Procedures/Guidelines For The Microbiology Laboratory
Procedures/Guidelines For The Microbiology Laboratory
Procedures/Guidelines For The Microbiology Laboratory
Procedures/Guidelines
for the Microbiology
Laboratory
January 2010
Table of Contents
Page 1
The most recent update of the following CLSI documents should be followed:
(i)
(ii)
(iii)
M2
(iv)
M7
Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria that Grow Aerobically
(v)
b)
c)
ATCC organisms shall be used for proper quality control of media and of
antimicrobial susceptibility testing.
CLSI: Clinical and Laboratory Standards Institute
ATCC: American Type Culture Collection
All laboratories performing microbiology shall demonstrate and document their efforts to
meet the standards established by CLSI guidelines and quality control using appropriate
organisms.
Page 2
Page 3
Grade
0
+1
+2
Mucous present
+1
1
2
10 SEC / LPF
Acceptable:
NOTE: if number of PMN is 10X the number of SECs and there is 3-4+ of a single
morphotype accept the specimen for culture.
Page 4
Description
Number/OIF
1+, rare
<1
2+, few
1-10
3+,
11-25
moderate
4+, many
>25
4+, many
>25
Low-power field (LPF) (100); high-power oil immersion field (OIF) (1000).
Numbers are based on an average of 10 fields.
A thorough examination of all areas of the Gram-stained smear should be made
microscopically first under LPF (100) to observe any inflammatory cells and then
under OIF (1000) to observe bacteria.
NOTE: For fluid specimens where cytospin or similar preparations are used, the
quantification guidelines do not apply.
3) QA Criteria for Gram Stain Interpretation:
For scoring Gram stains a standard error classification schema is recommended as
follows:
MINOR ERROR:
Report the presence of polymorphs when not present
MAJOR ERROR:
Report the presence of an organism not present or;
Failure to report the presence of 1+ to 2+ polymorphs
VERY MAJOR ERROR:
Failure to report the presence of a microorganism
Failure to report the presence of 3+ to 4+ polymorphs
References:
1.
2.
Bartlett, JG, KJ Ryan, TF Smith and WR Wilson. 1987. Cumitech 7B, Lower respiratory tract
infections. Coordinating ed., J.A. Washington II. American Society for Microbiology,
Washington, D.C.
Evangelista, AT and HR Beilstein. 1993. Cumitech 4A, Laboratory diagnosis of gonorrhea.
Coordinating ed., C. Abramson. American Society for Microbiology, Washington, D.C.
Page 5
3.
Kruczak-Filipov P and RG Shively. 1992. Gram stain procedure. In: Clinical Microbiology
Procedures Handbook, Isenberg HD (Ed.), p.1.5.1-1.5.18, American Society for Microbiology,
Washington, D.C.
4. Martin RS, RK Sumarah and EM Robart. 1978. Assessment of expectorated sputum for
bacteriological analysis based on polymorphs and squamous epithelial cells: six-month study. J.
Clin. Microbiol. 8:635-637.
5. Murray PR, and JA Washington. 1975. Microscopic and bacteriologic analysis of sputum. Mayo
Clin. Proc. 50:339-344.
6. Thompson Jr RB, Miller JM. Specimen Collection, Transport, and Processing : Bacteriology. In:
Murray PR, Baron EJ, Jorgenson JH, Pfaller MA, Yolken RH (eds). Manual of Clinical
Microbiology 8th ed. 2003. American Society for Microbiology. Washington DC.
7. Chuard C, CM Antley and LB Reller. 1998. Clinical utility of cardiac valve Gram stain and
culture in patients undergoing native valve replacement. Arch. Pathol. Lab. Med. 122:412-415.
8. Ketai L, T Washington, T Allen and J Rael. 2000. Is the stat Gram stain helpful during
percutaneous image-guided fluid drainage? Acad. Radiol. 7:228-231.
9. Nugent RP, MA Krohn and SL Hillier. 1991. Reliability of diagnosing bacterial vaginosis is
improved by a standardized method of Gram stain interpretation. J. Clin. Microbiol. 29:297-301.
10. Schreckenberger PC. 1992. Diagnosis of bacterial vaginosis by Gram-stained smears. Clin.
Micro News. 14(16): 121-128
11. Clinical Microbiology Procedures Handbook 2nd Ed 2007
Isenberg HD (ed) p 3.2.1.20
12. Zaidi A.K., and L.B.R.eller. 1996. Rejection criteria for endotracheal aspirates from pediatric
patients. J Clin. Microbiol. 34: 352-354
13. Thakker JC, Splaingard M, Zhu J, Babe lK, Bresnahan J , Havens PL. Crit Care Med 1997 Aug;
25(8): 1997 . Survival and functional outcome of children requiring endotracheal intubation during
therapy for severe traumatic brain injury.
Page 6
negative
report: coagulase
negative staphylococci
negative
positive
tube coag
colonial morphology
consistent with S. aureus?
positive
report: possible S. aureus (1, 3)
perform or refer out for Staph ID and sens
NO
YES
report: S. aureus (3)
tube coag 2
negative
report: possible S. lugdenensis (4)
1.
2.
3.
4.
positive
report: S. aureus (3)
Page 7
Page 8
S. schleiferi can also be misidentified as S. aureus if only the slide coagulase test is used.
It is slide coagulase positive, tube coagulase negative, PYR positive, and ornithine
decarboxylase negative. It too appears to be a significant opportunistic pathogen
associated with empyema, wound and IV catheter infections and bacteremia. As with S.
lugdenensis, many affected patients have defective immune systems due to underlying
disease.
S. haemolyticus has been reported to be the second most commonly isolated CNS and has
been isolated in endocarditis, septicemia, UTIs, bone, wound and joint infections.
Other CNS less commonly isolated include S. hominis, S. warneri, S. capitis, S. simulans,
S. cohnii, S. xylosus, and S. saccharolyticus.
Procedure:
A review of the literature suggests that CNS be identified to species:
o when isolated from normally sterile sites-blood, CSF and joint and other fluids.
Regarding blood cultures, a CNS isolated from one bottle only is considered to be a
contaminant. It is not necessary to identify or report susceptibility results. Laboratories
may store the isolate in case further testing is requested. Isolates from several bottles are
identified to species level. If the same species is isolated, it is a significant finding and
susceptibility testing should be performed. If different species are identified, it is
probably a contaminated set of cultures and susceptibility testing should not be
performed. CNS can be contaminants in CSF and other sterile fluids, therefore always
correlate with clinical information and direct gram smear results to determine the
significance of the isolate.
o when repeatedly isolated from wound specimens with a good gram smear (showing
the presence of GPC and WBC).
o when isolated from shunts, catheters, or prosthetic devices.
o to differentiate CNS from S. saprophyticus in midstream urines.
o when isolated from catheter or cystoscopy urine specimens
It is not appropriate or cost effective to do identification and susceptibility testing on
every CNS isolated. Each laboratory should look at their particular situation and
establish policies specific to their needs. If the CNS is isolated as part of the normal
flora, then it should be reported as such-normal flora. Although a review of the literature
and laboratory practice shows that there is consensus that full ID & susceptibility testing
is not always required, there is no absolute consensus as to when ID & susceptibility
testing should be performed. It remains the responsibility of the laboratory to look at
patient demographics, type of service, etc and decide on their policies. It is advisable to
consult with a Medical Microbiologist or Infectious Disease specialist when developing
these policies.
Page 9
References:
1. Identification of Commonly Isolated Aerobic Gram-Positive Bacteria pg 1.20.1-1.20.3 in
Handbook of Clinical Microbiology Procedures 1992. ASM Washington DC
2. Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A.
Pfaller, Manual of Clinical Microbiology, 9th ed., 2007 American Society
for Microbiology.
3. von Eiff C, Proctor, RA, Peters G. 2001. Coagulase-negative staphylococci in Post
Graduate Medicine online Vol 110 No 4 October 2001. www.postgradmed.com
4. Laboratory Detection of Oxacillin-resistant Coagulase-negative Staphylococcus spp. 1999.
Fact Sheet Issues in Healthcare Settings. CDC Atlanta GA.
5. Dowell EB, James. JF. Speciation of Coagulase Negative Staphylococci 2001 in
Contagious Comments, Vol XVI Number 2. The Childrens Hospital, Department of
Epidemiology, Denver CO.
6. Baron, EJ. Staphylococcus lugdenensis: Not your Fathers (or Mothers)
Coagulase-Negative Staphylococcus. 2003. www.antibiotic-consult.com
7. Ling, ML. Staphylococcus lugdenensis: Report of first case of skin and soft tissue
infection in Singapore. Singapore Med J 2000 Vol 41(4): 177-178
8. Canadian Paediatric Society. Coagulase negative staphylococci as pathogens:
Believe it or not? Canadian Journal of Paediatrics 1994: 1(2):61-3 Reaffirmed
April 2002.
9. CMPT Critique. M93-1 Urine Sample: Coagulase Negative Staphylococcus
(S. epidermidis) November 1999.
10. CMPT Critique M94-1 Deep Wound: Coagulase Negative Staphylococcus species
(Staphylococcus epidermidis) Feb 2000.
Page 10
Page 11
bacteria present, which may be helpful to the clinician, particularly if different organism
types are present.
Several methods of semi-quantitation are used (Figure 2a & b). Some laboratories use
very few, few, moderate or many; others use 1+ to 4+, based on the following criteria:
Score
1+
2+
3+
4+
Page 12
Reference:
Clinical and Pathogenic Microbiology
Page 13
Figure 1
Figure 2a
Figure 2b
Page 14
Page 15
3.
4.
Spread the urine over the surface of the agar plate as described in Bacteriology
Culture Planting Procedure. Media generally used are Blood Agar and
MacConkey agar or a single media that will support the growth of uropathogens.
Incubate plates aerobically overnight at 35oC.
Specimen Type
MSU
Catheterized urine
Bladder, Suprapubic or
Cystoscopy Urine
Calibrated Loop
Media
1 _l loop
Blood Agar (BA)
1 _l loop
MacConkey Agar
(MAC)
10 _l loop
Incubation
O2, 35C x 24 hrs
O2, 35C x 24 hrs
Inspect the plates and dipslides for growth and determine the colony count (refer
to Standardization of the Colony Count for Urine Cultures).
Identify isolates of potential pathogens present in significant numbers according
to laboratory identification protocol.
Perform antimicrobial susceptibility tests according to the Antibiotic Reporting
Guideline and laboratory protocol.
Potential Pathogens in Urine Specimens:
Enterobacteriaceae such as E. coli, P. mirabilis. Klebsiella sp.
Pseudomonas aeruginosa
Staphylococcus aureus
Staphylococcus saprophyticus
Coagulase Negative Staphylococcus
-haemolytic Streptococci
Streptococcus pneumoniae
Page 16
Enterococcus spp.
Listeria monocytogenes
Yeast Candida species
These organisms are normally considered contaminants, but may be
significant upon repeat isolation:
Lactobacillus spp.
_-haemolytic Streptococci
Most Gram positive rods such as Diphtheroids
NOTE: ANY FEMALE WITH GBS BACTERIURIA IN ANY CONCENTRATION
DURING HER PREGNANCY SHOULD RECEIVE INTRAPARTUM ANTIBIOTICS.
(SOGC Clinical Practice Guideline No. 149, J. Obstet. Gynaecol. Can. 2004; 26:826832)
References:
Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller,
Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology, 2007.
Page 17
In-dwelling Catheter
Midstream Urine
1l loop
1 organism isolated
<107 cfu/L
No significant
growth**
107 cfu/L
ID & Sens
107cfu/L
2 probable pathogens
in equal amounts
ID & Sens both
ID & Sens
predominant org only
1 org >107 cfu/L
2nd org <107 cfu/L
>107 cfu/L
ID & Sens both
probable
pathogens
1 org >107 cfu/L
2nd org <107 cfu/L
2 organisms isolated
2 organisms with
one predominate
organism
>2 organisms
Bladder Urine
Suprapubic or
Cystoscopy
10 l loop
ID & Sens any
amount of growth
Page 18
References:
1.
2.
A Joint Accreditation Program of the College of Physicians & Surgeons of B.C. and the B.C.
Medical Association, Accreditation Protocol Microbiology, (Vancouver: Diagnostic Accreditation
Program), criteria 30, 34, page 7.
3.
Baron Ellen, Peterson Lance, Finegold Sydney, Bailey & Scotts Diagnostic Microbiology,
(Toronto: Mosby,1994), 252.
4.
5.
College of Physicians & Surgeons of Manitoba, (Winnipeg: Executive Assistant, April 1998).
6.
7.
Nicolle, Harding, Kennedy, McIntyre, Aoki, & Murray, Bacteriuria in Males, (Americian Society
for Microbiology, 1988), 1118.
8.
Shapiro Eugene, Infections of the Urinary Tract, (Yale University of Medicine, 1992), 165.
9.
Page 19
THROAT SWABS
Background:
Throat swab specimens are most commonly collected to diagnose infections caused by S.
pyogenes (Group A haemolytic streptococcus) and occasionally sequelae and should be
reported.
Throat swabs can also yield large (not pinpoint) colony-forming beta-hemolytic
streptococci harboring the Lancefield Group C or Group G antigen belonging to
Streptococcus dysgalactiae subsp. equisimilis.
They are associated with upper
respiratory tract infections similar to S. pyogenes, infections.
Cases of
glomerulonephritis and acute rheumatic fever have been reported. In work up of throat
swabs submitted for acute pharyngitis, Group C and Group G need to be reported.
Culture of any other suspected agents of disease such as: Arcanobacterium hemolyticum,
N. gonorrhoeae, C. diphtheriae shall be performed only at the request of the ordering
physician.
Specimen Collection:
A sterile swab is used to collect the throat swab from the posterior pharynx, tonsils and/or
inflamed areas. The swab is placed into the appropriate transport media, for C & S use
Modified Amies.
Storage Requirements: Place specimens at room temperature if to be processed within 2
hours, if processing to be delayed place into 4 C fridge.
Procedure\Media:
A. Processing of Specimens:
Culture: On the following media, and incubate plate as indicated:
Media
Blood Agar(BA) or selective media
such as Beta Strep Agar
Incubation
CO2 35C x 48 hours
AnO2 35C x 48 hours or /
AnO2 35C x 24 hours and then
CO2 35C x 24 hours
* Some institutions have better recovery incubating anaerobically.
Page 20
Commercial kits for Group A streptococcus (rapid direct tests) are also available. If a
laboratory implements the rapid direct test, two throat swabs should be collected. If the
rapid test is positive the second swab may be discarded; but if the rapid test is negative
the second swab should be cultured, because the sensitivity of these direct tests are
generally <100%.
B. Interpretation of Cultures:
The medium used to isolate S.pyogenes and other beta-hemolytic streptococci is sheep
blood agar with 5% blood, or selective agar containing agents that will suppress the
normal flora of the throat. Anaerobic incubation of the plates may enhance the betahemolysis, thereby making the colonies more easily detectable. If there are no colonies
demonstrating beta-hemolysis after 24 hours incubation, reincubate plates for another 24
hours. If beta-hemolysis is present, perform catalase and if negative, perform PYR &
latex agglutination testing.
C. Susceptibility Testing:
Antimicrobial susceptibility testing of these isolates is not required unless the patient is
known to be allergic to penicillin. Perform antimicrobial susceptibility tests according to
the Antibiotic Reporting Guideline and laboratory protocol.
Reporting Results:
Culture: Quantitate all significant isolates.
i.
ii.
iii.
References
1. Isenberg et al, eds. Clinical Microbiology Procedures Handbook, ASM Press
2. Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A.
Pfaller, Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology, 2007.
Page 21
CONJUNCTIVAL SWABS
Background:
Conjunctival swabs are collected for the diagnosis of conjunctivitis. Swabs should be
sent from both the infected and non-infected eye. This allows comparison of flora and
helps determine pathogenity of any organisms isolated.
Specimen Collection and Transport:
Swabs should be collected prior to the administration of drugs such as topical anesthetics
or antibiotics. The swab should be sent in Amies transport medium.
Procedure\Media:
A. Processing of Specimens:
a) Direct Examination of Gram stained
smear Note the presence of
polymorphonuclear cells and organisms. Quantitate as per Guideline for
Quantitative Interpretation of Gram Stains.
b) Culture on the following media, and incubate the plates as indicated.
Media
Blood Agar (BA)
ChocolateAgar (CHOC)
G. C. Selective Agar (neonates)
Incubation
CO , 35 C x 48 hours
CO2, 35oC x 48 hours
CO2, 35oC x 72 hours
2
Interpretation of Cultures:
Examine the BA and CHOC plates after 24 hours and 48 hours incubation and the G.C.
plate after 48 and 72 hours incubation. Compare growth from infected and non-infected
eye cultures. This comparison will often provide hints of which organisms are pathogens.
Potential pathogens: S aureus. H.influenzae, M, catarrhalis, N. gonorrhoeae, S.
pyogenes, S. pneumoniae, Moraxella species, and P. aeruginosa . For other organisms, a
significant result is determined by the isolation of a moderate or heavy growth of an
organism that is seen as the predominant organism on the Gram stain. There should be
>1+ polymorphonuclear cells on the Gram smear. Full identification is required to the
species level for all significant organisms.
B. Susceptibility Testing:
Perform antimicrobial susceptibility tests according to the Antibiotic Reporting
Page 22
References:
1. Cumitech BA. Sep 1994. ASM Press, Laboratory Diagnosis of Ocular Infections
2. Bailey and Scott's Diagnostic Microbiology, 11th Edition 2002. Mosby Inc.
Page 23
Incubation
CO2,
35C x 48 hours
O2/CO2, 35C x 48 hours
CO2,
35C x 48 hours
Using a sterile swab or loop enter the specimen container and obtain a purulent portion of
the specimen. Plant each plate with the specimen following Bacteriology Culture
Planting Procedures.
B. Interpretation of Cultures:
After 24 hours incubation the plates are examined and if needed, tests are performed to
determine the organism(s) present. Any plates with significant growth of a potential
pathogen must be quantified, identified, and have susceptibility testing done. Plates
showing only normal flora are then reincubated for another 24 hours and re-read the
Page 24
following day. If any changes are noted do the appropriate testing required to identify the
organism(s) and perform sensitivity testing.
C. Susceptibility Testing:
Perform antimicrobial susceptibility tests according to the Antibiotic Reporting Guideline
and laboratory protocol.
Reporting Results
a) Gram stained smear: If the specimen is suitable for culture, report the gram stain
smear result. Report with quantification the presence of polymorphonuclear cells,
squamous epithelial cells and organisms.
If the specimen is unsuitable for culture according to the Q score, report the gram
smear result and also comment: Gram smear indicates the sputum specimen is not
suitable for culture. Please resubmit. (refer to: Guideline for Quantitative
Interpretation of Gram Stains)
b) Culture: Quantify all significant pathogens and report with appropriate antimicrobial
susceptibilities. If only normal oral flora present, report with quantification:
"Growth of Oropharyngeal Flora.
Limitations Of The Procedure:
1) Poor quality specimen will not be cultured.
2) Procedure does not allow for isolation of other agents such as Legionella sp. Submit
urine specimen for antigen detection to the Saskatchewan Disease Control Laboratory
(SDCL). SDCL will culture Legionella sp. and/or bronchoalveolar lavage (BAL).
References:
Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller,
Manual of Clinical Microbiology, 9th ed., 2007: American Society of Microbiology
Page 25
EAR SWABS
Background:
Ear swabs are collected for the diagnosis of otitis externa. Otitis externa is a bacterial
infection of the external auditory canal usually caused by P. aeruginosa, S. aureus,
Group A streptococcus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella
catarrhalis or fungus/yeast. Tympanocentesis fluid is required for the diagnosis of otitis
media, which is cultured as a deep wound.
Specimen Collection and Transport:
The ear swab should be collected using sterile swab and sent in Amies transport medium.
Procedure\Media:
A. Processing of Specimens:
a. Direct examination: Gram stained smear note presence of polymorphonuclear
cells organisms. Quantitate as per Guideline for Quantitative Interpretation of
Gram Stains.
b. Culture on the following media, and incubate plate as indicated.
Media
Blood Agar(BA)
MacConkey Agar (MAC)
Fungal Plate (ie: Sabouraud Dextrose
Agar, Fungal Selective Agar, Phytone)
Chocolate Agar (CHOC)
Incubation
CO2, 35C x 48 hours
O2/CO2, 35C x 48 hours
O2, 30C x 48 hours
optional up to 4 weeks
CO2, 35C x 48 hours
B. Interpretation of Cultures:
Examine the culture plates after 24 and 48 hours incubation. Potential pathogens are S.
aureus, P. aeruginosa, Group A streptococcus, Haemophilus influenzae, Streptococcus
pneumoniae, Moraxella catarrhalis or yeast. For specimens from neonates only, identify
and report Gp. B. Strep. For other organisms, a significant result is determined by the
presence of a moderate to heavy growth of an organism which is seen as the predominant
organism on the Gram smear.
The Gram smear should also show 1+
polymorphonuclear cells. Fungal plates may be incubated at 30 for 4 weeks.
C. Susceptibility Testing:
Perform antimicrobial susceptibility tests according to the Antibiotic Reporting Guideline
and laboratory protocol.
Page 26
Reporting Results:
a) Gram Stained smear: Report with quantitation the presence of polymorphonuclear
cells and organisms.
b) Culture: Quantitate all significant isolates and report with appropriate antimicrobial
susceptibilities. If normal skin flora is also present, report as normal.
References:
1. Cumitech 10. Dec 1979. ASM. Press, Laboratory Diagnosis of Upper Respiratory
Tract Infection
2. Bailey and Scott's Diagnostic Microbiology, 11th Edition 2002. Mosby Inc.
3. see Antibiotic Reporting Guideline - www.quadrant.net/cpss
Page 27
Page 28
Genital child
Cervix
Urethral
Rectal
Throat
Combined
Vaginal/Anorectal for
Group B Streptococcus
Media
Not cultured: direct stain
Blood Agar (BA)
Sabourauds Agar with Chloramphenicol
(Phytone)
Other media (Choc, MML) added per
clinical information post partum, post
op, recurrent vaginitis
Blood agar
Sabouraud agar
Chocolate Agar (CHOC)
Modified Martin Lewis (MML)
Chocolate Agar (CHOC)
Modified Martin Lewis (MML)
Group B Streptococcus Broth
Blood Agar (BA) subculture from broth
Incubation
CO2, 35C x 48 hours
O2, 30C x 48 hours
B. Interpretation of Cultures:
Vaginal Cultures:
BA plates are examined at 24 and 48 hours: Growth of N. gonorrhoeae in any number is
significant. The significance of other organisms, is determined by pure or heavy growth
correlated with their presence as predominant organisms in the Gram smear. The Gram
smear should also show the presence of polymorphonuclear cells.
Cervical and Urethral Cultures:
CHOC and MML plates are examined at 48 hours and 72 hours for N. gonorrhoeae
colonies. Growth of N. gonorrhoeae in any number is significant.
Group B Streptococcus Screening:
The swab is inoculated into the GBS broth and incubated for 24 hours. Subcultures are
made from the broth to the BA plate and the plate incubated for 24 hours. The BA plates
are examined for strep-like colonies at 24 and 48 hours and the organism identified using
the laboratory protocol.
Page 29
C. Susceptibility Testing:
Susceptibility testing is not performed for N. gonorrhoeae, Group B Streptococcus and
Yeast. For other organisms, follow the antimicrobial susceptibility tests according to the
Antibiotic Reporting Guideline and laboratory protocol, where appropriate.
All isolates of N. gonorrhoeae are referred to Saskatchewan Disease Control Laboratory
for susceptibility testing.
Isolates of Group B. Streptococcus from patients with a history of penicillin allergy will
need to be tested for susceptibility to erythromycin.
Reporting Results:
a.
Gram stained smear report with quantitation of polymorphonuclear cells and
organisms.
b.
Culture Report pathogens and normal flora with quantitation. Report
susceptibilities where appropriate.
c.
All isolates of N. gonorrhoeae must be confirmed by two methods.
References:
1. Bailey and Scott Diagnostic Microbiology, 11th Ed., Mosby Inc.
2. Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A.
Pfaller, Manual of Clinical Microbiology, 9th ed., 2007: American Society of Microbiology
Page 30
Page 31
categories is of less value, if the microscopic picture is consistent with BV, but the
specimen specified as having been collected from a prepubescent individual, further
investigation is warranted and the results should not be ignored and be discussed with
the physician.
NOTE: The Nugent system was validated using smears made with a modified Gram
Stain4,5,6. This modified staining method recommended by Spiegel, utilizes a basic fuchsin
counterstain to minimize over-decolorization of gram-positive bacteria and to enhance
detection of gram-negative bacteria. It is acknowledged however, that the use of this
modified Gram Stain is not common practice in Canada.
NOTE: The wording of the score interpretations was suggested by Nugent and
subsequently endorsed.
Nugent Scoring System:
Gram-stained slides are examined under oil immersion (1000). Smears are observed
and quantitated for the presence of the following morphotypes:
Large gram-positive bacilli (Lactobacillus morphotypes)
Small gram-variable bacilli (Gardnerella morphotypes)
Curved gram-negative or gram-variable bacilli (Mobiluncus morphotypes)
The number of organisms seen is quantitated according to the following scale:
1 + = <1 organism per field
2 + = 14 organisms per field
3 + = 530 organisms per field
4 + = >30 organisms per field
A total numerical score is calculated by summing the scores for the three components
as indicated in the following table:
Lactobacillus
4+
Score
0
Gardnerella
4+
Score
4
Mobiluncus
4+
Score
2
3+
3+
3+
2+
2+
2+
1+
1+
1+
Page 32
= TOTAL SCORE
46
Gram stain reveals altered vaginal flora that is not consistent with
bacterial vaginosis. This frequently represents a transitional stage. If signs
and/or symptoms persist, repeat testing is warranted
7 10 Gram stain consistent with bacterial vaginosis
References:
1.
2.
3.
4.
5.
6.
Gardner HL, Dukes CD. Haemophilus vaginalis vaginitis. A newly defined specific infection
previously classified "nonspecific" vaginitis. Am J Obstet Gynecol 1955;69:962-976.
Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis:
diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983;74(1):14-22.
Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a
standardized method of gram stain interpretation. J Clin Microbiol 1991;29(2):297-301.
Spiegel CA, Amsel R, Holmes KK. Diagnosis of bacterial vaginosis by direct gram stain of
vaginal fluid. J Clin Microbiol 1983;18(1):170-7.
Spiegel CA. Bacterial vaginosis. Clin Microbiol Rev 1991;4(4):485-502.
Spiegel CA. Bacterial vaginosis: changes in laboratory practice. Clin Microbiol Newsletter
1999;21:33-37.
Page 33
STOOL CULTURES
Background:
Enteric pathogens include Salmonella and Shigella sp., Campylobacter, E. coli 0157:H7,
Yersinia enterocolitica, Clostridium difficile, Aeromonas and Plesiomonas. Vibrio
(cholerae and parahemolyticus) are an important cause of diarrhea in other parts of the
world. S. aureus and Candida albicans may be found in pure cultures from patients on
broad-spectrum antibiotic therapy.
In adult patients who develop diarrhea 3 days after hospitalization,
C. difficile is the most common pathogen. Stools from these patients are tested for the
presence of C. difficile toxin only.
Specimens from outbreaks, as defined by Public Health must be sent to Saskatchewan
Disease Control Laboratory with the outbreak number.
When investigating patients suspected of being in a carrier state refer to the
Saskatchewan Disease Control Laboratory.
Specimen Collection and Transport:
Freshly passed stool specimens should be transported to the laboratory within 30 minutes
of collection, and processed in 2 hours. If there is a delay in transport and/or processing,
the specimen should be transferred into a transport container with Cary-Blair medium.
Specimens should be up to the fill line in the transport container. Rectal swabs may be
adequate for the detection of pathogens in acute infections, but are NOT the preferred
specimens. Ensure that rectal swabs are for C & S, and not for isolation of N.
gonorrhoeae. Submit one specimen per stool sample, to a maximum of 2 stool samples,
ideally on separate days.
Stools for C. difficile toxin testing should be sent in a sterile container without transport
medium.
Unacceptable Specimens:
1. Specimens in containers with external contamination.
2. Specimens with contaminants such as urine or tissue paper.
3. Specimens not correctly identified will be rejected if the discrepancy cannot be
rectified.
4. Stool in SAF.
In all cases the ward or physician will be notified and a repeat specimen requested.
Page 34
Procedure/Media:
A. Processing of Specimens:
Culture on the following media, and incubate the plates as indicated.
Media
MacConkey Agar (MAC)
Xylose Lysine Deoxycholate (XLD),
Hektoen, Salmonella-Shigella (SS), or
Decoxycholate Agar (DCA) - selective for
Salmonella
Sorbitol MacConkey (SMA)
Campylobacter Agar
When requested or clinically indicated culture to:
Media
Blood Agar (BA) or Aeromonas selective
Thiosulfate Citrate Bile Salts Sucrose (TCBS)
- selective for Vibrio
Yersinia Agar (CIN)
Incubation
O2, 35C x 24 hours
O2, 35C x 24 hours
Suspicious colonies of enteric pathogens should be subcultured and identified using the
laboratory identification system. i.e. conventional or automated methods as applicable.
Reporting Results:
Isolation of Salmonella, Shigella, Campylobacter, E. coli 0157:H7, Yersinia
enterocolitica, Plesiomonas, Aeromonas, and Vibrio are considered significant. A
pathogen specific negative report should be provided for each pathogen ruled out.
Antibiotic susceptibility testing is not done on E. coli 0157:H7 since its treatment is
associated with an increased risk of HUS and it is a self-limited disease. It is also not
done on Campylobacter and Yersinia sp, Aeromonas, Plesiomonas and Vibrio sp. For
Salmonella sp, the illness is also self-limited and the antibiogram is done and released
only in specific circumstances (elderly, immunosuppressed, infant (<2 years), severe
disease, presence of S. typhi).
If there is a concomitant isolate from a blood culture, antimicrobial susceptibilities should
be reported on the blood isolate.
* see Antibiotic Guidelines for more information
Page 35
References:
1. Bailey and Scotts Diagnostic Microbiology, 11th Edition., Mosby Inc.
2. Cumitech 12A Diagnosis of Bacterial Diarrhoea. 1992. ASM Press.
Page 36
Page 37
Page 38
Page 39
No
Are any of
them
listed?*
>3
How many
potential
pathogens?
Yes
<3
Minimal identification
and sensitivity screen
Is it a good
quality
specimen?
No
Yes
Full Work-up
ID and SENS as
appropriate
Report
Yes
Workup
required?
No
End
Potential Pathogens:
S. aureus
Beta haemolytic streptococci
Pasteurella sp.
Capnocytophaga sp. (animal bites)
Eikenella sp. (human bites)
Enterobacteriaceae,
P. aeruginosa
anaerobes
*Listed Organisms:
S. aureus - rule out MRSA
beta-hemolytic streptococci (groups A,
B, C, F, and G)
Yeast only when isolated in moderate to
abundant amounts (consult with MOC
or supervisor before reporting)
Pseudomonas species
Enterococcus species rule out VRE
Bacteroides fragilis group
Actinomyces israelii
Clostridium perfringens - if predominant
Page 40
C. Susceptibility Testing:
Perform antimicrobial susceptibility tests according to the Antibiotic Reporting Guideline
and laboratory protocol.
Reporting Results:
Gram stained smear: Report with quantitation the presence of polymorphonuclear cells,
squamous epithelial cells and organisms.
Culture:
a. Negative report : No growth , Mixed aerobic flora, Mixed anaerobic flora, Mixed
aerobic and anaerobic flora Including Listed Organisms or Type of flora,
depending on site
b. Positive report: Report potential pathogens and sensitivities (as determined by
organism). Quantitate. Single isolates of anaerobes may require susceptibility
testing.
c. If organisms seen in the direct smear do not appear in culture, and anaerobic
specimen was not submitted, it might suggest that the organisms seen failed to
grow indicating the presence of fastidious or anaerobic organisms. It could also
indicate that the gram stain was interpreted incorrectly. It is helpful to re-check
the direct smear. If the gram stain is correct, the comment Organism(s) seen in
direct smear did not grow on aerobic culture, suggesting the presence of anaerobic
or other fastidious organisms or previous antibiotic therapy.
References:
1. Isenberg, H. 1992. Clinical Microbiology Procedures Handbook.
Microbiology. Washington DC.
Page 41
Attach Q-values to squamous cell and neutrophil quantities. Add the two Q-values
together. Specimens with positive Q-values are more likely to contain increased numbers
of potential pathogens and decreased numbers of potential contaminants. Specimens with
negative Q-values (or a Q-value of zero) are likely to be contaminated with the local
flora. Specimens with no squamous cells or neutrophils are scored as 1, allowing
neutropenic patients or those with necrotic or serous secretions to be processed as
acceptable.
NOTE: The first row of numbers in the table is the Q-value for squamous epithelial cells
only. The following four rows are the Q-values for the sum of the neutrophils and
squamous epithelial cells.
Page 42
CO2
O2/CO2
CO2
CO2
Incubation
35C x 48 hrs
35C x 48 hrs
35C x 48 hrs
35C x 48 hrs
B. Interpretation of Cultures:
Examine the plates after 24 and 48 hrs incubation. Potential pathogens include S. aureus,
Beta hemolytic Streptococcus species, P. aeruginosa. A heavy, pure growth of other
organisms that is consistent with the predominant organism seen in the direct smear is
significant if there are 1+ polymorphonuclear cells seen.
C. Susceptibility Testing:
Perform antimicrobial susceptibility tests according to the Antibiotic Reporting Guideline
and laboratory protocol.
Reporting Results:
A. Gram stained smear: Report with quantitation the presence of polymorphonuclear
cells, squamous epithelial cells and organisms.
If Q-score has a negative value, add a report comment such as: Microscopy of
Gram-stained smear indicates potential contamination with skin flora.
B. Culture:
a. Negative report: No growth or other normal type of flora dependent on site
as per laboratory protocol.
b. Positive report: Quantitate all significant isolates and report with appropriate
antimicrobial susceptibilities. If normal skin flora also present, report with
quantitation. Or, if other normal flora, report with quantitation.
References:
1. Isenberg, H. 1992. Clinical Microbiology Procedures Handbook, American Society for
Microbiology. Washington DC.
2. Miller, J.M. 1996. A Guide to Specimen Management in Clinical Microbiology, American
Society for Microbiology. Washington DC.
Page 43
3. Murray, P.R. 1996. ASM Pocket Guide to Clinical Microbiology, American Society for
Microbiology. Washington DC.
4. Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A.
Pfaller, Manual of Clinical Microbiology, 9th ed., 2007: American Society of Microbiology;
Washington, DC.
Page 44
Adult
Routine
or
ANY adult on
antibiotics
Adult
Endocarditis
FUO* on
antibiotics
Neonates
Children < 4 yrs.
Site 1:
1 aerobic
1 anaerobic
Volume/Bottle
10 ml
7 ml
Site 2:
1 aerobic
Site 1:
1 aerobic
1 anaerobic
10 ml
Site 2:
1 aerobic
Site 1:
1 pediatric
10 ml
Site 2:
1 pediatric
1 aerobic
0.5-3 ml
10 ml
7 ml
# of Sets
0.5-3 ml
Timing
No time required between
draws from site 1 and site
2
1 hour**
The first set of three
bottles is drawn as above,
and an hour later, the
second set of three bottles
is drawn
NOTE: * FUO Fever of Unknown Origin (ie. 3 weeks of fever above 38C)
** In severe cases antibiotics may need to be started immediately after collection
of the first set.
Page 45
Collect a maximum of 30 mls for adults, maximum of 5 mls for children, and any
obtainable amount for infants.
Unacceptable Specimens:
1. Bottles with external contamination.
2. Specimens incorrectly identified. In these cases the ward or physician must be
notified immediately and a repeat specimen requested.
Incubation:
For patients presenting with an unknown fever and if endocarditis is suspected, up to 3
weeks incubation may be required.
Materials, Equipment and Reagents: As per your laboratory procedure.
Procedure:
A.
Collection of Specimen
1. Remove flip caps from the blood culture vials and clean vial tops with an
alcohol swab.
2. Select the vein and prepare the arm using a double application of 70% alcohol
starting at the centre of the site and swabbing concentrically for 1 minute.
3. Allow the venipuncture site to dry.
Page 46
Subculture:
Media
Blood Agar (BA)
ChocolateAgar (CHOC)
MacConkey Agar (MAC)
Anaerobic media eg. Brucella
Incubation
CO , 35 C x 48 hours
CO2, 35oC x 48 hours
CO2, 35oC x 48 hours
AnO2, 35oC x 72 hours
2
Identify isolates and perform susceptibility testing as per your laboratory protocol.
Reporting:
Negative No growth after 5 days incubation, or as per manufacturers instructions.
Positive Report organism with corresponding antimicrobial susceptibility.
NOTE: Organisms that are normal skin commensals such as coagulase negative
staphylococci, may be contaminants. The isolation of this type of organism in one
bottle/culture only may indicate contamination. This will need to be resolved by
discussion with the clinician. Reporting of contaminants may result in the inappropriate
use of antibiotics.
References:
1. CUMITECH 1C Blood Cultures 111 (2005)
2. Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A.
Pfaller, Manual of Clinical Microbiology, 9th ed., 2007: American Society of Microbiology
Page 47
Record volume and gross appearance of CSF, i.e., clear, bloody, cloudy,
xanthochromic. If the specimen is cloudy, a gram stain should be done
before the centrifugation.
2. Centrifuge the CSF specimen for Gram stain.
3. Centrifuge the specimen:
a. If the volume of fluid is >1 ml, centrifuge for 15 min. at 2500-3000 rpm
(1500 x g). The higher speed is preferred to sediment the bacteria.
b. Remove supernatent and vortex the sediment vigorously for at least 30
secs. This step is critical. Do not use a pipette to mix the sediment,
because the bacteria and cells may adhere to the sides of the tube and
cause false-negative findings.
c. If 1 ml is received, do not centrifuge but vortex the specimen.
4. Using a sterile pipette, inoculate the media and streak for isolated colonies. In
addition, add a single drop of fluid to the 4th quadrant of the plate and allow to
air dry. Do not streak out this drop of fluid.
Page 48
Incubation
CO2 35C x 72 hrs
CO2 35 C x 72 hrs
O2/CO2 35C x 72 hrs
CO2 35C x 72 hrs
B. Interpretation of Cultures:
a. CSF specimens: Examine the BA, MAC, CHOC and BRUC plates daily for 72
hours. In shunt infections, examine the THIO broth media daily for 7 days.
b. Sterile Body Fluids: Examine the BA, MAC, CHOC and BRUC plates daily for 48
hours. Anaerobic jars - examine at 48 hours and 72-96 hours before discarding.
Broth can be examined daily.
Page 49
c. Potential Pathogens include the organisms listed in the table below. Identify and
provide antimicrobials susceptibility on all microorganisms, regardless of the
amount isolated. Correlate with the results of the direct Gram smear.
CSF - Common organisms causing acute bacterial meningitis:
Age or condition
Organism(s)
Neonate < 30 d
Escherichia coli, Group B Streptococcus, Listeria monocytogenes,
Gram-negative bacilli, S. aureus
1-3 months
Gr. B Streptococcus, E.coli, Streptococcus pneumoniae, Neisseria
meningitidis, Haemophilus influenzae, L. monocytogenes
3 mo-18 yrs
Streptococcus pneumoniae, Neisseria meningitides, Haemophilus
influenzae
Young adult
Neisseria meningitidis
Adult
S. pneumoniae, N. meningitidis
Elderly
S. pneumoniae, N. meningitidis, Gram-negative bacilli, L.
monocytogenes
AIDS patient
Cryptococcus neoformans (fungi)
CNS Shunts
Coagulase-negative Staphylococci, Corynebacterium,
Propionibacterium, Gram-negative bacilli
NOTE: For CSF and Sterile Body Fluids Potential Pathogens include all isolates,
especially in immunocompromised patients.
C. Susceptibility Testing
As per laboratory protocol. For CSF follow the Antibiotic Reporting Guideline from
Microbiology Quality Asurance Committee.
Reporting Results:
A. Gram stained smear: Report with quantitation the presence of polymorphonuclear
cells, epithelial cells and organisms. Phone the results to the ward/doctor.
B. Culture: Report all isolates with quantitation and with the appropriate antimicrobial
susceptibilities. Note: subcultures from broths cannot be quantitated.
NOTE: A positive culture is a critical value, and must be phoned to the doctor STAT.
Procedure notes for sterile body fluids:
Although in the past the use of blood bottles for fluid collection has not been
recommended, recent studies suggest that the large sample volume that can be cultured
may improve the recovery of low numbers of organisms in fluids such as ascites. As
with any broth system, however, the fastest growing organism is often the only one
Page 50
isolated, jeopardizing the recovery of slow growers. When a broth is used, no direct
smear information is available, and, therefore, no assessment of the initial distribution of
organisms or inflammatory cells can be made. A smear can be prepared, however, at the
time of specimen procurement and submitted with the broth medium. Also, unless a
biphasic system is available, a broth culture delays by at least 24 h the preliminary
workup of organisms possible only from growth on solid media.
Limitations of the Procedure:
This procedure is not suitable for optimal recovery of all pathogenic microorganisms
found in sterile body fluids, including fungi, mycobacteria, parasites, or viruses.
Culture Negative CSF specimens from patients who have received antibiotics may be
sent to Saskatchewan Disease Control Laboratory for PCR, upon request of the MHO.
References:
1. Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A.
Pfaller, Manual of Clinical Microbiology, 9th ed., 2007: American Society of Microbiology
2. Meredith F.T., Phillips H.K, and L. B. Reller, Clinical Utility of Broth Cutures of Cerebral
Spinal Fluid from Patients at Risk for Shunt Infections, 1997, JCM, 35:3109-3111
3. Dunbar, S.A., Eason R.A., Musher D.M., & J.E. Clarridge, Microscopic Examination and
Broth Culture of cerebral Spinal Fluid in Diagnosis of Meningitis, 1998 JCM, 36:1617
1620.
4. Sturgis C.D., Petersen L.R. & J.R. Warren, Cerebral Fluid Broth Culture Isolates: Their
Significance to Antibiotic Treatment, Am J Clin Pathol. 1997, 108(2): 217-221.
5. Performance Standards for Antimicrobial Susceptibility Testing; M100-S11 Vol. 21. No.1.
CLSI.
Page 51
Organism
Site
Staphylococcus species
Wound/Sputum
Blood
CSF
Urine
Eye/ear3
Blood
CSF
Sputum
Sterile sites4
Penicillin, Oxacillin
Penicillin, Oxacillin
Penicillin, Oxacillin, Cefazolin, Nitrofurantoin
Penicillin, Erythromycin, SXT
Wound
Blood
Urine
Wound
Sputum
Blood
CSF5
Sterile sites
Throat
Vaginal/Rectal
Ampicillin
Ampicillin, Gentamicin synergy12, Streptomycin synergy12
Ampicillin, Fluoroquinolone2, Nitrofurantoin
Urine
Wound/Sputum
Streptococcus pneumoniae
Enterobacteriaceae
Blood
Urine
Pseudomonas aeruginosa
Shigella species
Salmonella and Shigella species
Haemophilus species
Stenotrophomonas maltophilia
CSF
All sites
Stool
Systemic
Sputum
Eye/Ear3
Blood
CSF
CSF/Sterile
sites
Page 52
Penicillin
Penicillin, Erythromycin, SXT, Fluoroquinolone2
Penicillin, 3rd Gen Cephalosporin
Alternate
SXT1, Clindamycin9,
Vancomycin
Vancomycin, Clindamycin9
Vancomycin
SXT, Fluoroquinolone2
Cefuroxime, Clindamycin
Cefotaxime, Vancomycin
Cefotaxime8, Vancomycin8
Cefuroxime, Clindamycin
Vancomycin
Vancomycin
Vancomycin
Clindamycin7,9, Erythromycin7,
Vancomycin
Cefotaxime5, Meropenem,
Piperacillin, Tobramycin, PipTazobactam
Ceftazidime, Tobramycin,
Meropenem, Pip-Tazobactam
Cefixime if resistant to SXT
Cefotaxime
Fluoroquinolone2,
Clarithromycin
Meropenem
NOTES:
Alternate antibiotics should be reported, only if the organism is resistant to two or
more first choice antibiotics, if the patient cannot tolerate those antibiotics or if the
patient is already on the antibiotic of choice.
For Entercoccus species, cephalosporins, aminoglycosides, clindamycin and
trimethoprim/sulfamethoxazole may appear susceptible in vitro but are not effective
clinically and should not be reported.
Oxacillin agar screen plate should be used for detection of Methicillin Resistant
Staphylococcus aureus (MRSA)
Vancomycin agar screen plate should be used for detection of Vancomycin Resistant
Enterococcus (VRE)
All enteric pathogens other than Shigella and Salmonella typhi: do not report
susceptibility, unless the patient is less than two years old, or is immunosuppressed.
For Salmonella and Shigella species, first and second generation cephalosporins may
appear susceptible in vitro but are not effective clinically and should not be reported.
-lactamase testing should be done on all isolates of: (1) Penicillin susceptible Staph.
aureus; (2) Haemophilus sp.; (3) N. gonorrhoeae; (4) M. catarrhalis; (5)
Enterococcus sp.isolated from sterile body sites; (6) anaerobic gram negative bacilli
(B. fragilis group).
1
Page 53
CLSI recommends that all CSF isolates of S. pneumoniae should immediately be tested
by an MIC method for susceptibility for penicillin, 3rd gen Ceph and Vancomycin. Only
report 3rd gen Ceph and Vancomycin when penicillin is resistant.
9
Strains of Staphylococci and Streptococcus that are susceptible to Clindamycin, but
resistant to Erythromycin should be tested for inducible Clindamycin resistance using the
D-zone test. Erythromycin should be included in testing for this purpose, even if not
reported.
10
Resistance of Salmonella species to Nalidixic Acid may be used to predict the potential
for resistance to Fluoroquinolones.
11
Highly inherently resistant organism. SXT in high doses plus other antibiotics (if
susceptible) used.
12
Use of high-level aminoglycoside screening to predict synergy.
There are several reasons for prescribing two antimicrobial drugs together and one is to
achieve a synergistic effect. Bacterial synergy is seen between two drugs, which
individually are incompletely bactericidal, although acting together they completely kill
the inoculum. Enterococci are inherently resistant to aminoglycosides. However, strains
of enterococci with medium-level resistance (MIC < 2,000 g/ml) show synergistic
killing by a combination of penicillin/ampicillin and gentamicin. For enterococcal
infections of sterile sites such as blood the recommended therapy is a combination of
beta-lactam (e.g., ampicillin) or vancomycin combined with an aminoglycoside
(gentamicin or streptomycin). In the JCM 25:2443 - 2444 (1987)
Zervos et al.
demonstrated that growth in a single broth microdilution well containing gentamicin at a
concentration of 500g/ml predicted high level resistance to gentamicin (MIC > 2,000
g/ml).
Antimicrobial testing panels use a single concentration high-level
aminoglycoside as a synergy screen.
The ATB ENTEROC5 susceptibility test strip tests for high-level
gentamicin and
streptomycin sensitivity. The MicroScan choice is also based on the observation that in
the presence of high-level gentamicin resistance many isolates can be sensitive to
streptomycin. High-level gentamicin resistance indicates that there is resistance to
synergy to gentamicin, tobramycin, sisomicin and netilmicin, due to the presence of the
common bifunctional 6'-N-acetyltransferase and 2"-O-phosphotransferase enzyme. This
enzyme does not affect streptomycin. Thus streptomycin has its own screen.
From the January 2005, Performance Standards for Antimicrobial Susceptibility
Testing from CLSI (formerly CLSI) document M100 S15; Table 2D recommends the
following parameters for screening for high-level resistance
(HLR) to aminoglycosides among enterococci: ( i) agar dilution concentrations
gentamicin 500 g/ml and streptomycin 2000 gm/ml; endpoint any growth greater than
1 colony, (ii) broth microdilution, gentamicin 500 g/ml and streptomycin 1000 g/ml;
endpoint - any growth; (iii) disk diffusion concentrations gentamicin 120 gm disk and
streptomycin 300 gm/disk; endpoint 5mm = HLR, 7-9 mm inconclusive (use agar
dilution method), > 10 mm sensitive. In summary, enterococci isolated from sterile sites
should have antimicrobial susceptibility testing for ampicillin MIC, high-level screen for
gentamicin and streptomycin, and vancomycin screen.
Page 54
EXCEPTIONS
Any organism that does not present the typical antibiotic pattern for that organism should
have repeat testing (identification and susceptibility) performed +/or sent to reference
laboratory (ie. SDCL).
Page 55
LIMITATIONS
The following organisms do not have CLSI standards for susceptibility and interpretation.
If testing is performed, an MIC method should be used, and all interpretations must be
reported as "non-standardized".
These guidelines are for the use of various antibiotics in treatment of infections caused by
these organisms.
Pasteurella multocida
Penicillin remains the drug of choice. Ampicillin, SXT, Cefuroxime, Amoxicillinclavulanic Acid, Tetracycline and Fluoroquinolones also demonstrate good in vitro
activity.
For Pasteurella species Oxacillin, Macrolides, Aminoglycosides and 1st gen
Cephalosporins are not effective and should not be reported.
Listeria monocytogenes
Ampicillin, or Amoxicillin combined with an Aminoglycoside is the primary choice for
treatment. Co-trimoxazole is the alternative drug. Resistant to Cephalosporins
Moraxella catarrhalis
Most M. catarrhalis strains are beta-lactamase positive. Ampicillin, amoxicillin,
penicillin, and 1st generation cephalosporin therapy is not suggested. Recommended
therapy: generally susceptible to macrolides, SXT, cefuroxime, ceftriaxone, quinolones,
and amoxicillin/clavulanic acid. Flouroquinolones are not recommended in pregnancy or
individuals <17 years.
Bacillus sp.
Corynebacterium sp.
Eikenella sp.
Micrococcus sp.
Page 56
Buttiauxella species.
Cedecea species.
Citrobacter amalonaticus
Citrobacter freundii
Citrobacter diversus (C. koseri)
Edwardsiella tarda
Enterobacter cloacae
Enterobacter aerogenes.
Many other Enterobacter species.
Escherichia hermannii
Ewingella americana
Hafnia alvei
Klebsiella pneumoniae
Kluyvera ascorbata
Kluyvera cryocrescens
Proteus mirabilis
Proteus vulgaris
Cephalothin
Polymyxins, ampicillin, cephalothin
Ampicillin
Cephalothin
Cephalothin, carbenicillin
Colistin
Cephalothin
Cephalothin
Cephalothin
Ampicillin, carbenicillin
Cephalothin
Cephalothin
Ampicillin, carbenicillin
Ampicillin
Ampicillin
Polymyxins, tetracycline, nitrofurantoin
Polymyxins, ampicillin, nitrofurantoin,
tetracycline
Polymyxins, ampicillin, cephalothin
Polymyxins, cephalothin, nitrofurantoin,
tetracycline
Polymyxins, nitrofurantoin
Polymyxins, cephalothin, nitrofurantoin
Ampicillin, carbenicillin, cephalothin
Polymyxins,c cephalothin
Morganella morganii
Providencia rettgeri
Other Providenciaa species.
Serratia marcescensb
Serratia fonticola
Other Serratia species.
a
Most strains of Providencia stuartii are also resistant to cephalothin and tetracycline.
Serratia marcescens can also be resistant to ampicillin, carbenicillin, streptomycin, and
tetracycline.
c
Most Serratia species are resistance to polymyxins, but some strains have unusual
zones of inhibition, 10 to 12 mm or larger , even though they are resistant when tested
by other methods.
b
Page 57
Definition:
CLSI or International Federation of Clinical
Chemistry (IFCC)
1. Imprecision
Within run
Between run
2. Patient correlation
3. Linearity
4. Reference range
validation
5. Accuracy
Page 58
Applicability:
Qualitative
Quantitative
_
n=20
n=40
6. Sensitivity
7. Specimen Stability
8. Interference
9. Organism
Identification
10. Antibiotic
sensitivity
Quantitative:
Microbiology Procedures/Guidelines 2010 Edition
Page 59
_
semi-quantitative only
_
semi-quantitative only
Work-up requirements when an instrument is moved from site A to site B: (it is assumed that the instrument has been in recent
use with acceptable performance).
Work-up Element:
1. Imprecision studies, QC only
2. Patient correlation
Qualitative or semi-qualitative:
If site B has no previous experience
Work-up Element:
1. Organism Identification
2. Antibiotic susceptibility
Page 60