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Susceptibility testing of fastidious and unusual pathogens
A N N L D O zt(6)47-56, 1987
ISSN 0738-1751
VOLUME 4, NUMBER 6, JUNE 1987
EDITORIAL BOARD Editor
Associate Editors
DANIEL AMSTERDAM, PhD,
RONALD N. JONES, MD,
CLYDE THORNSBERRY, PhD,
State University of New York at Buffalo and Erie County Medical Center Buffalo, New York
Clinical Microbiology Institute Tualatin, Oregon
Center for Infectious Diseases Centers for Disease Control Atlanta, Georgia
HAROLD C. NEU, MD,
LOWELL S. YOUNG, MD,
College of Physicians and Surgeons, Columbia University New York, New York
Kuzell Institute for Arthritis and Infectious Diseases Medical Research Institute ef San Francisco Pacific Presbyterian Medical Center San Francisco, California
SUSCEPTIBILITY T E S T I N G OF F A S T I D I O U S AND UNUSUAL PATHOGENS EDITOR'S N O T E
47
D. AMST E RDAM
Susceptibility Testing of Fastidious and Unusual Pathogens C. J. C. L. S. B.
47
THORNSBERRY M. SWE NSON N. BAKER K. MCDOUGAL A. STOCKER C. HILL
EDITOR'S NOTE In this A M N publication, the staff at the Antimicrobics Investigations Branch, headed by Dr. Clyde Thornsberry, pool their experience and knowledge to present their recommendations for antimicrobic susceptibility evaluations of 25 groups and bacterial species for which standards have not yet been completely developed or there is ongoing modification and --Anaerobes --MRSA --H. influenzae --Mycobacteria --Nonenterococcal Streptococci --Branhamella
ELSEVIER
CLYDE T H O R N S B E R R Y J A N A M. S W E N S O N C A R O L Y N N. BAKER L I N D A K. M c D O U G A L S H E I L A A. STOCKER, a n d B E R T H A C. HILL Antimicrobics Investigations Branch, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia
reinterpretation of standards. For several species, notably Mycobacterium, Branhamella, and Neisseria, and for the anaerobes, NCCLS recommendations are currently being evaluated and may soon be available. In recent issues, The A M N has presented problems associated with the testing and interpretation of some of these fastidious or unusual isolates. The reader may wish to refer to the following: The AMN, The AMN, The AMN, The AMN, The AMN, The AMN, The AMN,
Vol. Vol. Vol. Vol. Vol. Vol. Vol.
2, 2, 2, 2, 3, 3, 3,
No. No. No. No. No. No. No.
6, 1985 7, 1985 8, 1985 12, 1985 8, 1986 2, 1986 12, 1986
Some clinically significant bacteria have characteristics that preclude their being tested by the standardized disk diffusion method. 3's'15'19 They may grow too slowly, require special nutrients, require special atmospheres, or simply may not have been tested adequately to show that they can be tested accurately and reproducibly by the standard NCCLS method. ~'~9 We have called these bacteria "fastidious" and/or "unusual" isolates. More specifically, the "fastidious" organism will not grow on Mueller-Hinton medium without supplementation. The "unusual" organism however, may grow well on Mueller-Hinton medium but studies have not been done to show that it can be reliably tested by standard methods. The "unusual" organism may also present special problems in testing its susceptibility to antimicrobial agents. In previous years, many of these organisms did not require susceptibility tests because they were known to be universally susceptible to an appropriate antimicrobial agent(s). Resistant strains have emerged, however. Resistance to 13-1actams is most often due to the production of ~lactamase which may be constitu-
o73s-
2.2°
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THE A N T I M I C R O B I C NEWSLETTER, V O L U M E 4, N U M B E R 6, JUNE 1987
tive or inducible and m a y be mediated by either c h r o m o s o m a l or plasmid genes. 2''22 In some of these strains, such as Haemophilus influenzae and Neisseria gonorrhoeae, most, but not all, of the penicillin and ampicillin resistance has been caused by the acquisition of plasraids that mediate constitutive 13lactamase p r o d u c t i o n by the organism, 6'7 but in others, such as Streptococcus pneumoniae, penicillin resistance has not been due to 13lactamase or a plasmid but is chromosomally mediated and is due to alteration of penicillin-binding proteins,
33
These d e v e l o p m e n t s have dictated that, for infections caused by some of these "fastidious" or " u n usual" organisms, the clinician has had to realize that some previously e m p l o y e d empirical t h e r a p y might not be adequate, and the clinical microbiologist has had to begin testing the antimicrobial susceptibility of organisms that were previously not tested. Antimicrobial susceptibility tests should be p e r f o r m e d only on microorganisms that are etiologic
agents of the infection in question. W h e n it has been d e t e r m i n e d that an organism is involved in the infection, the next step is to determine if a susceptibility test is necessary. For some organisms, such as G r o u p A streptococci, susceptibility tests are not necessary because these organisms are still universally susceptibile to penicillin, the drug of choice. H o w e v e r , if the organism is S. pneumoniae, and especially if it was isolated from the cerebrospinal fluid, a test to determine the susceptibility to penicillin is indicated because some of these bacteria are either relatively resistant or resistant to penicillin. If it is d e e m e d necessary to perform susceptibility tests on a clinical isolate for which a m e t h o d has not been described, it is usually best to determine a minimal inhibitory concentration (MIC) using the general MIC m e t h o d described in NCCLS Standard M7-A.'7 For testing some organisms, modification of the basic m e t h o d s is not necessary, but for others, additional nutrients, different
atmospheres, or other changes m a y be necessary. If supplementation of the m e d i u m is required, only essential nutrients should be added, and the a t m o s p h e r e and t e m p e r a t u r e of incubation should not be changed unless necessary. But if, for example, the organism requires blood and will not grow at t e m p e r a t u r e s higher than 25°C, then the MIC should be determined u n d e r these conditions. H o w e v e r , the clinician should be informed that the MIC could not be d e t e r m i n e d by a standardized m e t h o d and the results, therefore, may be the best approximation obtainable. Following are some antimicrobial susceptibility testing m e t h o d s that can be used for some of these fastidious or unusual organisms. It should be pointed out, however, that these m e t h o d s are, for the most part, tests that we have used in our laboratory and are not meant to be standard methods. For some of the organisms, intensive s t u d y of m e t h o d o l o g y is n o w ongoing and standard r e c o m m e n dations m a y soon be available.
TABLE 1. Susceptibility Testing of Fastidious and Unusual Pathogens""
Organism Anaerobes
Method Broth microdilution using reduced plates and water blanks for inoculum d i l u t i o n '~,3"
NCCLS reference agar dilution '"
Media~
Incubation
Schaedler broth + 0.1 ixg/mL vitamin K and 10 ixg/mL hemin 35°C, 48 h in 85% N2, 5% CO2, 10% H2, Wi|kinsor the Gas Pak, Chalgren agar or equivalent
Comments May also use other broths shown to support growth of anaerobes, eg, supplemented BHI. Commercial microdilution tests now available. Microdilution MICs for cephalosporins tend to be lower than MICs by the standard reference agar dilution method.
Wilkins-Chalgren agar may not support growth of all anaerobes. For some cephalosporins, results obtained by microdilution and agar dilution may not agree.
The Antmlhrobh Nt~l,lsetter (ISSN 0738-1751) is issued monthly in one indexed volume per year by Elsevier Science Publishing Co.. Inc., 52 Vanderbilt Avenue, New York, NY 10017. Printed in
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THE ANTIMICROBICNEWSLETI~ER,VOLUME4, NUMBER6, JUNE 1987
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TABLE 1. Susceptibility Testing of Fastidious and Unusual Pathogens a~ (Continued)
Organism
Method
Media'
Broth-Disk elution Pre-reduced (Wilkins-Thiel) ,8,30,32 BHI supplemented with 0.0005% hemin, 0.002% menadione, 0.5% yeast extract Broth-Disk elution (Kurzynski et al) '''''~'3"
Thioglycolate (boiled and cooled prior to use)
Incubation As above except 18--24 h
Comments Preparation of tubes for tests (5 ml broth) Antimicrobial
Carbenicillin Cefoperazone Cefotaxime Other cephalosporins* Clindamycin 35°C, 18--24 h, Chloramphenicol aerobically Erythromycin (caps tightened) Metronidazole Mezlocillin Moxalactam Penicillin G Piperacillin Tetracycline Ticarcillin
Disk (Ixg) 100 75 30 30 2 30 15 80 75 30 6 (10 U) 100 30 75
Disks/ Final Conc. tube (Ixg/mL) 6 4 5 5 10 3 1 1 4 5 8 3 1 4
120 60 30 30 4 18 3 16 60 30 9.6 (16 U) 60 6 60
*Includes cephalothin, cefazolin, cefamandole, cefoxitin.
Branhamella catarrhalis
Campylobacter spp
Limited agar dilution TM
WilkinsChalgren agar
As for broth microdilution
Category 2 ~ ' ~ '
Schaedler broth (as above)
35°C, 18-24 h Concentrations to Test in 85% N2, 5% Antimicrobial Conc. to test (~g/mL) CO2 10% H2, or 16, 128, 512 the Gas-Pak or Carbenicillin Cefotaxime 8, 32, 64 equivalent Cefoxitin 4, 16, 64 Clindamycin 2, 8, 64 Chloramphenicol 1, 12 Metronidazole 4, 16, 64 Mezlocillin 16, 128, 512 Moxalactam 8, 32, 64 Penicillin G 0.25, 16, 128 Piperacillin 16, 128, 512
Broth microdilution 17
CSMHB
Agar dilution ~7
MHA
Disk diffusion '2 (ampicillin only)
MHA
13-1actamase'"'2'2"
Isolation media Room temp. u p Use only the nitrocefin method. Other methods to 1 h must be proven to be equivalent. 80% or more of strains are 13-1actamase + . This (these) 13-1actamase(s) is (are) not the TEM type but is (are) inhibited by clavulanic acid and sulbactam.
Broth microdilution ~7
CSMHB + 5% lysed horse blood"
Agar dilution ~7
M H A + 5% blood
35°C, 24 h
35°C, 24 h in 85% N2, 10% CO2, 5% 02
Corynebacterium Broth spp
Two concentrations tested. See NCCLS M17-P for recommendations '~
microdilution ~7
CSMHB
Agar dilution '7
MHA
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35°C, 18-24 h
Some strains may require a more nutritive medium. Ampicillin disk diffusion breakpoint: S I>29 m m R ~<28 m m
Some strains may require 48 h for adequate growth. Does not require routine testing since empirical treatment with erythromycin is usually successful.
Some strains may require a more nutritive medium, eg, blood or serum additives. Test JK against vancomycin and rifampin. a~ 1987 BY ELSEVIERSCIENCE PUBLISHINGCO., INC.
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T H E A N T I M I C R O B I C N E W S L E T T E R , V O L U M E 4, N U M B E R 6, J U N E 1987
T A B L E 1. Susceptibility Testing of Fastidious and Unusual Pathogens at" (Continued)
Organism
Method
Media'
Incubation
Comments
Francisella tularensis
(See reference 2)
Do not test
Susceptibility test should be performed only in appropriate reference laboratories. Streptomycin is usually considered drug of choice. See reference 2 for susceptibility patterns to various antimicrobial agents including newer cephalosporins.
Haemophilus influenzae
Broth microdilution ' ' ' ' 7 ' " ~>
CSMHB + lysed horse blood and 10 lag/mL NAD
Disk diffusion or agar dilution I1, I~. 17. 19,2t~
Mueller-Hinton agar + 1% hemoglobin + 1% IsoVitaleX, pH of medium should be 7.2
See the most recent NCCLS disk diffusion and dilution standards for recommendations on drugs and breakpoints. NCCLS is now considering revision of the breakpoints for erythromycin. Some [3lactamase + isolates may have ampicillin MICs of 2 p,g/mL (susceptible by NCCLS). Some isolates with chloramphenicol MICs of 4 and 8 produce acetyltransferase (resistant?). A report has indicated that the NCCLS breakpoints may not detect ampicillin resistance in some [3-1actamase-negative strains.'4
[8_lactamaseL~ ,2,_*~
Isolation medium
35°C, 24 h
Do not need induction. Any 6-1actamase method can temperature up be used. Some strains may be ampicillin-resistant to1 h but [3-lactamase negative. Room
Legionella spp
Do not test routinely
Therapy can be empirical (erythromycin and/or rifampin). Susceptibility testing should be done only in reference laboratories.
Listeria Broth monocytogenes microdilution j7....
CSMHB + 5% lysed horse blood
Ampiciili,, MICs are usually 0.5-1.0 lag/mL. Gentamicin and ampicillin are usually the drugs of choice in meningitis. The NCCLS is presently considering appropriate disk diffusion breakpoints.
Agar dilution ~7'''
MHA + 5% sheep blood
Disk diffusion '~"'
MHA + 5% sheep blood
Mycobacterium fi~rtuitum and M. chelonae
Broth microdilution. CSMHB Inoculum is prepared from overnight growth in MHB + 0.02% Tween 80 >
Neisseria meningitidis
Broth microdilution 'r'''
CSMHB
Agar dilution '~ "
MHA
35°C, 18-24 b
35°C, 72 h
Avoid creating aerosols. Test aminoglycosides, doxycycline, cefoxitin, erythromycin, and sulfonamides. Ciprofloxacin, a new investigational quinolone, also has activity against M. fortuitum. Other Mycobacterium spp are not tested by this method. Test penicillin, rifampin, and sulfonamide. Organisms in broth may lyse after 24 h. Rifampin and sulfa data are for decisions on prophylaxis and not therapy.
35°C, 18-24 h in CO2
Disk diffusion breakpoints
Neisseria y,onorrhoeae
Disk diffusion (sulfathiazole and rifampin only) '~
MHA
Agar dilution using 10-fold less inoculum than for other aerobic organisms (final inoculum of 10 ~ CFU/spot)~ ,777,~
Proteose #3 agar + 1% hemoglobin + 1% Kellogg's supplement"
Sulfathiazole (300 tag) Rifampin (5 p,g)
S
R
>140 mm ~25 mm
~<36 mm -<-24 mm
Do not test sulfonamides in this medium. Method using s u p p l e m e n t e d GC agar base and a standard inoculum is now being investigated.
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TABLE 1. Susceptibility Testing of Fastidious and Unusual Pathogens ~h (Continued) Organism
Method Agar dilution as above ~"
Media L.
Incubation
Oxoid Diagnostic Sensitivity Test 35°C, 24 h in agar + 5% CO2 lysed horse blood + 1% Kellogg's supplement
Comments Use to test sulfonamides.
Disk diffusion breakpoint Disk diffusion 4,,.,~.2~
S
GC agar base + 1% IsoVitaleX
~_lactamase 4.~~, 27,28
Nocardia spp
From isolation media or media listed above
Room temperature up to 1 h
Broth microdilution~7"
CSMHB
I
R
Penicillin />20 m m ~<19 m m Spectinomycin I>18 m m 15-17 m m ~<14 m m Disk tests for several antimicrobics now being investigated. Based on their studies with pencillin resistant f~-lactamase strains, the CDC STD Laboratory suggests the use of ~<25 mrn as the resistant breakpoint for penicillin rather than ~<20 m m that we and the NCCLS have recommended; this is also being investigated. Occasional strains m a y be resistant to penicillin but 13-1actamase negative. Do not need induction. A n y 13-1actamase m e t h o d can be used. 35°C, 48 h
N. asteroides has four or five different susceptibility patterns; N. braziliensis has one. Sulfas are generally drugs of choice but intolerance or allergy fairly common. Test a variety of antimicrobials including 3rd generation cephalosporins and 13-1actamq3-1actamase inhibitor combinations. (Above information from personal communication with Dr. Richard Wallace, University of Texas Health Science Center, Tyler, Texas).
Nonfermentative Broth bacteria (other microdilution'7"~ than
Acinetobacter spp)
Agar dilution 17''~
CSMHB or CSMHB + s u p p l e m e n t s if needed
35°C, 18-24 h or longer, use MHA + CO2 if s u p p l e m e n t s if necessary needed
"Organisms See Streptococcus spp causing section for MIC endocarditis": methods. Nonenterococcal streptococci
Enterococcus spp
If penicillin M1C > 0.1 wg/mL, 13-1actam plus aminoglycoside (streptomycin or gentamicin) therapy should be considered.
See Streptococcus spp section for MIC methods High level aminoglycoside test for synergy
Some of these isolates may require a more enriched medium.
Use ~-lactarn and aminoglycoside (streptomycin or gentamicin) for therapy. Some strains will not respond synergistically to the combination (see below). CSMHB + 5% lysed horse blood
35°C, 18--24 h Test streptomycin and gentamicin at 2000 v,g/mL CO, if necessary (some microbiologists use 500 wg for gentamicin). If there is no growth, synergy between the 13-1actam and the aminoglycoside is likely to occur; if there is growth, synergy is unlikely. Blood may influence susceptibility tests with aminoglycosides and cephalosporins and some enterococci.
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THE ANTIMICROBICNEWSLETTER,VOLUME4, NUMBER6, JUNE 1987
TABLE 1. Susceptibility Testing of Fastidious and Unusual Pathogens ~b (Continued)
Organism
Method
Media"
Comments
Incubation
Staphylococcus spp See Staphylococcus spp section for MIC methods. Includes a variety of gram-positive and gram-negative bacteria. No one medium or atmosphere is adequate for all species. Determine nutritional and atmospheric needs and do an MIC.
Other species
Staphylococcus aureus
Agar screen 13.29
Oxacillin resistant Methicillin resistant
MHA + 4% 35°C, full 24 h NaCl and 6 p.g/ but no longer mL oxacillin or 10 p.g/mL methicillin
CSMHB + 2% Broth NaC1 microdilution--for methicillin and oxacillin only "''7'2~ (cephalothin or other B-lactams, excluding cefamandole, if desired)
Agar dilution ~7'~"
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CDC Methicillin Oxaclllin
S 44 ~2
I 8 4
NCCLS R />16 ~> 8
S ~8 ~2
1 ---
R ~ 16 ~ 4
The above breakpoints are to be used only with this MIC method. Oxacillin is the recommended and preferred test agent in most institutions. To prepare inoculum suspend colonies from an overnight agar plate into broth or saline to equal the turbidity of a 0.5 McFarland standard. ,.,~.,7 Final inoculum should be 3-5 x 10s CFU/mL. Some strains are hyperproducers of ~-lactamase and yield resistant or borderline MICs to oxacillin and methicillin but will not be intrinsically resistant. '3 These strains are not usually multiresistant to drugs other than ~qactams. These strains are usually susceptible to amoxicillin-clavulanicacid (intrinsically oxacillin-resistant strains are not).
1. Multiple resistance to any or all of the following: erythromycin, aminoglycosides, chloramphenicol, tetracycline, or clindamycin. 2. Intermediate MIC to methicillin or oxacillin. 3. Failure to show cross-resistance between methiciilin, nafcillin, and oxacillin.
MHA
MIC Breakpoints (~g/mL)
35°C, full 24 h but no longer
Clues (flags) indicating resistance to methicillin, oxacillin, or nafcillin
Disk diffusion 's'~"
Use as a screening test for methicillin, oxacillin, nafcillin, or cloxacillin resistance. Oxacillin is the preferred test agent in most institutions. Inoculate plate by "pie-plating" or by "spot" inoculation with swab. Growth indicates chromosomal intrinsic resistance. Use as a second test in addition to the broth microdilution or disk diffusion test.
35°C, full 24 h but no longer
Do not use above MIC breakpoints for correlations with this test. The MIC breakpoint correlates for susceptible for this method are ~3 p.g/mL for methicillin and ~1.0 p.g/mL for oxacillin and nafcillin. Oxacillin is the preferred test agent in most institutions. Inoculum should be prepared as above. Look for growth within a zone of inhibition. Some strains are hyperproducers of 13-1actamase and may yield resistant zone sizes; these strains yield either susceptible, borderline, or resistant MICs. '~ These strains are usually susceptible to amoxicillin-clavulanic acid (intrinsically oxaciUin-resistant strains are not). Although we assume that most oxacillin-resistant staphylococci can be detected by agar dilution, we have not studied it. We do not have any recommendations for adding NaCI.
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THE ANTIMICROBICNEWSLETFER,VOLUME4, NUMBER6, JUNE 1987
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T A B L E 1. Susceptibility Testing of Fastidious and Unusual Pathogens ~b (Continued)
Orc,anism S. epidermidis
Method
Media"
Incubation
Comments
Same tests that are used for S. aureus. We have much less experience with coagulase negative staphylococci (CNS) than with S. aureus. Recommendations are provisional. Approximately 2A of clinical isolates of CNS will be S. epidermidis. Approximately V2 of clinical CNS will be oxadllinresistant, and the percentage is even higher for S. epidermidis. Most disk diffusion experience has been with S. aureus, not CNS. Do not use nafdllin as a test agent. Most oxicillin-resistant strains are multiresistant. Use oxacillin and/or methicillin as the test agent(s); there is some advantage to using both with CNS. Use the agar screen test as a second test in addition to the microdilution or disk diffusion tests. Induce with oxacillin or methicillin prior to testing for 13-1actamase.
Clues (flags) indicatin,¢~ resistance to methicillin, oxacillin, or nafcillin 1. Multiple resistance to any or all of the following: erythromydn, aminoglycosides, chloramphenicol, tetracycline, or clindamycin. 2. Intermediate MIC to methicillin or oxacillin. 3. Failure to show cross-resistance between methicillin, nafcillin, and oxadllin.
S. saprophyticus
Same tests that are used for S. aureus.
May be a common urinary tract isolate. May produce small amounts of 13-1actamase. Usually intermediate in its susceptibility to oxacillin and methicillin so results may flip-flop from test to test, especially with disk diffusion tests. This resistance appears to be different from that seen with S. aureus and S. epidermidis and the screen test will be negative (no growth). These isolates often show test results such as pencillinsusceptible (13-1actamase negative) but oxicillin-resistant and negative (no growth) for the screen test. Penicillin MICs are usually 0.12 or 0.25 p~g/mL. The 13-1ac test usually requires/> 1 h to become positive and the color is usually not intense (induction does not change the reaction time or color intensity).
S. haemolyticus
Same tests that are used for S. aureus.
These isolates are usually the most resistant of the coagulase negative staphylococci. Induce with oxacillin or methicillin prior to testing for [3-1actamase.
Streptococcus pneumoniae
Broth microdilution'7'2"3'
CSMHB + 5% lysed horse blood
35°C, 18-24 h
MIC breakpoints for penicillin are: susceptible, <~0.06; relatively resistant, 0.12-1.0; and resistant, ~>2 ~g/mL. Oxacillin MICs of -~0.25 #,g/mL indicate susceptibility to penicillin; oxacillin MICs of ~>1.0 indicate resistance or relative resistance to penicillin; oxacillin MICs of 0.5 i~g/mL are equivocal.
Disk diffusion screen MHA + 5% for penidUin sheep blood resistance '~,2.,3~
Using a 1-p,g oxacillin disk, a zone of ~<19 mm almost always indicates penicillin resistance or relative resistance; an oxacillin zone of/>20 mm indicates susceptible. Oxacillin is preferred over methicillin as the test agent. 2" Do not report result in terms of susceptibility to oxacillin, but rather as penicillin susceptibility.
Disk diffusion '~''~
Chloramphenicol breakpoints are the same as the NCCLS: R ~< 12 mm, S I> 18 mm. '~'' Testing penicillin by disk diffusion may lead to reporting of false susceptibility (use the oxacillin screen test above). It is important to test for chloramphenicol resistance, especially if penicillin resistance is also detected.
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MHA + 5% sheep blood
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THE ANTIMICROBICNEWSLETTER,VOLUME4, NUMBER6, JUNE 1987
TABLE
1. Susceptibility Testing of Fastidious and Unusual Pathogens ~b (Continued)
Organism
Method
Streptococcus spp. Broth microdilution' 7.~,
Streptococci, pyridoxal dependent
Broth microdilution ~''7''~
Broth dilution ~7'~ Fastidious or Agar dilution '7"~ unusual organisms not m e n t i o n e d above
Media ~ CSMHB + 5% lysed horse blood
Incubation
Comments
35°C, 18-24 h in Nonenterococcal streptococci that grow well in this CO2 if necessary m e d i u m (and may require CO2) may also be tested by the standard NCCLS disk diffusion methods. ~~" The oxacillin screen test as u s e d for pneumococci does not w o r k (ie, to indicate pencillin resistance) for these organisms. Pencillin resistance occurs most often in S. mitis but does occur in other species. For G r o u p A streptococci, erythromycin resistance occurs; zones of 318 m m correlate well with MICs of 40.12 ~g/mL and zones of <18 m m w i t h MICs of 3 2 p,g/mL (unpublished data).
CSMHB + 5% 35°C, 18-24 h in G r o w organisms on agar containing 0.001% pyridoxal lysed horse CO2 if necessary HC1. Alternatively, 1% pyridoxal HCi can be swabbed onto the surface of a blood agar plate prior to blood + 0.001% inoculation. pyridoxal HCi Prepare i n o c u l u m suspension from growth on plate.' Use MuellerHinton and s u p p l e m e n t s as necessary. Trial and error m a y be necessary to determine needs,
35°C, 18-24 h or If unusual s u p p l e m e n t s or a t m o s p h e r e is necessary, report M1C as not d o n e with a standardized test. longer. Use required atmosphere. Trial and error m a y be necessary to determine needs.
" For this document the following definitions are used: A fastidious organism is one that does not grow adequately in or on unsupplemented Mueller-Hinton media for antimicrobial susceptibility tests. An unusual organism is one for which standard methods have not been developed or for which it has not been established that it can be tested with a standard method such as the NCCLS disk-diffusion standard. b In general, the inoculum for most of these tests is preferably prepared directly from growth off of agar plates.~ It is best to use as fresh a plate as possible but some organisms may l~Kluire 48 h for adequate growth. Suspend the growth into a clear broth and standardize the suspension to match a 0.5 McFarland Standard. Then make appropriate dilutions de pesnding on type of system or method being used (see directions for specific organisms). Inoculum for broth microdilution should be 1-5 x 10 CFU/mL, and for agar, 1-5 x 1 0 4 CFU/spot. Abbreviations for media or reagents: MHA - Mueller-Hinton agar CSMHB - Cation-supplemented Mueller-Hinton broth ~7 BHI - Brain heart infusion broth NAD - Nicotinamide adenine dinucleotide SXT - Sulfamethoxazole-trimethoprim ,t The lysed blood used in these tests is lysed by freezing, thawing at least 6 times and then adding an equal volume of sterile distilled water. The solution is clarified by centrifugation (10,000 x g) for 20 min and subsequent decantation of the supemate (not necessary if it is to be added to agar). In some tests, the lysed horse blood could probably be replaced with blood from other species, but we have always used horse blood. We do not recommend sheep blood for tests with Haemophilus or with sulfonamides. ' Kellogg's Supplement: By stirring, dissolve in' 100 mL distilled water, 40 g dextrose, 1 g glutamine, and 1 mL of 0.2% thiamine pyrophosphate. Then add 85 mg Fe(NO3)3 • 9 1-120and stir until dissolved. Filter, sterilize, and freeze in appropriate portions.
REFERENCES
1. Baker CN, T h o r n s b e r r y C, H a w k i n s o n RW: I n o c u l u m stand a r d i z a t i o n in antimicrobial susceptibility testing: Evaluation of o v e r n i g h t agar cultures and the Rapid I n o c u l u m S t a n d a r d i z a t i o n System. J Clin Microbiol 17:450457, 1983.
(c) 1987 BY ELSEVIERSCIENCE PUBLISHINGCO., INC.
2. Baker CN, Hollis G, T h o r n s b e r r y C: Antimicrobial susceptibility testing of Francisella tularensis w i t h a m o d i f i e d M u e l l e r - H i n t o n broth. J Clin Microbiol 22:212-215, 1985. 3. Bauer AW, Kirby W M M , Sherris JC, et al: Antibiotic susceptibility by a s t a n d a r d i z e d single disk m e t h o d . A m J Clin Pathol 45:493496, 1966.
4. Biddle JW, S w e n s o n JM, Thornsberry C: Disc agar diffusion antimicrobial susceptibility tests w i t h ~-iactamase p r o d u c i n g Neisseria gonorrhoeae. J Antibiot 31:352-358, 1978. 5. C o o k s e y RC, S w e n s o n JM: In vitro antimicrobial inhibition p a t t e r n s of nutritionally variant streptococci. Antimicrob Agents Chemother
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THE ANTIMICROBICNEWSLETFER,VOLUME4, NUMBER6, JUNE1987
16:514-518, 1979. 6. Elwell LP, DeGraaff J, Seibert D, Falkow S: Plasmid-linked ampicillin resistance in Haemophilus influenzae type b. Infect Immun 12:404410, 1975. 7. Elwell LP, Roberts M, Mayer L, et al: Plasmid-mediated 13-1actamase production in Neisseria gonorrhoeae. Antimicrob Agents Chemother 11:538-533, 1977. 8. Gavan TL, Jones RN, Barry AL, et al: Quality control limits for ampicillin, carbenicillin, mezlocillin, and piperacillin disk diffusion susceptibility tests: A collaborative study. J Clin Microbiol 14:67-72, 1981. 9. Kirven LA, Thornsberry C: Minimum bactericidal concentrations of sulfamethoxazole-trimethoprim for Haemophilus influenzae: Correlation with prophylaxis. Antimicrob Agents Chemother 14:731-736, 1978. 10. Kurzynski TA, Yrios JW, Helstad AG, et al: Aerobically incubated thioglycolate broth disk method for antibiotic susceptibility testing of anaerobes. Antimicrob Agents Chemother 10:727-732, 1976. 11. Lennette EH, Balows A, Hausler WJ, et al: Manual of Clinical Microbiology, 3rd ed. American Society for Microbiology, Washington, D.C., 1980. 12. Luman I, Wilson RW, Wallace RJ, et al: Disk diffusion susceptibility of Brahamella catarrhalis and relationship of 13-1actam zone size to 13-1actamase production. Antimicrob Agents Chemother 30:774776, 1986. 13. McDougal LK, Thornsberry C: The role of 13-1actamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporins. J Clin Microbiol 23:832-839, 1986. 14. Mendelman PM, Chaffin DO, Clausen C, et al: Failure to detect ampicillin-resistant, non-13-1actamase-producing Haemophilus influenzae by standard disk diffusion testing. Antimicrob Agents Chemother 30:274-280. 15. National Committee for Clinical Laboratory Standards. Approved Standard M2-A3. Performance standards for antimicrobic disk susceptibility tests, 3rd edition,
0738-1751/87/$0.00 + 2.20
16.
17.
18.
19.
20.
21.
22.
23.
24.
55
1984. National Committee for Clinical Laboratory Standards. Villanova, PA. National Committee for Clinical Laboratory Standards. Approved Standard Mll-A. Approved reference dilution procedure for antimicrobic susceptibility testing of anaerobic bacteria, 1985. National Committee for Clinical Laboratory Standards, Villanova, PA. National Committee for Clinical Laboratory Standards. Approved standard M7-A. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 1985. National Committee for Clinical Laboratory Standards, Vilanova, PA. National Committee for Clinical Laboratory Standards. Proposed Standard M17-P. Alternative methods for antimicrobial susceptibility testing of anaerobic bacteria, 1985. National Committee for Clinical Laboratory Standards, Villanova, PA. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing: First informational supplement, 1986. National Committee for Clinical Laboratory Standards, Villanova, PA. Rein ME, Elliott WC, Swenson JM, et al: Sulfamethoxazole-trimethoprim synergism for Neisseria gonorrhoeae. Antimicrob Agents Chemother 17:247-250, 1980. Richmond MH, Sykes RB: The 13lactamases of gram-negative bacteria and their possible physiological role, in Rose AH, Wilkinson JF (eds): Advances in Microbiology and Physiology, vol. 9. New York, Academic Press, 1973. Sanders CC, Sanders WE: Emergence of resistance to cefamandole: Possible role of cefoxitin-inducible beta-lactamases. Antimicrob Agents Chemother 7:15-21, 1979. Stalons DR, Thornsberry C: Brothdilution method for determining the antibiotic susceptibility of anaerobic bacteria. Antimicrob Agents Chemother 7:15-21, 1975. Swenson JM, Thornsberry C: Susceptibility tests for sulfamethoxazole-trimethoprim by a broth microdilution procedure. Current Microbiology 1:189-193, 1978.
25. Swenson JM, Thornsberry C, Silcox VA: Rapidly growing mycobacteria: Testing of susceptibility to 34 antimicrobial agents by broth microdilution. Antimicrob Agents Chemother 22:186-192, 1982. 26. Swenson JM, Hill BC, Thornsberry C: Screening pneumococci for penicillin resistance. J Clin Microbiol 24:749-752, 1986. 27. Thornsberry C, Biddle JW, Kirven LA, et al: Penicillin resistance in Neisseria gonorrhoeae due to 13-1actamase production. Microbios 20:3946, 1978. 28. Thornsberry C, Gavan TL, Gerlach EH: New developments in antimierobial agent susceptibility testing. Cumitech 6, American Society for Microbiology, Washington, D.C., 1977. 29. Thornsberry C, McDougal LK: Successful use of broth microdilution in susceptibility tests for methicillin-resistant (heteroresistant) staphylococci, l Clin Microbiol. 18:1084-1091, 1983. 30. Thornsberry C, Swenson IM: Antimicrobial susceptibility testing of anaerobes. Laboratory Medicine 9:43--48, 1978. 31. Thornsberry C, Swenson JM: Antimicrobial susceptibility tests for Streptococcus pneumoniae. Laboratory Medicine 11:83-86, 1980. 32. Wilkins TD, Thiel T: Modified broth-disc method for testing the antibiotic susceptibility of anaerobic bacteria. Antimicrob Agents Chemother 3:350--356, 1973. 33. Zigheiboin S, Tomasz A: Penicillin binding proteins of multiply antibiotic-resistant South African strains of Streptococcus pneumoniae. Antimicrob Agents Chemother 17:434 441, 1980.
GENERAL REFERENCES
1. Thornsberry C: (section editor). Section XI. Laboratory tests in chemotherapy, in Lennette EH, Balows A, Hausler WJ, et al (eds): Manual of Clinical Microbiology, 4th ed, American Society for Microbiology, 1985. 2. Lorian V (ed.): Antibiotics in Laboratory Medicine, 2nd ed., Williams and Wilkins, Baltimore, MD, 1986.
© |987 BYELSEVIERSCIENCEPUBLISHINGCO., INC.
ISSN 0738-1751
VOLUME 4, NUMBER 6, JUNE 1987
EDITORIAL BOARD Editor
Associate Editors
DANIEL AMSTERDAM, PhD,
RONALD N. JONES, MD,
CLYDE THORNSBERRY, PhD,
State University of New York at Buffalo and Erie County Medical Center Buffalo, New York
Clinical Microbiology Institute Tualatin, Oregon
Center for Infectious Diseases Centers for Disease Control Atlanta, Georgia
HAROLD C. NEU, MD,
LOWELL S. YOUNG, MD,
College of Physicians and Surgeons, Columbia University New York, New York
Kuzell Institute for Arthritis and Infectious Diseases Medical Research Institute ef San Francisco Pacific Presbyterian Medical Center San Francisco, California
SUSCEPTIBILITY T E S T I N G OF F A S T I D I O U S AND UNUSUAL PATHOGENS EDITOR'S N O T E
47
D. AMST E RDAM
Susceptibility Testing of Fastidious and Unusual Pathogens C. J. C. L. S. B.
47
THORNSBERRY M. SWE NSON N. BAKER K. MCDOUGAL A. STOCKER C. HILL
EDITOR'S NOTE In this A M N publication, the staff at the Antimicrobics Investigations Branch, headed by Dr. Clyde Thornsberry, pool their experience and knowledge to present their recommendations for antimicrobic susceptibility evaluations of 25 groups and bacterial species for which standards have not yet been completely developed or there is ongoing modification and --Anaerobes --MRSA --H. influenzae --Mycobacteria --Nonenterococcal Streptococci --Branhamella
ELSEVIER
CLYDE T H O R N S B E R R Y J A N A M. S W E N S O N C A R O L Y N N. BAKER L I N D A K. M c D O U G A L S H E I L A A. STOCKER, a n d B E R T H A C. HILL Antimicrobics Investigations Branch, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia
reinterpretation of standards. For several species, notably Mycobacterium, Branhamella, and Neisseria, and for the anaerobes, NCCLS recommendations are currently being evaluated and may soon be available. In recent issues, The A M N has presented problems associated with the testing and interpretation of some of these fastidious or unusual isolates. The reader may wish to refer to the following: The AMN, The AMN, The AMN, The AMN, The AMN, The AMN, The AMN,
Vol. Vol. Vol. Vol. Vol. Vol. Vol.
2, 2, 2, 2, 3, 3, 3,
No. No. No. No. No. No. No.
6, 1985 7, 1985 8, 1985 12, 1985 8, 1986 2, 1986 12, 1986
Some clinically significant bacteria have characteristics that preclude their being tested by the standardized disk diffusion method. 3's'15'19 They may grow too slowly, require special nutrients, require special atmospheres, or simply may not have been tested adequately to show that they can be tested accurately and reproducibly by the standard NCCLS method. ~'~9 We have called these bacteria "fastidious" and/or "unusual" isolates. More specifically, the "fastidious" organism will not grow on Mueller-Hinton medium without supplementation. The "unusual" organism however, may grow well on Mueller-Hinton medium but studies have not been done to show that it can be reliably tested by standard methods. The "unusual" organism may also present special problems in testing its susceptibility to antimicrobial agents. In previous years, many of these organisms did not require susceptibility tests because they were known to be universally susceptible to an appropriate antimicrobial agent(s). Resistant strains have emerged, however. Resistance to 13-1actams is most often due to the production of ~lactamase which may be constitu-
o73s-
2.2°
48
THE A N T I M I C R O B I C NEWSLETTER, V O L U M E 4, N U M B E R 6, JUNE 1987
tive or inducible and m a y be mediated by either c h r o m o s o m a l or plasmid genes. 2''22 In some of these strains, such as Haemophilus influenzae and Neisseria gonorrhoeae, most, but not all, of the penicillin and ampicillin resistance has been caused by the acquisition of plasraids that mediate constitutive 13lactamase p r o d u c t i o n by the organism, 6'7 but in others, such as Streptococcus pneumoniae, penicillin resistance has not been due to 13lactamase or a plasmid but is chromosomally mediated and is due to alteration of penicillin-binding proteins,
33
These d e v e l o p m e n t s have dictated that, for infections caused by some of these "fastidious" or " u n usual" organisms, the clinician has had to realize that some previously e m p l o y e d empirical t h e r a p y might not be adequate, and the clinical microbiologist has had to begin testing the antimicrobial susceptibility of organisms that were previously not tested. Antimicrobial susceptibility tests should be p e r f o r m e d only on microorganisms that are etiologic
agents of the infection in question. W h e n it has been d e t e r m i n e d that an organism is involved in the infection, the next step is to determine if a susceptibility test is necessary. For some organisms, such as G r o u p A streptococci, susceptibility tests are not necessary because these organisms are still universally susceptibile to penicillin, the drug of choice. H o w e v e r , if the organism is S. pneumoniae, and especially if it was isolated from the cerebrospinal fluid, a test to determine the susceptibility to penicillin is indicated because some of these bacteria are either relatively resistant or resistant to penicillin. If it is d e e m e d necessary to perform susceptibility tests on a clinical isolate for which a m e t h o d has not been described, it is usually best to determine a minimal inhibitory concentration (MIC) using the general MIC m e t h o d described in NCCLS Standard M7-A.'7 For testing some organisms, modification of the basic m e t h o d s is not necessary, but for others, additional nutrients, different
atmospheres, or other changes m a y be necessary. If supplementation of the m e d i u m is required, only essential nutrients should be added, and the a t m o s p h e r e and t e m p e r a t u r e of incubation should not be changed unless necessary. But if, for example, the organism requires blood and will not grow at t e m p e r a t u r e s higher than 25°C, then the MIC should be determined u n d e r these conditions. H o w e v e r , the clinician should be informed that the MIC could not be d e t e r m i n e d by a standardized m e t h o d and the results, therefore, may be the best approximation obtainable. Following are some antimicrobial susceptibility testing m e t h o d s that can be used for some of these fastidious or unusual organisms. It should be pointed out, however, that these m e t h o d s are, for the most part, tests that we have used in our laboratory and are not meant to be standard methods. For some of the organisms, intensive s t u d y of m e t h o d o l o g y is n o w ongoing and standard r e c o m m e n dations m a y soon be available.
TABLE 1. Susceptibility Testing of Fastidious and Unusual Pathogens""
Organism Anaerobes
Method Broth microdilution using reduced plates and water blanks for inoculum d i l u t i o n '~,3"
NCCLS reference agar dilution '"
Media~
Incubation
Schaedler broth + 0.1 ixg/mL vitamin K and 10 ixg/mL hemin 35°C, 48 h in 85% N2, 5% CO2, 10% H2, Wi|kinsor the Gas Pak, Chalgren agar or equivalent
Comments May also use other broths shown to support growth of anaerobes, eg, supplemented BHI. Commercial microdilution tests now available. Microdilution MICs for cephalosporins tend to be lower than MICs by the standard reference agar dilution method.
Wilkins-Chalgren agar may not support growth of all anaerobes. For some cephalosporins, results obtained by microdilution and agar dilution may not agree.
The Antmlhrobh Nt~l,lsetter (ISSN 0738-1751) is issued monthly in one indexed volume per year by Elsevier Science Publishing Co.. Inc., 52 Vanderbilt Avenue, New York, NY 10017. Printed in
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© 1987 BY ELSEVIER SCIENCE P U B L I S H I N G CO., INC.
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THE ANTIMICROBICNEWSLETI~ER,VOLUME4, NUMBER6, JUNE 1987
49
TABLE 1. Susceptibility Testing of Fastidious and Unusual Pathogens a~ (Continued)
Organism
Method
Media'
Broth-Disk elution Pre-reduced (Wilkins-Thiel) ,8,30,32 BHI supplemented with 0.0005% hemin, 0.002% menadione, 0.5% yeast extract Broth-Disk elution (Kurzynski et al) '''''~'3"
Thioglycolate (boiled and cooled prior to use)
Incubation As above except 18--24 h
Comments Preparation of tubes for tests (5 ml broth) Antimicrobial
Carbenicillin Cefoperazone Cefotaxime Other cephalosporins* Clindamycin 35°C, 18--24 h, Chloramphenicol aerobically Erythromycin (caps tightened) Metronidazole Mezlocillin Moxalactam Penicillin G Piperacillin Tetracycline Ticarcillin
Disk (Ixg) 100 75 30 30 2 30 15 80 75 30 6 (10 U) 100 30 75
Disks/ Final Conc. tube (Ixg/mL) 6 4 5 5 10 3 1 1 4 5 8 3 1 4
120 60 30 30 4 18 3 16 60 30 9.6 (16 U) 60 6 60
*Includes cephalothin, cefazolin, cefamandole, cefoxitin.
Branhamella catarrhalis
Campylobacter spp
Limited agar dilution TM
WilkinsChalgren agar
As for broth microdilution
Category 2 ~ ' ~ '
Schaedler broth (as above)
35°C, 18-24 h Concentrations to Test in 85% N2, 5% Antimicrobial Conc. to test (~g/mL) CO2 10% H2, or 16, 128, 512 the Gas-Pak or Carbenicillin Cefotaxime 8, 32, 64 equivalent Cefoxitin 4, 16, 64 Clindamycin 2, 8, 64 Chloramphenicol 1, 12 Metronidazole 4, 16, 64 Mezlocillin 16, 128, 512 Moxalactam 8, 32, 64 Penicillin G 0.25, 16, 128 Piperacillin 16, 128, 512
Broth microdilution 17
CSMHB
Agar dilution ~7
MHA
Disk diffusion '2 (ampicillin only)
MHA
13-1actamase'"'2'2"
Isolation media Room temp. u p Use only the nitrocefin method. Other methods to 1 h must be proven to be equivalent. 80% or more of strains are 13-1actamase + . This (these) 13-1actamase(s) is (are) not the TEM type but is (are) inhibited by clavulanic acid and sulbactam.
Broth microdilution ~7
CSMHB + 5% lysed horse blood"
Agar dilution ~7
M H A + 5% blood
35°C, 24 h
35°C, 24 h in 85% N2, 10% CO2, 5% 02
Corynebacterium Broth spp
Two concentrations tested. See NCCLS M17-P for recommendations '~
microdilution ~7
CSMHB
Agar dilution '7
MHA
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35°C, 18-24 h
Some strains may require a more nutritive medium. Ampicillin disk diffusion breakpoint: S I>29 m m R ~<28 m m
Some strains may require 48 h for adequate growth. Does not require routine testing since empirical treatment with erythromycin is usually successful.
Some strains may require a more nutritive medium, eg, blood or serum additives. Test JK against vancomycin and rifampin. a~ 1987 BY ELSEVIERSCIENCE PUBLISHINGCO., INC.
50
T H E A N T I M I C R O B I C N E W S L E T T E R , V O L U M E 4, N U M B E R 6, J U N E 1987
T A B L E 1. Susceptibility Testing of Fastidious and Unusual Pathogens at" (Continued)
Organism
Method
Media'
Incubation
Comments
Francisella tularensis
(See reference 2)
Do not test
Susceptibility test should be performed only in appropriate reference laboratories. Streptomycin is usually considered drug of choice. See reference 2 for susceptibility patterns to various antimicrobial agents including newer cephalosporins.
Haemophilus influenzae
Broth microdilution ' ' ' ' 7 ' " ~>
CSMHB + lysed horse blood and 10 lag/mL NAD
Disk diffusion or agar dilution I1, I~. 17. 19,2t~
Mueller-Hinton agar + 1% hemoglobin + 1% IsoVitaleX, pH of medium should be 7.2
See the most recent NCCLS disk diffusion and dilution standards for recommendations on drugs and breakpoints. NCCLS is now considering revision of the breakpoints for erythromycin. Some [3lactamase + isolates may have ampicillin MICs of 2 p,g/mL (susceptible by NCCLS). Some isolates with chloramphenicol MICs of 4 and 8 produce acetyltransferase (resistant?). A report has indicated that the NCCLS breakpoints may not detect ampicillin resistance in some [3-1actamase-negative strains.'4
[8_lactamaseL~ ,2,_*~
Isolation medium
35°C, 24 h
Do not need induction. Any 6-1actamase method can temperature up be used. Some strains may be ampicillin-resistant to1 h but [3-lactamase negative. Room
Legionella spp
Do not test routinely
Therapy can be empirical (erythromycin and/or rifampin). Susceptibility testing should be done only in reference laboratories.
Listeria Broth monocytogenes microdilution j7....
CSMHB + 5% lysed horse blood
Ampiciili,, MICs are usually 0.5-1.0 lag/mL. Gentamicin and ampicillin are usually the drugs of choice in meningitis. The NCCLS is presently considering appropriate disk diffusion breakpoints.
Agar dilution ~7'''
MHA + 5% sheep blood
Disk diffusion '~"'
MHA + 5% sheep blood
Mycobacterium fi~rtuitum and M. chelonae
Broth microdilution. CSMHB Inoculum is prepared from overnight growth in MHB + 0.02% Tween 80 >
Neisseria meningitidis
Broth microdilution 'r'''
CSMHB
Agar dilution '~ "
MHA
35°C, 18-24 b
35°C, 72 h
Avoid creating aerosols. Test aminoglycosides, doxycycline, cefoxitin, erythromycin, and sulfonamides. Ciprofloxacin, a new investigational quinolone, also has activity against M. fortuitum. Other Mycobacterium spp are not tested by this method. Test penicillin, rifampin, and sulfonamide. Organisms in broth may lyse after 24 h. Rifampin and sulfa data are for decisions on prophylaxis and not therapy.
35°C, 18-24 h in CO2
Disk diffusion breakpoints
Neisseria y,onorrhoeae
Disk diffusion (sulfathiazole and rifampin only) '~
MHA
Agar dilution using 10-fold less inoculum than for other aerobic organisms (final inoculum of 10 ~ CFU/spot)~ ,777,~
Proteose #3 agar + 1% hemoglobin + 1% Kellogg's supplement"
Sulfathiazole (300 tag) Rifampin (5 p,g)
S
R
>140 mm ~25 mm
~<36 mm -<-24 mm
Do not test sulfonamides in this medium. Method using s u p p l e m e n t e d GC agar base and a standard inoculum is now being investigated.
THE ANTIMICROBICNEWSLETFER,VOLUME4, NUMBER6, JUNE 1987
51
TABLE 1. Susceptibility Testing of Fastidious and Unusual Pathogens ~h (Continued) Organism
Method Agar dilution as above ~"
Media L.
Incubation
Oxoid Diagnostic Sensitivity Test 35°C, 24 h in agar + 5% CO2 lysed horse blood + 1% Kellogg's supplement
Comments Use to test sulfonamides.
Disk diffusion breakpoint Disk diffusion 4,,.,~.2~
S
GC agar base + 1% IsoVitaleX
~_lactamase 4.~~, 27,28
Nocardia spp
From isolation media or media listed above
Room temperature up to 1 h
Broth microdilution~7"
CSMHB
I
R
Penicillin />20 m m ~<19 m m Spectinomycin I>18 m m 15-17 m m ~<14 m m Disk tests for several antimicrobics now being investigated. Based on their studies with pencillin resistant f~-lactamase strains, the CDC STD Laboratory suggests the use of ~<25 mrn as the resistant breakpoint for penicillin rather than ~<20 m m that we and the NCCLS have recommended; this is also being investigated. Occasional strains m a y be resistant to penicillin but 13-1actamase negative. Do not need induction. A n y 13-1actamase m e t h o d can be used. 35°C, 48 h
N. asteroides has four or five different susceptibility patterns; N. braziliensis has one. Sulfas are generally drugs of choice but intolerance or allergy fairly common. Test a variety of antimicrobials including 3rd generation cephalosporins and 13-1actamq3-1actamase inhibitor combinations. (Above information from personal communication with Dr. Richard Wallace, University of Texas Health Science Center, Tyler, Texas).
Nonfermentative Broth bacteria (other microdilution'7"~ than
Acinetobacter spp)
Agar dilution 17''~
CSMHB or CSMHB + s u p p l e m e n t s if needed
35°C, 18-24 h or longer, use MHA + CO2 if s u p p l e m e n t s if necessary needed
"Organisms See Streptococcus spp causing section for MIC endocarditis": methods. Nonenterococcal streptococci
Enterococcus spp
If penicillin M1C > 0.1 wg/mL, 13-1actam plus aminoglycoside (streptomycin or gentamicin) therapy should be considered.
See Streptococcus spp section for MIC methods High level aminoglycoside test for synergy
Some of these isolates may require a more enriched medium.
Use ~-lactarn and aminoglycoside (streptomycin or gentamicin) for therapy. Some strains will not respond synergistically to the combination (see below). CSMHB + 5% lysed horse blood
35°C, 18--24 h Test streptomycin and gentamicin at 2000 v,g/mL CO, if necessary (some microbiologists use 500 wg for gentamicin). If there is no growth, synergy between the 13-1actam and the aminoglycoside is likely to occur; if there is growth, synergy is unlikely. Blood may influence susceptibility tests with aminoglycosides and cephalosporins and some enterococci.
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THE ANTIMICROBICNEWSLETTER,VOLUME4, NUMBER6, JUNE 1987
TABLE 1. Susceptibility Testing of Fastidious and Unusual Pathogens ~b (Continued)
Organism
Method
Media"
Comments
Incubation
Staphylococcus spp See Staphylococcus spp section for MIC methods. Includes a variety of gram-positive and gram-negative bacteria. No one medium or atmosphere is adequate for all species. Determine nutritional and atmospheric needs and do an MIC.
Other species
Staphylococcus aureus
Agar screen 13.29
Oxacillin resistant Methicillin resistant
MHA + 4% 35°C, full 24 h NaCl and 6 p.g/ but no longer mL oxacillin or 10 p.g/mL methicillin
CSMHB + 2% Broth NaC1 microdilution--for methicillin and oxacillin only "''7'2~ (cephalothin or other B-lactams, excluding cefamandole, if desired)
Agar dilution ~7'~"
© 1987BY ELSEVIERSCIENCEPUBLISHINGCO., INC.
CDC Methicillin Oxaclllin
S 44 ~2
I 8 4
NCCLS R />16 ~> 8
S ~8 ~2
1 ---
R ~ 16 ~ 4
The above breakpoints are to be used only with this MIC method. Oxacillin is the recommended and preferred test agent in most institutions. To prepare inoculum suspend colonies from an overnight agar plate into broth or saline to equal the turbidity of a 0.5 McFarland standard. ,.,~.,7 Final inoculum should be 3-5 x 10s CFU/mL. Some strains are hyperproducers of ~-lactamase and yield resistant or borderline MICs to oxacillin and methicillin but will not be intrinsically resistant. '3 These strains are not usually multiresistant to drugs other than ~qactams. These strains are usually susceptible to amoxicillin-clavulanicacid (intrinsically oxacillin-resistant strains are not).
1. Multiple resistance to any or all of the following: erythromycin, aminoglycosides, chloramphenicol, tetracycline, or clindamycin. 2. Intermediate MIC to methicillin or oxacillin. 3. Failure to show cross-resistance between methiciilin, nafcillin, and oxacillin.
MHA
MIC Breakpoints (~g/mL)
35°C, full 24 h but no longer
Clues (flags) indicating resistance to methicillin, oxacillin, or nafcillin
Disk diffusion 's'~"
Use as a screening test for methicillin, oxacillin, nafcillin, or cloxacillin resistance. Oxacillin is the preferred test agent in most institutions. Inoculate plate by "pie-plating" or by "spot" inoculation with swab. Growth indicates chromosomal intrinsic resistance. Use as a second test in addition to the broth microdilution or disk diffusion test.
35°C, full 24 h but no longer
Do not use above MIC breakpoints for correlations with this test. The MIC breakpoint correlates for susceptible for this method are ~3 p.g/mL for methicillin and ~1.0 p.g/mL for oxacillin and nafcillin. Oxacillin is the preferred test agent in most institutions. Inoculum should be prepared as above. Look for growth within a zone of inhibition. Some strains are hyperproducers of 13-1actamase and may yield resistant zone sizes; these strains yield either susceptible, borderline, or resistant MICs. '~ These strains are usually susceptible to amoxicillin-clavulanic acid (intrinsically oxaciUin-resistant strains are not). Although we assume that most oxacillin-resistant staphylococci can be detected by agar dilution, we have not studied it. We do not have any recommendations for adding NaCI.
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T A B L E 1. Susceptibility Testing of Fastidious and Unusual Pathogens ~b (Continued)
Orc,anism S. epidermidis
Method
Media"
Incubation
Comments
Same tests that are used for S. aureus. We have much less experience with coagulase negative staphylococci (CNS) than with S. aureus. Recommendations are provisional. Approximately 2A of clinical isolates of CNS will be S. epidermidis. Approximately V2 of clinical CNS will be oxadllinresistant, and the percentage is even higher for S. epidermidis. Most disk diffusion experience has been with S. aureus, not CNS. Do not use nafdllin as a test agent. Most oxicillin-resistant strains are multiresistant. Use oxacillin and/or methicillin as the test agent(s); there is some advantage to using both with CNS. Use the agar screen test as a second test in addition to the microdilution or disk diffusion tests. Induce with oxacillin or methicillin prior to testing for 13-1actamase.
Clues (flags) indicatin,¢~ resistance to methicillin, oxacillin, or nafcillin 1. Multiple resistance to any or all of the following: erythromydn, aminoglycosides, chloramphenicol, tetracycline, or clindamycin. 2. Intermediate MIC to methicillin or oxacillin. 3. Failure to show cross-resistance between methicillin, nafcillin, and oxadllin.
S. saprophyticus
Same tests that are used for S. aureus.
May be a common urinary tract isolate. May produce small amounts of 13-1actamase. Usually intermediate in its susceptibility to oxacillin and methicillin so results may flip-flop from test to test, especially with disk diffusion tests. This resistance appears to be different from that seen with S. aureus and S. epidermidis and the screen test will be negative (no growth). These isolates often show test results such as pencillinsusceptible (13-1actamase negative) but oxicillin-resistant and negative (no growth) for the screen test. Penicillin MICs are usually 0.12 or 0.25 p~g/mL. The 13-1ac test usually requires/> 1 h to become positive and the color is usually not intense (induction does not change the reaction time or color intensity).
S. haemolyticus
Same tests that are used for S. aureus.
These isolates are usually the most resistant of the coagulase negative staphylococci. Induce with oxacillin or methicillin prior to testing for [3-1actamase.
Streptococcus pneumoniae
Broth microdilution'7'2"3'
CSMHB + 5% lysed horse blood
35°C, 18-24 h
MIC breakpoints for penicillin are: susceptible, <~0.06; relatively resistant, 0.12-1.0; and resistant, ~>2 ~g/mL. Oxacillin MICs of -~0.25 #,g/mL indicate susceptibility to penicillin; oxacillin MICs of ~>1.0 indicate resistance or relative resistance to penicillin; oxacillin MICs of 0.5 i~g/mL are equivocal.
Disk diffusion screen MHA + 5% for penidUin sheep blood resistance '~,2.,3~
Using a 1-p,g oxacillin disk, a zone of ~<19 mm almost always indicates penicillin resistance or relative resistance; an oxacillin zone of/>20 mm indicates susceptible. Oxacillin is preferred over methicillin as the test agent. 2" Do not report result in terms of susceptibility to oxacillin, but rather as penicillin susceptibility.
Disk diffusion '~''~
Chloramphenicol breakpoints are the same as the NCCLS: R ~< 12 mm, S I> 18 mm. '~'' Testing penicillin by disk diffusion may lead to reporting of false susceptibility (use the oxacillin screen test above). It is important to test for chloramphenicol resistance, especially if penicillin resistance is also detected.
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MHA + 5% sheep blood
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THE ANTIMICROBICNEWSLETTER,VOLUME4, NUMBER6, JUNE 1987
TABLE
1. Susceptibility Testing of Fastidious and Unusual Pathogens ~b (Continued)
Organism
Method
Streptococcus spp. Broth microdilution' 7.~,
Streptococci, pyridoxal dependent
Broth microdilution ~''7''~
Broth dilution ~7'~ Fastidious or Agar dilution '7"~ unusual organisms not m e n t i o n e d above
Media ~ CSMHB + 5% lysed horse blood
Incubation
Comments
35°C, 18-24 h in Nonenterococcal streptococci that grow well in this CO2 if necessary m e d i u m (and may require CO2) may also be tested by the standard NCCLS disk diffusion methods. ~~" The oxacillin screen test as u s e d for pneumococci does not w o r k (ie, to indicate pencillin resistance) for these organisms. Pencillin resistance occurs most often in S. mitis but does occur in other species. For G r o u p A streptococci, erythromycin resistance occurs; zones of 318 m m correlate well with MICs of 40.12 ~g/mL and zones of <18 m m w i t h MICs of 3 2 p,g/mL (unpublished data).
CSMHB + 5% 35°C, 18-24 h in G r o w organisms on agar containing 0.001% pyridoxal lysed horse CO2 if necessary HC1. Alternatively, 1% pyridoxal HCi can be swabbed onto the surface of a blood agar plate prior to blood + 0.001% inoculation. pyridoxal HCi Prepare i n o c u l u m suspension from growth on plate.' Use MuellerHinton and s u p p l e m e n t s as necessary. Trial and error m a y be necessary to determine needs,
35°C, 18-24 h or If unusual s u p p l e m e n t s or a t m o s p h e r e is necessary, report M1C as not d o n e with a standardized test. longer. Use required atmosphere. Trial and error m a y be necessary to determine needs.
" For this document the following definitions are used: A fastidious organism is one that does not grow adequately in or on unsupplemented Mueller-Hinton media for antimicrobial susceptibility tests. An unusual organism is one for which standard methods have not been developed or for which it has not been established that it can be tested with a standard method such as the NCCLS disk-diffusion standard. b In general, the inoculum for most of these tests is preferably prepared directly from growth off of agar plates.~ It is best to use as fresh a plate as possible but some organisms may l~Kluire 48 h for adequate growth. Suspend the growth into a clear broth and standardize the suspension to match a 0.5 McFarland Standard. Then make appropriate dilutions de pesnding on type of system or method being used (see directions for specific organisms). Inoculum for broth microdilution should be 1-5 x 10 CFU/mL, and for agar, 1-5 x 1 0 4 CFU/spot. Abbreviations for media or reagents: MHA - Mueller-Hinton agar CSMHB - Cation-supplemented Mueller-Hinton broth ~7 BHI - Brain heart infusion broth NAD - Nicotinamide adenine dinucleotide SXT - Sulfamethoxazole-trimethoprim ,t The lysed blood used in these tests is lysed by freezing, thawing at least 6 times and then adding an equal volume of sterile distilled water. The solution is clarified by centrifugation (10,000 x g) for 20 min and subsequent decantation of the supemate (not necessary if it is to be added to agar). In some tests, the lysed horse blood could probably be replaced with blood from other species, but we have always used horse blood. We do not recommend sheep blood for tests with Haemophilus or with sulfonamides. ' Kellogg's Supplement: By stirring, dissolve in' 100 mL distilled water, 40 g dextrose, 1 g glutamine, and 1 mL of 0.2% thiamine pyrophosphate. Then add 85 mg Fe(NO3)3 • 9 1-120and stir until dissolved. Filter, sterilize, and freeze in appropriate portions.
REFERENCES
1. Baker CN, T h o r n s b e r r y C, H a w k i n s o n RW: I n o c u l u m stand a r d i z a t i o n in antimicrobial susceptibility testing: Evaluation of o v e r n i g h t agar cultures and the Rapid I n o c u l u m S t a n d a r d i z a t i o n System. J Clin Microbiol 17:450457, 1983.
(c) 1987 BY ELSEVIERSCIENCE PUBLISHINGCO., INC.
2. Baker CN, Hollis G, T h o r n s b e r r y C: Antimicrobial susceptibility testing of Francisella tularensis w i t h a m o d i f i e d M u e l l e r - H i n t o n broth. J Clin Microbiol 22:212-215, 1985. 3. Bauer AW, Kirby W M M , Sherris JC, et al: Antibiotic susceptibility by a s t a n d a r d i z e d single disk m e t h o d . A m J Clin Pathol 45:493496, 1966.
4. Biddle JW, S w e n s o n JM, Thornsberry C: Disc agar diffusion antimicrobial susceptibility tests w i t h ~-iactamase p r o d u c i n g Neisseria gonorrhoeae. J Antibiot 31:352-358, 1978. 5. C o o k s e y RC, S w e n s o n JM: In vitro antimicrobial inhibition p a t t e r n s of nutritionally variant streptococci. Antimicrob Agents Chemother
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16:514-518, 1979. 6. Elwell LP, DeGraaff J, Seibert D, Falkow S: Plasmid-linked ampicillin resistance in Haemophilus influenzae type b. Infect Immun 12:404410, 1975. 7. Elwell LP, Roberts M, Mayer L, et al: Plasmid-mediated 13-1actamase production in Neisseria gonorrhoeae. Antimicrob Agents Chemother 11:538-533, 1977. 8. Gavan TL, Jones RN, Barry AL, et al: Quality control limits for ampicillin, carbenicillin, mezlocillin, and piperacillin disk diffusion susceptibility tests: A collaborative study. J Clin Microbiol 14:67-72, 1981. 9. Kirven LA, Thornsberry C: Minimum bactericidal concentrations of sulfamethoxazole-trimethoprim for Haemophilus influenzae: Correlation with prophylaxis. Antimicrob Agents Chemother 14:731-736, 1978. 10. Kurzynski TA, Yrios JW, Helstad AG, et al: Aerobically incubated thioglycolate broth disk method for antibiotic susceptibility testing of anaerobes. Antimicrob Agents Chemother 10:727-732, 1976. 11. Lennette EH, Balows A, Hausler WJ, et al: Manual of Clinical Microbiology, 3rd ed. American Society for Microbiology, Washington, D.C., 1980. 12. Luman I, Wilson RW, Wallace RJ, et al: Disk diffusion susceptibility of Brahamella catarrhalis and relationship of 13-1actam zone size to 13-1actamase production. Antimicrob Agents Chemother 30:774776, 1986. 13. McDougal LK, Thornsberry C: The role of 13-1actamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporins. J Clin Microbiol 23:832-839, 1986. 14. Mendelman PM, Chaffin DO, Clausen C, et al: Failure to detect ampicillin-resistant, non-13-1actamase-producing Haemophilus influenzae by standard disk diffusion testing. Antimicrob Agents Chemother 30:274-280. 15. National Committee for Clinical Laboratory Standards. Approved Standard M2-A3. Performance standards for antimicrobic disk susceptibility tests, 3rd edition,
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16.
17.
18.
19.
20.
21.
22.
23.
24.
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1984. National Committee for Clinical Laboratory Standards. Villanova, PA. National Committee for Clinical Laboratory Standards. Approved Standard Mll-A. Approved reference dilution procedure for antimicrobic susceptibility testing of anaerobic bacteria, 1985. National Committee for Clinical Laboratory Standards, Villanova, PA. National Committee for Clinical Laboratory Standards. Approved standard M7-A. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 1985. National Committee for Clinical Laboratory Standards, Vilanova, PA. National Committee for Clinical Laboratory Standards. Proposed Standard M17-P. Alternative methods for antimicrobial susceptibility testing of anaerobic bacteria, 1985. National Committee for Clinical Laboratory Standards, Villanova, PA. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing: First informational supplement, 1986. National Committee for Clinical Laboratory Standards, Villanova, PA. Rein ME, Elliott WC, Swenson JM, et al: Sulfamethoxazole-trimethoprim synergism for Neisseria gonorrhoeae. Antimicrob Agents Chemother 17:247-250, 1980. Richmond MH, Sykes RB: The 13lactamases of gram-negative bacteria and their possible physiological role, in Rose AH, Wilkinson JF (eds): Advances in Microbiology and Physiology, vol. 9. New York, Academic Press, 1973. Sanders CC, Sanders WE: Emergence of resistance to cefamandole: Possible role of cefoxitin-inducible beta-lactamases. Antimicrob Agents Chemother 7:15-21, 1979. Stalons DR, Thornsberry C: Brothdilution method for determining the antibiotic susceptibility of anaerobic bacteria. Antimicrob Agents Chemother 7:15-21, 1975. Swenson JM, Thornsberry C: Susceptibility tests for sulfamethoxazole-trimethoprim by a broth microdilution procedure. Current Microbiology 1:189-193, 1978.
25. Swenson JM, Thornsberry C, Silcox VA: Rapidly growing mycobacteria: Testing of susceptibility to 34 antimicrobial agents by broth microdilution. Antimicrob Agents Chemother 22:186-192, 1982. 26. Swenson JM, Hill BC, Thornsberry C: Screening pneumococci for penicillin resistance. J Clin Microbiol 24:749-752, 1986. 27. Thornsberry C, Biddle JW, Kirven LA, et al: Penicillin resistance in Neisseria gonorrhoeae due to 13-1actamase production. Microbios 20:3946, 1978. 28. Thornsberry C, Gavan TL, Gerlach EH: New developments in antimierobial agent susceptibility testing. Cumitech 6, American Society for Microbiology, Washington, D.C., 1977. 29. Thornsberry C, McDougal LK: Successful use of broth microdilution in susceptibility tests for methicillin-resistant (heteroresistant) staphylococci, l Clin Microbiol. 18:1084-1091, 1983. 30. Thornsberry C, Swenson IM: Antimicrobial susceptibility testing of anaerobes. Laboratory Medicine 9:43--48, 1978. 31. Thornsberry C, Swenson JM: Antimicrobial susceptibility tests for Streptococcus pneumoniae. Laboratory Medicine 11:83-86, 1980. 32. Wilkins TD, Thiel T: Modified broth-disc method for testing the antibiotic susceptibility of anaerobic bacteria. Antimicrob Agents Chemother 3:350--356, 1973. 33. Zigheiboin S, Tomasz A: Penicillin binding proteins of multiply antibiotic-resistant South African strains of Streptococcus pneumoniae. Antimicrob Agents Chemother 17:434 441, 1980.
GENERAL REFERENCES
1. Thornsberry C: (section editor). Section XI. Laboratory tests in chemotherapy, in Lennette EH, Balows A, Hausler WJ, et al (eds): Manual of Clinical Microbiology, 4th ed, American Society for Microbiology, 1985. 2. Lorian V (ed.): Antibiotics in Laboratory Medicine, 2nd ed., Williams and Wilkins, Baltimore, MD, 1986.
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