SIXTH EDITION
`
`Manual of
`Clinical Microbiology
`
`EDITOR IN CHIEF
`PATRICK R. MURRAY
`Departments of Pathology and Medicine, Washington University
`School of Medicine, St. Louis, Missouri
`
`EDITORS
`ELLEN JO BARON
`Department of Medicine, University of California,
`Los Angeles, California
`
`MICHAEL A. PFALLER
`Department of Pathology, University of Iowa Hospitals and Clinics,
`Iowa City, Iowa
`
`FRED C. TENOVER
`Hospital Infections Program, Centers for Disease Control and Prevention,
`Atlanta, Georgia
`
`ROBERT H. YOLKEN
`Department of Pediatric Infectious Diseases, Johns Hopkins Hospital,
`Baltimore, Maryland
`
`•''""""
`
`ASMPRESS
`Washington, D.C.
`
`M
`
`

`

`Copyright© 1970, 1974, 1980, 1985, 1991, 1995
`American Society for Microbiology
`1325 Massachusetts Avenue, N.W.
`Washington, DC 20005
`
`Library of Congress Cataloging-in-Publication Data
`
`Manual of clinical microbiology.-6th ed./editor in chief, Patrick R. Murray:
`editors, Ellen Jo Baron ... [et a!.]
`p. em.
`h}cludes bibliographical references and index.
`ISBN 1-55581-086-1 (hard cover)
`.Z. Medical microbiology-Handbooks,
`1. Diagnostic microbiology-Handbooks, manuals, etc.
`II. American Society for Microbiology.
`I. Murray, Patrick R.
`manuals, etc.
`[DNLM: 1. Microbiology.
`QW 4 M294 1995]
`QR67.M36 1995
`616'.01-dc20
`DNLM/DLC
`for Library of Congress
`
`94-44246
`CIP
`
`10 9 8 7 6 5 4 3 2
`
`All Rights Reserved
`Printed in the United States of America
`
`Cover figure: Nocardia asteroides complex in an expectorated sputum specimen stained with fluorescein.
`The photograph was taken with a Zeiss fluorescent microscope at a magnification of X 1 ,000.
`(Courtesy of Patrick R. Murray.)
`
`

`

`Antibacterial Susceptibility Tests: Dilution and Disk
`Diffusion Methods
`GAIL L. WOODS AND JOHN A. WASHINGTON
`
`Antibacterial susceptibility testing may be performed by
`either dilution or diffusion methods. The choice of meth(cid:173)
`odology is often based on many factors, including relative
`ease of performance, flexibility, use of automated or semi(cid:173)
`automated devices for both identification and susceptibility
`testing, and, in some instances, misconceptions regarding
`the clinical importance of MICs versus the interpretive
`results that can be provided by the disk diffusion method.
`These misconceptions are often based on an assumption
`that the dilution test is the more accurate of the two
`methods and that physicians prefer a quantitative result.
`Since there is a direct relationship between the zone of
`inhibition diameter and the MIC and since MICs and zone
`of inhibition diameters with reference strains are reproduc(cid:173)
`ible on an interlaboratory and intralaboratory basis, there is
`no evidence to support the impression that one method is
`more accurate than the other. The fact is that a numerical
`or quantitative result reported from a conventional dilution
`test should not be misconstrued as representing greater
`accuracy, since the actual MIC may be a concentration
`somewhere between the concentration inhibiting the or(cid:173)
`ganism and the next lowest concentration tested (i.e., 1
`log2 dilution lower). For example, an organism may grow in
`the presence of an antimicrobial agent at a concentration of
`4 M-g/ml and be inhibited at the next higher concentration
`tested, 8 M-g/ml. The actual MIC could be anywhere be(cid:173)
`tween 4.1 and 8 M-g/ml. Moreover, few physicians other
`than those specializing in the field of infectious diseases can
`interpret MICs, so the laboratory is obliged to provide an
`interpretation of MICs anyway to avoid any risk of their
`misinterpretation. Specific ind\s:;ations for performing dilu(cid:173)
`tion tests are not well agreed upon, but they may be limited
`to isolates from patients with endocarditis, osteomyelitis,
`and meningitis. MICs can be useful for evaluating relative
`degrees of susceptibility of bacteria to various antimicrobial
`agents and for comparing the relative activities of drugs
`against various species. Finally, MICs can be useful in the
`delineation of relative resistance and the intermediate cat(cid:173)
`egory. In general, however, the decision as to whether to
`perform dilution or disk diffusion testing is usually based on
`logistical reasons, such as the laboratory's selection of a
`system for providing both identification ~nd susceptibility
`testing. Many such systems are now commercially available;
`however, the major challenge posed by such systems is the
`flexibility available in the panel of antimicrobial agents to
`
`be tested. Limited flexibility of commercially available
`products has led many laboratories back to the use of disk
`diffusion testing, which is highly flexible in this regard.
`The selection of antibacterial agents is complicated by
`the sheer number of agents available today. Many of these
`compounds, however, exhibit similar if not identical activ~
`ities in vitro, so that one compound representing the par(cid:173)
`ticular spectrum class of agents can be selected for testing
`purposes. It is important in this regard that the microbiol(cid:173)
`ogist work with the hospital formulary committee to ensure
`that the antibacterial agents being tested in the laboratory
`reflect those in the hospital's formulary. Failure to do so can
`contribute to antibiotic misuse (29). Guidelines for the
`selection of antibacterial agents to be tested have been
`published by the National Committee for Clinical Labora(cid:173)
`tory Standards (NCCLS) (Table 1) (24, 25). While this
`listing is sometimes regarded as the standard for selection of
`those agents to be tested, it should be emphasized that it is
`a guideline only and that many variables go into the deci(cid:173)
`sion as to what agents should be tested in any particular
`setting. The NCCLS also cautions that the decision as to
`which agents should be tested and reported on selectively
`should be made by the clinical microbiologist in conjunc(cid:173)
`tion with the infectious disease practitioner, the pharmacy,
`and/or the infection control committee. (For further dis(cid:173)
`cussion of the selection of antimicrobial agents to be tested,
`see chapter 110 of this Manual.)
`
`DILUTION METHODS
`Dilution susceptibility testing methods are used to deter(cid:173)
`mine the minimal concentration, usually expressed· in mi(cid:173)
`crograms per milliliter, of antimicrobial agents required to
`inhibit or kill a microorganism. Procedures for determining
`antimicrobial inhibitory activity are carried out by either
`agar- or broth-based methods. Antimicrobial agents are
`usually tested at log2 (twofold) serial dilutions, and the
`lowest concentration that inhibits visible growth of an
`organism is recorded as the MIC. The concentration range
`used may vary with the drug, organism identification, and
`site of infection. Ranges should include concentrations that
`allow determinations of the interpretive categories (i.e.,
`susceptible, intermediate, and resistant) and also the ac(cid:173)
`ceptable ranges for quality control reference strains. Other
`dilution methods include those that test a single or a
`
`1327
`
`

`

`1328 • ANTIMICROBIAL AGENTS AND SUSCEPTIBILITY TESTING
`
`TABLE 1 Antimicrobial agents recommended for routine dilution and disk diffusion susceptibility testinga
`
`Groupb recomm~nded for testing:
`
`Antimicrobial agent
`
`Enterobacteriaceae
`
`Pseudomonas spp.
`and other non(cid:173)
`Enterobacteriaceae
`
`Staphylococci
`
`Enterococci
`
`Streptococc;i
`other than S.
`jJneumoniae
`
`Penicillins
`Penicillin G
`Ampicillin
`Oxacillin ot methicillin
`Ticarcillind
`Mezlocillind
`Piperacillin
`Azlocillind
`Ampicillin-sulbactam
`Amoxicillin-clavulanic acid
`Piperacillin-tazobactam
`Ticarcillin-clavulanic acid
`
`A
`
`~
`
`Cephalosporins
`Cephalothin
`Cefazolin
`Cefamandole
`Cefonicid
`Cefuroxime
`Cefmetazole
`Cefoperazone
`Cefoxitin
`Cefotetan
`Cefotaxime
`Ceftriaxone
`Ceftizoxime
`Ceftazidime
`Cefepime
`
`Other beta-lactams
`lmipenem
`Aztreonam
`
`Aminoglycosides
`Gentamicin
`Netilmicin
`Tobramycin
`Amikacin
`
`Macro !ides
`Azithromycin
`Clarithromycin
`Erythromycin
`Quinolones
`Ciprofloxacin
`Lomefloxacin
`Norfloxacin
`Ofloxacin
`
`Miscellaneous
`Chloramphenicol
`Clindamycin
`Nitrofurantoin
`Rifampin
`Sulfisoxazole
`Tetracycline
`Trimethoprim-sulfamethoxazole
`Trimethoprim
`Vancomycin
`
`[]
`~ B
`[[]
`~ c
`
`B
`
`c
`
`B
`B
`
`A
`c
`c
`c
`
`B
`[g_J
`c
`
`c
`
`D
`
`D
`c
`B
`D
`
`B
`11
`
`B
`
`u
`c
`
`B
`B
`
`A
`c
`B
`B
`
`B
`
`[gJ
`c
`
`c
`
`i<l~•·.- I
`
`ce
`
`B
`B
`B
`c
`[g_J
`c
`
`c
`B
`D
`c
`D
`C'
`B
`D
`A
`
`B
`
`[g_J
`c
`
`B
`D
`
`c
`
`u
`[g_J
`c
`
`D
`
`D
`
`B
`(Footnotes oh next page)
`
`

`

`113. Dilution and Disk Diffusion Testing Methods •
`
`1329
`
`selected few concentrations of antimicrobial agents (i.e.,
`breakpoint susceptibility testing and single-drug-concentra(cid:173)
`tion screens).
`Dilution methods offer flexibility in the sense that the
`standard medium used to test routinely encountered micro(cid:173)
`organisms (e.g., staphylococci, members of the family En(cid:173)
`terobacteriaceae, Pseudomonas aeruginosa) may be readily
`supplemented or even replaced with another medium to
`allow accurate testing of various fastidious bacteria not
`reliably tested by disk diffusion. Dilution methods are also
`adaptable to automated systems. In addition, if plates or
`panels are prepared in-house, the combination of antibiot(cid:173)
`ics to be included is not limited. Any drug available in
`powder form may be used.
`The flexibility of dilution testing is also evident in the
`reporting formats that may be used. Quantitative results
`(MICs in micrograms per milliliter), category results (sus(cid:173)
`ceptible, intermediate, or resistant), or both may be used.
`
`DILUTION TESTING: AGAR METHOD
`
`Dilution of Antimicrobial Agents
`The solvents and diluents needed to prepare stock solutions
`of most commonly used antimicrobial agents are presented
`ig_the NCCLS document on dilution testing (25).
`
`Preparation and Storage of Antimicrobial Solutions
`A twofold dilution scheme for an antimicrobial agent that
`starts with an antimicrobial stock concentration of 5,120
`[Lg/ml is shown in Table 2 (17, 25). Depending on the
`number of tests to be prepared, the actual volumes used are
`proportionally increased. Certain intermediate concentra(cid:173)
`tions are also used as the starting concentrations for the
`preparation of subsequent dilutions, and this should be
`taken into account when actual volume needs are being
`calculated. Although there are other approaches to obtain(cid:173)
`ing a range of doubling dilutions, the scheme outlined in
`Table 2 is convenient, is economical in pipette use, and
`avoids some of the cumulative error inherent in traditional
`serial dilution methods.
`
`Preparation, Supplementation, and Storage
`of Media
`Mueller-Hinton agar is the recommended medium for test(cid:173)
`ing most commonly encountered aerobic and facultatively
`anaerobic bacteria. The dehydrated agar base is commer(cid:173)
`cially available and should be prepared as described by the
`
`manufacturer. Before sterilization, the molten agar is dis(cid:173)
`tributed into screw-cap containers in exact aliquots suffi(cid:173)
`cient to dilute the intermediate antimicrobial concentra(cid:173)
`tions 10-fold. Containers, one for each drug concentration
`to be tested, are sterilized by autoclaving at 121 oc for 15
`min and allowed to equilibrate to 48 to 50oC in a preheated
`water bath. Once the containers are equilibrated, the ap(cid:173)
`propriate volume of intermediate antimicrobial concentra(cid:173)
`tion is added, and the container contents are mixed by
`gentle inversion, poured into 100-mm round or square
`sterile plastic plates set on a level surface, and allowed to
`solidify. For growth controls, plates containing drug-free
`agar are also prepared. All plates should be poured to a
`depth of 3 to 4 mm (20 to 25 ml of agar per plate), and the
`pH of each batch should be checked to confirm the appro(cid:173)
`priate pH range of 7.2 to 7.4.
`After sterilization and temperature equilibration of the
`molten agar, any necessary supplements are aseptically
`added to the Mueller-Hinton agar. For testing streptococci,
`supplementation with 5% defibrinated sheep or horse blood
`is recommended (25). However, sheep blood supplementa(cid:173)
`tion may antagonize the activities of sulfonamide and tri(cid:173)
`methoprim ( 8); therefore, if these drugs are to be tested, 5%
`lysed horse blood should be used. The presence of blood
`also affects results with novobiocin and nafcillin as well as
`the in vitro activities of cephalosporins against enterococci
`(9, 10, 31, 34); therefore, blood supplementation should
`not be used unless necessary for bacterial growth. Perfor(cid:173)
`mance standards of Mueller-Hinton agar have been defined
`sufficiently that calcium and magnesium suppletnentation
`should not be done. The agar should be supplemented with
`2% NaCl for testing of methicillin or oxacillin against
`staphylococci (21).
`Prepared plates should be sealed in plastic bags and
`stored at 4 to 8°C. If the plates are for research or reference
`studies, they should be used within 5 days of preparation;
`for routine clinical laboratory purposes, there may be no
`time limit as long as control strains that are tested routinely
`give accurate results within acceptable ranges. Before inoc(cid:173)
`ulation, plates that have been stored under refrigeration
`should be allowed to equilibrate to room temperature, and
`the agar surface should be dry.
`
`Inoculation Proc{)dures
`Variations in inoculum size may substantially affect MIC
`endpoint determinations; therefore, careful preparation is
`required to obtain accurate results. The recommended final
`
`(Footnotes to Table 1)
`
`"Modified from NCCLS data (24, 25) with permission. Current standards and supplements to them may be obtained from NCCLS, 771 East Lancaster Ave.,
`Villanova, PA 19085.
`bGroup A comprises primary drugs to be tested and reported; group B comprises those to be tested as primary drugs but reported selectively; group C comprises
`supplemented drugs to be reported selectively; and group D comprises drugs to be tested against urinary isolates only. Groups known to have nearly identical
`antimicrobial activities are enclosed in boxes. Because the clinical efficacies of the agents within each group should be similar, only one from each group need be tested.
`'Results of testing penicillin apply to other penicillins (e.g., ampicillin, amoxicillin, carboxy- and ureidopenicillins) versus {3-lactamase-negative enterococci.
`dCombination therapy consisting of penicillin, ampicillin, or vancomycin and an aminoglycoside is recommended.
`"Staphylococci resistant to the penicillinase-resistant penicillins should also be considered resistant to penicillins, beta-lactam-{3-lactamase inactivating combi(cid:173)
`nations, cephalosporlns, and imipenetn.
`fTicarcillin-clavulanic acid should not be considered as a therapeutic alternative for P. aeruginosa isolates resistant to carboxy- or ureidopenicillins.
`I· gCephalothin test results may also be used to represent cephapirin, cephradine, cephalexin, cefaclor, cefadroxil, and cefazolin (except against the Enterobacteriaceae).
`Cefuroxime, cefpodoxime, cefproxil, and loracarbef may be tested separately on a supplemental basis because their activities may be greater against the Enterobacteriaceae
`than are those of cephalothin or cefazolin.
`.
`hFor use of aminoglycosides to screen enterococci for $ynergy resistance, see sectiol! on Breakpoint Susceptibility Testing and Single-Drug-Concentration Screens
`for Resistance.
`'Doxycycline or minocycline may be tested on a supplemental basis against some nonfennentative gram-negative bacilli and staphylococci.
`
`

`

`1330 • ANTIMICROBIAL AGENTS AND SUSCEPTIBILITY TESTING
`
`TABLE 2 Antibacterial agent dilution schedule for agar and broth susceptibility methodsa
`
`Step
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`
`Starting solution of
`antimicrobial agent
`
`Concn
`(J.tg/ml)
`
`5,120
`5,120
`5,120
`1,280
`1,280
`1,280
`160
`160
`160
`20
`20
`20
`2.5
`
`No. of
`volb
`
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`1
`
`No. of volb ·of
`distilled water
`
`il
`
`Intermediate
`concn (f.tg/ml)c
`
`0
`1
`3
`1
`3
`7
`1
`3
`7
`1
`3
`7
`1
`
`5,120
`2,560
`1,280
`640
`320
`160,,
`so"·
`40
`20
`10
`5
`2.5
`1.25
`
`Final concn
`( p,g/ml) after
`1: 10 dilution h\
`agar or broth
`
`512 (256)
`256 (128)
`128 (64)
`64 (32)
`32 (16)
`16 (8)
`8 (4)
`4 (2)
`2 (1)
`1 (0.5)
`0.5 (0.25)
`0.25 (0.125)
`0.125 (0.0625)
`
`"Adapted from references 17 arid 25.
`bVolume proportions remain constant, but the actual number of volumes selected depends on the number of plates, tubes, or trays to be prepared.
`cconcentrations in boldface type are dilutions used for preparing the correspo11ding final concentration and as starting concentrations in the preparation of
`subsequent dilutions.
`dFor agar dilution and broth microdilution methods that use inoculum volumes of 0.001 to 0.005 ml to inoculate 0.1 ml of drug-containing broth, the final
`concentration is also the actual testing concentration. For macrodilution and microdilution methods in which the inoculum dilutes the final drug concentration 1:2,
`the actual testing concentrations are given in parentheses (see text for discussion of macrodilution inoculation procedures).
`
`inoculum for agar dilution is 104 CFU per spot, and it may
`be prepared in either of two ways. Four or five colonies are
`picked from overnight growth on agar-based medium and
`inoculated into 4 to 5 ml of suitable broth that will support
`good growth (Mueller-Hinton, brain heart infusion, Tryp(cid:173)
`ticase soy [BBL Microbiology Systems, Cockeysville, Md.],
`or tryptic soy [Difco Laboratories, Detroit, Mich.]). Broths
`are incubated at 35°C until turbid, and the turbidity is
`adjusted to match that .of a 0.5 McFarland standard (ca. 108
`CFU/ml). Alternatively, colonies from overnight growth
`may be suspended in broth to a turbidity that matches the
`McFarland standard, eliminating the time needed for grow(cid:173)
`ing the inoculum in broth (25). By using either sterile broth
`or normal saline, a 1:10 dilution of the suspension is made
`to give an adjusted concentration of 107 CFU/ml (25),
`Once the adjusted inoculum is prepared, inoculation of
`the antimicrobial agent plates should be accomplished
`within 30 min, since longer delays may lead to significant
`changes in inoculum size. By using either a calibrated loop
`or an inoculum replicating device, 0.001 to 0.002 ml of the
`107 -CFU/ml suspension is delivered to the agar surface
`resulting in the final desired inoculum of approximately 104
`CFU per spot. For convenience, use of a replicator is
`preferred, because a-consistent inoculum volume for up to
`36 different isolates is simultaneously delivered. To use this
`device, an aliquot of the adjusted inoculum for each isolate
`is pipetted into the appropriate well of the seed plate, and
`a multiprong inoculator is used to pick up and gently
`transfer 0.002 ml from the wells to the agar surfaces. The
`surface of the agar plates must be dry before inoculation,
`which should begin with the lowest drug concentration. To
`check for viability of each test isolate and also as an added
`check for purity, control plates not containing drug are
`inoculated last. Finally, plates should be clearly marked so
`that orientations of the different isolates being tested on
`each plate are known.
`
`Incubation
`Inoculated plates are allowed to stand until inocula have
`been completely absorbed by the medium; then they are
`inverted and incubated in air at 35°C for 16 to 20 h before
`being read. To facilitate detection of vancomycin-resistant
`enterococci and methicillin-resistant staphylococci, plates
`containing vancomycin and either oxacillin or methicillin,
`respectively, should be incubated for a full24 h before being
`read (see chapter 116 of this Manual). Incubation in the
`presence of increased C02 is not recommended.
`---
`
`Interpretation and Reporting of Results
`Before reading and recording the results obtained with
`clinical isolates, those obtained with quality control strains
`should be checked to ensure that they are within the
`acceptable accuracy ranges (see Quality Control below),
`and drug-free control plates should be examined for isolate
`viability and purity. Endpoints for each antibiotic are best
`determined by placing plates on a dark background and
`observing for the lowest concentration that inhibits visible
`growth, which is recorded as the MIC. A single colony or a
`faint haze left by the initial inoculum should not be re(cid:173)
`garded as growth. If two or more colonies persist at antimi(cid:173)
`crobial concentrations beyond an otherwise obvious end(cid:173)
`point or if there is no growth at lower concentrations but
`growth at higher concentrations, the isolate should be
`subcultured to confirm purity, and the test should be re(cid:173)
`peated. Substances that may antagonize the antibacterial
`activities of sulfonamides and trimethoprim may be carried
`over with the inoculum and cause "trailing," or less definite
`endpoints (8, 9). Therefore, the MICs for these antimicro(cid:173)
`bial agents should be interpreted as the endpoint at which
`an obvious 80 to 90% diminution of growth occurs.
`The MIC of each antimicrobial agent is usually·recorded
`in micrograms per milliliter. These quantitative results may
`be reported with the appropriate corresponding interpretive
`
`

`

`113. Dilution and Disk Diffusion Testing Methods •
`
`1331
`
`i il
`
`categories (susceptible, intermediate, or resistant), or the
`interpretive category may be reported alone. The MIC
`interpretive standards for these susceptibility categories, as
`recommended by the NCCLS, are provided in Table 3. For
`detailed instructions concerning the use of these criteria
`and categories, the latest NCCLS standards for dilution
`testing methods should be consulted. Note that the inter(cid:173)
`pretive standards for most of the penicillin-class drugs vary
`with the organism being tested.
`The three interpretive categories are defined as follows.
`Susceptible indicates that an infection caused by the tested
`microorganism may be appropriately treated with the usu(cid:173)
`ally recommended dose of antibiotic. Intermediate indi(cid:173)
`cates that the isolate may be inhibited by attainable con(cid:173)
`centrations of certain drugs (e.g., beta-lactams) if higher
`dosages can be used or if the infection involves a body site
`in which the drug is physiologically concentrated (e.g.,
`urinary tract). The intermediate category also serves as a
`buffer zone that prevents technical artifacts from causing
`major interpretive discrepancies. Resistant isolates are not
`inhibited by the concentration of antibiotic achievable
`with the recommended dose and/or yield results that fall
`within a range in which specific resistance mechanisms are
`likely to be present (25).
`
`Achrantages and Disadvantages
`Dilution testing by the agar method is a well-standardized,
`reliable susceptibility-testing technique that may be used as
`a reference for evaluating the accuracy of other testing
`systems. In addition, the simultaneous testing of several
`isolates is possible, and microbial contamination or heter(cid:173)
`ogeneity is more readily detected than by broth methods.
`The major disadvantages of the agar method are associated
`with the time-consuming and labor-intensive tasks of pre(cid:173)
`paring the plates, especially as the number of different
`antimicrobial agents to be tested against each isolate in(cid:173)
`creases.
`
`DILUTION TESTING: BROTH METHODS
`The general approaches for broth methods include macro(cid:173)
`broth dilution, in which the broth volume for each anti(cid:173)
`microbial concentration is 2:1.0 ml contained in 13- by
`100-mm tubes, and mlcrobroth dilution, in which antimi(cid:173)
`crobial dilutions are in 0.05- to 0.1-ml volumes contained
`in wells of microtiter trays.
`
`Macrobroth Dilution Methods
`
`Dilution of Antimicrobial Agefits
`Stock solutions are prepared as discussed in the NCCLS
`document on dilution testing (25), and a typical dilution
`scheme that may be used to obtain full-range doubling
`dilutions is outlined in Table 2. As in the agar method, the
`actual volumes used for the dilutions would be proportion(cid:173)
`ally increased according to the number of tests being pre(cid:173)
`pared, with a minimum of 1.0 ml needed for each drug
`cqncentration. Because addition of the inoculum results in
`a 1:2 dilution of each concentration, all final drug concen(cid:173)
`trations must be prepared at twice the actual desired testing
`concentration (see Inoculation Procedures''below ). If not,
`the actual testing concentrations will be those given par(cid:173)
`enthetically in Table 2.
`
`Preparation, Supplementation, and Storage of Media
`Cation-adjusted Mueller-Hinton broth (CAMHB) is rec(cid:173)
`ommended for routine testing of commonly encountered
`nonfastidious organisms (25). AdJustment with the cations
`Ca2+ (20 to 25 mg/liter) and Mg + (10 to 12.5 mg/liter) is
`required to ensure acceptable results when P. aeruginosa
`isolates are tested against aminoglycosides and when tetra(cid:173)
`cycline is tested against other bacteria. Many manufacturers
`provide Mueller-Hinton broth that already has appropriate
`concentrations of divalent cations, so care must be taken
`not to supplement such products with additional cations. If
`cation concentrations are below the rec;ommended levels,
`adjustment is accomplished by the addition of suitable
`volumes of filter-sterilized, chilled CaCl2 stock (3.68 g of
`CaC12 • 2H20 dissolved in 100 ml of deionized water for a
`concentration of 10 mg of Ca2+ per ml) and MgCl2 stock
`(8.36 g of MgC12 • 6H20 in 100 ml of deionized water for a
`concentration of 10 mg of MgZ+ per ml) to the cooled
`broth (6, 25). Insufficient cation concentrations result in
`increased aminoglycoside activity (13, 19, 30, 35), while
`excess cation content results in decreased aminoglycoside
`activity against P. aeruginosa (5, 13, 19, 30). Reliable de(cid:173)
`tection of staphylococcal resistance to oxacillin, methicil(cid:173)
`lin, or nafcillin requires that the CAMHB used for testing
`these drugs be supplemented with 2% NaCl (21, 25).
`Appropriately adjusted and supplemented Mueller-Hin(cid:173)
`ton broth is used to prepare the final antimicrobial agent
`concentrations according to the scheme shown in Table 2.
`To minimize evaporation and antibiotic deterioration,
`tubes should be tightly capped and stored at 4 to 8°C. For
`reference studies, the dilutions should be used within 5 days
`of preparation; for routine use, there may be no time limit
`as long as quality control accuracy ranges are maintained
`(see Quality Control below).
`
`Inoculation Procedures
`The recommended final inoculum is 5 X 105 CFU/ml.
`Isolates are inoculated into a broth that will support good
`growth and incubated until turbid, and the turbidity is
`adjusted to match that of a 0.5 McFarland standard (ap(cid:173)
`proximately 108 CFU/ml). Alternatively, four or five colo(cid:173)
`nies from overnight growth on a blood agar plate may be
`directly suspended in broth to match the turbidity of the
`McFarland standard (25). This alternative is recommended
`for testing methicillin, oxacillin, or nafcillin against staph(cid:173)
`ylococci but may also be used with other bacteria (25). A
`portion of the suspension is diluted 1:100 (106 CFU/ml)
`with broth or 0.85% saline. When 1 ml of this dilution is
`added to each tube containing 1 ml of the drug diluted in
`CAMHB, a final inoculum of 5 X 105 CFU/ml is achieved.
`Broth not containing antimicrobial agent is inoculated as a
`control for organism viability (growth control). All tubes
`should be inoculated within 15 to 20 min of inoculum
`preparation, and an aliquot of the inoculum should be
`plated to check for purity and inoculum size.
`
`Incubation
`Tubes are incubated in air at 35°C for 16 to 20 h before
`being read. Incubation should be extended to a full 24 h for
`the detection of vancomycin-resistant enterococci and me(cid:173)
`thicillin-resistant staphylococci (25). Use of increased C02
`is not recommended.
`
`Interpretation and Reporting of Results
`Before MICs for the test strains are read and recorded, the
`growth controls should be examinedfor viability, inoculum
`
`

`

`TABLE 3
`
`Antimicrobial agent and
`organism
`
`Interpretive standards fot dilution and disk diffusion susceptibility testinga
`,,
`
`MIC (J-Lg/ml)
`
`'''I
`. ::·~~·~ ..
`
`--·----.-.•
`
`Zone diam (mm)
`
`1332 • ANTIMICROBIAL AGENTS AND SUSCEPTIBILITY TESTING
`
`Susceptible
`
`Intermediate
`
`Resistant
`
`Susceptible
`
`Intermediate
`
`Resistant
`
`Penicillins
`Penicillin G
`Staphylococcib
`Enterococcic
`Listeria monocytogenes
`Streptococci other than
`Streptococcus pneumoniae
`Methicillind
`Oxacillin-nafcillind
`Ampicillin
`Enterobacteriaceae
`Staphylococci
`L monocytogenes
`Enterococcic
`Streptococci other than S.
`pneumoniae
`Amoxicillin-clavulanic acid
`Staphylococci
`Other organisms
`Ampicillin-sulbactam
`Azlocillin
`Pseudomonas aeruginosa
`Carbenicillin
`P. aeruginosa
`Other gram-negative bacilli
`Mezlocillin
`P. aeruginosa
`Other gram-negative bacilli
`Plperacillin
`P. aeruginosa
`Other gram-negative bacilli
`Piperacillin-tazobactam
`P. aeruginosa
`Other gram-negative bacilli
`Staphylococci
`Ticarcillin
`P. aeruginosa
`Other gram-negative bacilli
`Ticarcillin-clavulanic acid
`P. aeruginosa
`Other gram-negative bacilli
`
`Cephalosporins
`Cefaclor
`Cefamandole
`Cefazolin
`Cefepime
`Cefetamet
`Cefixime
`Cefmetazole
`Cefonicid
`Cefoperazone
`Cefotaxime
`Cefotetan
`Cefoxitin
`Cefpodoxime
`Cefprozil
`Ceftazidime
`Ceftlzoxime
`Ceftriaxone
`Cefuroxime axetil
`
`o:;OJ2
`s8
`o:;2
`o:;OJ2
`
`o:;8
`o:;2
`o:;8
`o:;8
`s0.25
`,;2
`o:;8
`o:;0.12
`
`s4/2
`o:;8j4
`o:;8j4
`o:;64
`
`o:;128
`o:;16
`
`o:;64
`o:;16
`
`o:;64
`o:;16
`
`:S64/4
`o:;16/4
`s8/4
`
`o:;64
`o:;16
`
`:S64/2
`:S16/2
`
`o:;8
`o:;8
`:S8
`o:;8
`,;4
`o:;1
`o:;16
`o:;8
`o:;16
`o:;8
`:S16
`o:;8
`o:;2
`o:;8
`o:;8
`:S/3
`,;8
`,;4
`
`20.25
`216
`24
`24
`
`216
`24
`216
`232
`20.5
`24
`216
`24
`
`28/4
`232/16
`232/16
`2128
`
`2512
`264
`
`2128
`~128
`
`2128
`2:128
`
`2:128/4
`2128/4
`2:16/4
`
`2:128
`2128
`
`2128/2
`2128/2
`
`2:32
`2:32
`232
`232
`2:16
`24
`264
`2:32
`2:64
`264
`264
`232
`28
`232
`232
`2:64
`2:64
`2:32
`
`0.25-2.0
`
`16
`
`0.25-2
`
`16/8
`16/8
`
`256
`32
`
`32-64
`
`32-64
`
`32/4-64/4
`
`32-64
`
`32/2-64/2
`
`16
`16
`16
`16
`8
`2
`32
`16
`32
`16-32
`32
`16
`4
`16
`16
`16-32
`16-32
`8-16
`
`229
`215
`220
`228
`
`214 ~~~ .. ;
`213
`214
`2':17
`229
`220
`217
`230
`
`220
`218
`215
`218
`
`2':17
`223
`
`216
`2:21
`
`218
`221
`
`218
`221
`2:18
`
`215
`220'
`
`215
`220
`
`218
`2:18
`218
`218
`218
`219
`216
`218
`221
`223
`216
`218
`221
`218
`218
`2:20
`2:21
`223
`
`20-27
`
`10-13
`11-12
`
`14-16
`
`22-29
`
`14-17
`12-14
`
`14-16
`20-22
`
`18-20
`
`18-20
`
`18-20
`
`15-19
`
`15-19
`
`15-17
`15-17
`15-17
`15-17
`15-17
`16-18
`13-15
`15-17
`16-20
`15-22
`13-15
`15-17
`18-20
`15-17
`15-17
`15-19
`14-20
`15-22
`
`528
`o:;14
`,;19
`,;[9
`
`o:;9
`o:;10
`o:;9
`o:;1J
`s28
`o:;19
`s16
`s21
`
`s19
`sl3
`o:;U
`s17
`
`o:;l3
`o:;19
`
`,;15
`o:;17
`
`o:;17
`o:;17
`
`o:;17
`o:;17
`o:;17
`
`o:;14
`o:;14
`
`o:;14
`o:;14
`
`o:;14
`s14
`o:;14
`o:;14
`o:;14
`,;15
`o:;12
`o:;14
`s15
`o:;14
`o:;12
`,;14
`s17
`,;14
`214
`s14
`,;13
`,;14
`
`(Continued on next page)
`
`~
`
`

`

`TABLE 3 1 (Continued)
`
`Antimicrobial agent and
`organism
`
`Cefuroxime sodium
`Cephalothin
`Loracarbef
`Moxalactam
`
`Other beta-lactams
`Aztreonam
`Imipenem
`
`Aminoglycosides
`Amikacin
`Gentamicin
`Enterococci (high-level
`resistance)
`Netilmicin
`Tobrmnycin
`
`Streptomycin
`Enterococci (high-level
`resistance)
`Broth microdilution
`Agar based
`
`Glycopeptides
`Teicoplanin
`Vancomycin
`Enterococci
`Other organisms
`
`Macro !ides
`Azithromycin
`Clarithromycin
`Erythromycin
`
`Quinolones
`Ciprofloxacin
`Enoxacin
`Fleroxacin
`Lomefloxacin
`Norfloxacine
`Ofloxacin
`
`Other
`Chloramphenicol
`Clindamycin
`Nitrofurantoin
`Rifampin
`Sulfonamide
`Tetracycline
`Trimethoprime
`Trimethoprim-
`sulfamethoxazole
`
`113. Dilution and Disk Diffusion Testing Methods
`
`•
`
`1333
`
`MIC (!-Lg/ml)
`
`Zone diam (mm)
`
`Susceptible
`
`Intermediate
`
`Resistant
`
`Susceptible
`
`Intermediate
`
`Resistant
`
`s8
`s8
`s8
`s8
`
`s8
`s4
`
`s16
`s4
`s500
`
`s8
`s4
`
`s1,000
`s2,000
`
`s8
`s4
`
`s4
`
`s2
`s2
`s0.5
`
`::s1
`::s2
`s2
`s2
`s4
`s2
`
`s8
`s0.5
`s32
`s1
`s256
`::s4
`s8
`s2/38
`
`,p~r.
`
`16
`16
`16
`16-32
`
`16
`8
`
`32
`8
`
`16
`8
`
`16
`8-16
`
`8-16
`
`4
`4
`1-2
`
`2
`4
`4
`4
`8
`4
`
`16
`1-2
`64
`2
`
`8
`
`2::32
`2::32
`2::32
`2::64
`
`2::32
`2::16
`
`2::64
`2::16
`>500
`
`2::32
`2::16
`
`>1,000
`>1,000
`
`2::32
`2::32
`
`2::32
`
`2::8
`2::8
`2::8
`
`2::4
`2::8
`2::8
`2::8
`2::16
`2::8
`
`2::32
`2::4
`2::128
`2::4
`2::512
`2::16
`2::16
`