Medium and Antimicrobial Agents.

With in vitro susceptibility testing methods, certain conditions must be altered when examining fastidious organisms to optimize growth and facilitate expression of bacterial resistance. For example, the Mueller-Hinton preparation is the standard medium used for most broth dilution testing, and conditions in the medium (e.g., pH, cation concentration, thymidine content) are well controlled by commercial manufacturers. However, media supplements or different media are required to obtain good growth and reliable susceptibility profiles for bacteria such as S. pneumoniae and H. influenzae. Although staphylococci are not considered fastidious organisms, media supplemented with sodium chloride (NaCl) enhance the expression and detection of methicillin-resistant isolates (see Table 12-1).

Broth dilution testing is divided into two general categories: microdilution and macrodilution. The principle of each test is 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 of several antibiotics at several different concentrations, the smaller volume used in microdilution allows this to be conveniently accomplished in a single microtiter tray (Figure 12-2).

The need for multiple large test tubes in the macrodilution method makes that technique substantially cumbersome and labor intensive when several bacterial isolates are tested simultaneously. For this reason, macrodilution is rarely used in most clinical laboratories, and subsequent comments about broth dilution focuses on the microdilution approach.

A key component of broth testing is proper preparation and dilution of the antimicrobial agents 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 are not commonly carried out in most clinical laboratories (the details of this procedure are outlined in the CLSI M07-A6 document). In most instances, each antimicrobial agent is included in the microtiter 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 immediately before use. Once thawed the panels should never be refrozen, which may result in substantial loss of antimicrobial action and potency. Alternatively, the antimicrobial agents may be lyophilized or freeze dried with the medium or drug in each well; 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 McFarland standard (i.e., 1.5 × 10⁸ CFU/mL) usually serve as the starting point for dilutions ultimately achieving the required final standard bacterial concentration of 5 × 10⁵ CFU/mL in each microtiter well. It is essential to prepare the standard inoculum from a fresh, overnight, pure culture of the test organism. Inoculation of the microdilution panel is accomplished using manual or automated multiprong inoculators calibrated to deliver the precise volume of inoculum to each well in the panel simultaneously (see Figure 12-2).

Inoculated trays are incubated under optimal environmental conditions to optimize bacterial growth without interfering with the antimicrobial activity (i.e., avoiding environmentally mediated results). For the most commonly tested bacteria (e.g., Enterobacteriaceae, P. aeruginosa, staphylococci, and enterococci), the environmental condition consists of room air at 35°C (see Table 12-1). Fastidious bacteria, such as H. influenzae, require incubation in 5% to 10% carbon dioxide (CO₂). 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 result in false or elevated resistance patterns. This is a primary factor that limits the ability to perform accurate testing with some slow-growing bacteria.

Reading and Interpretation of Results.

After 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. The detection 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, bacterial growth, 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 for each antibiotic is inspected, the microdilution well containing the lowest drug concentration that completely inhibits visible bacterial growth is recorded as the MIC. Once the MICs for the antimicrobials in the test battery for an organism have been recorded, they are usually translated into one of the interpretive categories, specifically susceptible, intermediate, or resistant (Box 12-2). The interpretive criteria for these categories are based on extensive studies that correlate the 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, “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 µg/mL would be classified as susceptible; one with an MIC of 8 µg/mL would be classified as intermediate; and one with an MIC of 16 µg/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 the 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.

In some settings, the full range of antimicrobial dilutions is not used; only the concentrations that separate the categories of susceptible, intermediate, and resistant are used. The specific concentrations that separate or define the different categories are known as 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 methods provide data for both quantitative results (i.e., MIC) and qualitative results (i.e., category interpretation). Whether this is an advantage is the subject of debate. On one hand, the MIC can be helpful in establishing the level of resistance of a particular bacterial strain and can substantially affect the decision to treat a patient with a specific antimicrobial agent. For example, the penicillin MIC for S. pneumoniae may determine whether penicillin or alternative agents will be used to treat a patient with meningitis. On the other hand, for most antimicrobial susceptibility testing methods, a category report is sufficient and the actual MIC data are superfluous. This is one reason other methods (e.g., disk diffusion) that focus primarily on producing interpretive categories have been maintained among clinical microbiologists.

Medium and Antimicrobial Agents.

The Mueller-Hinton preparation is the standard agar-base medium used for testing of most bacterial organisms, although certain supplements and substitutions are required for testing of fastidious organisms. In addition to factors such as the pH and cation content, the depth of the agar medium can affect test accuracy and must be carefully controlled. Because antimicrobial agents diffuse in all directions from the surface of the agar plate, the thickness of the agar affects the antimicrobial drug concentration gradient. If the agar is too thick, the antimicrobial agent diffuses down through the agar as well as outward, resulting in smaller zone sizes; if the agar is too thin, the inhibition zones are larger. For many laboratories that perform disk diffusion testing, commercial manufacturers are reliable sources for properly prepared and controlled Mueller-Hinton plates.

The appropriate concentration of drug for each disk is predetermined and set by the U.S. Food and Drug Administration (FDA). The disks are available from various commercial sources and should be stored at the recommended temperature in a desiccator until used. Inappropriate storage can lead to deterioration of the antimicrobial agents and result in misleading zone size results.

To ensure equal diffusion of the drug into the agar, the disks must be placed flat on the surface and be firmly applied to ensure adhesion. This is most easily accomplished by using any one of several disk dispensers that are available through commercial disk manufacturers. With these dispensers, all disks in the test battery are simultaneously delivered to the inoculated agar surface and are adequately spaced to minimize the chances for inhibition zone overlap and significant interactions between antimicrobials. In most instances, a maximum of 12 antibiotic disks may be applied to the surface of a single 150-mm Mueller-Hinton agar plate (see Figure 12-5).

Inoculation and Incubation.

Before disk placement, the plate surface is inoculated using a swab that has been submerged in a bacterial suspension standardized to match the turbidity of the 0.5 McFarland turbidity standard, equivalent to 1.5 × 10⁸ CFU/mL. The surface of the plate is swabbed in three directions to ensure even and complete distribution of the inoculum over the entire plate. Within 15 minutes of inoculation, the antimicrobial disks are applied and the plates are inverted for incubation to prevent the accumulation of moisture on the agar surface, which would interfere with the interpretation of test results.

Most organisms are incubated at 35°C in room air, but increased CO₂ is used for testing of specific fastidious bacteria (see Table 12-3). Similarly, the incubation time may be increased beyond 16 hours to enhance detection of certain resistance patterns (e.g., methicillin resistance in staphylococci and vancomycin resistance in enterococci) and to ensure accurate results in general for fastidious organisms such as N. gonorrhoeae.

The dynamics and timing of antimicrobial agent diffusion required for establishing a concentration gradient, in addition to growth of the organisms over 18 to 24 hours, are critical for reliable results. Therefore, incubation of disk diffusion plates beyond the allotted time should be avoided, and disk diffusion generally is not an acceptable method for testing slow-growing organisms that require extended incubation such as mycobacteria and anaerobes.

Reading and Interpretation of Results.

Before results with individual antimicrobial agent disks are read, the plate is examined to confirm that a confluent lawn of growth has been obtained (see Figure 12-5). If growth between inhibitory zones around each disk is poor and nonconfluent, the test should not be interpreted and should be repeated. The lack of confluent growth may be due to insufficient inoculum. Alternatively, a particular isolate may have undergone mutation, and growth factors supplied by the standard medium are no longer sufficient to support robust growth. In the latter case, medium supplemented with blood and/or incubation in CO₂ may enhance growth. However, caution in interpreting results is required when extraordinary measures are used to obtain good growth and the standard medium recommended for testing a particular type of organism is not used. Plates should also be examined for purity. Mixed cultures are evident through the appearance of different colony morphologies scattered throughout the lawn of bacteria (Figure 12-7). Mixed cultures require purification and repeat testing.

A dark background and reflected light are used to examine a disk diffusion plate (Figure 12-8