Fastidious Organisms: Brett Crawley MASM, MACTM, MAIMS Reference: Manual of Clinical Microbiology, 11 Ed. ASM Press

Fastidious Organisms
Brett Crawley MASM, MACTM, MAIMS
Reference: Manual of Clinical Microbiology, 11th Ed. ASM Press
PIONEERING DIAGNOSTICS
DEFINITION
A fastidious organism is any organism that has complex or particular nutritional

requirements.
Thus fastidiousness is often practically defined as being difficult to culture, by any

method yet tried. E.g. Neisseria gonorrhoeae, which requires blood or
haemoglobin and several amino acids and vitamins to grow.
Fastidious organisms are not "weak"—they can flourish in their particular

ecological niche with its nutrients, temperature and absence of competitors. They
can be quite difficult to kill.
But they are difficult to culture because it is difficult to accurately simulate their
natural conditions in a culture medium, e.g.Treponema pallidum is not easy to
culture, yet it is resilient, being difficult to eradicate from all tissues of a person
with syphilis.
2
HAEMOPHILUS SPP.
DESCRIPTION
Haemophilus spp. are members of the family Pasteurellaceae
Eight species affecting humans

• H. influenzae,
• H. aegyptius,
• H. ducreyi,
• H. pittmaniae,
• H. parainfluenzae,
• H. haemolyticus,
• H. parahaemolyticus,
• H. paraphrohaemolyticus.
• Recently a new taxon, H. sputorum,
4
DESCRIPTION
Haemophilus genus are small, non-motile, non-spore forming, non-acid fast,

pleomorphic, Gram negative bacilli with fastidious growth requirements.
Facultative anaerobes, with requirements for X and/or V factors.

• X factor is protoporphyrin IX, a metabolic intermediate in the haemin biosynthetic pathway
• V factor is composed of nicotinamide, complexed as NAD or NADP.
• Both X and V factors are present in erythrocytes
• “haemophilus” means “blood loving” in Greek.
Optimal growth at 35 to 37oC with 5 - 7% CO2.
All species are CAMP negative
Produce alkaline phosphatase.

5
DESCRIPTION
Grow on chocolate agar

• Colonies are usually smooth, with a flat or convex shape.
• Non-pigmented (i.e., buff or light tan) or slightly yellow
• 0.5 to 2.0 mm in diameter.
• Some produce beta-haemolysis on sheep blood agar
H. influenzae strains may produce one of six capsular polysaccharides (“a” to “f”)
or be non-encapsulated.
Non-encapsulated H. influenzae strains are referred to as non-typeable (NTHi)
6
EPIDEMIOLOGY AND TRANSMISSION
Haemophilus species can be part of the normal flora of mucous membranes in

humans.
• Colonisation of the oral cavity, superior to the palatal arches, by H. parainfluenzae and H.
pittmaniae is common.
• H. parahaemolyticus and H. haemolyticus colonisation in healthy individuals is rare.
• Colonisation of the cervix with H. ducreyi has been documented.
Disseminate to various sites, including the

• Meninges
• Subcutaneous tissue
• Joints
• Pleura
• Pericardia
• Lungs
7
CLINICAL SIGNIFICANCE: H. INFLUENZAE
Invasive infections caused by H. influenzae, such as meningitis, epiglottitis,

orbital cellulitis and bacteraemia, are usually caused by capsular type b (HiB)
biotypes I and II.
Biotype IV strains often found to cause systemic infections in neonates as well as

aggressive infections of the genital tract in postpartum women.
Vast majority of infections today are caused by NTHi, causing acute

• Conjunctivitis
• Otitis media
• Maxillary sinusitis
• Exacerbation of chronic bronchitis
• Pneumonia
8
CLINICAL SIGNIFICANCE: H. INFLUENZAE
Persons at risk for systemic NTHi infection:

• The elderly
• Functional or anatomic asplenism
• Sickle cell disease
• Complement deficiencies
• Hodgkin’s lymphoma
• Hypo-gammaglobulinaemia
• T-cell immunodeficiency states (e.g., HIV infection).
9
CLINICAL SIGNIFICANCE: H. DUCREYI
Chancroid is a sexually transmitted disease caused by H. ducreyi.
Characterized by a single painful genital ulcer, with associated inguinal

lymphadenopathy 2 to 7 days following exposure.
Mostly in developing countries, including Asia, Africa and Latin America.
Epidemics are associated with low socioeconomic status, poor hygiene,

prostitution and drug abuse.
10
CLINICAL SIGNIFICANCE: OTHERS
H. parainfluenzae
Colonises the Upper Respiratory Tract, 75% of the Haemophilus normal flora in
the oral cavity and pharynx.
Some cases of acute otitis media, acute sinusitis, acute bacterial exacerbation of
chronic bronchitis and subacute bacterial endocarditis.
H. aegyptius, (Koch-Weeks bacillus)
Important cause of acute purulent conjunctivitis (pinkeye), occurs in younger

children, especially having contact with other children in closed settings, such as
day care centres.
11
CLINICAL SIGNIFICANCE: OTHERS
Brazilian purpuric fever

• Occurs in South America
• Characterised by rapid onset of high fevers, hypotension, diffuse cutaneous haemorrhaging
and abrupt vascular compromise.
• The causative agent is often mistaken to be identified as H. aegyptius but is instead it is
classified as H. influenzae biogroup III (aegypticus).
H. haemolyticus is a cause of invasive disease, often misidentified as H.

influenzae.
• Associated with bacteraemia, septic arthritis and peritonitis.
Other Haemophilus species

• Rarely implicated in infection of humans
• Lower respiratory tract infection, sinusitis, conjunctivitis, bacteraemia, meningitis, wound
infections, peritonitis, arthritis, osteomyelitis, and brain abscess have been documented.
12
SPECIMEN COLLECTION & TRANSPORTATION
The collection of specimens for the diagnosis of Haemophilus infections is

dependent upon the nature of the infection:
Infection Specimens
Meningitis Blood culture and CSF cultures
Otitis media Middle ear fluid obtained by tympanocentesis
Perforated tympanic membranes Aspirate of middle ear fluid from the external auditory canal.
and otorrhea
Maxillary sinusitis Direct sinus aspirates or middle meatal swab
Conjunctivitis Conjunctival swab
Bronchopulmonary infections Broncho alveolar lavage (BAL) or bronchial washing, sputum and
tracheal aspirates
Bacterial pneumonia Blood culture in addition to respiratory collection
Chancroid specimens for culture of H. ducreyi should be collected from the

13 margins of the genital lesions with a saline moistened swab
Swabs should be immediately transported to the laboratory and plated without

delay.
When refrigerated, Amies transport medium has been demonstrated to maintain

the viability of Haemophilus, including H. ducreyi for up to 3 days.
Long-term storage of Haemophilus spp.

• Lyophilisation
• Freezing isolates at −60 to −80oC in tryptic soy broth with 15% glycerol
• Porous beads (Cryobeads)
14
GRAM STAIN
Small, pleomorphic, Gram negative coccobacilli with coccoid, cocco-bacillary,

rod-shaped, or filamentous forms.
Gram stain of Haemophilus influenzae in a CSF
15
ANTIGEN DETECTION
Commercial immunochemical techniques are available for the detection of S.

pneumoniae, Streptococcus dysgalactiae, H. influenzae, and N. meningitidis
directly from CSF and other body fluids.
These techniques provide rapid identification of the pathogen, but lack sensitivity
and specificity
16
MEDIA
Chocolate agar
• Home made by adding blood to the base media as it cools to 80oC after being autoclaved.
• Commercial e.g. Chocolate PolyViteX
Encapsulated and non-encapsulated Haemophilus influenzae on chocolate agar
17
MEDIA
Selective Chocolate agar

• Home made chocolate agar with bacitracin, vancomycin and/or clindamycin to inhibit
normal flora
• Commercial e.g. Haemophilus Chocolate 2 Agar
Sputum containing Haemophilus influenzae non-selective chocolate vs selective

chocolate
18
INCUBATION
Plates should be incubated at 35oC in 5 to 7% CO2.
most Haemophilus spp. grow within 24 to 48 h.
For H. ducreyi and H. aegyptius Incubate up to 5 days
For H. ducreyi, incubate 30 to 33oC in 5 to 7% CO2 for better growth.
19
COLONY MORPHOLOGY
Colonies of Haemophilus spp. on solid media, in general, are non-pigmented or

slightly yellow and flat to convex and have a diameter of 0.5 to 2 mm after 48 h of
incubation. Certain species of Haemophilus produce beta-haemolysis on blood
agar
Colonies of H. influenzae on chocolate agar are smooth, low, convex, greyish,

and translucent.
• Encapsulated strains often have a mucoid appearance
• Non-encapsulated strains produce smaller, buff colonies.
• Most strains of H. influenzae produce indole, emitting a strong amine-like odour.
• Non-indole-producing strains emit a “mousy” odour.
• Colonies are 1 to 2 mm in diameter and often grow within 24 h.
20
IDENTIFICATION: MANUAL
Manual ientification of Haemophilus spp. is by the following tests:

• X and V factor requirements for growth
• Porphyrin test;
• Haemolysis
• Carbohydrate fermentation
• Indole
• Ornithine decarboxylase
• Urease
• Catalase
• beta-Galactosidase
21
22
X and V factor discs in determining the growth factor requirements of

Haemophilus spp. e.g. Haemophilus influenzae.
23
24
IDENTIFICATION: MANUAL COMMERCIAL
API NH indentification in 2 hrs
25
API NH
26
Other commercial manual identification kits

• RapID NH system (Remel)
• BBL Crystal Neisseria/Haemophilus ID system (BD)
27
IDENTIFICATION: AUTOMATED
Vitek 2 NH identification in 6 hrs
28
VITEK NH CARD ORGANISMS VITEK NH CARD ORGANISMS
Actinobacillus pleuropneumoniae • Histophilus somni

• Actinobacillus suis • Kingella denitrificans
• Actinobacillus ureae • Kingella kingae
• Aggregatibacter actinomycetemcomitans • Moraxella (Branhamella) catarrhalis
• Aggregatibacter aphrophilus • Moraxella (Neisseria) ovis
• Aggregatibacter segnis • Neisseria cinerea
• Campylobacter coli • Neisseria elongata
• Campylobacter fetus ssp. fetus • Neisseria gonorrhoeae
• Campylobacter jejuni ssp. jejuni • Neisseria lactamica
• Capnocytophaga spp. • Neisseria meningitidis
• Cardiobacterium hominis • Neisseria sicca
• Eikenella corrodens • Neisseria weaveri
• Gardnerella vaginalis • Oligella urethralis
• Haemophilus haemolyticus • Riemerella anatipestifer
• Haemophilus influenzae • Suttonella indologenes
• Haemophilus parahaemolyticus
• Haemophilus parainfluenzae
29
Vitek Mass Spectrometer (MALDI-TOF) 1 minute per isolate after vacuum
> 1,300 organisms in IVD database
> 2,000 organisms in RUO database
30
IDENTIFICATION: MOLECULAR
Molecular Identification Methods include:

• 16S rRNA gene sequencing,
• Next-generation sequencing,
• PCR
• Microarrays,
• Florescence in situ hybridization
Multiplex disease-state panels for direct inoculation with a positive blood culture,
has demonstrated a reduction in length of stay and enhanced antimicrobial
stewardship:
• FilmArray (bioMérieux’ BioFire Diagnostics, Salt Lake City, UT)
• Verigene (Nanosphere, Inc., Northbrook, IL)
31
IDENTIFICATION: FILMARRAY
Blood Culture Identification Panel
32
33
ANTIMICROBIAL SUSCEPTIBILITIES
H. influenzae may produce one of two beta-lactamases, TEM-1 and ROB-1. Both
enzymes are plasmid associated, extracellular and are produced constitutively in
large amounts.
Beta lactamase producing strains should be considered resistant to ampicillin and

amoxicillin, as these drugs typically have MICs of ≥128 μg/ml against these
strains
Beta-lactamase producing strains of H. influenzae remain susceptible to oral and

parenteral cephalosporins and carbapenems. They are also susceptible to
amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam.
Beta-lactamase production by Haemophilus spp. can be determined rapidly with

a chromogenic cephalosporin spot test (beta-lactamase test).
34
Disc diffusion susceptibility tests can be performed using Haemophilus test

medium (HTM) agar, with incubation of 16 - 18 hrs at 35oC in 5 - 7% CO2. CLSI
zone diameter breakpoints are available for 39 different antibiotics.
MICs can be determined with Haemophilus spp. by broth micro-dilution (BMD).

CLSI recommends the use of HTM broth. After inoculation, trays are incubated
for 20 to 24 hrs in ambient air at 35oC prior to reading the MICs. CLSI has
breakpoints for 43 different antibiotics.
Susceptibility tests of H. ducreyi should not be performed in routine clinical

microbiology laboratories, as standardised test methods, of proven reliability,
have not yet been developed.
35
CASE STUDY
• A 6 year-old girl with a history of posterior fossa ependymoma presented with a one month
history of fever, headaches, vomiting and more recently, neck stiffness.
• Additional history includes remote tumour resection followed by radiation and
chemotherapy resulting in remission, with a residual ventriculo peritoneal shunt (VPS).
• Her parents reported she was in good health until approximately 1 month prior to
presentation and is up to date on her immunisations.
• Seen by her primary care physician for her symptoms and treated with amoxicillin for
suspected strep throat.
• Upon admission, she received supportive therapy for her symptoms after she was found to
have tumour recurrence on imaging.
• The patient was scheduled for resection approximately two weeks after discharge and on
post-operative day two she developed fever, vomiting and neck stiffness again.
• At this time, blood cultures were drawn and a lumbar puncture (LP) was performed.
Cerebrospinal fluid (CSF) from both the LP and VPS submitted for fluid analysis (Table 1)
and culture.
36
CASE STUDY
Spinal Fluid LP VPS

Appearance Clear Clear
Nucleated cells 1075 cells/μL 628 cells/μL
RBC 150 cells/μL 35 cells/μL
Polys 94% 87%
Lymphs 2% 6%
Mono/Macrophage 4% 7%
Glucose 68 mg/dL 13 mg/dL
Protein 69 mg/dL 164 mg/dL
37
CASE STUDY
Gram stain of the pathogen

isolated from aerobic blood
culture, showing gram-negative
coccobacilli, sometimes in pairs.
The same organism was seen on
the patient’s CSF Gram stain.
38
CASE STUDY
Aerobic blood culture on (A) 5% sheep blood agar plate (BAP), showing no
growth and on (B) chocolate agar plate (CAP), showing round, smooth, opaque
grey-yellow colonies.
39
CASE STUDY
• The CSF Gram stain showed rare, paired, Gram negative diplococci, which could raise
suspicion for Neisseria meningitidis, however the typical flattened sides of adjacent bacteria
were not observed.
• The morphology was more consistent with Gram-negative cocco-baccilli, which is better
demonstrated on Gram stain of the blood culture.
• Culture of both the CSF and blood specimens grew fairly large, smooth, round, opaque
grey-yellow colonies on chocolate agar, however showed no growth on blood agar,
suggesting a fastidious organism requiring growth factors.
• The colonies were both catalase and oxidase positive. The organism was identified
as Haemophilus influenzae by MALDI-TOF MS (matrix-assisted laser desorption/ionizations
time-of-flight mass spectrometry).
• This H. influenzae isolate was non-typeable by slide agglutination serotyping performed at
the state public health laboratory.
40
NEISSERIA SPP.
DESCRIPTION
Neisseria belongs to the family Neisseriaceae of the order Neisseriales, which is

placed in the class Betaproteobacteria.
Most Neisseria are Gram negative cocci with a diameter of up to 2 μm,

presenting as single bacteria or in pairs.
Neisseria elongata, N. weaveri, N. bacilliformis and N. shayeganii ssp. nov. are

exceptions and are short rods, arranged as diplobacilli or in chains.
Neisseria species are Gram negative, but they occasionally have a tendency to
with stand decolourisation, appearing Gram positive.
Capsules (N. meningitidis) and pili (N. meningitidis and N. gonorrhoeae) may be
present, but not flagella.
42
DESCRIPTION
N. meningitidis is the only species having a polysaccharide capsule, of which

there are 12 different serotypes.
Several species, N. flavescens, N. sicca and N. subflava, produce a yellowish

pigment.
Neisseria spp. grow optimally under aerobic conditions and at 35 to 37oC.
N. gonorrhoeae and N. meningitidis are fastidious, showing susceptibility to

unfavourable factors, such as extreme temperatures, desiccation and alkaline or
acidic conditions and are exclusively human pathogens.
All are oxidase positive and, with the exception of N. elongata subsp.
nitroreducens and some N. bacilliformis strains, catalase positive.
43
DESCRIPTION
Neisseria species produce acid from carbohydrates by oxidation, not

fermentation.
Some species, N. elongata and N. cinerea, are asaccharolytic.
Natural habitats of Neisseria species are the mucous membranes of animals and
humans.
44
N. gonorrhoeae causes gonorrhoea, a commonly reported notifiable disease in

most countries.
N. gonorrhoeae is always considered pathogenic and humans are the only host.
Transmitted mainly through sexual practices and infects the mucosal surfaces of
the urethra, cervix, rectum, pharynx and eyes.
Eyes can be infected as an intrapartum event during passage of the foetus

through the birth canal.
45
N. meningitidis occurs exclusively in humans and is both a commensal and a

potentially invasive pathogen.
On average, the mucosal surfaces of the oro- and nasopharynges of 10% of the
population are colonised by this bacterium.
Carriage is strongly age dependent:

• Adolescents and young adults with > 30%
• Infants 1-2%
Transmission occurs through large droplet secretions from the oropharynx,

favoured by repeated or close contact.
Disease occurs in only a small proportion of individuals and follows a typical age
distribution, with infants and adolescents having the highest incidences in
46
developed countries.
People with underlying conditions, such as properdin deficiencies, late

complement deficiencies and splenic impairment, including asplenia, are at
increased risk for invasive meningococcal disease (IMD).
A number of vaccines have been developed for the prevention of IMD.
47
CLINICAL SIGNIFICANCE
Neisseria have a high affinity for mucosal membranes.
A wide variety of species can be isolated from humans, including:

• N. gonorrhoeae, N. cinerea, N. elongata, N. flavescens, N. lactamica, N. meningitidis, N.
mucosa, N. polysaccharea, N. sicca, and N. subflava.
Several species are predominantly recovered from animals:

• N. animalis, N. animaloris, N. zoodegmatis (from throats of cats and dogs),
• N. denitrificans (from throats of guinea pigs),
• N. dentiae (from dental plaques of domestic cows),
• N. macacae (from oropharynges of rhesus monkeys),
• N. weaveri (from the oral flora of dogs).
48
N. meningitidis is a commensal of the human oropharynx yet can cause life-

threatening, acute disease in previously healthy individuals.
N. gonorrhoeae, is always considered a pathogen, even if obvious signs of

disease are absent.
Uncomplicated infection by N. gonorrhoeae (gonorrhoea) appears as acute

urethritis in men. Symptoms are a urethral discharge, sometimes associated with
dysuria, without frequency or urgency.
Co-infection of the preputial, urethral and bulbo-urethral glands is possible.
Asymptomatic infections occur in up to 10% of cases.
Most cases of untreated urethritis resolve spontaneously after several weeks.

49
In women, the endocervix is the primary site of genital infection.
N. gonorrhoeae may infect the urethra, the rectum, the peri-urethral glands, and
the ducts of the greater vestibular (Bartholin’s) glands.
Asymptomatic infection in women is common.
If symptoms appear, they often cannot clearly be attributed to infection by N.

gonorrhoeae, as concurrent infection by Chlamydia trachomatis and Mycoplasma
genitalium is common.
Symptoms include increased vaginal discharge, dysuria, and intermenstrual

bleeding.
50
Ascension of the infection may result in pelvic inflammatory disease, which

manifests as various combinations of endometritis, salpingitis, tubo-ovarian
abscess, and peritonitis.
Acute peri-hepatitis (Fitz-Hugh- Curtis syndrome) can develop following direct

extension of N. gonorrhoeae from the fallopian tube to the liver capsule and the
surrounding peritoneum.
Pharyngeal infection is acquired by oral sexual exposure and is mostly

asymptomatic but can also cause pharyngitis or tonsillitis.
Gonococcal conjunctivitis in adults usually results from auto-inoculation or oculo-

genital or oro-genital exposure. If not treated promptly, corneal ulceration may
develop.
51
Conjunctivitis of the newborn (ophthalmia neonatorum) is transmitted during birth

and is favoured by premature rupture of the membranes and preterm delivery.
Disseminated gonococcal infections manifests as septic arthritis and a

characteristic syndrome of polyarthritis and dermatitis, and should be suspected
in patients presenting with tenosynovitis, arthritis and vasculitic skin lesions.
IMD presents as meningitis, acute sepsis, or a combination of both. Unusual

presentations:
• Transient mild bacteraemia,
• Chronic meningococcal sepsis, septic arthritis,
• Pneumonia (mainly by serogroup Y),
• Endocarditis.
Symptoms can include a stiff neck, headache, confusion, and photophobia.

52
Sequelae, such as senso-rineural hearing loss, developmental delay, and speech

defects, afflict a substantial portion of survivors.
Petechial lesions are tell-tale signs of meningococcal sepsis
Meningococcal septic shock can take a fulminant course with a mortality of 30%,
and plasma concentrations can reach up to 108 cfu/ml, which leads to massive
activation of cytokines and vasoactive anaphylatoxins.
Complications of Invasive Meningococcal Disease include:

• Arthritis
• Cranial nerve dysfunction
• Meningococcal pericarditis
• Cerebral or spinal infarction.
53
Meningococcal pneumonia has been recognised as a clinical syndrome for more

than 100 years. Most meningococcal pneumonias are caused by serogroup Y
and affect adults. A preceding viral illness, notably pandemic influenza, has been
reported to promote its development.
N. meningitidis is an uncommon cause of acute bacterial conjunctivitis and can

be the aetiological agent of urethritis in men.
54
Neisseria gonorrhoeae
The male urethra is sampled by introducing a swab up to 2 cm in and rotating.
Endocervical swabs are obtained by introducing a swab after removal of mucus

plugging the cervical orifice. Vaginal swabs are inadequate for culture-based
diagnosis for sexually mature women yet can be used for prepubescent females.
For men who have sex with men (MSM) and women who practice anal
intercourse, rectal swabs. Swabs heavily contaminated with faeces should be
discarded.
A pharyngeal swab should be obtained from individuals engaging in oral sex.
55
Dacron or rayon tipped swabs are preferable for culture-based diagnosis of

gonorrhoea.
Calcium alginate swabs should be avoided due to reported toxicity and

interference with PCR.
Cotton swabs and oil-based lubricants contain unsaturated fatty acids, which
inhibit N. gonorrhoeae.
Amies based semi-solid transport media can be used to transport swabs to the
laboratory at room temperature
Plate swabs transported in these media within 6 hrs after collection.
56
Survival and transport of gonococci over 24 hrs can be achieved by culture

medium transport systems which allow direct plating of specimens in a clinical
environment.
These usually consist of a solid medium, onto which swabs are inoculated directly
after collection, and a CO2 generating system within a resealable container.
57
Neisseria meningitidis
Types of specimens that can be used for the detection of N. meningitidis include:
• Blood cultures
• Cerebrospinal fluid (CSF)
• Nasopharyngeal and oropharyngeal swabs
• Broncho alveolar lavage (BAL)
• Joint aspirates
• Urethral and endocervical swabs
• Petechial aspirates
• Biopsies
Swabs placed in Amies-based transport media and plated within 5 hrs after
collection.
58
GRAM STAIN
Gram-negative diplococci associated with or within polymorphonuclear

leucocytes.
Neisseria gonorrhoeae Neisseria meningitidis
59
ANTIGEN DETECTION
Commercial immunochemical techniques are available for the detection of S.

pneumoniae, Streptococcus dysgalactiae, H. influenzae, and N. meningitidis
directly from CSF and other body fluids.
These techniques provide rapid identification of the pathogen, but lack sensitivity
and specificity
Currently, no commercially available antigen detection kits with adequate

sensitivity and specificity exist for Neisseria gonorrhoeae.
60
MEDIA
Cultivation of N. gonorrhoeae requires the use of chocolate agar, which supports

the growth of many other commensal bacteria.
To isolate N. gonorrhoeae from mucosal and other nonsterile body sites, several
selective media containing a mixture of inhibitory agents have been developed.
Specimens should be inoculated on warmed or room temperature plates and

61 incubated at 35 to 37oC with 5 to 7% CO2
MEDIA
Detection of N. meningitidis from sterile specimens, such as CSF or joint fluid,

inoculate sheep blood and chocolate agar.
Specimens from mucosal surfaces (e.g., respiratory material) inoculate

additionally on selective media (see previous slide) which exclude growth of most
commensal Neisseria spp.
Incubation conditions are identical to those for N. gonorrhoeae.
Nevertheless, in contrast to N. gonorrhoeae, N. meningitidis tends to grow more

readily on solid media and almost invariably grows on blood agar plates.
62
COLONIAL MORPHOLOGY
After 48 hrs on chocolate agar, colonies of N. gonorrhoeae are up to 1 mm in

diameter, opaque, greyish-white, glistening and convex.
• Colonies expressing pili and opacity proteins are wrinkled and well defined, with a clear
edge
• Non-piliated colonies have more diffuse edges and are more glistening.
63
COLONIAL MORPHOLOGY
Colonies of N. meningitidis have smooth entire edges and are about 1 mm in

diameter after 18 hrs on blood agar.
They are grey, convex, glistening and occasionally mucoid. Blood agar beneath
the colonies may display a grey-green discolouration.
64
Both N. gonorrhoeae and N. meningitidis are oxidase positive.
Neisseria species produce acid from carbohydrates by oxidation. The only

carbohydrate used by N. gonorrhoeae is glucose, while N. meningitidis
catabolises both glucose and maltose.
The traditional cystine tryptic agar sugar method has been replaced by rapid
carbohydrate utilization tests in most routine laboratories which can be read after
4 hrs.
65
66
67
API NH
68
Other commercial manual identification kits

• RapID NH system (Remel) - glucose and sucrose only
• BBL Crystal Neisseria/Haemophilus ID system (BD)
69
70

71
72

• PCR
• Microarrays,
Multiplex disease-state panels for direct inoculation with a positive blood culture,
has demonstrated a reduction in length of stay and enhanced antimicrobial
stewardship:
• FilmArray (bioMérieux’ BioFire Diagnostics, Salt Lake City, UT)
• Verigene (Nanosphere, Inc., Northbrook, IL)
73
IDENTIFICATION: FILMARRAY
Blood Culture Identification Panel
74
75
MOLECULAR DETECTION
76
The CLSI recommends for disc diffusion:

• GC (gonococcal) agar containing 1% growth supplement for N. gonorrhoeae
• Suspensions of 0.5 McFarland
CLSI recommends agar dilution for the measurement of MICs, yet due to their
ease of use, gradient test systems (e.g., Etest) are an acceptable and frequently
used alternative.
Due to the global increase in resistance, guidelines now list a combination of

intramuscular ceftriaxone and oral azithromycin as a first-line treatment.
Ceftriaxone may be replaced by cefixime if ceftriaxone is not available.
Fluoroquinolones may be used for treatment only after antimicrobial susceptibility

77
is documented by culture.
According to the CLSI, testing should be performed by:

• Disc diffusion on Mueller-Hinton agar with 5% defibrinated sheep blood
• Broth micro-dilution using cation adjusted Mueller-Hinton broth with 2.5 to 5.0% lysed horse
blood.
Alternatively, gradient test systems (e.g., Etest) are frequently used.
Unlike N. gonorrhoeae, N. meningitidis is usually penicillin susceptible and beta-

lactamase production is rare. Although reduced susceptibility, resulting from
modification of penicillin binding protein 2, has been recorded in several countries
Rifampin and ciprofloxacin are used for chemoprophylaxis in close contacts of

patients.
78
REPORTING RESULTS
Due to the imperfect specificity of many diagnostic methods used for identification
of N. gonorrhoeae, results have to be interpreted in the context of local disease
prevalence.
This particularly applies to NAATs, which, due to varying specificities, cannot be

recommended uniformly for use in low-prevalence settings.
If medico-legal ramifications are likely to result from a positive test, as is the case,
e.g., in victims of sexual assault, special scrutiny has to be applied to all
laboratory procedures involved in the issuing of a positive result.
79
REPORTING RESULTS
Special protocols should be in place to ensure confirmation of results. This is also

important for specimens from children and adolescents and for the
documentation of sexual abuse.
Suspected N. gonorrhoeae should be confirmed by at least two different

methods:
• (i) Multi-test identification systems
• (ii) Immunologic methods
• (iii) DNA probe culture confirmation
• (iv) Sequencing of the 16S rRNA gene
• (v) MALDI-TOF mass spectrometry.
Additionally, strains and DNA need to be preserved.
80
REPORTING RESULTS
A definitive diagnosis requires

• (i) Isolation of oxidase-positive Gram-negative diplococci from sites of exposure (e.g.,
urethra, endocervix, throat, or rectum) by culture on selective media and
• (ii) Confirmation by biochemical or molecular methods.
Laboratories that rarely encounter N. gonorrhoeae should prefer test kits which
give a full identification to the species level.
81
REPORTING RESULTS
Is always considered a pathogen when isolated from sterile body fluids, such as
blood or CSF.
If it is isolated from the urethra, cervix, or conjunctiva, a pathogenic role is likely.
N. meningitidis should always be reported and the strain be forwarded to a

reference laboratory.
Detection of N. meningitidis from broncho-alveolar lavage fluid or sputum has to

be interpreted with the clinician, as it may or may not have a pathogenic role.
Growth from oro-pharyngeal or naso-pharyngeal specimens usually reflects

asymptomatic carriage and may not be reported.
82
CASE STUDY
• A 22 year old female with no significant past medical history presented with a fever, joint
pain and a petechial rash.
• She endorsed having cold/flu symptoms for two weeks prior.
• The patient was admitted to the hospital where blood cultures were drawn and antibiotics
were initiated.
• One set of blood cultures from the patient flagged positive at 14 hours of incubation with the
following gram stain and colony morphology.
83
CASE STUDY
• The patient’s blood was cultured on aerobic blood agar and chocolate agar plates.
• The gram stain revealed gram negative diplococci.
• Medium sized, round, grey to white, slightly mucoid colonies grew on blood and chocolate
agars.
• The organism was definitively identified as Neisseria meningitidis by VITEK-MS.
• Prior to adoption of mass spectrometry, biochemical tests were performed for further
characterization of the organism.
• Neisseria meningitidis is catalase positive, ferments glucose and maltose but not lactose, is
oxidase positive, and does not reduce nitrate.
84
AGGREGATIBACTER, CAPNOCYTOPHAGA,
EIKENELLA, KINGELLA,
PASTEURELLA, AND OTHER FASTIDIOUS
GRAM-NEGATIVE RODS
DESCRIPTION
Taxonomically diverse group, belong to the families Cardiobacteriaceae,

Flavobacteriaceae, Leptotrichiaceae, Neisseriaceae, Pasteurellaceae and
Porphyromonadaceae, but common traits justify grouping together.
Isolated infrequently
With the exception of Chromobacterium and some Pasteurella species, for

aerobic growth they require:
• Supplemented media, grow slowly (48 h at 35 to 37oC)
• Fail to grow on enteric media.
• 5 to 7% CO2 atmosphere.
With the exception of Chromobacterium, do not possess flagella but may show
gliding or twitching motility, resulting in limited spread of colonies and pitting of
the agar surface.
86
Actinobacillus spp.
• A. lignieresii causes actinobacillosis, a granulomatous disease in cattle and sheep in which,
sulphur granules form in tissues. A few human soft tissue infections after a cow or sheep
bite have been reported.
• A. equuli and A. suis have caused a variety of diseases in horses and pigs; human
infections are generally due to horse or pig bites or contact. Both species have also been
isolated from the human upper respiratory tract.
• A. ureae is most often a commensal in the human respiratory tract, particularly in patients
with lower respiratory tract disease, but has also been found as the cause of meningitis
following trauma or surgery and of other infections in immunocompromised patients as with
A. hominis.
87
Aggregatibacter spp.
• A. actinomycetemcomitans is one of the major agents of juvenile and adult periodontitis and
may occur together with Actinomyces spp. in actinomycotic sulphur granules.
• It may cause HACEK (Haemophilus, Aggregatibacter, Cardiobacterium, E. corrodens, and
Kingella spp.) endocarditis, soft tissue infections, and other infections.
• A. aphrophilus may cause systemic disease, particularly bone and joint infections,
spondylodiscitis, and endocarditis.
• A. segnis, may cause endocarditis. It may be an important cause of bacteraemia and
sometimes of other infections, e.g., pyelonephritis
88
Capnocytophaga spp.
• C. ochracea, C. gingivalis, C. sputigena, C. haemolytica, and C. granulosa have been
reported as agents of septicaemia and other endogenous infections (endocarditis,
endometritis, osteomyelitis, soft tissue infections, peritonitis, ophthalmic lesions).
• C. canimorsus and C. cynodegmi are associated mainly with dog or cat bites. C.
canimorsus most often present with septicaemia of splenectomised patients or alcoholics.
• In fulminant cases with poor prognosis, disseminated intravascular coagulation, acute renal
failure, respiratory distress syndrome, and shock may develop. Haemolytic uremic
syndrome and thrombotic thrombocytopenic purpura are other possible sequelae.
Meningitis, keratitis, and endocarditis have been reported.
Cardiobacterium spp.
• Disease by Cardiobacterium species is mainly HACEK (Haemophilus, Aggregatibacter,
Cardiobacterium, E. corrodens, and Kingella spp.) endocarditis; C. hominis prosthetic valve
endocarditis and on rare occasions, from other body sites. Most reported cases of C.
valvarum endocarditis are related to periodontal diseases. In blood culture negative cases,
89
the diagnosis has been made by PCR applied to valve tissue.
Chromobacterium violaceum
• Localised infections usually arise from contaminated wounds and septicaemia and multiple
organ abscesses may follow. Significantly associated with neutrophil dysfunction (glucose-
6-phosphate dehydrogenase deficiency, chronic granulomatous disease). Children without
these conditions and those with bacteraemia show a high fatality rate. Virulence factors
other than endotoxin, i.e., adhesins, invasins, and cytolytic proteins, have been described.
Dysgonomonas spp.
• Diarrhoea was reported to have occurred patients with faecal isolates of D.
capnocytophagoides. Bacteraemia occurs as well. One strain of D. mossii was isolated
from intestinal juice of a patient with pancreatic carcinoma. D. gadei has been isolated from
a human gallbladder and from blood, and D. hofstadii has been isolated from a wound.
90
Eikenella corrodens
• E. corrodens is associated with juvenile and adult periodontitis but is also an agent of
infections of the upper respiratory tract, pleura and lungs, abdomen, joints, bones, wounds
(e.g., from a human bite), and other infections, like noma. These organisms are often found
mixed with other members of the oropharyngeal microbiota, particularly staphylococci and
streptococci. Risk factors are dental manipulations and intravenous drug abuse.
Endocarditis is of the HACEK type if mono-microbial, but poly-microbial non-HACEK cases
are known.
Kingella spp.
• Infections with K. kingae show a predilection for bones and joints of healthy children <4
years of age. Most common cause of osteo-arthritic infection in this age group. Septic
arthritis, discitis, and osteomyelitis of the lower extremities as well as bacteraemia occur.
Stomatitis and/ or upper respiratory infections may precede systemic disease. In adults,
systemic infections occur in immunocompromised individuals or may present as HACEK
endocarditis. Virulence factors are an RTX toxin and type IV pili. K. denitrificans has been
reported as an agent of endocarditis.
91
Pasteurella spp.
• Human isolates of P. multocida are mostly found in wound or soft tissue infections. Less
frequent are colonisation or infection of the respiratory tract and systemic disease, such as
meningitis, dialysis-associated peritonitis, endocarditis, osteomyelitis, urinary tract infection,
and septicaemia, with cirrhosis of the liver being a particular risk factor. The subspecies
most frequently encountered is subsp. multocida, which is also more frequent in respiratory
infections and bacteraemia than subsp. septica, which is most often associated with wound
infections and central nervous system infections.
• Human infection with P. canis (from dogs), P. dagmatis, and P. stomatis (from dogs or cats)
are infrequent. P. canis is the most common species from infected dog bite wounds. P.
dagmatis has been isolated in systemic infections such as pneumonias, peritonitis,
septicaemia, and endocarditis and can cause exacerbation in chronic obstructive
pulmonary disease patients.
92
Streptobacillus moniliformis
• Rat bite fever is a systemic illness beginning with fever and chills, followed by migratory
polyarthritis and a maculopapular rash on the extremities. Rare complications include
endocarditis, myo- or pericarditis, pneumonia, septicaemia, and abscess formation.
93
The low viability of many species makes the use of transport media, e.g., Aimes
swab, or eSwab, mandatory.
Cultures of most bacteria can be stored at room temperature for 1 to 2 weeks.

However, for some other very fastidious bacteria, e.g., Capnocytophaga
canimorsus, subcultures must be performed frequently.
For keeping strains in a culture collection, the isolates should be frozen in a

cryoprotective solution, e.g., Cryobeads, at –70oC.
94
GRAM STAIN
Aggregatibacter spp.
• Coccoid to rod-shaped, Gram negative bacteria, occasionally exhibiting filamentous
Capnocytophaga spp.
• Mainly fusiform, Gram negative, medium-to long cells with tapered ends.
• Pleomorphic on blood agar. C. hominis stains irregularly and appears as straight, Gram
negative rods in short chains, pairs, or rosettes, sometimes with bulbous ends;
occasionally, filaments are formed. On chocolate agar, this morphology is less distinct than
on blood agar.
• Medium-sized, Gram-negative rod or coccus that is motile by one polar flagellum and 1-4
lateral flagella.
95
GRAM STAIN
Dysgonomonas spp.
• Small, Gram-negative rods or cocci similar to Moraxella spp
E. corrodens
• Sender, straight, small, Gram-negative rod with rounded.
Kingella spp.
• short, Gram-negative rods with square ends that lie together in pairs or clusters. They
• tend to decolourise unevenly on Gram stains.
Pasteurella spp.
• Small, Gram-negative rods or cocci that occur singly, in pairs, or in short chains. Bipolar
staining is frequent.
96
GRAM STAIN
Simonsiella spp.
• Gram-negative, crescent-shaped rods (0.5 to 1.0 μm long) arranged in multicellular
filaments (10 to 50 μm by 2 to 8 μm) and segmented into groups of mostly eight cells,
resulting in a caterpillar-like appearance. Incomplete decolourisation on Gram stains is
common.
Streptobacillus moniliformis
• Gram-negative rod with a variable morphology. Depending on age and culture conditions,
cells may appear as straight, small to medium-size rods or as 100- to 150-μm-long tangled
chains and filaments with bulbar swellings.
• S. indologenes is a plump, irregularly staining, Gram negative rod; occasionally, pairs,
chains, or rosettes are formed.
97
MEDIA
Culture of members of this group use:

• Blood agar
• Chocolate agar
• Selective media wherever normal flora and specific bacteria are suspected.
- Thayer-Martin
- Martin-Lewis
They grow better in a 5 to 7% CO2 atmosphere at 35oC.
Most of them are facultative anaerobes and do not grow on MacConkey agar.
HACEK members do not need more than 5 days of incubation in automated

blood culture systems.
98
COLONY MORPHOLOGY
Actinobacillus spp.
• Are ~2 mm in diameter after 24 h of growth at 35oC, smooth or rough, viscous and often
adherent to the agar.
• Smooth colonies are dome shaped and have a bluish hue when viewed by transmitted light.
Aggregatibacter actinomycetemcomitans
• Colonies initially show a central dot and a slightly irregular edge and, on further incubation,
develop a star-like configuration resembling “crossed cigars” and pit the agar. After several
subcultures, this rough morphology may give way to smooth and opaque, nonpitting
colonies, reflecting loss of fimbriae.
Capnocytophaga spp.
• Colonies on blood agar are very small after 24 h at 35oC and reach 2 to 4 mm in diameter
after 2 to 4 days; they are convex or flat and often slightly yellow when scraped off agar and
adhere to the agar surface.
99
COLONY MORPHOLOGY
• Colonies attain a diameter of ~1 mm after 48 h at 35oC on blood agar; they are circular,
smooth, and opaque, and they may pit the agar.
• Colonies measure 1 to 2 mm in diameter after 24 h of growth, are round and smooth, have
an almond like smell, and may be beta-haemolytic. Most strains produce a violet pigment
called violacein. Identification is easy if this pigment is produced, although the positive
oxidase reaction will be detected only by a modified technique
Dysgonomonas spp.
• Colonies are entire, measure 1 to 2 mm in diameter after 24 h of growth, have a strawberry
like odour, and do not spread or adhere.
100
COLONY MORPHOLOGY
E. corrodens
• Colonies are 1 to 2 mm in diameter after 48 h of growth, show clear centres that are often
surrounded by spreading growth, may pit the agar, and assume a slightly yellow hue after
several days.
Kingella
• Colonies on blood agar in 5 to 10% CO (which enhances growth) are 1 to 2 mm in diameter
2
after 48 h of growth. One type is smooth with a central papilla, and the other spreads and
pits the medium.
• Only K. kingae shows a small but distinct zone of haemolysis on blood agar.
• Colonies have a short viability and have to be subcultured frequently.
101
COLONY MORPHOLOGY
Pasteurella spp.
• Colonies are 1 to 2 mm in diameter after 24 h of growth at 35oC and are opaque and
greyish.
• Encapsulated strains tend to be mucoid. A slight greening underneath the colonies may be
noted.
Simonsiella spp.
• Colonies are 1 to 2 mm in diameter after 24 h, may show gliding motility and produce a pale
yellow pigment. S. muelleri is beta-haemolytic.
S. moniliformis
• May show wild-type and L-phase colonies in the same culture. The wild-type are 1 to 3 mm
in diameter after 48 to 72 h of growth on blood agar and are round and smooth. L-phase
colonies grow better on clear media, yielding the “fried egg” appearance, with irregular
outlines and coarse lipid globules.
102
IDENTIFICATION
Phenotypic identification of fastidious Gram-negative rods presents several

challenges:
• Triple sugar iron or Kligler’s agar does not support the growth of fastidious genera (e.g.,
Eikenella).
• Media should be rich in peptones (e.g., cysteine Trypticase agar);
• Serum (except rabbit serum) should not be used, because it may split maltose.
• The inoculum should be large.
• Un-supplemented media used to check acid formation from carbohydrates may yield false
negative reactions.
• Gas formation from carbohydrates is scant or absent in most species.
• Indole may have to be extracted with xylene.
• Correct identification to the species level often requires multiple substrates that may not be
available in routine laboratories.
103
IDENTIFICATION
VITEK 2 Neisseria-Haemophilus identification card allows for the identification of

most bacteria of the HACEK group.
Pasteurella spp. are not in the database of the VITEK 2 ID-NH card, but they are
in the database of the identification card for Gram-negative bacilli, VITEK 2 ID-
GN card.
Determination of key reactions may be used for a rapid presumptive identification,

e.g., with API NH within 2 hrs.
Newer identification methods like MALDI-TOF MS shows good results regarding

the identification of HACEK group, i.e., VITEK MS (bioMérieux) and MALDI
Biotyper (Bruker).
104
105
106
107
108
109
110
API NH
111
112

113
VITEK GN CARD IDENTIFICATIONS
NON-ENTEROBACTERIACEAE NON-ENTEROBACTERIACEAE
• Achromobacter denitrificans • Aeromonas veronii

• Achromobacter xylosoxidans • Alcaligenes faecalis ssp. faecalis
• Acinetobacter baumannii complex • Bordetella bronchiseptica
• Acinetobacter haemolyticus • Bordatella hinzii
• Acinetobacter junii • Bordetella trematum
• Acinetobacter lwoffii • Brevundimonas diminuta/vesicularis
• Acinetobacter radioresistens • Brucella melitensis
• Acinetobacter ursingii • Burkholderia cepacia group+
• Actinobacillus ureae • Burkholderia gladioli*
• Aeromonas hydrophila/Aeromonas caviae • Burkholderia mallei
• Aeromonas salmonicida • Burkholderia pseudomallei
• Aeromonas sobria • Chromobacterium violaceum
114
• Chryseobacterium gleum • Neisseria animaloris/zoodegmatis

• Chryseobacterium indologenes • Ochrobactrum anthropi
• Comamonas testosteroni • Oligella ureolytica
• Cupriavidus pauculus • Pandoraea species
• Delftia acidovorans • Paracoccus yeei
• Elizabethkingia meningoseptica • Pasteurella aerogenes
• Francisella tularensis • Pasteurella canis
• Grimontia hollisae • Pasteurella dagmatis
• Mannheimia haemolytica • Pasteurella multocida
• Methylobacterium spp. • Pasteurella pneumotropica
• Moraxella group • Pasteurella testudinis
• Myroides spp. • Photobacterium damselae
115
• Pseudomonas aeruginosa* • Rhizobium radiobacter

• Pseudomonas alcaligenes • Roseomonas gilardii
• Pseudomonas fluorescens* • Shewanella algae
• Pseudomonas luteola • Shewanella putrefaciens
• Pseudomonas mendocina • Sphingobacterium multivorum
• Pseudomonas oleovorans • Sphingobacterium spiritivorum
• Pseudomonas oryzihabitans • Sphingobacterium thalpophilum
• Pseudomonas putida • Sphingomonas paucimobilis
• Pseudomonas stutzeri • Stenotrophomonas maltophilia
• Ralstonia insidiosa • Vibrio alginolyticus*
• Ralstonia mannitolilytica • Vibrio cholerae*
• Ralstonia pickettii • Vibrio fluvialis*
116
NON-ENTEROBACTERIACEAE
• Vibrio metschnikovii*
• Vibrio mimicus*
• Vibrio parahaemolyticus*
• Vibrio vulnificus*
117
118

• PCR
• Microarrays,
119
Approved guidelines for broth microdilution susceptibility testing of the HACEK

group and for antimicrobial dilution and disk susceptibility testing of Pasteurella
spp. have been published by the Clinical and Laboratory Standards Institute
(CLSI).
Beta-lactamase production among members of the HACEK group is well

documented and b-lactamase producing isolates are ampicillin resistant; some
isolates may be resistant to ampicillin due to mechanisms other than beta-
lactamase production;
Routine use of a beta-lactamase test is recommended for all HACEK isolates
120
Susceptibility studies of Actinobacillus spp. exist for a few isolates of the human
pathogenic species that are susceptible to many antimicrobials, including
penicillin.
Aggregatibacter spp. are susceptible to cephalosporins, tetracyclines, and

aminoglycosides. Resistance to ampicillin is not uncommon, but amoxicillin
combined with a beta-lactamase inhibitor has been effective.
Capnocytophaga spp. are usually susceptible to broad spectrum cephalosporins,

carbapenems, clindamycin, macrolides, tetracyclines, and fluoroquinolones but
are resistant to aminoglycosides. Multidrug-resistant isolates have occasionally
been encountered.
121
Cardiobacterium hominis and C. valvarum are susceptible to many antimicrobials,

including penicillin. Beta-lactamase production is rare, and its effect can be
neutralised by clavulanic acid.
Chromobacterium violaceum is resistant to many antimicrobials (beta-lactams

and colistin) but is mostly susceptible to imipenem, fluoroquinolones, gentamicin,
tetracyclines and co-trimoxazole.
Dysgonomonas capnocytophagoides strains are susceptible to tetracyclines,

clindamycin, macrolides, and co-trimoxazole, whereas they are resistant to
cephalosporins, aminoglycosides and fluoroquinolones and variably susceptible
to other beta-lactam antibiotics and to Imipenem.
122
E. corrodens is generally susceptible to penicillin, cephalosporins, carbapenems,

doxycycline, azithromycin, and fluoroquinolones but is often resistant to narrow-
spectrum cephalosporins, macrolides, and clindamycin. Beta-lactamase positive
strains have been reported, but the enzyme was inhibited by b-lactamase
inhibitors.
Kingella spp. are generally susceptible to beta-lactam antibiotics, macrolides,

tetracyclines, co-trimoxazole, and quinolones. Beta –lactamase positive isolates
have been reported to be susceptible to combinations with b-lactam inhibitors.
Pasteurella spp. are generally susceptible to penicillin, broad-spectrum

cephalosporins, tetracyclines, quinolones, co-trimoxazole, and azithromycin;
resistance to erythromycin can occur.
123
Streptobacillus moniliformis is susceptible to penicillin and tetracyclines, the

mainstays of treatment, and to cephalosporins, carbapenems, aztreonam,
clindamycin, erythromycin and tetracycline; it shows intermediate susceptibility to
aminoglycosides and fluoroquinolones and is resistant to colistin and co-
trimoxazole.
Patients with S. moniliformis endocarditis require dual therapy with high-dose

penicillin G in combination with an aminoglycoside; the use of an aminoglycoside
appears to enhance activity against the cell wall-deficient L forms of S.
moniliformis
124
REPORTING
Some bacteria of the group are colonisers of the human or animal oral cavity;
therefore, the evaluation of their isolation may be difficult.
All should be identified to the species level if isolated as pure cultures from
normally sterile body sites.
Interpretation as infectious agents and results of susceptibility testing should be

clearly reported to the physician.
With specimens normally colonised with aerobic and anaerobic bacteria, as well
as with specimens from wounds, e.g., bite wounds, the significance of the
bacteria depends on their predominance and the absence of other potentially
pathogenic bacteria.
125
CASE STUDY
• A 63-year-old male was admitted to the emergency department with a complaint of left
shoulder pain for one month and chest tightness for 3 days.
• The patient had a history of diabetes mellitus and his glucose was well regulated.
• Six years prior the patient had been treated with oesophagectomy and radiotherapy for
oesophageal cancer.
• After the surgery, he had 3 times of following-up gastroscopy examinations, which indicated
no evidence of recurrence, while the white blood cell count (WBC) kept in relatively lower
level around 3.5 × 109 /L.
• At admission he had no cough, vomiting and abdominal pain.
• The patient had a temperature of 37.6 °C, a blood pressure of 123/87 mmHg, a pulse of
103 bpm, a respiratory rate of 20 per minute and an oxygen saturation of 97% at admission.
• Laboratory examination indicated WBC of 12 × 109/L and serum procalcitonin level of
1.8 ng/ml, while the liver function tests and serum myocardial markers level (troponin T and
pro-b-type natriuretic peptide) were slightly above normal.
• Chest computed tomography revealed a massive pyopneumopericardium, a bilateral
126 pleural effusion, and a collapse of the lower lobe of left lung.
CASE STUDY
• Analysis of specimens of pericardium pus indicated WBC of 20 × 109/L (100% neut), lactic
dehydrogenase (LDH) > 17,000 IU/L and glucose of 0.05 mmol/L.
• Parenteral treatment with ceftriaxone 2000 mg IV every 24 h was given at admission, but no
clinical improvement was achieved.
• On the day after his admission the pericardial fluid culture identified E. corrodens
and Streptococcus anginosus.
• The antibiotic therapy was changed to imipenem 500 mg IV every 8 h, and mechanical
ventilation was provided.
• But the patient’s condition still had no improvement.
• The antimicrobial susceptibility test showed that the isolated strain was sensitive to
imipenem and ceftriaxone, while resistant to clindamycin and amikacin.
• The patient died three days after admission.
127
PIONEERING DIAGNOSTICS

Fastidious Organisms: Brett Crawley MASM, MACTM, MAIMS Reference: Manual of Clinical Microbiology, 11 Ed. ASM Press

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Fastidious Organisms

Brett Crawley MASM, MACTM, MAIMS

Reference: Manual of Clinical Microbiology, 11th Ed. ASM Press

A fastidious organism is any organism that has complex or particular nutritional

Thus fastidiousness is often practically defined as being difficult to culture, by any

Fastidious organisms are not "weak"—they can flourish in their particular

Haemophilus spp. are members of the family Pasteurellaceae

Eight species affecting humans

Haemophilus genus are small, non-motile, non-spore forming, non-acid fast,

Facultative anaerobes, with requirements for X and/or V factors.

Optimal growth at 35 to 37oC with 5 - 7% CO2.

All species are CAMP negative

Produce alkaline phosphatase.

Grow on chocolate agar

Non-encapsulated H. influenzae strains are referred to as non-typeable (NTHi)

Haemophilus species can be part of the normal flora of mucous membranes in

Disseminate to various sites, including the

Invasive infections caused by H. influenzae, such as meningitis, epiglottitis,

Biotype IV strains often found to cause systemic infections in neonates as well as

Vast majority of infections today are caused by NTHi, causing acute

Persons at risk for systemic NTHi infection:

Chancroid is a sexually transmitted disease caused by H. ducreyi.

Characterized by a single painful genital ulcer, with associated inguinal

Mostly in developing countries, including Asia, Africa and Latin America.

Epidemics are associated with low socioeconomic status, poor hygiene,

H. aegyptius, (Koch-Weeks bacillus)

Important cause of acute purulent conjunctivitis (pinkeye), occurs in younger

Brazilian purpuric fever

H. haemolyticus is a cause of invasive disease, often misidentified as H.

Other Haemophilus species

The collection of specimens for the diagnosis of Haemophilus infections is

Meningitis Blood culture and CSF cultures

Otitis media Middle ear fluid obtained by tympanocentesis

Conjunctivitis Conjunctival swab

Chancroid specimens for culture of H. ducreyi should be collected from the

Swabs should be immediately transported to the laboratory and plated without

When refrigerated, Amies transport medium has been demonstrated to maintain

Long-term storage of Haemophilus spp.

Small, pleomorphic, Gram negative coccobacilli with coccoid, cocco-bacillary,

Gram stain of Haemophilus influenzae in a CSF

Commercial immunochemical techniques are available for the detection of S.

Encapsulated and non-encapsulated Haemophilus influenzae on chocolate agar

Selective Chocolate agar

Sputum containing Haemophilus influenzae non-selective chocolate vs selective

Plates should be incubated at 35oC in 5 to 7% CO2.

most Haemophilus spp. grow within 24 to 48 h.

For H. ducreyi and H. aegyptius Incubate up to 5 days

For H. ducreyi, incubate 30 to 33oC in 5 to 7% CO2 for better growth.

Colonies of Haemophilus spp. on solid media, in general, are non-pigmented or

Colonies of H. influenzae on chocolate agar are smooth, low, convex, greyish,

Manual ientification of Haemophilus spp. is by the following tests:

X and V factor discs in determining the growth factor requirements of

API NH indentification in 2 hrs

Other commercial manual identification kits

Vitek 2 NH identification in 6 hrs

VITEK NH CARD ORGANISMS VITEK NH CARD ORGANISMS

Actinobacillus pleuropneumoniae • Histophilus somni

Vitek Mass Spectrometer (MALDI-TOF) 1 minute per isolate after vacuum

> 1,300 organisms in IVD database