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The Role of the Microbiology Laboratory in Antimicrobial Susceptibility Testing
Introduction
One of the major functions of the microbiology laboratory is to determine the antimicrobial susceptibility of clinically significant isolates. While the susceptibility of the ß-hemolytic streptococci to penicillins and cephalosporins remains unchanged, the resistance of other bacteria has increased in recent years as novel resistance mechanisms have emerged apace. It is therefore necessary to determine the susceptibility of streptococci other than ß-hemolytic groups, staphylococci, enterococci, and gram-negative bacilli to various antimicrobial agents.
Antimicrobials to Be Tested
The microbiologist must work with the hospital's formulary committee to ensure that the antibiotics being tested are the same or are in the same spectrum class as those in the formulary. The laboratory should then ensure that the antibiotics selected for testing are appropriate to the type of organism isolated and to the site of infection. For example, cefaclor and erythromycin would not be appropriate for an isolate of Streptococcus pneumoniae from the spinal fluid of a child with meningitis; they would be appropriate if this organism were isolated from a respiratory tract specimen. Because enterococci utilize exogenous folate, which is present in urine but not in the medium used for susceptibility testing, they will appear falsely susceptible to trimethoprim/sulfamethoxazole (TMP-SMX) in vitro, and the laboratory should not test this group of organisms against this antimicrobial combination. Systemic enterococcal infections have developed during treatment of an enterococcal urinary tract infection with TMP-SMX. Whatever is known about antibiotic resistance in the community or in the hospital is an additional consideration in the selection of antibiotics to be tested.
There is a basic set of antibiotics that needs to be tested routinely, depending on the organism's colonial and microscopic characteristics. For example, penicillin and oxacillin should be tested against staphylococci; ampicillin, cephalothin or cefazolin, a third-generation cephalosporin, an expanded spectrum penicillin, and gentamicin against the Enterobacteriaceae; ceftazidime, a carboxypenicillin or ureidopenicillin and gentamicin against Pseudomonas aeruginosa; penicillin and ceftriaxone against pneumococci. These basic sets may be supplemented as necessary according to the formulary and local patterns of antibiotic resistance.
The concept of spectrum class is important to understand because the laboratory ordinarily tests only one representative of a particular spectrum class. For example, oxacillin is tested against staphylococci as the representative for the penicillinase-resistant penicillins (ie, methicillin and nafcillin). Tetracycline is used to represent doxycycline and minocycline. Cefotaxime can be used to represent the third-generation cephalosporins against the Enterobacteriaceae. Susceptibility to the tested antibiotic predicts susceptibility or resistance to the other antibiotics in the same spectrum class, and there is no need to test each member of that class separately.
Susceptibility Testing Method
Two basic test methods are used today -- disk diffusion and dilution. Although the dilution method provides a quantitative result, the minimum inhibitory concentration (MIC, which is expressed in micrograms per milliliter), this value requires interpretation. The choice of method is made by the laboratory director based on a number of technical and logistical considerations. The methods are of comparable accuracy. In both cases, the results are reported as susceptible, intermediate, or resistant. If the dilution test is being used, the MICs are usually reported.
Interpretation of Results
One of the most challenging tasks facing the microbiologist is to ensure the proper interpretation of results of susceptibility testing. Several principles concerning the susceptibility test need to be understood.[1] First, the MIC is not a chemical or physical measurement, as is the case with a serum creatinine or a hemoglobin assay. Second, host factors may be a more important determinant of clinical outcome than the results of susceptibility testing. Susceptibility of an organism to an antibiotic, therefore, may not predict successful therapy; however, resistance in vitro should indicate a high probability of therapeutic failure.
The dilution test is reproducible to within ± 1 dilution. Thus, a ticarcillin MIC of 64 µg/mL for an isolate of P aeruginosa may be 32 or 128 µg/mL when tested against subsequent isolates of the same organism from the same site in the same patient. In this example, the variation in results is problematic because susceptibility of P aeruginosa to ticarcillin is defined by an MIC of 64 µg/mL or less and resistance by an MIC of 128 µg/mL. Such single-dilution differences between susceptibility and resistance are uncommon; in most cases there is an intermediate category.
Despite standardization of the inoculum size, test medium composition, disk content of antibiotic, incubation conditions, and interpretative criteria by the National Committee for Clinical Laboratory Standards (NCCLS), minor variations, particularly of inoculum size, may affect the test result.
It is also important to bear in mind that the conditions in vivo may differ substantially from those in vitro. For example, the number of organisms at the infected site may differ from that used in the test (5 x 105 colony-forming units per milliliter). The pH at the site of infection may also differ from that in the test (pH 7.4). Therefore, the MIC of an antibiotic for a specific organism must be considered in relation to the MICs for other isolates that are known to be susceptible on the basis of clinical information. Add to this host factors, pharmacokinetics, the conditions at the infected site, and specific microbial pathogenic characteristics, and it should be apparent that the definition of susceptibility is not easily made. As Baquero[2] stated, the "criteria for antibiotic susceptibility have frequently resulted from a blend of science, faith and business."
The breakpoint for resistance is more easily defined by the knowledge of resistance mechanisms and therapeutic failure in animal model infections (for example, endocarditis, meningitis, peritonitis, osteomyelitis) and in subsequent human clinical trials. In the latter case, the endpoints being sought are clinical cure and/or bacterial eradication. In the final analysis, the definition of breakpoints is based on a composite of what is known about the antibiotic's pharmacokinetics, toxicity profile, in vitro activity, stability to known resistance mechanisms, in vivo activity, and claims made by the manufacturer.
The intermediate category is often confusing to physicians. For the ß-lactams, which have a broad toxic-therapeutic ratio, it usually indicates that the organism causing the infection may be treated with a higher dose of the antibiotic or the drug may be used in situations in which the infection is in a site where the antibiotic is concentrated (for example, the urinary tract). With aminoglycosides, which have a narrow toxic to therapeutic ratio, the intermediate category is a buffer zone that takes into account the normal variability of the test.
In conclusion, the microbiology laboratory has the responsibility for susceptibility testing clinically significant isolates using standardized methodology and antibiotics that are appropriate for the treatment of organisms causing infection at specific sites; it also has responsibility for providing interpretative information to help guide treatment.
References
1. Rex JA, Pfaller MA, Galgiani JN, et al. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and Candida infections. Clin Infect Dis. 1997; 24:235-247.
2. Baquero F. European standards for antimicrobial susceptibility testing: towards theoretical consensus. Eur J Clin Microbiol. 1990;9:492-495.
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The Role of the Microbiology Laboratory in Antimicrobial Susceptibility Testing
Introduction
One of the major functions of the microbiology laboratory is to determine the antimicrobial susceptibility of clinically significant isolates. While the susceptibility of the ß-hemolytic streptococci to penicillins and cephalosporins remains unchanged, the resistance of other bacteria has increased in recent years as novel resistance mechanisms have emerged apace. It is therefore necessary to determine the susceptibility of streptococci other than ß-hemolytic groups, staphylococci, enterococci, and gram-negative bacilli to various antimicrobial agents.
Antimicrobials to Be Tested
The microbiologist must work with the hospital's formulary committee to ensure that the antibiotics being tested are the same or are in the same spectrum class as those in the formulary. The laboratory should then ensure that the antibiotics selected for testing are appropriate to the type of organism isolated and to the site of infection. For example, cefaclor and erythromycin would not be appropriate for an isolate of Streptococcus pneumoniae from the spinal fluid of a child with meningitis; they would be appropriate if this organism were isolated from a respiratory tract specimen. Because enterococci utilize exogenous folate, which is present in urine but not in the medium used for susceptibility testing, they will appear falsely susceptible to trimethoprim/sulfamethoxazole (TMP-SMX) in vitro, and the laboratory should not test this group of organisms against this antimicrobial combination. Systemic enterococcal infections have developed during treatment of an enterococcal urinary tract infection with TMP-SMX. Whatever is known about antibiotic resistance in the community or in the hospital is an additional consideration in the selection of antibiotics to be tested.
There is a basic set of antibiotics that needs to be tested routinely, depending on the organism's colonial and microscopic characteristics. For example, penicillin and oxacillin should be tested against staphylococci; ampicillin, cephalothin or cefazolin, a third-generation cephalosporin, an expanded spectrum penicillin, and gentamicin against the Enterobacteriaceae; ceftazidime, a carboxypenicillin or ureidopenicillin and gentamicin against Pseudomonas aeruginosa; penicillin and ceftriaxone against pneumococci. These basic sets may be supplemented as necessary according to the formulary and local patterns of antibiotic resistance.
The concept of spectrum class is important to understand because the laboratory ordinarily tests only one representative of a particular spectrum class. For example, oxacillin is tested against staphylococci as the representative for the penicillinase-resistant penicillins (ie, methicillin and nafcillin). Tetracycline is used to represent doxycycline and minocycline. Cefotaxime can be used to represent the third-generation cephalosporins against the Enterobacteriaceae. Susceptibility to the tested antibiotic predicts susceptibility or resistance to the other antibiotics in the same spectrum class, and there is no need to test each member of that class separately.
Susceptibility Testing Method
Two basic test methods are used today -- disk diffusion and dilution. Although the dilution method provides a quantitative result, the minimum inhibitory concentration (MIC, which is expressed in micrograms per milliliter), this value requires interpretation. The choice of method is made by the laboratory director based on a number of technical and logistical considerations. The methods are of comparable accuracy. In both cases, the results are reported as susceptible, intermediate, or resistant. If the dilution test is being used, the MICs are usually reported.
Interpretation of Results
One of the most challenging tasks facing the microbiologist is to ensure the proper interpretation of results of susceptibility testing. Several principles concerning the susceptibility test need to be understood.[1] First, the MIC is not a chemical or physical measurement, as is the case with a serum creatinine or a hemoglobin assay. Second, host factors may be a more important determinant of clinical outcome than the results of susceptibility testing. Susceptibility of an organism to an antibiotic, therefore, may not predict successful therapy; however, resistance in vitro should indicate a high probability of therapeutic failure.
The dilution test is reproducible to within ± 1 dilution. Thus, a ticarcillin MIC of 64 µg/mL for an isolate of P aeruginosa may be 32 or 128 µg/mL when tested against subsequent isolates of the same organism from the same site in the same patient. In this example, the variation in results is problematic because susceptibility of P aeruginosa to ticarcillin is defined by an MIC of 64 µg/mL or less and resistance by an MIC of 128 µg/mL. Such single-dilution differences between susceptibility and resistance are uncommon; in most cases there is an intermediate category.
Despite standardization of the inoculum size, test medium composition, disk content of antibiotic, incubation conditions, and interpretative criteria by the National Committee for Clinical Laboratory Standards (NCCLS), minor variations, particularly of inoculum size, may affect the test result.
It is also important to bear in mind that the conditions in vivo may differ substantially from those in vitro. For example, the number of organisms at the infected site may differ from that used in the test (5 x 105 colony-forming units per milliliter). The pH at the site of infection may also differ from that in the test (pH 7.4). Therefore, the MIC of an antibiotic for a specific organism must be considered in relation to the MICs for other isolates that are known to be susceptible on the basis of clinical information. Add to this host factors, pharmacokinetics, the conditions at the infected site, and specific microbial pathogenic characteristics, and it should be apparent that the definition of susceptibility is not easily made. As Baquero[2] stated, the "criteria for antibiotic susceptibility have frequently resulted from a blend of science, faith and business."
The breakpoint for resistance is more easily defined by the knowledge of resistance mechanisms and therapeutic failure in animal model infections (for example, endocarditis, meningitis, peritonitis, osteomyelitis) and in subsequent human clinical trials. In the latter case, the endpoints being sought are clinical cure and/or bacterial eradication. In the final analysis, the definition of breakpoints is based on a composite of what is known about the antibiotic's pharmacokinetics, toxicity profile, in vitro activity, stability to known resistance mechanisms, in vivo activity, and claims made by the manufacturer.
The intermediate category is often confusing to physicians. For the ß-lactams, which have a broad toxic-therapeutic ratio, it usually indicates that the organism causing the infection may be treated with a higher dose of the antibiotic or the drug may be used in situations in which the infection is in a site where the antibiotic is concentrated (for example, the urinary tract). With aminoglycosides, which have a narrow toxic to therapeutic ratio, the intermediate category is a buffer zone that takes into account the normal variability of the test.
In conclusion, the microbiology laboratory has the responsibility for susceptibility testing clinically significant isolates using standardized methodology and antibiotics that are appropriate for the treatment of organisms causing infection at specific sites; it also has responsibility for providing interpretative information to help guide treatment.
References
1. Rex JA, Pfaller MA, Galgiani JN, et al. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and Candida infections. Clin Infect Dis. 1997; 24:235-247.
2. Baquero F. European standards for antimicrobial susceptibility testing: towards theoretical consensus. Eur J Clin Microbiol. 1990;9:492-495.
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