Methods That Directly Detect Specific Resistance Mechanisms

As an alternative to detecting resistance by measuring the effect of antimicrobial presence on bacterial growth, some strategies focus on assaying for the presence of a particular mechanism. When the presence or absence of the mechanism is established, the resistance profile of the organism can be generated without having to test several different antimicrobial agents. The utility of this approach, which can involve phenotypic and genotypic methods, depends on the presence of a particular resistance mechanism as being a sensitive and specific indicator of clinical resistance.

Phenotypic Methods

The most common phenotypic-based assays are those that test for the presence of beta-lactamase enzymes in the clinical bacterial isolate of interest. Less commonly used are tests to detect the chloramphenicol-modifying enzyme chloramphenicol acetyltransferase.

Beta-Lactamase Detection. Beta-lactamases play a key role in bacterial resistance to beta-lactam agents, and detection of their presence can provide useful information (see Chapter 11). Various assays are available to detect beta-lactamases, but the most useful one for clinical*laboratories is the chromogenic cephalo-sporinase test Beta-lactamases exert their effect by opening the beta-lactam ring (see Figure 11-9), With the use of a chromogenic cephalosporin as the substrate, this process results in a colored product. One such assay

Figure 12-15 The chromogenic cephalosporin test allows direct detection of beta-lactamase production. When the beta-lactam ring of the cephalosporin substrate in the disk is hydrolyzed by the bacterial inoculum, a deep pink color is produced (A). Lack of color production indicates the absence of beta-lactamase (B).

Figure 12-15 The chromogenic cephalosporin test allows direct detection of beta-lactamase production. When the beta-lactam ring of the cephalosporin substrate in the disk is hydrolyzed by the bacterial inoculum, a deep pink color is produced (A). Lack of color production indicates the absence of beta-lactamase (B).

is the cefinase disk (BD Microbiology Systems, cock-eysville, Md) test shown in Figure 12-15.

Useful application of tests to directly detect beta-lactamase production is limited to those organisms for which the list of beta-Iactams significantly affected by the enzyme are known. Furthermore, this list must include the beta-lactams commonly considered for therapeutic eradication of the organism. Examples of useful applications include detection of:

  • N. gonorrhoeae resistance to penicillin
  • H. influenzae resistance to ampicillin
  • Staphylococcal resistance to penicillin

The actual utility of this approach even for the organisms listed is decreasing. As beta-lactamase-mediated resistance has become widespread among N. gonorrhoeae, H. influenzae, and staphylococci, other agents not affected by the beta-lactamases have become the antimicrobials of choice for therapy. Therefore, the need to know the beta-lactamase status of these bacterial species has become substantially less urgent. Whereas several Enterobacteriaceae and P, aeruginosa produce beta-lactamases, the effect of these enzymes on the various beta-lactams depends on which enzymes are produced. Therefore, even though such organisms would frequendy produce a positive beta-lactamase assay, very little, if any, information regarding which antimicrobial agents are affected would be gained. Detection of beta-lactam resistance among these organisms is best accomplished using conventional and commercial systems for direcdy evaluating antimicrobial agent-organism interactions.

Chloramphenicol Acetyltransferase Detection. Chloramphenicol modification by chloramphenicol acetyl-transferase (CAT) detection is only one mechanism by which bacteria may express resistance to this agent. This, coupled with the substantially diminished use of chloramphenicol in today's clinical settings, significantiy limits the utility of this test. Commercial colorimetric assays, such as that produced by REMEL (Lenexa, Kan), do provide a convenient method for establishing the presence of this enzyme. If positive, Chloramphenicol resistance can be reported, but a negative test does not rule out resistance that may be mediated by other mechanisms such as decreased uptake.

Genotypic Methods

The genes that encode many of the clinically relevant acquired resistance mechanisms are known, as is all or part of their nucleotide sequences, This has allowed for the development of molecular methods involving nucleic acid hybridization and amplification for the study and detection of antimicrobial resistance (for more information regarding molecular methods for the characterization of bacteria, see Chapter 8). The ability to definitively determine the presence of a particular gene that encodes antimicrobial resistance has several advantages. However, as with any laboratory procedure, certain disadvantages and limitations also exist.

From a research and development perspective, molecular methods are extremely useful for more thoroughly characterizing the resistances of bacterial collections used to establish and evaluate conventional standards recommended by CLSI. Phenotype-based commercial (automated and nonautomated) susceptibility testing methods and systems can also be evaluated.

Molecular methods also may be direcdy applied in the clinical setting as an important backup resource to investigate and arbitrate equivocal results obtained by phenotypic methods. For example, the clinical importance of accurately detecting methidUin resistance among staphylococci coupled with the inconsistencies of phenotype-based methods is problematic In doubdul situations, molecular detection of the mec gene that encodes methicillin resistance can be usefully applied to definitively establish an isolate's methicillin resistance. Similarly, doubt raised by equivocal phenotypic results obtained with potentially vancomycin-resistant entero-cocri can be definitively resolved by establishing the presence and classification of van genes that mediate this resistance.

Although molecular methods have been and will continue to be extremely important in antimicrobial resistance detection, numerous factors still complicate their use beyond supplementing phenotype-based susceptibility testing protocols. These factors include the following:

  • Use of probes or oligonucleotides for specific resistance genes only allows those particular genes to be found. Resistance mediated by divergent genes or totally different mechanisms could be missed (i.e„ the absence of one gene may not guarantee antimicrobial susceptibility).
  • Phenotypic resistance to a level that is clinically significant for any one antimicrobial agent may
  • be due to a culmination of processes that involve enzymatic modification of the antimicrobial, decreased uptake, altered affinity of the target for the drug, or some combination of these mechanisms (i.e., the presence of one gene does not guarantee resistance).
  • The presence of a gene encoding resistance does not provide information regarding the status of the control genes necessary for expression of resistance. That is, although present, the genes may be silent or nonfunctional and the organism may be incapable of expressing die resistance encoded by the detected gene.
  • From a clinical laboratory perspective, there is practical difficulty in adopting molecular methods specific for only a few resistance mechanisms when the vast majority of the susceptibility testing still will be accomplished using phenotypic-based methods.

Even though there are challenges to the widespread adoption of molecular methods for routine antimicrobial susceptibility testing, the significant contributions that this approach has made to resistance detection will continue to expand.

special methods for complex antimicrobial-organism interactions

Certain in vitro tests have been developed to investigate aspects of antimicrobial activity not routinely addressed by commonly used susceptibility testing procedures. Specifically, these are tests designed to measure bactericidal activity (i.e., bacterial killing) or to measure the antibacterial effect of antimicrobial agents used in combination.

These tests are often labor intensive, fraught with the potential for technical problems, frequently difficult to interpret, and of uncertain clinical utility. For these reasons, their use should be substantially limited. If performed at all, they should be done with the availability of expert microbiology and infectious disease consultation.

Bactericidal Tests

Bactericidal tests are designed to determine the ability Of antimicrobial agents to kill bacteria. The killing ability of most drugs is already known, and they are commonly classified as bacteriostatic or bactericidal agents. However, many variables, including the concentration of antimicrobial agent and the species of targeted organism, can influence this classification. For example, beta-Iactams, such as penicillins, typically are bactericidal against most gram-positive cocci but are usually only bacteriostatic against enterococd. If bactericidal tests are clinically appropriate, they should only be applied to evaluate antimicrobials usually considered to be bactericidal (e.g., beta-lactams and vancomycin) and not to those agents known to be bacteriostatic (e.g., maaolides).

Key clinical situations in which achieving bactericidal activity is of greatest clinical importance include severe and life-threatening infections, infections in the immunocompromised host, and infections in body sites where assistance from the patient's own defenses is minimal (e.g., endocarditis or osteomyelitis). Based on research trials in animal models and clinical trials in humans, the most effective therapy for these types of infections is often already known. However, occasionally the laboratory may be asked to substantiate that bactericidal activity is being achieved or is achievable. The methods available for this include minimal bactericidal concentration (MBC) testing, time-kill studies, and serumcidal testing. Regardless of which method is used, the need to interpret the results cautiously with the understanding of uncertain clinical correlation and the potential for substantial technical artifacts cannot be overemphasized.

Minimal Bactericidal Concentration. The MBC test involves continuation of the procedure for conventional broth dilution testing. After incubation and determination of the antimicrobial agent's MIC, an aliquot from each tube or well in the dilution series showing inhibition of visible bacterial growth is subcultured to an enriched agar medium (usually sheep blood agar). Following overnight incubation, the plates are examined and the CFUs counted. Knowing the volume of the aliquot sampled and the number of CFUs obtained, the number of viable cells per milliliter for each antimicrobial dilution well can be calculated. This number is compared with the known CFU/ml in the original inoculum. The antimicrobial concentration that resulted in a 99.9% reduction in CFU/ml compared with the organism concentration in the original inoculum is recorded as the MBC.

Although the clinical significance of MBC results is uncertain, applications of this information include considering whether treatment failure could be occurring because an organism's MBC exceeds the serum achievable level of the antimicrobial agent. Alternatively, if an antibiotic's MBC is greater than or equal to 32 times higher than the MIC, the organism may be tolerant to that drug. Tolerance is a phenomenon most commonly associated with bacterial resistance to beta-lactam antibiotics and reflects an organism's ability to be only inhibited by an agent that is usually bactericidal. Although the physiologic basis of tolerance has been studied in several bacterial species, the actual clinical relevance of this phenomenon has not been well established.

Time-Kill Studies. Another approach to examine bactericidal activity involves exposing a bacterial isolate to a concentration of antibiotic in a broth medium and measuring the rate of killing over a specified period. By this time-kill analysis, samples are taken from the antibiotic-broth solution immediately after the inoculum was added and at regular intervals afterward. Each time-sample is plated to agar plates; following incubation, CFU counts are performed as described for MBC testing. The number of viable bacteria from each sample is plotted over time so that the rate of killing can be determined. Generally, a 1000-fold decrease in the number of viable bacteria in the antibiotic-containing broth after a 24-hour period compared with the number of bacteria in the original inoculum is interpreted as bactericidal activity. Although time-kill analysis is frequently used in the research environment to study the in vitro activity of antimicrobial agents, the labor intensity and technical specifications of the procedure preclude its use in most clinical microbiology laboratories for the production of data used to manage a patient's infection.

Serum Bactericidal Test. The serum bactericidal test (SBT) is analogous to the MIC-MBC test except that the medium used is patient's serum that contains the therapeutic antimicrobial agents that the patient has been receiving. By using patient serum to detect bacteriostatic and bactericidal activity, the antibacterial impact of factors other than the antibiotics (e.g., antibodies and complement) also are observed.

For each test, two serum samples are required. One is collected just before the patient is to receive the next antimicrobial dose and is referred to as the trough specimen. The second sample is collected when the serum antimicrobial concentration is highest and is referred to as the peak specimen. When to collect the peak specimen varies with the pharmacokinetic properties of the antimicrobial agents and the route through which they are being administered. Peak levels for intravenously, intramuscularly, and orally administered agents are generally obtained 30 to 60 minutes, 60 minutes, and 90 minutes after administration, respectively. The trough and peak levels should be collected around the same dose and tested simultaneously.

Serial twofold dilutions of each specimen are prepared and inoculated with the bacterial isolate (final inoculum of 5 x 105 CFU/mL) that is causing the patient's infection. Dilutions are incubated overnight, and the highest dilution inhibiting visibly detectable growth is the serumstatic titre (e.g., 1:8,1:16,1:32).

Aliquots of known size are then taken from each dilution at or below the serumstatic titre (i.e., those dilutions that inhibited bacterial growth) and are plated on sheep blood agar plates. Following incubation, the CFUs per plate are counted and the serum dilution that results in a 99.9% reduction in the CFU/mL as compared with the original inoculum is recorded as the serumcidal titre. For example, if a bacterial isolate showed a serumstatic titre of 1:32, then the tubes containing dilutions of 1:2, 1:4, 1:8, 1:16, and 1:32 would be subcultured. If the 1:8 dilution was the highest dilution to yield a 99.9% decrease in CFUs, then the serumddal titre would be recorded as 1:8.

The SBT was originally developed to help predict the clinical efficacy of antimicrobial therapy for staphylococcal endocarditis. Peak serumcidal litres of 1:32 to 1:64 or greater have been thought to correlate best with a positive clinical outcome. However, even though the test is performed in patient serum, there are still many differences not accounted for between the in vitro test environment and the in vivo site oi infection. Therefore, although the test is used to evaluate whether effective bactericidal concentrations are being achieved, the predictive clinical value for staphylococcal endocarditis or any other infection caused by other bacteria is still uncertain.

Details regarding the performance of these bactericidal tests are provided in the CLSI document M26-A titled, "Methods for Determining Bactericidal Activity of Antimicrobial Agents."

Tests for Activity of Antimicrobial Combinations

Therapeutic management of bacterial infections often requires simultaneous use of more than one antimicrobial agent. Multiple therapies are used for reasons that include:

  • Treating polymicrobial infections caused by organisms with different antimicrobial resistance profiles
  • Achieving more rapid bactericidal activity than could be achieved with any single agent
  • Achieving bactericidal activity against bacteria for which no single agent is lethal (e.g., enterococci)
  • Minimizing the chance of resistant organisms emerging during therapy (e.g., M. tuberculosis)

Testing the effectiveness of antimicrobial combinations against a single bacterial isolate is referred to as synergy testing. When combinations are tested, three outcome categories are possible:

• Synergy: the activity of the antimicrobial combination is substantially greater than the activity of the single most active drug alone

Relevance

1 Clinical significance of bacterial isolate 1 Predictability of isolate's susceptibility Availability of standardized test methods Selection of appropriate antimicrobial agents

Figure 12-16 Goals of effective antimicrobial susceptibility testing strategies.

Accuracy

1 Use of reliable methods 1 Prompt and thorough review of results Prompt resolution of unusual results

Communication

  • Augment susceptibility reports with messages that help clarify and explain potential therapeutic problems not necessarily evident by data alone
  • Indifference: the activity of the combination is no better or worse than the single most active drug alone
  • Antagonism: the activity of the combination is substantially less than the activity of the single most active drug alone (an interaction to be avoided)

The checkerboard assay and the time-kill assay are two basic methods for synergy testing. In the checkerboard method, MIC panels are set up that contain two antimicrobial agents serially diluted alone and in combination with each other. Following inoculation and incubation, the MICs obtained with the single agents i d the various combinations are recorded. By calculating the MIC ratios obtained with single and combined agents, the drug combination in question is classified as synergistic, indifferent, or antagonistic.

Using the time-kil] assay, the same procedure described for testing bactericidal activity is used except the killing curve obtained with a single agent is compared with the killing curve obtained with antimicrobial combinations. Synergy is indicated when the combination exhibits 100-fold or more greater killing than the most active single agent tested alone following 24 hours of incubation. Similar killing rates between the most active agent and the combination is interpreted as indifference. Antagonism is evidenced by the combination being less active than the most active single agent.

The decision to use more than one antimicrobial agent may be based on antimicrobial resistance profiles or identifications of particular bacterial pathogens reported by the clinical microbiology laboratory. However, the decision regarding which antimicrobial agents to combine should not rely on the results of complex synergy tests performed in the clinical laboratory. Most clinically useful antimicrobial combinations have been investigated in a clinical research setting and are well described in the medical literature. These data should be used to guide the decision for combination therapy. The technical difficulties associated with performing and interpreting synergy tests, which at most would only be performed rarely in the clinical laboratory, precludes their reliable utility in the diagnostic setting.

laboratory strategies for antimicrobial susceptibility testing

The clinical microbiology laboratory is responsible for maximizing the positive impact that susceptibility testing information can have on the use of antimicrobial agents to therapeutically manage infectious diseases. However, meeting this responsibility is difficult because of demands for more efficient use of laboratory resources, the increasing complexities of important bacterial resistance profiles, and the continued expectations for high-quality results. To ensure quality in the midst of dwindling resources and expanding antimicrobial resistance, strategies for antimicrobial susceptibility testing must be carefully developed. These strategies should target relevance, accuracy, and communication as their goals (Figure 12-16).

relevance

Antimicrobial susceptibility testing should only be performed when there is sufficient potential for providing clinically useful and reliable information regarding those antimicrobial agents that are appropriate for the bacterial isolate in question. Therefore, for the sake of relevance, two questions must be addressed:

Table 12-5 Categorization of Bacteria According to Need for Routine Performance of Antimicrobial Susceptibility Testing*

Testing Commonly Required

Testing Occasionally Required*

Testing Rarely Required

Staphylococci

Haemophilus influenzae

Bela-nemoiyb'c streptococci (groups A, B, C, F, and G)

Streptococcus pneumoniae

Neisseria gonorrhoeae

Neisseria meningltldes

Viridans streptococci*

Moraxella catarrhalis

Listeria monocytogenes

Enterococci

Anaerobic bacteria

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    How to perform mechanism specific test?
    3 years ago

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