• Enterococeus spp.
  • Streptococcus spp. (not including S. pneumoniae)
  • Streptococcus pneumoniae
  • Haemophilus influenzae
  • Neisseria gonorrhoeae

Conventional Testing Methods: Broth Dilution

Broth dilution testing involves challenging the organism of interest with antimicrobial agents in a broth environment. Each antimicrobial agent is tested using a range of concentrations commonly expressed as ng (micrograms) of active drug/mL (milliliter) of broth (i.e., (xg/mL). The concentration range tested for a particular drug depends on various criteria, including the concentration that is safely achievable in a patient's serum. Therefore, the concentration range tested will often vary from one drug to the next, depending on the pharmacologic properties of each. Additionally, the concentration range tested may be based on the level of drug that is needed to most reliably detect a particular underlying resistance mechanism. In this case, the test concentration for a drug may vary depending on the organism and its associated resistances that the test is attempting to detect. For example, to detect clinically significant resistance to cefotaxime in S. pneumoniae, the dilution scheme need only go as high as 2 pg/mL, but to detect cefotaxime resistance in Escherichia coli, the scheme must go up to 16 pg/mL or beyond.

Typically, the range of concentrations tested for each antibiotic are a series of doubling dilutions (e.g., 16, 8, 4, 2, 1, 0.5, 0.25 pg/mL); the lowest antimicrobial concentration that completely inhibits visible bacterial growth, as detected visually or via an automated or semiautomated method, is recorded as the minimal inhibitory concentration (MIC).

Procedures. The key features of broth dilution testing procedures are given in Table 12-1. Because changes in procedural recommendations occur, the CLSI M07 series titled, "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically" should be consulted annually.

Medium and Antimicrobial Agents. For any in vitro susceptibility testing method, it is necessary to alter certain conditions when testing particular types of organisms in order to optimize growth of some fastidious bacteria and facilitate expression of certain types of bacterial resistance. For example, Mueller-Hinton is the standard medium used for most broth dilution testing, and conditions of this medium (e.g., pH, cation concentration, thymidine content) are well controlled by commercial media manufacturers. However, media supplements or different media altogether are required to obtain good growth and reliable susceptibility profiles with relatively fastidious bacteria such as S. pneumoniae and Haemophilus influenzae. Also, although staphylococci are not fastidious organisms, media supplementation with NaCl is needed to enhance expression and detection of methicillin-resistant isolates (see Table 12-1).

Broth dilution testing is divided into two categories: microdilution and macrodilution. The principles of the tests are the same; the only difference is the volume of broth in which the test is performed. For microdilution testing, the total broth volume is 0.05 to 0.1 mL; for macrodilution testing, the broth volumes are usually 1 mL or greater. Because most susceptibility test batteries require testing several antibiotics at several different concentrations, the smaller volume used in microdilution allows this to be conveniently accomplished in a single microtitre tray format (Figure 12-2).

Use of test tubes as required by the macrodilution method becomes substantially cumbersome and labor intensive, especially because most laboratories must test

Figure 12-2 Microtitre tray for broth microdilution testing. Doubling dilutions of each antimicrobial agent in test broth occupies one vertical row of wells.

Figure 12-2 Microtitre tray for broth microdilution testing. Doubling dilutions of each antimicrobial agent in test broth occupies one vertical row of wells.

several bacterial isolates daily. For this reason, macrodilution is rarely used in most clinical laboratories and subsequent comments regarding broth dilution focus on the microdilution approach.

A key component to broth dilution testing is proper preparation and dilution of the antimicrobial agents that will be incorporated into the broth medium. Most laboratories that perform broth microdilution use commercially supplied microdilution panels in which the broth is already supplemented with appropriate antimicrobial concentrations. Therefore, antimicrobial preparation and dilution is not commonly done in most clinical laboratories, but details of this procedure are outlined in the CLSIM07-A6 document. In most instances, each antimicrobial agent is presented in the microtitre trays as a series of doubling twofold dilutions. To ensure against loss of antibiotic potency, the antibiotic microdilution panels are stored at -20° C or lower, if possible, and are thawed just before inoculation and use. Once thawed the panels should never be refrozen because doing so can result in substantial loss of antimicrobial potency. Alternatively, the antimicrobial agents may be lyophilized or freeze-dried with the medium or drug in each well and upon inoculation with the bacterial suspension the medium and drug are simultaneously reconstituted to the appropriate concentration.

Inoculation and Incubation. Standardized bacterial suspensions that match the turbidity of the 0.5 McFariand standard (Le., 1.5 x 10s CFU/mL) usually serve as the starting point for dilutions that ultimately allow the final standard bacterial concentration of 5 x 10s CFU/mL in each microtitre well to be achieved. Of importance, the standard inoculum should be prepared from a fresh, overnight, pure culture of the test organism. Inoculation of the microdilution panel is readily accomplished using manual or automated multiprong inoculators that are calibrated to deliver the precise volume of inoculum to each well in the panel simultaneously (see Figure 12-2).

Inoculated trays are incubated under environmental conditions that optimize bacterial growth but do not interfere with antimicrobial activity (i.e., avoiding environmentally mediated results). For the most commonly tested bacteria (e.g., Enterobacteria-ceae, P. aeruginosa, staphylococci, and enterococci) the environment is air at 35° C (see Table 12-1). Exceptions exist for the sake of testing more fastidious bacteria (e.g., N. meningitidis optimally requires 5% to 7% C02). Similarly, incubation durations for some organisms may need to be extended beyond the usual 16 to 20 hours (see Table 12-1). However, prolonged incubation times beyond recommended limits should be avoided because antimicrobial deterioration may

Figure 12-3 Bacterial growth profiles in a broth microdilution tray. The wells containing the lowest concentration of an antibiotic that completely inhibits visible growth (arrow) is recorded, in (ig/mL, as the minimal inhibitory concentration (MIC).

result iB false resistance interpretations. This is a primary factor that limits our ability to perform accurate testing with some slow-growing bacteria.

Reading and Interpretation of Results. Following incubation, the microdilution trays are examined for bacterial growth. Each tray should include a growth control that does not contain antimicrobial agent and a sterility control that was not inoculated. Once growth in the growth control and no growth in the sterility control wells have been confirmed, the growth profiles for each antimicrobial dilution can be established and the MIC determined. Detecting the presence of growth in microdilution wells is often augmented through the use of light boxes and reflecting mirrors. When a panel is placed in these devices, the presence of bacterial growth, which may be manifested as light to heavy turbidity or a button of growth on the well bottom, is more reliably visualized (Figure 12-3).

When the dilution series of each antibiotic is inspected, the microdilution well containing the lowest drug concentration that completely inhibited visible bacterial growth is recorded as the MIC. Once the MICs for the antimicrobials in the test battery for a particular organism have been recorded, they are usually translated into interpretive categories of sus ceptible, intermediate, or resistant (Box 12-2). The interpretive criteria for these categories are based on extensive studies that correlate MIC with serum achievable levels for each antimicrobial agent, particular resistance mechanisms, and successful therapeutic outcomes. The interpretive criteria for an array of antimicrobial agents are published in the CLSI M07 series document titled, "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically (M100 supplements)." For example, using these standards, an isolate of P. aeruginosa with an imipenem MIC of less than or equal to 4 ng/mL would be classified as being susceptible, one with an MIC of 8 ng/mL would be considered intermediate, and one with an MIC of 16 ng/mL or greater would be classified as resistant to imipenem.

After the MICs are determined and their respective and appropriate interpretive categories assigned, the laboratory may report the MIC, the category, or both. Because die MIC alone will not provide most physicians with a meaningful interpretation of data, either the category result with or without the MIC is usually reported.

hi some settings, the full range of antimicrobial dilutions is not used; only the concentrations that separate the categories of susceptible, intermediate,

BOX 12-2 Definitions of Susceptibility Testing _Interpretive Categories*_


m category indicates that the antimicrobial agent in question may be an appropriate choice for treating the infection caused by the bacterial isolate tested. Bacterial resistance is absent or a clinically insignificant level. INTERMEDIATE:

Thi category is used to indicate a number of possibilities, Including:

j The potential utility of the antimicrobial agent in body sites where it may be concentrated (e,g., the urinary tract) or if high concentrations of the drug are used t* The antimicrobial agent may still be effective against the tested isolate but possibly less so than against a susceptible isolate

As an interpretive safety margin to prevent relatively small changes in test results from leading to major swings in interpretive category (e.g„ resistant to susceptible or vice versa) RESISTANT:

his category indicates that the antimicrobial agent-in question may not be an appropriate choice for treating the infection caused y the bacterial isolate tested, either because the organism is not ihibrt' by serum-achievable levels of the drug or because the ¡st result highly correlates with a resistance mechanism that renders treatment success doubtful.

'Although definitions are adapted from CLSIM7-A3 titled, "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically," they are commonly applied to results obtained by various susceptibility testing methods.

and resistant are used. The specific concentrations that separate or define the different categories are known is breakpoints, and panels that only contain these antimicrobial concentrations are referred to as breakpoint panels. In this case, only category results are produced; precise MICs are not available because the full range of dilutions is not tested.

Advantages and Disadvantages. Broth dilution testing allows the option of providing both quantitative (i.e., MIC) and qualitative (i.e., category interpretation) results. Whether this is an advantage or not is controversial. On one hand, an MIC can be helpful in establishing the level of resistance of a particular bacterial strain and can substantially affect the decision to use certain antimicrobial agents. For example, the penicillin MIC for S. pneumoniae may determine whether penicillin or alternative agents will be used to treat a case of meningitis. On the other hand, for most antimicrobial susceptibility testing methods, a category report is Sufficient so that once this determination is made the actual MIC data are superfluous. This is one reason why other methods (e.g., disk diffusion) that focus only on producing interpretive categories have been maintained in the clinical microbiology community.

Figure 12-4 Growth pattern on an agar dilution plate. Each plate contains a single concentration of antibiotic, and growth is indicated by a spot on the agar surface. No spot is seen for isolates inhibited by the concentration of antibiotic incorporated into the agar of that particular plate.

Conventional Testing Methods: Agar Dilution

With agar dilution the antimicrobial concentrations and organisms to be tested are brought together on an agar-based medium rather than in broth. Each doubling dilution of an antimicrobial agent is incorporated into a single agar plate so that testing a series of six dilutions of one drug would require the use of six plates, plus one positive growth control plate without antibiotic. The standard conditions for agar dilution are given in Table 12-2 and, as shown in Figure 12-4, the surface of each plate is inoculated with 1 x 104 CFU. By this method, one or more bacterial isolates are tested per plate. After incubation, the plates are examined for growth and the MIC is the lowest concentration of an antimicrobial agent in agar that completely inhibits visible growth. The same MIC breakpoints and interpretive categories used for broth dilution are applied for interpretation of agar dilution methods. Similarly, test results may be reported as MICs only, as category only, or as both.

Preparing agar dilution plates (see CLSI M07-A6 series document titled, "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically") is suffidentiy labor intensive to predude the use of this method in most clinical laboratories in which multiple antimicrobial agents must be tested even though several isolates may be tested per plate. As with broth dilution, the standard medium is Mueller-Hinton, but supplements and substitutions are made as needed to facilitate growth of more fastidious organisms. (See Table 12-3 for agar media used for agar dilution and disk diffusion testing.) In fact, one advantage of this method is that it provides a means for determining MICs for N. gonorrhoeae, which does not grow suffidentiy in broth to be tested by broth dilution methods.

Table 12-2 Summary of Agar Dilution Susceptibility Testing Conditions

Organism Groups

Test Medium

Inoculum Size (CFU/spot)

Incubation Conditions

Incubation Duration



1 x 104

35° C; air

16-20 hr

Pseudomonas aeruginosa

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