Cez

Figure 12-5 A, By the disk diffusion method, antibiotic disks are placed on the surface just after the agar surface was inoculated with the test organism. B, Zones of growth inhibition around various disks are apparent following 16 to 18 hours of incubation.

Susceptible zone

Susceptible zone

Figure 12-6 Example of a regression analysis plot to establish zone-size breakpoints for defining the susceptible, intermediate, and resistant categories for an antimicrobial agent. In this example, the maximum achievable serum concentration of the antibiotic is 8 pg/mL. Disk inhibition zones less than or equal to 18 mm in diameter indicate resistance, zones greater than or equal to 26 mm indicate susceptibility, and the intermediate category is indicated by zones ranging from 19 to 25 mm.

10 15 20 25

Zone of inhibition surrounding disk (mm)

10 15 20 25

Zone of inhibition surrounding disk (mm)

Figure 12-6 Example of a regression analysis plot to establish zone-size breakpoints for defining the susceptible, intermediate, and resistant categories for an antimicrobial agent. In this example, the maximum achievable serum concentration of the antibiotic is 8 pg/mL. Disk inhibition zones less than or equal to 18 mm in diameter indicate resistance, zones greater than or equal to 26 mm indicate susceptibility, and the intermediate category is indicated by zones ranging from 19 to 25 mm.

the zone of inhibition around each disk is measured in millimeters (see Figure 12-5).

To establish reference inhibitory zone-size breakpoints to define susceptible, intermediate, and resistant categories for each antimicrobial agent-bacterial species combination, hundreds of strains are tested. The inhibition zone sizes obtained are then correlated with MICs obtained by broth or agar dilution, and a regression analysis (i.e., line of best fit) is performed by plotting the zone size in millimeters against the MIC (Figure 12-6). As the MIC of the bacterial strains tested increase (i.e., more resistant bacterial strains), the corresponding inhibition zone sizes (i.e., diameters) decrease. Using Figure 12-6 to illustrate, horizontal lines are drawn from the MIC resistant breakpoint and the susceptible MIC breakpoint, 8 pg/mL

and 2 pg/mL, respectively. Where the horizontal lines intersect the regression line, vertical lines are drawn to delineate the corresponding inhibitory zone size breakpoints (in millimeters). Using this approach, zone size interpretive criteria have been established for most of the commonly tested antimicrobial agents and are published in the CLSI M02 series titled, "Performance Standards for Antimicrobial Disk Susceptibility Tests."

Procedures. The key features of disk diffusion testing procedures are summarized in Table 12-3, with more details and updates available through CLSI.

Medium and Antimicrobial Agents. Mueller-Hinton is the standard agar base medium for testing most bacterial organisms, with certain supplements and substitutions again required for testing more fastidious organisms. In addition to factors such as pH and cation content, the depth of the agar medium can also 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, zone sizes would be smaller; if too thin, the inhibition zones would be larger. Por many laboratories that perform disk diffusion, 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 Food and Drug Administration. The disks are available from various commercial sources and should be kept at -20° C in a desiccator until used. Thawed, unused disks may be stored at 4° to 8° C for up to a week, 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 (i.e„ 1.5 x 10® CFU/mL). The surface of the plate is swabbed in three directions to ensure an even and complete distribution of the inoculum over the entire plate. Within 15 minutes of inoculation, the antimicrobial agent disks are applied and the plates are inverted for incubation to avoid accumulation of moisture on the agar surface that can interfere with interpretation of test results.

For most organisms, incubation is at 35° C in air, but increased C02 is used when testing certain 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 certain fastidious organisms such as N. gonorrhoeae.

Figure 12-7 Disk diffusion plate that was inoculated with a mixed culture as evidenced by different colony morphologies (arrows) appearing throughout the lawn of growth.

The dynamics and timing of antimicrobial agent diffusion to establish a concentration gradient coupled with the growth of organisms over an 18- to 24-hour duration is 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 organisms that require extended incubation times to grow.

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 good growth has been obtained (see Figure 12-5). If growth between inhibitory zones around each disk is poor and nonconfluent, then the test should not be interpreted but should be repeated. The lack of confluent growth may be due to insufficient inoculum. Alternatively, a particular isolate may have undergone mutation so that growth factors supplied by the standard susceptibility testing medium are no longer sufficient for supporting robust growth. In the latter case, medium supplemented with blood and/or incubation in C02 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 because mixed cultures are most evident as different colony morphologies scattered throughout the lawn of bacteria that is being tested (Figure 12-7). Mixed cultures require purification and repeat testing of the bacterial isolate of interest.

Using a dark background and reflected light (Figure 12-8), the plate is situated so that a ruler or

Reflected

Transmitted light

Figure 12-8 Examination of a disk diffusion plate by transmitted and reflected light.

caliper may be used to measure inhibition zone dia-eters for each antimicrobial agent. Certain motile organisms, such as Proteus spp., may swarm over the surface of the plate and complicate clear interpretation of the zone boundaries. In these cases, the swarming haze is ignored and zones are measured at the point where growth is obviously inhibited. Similarly, hazes of bacterial growth may be observed when testing sulfonamides and trimethoprim as a result of the organism population going through several doubling generations before inhibition; the resulting haze of growth should be ignored for disk interpretation with these agents.

In instances not involving swarming organisms or the testing of sulfonamides and trimethoprim, hazes c growth that occur within more obvious inhibition zones should not be ignored. In many instances, this is the only way in which clinically relevant resistance patterns are manifested by certain bacterial isolates when tested using the disk diffusion method. Key examples in which this can occur include cephalosporin resistance among several species of Enterobacteriaceae, methicillin resistance in staphylococci, and vancomycin resistance in some enterococci. In fact, detection of the haze produced by some staphylococci and enterococci can best be accomplished using transmitted rather than reflected light. In these cases, the disk diffusion plates are held in front of the light source when methicillin and Vancomycin inhibition zones are being read (see Figure 12-S). Still other significant resistances may be subtly

Figure 12-9 Individual bacterial colonies within a more obvious zone of inhibition (arrows). This could indicate inoculation with a mixed culture. However, emergence of resistant mutants of the test isolate is a more likely reason for this growth pattern.

evident and appear as individual colonies within an obvious zone of inhibition (Figure 12-9). When such colonies are seen, purity of the test isolate must be confirmed. If purity is confirmed, the individual colonies are variants or resistant mutants of the same species and the test isolate should be considered resistant.

Once zone sizes are recorded, interpretive categories are assigned. Interpretive criteria for antimicrobial agent-organism combinations that can be tested by disk diffusion are provided in the CLSI-M2 series titled, "Performance Standards for Antimicrobial Disk Susceptibility Tests (Ml00 supplements).' Definitions for susceptible, intermediate, and resistant are the same as those used for dilution methods (see Box 12-2). For example, using the CLSI interpretive standards, an E. coli isolate that produces an ampicillin inhibition zone diameter of 13 mm or less is classified as resistant; if the zone is 14 to 16 mm, the isolate is considered intermediate to ampicillin; if the zone is 17 mm or greater, the organism is categorized as susceptible.

Unlike MICs, inhibition zone sizes are only used to produce a category interpretation and have no clinical utility in and of themselves. Therefore, when testing is performed by disk diffusion, only the category interpretation of susceptible, intermediate, or resistant is reported.

Advantages and Disadvantages. One of the greatest advantages of the disk diffusion test is convenience and user friendliness. Up to 12 antimicrobial agents can be tested against one bacterial isolate with minimal use of extra materials and devices. Because results are generally accurate and most commonly encountered bacteria are reliably tested, the disk diffusion test is still among the most commonly used methods for antimicrobial susceptibility testing, the major disadvantages of this method include the lack of interpretive criteria for organisms not included in Table 12-3 and the inability to provide more precise data regarding the level of an organism's resistance or susceptibility as can be provided by MIC methods.

Commercial Susceptibility Testing Systems

The variety and widespread use of commercial susceptibility testing methods reflect the key role that resistance detection plays among the responsibilities of clinical microbiology laboratories. In many instances, the commercial methods are variations of the conventional dilution or disk diffusion methods, and their accuracies have been evaluated by comparison of results with those obtained by conventional methods. Additionally, many of the media and environmental conditions standardized for conventional methods are maintained with the use of commercial systems. The goal of detecting resistance is the same for all commercial methods, but the principles and practices that are applied to achieve that goal vary with respect to:

  • Format in which bacteria and antimicrobial agents are brought together
  • Extent of automation for inoculation, incubation, interpretation, and reporting
  • Method used for detection of bacterial growth inhibition
  • Speed with which results are produced
  • Accuracy

Accuracy is an extremely important aspect of any susceptibility testing system and is addressed in more detail later in this chapter.

Broth Microdilution Methods. Several systems have been developed that provide microdilution panels already prepared and formatted according to the guidelines for conventional broth microdilution methods (e.g., BEL Sceptor, BD Microbiology Systems, Cockey-sville, Md; Sensititre, Trek Diagnostics Systems, file., Westlake, Ohio; Micro Scan touchSCAN-SR, Dade Behring, Inc., West Sacramento, Calif). These systems enable laboratories to perform broth microdilution without having to prepare their own panels.

The systems may differ to some extent regarding volume within the test wells, how inocula are prepared and added, the availability of different supplements for the testing of fastidious bacteria, the types of antimicrobial agents and the dilution schemes used, and the format of medium and antimicrobial agents (e.g., dry-Iyophilized or frozen), Furthermore, the degree of automation for inoculation of the panels and the devices available for reading results vary among the different products. In general, these commercial panels are designed to receive the standard inoculum and are incubated using conditions and durations recommended for conventional broth microdilution. They are growth-based systems that require overnight incubation, and CLSI interpretive criteria apply for interpretation of most results. Reading of these panels is frequently augmented by the availability of semiautomated reading devices.

Agar Dilution Derivations. One commercial system (Spiral Biotech Inc., Bethesda, Md) uses an instrument to apply antimicrobial agent to the surface of an already prepared agar plate in a concentric spiral fashion. Starting in the center of the plate, the instrument deposits the highest concentration of antibiotic and from that point drug application proceeds to the periphery of the plate. Diffusion of the drug in the agar establishes a concentration gradient from high (center of plate) to low (periphery of plate). Starting at the periphery of the plate, bacterial inocula are applied as a single streak perpendicular to the established gradient in a spoke-wheel fashion. Following incubation, the distance from where growth is noted at the edge of the plate to where growth is inhibited toward the center of the plate is measured (Figure 12-10). This value is used to calculate the MIC of the antimicrobial agent against each of the bacterial isolates streaked on the plate.

Diffusion in Agar Derivations. One test has been developed that combines the convenience of disk diffusion with the ability to generate MIC data. The Etest (AB BIODISK, Solna, Sweden) uses plastic strips; one side of the strip contains the antimicrobial agent concentration gradient and the other contains a numeric scale that indicates the drug concentration (Figure 12-11). Mueller-Hinton plates are inoculated as for disk diffusion and the strips are placed on the inoculum lawn. Several strips may be placed radially on the same plate so that multiple antimicrobials can be tested against a single isolate. Following overnight incubation, the plate is examined and the number present at the point at which the border of growth inhibition intersects the E-strip is taken as the MIC (see Figure 12-11). The same MIC interpretive criteria used for dilution methods as provided in CLSI guidelines are used with the E-test value to assign an inierpretive category of susceptible, intermediate, or resistant. This method provides a means for producing MIC data in those situations in which the level of resistance can be clinically important (e.g., penicillin or cephalosporins against S. pneumoniae).

Another method (BIOMIC, Giles Scientific, Inc., New York) combines the use of conventional disk diffusion methodology with video digital analysis to

Figure 12-10 Growth patterns on a plate containing an antibiotic gradient (concentration decreases from center of the plate to the periphery) applied by the Spiral Gradient instrument. The distance from where growth is noted at the edge of the plate to where growth is inhibited toward the center of the plate is measured. This value is used in a formula to calculate the MIC of the antimicrobial agent against each of the bacterial isolates streaked on the plate. (Courtesy Spiral Biotech, Inc., Bethesda, Md.)

Figure 12-10 Growth patterns on a plate containing an antibiotic gradient (concentration decreases from center of the plate to the periphery) applied by the Spiral Gradient instrument. The distance from where growth is noted at the edge of the plate to where growth is inhibited toward the center of the plate is measured. This value is used in a formula to calculate the MIC of the antimicrobial agent against each of the bacterial isolates streaked on the plate. (Courtesy Spiral Biotech, Inc., Bethesda, Md.)

automate interpretation of inhibition zone sizes. Automated zone readings and interpretations are combined with computer software to produce MIC values and to allow for data manipulations and evaluations for detecting unusual resistance profiles and producing antibiogram reports.

Automated Antimicrobial Susceptibility Test Systems.

The automated antimicrobial susceptibility test systems available for use in the United States include the Vitek Legacy system and Vitek 2 Systems (bioMérieux, Inc., Hazelwood, Mo), the MicroScan WalkAway System (Dade International, Sacramento, Calif), and the Phoenix System (BD Microbiology Systems, Cockeysville, Md). These different systems vary with respect to the extent which inoculum preparation and inoculation are automated, the methods used to detect growth, and the algorithms used to interpret and assign MIC values and categorical (i.e., susceptible, intermediate, resistant) findings.

For example, the Vitek 2 AST inoculum is automatically introduced via a filling tube into a miniaturized plastic 64-well, closed card containing specified concentrations of antibiotics (Figure 12-12). Cards are incubated in a temperature-controlled compartment.

Optical readings are performed every 15 minutes to measure the amount of light transmitted through each well, including a growth control well. Algorithmic analysis of the growth kinetics in each well is performed by the system's software to derive the MIC data. The MIC results are validated with the Advanced Expert System (AES) software, a category interpretation is assigned, and the organism's antimicrobial resistance patterns are reported. Resistance detection is enhanced with the sophisticated AES software, which can recognize and report resistance patterns utilizing MICs. In summary, this system facilitates standardized susceptibility testing in a closed environment with validated results and recognition of an organism's antimicrobial resistance mechanism in 6 to 8 hours for most clinically relevant bacteria (Figure 12-13).

The WalkAway System uses the broth microdilution panel format that is manually inoculated with a multiprong device. Inoculated panels are placed in an incubator-reader unit, where they are incubated for the required length of time, and then the growth patterns are automatically read and interpreted. Depending on the microdilution tray used, bacterial growth may be detected spectrophotometrically or fluorometrically (Figure 12-14).

Figure 12-11 Elest uses the principle of a predefined antibiotic gradient on a plastic strip to generate an MIC value. It is processed like the disk diffusion. A, Individual antibiotic strips are placed on an inoculated agar surface. B, After incubation, the MIC is read where the growth/inhibition edge intersects the strip graduated with an MIC scale across 15 dilutions (arrow). Several antibiotic strips can be tested on a plate. (Courtesy AB BIODISK, Solna, Sweden.)

Figure 12-11 Elest uses the principle of a predefined antibiotic gradient on a plastic strip to generate an MIC value. It is processed like the disk diffusion. A, Individual antibiotic strips are placed on an inoculated agar surface. B, After incubation, the MIC is read where the growth/inhibition edge intersects the strip graduated with an MIC scale across 15 dilutions (arrow). Several antibiotic strips can be tested on a plate. (Courtesy AB BIODISK, Solna, Sweden.)

Spectrophotometrically analyzed panels require overnight incubation, and the growth patterns may be read manually as described for routine microdilution testing. Fluorometric analysis is based on the degradation of fluorogenic substrates by viable bacteria as the means for detecting bacterial inhibition by the antimicrobial agents. The fluorogenic approach can provide susceptibility results in 3.5 to 5.5 hours. Either full dilution schemes or breakpoint panels are available. In addition to speed and facilitating work flow, the auto mated systems also provide increasingly powerful computer-based data management systems that can be used to evaluate the accuracy of results, manage larger databases, and interface with pharmacy areas to enhance the utility of antimicrobial susceptibility testing data.

The Phoenix System provides a convenient, albeit manual, gravity-based inoculation process. Growth is monitored in an automated fashion based on a redox indicator system and interpretation of results of augmented by a rules-based data management expert system.

Figure 12*12 The Vitek 2 antimicrobial susceptibility test card contains 64 wells with multiple concentrations of up to 22 antibiotics. The antibiotic is rehydrated when the organism suspension is introduced into the card during the automated tilling process. (Courtesy bioMerieux, Inc., Hazelwood, Mo.)

Figure 12*12 The Vitek 2 antimicrobial susceptibility test card contains 64 wells with multiple concentrations of up to 22 antibiotics. The antibiotic is rehydrated when the organism suspension is introduced into the card during the automated tilling process. (Courtesy bioMerieux, Inc., Hazelwood, Mo.)

Figure 12-14 Microdilution tray format (A) used with the MicroScan WalkAway instrument (B) for automated incubation, reading, and interpretation of antimicrobial susceptibility tests. (Courtesy Dade International, Sacramento, Calif.)

Figure 12-13 The components of the Vitek 2 System consist of the instrument housing; the sample processing and reader/incubator; the computer workstation, which provides data analysis, storage, and epidemiology reports; the Smart Carrier Station, which is the direct interface between the microbiologist on the bench and the instrument; and a bar code scanner to facilitate data entry. (Courtesy bioMerieux, Inc., Hazelwood, Mo.)

Figure 12-14 Microdilution tray format (A) used with the MicroScan WalkAway instrument (B) for automated incubation, reading, and interpretation of antimicrobial susceptibility tests. (Courtesy Dade International, Sacramento, Calif.)

Alternative Approaches for Enhancing Resistance Detection

Although the various conventional and commercial antimiaobial susceptibility testing methods provide accurate results in most instances, certain clinically relevant resistance mechanisms can be difficult to detect. Tn these instances supplemental tests and alternative approaches are needed to ensure reliable detection of resistance. Also, as new and clinically important resistance mechanisms emerge and are recognized, they will be a "lag time" during which conventional and commercial methods are being developed to ensure accurate detection of these new patterns. During such lag periods, special tests may be used until more conventional or commercial methods become available. Key examples of such alternative approaches are discussed in this section.

Supplemental Testing Methods. Table 12-4 highlights some of the features of supplemental tests that may be used to enhance resistance detection. For certain strains of staphylococci, conventional and commercial systems may have difficulty detecting resistance to oxacillin and the related drugs methicillin and nafcillin. The oxacillin agar screen provides a backup test that may be used when other methods provide equivocal or

Figure 12-13 The components of the Vitek 2 System consist of the instrument housing; the sample processing and reader/incubator; the computer workstation, which provides data analysis, storage, and epidemiology reports; the Smart Carrier Station, which is the direct interface between the microbiologist on the bench and the instrument; and a bar code scanner to facilitate data entry. (Courtesy bioMerieux, Inc., Hazelwood, Mo.)

Table 12-4 Supplemental Methods for Detection of Antimicrobial Resistance

Test

Purpose

Conditions

Interpretation

Oxacillin agar screen

Detection of staphylococcal resistance to penicllllnase-resistant penicillins (e.g., oxacillin, mettiicillin,or nafcillin)

Medium: Mueiler-Hinton agar plus G |ig oxacillin/mL plus 4% NaCL Inoculum: Swab or spot from 1.5 x 10®

standard suspension Incubation: 30°-35° C 24 hr, up to 48 hr for nor\-Staphylococcus aureus

Growth = Resistance No growth = Susceptible

Vancomycin agar screen

Detection of enterococcal resistance to vancomycin

Medium: Brain-heart infusion agar plus 6 nfl vancomycln/mL Inoculum: Spot of 105-106 CFU Incubation: 35° C, 24 hr

Growth^ Resistance No growth=Susceptible

Aminoglycoside screens

Detection of acquired enterococcal high-level resistance to aminoglycosides that would compromise synergy with a cell wall-active agent such as ampicillin or vancomycin

Medium: Brain-heart infusion broth: 500 [ig/mL serrtamicin; 1000 ng/mL

streptomycin Agar. 500 ng/mL gentamictn; 2000 ug/mL

streptomycin Inoculum: Broth; 5 x 105 CFtl/mL agar;

106CFU/spot Incubation: 35° C, 24 hr; 48 hr for streptomycin, only if no growth at 24 hr

Growth - Resistance No growth = Susceptible

Oxacillin disk screen

Detection of Streptococcus pneumoniae resistance to penicillin

Medium: Mueiler-Hinton agar plus 5% sheep blood plus 1 ng oxacillin disk Inoculum: as for disk diffusion Incubation; 5%-7% C02 35° C; 20-24 hr

inhibition zone s20 mm; penicillin susceptible Inhibition zone <20 mm; penicillin resistant, intermediate, or susceptible. Further testing by MIC method is needed

"D"test

Differentiate clindamycin resistance among S. aureus resulting from efflta (mcrA orMLSa)

Approximation clindamycin and erythromycin disk to look for blunting of clindamycin zone

Blunting of clindamycin zone to give "D" pattern, indicating inducible clindamycin resistance

uncertain profiles. Growth on the screen correlates highly with the presence of oxacillin (or methidllin) resistance, and no growth is strong evidence that an isolate is susceptible. This is an important determination; strains that are classified as resistant are considered resistant to all other currently available beta-lactam antibiotics so that therapy must include the use of vancomycin. The agar screen plates can be made in-house, and they are available commercially (e.g., REMEL, Lenexa, Kan; BBL, Cockeysville, Md). Additionally, other commercial tests designed to detect oxacillin resistance more rapidly (i.e., 4 hours) have been developed and may provide another approach to supplemental testing (e.g.. Crystal MRSA ID System, BBL, Cockeysville, Md). In addition to the agar screen, use of the 30-ng cefoxitin disk has been developed to use by disk diffusion for assisting in the detection of oxacillin-resistant staphylococci (CLSIM100-15). By this method cefoxitin inhibitory zones of less than or equal to 24 mm indicates oxacillin resistance among staphylococci. The cefoxitin disk test is especially helpful in detecting oxacillin resistance among coagulase-negative staphylococci.

Similarly, detection of reduced staphylococcal susceptibility to vancomycin (i.e., MICs from 4 to 16fig/mL) can be difficult by disk diffusion and some commercial methods. Although the therapeutic relevance of staphylococci with vancomycin MICs in this range is currendy uncertain, this diminished susceptibility is outside the normal MIC range for fully susceptible strains; therefore, there is a need to have the capability of detecting this phenotype. The agar screen used for this purpose is outlined in Table 12-4 and is essentially the same as that outlined for enterococci, also in Table 12-4. Strains that grow on the screen should be tested by broth microdilution to obtain a definitive MIC value.

In the same fashion, detection of enterococcal resistance to vancomycin can be difficult by some conventional and commercial methods, and the agar screen can be helpful in confirming this resistance pattern (see Table 12-4). However, as a screen, not all enterococcal isolates that grow are resistant to vancomycin at clinically relevant levels. Therefore, strains that are detected using this method should be more fully characterized using a broth microdilution method to determine the isolate's MIC.

Aminoglycosides also play a key role in therapy for serious enterococcal infections, and acquired high-level resistance, which essentially destroys the therapeutic value of these drugs for combination therapy with ampicillin or vancomycin, is not readily detected by conventional methods. Therefore, screens (Table 12-4) that use high concentrations of aminoglycosides have been developed specifically for detecting this resistance and are available commercially (e.g., REMEL, Lenexa, Kan; or BBL, Cockeysville, Md).

With the emergence of penicillin resistance among S. pneumoniae, the penicillin disk diffusion test was not sufficiently sensitive to detect subtle but significant changes in susceptibility to this agent. To address this issue, the oxacillin disk screen described in Table 12-4 is useful but does have a notable limitation. Although organisms that give zones greater than or equal to 20 mm can be accurately characterized as penicillin susceptible, the penicillin susceptibility status of those with zones less than 20 mm remains uncertain and use of some method that produces an MIC value is required.

With regard to macrolide (e.g., erythromycin, azithromycin, clarithromycin) and lincosamide (e.g., clindamycin) resistance among staphylococci, interpretation of in vitro results can also be complicated by the different underlying mechanisms of resistance that have very different therapeutic implications. Isolates that produce a profile that demonstrates resistance to a macrolide (e.g., erythromycin) and susceptibility to clindamycin may do so as a result of two different resistance mechanisms. If this profile is the result of the efflux (msrA gene) mechanism, then the isolate can be considered susceptible to clindamycin. However, if this profile resulted from the inducible MLSB mechanism, which results in an altered ribosomal target, then clindamycin-resistant mutants may readily arise during therapy with this agent. Currently such strains should be reported as resistant to clindamycin. The "D" test that is used to discern between these two different resistance mechanisms is outlined in T^ble 12-4.

Undoubtedly, as complicated resistance mechanisms that require laboratory detection continue to emerge, screening and supplemental testing methods will continue to be developed. Some of these will be maintained as the primary method for detecting a particular resistance mechanism, while others may tend to fade away as adjustments in conventional and commercial procedures enhance resistance detection and preclude the need to use a supplemental test.

Predictor Antimicrobial Agents. Another approach that may be used to ensure accuracy in resistance detection is the use of "predictor" antimicrobial agents in the test batteries. The basic premise of this approach is to use antimicrobial agents (i.e., the predictor drugs) that are the most sensitive indicators of certain resistance mechanisms. The profile obtained with such a battery of agents is then used to deduce the underlying resistance mechanism. A susceptibility report is subsequently produced based on the likely effect that the resistance mechanisms would have on the antimicrobials being considered for therapeutic management of the patient. Use of predictor drugs is not a new concept; this approach has already been used in various situations. For example:

  • Staphylococcal resistance to oxacillin is used to determine and report resistance to all currently available beta-lactams, including penicillins, cephalosporins, and carbapenems (e.g., imipenem, meropenem, ertapenem)
  • Enterococcal high-level gentamicin resistance predicts resistance to nearly all other currently available aminoglycosides, including amikacin, tobramycin, netilmicin, and kanamydn
  • Enterococcal resistance to ampicillin predicts resistance to all penicillin derivatives

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