Transmission
 Determines the ability of the organism to produce PYRase
 Human vagina and rectum (pyrrolidonyl arylamidase)
 Transmitted during birth  important during the neonatal and perinatal
 Reaction liberates β – naphthylamide; upon the addition of
periods since the organism is part of the normal flora in the vagina dimethylcinnamaldehyde produces a red color

29 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  (+) – red color – complex of β – naphthylamde – p – dimethylamino -  Sodium taurocholate
cinammaldehyde  Indicator – ferric chloride
 Hydrolysis of esculin  esculetin
 Reacts with ferric chloride to form a black diffusible complex
 (+) black medium – evidence that esculin is hydrolyzed
 (+) – Enterococcus species
Group C Streptococci  Growth on the medium, with blackening of the medium
 Cause diseases in animals and zoonotic infections in man  Salt Tolerance Test
 Streptococcus equisimilis  Utilizes brain heart infusion broth
 Most common human isolate  Incorporated with 6.5% NaCl
 Exudative pharyngitis and tonsillitis  Determines the tolerance of the organism in an environment with a high
 Severe infections are seen in neutropenic hosts, leading to sepsis concentration of salt with bromcresol purple as an indicator
 Opportunistic infections with underlying conditions (ex. Diabetes,  False – positive reactions occur by heavily inoculating the media too
cardiovascular diseases) heavily
 Streptococcus zooepidimicus – seen in unpasteurized cow milk and homemade  (+) – turbidity – Enterococcus species
cheese  Leucine Aminopeptidase Test
 Streptococcus equi – causes respiratory infections in horses  Detects the ability of the organism to produce leucine aminopeptidase
 Streptococcus dysagalactiae – causes bovine mastitis  A filter paper disk is impregnated with leucine β – naphthylamide
 Substrate is cleaved or hydrolyzed to liberate β – naphthylamide
 Dimethyl cinnamaldehyde is added to produce a red color
Group D Streptococci and Enterococcus
 (+) – red color – Enterococcus species (E. faecalis)
 Characteristics
 Gram – positive cocci in chains
 Catalase – negative Group F Streptococci
 Variable hemolytic reactions
Streptococcus anginosus
 Enterococcus species
 Intrinsically resistant to cephalosporin due to abusing of antimicrobial  Produces minute colonies on BAM
drugs  Causes severe suppurative infections in humans
 Most strains are γ – hemolytic and α – hemolytic, but may be β - hemolytic  Cellulitis
 Habitat and Transmission  Deep tissue abscesses
 Human colon, urethra and female genital tract  Endocarditis
 Infections are disseminated via endogenous spread  Osteomyelitis
 Diseases – Urinary tract infections, septicemia and endocarditis (complication of  Septicemia and bacteremia
septicemia; organism may lodge into the heart and cause the destruction of the
heart muscles) Group G Streptococci
 Laboratory Diagnosis
 Normal flora of the human gastrointestinal tract, oropharyngeal, vagina and skin
 Bile Esculin Test
 Causes rare opportunistic infections
 Determines the ability of the organism to grow in a medium with a high
concentration of bile salts (4% or 40% bile salts)
 The organism hydrolyzes esculin in the presence of increased
concentration of bile salts
 Sodium desoxycholate

30 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Viridans Streptococci  Determinants of pathogenicity of Streptococcus pneumoniae
 Colonization and migration
 Characteristics
 Due to the surface protein adhesins which bind to the pharyngeal
 a – hemolysis
epithelial cells
 Normal flora of the mouth and the upper respiratory tract
 Secretory IgA proteases
 Diseases
 Pneumolysin O
 Dental caries – ‘cavities’
 Tissue damage
 S. mutans produces acid from sucrose present in food leftovers which
 Teichoic acid – serves as a virulence factor which activate the alternative
damages the enamel of the teeth
pathway of the complement cascade; it may produce hydrogen
 Septicemia and endocarditis
peroxide
 The organism demonstrates unusual tropism to the muscles of the heart.
 Phosphoryl choline – binds with the phospholipids and destroy hose
 Sequelae to dental procedures performed – access of the organism to
cell
the blood through wounds
 Loss of natural resistance
 Presence of viral and other respiratory tract infections, in which host cells
Streptococcus pneumoniae are already damage and provide entry into the bloodstream for the
 Characteristics organism
 Gram – positive lancet – shaped diplococci (Streptococcus lancealatus) –  Abnormal accumulation of mucus, which may surround the organism and
usually appears in pairs or as diplococcus; once they undergo binary fission, protect it from phagocytosis
they do not completely separate  Bronchial destruction
 Encapsulated, non – motile, non – spore forming  Respiratory tract injury
 Facultative anaerobe  Alcohol and drug intoxication, due to (1) destructor of host enzymes, (2)
 Requires increased carbon dioxide to be able to grow alcohol depresses phagocytic activity, (3) alcohol depresses cough reflex
 Habitat and Transmission and (4) it facilitates aspiration of foreign materials
 Human throat and nasopharynx  Abnormal circulatory dynamics
 Via respiratory droplets  Other mechanisms such as
 Virulence Factors  Malnutrition
 Polysaccharide capsule – inhibits phagocytosis and promotes adherence to  Sickle cell anemia
surfaces  Hyposplenism
 Pneumolysin O  Nephrosis
 Potent cytotoxin that binds to the cholesterol in the cytoplasmic  Complement deficiency
membrane of ciliated epithelial cells  transmembrance pores on the  Pathogenesis of Streptococcus pneumoniae
membrane of cells  influx of water  lysis of cells  Pneumonia – produces digestive enzymes in the alveoli, which dissolves the
 Suppresses digestion of phagocytized bacterial cells by interfering with the lining of the alveoli  accumulation of plasma, leukocytes and erythrocytes in
action of lysosomes in phagocytic cells the alveoli leads to the formation of fluid in the lungs (viscous, purulent fluid –
 Responsible for the β – hemolytic patterns seen in BAM EMPYEMA)  prevents the distention of the lungs to accommodate oxygen 
 Neuraminidase – hydrolyzes neuraminic acid (component of the mucous lining difficulty in breathing
of the respiratory tract, facilitating easier attachment to the epithelial lining of  Types of pneumonia
the respiratory tract)  Lobar pneumonia – both lobes of the lungs are filled with fluid
 Amidase – enables autolysis of the organism, liberating toxic substances  Pneumococcal pneumonia
 Purpura – producing principle – causes dermal hemorrhages  Fever, chills, congestion, cough and chest pain  rapid and
short breathing of the patient

31 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Blood enters the lungs  characteristic rust – colored sputum  All tests for laboratory diagnosis of the organism are performed on the
 Organism may enter the blood stream when untreated and growth on the culture medium.
cause meningitis in adults  Neufeld – Quellung Reaction – Capsular Swelling
 Diagnosis – Neutrophils in sputum  Directly test the presence of S. pneumoniae in CSF and sputum for early
 Treatment – Erythromycin, cephalosphorins detection
 May be fatal in children  Increases the refractive index of the capsule making it visible under the
 CDC promotes vaccine as prophylaxis against the organism microscope
 PPV 23 – pneumococcal polysaccharide vaccine – 23  Specimen is reacted with antiserum
 PCV 7 – pneumococcal conjugate vaccine – 7  Antibody reacts with the capsular antigen
 Otitis media  Antigen – antibody complexes are deposited on the capsule of the
 Sinusitis – due to loss of natural resistance organism, making it seemingly swell
 Laboratory Diagnosis  Methylene Blue – enhances the visualization
 Gram – staining – Gram – positive lancet – shaped diplococci
 Culture on BAM
 Small grayish mucoid colonies
 Hemolysis
 a – hemolysis in aerobic incubators
 β – hemolysis in anaerobic incubators
 48 hours of incubation – dome – shaped colonies with checker
appearance; donut – shaped colonies (autolysis of colonies)
 Bile Solubility Test – detects the enzyme AMIDASE (activated by the bile
present in the medium)
 (+) – dissolution of the colonies
 Broth medium – clearing (after addition of the bile)
 Agar medium – colonies completely dissolve (after pouring of the
bile into the medium)
 Optochin Susceptibility
 Ethyl hydrocupriene hydrochloride
 5 µg of optochin is impregnated (Taxo P)
 Results are correlated with the bile solubility test
 Differentiates S. pneumoniae from other α – hemolytic Streptococcus
 ZOGI ≥ 14 mm – susceptible – S. pneumoniae
 ZOGI ≤ 12 mm – resistant – Viridans Streptococci
 Inulin Fermentation
 (+) – acid production
 Mouse Virulence Test
 Animal inoculation test – organism is introduced intravenously (may be
CSF or sputum with the organism or pure culture of the organism)
 After several days of incubation, the blood and body fluids of the mice
are cultured

32 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 GRAM – NEGATIVE COCCI
 Allows inhibition of killing
Genus Neisseria  Opa (Opacity Protein); PII
 General Characteristics  Opacity of the colonies on the surface of the media
 Gram – negative diplococci  P3 or Rmp – diminish the antibacterial effect of the serum
 Non – motile, non – spore forming  Endotoxin
 Facultative anaerobe  IgA Protease – inactivates IgA found in the secretions
 Pathogenic species are fastidious organisms  Β – lactamase
 Capnophilic and mesophilic  Pathogenesis of Neisseria gonorrhoeae
 Generally catalase – positive  Gonorrhea
 Oxidase – positive (able to produce the enzyme cytochrome oxidase that  Risk factors – conditions or activities associated that contribute to the
oxidizes aromatic amines) acquisition of the disease
 Kovac’s reagent – tetra – methyl – p – phenylenediamine dihydrochloride   socioeconomic status
 McLeod’s reagent – dimethyl – p – phenylenediamine dihydrochloride  Urban areas
 Pathogenic species are seen intracellularly within polymorphonuclear cells  Past history of sexually – transmitted infections
 Types:
 Genital gonorrhea
Neisseria gonorrhoeae
 Males – Acute urethritis
 General Characteristics - Dysuria and purulent urethral discharge
 Gram – negative kidney or coffee bean – shaped that are intracellular – - If untreated  ascending infection and reach other
adjacent side flattened and other is concave organs of the genitourinary tract  epididimytis; prostitis
 Oxidase – positive and periurethral abscesses
 Glucose fermenter – only carbohydrate that the organism is able to use as a - May lead to infertility and sterility
source of energy  Females – Endocervicitis and pelvin inflammatory disease
 Habitat and Transmission - Infects the mucosal surfaces of the urogenital tract
 Human genital tract - Endocervicitis – intermenstrual bleeding; ascending
 Via sexual contact ingections
 Virulence Factors - PID – Salphingitis (inflammation of the uterine tubes) 
 Pili produces mucopurulent discharge; may lead to scarring of
 Hair – like protein polymer which projects from the surface of the bacterial the tubes, ectopic pregnancy, infertility and sterility
cell  Prepubescent girls – may cause vulvovaginitis
 Major virulence factor of N. gonnorhoeae  Extragenital gonorrhea
 Mediates the attachment to mucosal cell surfaces and initiate the  Opthalmia neonatorum
infection - Ocular gonococcal infection that affects neonates;
 Demonstrates antiphagocytic properties acquired through an infected birth canal
 Overcomes the electrostatic repulsion - Purulent conjunctiva, which may lead to blindness
 Able to undergo antigenic and phase variation - Greed’s method – administration of 1% silver nitrate to the
 Outer Membrane Protein eyes of the baby in order to prevent the complication
 Porin Protein – PI - Treatment – erythromycin and tetracycline
 Confers serum resistance  Pharyngitis or Proctitis – seen in bisexual and homosexual men

33 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Conjunctivitis Laboratory Diagnosis of Neisseria species
 Disseminated Gonococcal Infection – invasion of the bloodstream, N. gonorrhoeae N. meningitidis
causing fever, hemorrhagic and red skin lesions Gram – negative kidney or coffee bean
Microscopy
– shaped cells
Neisseria meningitidis Oxidase Test + +
Catalase Test + +
 General Characteristics
 Similar with Neisseria gonorrhoeae Growth on Selective Media + +
 Encapsulated (vs. N. gonorrhoeae)
 Habitat and Transmission GLU + +
 Human nasopharynx CHO Fermentation MAL – +
 Via respiratory droplets (Cystine – Tryptic
FRU – –
 Virulence Factors agar – yellow
colonies) SUC – –
 Pili
 Polysaccharide capsule LAC – –
 Lipopolysaccharides – causes vascular damage and hemorrhages
 IgA protease
 Pathogenesis of Neisseria meningitidis
 Meningococcemia
 Widespread dissemination of the organism in the body;
 Entry into the bloodstream causes skin hemorrhages (Petechiae)
 Waterhouse Friedrichsen – severe inflammation of the adrenal glands and
tissues; coalesce of Petechiae
 Meningitis – may result whether or not there is meningococcemia or
bacteremia
 Serotypes – A, B, C, HI, KL, W135, XYZ and Z’29E
 Meningitis – serotype A, B, C, W135
 Seen in underdeveloped countries
 Serotype A – meningococcemia outbreak in Baguio
 Serotype B – United States

34 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 GRAM – POSITIVE BACILLI
 Direct inoculation of spores in cuts, abrasions from soil and
Spore – forming Bacilli animal products
 Malignant pustule – black eschar surrounded by edema
1 to 3 days – non – painful papule
Genus Bacillus -
- 3 to 4 days – pustule with fluid erupts and spread
Bacillus anthracis  Black eschar – cytolysis of macrophages; necrosis of epithelial
cells where the organism is inoculated
 Characteristics
 Papule formation are the host’s immune response against the
 Gram – positive bacilli with square ends, occurring either singly or in pairs
organism
 Serpentine chain – smear preparation
 Mortality – 20% (in untreated cases)
 Bamboo fishing rod – smear culture
 Pulmonary Anthrax – ‘Wool’s Sorter Disease’
 Spores – ovoid, subterminal within the cell
 Inhalation of air – borne spores while handling animal products
 Encapsulated – polyglutamic acid
 Pneumonia
 Non – motile
- Colonization in the alveoli
 Facultative anaerobes
- Toxin production
 Habitat and Transmission
- Hemorrhagic necrosis – hemorrhagic mediastinal adenitis
 Soil – can survive for several decades due to sport production
- Leads to toxemia and eventually, death
 Via contact with infected animal; inhalation of spores from animal hides, hair,
 Mortality – 100% (untreated cases); 75% (treated cases)
wool and fur
 Gastrointestinal Anthrax – ‘Gastric Anthrax’
 Virulence Factors
 Reported in developing countries such as Asia and Africa
 Capsule – conferred by the transformation or transduction (plasmid); inhibits
 Ingestion of contaminated milk
phagocytosis; promotes adherence to cell surfaces of the host
 Violent enteritis – abdominal pain, vomiting and diarrhea
 Anthrax Toxin
 Prevention
 Types
 Vaccines
 Edema factor – fluid accumulation at the site of infection
 Contents of vaccines
 Lethal factor – cytolysis of macrophages in tissues
 Spore suspensions
 Not active when not bound to protective antigens; stimulates antibody
 Culture filtrates containing the protective antigen – Japan
production
 Attenuated bacilli – US and Britain
 Pathogenesis of Bacillus anthracis
 Soldiers are prioritized
 Anthrax
 Introduced subcutaneously
 Bioterrorism; zoonotic infection from herbivores
 Administered 2 weeks apart and boosters are annually administered
 Herbivores ingest the organism in the soil  germination occurs in the
 Laboratory Diagnosis
intestinal mucosa  progresses to the colonization in the bloodstream and
 Microscopy – Gram – positive bacilli with spores
lodged in the different organs  septicemia  death
 Endospores – colorless, ovoid structures located subterminally
 Types of Anthrax
 Writz – Conklin – uses malachite green and safranin to demonstrate the
 Cutaneous Anthrax
spores
 95% of human cases
 Culture on BAM – non – hemolytic colonies
 PLET Medium – Polymyxin – Lysozyme – EDTA – Thallous acetate

35 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Colonies appear as large, opaque, ground glass appearance with Bacillus cereus
irregular or curled margins
 Medusa head appearance – seen in dissecting microscope; comma –  Characteristics
shaped outgrowths that projects from the colonies  Similar characteristics with B. anthracis
 Incubation at 5% to 7% carbon dioxide at 37°C  Non – encapsulated
 Capsule demonstration  Motile
 Only visible in biological specimen  Habitat and Transmission
 Mucoid appearance – water content  Soil
 Capsules are induced by:  Via ingestion of the organism or the toxin produced by the organism
 Bicarbonate agar – 5% to 10% serum  Spores are widely distributed in food products grown on soil
 Sheep or Horse BAM – in vitro;  carbon dioxide tension  Virulence Factors
 MacFadyeah reaction – methylene blue  Exotoxin – Enterotoxin
 Capsule – pink to purple color  Acts on the gastrointestinal tract
 Bacilli – blue color  Types
 Gelatin Hydrolysis  Heat stable – found in cooked food
 Gelatin is acted upon by gelatinase  Heat labile – vegetables; production after ingestion of food
 Assess the motility of the organism  Pathogenesis of Bacillus cereus
 (+) – inverted fir tree appearance  Food Poisoning
 Starch Hydrolysis – (+)  Types
 Nitrate Reduction – (+)  Emetic Syndrome – results from food intoxication; induced by heat –
 Carbohydrate fermentation – glucose, sucrose, fructose, maltose and trehalose stable toxin
 String of Pearls Reaction  Infective dose of the preformed toxin is large, and all individuals
 Susceptibility to penicillin are susceptible
 Inoculation of the organism unto MHA with 10% penicillin disk  Signs and symptoms
 Incubated at 37°C for 3 to 6 hours - Produced after 1 to 6 hours of ingestion of contaminated
 The organism swells  circular form friend rice and pasta dishes
 Microscopically, it has round bodies appearance, producing a - Clinically resembles the signs and symptoms of
characteristic string of pearls Staphylococcal food poisoning
 Ascoli Test - Self – limiting; recovers within 24 hours
 Capsular antigen test  Diarrheal Syndrome – 10 to 12 hours of ingestion of heat – labile
 Hides or skin of animals are boiled for 10 minutes  extract toxin; profuse diarrhea, abdominal pain and cramps, resembling
produced from boiling contains capsular antigens Clostridium perfringens food poisoning
 Extract is placed in a test tube and overlayed with capsular  Eye infections – introduced in the eye through foreign objects; associated with
antibodies trauma
 (+) – precipitate will form at the junction of two fluids  Severe keratitis
 Gamma Phage Testing  Endolpthalmitis
 Gamma phage lysis test – virulent bacteriophage  Laboratory Diagnosis
 B. anthracis is susceptible to gamma phage  Microscopy – Gram – positive bacilli
 Lysis of B. anthracis cells  Culture on BAM – large zone of β – hemolysis; colonies appear as smooth
 Inoculation of the organism to a suspension of gamma phage  CHO fermentation – glucose, maltose and salicin
 (+) – formation of plaque due to lysis  Motility – swarming motility

36 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Bacillus anthracis vs. Bacillus cereus Genus Clostridium
 General Characteristics
Bacillus anthracis Bacillus cereus  Swollen sporangium
+  Thrives in anaerobic environment
Motility – (swarming  Sporulation occurs in anaerobic environment
motility)  Exposure to oxygen is lethal
Hemolysis on  4 classifications
γ β  Gas Gangrene – histotoxic
BAM
Growth at 45°C – +  Clostridium perfringnes
Growth on Phenyl  Clostridium septica
Ethyl Alcohol – +  Clostridium histolyticum
Agar  Clostridium bifermentans
Penicillin  Clostridium sporogenes
S R  Clostridium sordelli
Susceptibility
 Tetani – Clostridium tetani
Gelatin Hydrolysis – +
 Botulinum
Salicin
– +  Clostridium botulinum
Fermentation
 Clostridium bavati
Gamma Phage
+ –  Clostridium butyricum
Lysis
 Difficile – Clostridium difficile
CHO Glu, Suc, Fru, Mal,
Glu, Mal
Fermentation Tre
Clostridium perfringens
 General Characteristics
 ‘Welch’s bacillus’  C. welchi
 Gram – positive bacilli with blunt ends
 Sport formers; non – motile; encapsulated
 Anaerobic
 Spore – centrally or eccentrically located within the swollen sporangium  Box
– car appearance
 Habitat and Transmission
 Soil, normal flora of the colon of man and animals
 Via contamination of open wound with soil and feces; ingestion of
contaminated food
 Virulence Factors
 α – toxin – ‘Lecithinase enzyme’; Dissolves lecithin (normal component of the
cell wall of human hosts)  cell lysis
 Collagenase – dissolves collagen
 Protease – hydrolyzes protein
 Carbohydrate fermentation in tissues – crepitation with production of large
amounts of gas in tissues

37 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Enterotoxin  Neutraization of lecithinase by a specific antitoxin
 Predisposing factors  Egg yolk medium – Lambard Dowell test
 Surgical incisions  Lecithinase – opalescence
 Compound fractures  Lipase – Iridescence
 Diabetic ulcers  Egg yolk contains triglycerides, which are broken down by a – toxin
 Septic abortions to produce opalescence
 Puncture wounds  Glycerol and fatty acids are utilized by the organism  emulsion of
 Gunshot wounds olive oil  hydrolyzed halo surrounding the colonies
 Pathogenesis of Clostridium perfringens  Results
 Gas Gangrene  Wide zone of opalescence with no pearly iridescence
 Anaerobic myonecrosis; anaerobic tissue damage - C. perfringens
 Due to contamination or infection of an open wound - C. sordelli
 Spore (entry through traumatic wounds)  produce tissue necrosis - C. bifermentans
(polymicrobial in nature)  presence of tissue hypoxia or anoxia   Wide zone of opalescence with pearly iridescence
germination of spores  proliferation of the organism  production of - C. noyvi
virulence factors  acts on tissues  MYONECROSIS - C. botulinum
 Can be inhibited by penicillin, in combination with hyperbaric oxygen at  Narrow zone of opalescence and iridescence
the site of infection - C. sporogenes
 Infection spreads within 1 to 3 days, which produces crepitation to the  Reaction to Litmus Milk
subcutaneous later with foul smelling discharge, rapidly spreading  Speciate Clostridium species  detects specific Clostridium species
necrosis, hemolysis, toxemia, shock and death that can metabolize litmus milk
 Food Poisoning  Acid production – fermentation of lactose; (+) – blue to red
 Ingestion of poultry and meat products; contamination of enterotoxin –  Casein – coagulation; (+) – curd or solidify
producing strains  C. perfringens – stormy fermentation; pink medium; coagulate
 Toxins are preformed in the gut when it proliferates casein; production of gas (tear the curd apart)
 Signs and symptoms – diarrhea without vomiting and fever within 16 to 18  Reverse CAMP Test
hours  Presumptive identification of C. perfringens
 Self – limiting within1 to 2 days  Uses Streptococcus agalactiae
 Laboratory Diagnosis  (+) – arrowhead – shaped synergistic hemolysis at the area where
 Microscopy – Gram – positive large bacilli the two colonies are close
 Culture on BAM – Target hemolysis
 Double zone of hemolysis Clostridium tetani
 Inner zone of β – hemolysis (beta – toxin)
 Outer zone of wide zone of a – hemolysis (lecithinase)  General Characteristics
 Biochemical Tests  Lollipop bacillus, tennis racket bacillus, drumstick bacillus, tackhead bacillus
 Nagler’s Test – Lecithovetilline reaction  Gram – positive bacillus
 Presumptive test; detect the ability of the organism to produce  Spore – round, located terminally within the swollen sporangium
lecithinase and lipase  Motile, non – encapsulated
 Purpose  Anaerobic
 Separate Clostridium perfringens from other species of  Habitat and Transmission
Clostridium  Soil

38 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Via contamination of wound with soil  Habitat and Transmission
 Virulence Factor – Neurotoxin  Soil
 Tetanospasmin  Via ingestion of organism or the toxin present in improperly preserved food and
 Active in the CNS processed canned foods
 Attaches to the peripheral nerves of the CNS; binds to the GANGLIOSIDE  Virulence Factor and Pathogenesis of Clostridium botulinum
RECEPTORS  Neurotoxin – Botulinum toxin
 Affects the release of the neurotransmitter glycince  muscle spastic paralysis  Heat labile, phage – mediated toxin (facilitated by a phage in a virus
 Disease progresses to the diaphragm  respiratory failure through transduction)
 Pathogenesis of Clostridium tetani  Acts on the neuromuscular junction  inhibiting the release of
 Tetani acetylcholine
 Contamination of wounds with soil  presence of hypoxia or anoxia   Toxin types A to G
germination  proliferation  toxin production  attachment to  Muscle Flaccid Paralysis
peripheral nerves in the CNS  inhibition of the release of glycine   Death due to respiratory failure
muscle spastic paralysis  Toxin (preformed toxin) is ingested  absorbed in the blood  binds to
 Germination occurs when oxygen is reduced; pyogenic infections are the peripheral nervous system  muscles do not contract
caused by other bacteria (polymicrobial)  Signs and symptoms
 Muscle spastic paralysis  Nausea, vomiting constipation
 Lockjaw – Trismus  Disease worsens
 Difficulty in opening the jaw  Dyploppia – double visions
 Affects the masseter – inhibit the relaxation, continuous  Dysphonia – weakness of speech
contraction  Dysarthria – difficulty in movement
 Risus sardonicus – sustained trismus  Dysphagia – difficulty in swallowing
 Opisthanatus – persistent back spasms, causing backward arching  Peripheral muscle weakness
 Tetanus neonatorum – contaminated umbilical clamp  Symmetric descending paralysis
 Highest mortality rate is associated with respiratory failure  Respiratory failure
 Laboratory Diagnosis  Death
 Microscopy – Gram – positive large bacilli  Types of Botulism
 Culture on BAM – β – hemolysis with round small colonies with fimbriated  Classical or Food – borne Botulism – ingestion of canned goods and
margins vacuum packed foods
 CHO Fermentation – non – sacharolytic or asacharolytic; does not use CHO as  Infant Botulism – ingestion of spores, contaminated honey; toxin
a source of energy production in the gut of the infant; also termed as ‘floppy infant
syndrome’
Clostridium botulinum  Wound Botulism – spore contamination of wounds and production
of toxin; mediated by toxin type A, B and F
 General Characteristics  Laboratory Diagnosis – determination of toxin in the serum or food of the patient
 Canned good bacillus
 Gram – positive bacilli
Clostridium difficile
 Spores – oval; subterminally within the swollen sporangium; snow shoe
appearance  General Characteristics
 Motile, non – encapsulated  Gram – positive bacteria
 Anaerobe  Motile, non – encapsulated
 Anaerobe

39 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Habitat and Transmission Non – spore – forming Bacilli
 Normal flora of the air
 Via contamination or ingestion of fecally – contaminated food
 Virulence Factor and Pathogenesis of Clostridium difficile
Genus Corynebacterium
 Pseudomembranous colitis Corynebacterium diphtheriae
 Antibiotic – associated diarrhea – due to prolonged antibiotic
administration  General Characteristics
 Major cause of diarrhea in the hospitalized population but becoming  Kleb – Loeffler’s bacillus
increasingly more common as a community – acquired diarrhea  Medically significant – 30% to 60% mortality due to the effects in different parts
 Toxin mediated damage on the gut of the body
 Types of Toxin  Gram – positive bacilli
 Toxin A – Enterotoxin  Diarrhea  Non – spore former, non – motile, non – encapsulated
 Toxin B – Cytotoxin  Damage to the enteroeptihelial cells  Pleomorphic
 Yellow white plaques on the colonic mucosa  Snapping occurs when cells divide
 Pseudomembrane – necrotic debris, fibrin and white blood cells  Club – shaped; Chines – letter arrangements (at acute angles: X, Y, V and
 Laboratory Diagnosis L)
 CCFA – Cefoxitin Fructose Agar  Granules are located near the poles of the cells  swelling near the ends
 Horse manure – like odor after 48 hours of incubation  Volutin granules (Babes – Ernst granules) – metachromatic granules;
 Selective isolation for the organism made up of meta or polyphosphates, where the organism store its
 Yellow colonies with horse manure – like odor food source
 Fructose fermentation  neutral red (pH indicator)  Anaerobic or facultative anaerobes
 Catalase – positive
 Oxidase – positive
 Two types of C. diphtheriae strains
 Toxigenic strains
 Non – toxigenic strains – still able to produce diseases (localized infections)
 Habitat and Transmission
 Human pharynx and skin
 Via respiratory droplets or skin contact through lesions
 Virulence Factor – Diphtheria toxin
 Primary virulence factor of the organism
 Production of the toxin is conferred by the process of lysogeny
 Produced by the toxigenic strains of C. diphtheriae
 Heat labile, polypeptide toxin
 Lethal – 0.1 gram per kilogram  the ability of the organism to cause disease is
not due to the colonization of the organism but by the production of toxin
 Necrotizing and neurotoxic
 Pathogenesis of Corynebacterium diphtheriae
 Acute, contagious and febrile illness
 Virulence of the organism
 Capacity to establish infection

40 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Capacity to grow rapidly  Induces the production of antibodies (anti – toxoid) that will protect
 Quickly elaborate a reaction that is effectively absorbed the individual in an encounter in the natural environment
 Types of Diphtheria  Biotypes of C. diphtheriae
 Pharyngeal diphtheria  Var. gravis – produces the most severe infections
 Begins of the throat – sore throat and fever develops  Var. mitis
 Once the toxin is absorbed by the mucous membrane of the  Var. intermedius
pharynx  destruction of the epithelium accompanied by  Var. belfanti
inflammatory responses (superficial inflammatory reaction)  Laboratory Diagnosis
 Necrosis – embedded with exuding fibrin, red blood cells and white  Shick’s Test – used for the detection of the exotoxin
blood cells (grayish pseudomembrane) in the tonsils, pharynx and  Purposes
larynx  prostration, dyspnea  Determine an individual’s susceptibility or immunity against the toxin
 Extends to the trachea  obstruction of the trachea (removal of the  Determine an individual’s hypersensitivity to the toxin
pseudomembrane will arrest obstruction but will expose tears in the  Tests are done on the arms
capillaries causing bleeding and facilitate the spread of the  Test arm – 0.1 mL of diphtheria toxin
organism); suffocation due to membrane is not relieved by  Control arm – 0.1 mL of toxoid
intubation  Results are inspected after 24 hours up to the sixth day of injection
 Lymph nodes on the neck are enlarged with marked edema of  Results – presence of erythema or induration or necrosis
the neck  (+) – Erythema on test arm; no reaction for the control arm  the
 Toxic damage occurs  parenchymatous degeneration; fatty host is susceptible but NOT hypersensitive
infiltration and necrosis of the liver, heart (irregularities on  (-) – no reaction on both arms  individual is immune and
cardiac rhythm), kidneys, adrenal glands, accompanied by hypersensitive – negative
hemorrhage  The individual has already contracted the disease
 Nerve damage  paralysis of the soft palate, eye muscles and  The individual has vaccine and is still effective
extremities; difficulty with vision, speech, swallowing and movement  Combined – erythema reaction persists up to the 6th day on the test
of arms and legs arm; the reaction persists at 48 hours and subsides by the fifth day 
 Cutaneous diphtheria – Wound or skin diphtheria the individual is susceptible and hypersensitive to the toxin
 May be caused by insect bites  Pseudoreaction – reaction occurs but subside by the fifth day on
 Does not progress to systemic symptoms both the test and control arm  the individual is immune but
 Membrane will form on the wound it has been inoculated; fail to hypersensitive
heal (wound that fails to heal)  Microscopy – Gram – positive bacilli with Chinese character arrangement;
 Production of toxin will promote the development of anti – toxin pleomorphic appearance
antibodies once absorbed by the skin  Albert’s stain – methylene blue  reacts with the granules, producing a
 Prevention – Vaccine reddish or purple color (for visualization of volutin granules)
 DPT – Diphtheria, Pertussis, Tetanus  Culture
 Given to 2, 4 and 6 months old infants  BAM – routine enrichment medium
 Booster is given at 2 to 4 years of age  Potassium Tellurite Medium (Tynsdale Medium)
 Toxoid – pure culture emulsified in a broth medium + 0.3% formalin,  Selective – inhibits the growth of other Gram – negative bacteria
incubated at 37°C incubation until toxicity disappears  Differential – ability to reduce tellurite to tellurium  brown or black
 Purified and standardized, adsorbed unto aluminum hydroxide or precipitate within the colonies or surrounding colonies (black
aluminum phosphate colonies with brown halo)

41 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Differentiates the three biotypes of C. diphtheriae, based on the  A filter paper strip is soaked with diphtheria anti – toxin
growth characteristics  Covered with a liquefied solid medium – Elek’s medium  medium is
 Morphology of the colonies allowed to re – solidify
 Biochemical reactions exhibited  Anti – toxin diffuses outwards the filter paper unto the medium 
 Severity of the disease medium is saturated with anti – toxin
 A pure culture of the organism is heavily inoculated horizontally
Biotype Morphology against the width of the filter paper
Daisy head colonies – large,  Toxigenic strains will produce a V – shaped precipitin lines that forms
Var. gravis flat colonies with radial at an angle with the colonies of the organism
striations and irregular edges  In vivo – Animal Inoculation Test/ Animal Virulence Test
Coolie hats – medium sized,  Uses guinea pigs or white rabbits
Var. mitis convex, very black colonies  Emulsion or suspension of pure culture of the organism in a brain –
with regular edges heart infusion broth is incubated at 35°C to 37°C up to 48 hours
Frog’s eggs colonies – small,  Hair of the animal is clipped or shaved to expose the skin
Var. intermedius sometimes pinpoint, flat and  The skin is marked with 2 cm squares
gray  0.2 mL of the broth suspension is injected cutaneously
 5 hours after injection, 500 units of diphtheria anti – toxin will be
 Loeffler’s Serum Medium – poached egg appearance; enhances the injected intraperitoneally
visualization of the granules; contains horse serum and egg
 Biochemical Reactions Diphtheroids
 Glycogen and Starch Hydrolysis
 Corynebacterium species other than C. diphtheriae
 Hemolysis
 Mimics C. diphtheriae in infections and biochemical reactions
Glycogen and Starch
Nitrate
Biotype Hydrolysis Hemolysis Organism Urease Test Disease Others
Reduction
Glycogen Starch C. ulcerans – + Bovine mastitis
Var. gravis + + – Wood’s
C. xerosis + – Erythrasma
lamp
Var. mitis – – +
Opportuniustic
Var. intermedius – + – C. pseudodiphtheriae + +
infections
Isolated as
 Catalase – positive a multi –
 Oxidase – positive C. jaekium – – Septicemia drug
 CHO Fermentation – Glu, Mal resistant
 Urease Test – positive agent
 Nitrate Reduction – variable results C. minutissimum – –
 Virulence Test – Toxigenicity Test Notes:
 In vitro – Elek’s test  C. xerosis
 Purpose – for the detection of exotoxin from toxigenic strains of  Erythrasma – cutaneous skin infections  red rashes on toes, fingers, axillary
Corynebacterium diphtheriae and on the pubic area
 Uses serum or plasma

42 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Wood’s lamp – detects fluorescence; brick red fluorescence (porphyrin Listeria monocytogenes
production)
 General Characteristics
Propionibacterium acnes  Small, Gram – positive bacilli
 General Characteristics  Aerobic
 Uses propionic acid for carbohydrate fermentation  Non – spore forming, non – encapsulated
 Anaerobic Corynebacteria  Non – motile at 37°C, motile at 25°C (tumbling motility)
 Normal flora of the skin  Psychrophile – grows at refrigeration temperature
 Aerotrolerant – grows aerobically  Oxidase – negative
 Acne  Habitat and Transmission
 Splits the free fatty acids in the skin by lipases  release of FFA  tissue  Widespread in animals, soil and plants
inflammation  Human gastrointestinal tract and female genital tract
 Infections of prosthetic heart valves  Via in – utero infection, transferred transplacentally and during delivery;
 Infections of CSF shunts ingestion of contaminated milk products; direct contact with infected animal
 Strains – typed via surface antigens
Erysipelothrix rhusiopathiae  Ia
 Ib
 General Characteristics  IVb – epidemic listeriosis
 Gram – positive bacillus (somewhat looks like Gram – negative, because it  Virulence Factors
decolorizes easily) in short chains, randomly long, non – branching filaments  Intracellular – Internalin (i.e., within mononuclear phagocytes and epithelial
 Culture on BAM cells); cell wall surface protein which promotes adherence and invasion of non
 a – or γ – hemolysis – phagocytic cells
 Small translucent glistening colonies  Organizes host cell actin (Act A); the resulting actin trails propel Listeria directly
 Inert – catalase, oxidase and indole – negative into other cells, avoiding the extracellular environment; direct cell to cell
 TSIA – small amounts of H2S spread by the host’s actin polymerization
 Habitat and Transmission  Produces listeriolysin O, which facilitates the rapid phagosomal egress of
 Widely found in land and water animals (vertebrates and invertebrates) Listeria into the cytoplasm prior to phagosomal lysosome fusion, allowing
 Causes disease in swine, sheep, turkeys and ducks protected intracellular replication.
 Erysipelas – in swine  Pathogenesis of Listeria monocytogenes
 Can be transmitted to humans by direct inoculation to meat products  Ingestion of the organism  Internalin interacts with receptors on the epithelial
  risk – fishermen, fishes and fish handlers cell surface  induce phagocytosis by the epithelial cells  the organism is
 Erysipeloid – in humans; via fingers (direct inoculation) – ‘Seal finger’; ‘Whale secluded in a portion within the epithelial cell (phagolysosome), which has a
finger’ low pH  induces the production of listeriolysin O, which lyses the membrane
 2 to 7 days – the infected area is painful, swelling, lesion is raised and of the phagolysosome  organism is released into the cytoplasm of the
violaceous; purulent discharge  diffused cutaneous form of erysipeloid epithelial cells and proliferate within the epithelial cells  Act A induces the
 bacteremia and endocarditis host cell to polymerize actin and push the organism near the membrane of the
 Treatment – Penicillin G epithelial cell  organism will form filopods and allow the transfer of organism
 Organism demonstrates resistance to Vancomycin  Organism spreads via hematogenous spread
 Laboratory Diagnosis  Diseases
 Gelatin Stab Agar – bottle – brush growth  Granulomatosis infantiseptica
 Disseminated form of listeriosis; in – utero infection

43 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Early onset syndrome – infection in the fetus acquired  Colonies appear as small, translucent, slightly raised colonies with
transplacentally during pregnancy distinct blue green color
 Results to:  Semi – solid Medium (Hanging Drop Method)
 Abortion  Demonstrate the tumbling motility at 25°C
 Premature delivery  Umbrella – like zone of growth
 Sepsis during the peripartun period  Catalase – positive
- Neonatal sepsis – the organism is present in the blood  TSIA – does not produce H2S
- Pustular sepsis – viable organism in the granuloma  CHO Fermentation – glucose, trehalose, salicin
 Death of the infant may occur during or after delivery  CAMP test – positive
 Meningitis and Sepsis  Vogel – Proskauer – positive
 Colonized birth canal  Ocular Test of Anton – Anton’s Test
 Late onset syndrome – acquired during deliver; occurs 1 to 4 weeks  Pathogenicity test – differentiates pathogen strains from non – pathogenic
after delivery strains
 Caused by the serotype IVb  Instillation of 24 – hour broth culture of the organism in the conjunctiva of
 Predisposing factors – alcoholism, cirrhosis, hemochromatosis, ulcerative colitis, laboratory animals
asthma and AIDS, uncontrolled diabetes mellitus  (+) – purulent conjunctivitis within 24 to 36 hours (pathogenic strain)
 Treatment
 Ampicillin (intravenous) is given perinatally to infected pregnant women.
 The prognosis without treatment is poor, with a high fatality rate in newborns.
 Prevention
 The presence of Listeria in farm animal feces and its subsequent survival in the
environment and entry into processed meats and dairy products means there
is a relatively high risk of exposure. This is of concern mainly for pregnant
women and immunocompromised patients.
 Pasteurization kills Listeria in milk. Pregnant women and immunocompromised
patients should avoid raw cabbage and heat all processed meats before
eating.
 Laboratory Diagnosis
 Microscopy – Gram – positive coccobacillus (intracellular and/or extracellular);
resembles diptheroids
 Culture
 BAM – β – hemolysis (narrow zone of hemolysis that frequently do not
extend much beyond the edges of the colonies); forms small colonies of
about 1 to 2 mm in diameter
 Cold Enrichment Technique
 Enhances the isolation from clinical specimen
 Psychrophile – grows below its optimal temperature
 McBright Medium
 Selectively isolate Listeria monocytogenes from specimen
 Contains glycine, lithium chloride and ethyl phenol (bacteriocidal
agent) – inhibits Gram – positive and Gram – negative bacteria

44 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Gram – negative Bacilli
 Non – lactose fermenters
Enterobacteriaceae  Enteric pathogens, known cause opportunistic infections – Edwardsiella,
 Subdivided into tribes, according to Edward – Ewing’s Classification; each tribe is Proteus, Morganella and Providencia
composed of 1or 2 genera  Antigenic Structures
 Escherichieae – Escherichia and Shigella  O antigen – Somatic antigen (cell wall)
 Edwardsielleae –Edwardsiella  Most external part of the cell wall of the lipopolysaccharide layer
 Salmonelleae – Salmonella  It is an endotoxin, released when the cell lyses
 Citrobactereae – Citrobacter  Heat – stable, resistant to denaturation by heat and alcohol
 Klebsielleae – Klebsiella, Enterobacter, Pantoea, Hafnia, Serratia  K antigen – Envelope antigen (capsule)
 Proteae – Proteus, Morganella, Providencia  External to the O antigen
 Yersineae – Yersinia  Present in some but not all Enterobacteriaceae species
 Erwineae – Erwinia  Some capsular antigens are protein or polysaccharide in nature
 General Characteristics  Heat labile, prone to denaturation by heat and alcohol
 Gram – negative bacilli  H antigen – Flagellar antigen
 Non – spore forming  Composed of the flagellar protein, flagellin
 Some species are motile, with peritrichous flagella; others are non – motile  Heat labile, prone to denaturation by heat and alcohol
 Shigella, Yersinia – non – motile at 35°C to 37°C, motile at 25°C
 Some species are encapsulated (i.e. Klebsiella) General Laboratory Diagnosis for Enterobacteriaceae
 Facultative anaerobe, some are aerobes  Isolation Media
 Ubiquitous – found in the soil, water, vegetation, colons of humans and animals  Enrichment media – contains substances that enhance the growth of the
as part of the normal flora bacteria
 Normal enteric flora – incidentally cause opportunistic infections  Selenide Broth
 True enteric pathogens – regularly pathogenic (i.e. Shigella, Salmonella,  Tetrathionate Broth
Yersinia)  Gram – negative broth
 Oxidase – negative  Selective media
 Able to reduce nitrate to nitrite  Slightly selective – EMB, MacConkey
 Glucose fermenters at 35°C to 37°C, resulting to acid production and gas  Moderately selective – Salmonella –Shigella agar (SSA), Xylose – Lysine –
production Desoxycholate (XLD); Hektoen Enteric agar (HEA)
 Some are lactose fermenters  Highly selective – Bismuth Sulfite agar (BSA); Brilliant Green agar (BGA)
 Possess complex antigenic structures  Non – selective – BAM
 Produce a variety of toxins and virulence factors G(+) Colonies H 2S
 Compose of 25 genera, with more than 110 species; 20 to 25 species are Medium CHO pH Ind.
Agent Fer. N. Fer.
known to cause human infections Xylose
Phenol
 Classification XLD Agar Bile salts Lactose Yellow Red +
Red
 Colliforms Dextrose
 Lactose fermenters at 35°C to 37°C within 48 hours Yellow to
Salicin Blue –
orange;
 Normal enteric flora – Escherichia, Klebsiella, Enterobcter, Pantoea and HEA Bile salts Lactose BTB green; +
Salmon
Citrobacter Sucrose green
pink
 Non – coliforms BSA Brilliant Glucose +

45 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 green  Non – CHO fermenter – K/K
Citrate Thymol  Glucose fermenter, non – lactose and non – sucrose fermenter – K/A
TCBS Sucrose Yellow Colorless –
(pH 8.6) blue; BTB  iMVIC Reactions
Crystal
Neutral Pink to  Indole – Methyl red – Voges – Proskauer – Citrate
MAC violet; Lactose Colorless –
red red  Indole – production of tryptophanase
Bile salts
Eosin Y;
 Reagent
Dyes Pink to - Tryptophan broth
EMB Methylen Lactose Colorless –
(EMB) red
e blue - Xylene or chloroform (extractor)
Neutral Pink to - Kovak’s reagent – p – dimethylaminobenaldehyde
SSA Bile salts Lactose Colorless +
red red
BTB –Bromthymol blue

Notes: Tryptophan • Broken by

tryptophanase
 EMB and MAC – dyes will be precipitated in the acidic environment; the
precipitated dyes will be taken up by the colonies, resulting in the color of the
colonies
 Other mediums used • With pyruvic
 Moeller Decarboxylation Medium Indole acid and
ammonia
 Contains 1% amino acids; used if the organism cannot ferment glucose in
Lysine Iron gar (LIA)
 Sulfide Indole and Motility (SIM)
 Observes for H2S production and indole production Pink or wine • Xylene
- colored • Kovac's
 Motility in a semisolid medium – lateral growth away from the line of reagent
ring
inoculation (indicated by haziness on the medium)
 Biochemical Tests
 TSIA  Results
 Components - (+) – cherry red color at the junction of the two liquids
 0.1% glucose, 1% sucrose, 1% lactose - (-) – no color development
 Phenol red (pH indicator)  Spot Indole Test – modification of the Indole Broth test
 Beef extract, yeast extract, peptones  Methyl Red – Voges – Proskauer
 Ferrous sulfate, sodium thiosulfate  Citrate Utilization
 Reaction chambers  Determines the ability of the organism to use citrate as the sole
 Aerobic slant – oxidative decarboxylation; utilization of proteins, source of carbon and ammonium phosphate as the sole
forming alkaline by – products source of nitrogen
 Production of amines  RED (alkaline – K)  Simmon’s Citrate medium – Sodium citrate and ammonium
 Anaerobic deep – fermentation of carbohydrates phosphate
 Acid production  YELLOW (acid – A)
 Gas production – cracks of pulling away of the medium from the bottom
of the tube (g)
 H2S production – blackening of the medium; liberation of H2S can only be
produced in an ACIDIC medium
 Results
 Glucose, lactose or sucrose fermenter – A/A

46 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Deaminase Test
• Found in
Ammonium  Substrate specific test
Simmon's
phosphate medium  Examination of the ability of the organism to act on a specific substrate for
a specific enzyme
 Acts on the amino group of amino acids
• Alkaliniza
Ammonia tion  Phenylalanine deaminase test
 Usually performed on slants (presence of oxygen)
 Oxidative activation – ability of the organism to degrade the amino
Ammonium acid, phenylalanine
hydroxide

• Yellow at • Broken by
pH 6 Phenylalanine phenylalanine
Reaction • Bue at deaminase
with BTB pH
greater
than 7.6

 (+) – growth along the streak line or change in the color of the Phenylpyruvic
•(+) 10%
indicator acid (keto
FeCl3
acid)
- (+) Klebsiella pneumoniae
- (-) Escherichia coli
 ONPG Test
 O – nitrophenyl – β – D – galactopyranoside (+) Green
 Structure similar to lactose color
 Rapid identification of delayed lactose fermenters
 Enzymes used for lactose fermenters
 Permease – permit the penetration of lactose through the cell wall  Carboxylase Test
 Late lactose fermenters do not have the permease enzyme  Substrate specific test – acts on the carboxyl group of amino acids
 Β – galactoside – fermentation of lactose  Decarboxylation – removal of the carboxyl group
 Alkaline in nature, performed in an anaerobic medium (amine  alkaline)

• In the preence of
ONPG water and β -
galactosidase
•Lysine
Lysine decarboxylase

Galactose
+O- • Yellow color
Cadaverine
nitrophenol + CO2

 (+) Escherichia coli

 (-) Salmonella Lysine Decarboxylation

47 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Aerobic chamber – deamination process, in the presence of
oxygen
- (+) burgundy color
Ornithine •Ornithine
decarboxylase  Anaerobic chamber – decarboxylation process
- Fermentation of glucose  yellowing of the medium
- Decarboxylation process release of amines  neutralize
the acid formed during fermentation  yellow to purple
- (+) purple  yellow  purple (reversion of color)
Putrescine  Interpretation
+ CO2 -
-
R/K – (+) deamination; (+) decarboxylation
K/A – (+) deamination; (-) decarboxylation

Ornithine Decarboxylation

Arginine •Dehydrolase

Citrulline

Arginine Dehydrolation

 Lysine Decarboxylation Test

 Differentiates Citrobacter species from Salmonella species
 Assessed both in the slant and deep reaction chambers
 Biochemically resembles the iMVIC reactions
 Lysine Iron Agar (Edwards and Fife)
 For lysine deamtination and decarboxylation
 Components
- Lysine
- Ferrous sulfate for H2S production
- Bromcresol purple as pH indicator
- Glucose for carbohydrate fermentation; facilitate the
visualization of the decarboxylation process

48 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Escherichia coli  LT Toxin
- Heat – labile toxin
 General Characteristics - Activates adenyl cyclase, which catalyzes the addition of
 Gram – negative bacilli adenosine diphosphate ribose to G protein
 Lactose fermenter at 35°C to 37°C - Stimulate adenylate cyclase  increase intracellular cAMP
 Thermotolerant – growth beyond the optimal growth temperature (37°C to - Causes outpouring of fluid; affects the movement of ions
44°C) (K+ and Cl-) into the lumen of ileum and the jejunum
 Used for the index of fecal contamination of food and beverages - Results to diarrhea with watery stool
 Habitat and Transmission  ST Toxin
 Human colon, able to colonize the urethra and vagina - Heat stable, low molecular weight toxin
 Via fecal – oral route - Activates guanylate cyclase, increasing cGMP and
 Pathogenesis and associated Virulence Factors of Escherichia coli resulting in hypersecretion of fluids and electrolytes
 Urinary Tract Infections  Systemic Infections
 Caused by O serotypes  Virulence factors associated – capsule and endotoxin
 Virulence factors associated  Capsule interferes with phagocytosis and enhances the ability of the
 Pili with adhesins – adherence of the organism to specific receptor organism in various organs
sites found on the surface of the kidney and urinary tract epithelium  Endotoxin – lipopolysaccharides; associated with several signs and
 Motility – causes ascending infections; from the urinary bladder, the symptoms of Gram – negative sepsis
organism may reach the kidney  Hypotension
 Cystitis – inflammation of the urinary bladder  Fever
- Pain during urination (dysuria)  Disseminated intravascular coagulation
- Frequent urination  Neonatal meningitis
 Pyelonephritis – inflammation of the kidneys  E. coli is the second leading cause of neonatal meningitis
- Fever  Occurs in 25% of pregnant women due to the colonization of the
- Chills organism in the female reproductive system
- Flank pain  Sepsis
 Leading cause of community – acquired UTI, which occurs in females  Hospital – acquired sepsis
 Short urethra in females  Causes UTI, peritoneal and biliary infections (due to hematogenous
 Proximity of the urethra with the anus spread of the organism)
 Colonizer of the vagina  Diarrhea and Gastroenteritis
 Most frequent cause of nosocomial UTI, which equally occur in both male  Enterotoxigenic Escherichia coli (ETEC)
and females; commonly associated with in – dwelling catheters  Virulence factors associated
 Intestinal Tract Infections  LT Toxin
 Virulence factors associated  ST Toxin
 Pili – hair – like structures; promotes adherence to the cells of the  Adhesins
ileum, jejunum (small intestine)  Causes secretory diarrhea (Traveler’s diarrhea or Montezuma’s
 Enterotoxin – production of toxins when the cell has adhered to the revenge) in all age groups – similar to that of Vibrio cholera
intestinal mucosa; cell –specific toxins (attacks the cells of the ileum  Hypersecretion of water into the intestinal mucosa  watery, non –
and jejunum) bloody diarrhea
 Enterotoxigenic strains  Spreads through contaminated food and water
 Types  Signs and symptoms

49 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Vomiting
 Chills  Escherichia coli serotype O157:H7
 Headache  Most common serotype of EHEC; involved as a contaminant in
 Fevers undercooked meat (burger patties)
 Self – limiting for 1 to 3 days  Hemorrhagic colitis
 Due to the adhesion of the organism to the intestinal mucosa
 Enteroinvasive Escherichia coli (EIEC)  Signs and symptoms
 Causes bloody diarrhea in children; Shigella – like diarrhea - Abdominal cramps
 Penetration into the intestinal epithelium, producing an - Watery diarrhea
inflammatory diarrhea, similar to Shigella species - Lower gastrointestinal tract bleeding with little or no fever
 Serogroups 028, 0112, 0115, 0124, 0136, 0143, 0145 and 0146 are  Hemolytic Uremic Syndrome
most commonly implicated  EHEC complication
 No enterotoxin production  Toxin enters the bloodstream
 This strain of E. coli is suspected when observing blood, mucus and - Acute renal failure
segmented neutrophils in fecal smears - Thrombocytopenia
 Signs and symptoms - Microangiopathic hemolytic anemia
 Fever  Receptors on the kidney (Shiga –toxin receptors)  cell necrosis
 Abdominal cramps  platelets will migrate to the site of necrosis
 Dysenteric stool (thrombocytopenia), forming platelet plugs on the blood
 Necrosis, inflammation of the are bowel and ulcerations vessels  passage of red blood cells will cause distortion in its
morphology (schistocytes)  red blood cell destruction in the
 Enteropathogenic Escherichia coli (EPEC) liver
 Causes infantile diarrhea – epidemic in hospital nurseries  Uremia   urea (decreased clearance rate of urea due to
 Virulence factors associated – bundle – forming pilus, intimin and damaged kidneys; urea accumulates in the blood)
other factors that mediate organism adherence, resulting to
changes in cell surface  Enteroaggregative Escherichia coli
 Adhesins are produces causing adhesion to enterocytes, leading to  Newest strain causing diarrhea and gastroenteritis
the damage to villi  Virulence factors associated
 Moderately invasive  penetration into the intestinal epithelium  Adhesins – causing adherence to epithelial cells, resembling a
 Watery diarrhea – mucus with no blood pile of stacked bricks
 Heat stable – like toxins and hemolysins
 Enterohemorrhagic Escherichia coli (EHEC)  Signs and symptoms (which ultimately leads to DEHYDRATION)
 Verotoxic Escherichia coli (VTEC) – produces cytopathic effects  Watery diarrhea, that may be acute or chronic which lasts for
against the vero cells (seen in the green African monkeys)  more than 14 days
inflammation of the colonic mucosa and toxin damage to vascular  Vomiting and abdominal pain
endothelial cells, resulting to bloody diarrhea
 Shiga – toxin producing Escherichia coli (STEC) – exotoxin produced
is similar to Shigella species
 Acts by removing an adenine large (2BC) ribosomal RNA 
stopping protein synthesis
 Conferred by lysogeny (by temperate phages)

50 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Salmonella species  S. choleraesuis
 Habitat and Transmission
 General Characteristics  Found in human and animal colon
 Non – lactose fermenting  S. typhi – human colon (ingestion of animal products contaminated with
 H2S – positive human feces)
 Motile  S. choleraesuis – animal colon
 Antigens  Via fecal – oral route
 O and H – basis for the classification based on Kauffman and White  Pathogenesis and associated Virulence Factors of Salmonella species
 Vi antigen – seen in Salmonella typhi; capsular antigen (envelope  Enterocolitis
antigen)  Most common form of salmonellosis
 Taxonomy of Salmonella species  Most common agent – S. typhimurium
 Ewing’s Classification  Incubation period – 6 to 48 hours
 Salmonella typhi  Results from multiple sources of contamination, including food (most
 Salmonella choleraesius commonly poultry and poultry products), human carriers (particularly food
 Salmonella enteritidis handlers), and exotic pets (turtles and snakes)
 Contains 1500+ serotypes  Usually characterized by the following pattern:
 S. paratyphi A  Ingestion of organisms in contaminated food
 S. paratyphi B  Colonization of the ileum and cecum
 S. paratyphi C  Penetration of epithelial cells in the mucosa and invasion, resulting in
 S. puliorum acute inflammation and ulceration
 S. derbi  Release of prostaglandin by enterotoxins, resulting in activation of
 S. arizonae adenyl cyclase and increased cAMP
 S. typhimurium  Increased fluid secretion in the intestines
 Kauffman – White Classification  Signs and symptoms
 Contains several serotypes, with 1500+ species  Nausea
 Species are named after the city from which they have been isolated  Vomiting
 S. dublin – Salmonella enteritidis serotype dublin or var. dublin  Diarrhea (mild to severe with or without blood)  PEA SOUP STOOL
 DNA Hybridization Analysis  Abdominal pain
 Classification based on DNA relatedness  Ingestion of fecally contaminated food and beverages  invasion of the
 S. typhi – not a distinct specie of Salmonella gastrointestinal tract (epithelial subepithelial cells of the ileum and the
 Salmonella enteritica serovar. typhi (DNA composition is not cecum)  intracellular proliferation
similar to Salmonella)  Virulence factors associated
 Clinical Categorization  Cytotoxin – acts on the invaded cells, causing lysis of endosomes
 Typhoidal species – Salmonella species that cause typhoid fever within the invaded epithelial cells
 S. typhi – Eberth’s bacillus (Eberthella typhosa)  Enterotoxin – causes hypersecretion of water; movement of
 S. paratyphi electrolytes is affected  diarrhea
 Non – typhoidal species – Salmonella species that cause enterocolitis,  Enteric fevers
metastatic infections and osteomylelitis  Typhoid fever caused by S. typhi
 S. enteritidis – Gardner’s bacillus  Incubation period – 1 to 2 weeks
 S. paratyphi B – S. schottmuelleri  Signs and symptoms
 S. paratyphi C – S. hirschfeldii  1st week – fever and constipation

51 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  2nd week – sustained fever, splenomegaly and hepatomegaly; rose Shigella species
spots on the abdomen
 Blood specimen is used for the diagnosis of the disease  General Characteristics
 3rd week – fever declines  Non – lactose fermenters
 4th week – convalescence  Gas – negative
 Carrier state is established in the gallbladder  H2S – negative
 Stool specimen is used for the diagnosis of carrier state  Non-motile, non – encapsulated
 Ingestion  penetration of the intestinal mucosa by the organism   Most effective enteric pathogen
PHAGOCYTOSIS (by macrophages present in the GIT)  the organism  Infectious does of 10 to 100 bacilli
survives and multiply in the PEYER’S PATCHES  spread of the organism to  Ewing’s Classification – based on the cell wall surface O antigens
the liver, spleen and gallbladder  hematogenous spread  septicemia  Group A – Shigella dysenteriae (Shiga’s bacillus; causes Japanese dysentery)
 Complication – ulceration of the intestinal wall  Group B – Shigella flexneri (Flexneri’s bacillus, Strong’s bacillus; causes
 Carrier state Philippine dysentery)
 Predilection of the organism to the gallbladder (preference of the  Group C – Shigella boydi (Boyd’s bacillus; causes British dysentery)
organism with the gallbladder)  Group D – Shigella sonnei (Sonne – Duval’s bacillus; causes US dysentery)
 Infectious dose – number of the bacterial cells enough to establish  Habitat and Transmission
infection in a susceptible host  Human colon only
 100,000 bacterial cells  Via fecal – oral route
 Septicemia  Virulence Factors
 Most common agents – S. choleraesuis, S. typhi, S. paratyphi  Proteins
 Signs and symptoms  Invasion plasmid antigens (Ipa)
 Gastrointestinal symptoms  Surface presentation antigens (Spa)
 High, spiking fever  Membrane excretion proteins (Mxi)
 Present in patients with underlying diseases – sickle cell anemia and  Virulence proteins (Vir)
cancer  Endotoxin
 Osteomyelitis – seen in patients with sickle cell anemia; the organism is  Enterotoxin
present in the blood first before the bone  Neurotoxin (S. dysenteriae)
 Characterized by a precipitating incident that introduces bacteria (e.g.,  Pathogenesis of Shigella species
catheterization, contaminated intravenous fluids, abdominal or pelvic  Shigellosis – bacillary dysentery  bloody diarrhea
surgery), followed by a triad of chills, fever, and hypotension  Incubation period – 1 to 4 days
 May cause local abscesses, osteomyelitis, and endocarditis if the  Signs and symptoms
organisms are disseminated widely  Fever
 High mortality rate (30% to 50%), depending on the degree of preexisting  Abdominal cramps and pain
debilitation  Watery diarrhea followed by dysentery
 Tenesmus – little stool with much blood and mucus is produced
(indication of ineffectual defecation)
 Ingestion of fecally contaminated food and beverages  attachment of
the organism to the mucosa (via ATTACHMENT PROTEIN)  invasion of the
intestinal mucosa (ileum and colon)  proliferation within the intestinal
epithelium  intercellular spread by ACTIN POLYMERIATION 

52 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 degeneration and local inflammation of the site invaded, causing Enterobacter species
ulceration and bloody diarrhea (SHIGA – TOXIN)
 Shiga – toxin – conferred by lysogeny  Opportunistic pathogens – related to hospitalization
 No prolonged carrier state is established  Associated with invasive procedures – IV catheterization, respiratory
 Disease is confined in the gastrointestinal tract due to its non – motile intubation; urinary tract manipulation
characteristic  Resembles Klebsiella infections
 Samples used for the diagnosis of shigellosis are stool and rectal swab.  Motile in nature
 Significant species – Enterobacter cloacae; Enterobacter aerogenes

Klebsiella – Enterobacter – Serratia – Hafnia Group

Serratia marcescens
 General Characteristics
 Voges – Proskauer – positive  Associated with nosocomial infections
 Distinguishing characteristics between species are the biochemical reactions  Produces characteristic red pigments, which are more pronounced at room
and motility temperature incubation
 Produce large amounts of gas  Prodigiosin – non-water soluble (non – diffusible) dye which stays within the
 Habitat – human colon and soil colony
 Diseases caused – UTI, pneumonia, bacteremia  Pyrimine – water soluble (diffusible) dye, which spreads throughout the
 Most diseases associated are nosocomial and opportunistic infections medium (BAM and MAC)

Klebsiella pneumoniae Proteus – Morganella – Providencia Group

 Also known as Friedlander’s bacillus  General Characteristics
 Encapsulated (possess a very large polysaccharide capsule  bacillus mucosus  Non – lactose fermenters (glucose fermenter)
capsulatus), non – motile  Motile (hypermotile)
 Normal flora of the gastrointestinal tract (carried in the respiratory tract in 10% of  H2S – positive
healthy individuals, making them prone to pneumonia)  Grows on potassium cyanide medium (KCN)
 Predisposing factors to disease  Phenylalanine deaminase – positive
 Advanced age  Urease – positive
 Chronic respiratory disease  Habitat – found in the colon, soil and water
 Diabetes  Diseases caused – UTI and wound infections (nosocomial and community –
 Alcoholism acquired infections)
 Pneumonia  Associated with hypermotile characteristic of the organisms
 Produces a characteristic currant – jelly sputum (thick bloody sputum); lobar  Specimen for diagnosis – urine, wound swabs/discharge
pneumonia  Taxonomy
 Causes abscess formation in the lungs, necrosis, hemorrhage and death of the  Proteus vulgaris
ells in the lungs  Proteus mirabilis
 Produces a characteristics mucoid colonies – strikingly mucoid colonies; mucoid  Proteus morganii  Morganella morganii
watery colonies that tend to string out when touched by an inoculating loop  Proteus rettgeri  Providencia rettgeri
 Pathogenesis
 Urinary Tract Infection
 Urea in urine is converted to ammonia and carbon dioxide by the action
of urease, leading to the alkalization of the urine (due to ammonia)

53 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  The alkaline environment encourages the formation of renal calculi  Yersinia species
STRUVITES
 General Characteristics
 Causes obstruction of urine flow in the ureters  URINE STASIS
 Small, Gram – negative coccobacilli
 Hematuria and dysuria are due to the damage to the urinary epithelium
 Non – motile at 37°C, motile at room temperature (25°C to 30°C)
and struvites  NIDUS (recurrent infection due to trapping the bacteria in
 Non – lactose fermenter
the stones)
 Treatment – removal of the stones; antibiotics and acidification of the
urine Yersinia pestis
Proteus species  Characteristics
 Safety pin appearance (two poles are stained with a clear central area) –
 Swarming motility – periodic cycles of migration in BAM
enhanced by Wayson’s stain
 Produces concentric zones around the point of inoculation
 Encapsulated (seen in freshly – isolated culture)
 Diene’s phenomenon – differentiate species of Proteus
 Capsules disintegrate when left at room temperature, rendering the
 Different species are inoculated separately on the same medium
bacteria avirulent
 Swarm towards each other but do not mix, forming a demarcation
 Rodents are the principal reservoir
 Burnt gun powder odor in cultures
 Humans are incidental hosts of the organism; vectors bite men, resulting to
 Serology
infection
 OX2, OX19 and OXK – cross reactions with antigens of several species of
 Rodents are infected by being bitten by fleas
Rickettsiae
 Rat fleas (vector) – Xenopsylla cheopsis
 Detect antibodies from the patient serum against several species of
 Not a normal flora of the colon
Rickettsiae
 Pathogenesis – Black death
 Weil Felix Test
 Mode of transmission – bites of infected flea
 Flea performs a blood mean on a rodent infected with the organism  flea
Citrobacter species ingests the bacteria (blood is dissolved by the enzyme, coagulase, produced
 Associated with urinary tract infections, respiratory tract infections and sepsis by the organism)
 Found in the gastrointestinal tract and in water  Multiplication of the bacteria within the blood clot  blocking of the
 Biochemically and antigenically resembles Salmonella proventriculus of the flea  regurgitation occurs  flea injects the organism
 Significant species – Citrobacter freundii; Citrobacter koseri into the rat OR bites men due to loss of natural selectively for the host (no
nutrition in the rat)
 Types of Black Plague
 Bubonic plague
 Painful and swollen lymph nodes  BUBOES (seen in the groin and
axilla area)
 Progresses to septicemia  DIC
 Hemorrhagic – blue black rashes
 Necrotic – necrosis of many organs of the body
 Pneumonic plague
 Inhalation of infectious respiratory droplets (human to human)
 Septicemia plague
 Progression of bubonic and pneumonic plaque

54 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Yersinia enterocolitica Related Enteropathogens
 Characteristics
 Non – lactose fermenter (sucrose fermenter) Family Vibrionaceae
 True enteric pathogen – causes infections in the gastrointestinal tract
 General Characteristics
 Most common Yersinia species isolated
 Gram – negative curved or straight rods
 Pathogenesis
 Aerobic or facultative anaerobe
 Enterocolitis
 Non – spore forming
 Via fecal – oral route
 Motile (monotrichous flagella)
 Laboratory Diagnosis
 Oxidase – positive
 Cold Enrichment – inoculation of feces to a buffered saline solution at pH 7.6;
 Nitrate reduction – negative
incubated at 4°C for 2 weeks  subcultured to Cefsulodin – Irgasan-
Novobiocin (CIN) agar  incubated for 48 hours  exhibit BULL’S EYE colonies
Vibrio cholerae
 Characteristics
 Comma – shaped Gram – negative rods (komabacillus)
 Habitat – human colon (no animal reservoir)
 Transmission via fecal – oral route
 Biotypes – Classical and El Tor
Differentiation Classical El Tor
Hemolysis on SBA
γ Β
(Grieg Test)
Chicken RBC
– +
Hemagglutination
Polymyxin B (50
Susceptible Resistant
IU)
Voges –
– +
Proskauer

 Strains – classified by the nature of the O cell wall antigen

 O1 group – causes epidemic diseases
Serotypes of Vibrio cholerae O1
Serotype O Antigen
Ogawa (Philippines) A and B
Inaba (Japan) A and C
Hikojima (India) A, B and C
** Serotypes are named according to the country where they are commonly isolated
 Non – O1 group – causes sporadic diseases; non – pathogenic
 Virulence Factors
 Mucinase – dissolution of the protective glycoprotein covering of the mucosa

55 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Choleragen – enteroroxin; hypersecretion of water and electrolytes into the  Presence of bile salts inhibit the growth of Gram – positive
lumen of the mucosa  RICE WATERY STOOL organisms
 Pathogenesis of Vibrio cholerae  1% NaCl – optimum growth and metabolic process of
 Cholera (Asiatic cholera or Epidemic cholera) halotolerant organisms
 Disease process  Thiosulfate – source of sulfur, acts in combination with ferric
 Ingestion of the organism (infectious does of 108) via fecally citrate for H2S production
contaminated food and water  Sucrose – differential
 Adherence or attachment to the mucosa of the intestine via the pili  BTB or thymol blue – pH indicators
 Release of the enzyme, mucinase  dissolution of the protective  pH 8.5 to 9.5 – suppresses the growth of intestinal flora
glycoprotein covering of the mucosa  Yellow colonies
 Production of the enterotoxin (choleragen)  A and B subunit  Tellurite taurocholate gelatin agar (TTGA) – dark – centered colonies
 A subunit surrounded by cloudy zones
 Active  inserted into the cytosol of the enterocytes  Taurocholate gelatin agar – flat, transparent colonies surrounded by
 Stimulate the ADP ribose  GS subunit of the G protein  cloudy zones
persistent stimulation of adenylate cyclase  production of  Gelatin agar – transparent colonies surrounded by opaque halos
cAMP  loss of water and electrolytes  WATERY DIARRHEA  Biochemical tests
 B subunit  Oxidase – positive
 Binding  binds to the ganglioside receptors in the enterocytes  Nitrate reduction – positive
 proceed to the process of A subunit  Glucose, sucrose and lactose fermenters
 Ultimately leads to dehydration  excrete up to 1 liter of water per hour  TSIA – A/A
 Renal failure  Kriggler Iron Agar – K/A
 Cardiac arrest  Lysine decarboxylation – positive
 Death  Ornithine decarboxylation – positive
 Fatal in 40% of untreated cases  String test
 Infectious dose of 108 bacterial cells (the organism is susceptible to the  Organism is emulsified on a glass slide using 0.5% sodium desoxycholate
acids of the stomach)  Inoculating loop is used to pull away  string formation
  risk – individuals taking antacids and persons who underwent  (+) – long string becoming more tenacious after 60 seconds
gastrectomy (lack the acids that may neutralize the organism)  Cholera Red test – Nitroso Indole test
 Laboratory Diagnosis  APW with tryptophan and indole  addition of sulfuric acid  red color
 Microscopy – curved, Gram – negative rods  (+) red color (nitroso indole)
 Darkfield microscopy – darting motility (presumptive identification)  Pfeiffer’s phenomenon
 Culture  Immune guinea pig  inoculation of the organism  lysis of the organism
 Alkaline peptone water (APW)  0/129 susceptibility test
 Composition  2,4 –diamino – 6,7 – diisopropyl pteridine monophosphate  Vibriostatic
 1% peptone compound
 1% NaCl  (+) – zone of growth inhibition
 Specimen is inoculated
 Growth is subcultured within 12 to 18 hours using special agars
 Thiosulfate – Citrate – Bile Salts – Sucrose (TCBS) Agar
 Components

56 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Vibrio parahemolyticus Genus Campylobacter
 Found in marine organisms; halophilic in nature (able to grow at 2% to 4% NaCl) Campylobacter jejuni
 Gastroenteritis
 Disease commonly found in Japan – associated with the consumption of large  General Characteristics
amounts of raw fish meat  Slender, Gram – negative curved rods (seagull wings, S – shaped)
 Incubation time – 6 to 96 hours  Motile (polar flagellum; single, unsheathed)
 Pathogenesis is unknown; enterotoxin may be implicated (hypersecretion of  Microaerophilic, capnophilic – 5% oxygen, 10% carbon dioxide
water) and pili  Grows well at 42°C, fails to grow at 25°C
 Pili facilitates attachment to the intestinal mucosa  invasion of the organism  Oxidase – positive; catalase – negative
 Signs and symptoms  Does not utilize carbohydrates – asacharolytic
 Watery diarrhea  Habitat and Transmission
 Nausea  Human and animal feces
 Vomiting  Via fecal – oral route
 Abdominal cramps  Pathogenesis of Campylobacter jejuni
 Fever  Enterocolitis or Gastroenteritis
 Self – limiting within 3 days  Disease is limited in the gastrointestinal tract (extra – GIT infections are
 Laboratory Diagnosis rare)
 TCBS agar – blue – green colonies  Due to the invasion of the colon by the organism
 Kanagawa’s phenomenon  Enterotoxin – mediated – acts in the same manner as Cholera toxin
 Determine if the strain isolated produces a hemolysin  hemolysis  Signs and symptoms
indicates pathogenicity  Watery, foul – smelling diarrhea
 Hemolysin produced by V. parahemolyticus  cytotoxic and cardiotoxic  Blood stool
 Able to lyse human red blood cells in Wagatsuma BAM  Fever
 Hemolytic activity produced by strains of V. parahemolyticus  Severe abdominal pain
 (+) – hemolysis; hemolytic pattern under or surrounding the colony  Guillian –Barre Syndrome
 Medium with 8% NaCl - growth; differentiates V. cholerae from V.  Autoimmune disease associated with antibody production by the
parahemolyticus individual, which acts on the neurons  neuromuscular paralysis
 (+) V. parahemolyticus  Laboratory Diagnosis
 (-) V. cholerae  Specimen – blood, feces and rectal swab
 Darkfield microscopy – darting motility
 Culture – BAM with antibiotics (inhibit other normal flora of the GIT)
Vibrio vulnificus
 Incubation at 42°C with microaerophilic environment (5% oxygen, 25%
 Found in marine organisms; halophilic in nature (able to grow at 2% to 4% NaCl) nitrogen, 10% carbon dioxide)
 Causes cellulitis in shellfish handlers Medium Base AMA
 Septicemia – rapid and fatal among immunocompromised hosts who have eaten - Bacitracin
raw, contaminated shellfish 3% Fluid - Novobiocin
Butzler’s Selective
thioglycolate - Colisitin
Medium
agar - Cephalothin
- Actidine
Skirrow’s Blood Blood agar base – - Vancomycin

57 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Agar 7% lysed horse - Polymyxin B  Protease – modifies the gastric mucosa of the stomach, leading to the
blood - Trimethoprim decreased ability of the acid to diffuse
- Vancomycin  Urease – enzyme produced by the organism
Brucella agar - Polymyxin B  Breaks down urea to ammonia and carbon dioxide
Blaser’s Medium
base - Trimethoprim  Ammonia neutralizes that gastric mucosa, destroying the epithelium
(Campy – BAP)
10% Sheep blood - Cepahalothin of the stomach
- Ampothericin  Inflammatory response  damages the gastric mucosa
 Causes ulceration of the stomach  peptic ulcer
 Different species of Campylobacter
C. jejuni C. fetus  Laboratory Diagnosis
25°C – +  Gastric biopsy
Growth 37°C + +  Histologic examination
42°C + –  Via endoscopy
 Staining
Hippurate Hydrolysis + –
 Wartin Starry Silver Impregnation
Nalidix acid S R
Susceptibility - Seagull shaped, Gram – negative rods
Cepalothin R S
 Hematoxylin and Eosin
Urease test – –
- Seagull shaped, Gram – negative rods
Notes:
 Biopsy Urease test
 Campylobacter fetus
 In vitro; detects color change
 Sexually – transmitted infection animals  abortion
 Urea medium is inoculated at 37°C for 2 hours
 Rarely acquire infection from animals
 Urea, when acted upon by urease produces a magenta color
 Causes infections in humans (opportunistic infections in
 (+) – magenta color
immunocompromised patient)
 Culture
 Portal of entry in humans – GIT  diarrhea
 Brucella agar with 5% sheep blood – 35°C to 37°C incubation;
 May progress to systemic infections (bacteremia)
microaerophilic environment for 3 to 5 days; small translucent gray
colonies
Genus Helicobacter  Trypticase soy broth with 5% sheep blood
 Urea Breath test (UBT)
Helicobacter pylori
 Uses radioactive 14C – labeled urea
 General Characteristic – same characteristics as C. jejuni, differs in biochemical  Breath samples are used
reactions  Scintillation
 Pathogenesis of Helicobacter pylori  Ingestion of C – labeled urea  determines the amount of labeled
 Gastritis and Peptic ulcers radioactive by – product (carbon dioxide)
 Chronic and active inflammation characterized by infiltration of   CO2 – indicates Helicobacter infection
polymorphonuclear cells an monocytes in the lamina propria  Antibody detection – uses blood samples
 Optimum pH of the organism – pH 6.7  Antigen detection – uses stool samples
 Gastric mucosa – pH 1 – 2
 Epithelium – pH 7,4
 Organism resides near the epithelium of the stomach, where it is
physiologically suitable for the organism

58 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Helicobacter vs. Campylobacter Non – fermentative Gram – negative Bacilli
 Group of organisms that are either
Campylobacter Helicobacter  Asaccharolytic – organisms that are not able to utilize carbohydrates (ex.
Urease – + Alkaligenes, Moraxella)
Oxidase + +  Oxidative – organisms that utilize carbohydrates in the presence of oxygen
Catalase – +  (+) acids - production of weak or small amounts of acids
Motile – tuft of the  TSIA – K/K (reaction is not able to convert the pH indicator to another
Motile – polar, color)
flagella located
Flagella unsheathed  Ether – Duodoroff Pathway – pathway in which oxidative utilization of
at one end
(monotrichous) carbohydrates occur
(lophotrichous)
 Pseudomonas, Acinetobaacter, Stenotrophomonas
 Laboratory Diagnosis
 Carbohydrate Fermentation Medium
 Components
 Peptone – 10 grams
 Sodium chloride – 5 grams
 D –glucose – 5 grams
 Bromcresol purple – 0.02 grams
 Agar – 15 grams
 Hugh and Leifson O – F Medium
 Components
 0.2% peptones
 1.0% glucose
 0.3% agar
 Bromthymol blue
 Diphosphate buffer
  glucose;  peptone – adjust to the metabolic capacity
  peptone –  alkaline by – products by the oxidative process
  glucose –  acid by – products produced from glucose oxidation
  agar – determine the consistency of medium; semi –solid medium
(permit oxygen to penetrate the medium)
 Procedure
 Both tubes of O – F glucose are inoculated with test organism
 Mineral oil or paraffin – limits the entry of oxygen into the tube
 differentiates oxidative from fermentative medium
 Results
Open Tube Covered Tube Metabolism
Acid (YELLOW) Alkaline (GREEN) Oxidative
Acid (YELLOW) Acid (YELLOW) Fermentative
Alkaline (GREEN) Alkaline (GREEN) Asaccharolytic

59 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Genus Pseudomonas  Associated with hospital respirators, resulting to necrotizing pneumonia
 Alginate production – mechanism that surround the bacterial cells and
Pseudomonas aeruginosa protects it from phagocytosis
 Alginate gene – activated by cystic fibrosis
 General Characteristics
 Meningitis
 Gram – negative rods
 Associated with lumbar puncture (direct access of the organism) –
 Motile (polar flagella – monotrichous)
nosocomial infection
 Strict aerobes
 Sepsis
 Oxidase – positive (vs. Enterobacteriaceae)
 Seen in debilitated patients and infants
 Good growth on MAC (colorless colonies)
 Endocarditis – sequelae of sepsis seen in Iv drug users (due to
 Habitat and Transmission
contaminated syringes)
 Environmental water sources, hospital respirators and humidifiers (associated
 Mortality rate of >50%
with nosocomial infections); found in small amounts on the skin, upper
 Laboratory Diagnosis
respiratory tract and colon of about 10% of people
 Microscopy – Gram negative bacilli
 Via water aerosols, aspiration and fecal contamination
 Culture
 Virulence Factors
 BAM – β – hemolysis
 Pili – facilitates attachment to host cells
 EMB or MAC – colorless colonies (large, dry, flat colonies with ground –
 Slime – exopolysaccharide; forms mucoid colonies from patients with cystic
glass appearance
fibrosis
 Able to produce 2 pigments that give a characteristic fluorescence
 Endotoxin – lipopolysaccharide; has a direct role in the manifestation of signs
 Pyocyanin – blue pigment  facilitates damage of the ciliated mucosal
and symptoms (fever, shock, oliguria, leukocytosis, leukopenia, DIC and adult
cells of the respiratory tract
respiratory distress syndrome)
 Pyoverdin – yellow – green pigment – fluorescence under Wood’s lamp
 Exotoxin A – diphtheria – like toxin (inhibits protein synthesis)
 Biochemical tests
 Hemolysin
 TSIA – K/K
 Heat labile – phospholipase C
 Oxidase – positive
 Heat stable – glycolipid
 Characteristic grape- fruit odor or corn – taco odor
 Protease – degrades protein
 Collagenase and elastase – extracellular enzymes produced by the organism
which has histotoxic effects Burkholderia (Pseudomonas) mallei
 Alginate – surrounds the bacterial cell; protects it from phagocytosis; inhibits  General Characteristics
chemotaxis  Non – motile
 Pathogenesis of Pseudomonas aeruginosa  Does not grow at 42°C
 Wound infections  Weakly oxidase – positive
 The organism produces water soluble pigments  produces the  Glander’s Disease or Farcy
characteristic blue – green pus  Affects goats, horses, sheep and donkeys in Asia, Africa and the Middle East
 Ecthyma gangrenosum – black necrotic lesion surrounding the wound  Transmitted to man by:
(THIOMELANIN)  Direct contact
 May lead to sepsis  Trauma
 Urinary tract infections  Inhalation (from infected host)
 Associated with instrument or catheters that allowed the introduction of  Cutaneous involvement – thickening of the superficial lymphatics
the organism (nosocomial infection)
 Pneumonia

60 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Burkholderia (Pseudomonas) pseudomallei Genus Eikenella
 General Characteristics Eikenella corodens
 Motile (monotrichous flagella)
 Oxidase – positive  Pits or corrodes the surface of the agar medium
 Able to oxidize glucose, maltose, lactose, mannitol and cellubiose  Seen in blood culture after 2 extractions
 Vietnamese Time Bomb (Meloidosis)  Needs factor X (hemin) for growth – requires CAM
 Glander’s – like disease  Characteristics bleach – like odor
 Seen in soldiers who came from the Vietnam war  Produces alkaline reactions (asaccharolytic)
 Southeast Asia and Australia
 Severe pneumonia that could lead to death Genus Kingella
Kingella dentrificans
Genus Acinetobacter
 Rare pathogens to humans
 General Characteristics
 General Characteristics
 Oxidase – negative
 Oxidase – positive
 Catalase – negative
 Catalase – negative
 Non – hemolytic on BAM
 Indole – positive
 Non – motile
 Oxidizes glucose
 Significant species – Acinetobacter lwoffi, Acinetobacter baumanii
 Pits the surface of the agar
 Requires blood for growth
Acinetobacter lwoffi
 Acinetobacter calcoaceticus var. lwoffi
 Causes nosocomial infections (associated with hospital respirators and medical
equipment)
 Urinary tract infections
 Respiratory infections
 Wound infections – organism colonizes moist areas of the skin

Genus Alkaligenes
Alkaligenes faecalis
 Formerly known as Alkaligenes odorans
 Produces alkaline reactions on O – F medium (asaccharolytic)
 Has a characteristics fruity odor (apples, pears)
 Able to grow on 6.5% sodium chloride
 Causes opportunistic infections – CSF, blood, urinary tract, pleural cavity, wound
infections, abscesses

61 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Genus Haemophilus  Serotype B – causes the most severe and invasive diseases
 Habitat – respiratory tract
 General Characteristics  Transmission via respiratory droplets
 Small, Gram – negative bacilli  Virulence Factors
 Non – motile, non – spore forming  Polysaccharide capsule
 Aerobe or facultative anaerobe  Primary virulence factor of Haemophilus influenzae
 Requires growth factor from blood (for energy production)  Composed of polyribitol phosphate or polyribose phosphate
 Factor X  derived from red cell lysis, releasing hemoglobin  Classification
 Hemin, hematin or heme  Typeable strains
 Heat stable  Non – typeable strains – strains the can still cause disease but not as
 Factor V  derived from yeasts or potato extracts, Staphylococcus severe as serotype B
species, Neisseria species and pneumococci  Epiglottitis – characterized by cherry red, swollen epiglottis that
 Nicotinamide adenine dinucleotide (NAD) or CoE I may cause obstruction of the airways
 Nicotinamide adenine dinucleotide phosphate (NADP) or CoE II  Otitis media and sinusitis –characterized by pain on the
 Heat labile affected area (redness and bulging of the tympanic
membrane)
Identification of Haemophilus species  IgA Protease
Requirements  Degrades secretory IgA  facilitates attachment to the respiratory
Species Comments
Factor X Factor V mucosa  invasion of the bloodstream, leading to bacteremia and
H. influenzae + + meningitis
Causes subacute bacterial  β – lactamase
H. parainfluenzae – +
endocarditis (SBE)  Degrades β – lactam antimicrobial agents
H. haemolyticus + +  Renders treatment with penicillin – containing antimicrobial agents
H. parahaemolyticus – + ineffective
Also termed as Koch – Week’s  Endotoxin
H. aegyptius + + bacillus; causes pink eye  Pathogenesis of Haemophilus influenzae
(conjunctivitis or sore eyes)  Meningitis
H. aprophilus – –  Used to be a leading cause of meningitis
H. paraphilus – +  Causes meningitis in children from 6 months to 6 years of age
Also termed as Ducrey’s bacillus;  Peak ages – 6 months to 1 year
causes chancroid or soft chancre;  Attributed to the following factors
H. ducreyi + – Gram – negative pleomorphic  Decline in maternal IgG in the child
coccobacilli with school of fish  Inability of the child to generate sufficient antibody against the
appearance polysaccharide capsular antigen
 Signs and symptoms –fever, stiff neck and drowsiness
 May progress and produce neurologic sequelae  subdural empyema
Haemophilus influenzae
 Prevention  administration of a conjugate vaccine
 General Characteristics  Carrier protein or carrier
 Requires both factor X and V  Attenuated organism or poor antigen
 Encapsulated  Antigenic determinants
 Polysaccharide capsule with serotypes A, B, C, D, E and F  Otitis media

62 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Pneumonia – seen in patients with obstructive lung disease Genus Bordetella
 Laboratory Diagnosis
 Microscopy Bordetella pertussis
 Gram – negative bacilli or coccobacilli (in CSF and blood)
 General Characteristics
 Neufeld – Quellung Reaction  visualization of the capsule of the
 Very small, Gram – negative bacilli
organism in the CSF
 Non – motile
 Culture – requires increased carbon dioxide concentration
 Strict aerobe
 Chocolate agar medium – pinpoint to small gray mucoid colonies
 Has bipolar metachromatic granules which stain with toluidine blue
 Staphylococcal Streak Technique – utilizes blood agar medium
 Forms acid but NOT gas from glucose and lactose
 Haemophilus colonies appear as blue – gray, pinpoint, dew –drop
 Habitat  human respiratory tract
colonies around the Staphylococcus colonies (satellite
 Transmission via respiratory droplets
phenomenon or satelitism)
 Also known as Bordet – Gengou bacilli
 Other culture medium (enriched with factor X and V) – Casman’s agar,
 Virulence Factors
Fildes Enrichment Agar, Levinthal Agar
 Pili – facilitates attachment
 Other tests
 Filamentous Hemagglutinin (FHA)
 Test for X and V Factor Requirement – basis for the differentiation and
 Protein that mediates attachment of the organism to the cilia of the
identification
epithelial cells of the respiratory tract
 Utilizes BHI or TSIA
 Pertussis toxin
 Filter paper strips or disks are impregnated with X and V factor
 Resembles diphtheria toxin  stimulates adenylate cyclase
 Porphyrin Test – Porphyrin Production Test
 ADP – ribose + Gi (protein G inhibitory subunit)  prolonged stimulation of
 Alternative test for factor X requirement
adenylate cyclase   adenosine monophosphate activity and  cAMP
 Detects the ability of the organism to synthesize heme (X factor)
 ultimately inhibiting protein synthesis
 Differentiates H. influenzae from H. parahaemolyticus
 Characterized by striking lymphocytosis
 Medium – d – aminolevulinic acid (d – ALA)
 Inhibition of signal transduction of chemotactic receptors  inability
of lymphocytes to enter the lymphatic tissues
 Tracheal toxin - fragment of the peptidoglycan layer  damages the ciliated
d -ALA • Porphobilinog
en synthase
epithelial cells of the trachea
 Endotoxin
 Pathogenesis of Bordetella pertussis
 Whooping cough
Porphyrin • Erlich's
reagent  Incubation period – 7 to 10 days
 3 stages of whooping cough
 Catarrhal stage – lasts for 1 to 2 weeks
 Most infectious stage of the disease (via respiratory droplets 
Brick red sneezing, coughing)
color  Resembles common colds
 Mild coughing and sneezing
 Paroxysmal stage – 2 to 3 weeks
 Series of hacking coughs (continuous forcible coughs)
 Production of copious amounts of mucus

63 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Ends with the characteristic inspiratory “whoop”  due to the Genus Brucella
air rushing through the narrow epiglottis
 Convalescent stage -14 to 16 weeks  General Characteristics
 Laboratory diagnosis  Small, Gram – negative coccobacilli
 Microscopy  Non – motile, non – spore forming
 Gram – negative bacilli seen in nasal washings or nasopharyngeal swabs  Obligate intracellular – complex nutritional requirements  amino acids, salts,
 Culture vitamins and glucose
 Bordet – Gengou medium – selective for Bordetella pertussis  Saccharolytic – do not produce gas and acids
 Potato – blood – glycerol agar – smooth, transparent convex surface  Moderately sensitive to heat and acid
colonies with pearly sheen  MERCURY – LIKE DROPLETS with narrow zone  Catalase – positive
of hemolysis  Oxidase – porisitive
 Regan – Lowe agar  H2S – positive
 Composition  Nitrate reduction – positive
 Charcoal – absorb the fatty acid present in the agar medium  Habitat  domestic livestock
 10% horse blood  Transmission via:
 40 µg/mL cephalexin – inhibition of the growth of the normal  Ingestion of contaminated dairy products
flora in the specimen  Direct contact with infected anima
 Other tests  Inhalation
 Polymerase Chain Reaction (PCR)  Virulence Factors
 Fluorescent Antibody Staining (FAS)  Organisms are localized in reticuloendothelial (RES) cells
 Endotoxin
Species Urease Nitrate Motility Oxidase Citrate Disease  Pathogenesis of Brucella species  BRUCELLOSIS
Whooping  Causes sterility or abortion in animals – due to the predilection of the organism
B. pertussis – – – + – to the organs rich in ERYTHRITOL (placenta, uterus, breast, epididymis)
cough
 Causes undulant fever or Malta fever in humans (most implicated organism –
+ Milder form of
B. parapertussis – – – + Brucella melitensis)
(18 h) pertussis
 Laboratory Diagnosis
Respiratory
 Microscopy – small, Gram – negative coccobacilli in blood, bone marrow,
disease in
tissue aspirates or biopsy
+ dogs, swine
B. bronchiseptica + + + +  Culture – incubated in increased carbon dioxide tension (8% to 10% CO2)
(4 h) and
 TSB with enrichment
occasionally
 Liver infusion, calf serum or yeast hydrolysates C
humans
 Castañeda medium (biphasic medium) – utilizes tryptose agar (if solid) or
tryptose broth (if liquid)
 Brucella agar – produces pinpoint, convex colonies with smooth glistening
surface
 Biochemical test – Abortus or Bang’s Ring Probe Test
 Utilizes dairy products from infected animals + Hematoxidin stain Brucella
 Incubated for 30 to 45 minutes

64 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  (+) – formation of blue ring on the surface of the milk  indicates Brucella Genus Francisella
infection (reaction is due to the antibodies found in the milk of the
infected animal) Francisella tularensis
Growth on Dyes
Species CO2 Requirement Habitat  General Characteristics
Basic Fuchsin Thionine  Organism utilized for bioterrorism
B. abortus + + – Cattle  Small, pleomorphic Gram – negative rods
B. suis – +/– + Swine  Habitat – many species of wild animals (rabbits, dears and rodents)
B. melitensis – + + Goat, Sheep  Transmission via:
B. canis – – + Dogs  Ticks
 Aerosols
 Contact
 Ingestion
 Virulence Factors
 Organisms are localized in reticuloendothelial (RES) cells
 Endotoxin
 Pathogenesis of Francisella tularensis
 TULAREMIA – also known as rabbit fever or tick fever
Form Mode of Transmission Comments
At the site of inoculation,
an inflammatory ulcerating
Penetration of the skin (via
papule will be formed (2 to
Ulceroglandular cuts or wounds) or mucous
6 days) with enlarged
membranes
regional lymph nodes
leading to necrosis
Also known as pneumonic
tularemia; characterized
Inhalation of an infective
Pulmonary by peribronchial
aerosol
inflammation and localized
pneumonitis
Also known as typhoidal
Gastrointestinal Ingestion tularemia; may lead to
septicemia
Entry of contaminated
Oculoglandular
debris into the conjunctiva
Oropharyngeal Entry into the oropharynx
Liver, spleen, bone marrow
Glandular
and other glands
 Present with fever, malaise, headache, pain in the involved region and
pain in the regional lymph nodes

65 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Laboratory Diagnosis Genus Legionella
 Culture – requires special media
 Presents a high risk of acquiring the infection Legionella pneumophila
 Culture media are enriched with cysteine and are incubated in increased
 General Characteristics
carbon dioxide tension at 35°C to 37°C for 2 to 5 days
 Small, Gram – negative rods that stain poorly
 Buffered Charcoal Yeast Extract (BCYE) agar
 Requires increased iron and cysteine for growth in culture
 Modified Thayer – Martin (MTM) agar
 Habitat  environmental water sources (air conditioners, water cooling towers,
 Chocolate agar medium (CAM)
water taps, sinks and showers)
 Serologic tests for diagnosis
 Transmission via aerosols
 Predisposing factors
Genus Pasteurella  Age > 55 years old
 Smoking
Pasteurella multocoida
 High alcohol intake
 General Characteristics  Immunosuppressed and immunocompromised patients (AIDS, cancer and
 Small, Gram – negative bacilli organ transplant patients) – may occur as both hospital and community
 Habitat  mouths of many animals (cats and dogs) acquired disease
 Transmission via animal bites  Virulence Factors – endotoxin
 Virulence Factors – no exotoxins known, only that it spreads rapidly  Pathogenesis of Legionella pneumophila
 Causes wound infections in humans  Legionnaire’s Disease
 Laboratory Diagnosis  Pneumonia that occurred in the American Legion Convention
 Microscopy (Philadelphia, 1976), hence the name Legionnaire’s disease
 Culture – BAM  small (1 to 2 mm), smooth, moist to mucoid, non – hemolytic  Presents as an atypical pneumonia  may progress to severe pneumonia
colonies with a greenish or brownish discoloration after 48 hours  bacteremia  may cause damage to the vascular epithelium of
multiple organs due to hematogenous spread
 Accompanied by mental confusion, non – bloody diarrhea, hematuria
and proteinuria
 Clinical presentation
 Cough with scanty or absent septum
 Serum sodium levels of 130 mEq/L
 Pontiac Fever – milder form of Legionnaire’s disease presenting as mild upper
respiratory tract infection and does NOT progress to pneumonia
 Laboratory Diagnosis
 Microscopy – stains faintly with standard Gram – stain
 Silver impregnation technique – Diterle Silver Impregnation Technique
 Fluorescent antibody
 Culture
 BCYE agar
 Feely and Gorman agar

66 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 ACID FAST ORGANISMS

Genus Mycobacterium  Types of Lesions

 Exudative lesions
Mycobacterium tuberculosis Complex (MTBC) - Consists of inflammatory response, with edema fluid,
polymorphonuclear cells and later mononuclear cells
Mycobacterium tuberculosis
- May heal in healthy individuals or may develop into
 General Characteristics granulomatous type (central area of giant cells containing
 Acid fast, long, slender, straight rods the bacilli surrounded by epitheloid tissue  TUBERCLE
 High lipid content in the cell wall GRANULOMA)
 In X, Y, V and L arrangement (Chinese letter arrangement)  Granulomatous lesions
 Serpentine cord formation (due to cord factor) - Consists of central area of giant cells (Langhan’s giant
 Obligate aerobe – predilection with organs with high oxygen tension – bone cells) containing the tubercle bacilli surrounded by a zone
marrow, lungs, kidneys and brain of epitheloid cells
 Slow growth rate – generation time  15 to 2 hours  Tubercle – a granuloma surrounded by fibrous tissue that has
 Habitat – human lungs undergone central caseation necrosis
 Transmitted via respiratory droplets (almost always transmitted via active  In healthy individuals, tubercles may heal spontaneously by
respiratory droplets) fibrosis and calcification and persist as such for lifetime
 Virulence Factors  Appear as radio opaque nodules in chest x – rays
 Cord factor  Secondary Tuberculosis
 Composed of trehalose dimycolate  Lesions occur most commonly in the apex of the lungs
 Associated with the serpentine cord – like pattern of virulent strains  Dormant mycobacteria has been reactivated (viable non –
 Granulomas and caseation by cell –mediated immunity – primarily due to host proliferating bacilli within the healed lesions)
tissue reaction  Especially seen in immunocompromised hosts
 Wax D – enhancement of immune response in the host  Disseminated Tuberculosis
 Resistance to acids and dehydration  When tubercle lesion liquefies, its caseous contents may drain into
 Droplet nuclei – drying of the respiratory droplets  bacilli remains the bronchus
suspended in the air for indefinite periods of time  Facilitates spread of the organism to other parts of the lungs
 Pathogenesis of Mycobacterium tuberculosis  New tubercles are formed
 TUBERCULOSIS  Dissemination of the tubercle into the bloodstream forms MILIARY
 Classification TUBERCULOSIS
 Pulmonary tuberculosis  The blood carries the organism to many organs of the body
 Extrapulmonary tuberculosis – tuberculosis in the brain, kidney or  Clinical findings in TB  fever, fatigue, night seats and weight loss
bone marrow  Pulmonary TB – cough, chest pain, dyspnea and hemoptysis
 Primary Tuberculosis – Pulmonary TB  Scrofula – mycobacterial cervical adenitis; seen in children with
 Lesions usually occur in the LOWER LOBES of the lungs tuberculosis, caused by M. scrofulaceum
 Organisms are engulfed by alveolar macrophages  Gastrointestinal TB – abdominal pain, diarrhea, fever and weight
 Organisms survive and multiply inside alveolar macrophages  due loss; may cause intestinal obstruction and/or hemorrhage
to the sulfatides that enable the survival (prevention of the fusion of
lysosomes)

67 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Renal TB – fever, dysuria, hematuria (blood in the urine), flank pain,  Time exposure must be carefully controlled to no more than 15
sterile pyuria (excretion of urine filled with pus cells with negative minutes
bacterial culture)  Effects mucolytic action to promote concentration by
 TB of the bone or joints – most frequently affected is the spine, centrifugation
leading to the collapse of the vertebrae; paralysis may occur due to  N –acetyl – L – cysteine (NALC) + 2% NaOH
nerve compression  Mild decontamination olution with mucolytic agent  frees
 TB meningitis – mental deterioration, retardation, blindness and mycobacteria entrapped in mucus
deafness  Limit exposure to NaOH to 15 minutes
 Treatment  Dithiothreitol + 2% NaOH
 INH (isonicotinic acid hydrazine or ISONIAZID), rifampin pyrazinamide,  Very effective mucolytic agent used with 2% NaOH
ethambutomal  Reagent is more expensive than NALC
 Protracted therapy – long duration of treatment (3 months)  Limit exposure to NaOH to 15 minutes
 Multiple Drug Therapy – done to prevent the emergence of drug –resistance  13% Trisodium phosphate + Benzalkonium chloride (Zephiran)
mutants during the long duration of treatment  Zephiran should be neutralized with lecithin
 Prevention  Bacille Calmette et Guerin (BCG) Vaccine  Not inoculated to egg – based culture medium
 Vaccine containing live, attenuated strain of Mycobacterium bovis,  13% Trisodium phosphate
administered intracutaneously  Not as effective as TSP –Zephiran mixture
 Vaccine limits the extent of the disease but not prevent the diseases  5% Oxalic acid
 Laboratory Diagnosis  Most useful in processing specimen that contains Pseudomonas
 Microscopy – sputum microscopy aeruginosa as contaminant or present as coinfection with MTB
 Sputum specimen  1% Cetylpyridinium chloride + 2% NaCl
 Number  Tubercle bacilli have survived 8 – day transit without significant
 Case findings – 2 specimen loss
 On the same day with several hours interval (for spot  Culture media – incubated at 35°C to 37°C with 5 to 10% CO 2 tension;
collection); or examined weekly for growth up to 8 weeks; colonies appear as small, buff
 On two consecutive days in color, dry, scaly or warty (cauliflower – like)
 Quality Media Content of the Media
 Macroscopic – yellowish, mucopurulent Egg – based media
 Microscopic - >25 WBC per LPO or 5 WBC per OIO; presence of - Mycobacterium requires oleic acid, which is incorporated in egg – based
alveolar macrophages or dust cells medium (present in eggs).
 Staining – Ziehl – Neelsen Stain - Glycerol enhances the growth of Mycobacterium.
 Fluorescent dyes – Rhodamine auramine - Malachite green is inhibitory for other bacteria other than Mycobacterium.
 Culture Lowenstein – Jensen Coagulated whole eggs, salts, glycerol, potato flour,
 Decontamination, Digestion and Concentration (LJ) malachite green (0.025 g/dL)
 Components Coagulated whole eggs, egg yolks, whole milk, flour
 Decontamination – kill other contaminants Petragnani
glycerol, malachite green (0.052 g/dL)
 Digestion – mucoid material of the sputum is liquefied American Thoracic Coagulated whole eggs, potato flour, glycerol,
 Concentration – high speed centrifugation Society malachite green (0.02 g/dL)
 4% NaOH Non – egg – based medium
 Traditional decontamination and concentration solution Salts, vitamins, cofactors, oleic acid, albumin, catalase,
Middlebrook
biotin, glycerol, malachite green (0.0025 g/dL)

68 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

Middlebrook 7H10 Middlebrook + GLUCOSE  Reduction of nitrate to nitrate
Middlebrook 7H11 Middlebrook + CASEIN HYDROLASE •In the
Middlebrook 7H9 Broth medium with similar formulation as Middlebrook presence
Selective Media NO3 +H2 of
nitroreduct
Lowenstein – Jensen agar slant with RNA, penicillin and ase
Gruft Modified LJ
nalidixic acid
•By the
Lowenstein – Jensen agar wit cycloheximide, action of
Selective LJ Water + alpha -
lincomycin and nalidixic acid
Selective Middlebrook 7H10 with cycloheximide, lincomycin and NO2 napthylami
ne sulfanilic
Middlebrook 7H10 nalidix acid acid

Selective
Middlebrook 7H11 with carbenicillin, ampothericin B,
Middlebrook 7H11 p - sulfobenzene
polymyxin B and trimethoprim lactate - azo -a- •Red color
(Mitchison’s)
naphthylamine

 Biochemical tests – used to identify and differentiate atypical

Mycobacterium from tubercle Mycobacterium
 Niacin accumulation test
 Heat – stable catalase test

Niacin +
Cyanogen 30%
bromide Hydrogen • In the presence of heat
stable catalase

Peroxide
• Yellow color
• Normal metabolic by
– product of almost all
Niacin
Oxygen
Mycobacterium
species.Normal
ribonucleotide metabolic by – • Effervescence
product of almost all
Mycobacterium
and Water
species.

 Procedure
- Heat a suspension of bacterial isolate at 68oC for 20
minutes.
- The suspension is cooled and 30% hydrogen peroxide
(superoxol or superaxal) is added to the bacterial
suspension.
 (-) – Mycobacterium tuberculosis, Mycobacterium bovis,
Mycobacterium africanum

69 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Tripotassium
 Growth inhibition by Thiopene – 2 – Carboxylic Acid Hydrazine (T2H) salt of • In the
presence of
 5 µg or 1 µg of T2H is incorporated in Middlebrook 7H10 or 7H11 phenolphtha arylsulfatase

agar. lein
 The medium is incubated at 5% to 8% carbon dioxide for 14 to
21 days. • In the
Free presence of
 Allows other mycobacterium to grow, while Mycobacterium phenolphthalein sodium
carbonate
bovis is inhibited in the presence of T2H.
 Tween 80 hydrolysis
 Tween 80 – a strong detergent (polyoxythelene sorbitan
Purple or pink
monooleate) color

 Iron uptake test

Tween 80 + • In the presence of
lipase (from the
 Determines the ability of the organism to take up iron from
Neutral Red bacteria)
inorganic iron – containing reagents

• Addition of 2 to 3 drops
of ferric ammonium
Polyoxyethylated Growth in LJ agar citrate
• Incubate for 21 days
sorbitol + Oleci • Pink to red color

acid

Development of
 Arylsulfatase test rusty brown
 Used to differentiate group III Mycobacterium species from colonies
other Mycobacterium species.

 Tuberculin test
 In vivo testing; detects recent infection with Mycobacterium
tuberculosis complex; it is a skin test for tuberculosis
 Methods
- Mantoux intracutaneous method
- Von Pirquet method – introduced by scratching the skin of
the patient
- Volimer patch – cloth soaked in a solution with antigen
applied on the surface of the skin
 Intradermal inoculation of the bacteria’s antigens

70 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 - Original or Old Tuberculin (OT) – uses a filtrate of glycerol complete sensory loss patchy sensory loss
broth culture concentration by evaporation in water bath - Nerves visibly enlarged (leonine - Nerves not enlarged (saddle
- Purified Protein Derivative (PPD) – uses a purified face) face)
precipitation with trichloroacetic acid Histopathology Few AFB Numerous AFB
 (+) – induration at the site of introduction (>10 mm within 48 to Infectivity Low High
72 hours of injection) Lepromin Test + –
Prognosis Good Poor
Mycobacterium bovis  Laboratory Diagnosis
 Clinical presentation of the patient
 Also known as bovine tubercle bacillus and affects cows
 Acid – fast staining
 Causes gastrointestinal tuberculosis in man via ingestion of contaminated milk from
 Lepromin test - skin test for leprosy
cows
 (+) – good immune response

Mycobacterium africanum Atypical Mycobacterium Species

 Has similar mode of transmission, pathogenesis and clinical manifestations with
Mycobacterium tuberculosis

Optimum Growth

Negative Growth

Pathogenesis
Other Name
Bacteria

Others
Mycobacterium Other than Tubercle Bacilli
Mycobacterium leprae
 General Characteristics 37oC, 14 - Rapid Tween 80
Mycobacterium Yellow
 Aerobic, acid – fast rods to 28 42oC hydrolysis –
kansasii bacillus
 Can be grown on footpads of mice and armadillos – cannot be grown in days positive in 3 days
arterial culture media but in living cells Associated with skin
infections and
 Optimal growth at less than body temperature Mycobacterium Of the 30 to 35 to swimming pool
 Habitat  human skin and superficial nerves (spares the warm areas of the marinarum sea 32oC 37oC granuloma  skin
body) nodules that
ulcerate
 Transmission via prolonged contact with nasal secretions or skin lesions Causes cervical
 Also known as Hansen’s bacillus lymphadenitis
 Pathogenesis of Mycobacterium leprae (scrofula) especially
Mycobacterium No temperature
in children (1 to 5
 Leprosy – Hansen’s disease scrofulaceum preference
years old); inflamed
Tuberculoid Leprosy Lepromatous Leprosy lymph nodes in the
- Few, erythematous or - Many erythematous nodules; neck
- Photochromogen
Skin Lesions hypopigmented (fair) plaques extensive tissue destruction; Associated with
Mycobacterium at 25oC;
and Nerve with flat centers and raised associated with disfiguring skin pulmonary and
szulgai scotochromogen
Involvement demarcated borders lesions cutaneous infection
at 35 to 37oC
- Peripheral damage with - Diffuse nerve involvement with Mycobacterium Tap water Generally

71 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 gordonae bacillus nonpathogenic Mycobacterium avium –
- First isolated from – – + – – –
intracellulare
frogs (G. xenopi)
Mycobacterium terrae – triviale – – + + –/+ –
- Bird’s nest
Mycobacterium Mycobacterium malmoense – – + + – –
42oC 25oC Pulmonary infections appearance with
xenopi
stick – like Mycobacterium haemophilum – – – – – –
projections on Mycobacterium gastri – – – + – –
Middlebrook 7H10 GROUP IV – RAPID GROWERS
Mycobacterium 35 to
Battery Infections in AIDS Organisms that grow in 3 to 5 days in culture medium; they are non – photochromogens
avium – 37oC and 24oC
bacillus patients Mycobacterium fortuitum + + V + +
intracellulare 42oC –
28 to Mycobacterium chelonei – – + – + –
32oC, 24
Mycobacterium phlei – + + + – +
weeks
Mycobacterium Mycobacterium smegmatis – + + + – +
malmoense
25 to
35oC, 4 to
8 weeks
Runyon’s Classification of Mycobacteria Other than
Tubercle Bacilli (MOTT)
Heat – Stable

Arylsulfatase

Iron Uptake
Reduction

Hydrolysis
Tween 80
Catalase

(3 days)
Niacin

NO3

GROUP I – PHOTOCHROMOGENS
Organisms that develop yellow pigment when exposed to constant light source; non –
pigment in the dark
Mycobacterium kansasii – + + + – –
Mycobacterium marinarum V – – + V –
Mycobacterium simiae + – + Slow + – –
Mycobacterium asiaticum – – + + – –
GROUP II – SCOTOCHROMOGENS
Organisms that are pigmented yellow to orange in the dark; pigment intensifies to
orange or red when exposed to constant light source for 2 weeks
Mycobacterium scrofulaceum – – + – – –
Mycobacterium szulagi – + + Slow + – –
Mycobacterium gordonae – – + + – –
Mycobacterium flavescence – + + – –
GROUP III – NON – PHOTOCHROMOGENS
Organisms that cannot develop pigment on exposure to light; white to tan in color

72 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 ATYPICAL BACTERIA

Family Mycoplasmataceae  Cold agglutinins – IgM that can agglutinate red blood cells at 4oC,
and not at 37oC
Mycoplasma pneumoniae  A titer of 1:128 is indicative of recent infections with Mycoplasma
pneumoniae
 General Characteristics
 Also known as Eaton’s agent and is the causative agent of primary atypical
pneumonia (PAP) Other Mycoplasma Species
 It is a pleuro – pneumonia – like organism
 Smallest living free organisms, measuring 0.2 to 0.3µm. it is a filterable organism,
Other Mycoplasma species are classified as genital Mycoplasmas, as they generally
and is able to pass through HEPA filters
infect the genitourinary tract
 Wall – less bacteria
Organism Disease Association
 The cell membrane of this organism consists of sterols
 Habitat and Transmission Pyelonephritis, pelvic inflammatory disease
Mycoplasma hominis
 Habitat – human respiratory tract and post – partum fever
 Transmission – via respiratory droplets Mycoplasma genitalium Non – gonococcal urethritis
 Pathogenesis of Mycoplasma pneumoniae – causes atypical pneumonia Ureaplasma urealyticum Non – gonococcal urethritis
 Causes pneumonia commonly seen in young adults
 Bronchopneumonia, in conjunction with lobar pneumonia
 No known exotoxins and endotoxins associated with the pathogenesis Family Chlamydiaceae
 P1 adhesin mediates the adherence of the organism to epithelial cell surfaces
 General Characteristics
of the lungs
 Possesses two forms:
 P1 adhesins are proteins found on the organism shaped as rods with
 Elementary body (EB) – small, extracellular, infectious, inert form
tapered ends
 Reticulate body (RB) – larger, intracellular, pathogenic, metabolically
 Hydrogen peroxide and cytolytic enzymes damage the respiratory tract
active, replicating form
 Signs and symptoms
 Obligate intracellular bacteria, and grows within living cells
 Non – productive cough, sore throat and earache
 Possesses a rigid cell wall that lacks the peptidoglycan layer, resembling Gram
 Flu – like symptoms, such as fever, headache, malaise, myalgias
– negative cell wall, but lacks muramic acid
 The disease is self – limiting and resolves within 10 to 14 days.
 How infection is established
 Laboratory Diagnosis of Mycoplasma pneumoniae
 Elementary body attaches to the cell surface and endocytosis of the
 Microscopy  not useful for detection of the organism; use of fluorescent
elementary body occurs
stains, such as acridine orange for the staining of the nucleic acid is more
 Elementary body is contained in an endosome which does not fuse with
useful
the lysosome
 Culture
 The elementary body reorganizes into a reticulate body in the endosome,
 Specimen – throat washings or sputum
and multiplies by binary fission
 Colonies – fried egg appearance (spherical colonies with raised centers
 The reticulate bodies are reorganized into elementary bodies. Inclusion
and thinner outer edges developing 1 to 3 weeks)
granules within the cell contain both reticulate and elementary bodies.
 Serologic test
 Chlamydia psittaci – causes the lysis of the inclusion granule
 Detection of IgM antibodies produced upon infection with the organism

73 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

  Chlamydia trachomatis and Chlamydia pneumoniae – reverse
endocytosis  Laboratory Diagnosis
 Revere endocytosis – the host cell with lyse releasing the  Microscopy
contents of the endosomes and infect other host cells  Utilizes special stains such as Giemsa’s, Gimenez and Machiavello stains
 Habitat and Transmission  Immunofluorescence shows cytoplasmic inclusions directly from the
Organism Habitat Transmission specimen
Personal close contact (sexually) or by Organism Cytoplasmic Inclusion
Chlamydia trachomatis Humans
passage through an infected birth canal - Halberstadler – Prowazek bodies – round, vacuolar inclusions
Birds and many Inhalation of the organism in dried bird (and may be focused directly from the specimen)
Chlamydia psitacci Chlamydia trachomatis
mammals feces (parrots and birds) - Glycogen –filled bodies, as visualized by iodine staining, seen in
cell cultures
Chlamydia Human
Respiratory droplets Chlamydia psitacci - Levinthal – Cole – Lillie bodies – variable, dense inclusions
pneumoniae respiratory tract
Chlamydia pneumoniae - Round, dense inclusions
 Cell cultures
 Disease and Pathogenesis of Chlamydia species
 Treated with cycloheximide – inhibit protein synthesis in the host cell, but
Organism Disease Association
not the protein synthesis of the organism
- Trachoma (Tric agent) – chronic conjunctivitis results in
scarring of the conjunctiva and may lead to blindness;
transmission may be due to finger to eye contact or
fomite to eye contact
- Inclusion conjunctivitis (neonatal inclusion conjunctivitis) –
mucopurulent eye infection; in adults, it is associated with
genital tract infections; in infants, it is associated with
passage through an infected birth canal and becoming
apparent occurring 7 to 12 days after delivery
- Infant pneumonitis – infections occurring 2 to 12 weeks
after birth and often preceded by neonatal conjunctivitis
Chlamydia trachomatis
- Genital tract infections – urethritis, salphingitis, pelvic
inflammatory disease, cervicitis; it is often co – infected
with Neisseria gonorrheae
- Lymphogranuloma venereum – the primary stage consists
of a small, painless lesion on the genitalia; the second
stage consists of a marked inflammation of the lymph
nodes
- Reiter’s disease – autoimmune disease often preceded
by genitourinary tract infection; once infected with the
organism, antibodies that are produced attack other host
cells such as cells of the urethra, joints and the uveal tract
- Psittacosis (ornithosis, parrot fever) – may be
Chlamydia psitacci asymptomatic or symptomatic; manifested as high fever
with pneumonia
Chlamydia pneumoniae - Upper and lower respiratory tract infections

74 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 SPIROCHETES

 General Characteristics secondary stage

 Thin – walled, flexible, helical bacteria that may be visualized by silver - Early latent phase – initial 4 – year period; 90% of relapses
impregnation technique and by darkfield microscopy may occur during the first year
 Measures 0.1 to 0.3µm in diameter and 5 to 120µm in length - Late latent phase – subsequent period; indefinite period of
 Motile through the axial filaments (endoflagella) located within the periplasmic latency
space and moves through an undulation motion
 Congenital syphilis – occurs in the fetus of an infected pregnant woman
Genus Treponema after the third month of pregnancy; causes stillbirth or multiple fetal
abnormalities
Treponema pallidum subspecies pallidum  Laboratory Diagnosis
 Microscopy –for the detection of Treponema pallidum in the primary and
 General Characteristics
secondary lesions
 Characteristics
 Darkfield microscopy
 Spiral rods with numerous, tight rigid coils
 Immunofluorescence
 Not cultured in vitro
 Silver impregnation
 Maintained in the laboratory by growing the organism in rabbit’s testes,
 Levaditi’s, Fontana Tribondeau, Warthin – Starry, Dieterle
hamster and guinea pigs
 Microaerophilic organism
 Habitat and Transmission Non – Venereal Treponematoses
 Habitat – human genital tract
 Transmission
 Intimate contact with infected person through coitus from lesions of the Organism Transmission Disease
skin or mucous membranes (as the chancre contains live treponemes) Treponema pallidum Yaws, topical syphilis or Direct skin contact (in
 From mother to fetus across the placenta through hematogenous subspecies pertenue frambesia humans only)
dissemination and may cause stillbirth or abortion of the baby Direct mucosal contact (in
 Blood transfusion Treponema pallidum humans only); through
Bejel, or endemic syphilis
 Disease and Pathogenesis of Treponema pallidum subspecies endemicum contaminated eating or
 Dissemination to various organs of the body is the primary pathogenesis; no drinking vessels
toxins or enzymes produced Direct skin contact (in
Treponema carateum Pinta
 Venereal syphilis humans only)
 Other names – ‘Great pox’, ‘French disease’, ‘Italian pox’
 Stages of venereal syphilis Genus Borrelia
Phases Manifestation
Primary Syphilis - Painless chancre at the inoculation site
Borrelia recurrentis Borrelia burgdorferi
- Maculopapular rash on palms and soles
Secondary Syphilis - Irregularly, loosely coiled - Irregularly, loosely coiled
- Condylomata lata (condylomas)
spirochetes spirochetes
Tertiary or Late Syphilis - Granulomas (gummas) Characteristics
- Stains readily with Giemsa - Visualized by darkfield
Latent Syphilis - Subclinical (asymptomatic phase) phase following the
and other stains microscopy and by Giemsa’s

75 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 - Can be cultured on artificial and silver stains Genus Leptospira
culture media containing - Can be grown in culture
serum or tissue extracts - Microaerophilic Leptospira interrogans
- Microaerophilic
 General Characteristics
- Transmitted by arthropods
 Tightly coiled, fine spirochetes
- Antigens undergo variation
 One or both ends may be bent to form a question – mark hook
Habitat - Humans are the only hosts - Found in ticks (Ixodes)
 Not stained with dyes
- Transmitted from person to  Can be grown in artificial culture media containing serum
- Transmitted by tick bite primarily
Transmission person by the human body  Divided into two serogroups
from 2 reservoirs, mice and deer
louse (Pediculus humanus)  Var icterohemorrhagiae (rats)
Lyme disease  Var canicola (dogs)
- Eyrthema  Habitat and Transmission
chronicum  Habitat – Wild and domestic animals, such as rats and other rodents, domestic
migrans – a livestock and household pets
Primary
- Relapsing fever – spreading circular  Transmission – via urine of infected animals
stage
dissemination to various red rash with a  Ingestion of organism from contaminated foods and drinks
organs of the body; clear center at  Passage though the mucous membranes or skin when swimming or
multiplication in many the bite site wading in contaminated water
tissues producing fever, - Cardiac  Disease and Pathogenesis of Leptospira interrogans
Disease
chills, headaches and (myocarditis and  Dissemination of the organism to various organs in the body
multiple organ dysfunction; pericarditis) and  No toxins or enzymes produced
Secondary
antigenic variation is neurologic  Leptospirosis
stage
manifested and no toxins or (meningitis and  Composed of 2 phases
enzymes produced neuropathies)  First (bacteremic) phase
involvement  Organisms are found in the blood or in the cerebrospinal fluid
- Arthritis, usually in  Manifested as flu – like illness (7 –day fever, chills and intense
Tertiary
the large joints headache)
stage
(i.e. knees)  Conjunctival and scleral hemorrhage
- Microscopy – darkfield  Second (immune) phase
examination of blood or  Correlates with emergence of IgM
- Microscopy
Wright’s or Giemsa’s stain of  Aseptic meningitis
Laboratory - Culture – Kelly’s
peripheral blood smear  Weil’s disease – a severe case of leptospirosis with renal failure, hepatitis
Diagnosis - Serologic testing – detection of
- Culture – Kelly’s (and jaundice), mental status changes and hemorrhage in may organ
IgM antibodies
- Serologic tests are rarely  Laboratory Diagnosis
useful  Microscopy – darkfield microscopy and silver impregnation
 Culture – Fletcher’s medium
 First week of infection – blood or cerebrospinal fluid
 Second and subsequent weeks – urine
 Serological test – detecting rise in agglutinating antibody

76 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Taxonomic Approach in Microbiology

CELL MODE OF
STUDY CELL FORM CELL WALL MOVEMENT LOCATION OTHER FEATURES
ORGANIZATION REPRODUCTION

On plants and
BACTERIA Bacteriology Prokaryote Unicellular Peptidoglycan Asexual Flagella - Genetic materials – DNA and RNA
animals

Extreme - Genetic materials – DNA and RNA

ARCHAEA Prokaryote Unicellular Asexual Flagella
environment - Not known to cause infections in humans

Unicellular, Asexual and

FUNGI Mycology Eukaryote Chitin Environment
multicellular sexual

Asexual and Flagella, cilia, Environment,

PROTOZOA Protozoology Eukaryote Unicellular
sexual pseudopods host

Freshwater, - Obtains nutrition by photosynthesis

Phycology Unicellular, Asexual and
ALGAE Eukaryote Cellulose seawater, soil, - Requires light and air for survival
Alcology multicellular sexual
plants - Do not require organic compounds
- Small infectious agents
Living hosts - Capsid – protein coat of the virus
(obligate - Core – genetic material of the virus
VIRUS Virology Acellular
intracellular - May be enveloped or not
parasites) - Genetic materials – either DNA or RNA,
never both

77 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Enterobacteriaceae Biochemical Reactions

iMVIC
Species TSIA Gas H2S Mot Lys Ure Others
Ind MR VP Cit

Escherichia coli A/A + – + + – – + + –

Groups A, B and C K/A – – +/– + – – – – –

Shigella
Shigella sonnei K/A – – – + – – – – –
Edwardsiella tarda K/A + + + + – – + + –
Salmonella K/A + + – + – + + + –
A/A
Citrobacter freundii + + – + – + + – +/–
Citrobacter K/A
Citrobacter diversus K/A + – + + – + + – +/–
Klebsiella
A/A ++ – – – + + – + +
Klebsiella pneumoniae
Klebsiella oxytoca A/A ++ – + – + + – + +
Enterobacter
A/A ++ – – – + + + + – DNAse (+)
aerogenes
Enterobacter Lipase (+)
Enterobacter Gelatinse (+)
A/A ++ – – – + + + – +/–
cloacae
Hafnia alvei K/A + – – +/– + – + + –
A/A
Pantoea agglomerans +/– – +/– +/– +/– +/– + +/– +/–
K/A
Serratia marcescens K/A + – – +/– + + + + –
Proteus vulgaris K/A +/– + + + +/– + – ++
Proteus
Proteus mirabilis K/A + + – + +/– +/– + – ++
Morganella morganii K/A + – + + – – + – ++
Providencia rettgeri K/A – – + + – + + – ++
Providencia stuarti K/A – – + + – + +/– – +/–
Providencia
Providencia
K/A +/– – + + – + + – –
alcalifaciens
Yersinia enterolitica K/A – – +/– + – – – – +/–

78 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 Identification of Genus Staphylococcus

Gram – Positive Cocci

POSITIVE NEGATIVE
Micrococcaceae Streptococcaceae

POSITIVE
Staphylococcus aureus NEGATIVE
Staphylococcus lugdunensis Staphylococcus saprophyticus
Staphylococcus schleiferi Staphylococcus epidermidis
Staphylococcus intermedius

NEGATIVE
POSITIVE Staphylococcus
SUSCEPTIBLE RESISTANT NEGATIVE POSITIVE
lugdunensis Staphylococcus Staphylococcus Staphylococcus Staphylococcus
Staphylococcus aureus
Staphylococcus schleiferi epidermidis saprophyticus epidermidis saprophyticus

79 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015

 .
© 2015, Edited and completed, 2016
Notes by Ma’am Kathyren Estimada and Ma’am Janette Awisan
Bachelor in Medical Laboratory Science
School of Natural Sciences, Saint Louis University

80 MICROBIOLOGY – Bacteriology aabbbbyy :D 2015