Effect of incubation conditions on anaerobic susceptibility testing results.

We determined the effect of performing antimicrobial susceptibility tests in five different anaerobic incubation systems: GasPak jar, large GasPak jar, evacuated-gassed anaerobic jar, anaerobic chamber, and Bio-Bag. Growth of the anaerobes was equivalent in all five incubation systems. The results of testing 38 anaerobes against 11 antimicrobial agents were comparable for the anaerobic jars and anaerobic chamber. However, discordant results were observed for metronidazole and cefamandole tests when incubated in the Bio-Bag.     

Antimicrobial activity of cefmetazole (CS-1170) and recommendations for susceptibility testing by disk diffusion, dilution, and anaerobic methods

Cefmetazole, formerly CS-1170, was found to have antimicrobial activity slightly superior to that of cefoxitin but a clinically usable antimicrobial spectrum that should be considered identical to that of cefoxitin. Disk diffusion and dilution test methods with cefmetazole correlated highly (r, greater than or equal to 0.95) with cefoxitin results. The recommended 30-micrograms cefmetazole disk interpretive breakpoints for susceptibility and resistance were greater than or equal to 18 mm (MIC, less than or equal to 8.0 micrograms/ml) and less than or equal to 14 mm (MIC, greater than or equal to 32 micrograms/ml), respectively. Cefmetazole and cefoxitin should be considered to be in the same antimicrobial spectrum class, requiring separate testing for other cephalosporins such as cephalothin, cefamandole, cefuroxime, and cefotetan. Recommended interpretive criteria performed well for fastidious organisms (Haemophilus influenzae, Neisseria meningitidis, and Branhamella catarrhalis) and for broth microdilution tests with anaerobes. Cefmetazole and cefoxitin broth disk elution tests for anaerobic bacteria produced higher rates of false susceptibility results.     

Comparison of culture methods for monitoring Legionella species in hospital potable water systems and recommendations for standardization of such methods.

A lack of standardization of environmental monitoring techniques for Legionella spp. complicates the interpretation of results and comparisons of results from different institutions. A comparative assessment of techniques recommended by the Centers for Disease Control and Prevention, the Hygiene Institute (Graz, Austria), and our laboratory was performed. Variables investigated were sampling method (swabbing and collection of water samples [250 ml] before and after swabbing), method of concentration (none, filtration, and centrifugation), acid buffer treatment (no acid treatment, treatment for 3 min, and treatment for 15 min), and choice of medium (five formulations of buffered charcoal yeast extract agar with glycine, vancomycin, polymyxin B, anisomycin, or cycloheximide). Thirty-three sites in seven hospital buildings were studied. Recovery by swab correlated with recovery from water after swabbing (P < 0.05). However, the quantity of Legionella spp. recovered from swab specimens (mean, 3.0 x 10(4) CFU per swab) was greater than that recovered from water (mean, 4.7 x 10(3) CFU/250 ml). Filtration resulted in recovery rates (mean, 5.2 x 10(3) CFU/250 ml) higher than those by centrifugation (mean, 2.3 x 10(3) CFU/250 ml). Three minutes of acid buffer treatment to reduce overgrowth by commensal flora did not improve selectivity or sensitivity for Legionella spp. if glycine-containing selective media were used. Fifteen minutes of acid buffer treatment reduced recovery compared with that after a 3-min treatment. All glycine-containing media tested effectively inhibited background flora, but one selective medium containing dyes, glycine, vancomycin, and polymyxin B (DGVP) resulted in the greatest quantitative recovery of Legionella pneumophila.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of spiral gradient endpoint and agar dilution methods for susceptibility testing of anaerobic bacteria: a multilaboratory collaborative evaluation.

A multilaboratory collaborative study was carried out to assess the utility of the spiral gradient endpoint (SGE) method for the determination of the antimicrobial susceptibilities of anaerobes and to evaluate the equivalence of the MICs obtained by the SGE method with those obtained by the reference agar dilution method of the National Committee for Clinical Laboratory Standards. The standard deviation of the MIC obtained by the SGE method for the five participating laboratories was +/- 0.26 of a twofold dilution, whereas it was +/- 1 twofold dilution by the reference method. The interlaboratory reproducibility of the results for two control strains tested with imipenem, chloramphenicol, and metronidazole indicated that 96% of the measurements fell within +/- 1 twofold dilution of the mode. The equivalence of the SGE method with the agar dilution method was assessed with a wide variety of anaerobic organisms. The MICs by both methods were within 1 doubling dilution in 93% of the measurements (n = 1,074). Discrepancies generally occurred with those organism-drug combinations that resulted in tailing endpoints (Fusobacterium nucleatum, 86% agreement) or in cases of light growth (Peptostreptococcus spp., 86% agreement).     

Comparison of anaerobic susceptibility results obtained by two methods of inoculum preparation

We evaluated the use of inocula prepared directly from blood agar plates in agar dilution susceptibility tests of anaerobic bacteria and compared the results with susceptibility results obtained from the National Committee for Clinical Laboratory Standards proposed thioglycolate broth cultures. The objectives were to evaluate the reproducibility of each of the two methods of inoculum preparation and to compare the MICs obtained by each method. The reproducibility studies were conducted on 14 stock strains. The mode MICs obtained by the direct agar method were identical to those obtained by the reference broth method 74% of the time and within +/- 1 log2 dilution 100% of the time. The degree of reproducibility of each of the two methods was identical (93% +/- 1 log2 dilution). MIC results obtained by the direct agar method agreed with the MICs obtained by the reference broth culture method in 92.9% of 1,125 MIC data pair determinations performed on stock cultures. The reproducibility of the direct agar method within +/- 1 log2 dilution step for 115 fresh clinical isolates was 93%, including 93.4% of the results with the Bacteroides fragilis group. Only two very major discrepancies (false-susceptible by the agar method) were identified among the 708 MIC data pairs on these clinical isolates. Preparation of inocula directly from growth on agar plates provides a rapid and reproducible method for agar dilution susceptibility testing of anaerobes.     

Evaluation of three broth disk methods for testing the susceptibility of anaerobic bacteria to imipenem

Imipenem is a member of a new class of highly potent beta-lactam antibiotics, carbapenems, with an antibacterial spectrum that includes nearly all currently known aerobic and anaerobic bacterial species of clinical significance. Although relatively stable in most standard laboratory media used for antimicrobial susceptibility testing, imipenem undergoes rapid inactivation in thioglycolate broth, a recommended medium for susceptibility testing of anaerobic bacteria by the broth disk method. In the current study, a panel of 36 anaerobic bacteria consisting of 28 clinical isolates and eight quality control strains was used to determine the suitability and accuracy of the broth disk methods with brain heart infusion, Schaedler, and anaerobic broths, in comparison to the reference agar dilution method, for the anaerobic susceptibility testing of imipenem. To achieve single test concentrations of approximately 8, 16, and 64 micrograms/ml for imipenem, cefoxitin, and piperacillin, respectively, which correspond to the MIC breakpoints of the test drugs, four 10-microgram imipenem disks, three 30-microgram cefoxitin disks, and three 100-microgram piperacillin disks were used in 5 ml of broth. The correlation between the reference agar dilution method and each of the three broth disk elution procedures evaluated was excellent, for imipenem (100% agreement) and somewhat less so for cefoxitin and piperacillin. Therefore, brain heart infusion, Schaedler, and anaerobic broths, but not thioglycolate broth, are suitable for anaerobic susceptibility testing of imipenem by the disk elution method.     

Evaluation of broth disk elution methods for susceptibility testing of anaerobic bacteria with the newer beta-lactam antibiotics.

Broth disk elution procedures represent one of the most practical means for clinical laboratories to perform routine antibiotic susceptibility tests on anaerobic bacteria. The accuracy of five disk elution test methods and media (including the one to be proposed by the National Committee for Clinical Laboratory Standards) was evaluated for the testing of newer beta-lactam antibiotics, including cefoperazone, cefotaxime, cefoxitin, ceftazidime, ceftizoxime, moxalactam, and piperacillin. Various numbers of antibiotic disks were used to achieve disk elution test concentrations which approximated the highest MIC termed susceptible by the Food and Drug Administration. A group of 88 anaerobes representing many different species was tested in parallel by the five disk elution methods and the National Committee for Clinical Laboratory Standards reference agar dilution procedure. Overall, full agreement between the reference agar dilution MICs and the disk elution category results was 88.3% for PRAS BHI, 84.5% for Schaedler, 85.7% for thioglycolate, and 87.4% for Wilkins-Chalgren broth. Essential agreement (+/- 1 twofold MIC increment from the disk elution concentration) was achieved with 94.6% of PRAS BHI tests, 94.3% of Schaedler tests, 93.6% of thioglycolate tests, and 95.7% of Wilkins-Chalgren tests. Due to growth failures with a number of isolates and difficulties in interpreting results, the use of Wilkins-West broth was discontinued after approximately one-half of the isolates had been tested. The majority of errors with all of the disk elution methods occurred with isolates (most notably members of the Bacteroides fragilis group) having MICs near the single test concentrations used in the disk methods. With the notable exception of tests for the B. fragilis group, the disk elution methods offered acceptable accuracy with the newer beta-lactam antibiotics tested in this study.     

Evaluation of the E test for susceptibility testing of anaerobic bacteria.

The susceptibilities of 105 clinical isolates of anaerobic bacteria were determined by a new method, the E test (AB Biodisk, Solna, Sweden), and were compared with the MICs for these organisms obtained by the reference agar dilution method by using supplemented brucella and Wilkins-Chalgren agars. The E test is a plastic strip with a predefined antibiotic gradient immobilized on one side and a MIC interpretive scale printed on the other side. Strips with cefoxitin, cefotaxime, imipenem, penicillin, metronidazole, and clindamycin were used in this study. A suspension of the test strain equal to the visual turbidity of a no. 0.5 McFarland standard was prepared and swabbed onto a 150-mm-diameter plate. The strips were applied in a radial fashion, and the plates were incubated under anaerobic conditions. After growth had occurred, an ellipse of inhibition was seen around each strip. At the point of intersection of the ellipse with the strip, the MIC was read from the interpretive scale. For most antibiotic-organism combinations, the ellipse was clear and the endpoint was sharp. The E-test MICs were interpreted after overnight and 48-h incubation for 58 of the strains. After overnight incubation, 87% of the E-test MICs were within 1 dilution of the agar dilution MICs, and 98% were within 2 dilutions. After 48 h of incubation, agreement was 86 and 97% respectively. E-test MICs obtained for the Bacteroides fragilis group after overnight incubation were more comparable than those obtained after 48 h of incubation to agar dilution MICs determined at 48 h for all drugs except clindamycin. On brucella agar, there was a 2% categorical discrepancy rate between the E-test MICs and agar dilution MICs, which occurred mostly with cefoxitin. The E test is easy to perform and read, is suitable for all anaerobes, can be used to test single patient isolates as needed, and offers the laboratory a reliable method for susceptibility testing of anaerobic bacteria.     

Antibiotic susceptibility of blood culture isolates of anaerobic bacteria at a Norwegian university hospital

In the present study the susceptibility of 200 blood culture isolates of anaerobic bacteria to benzylpenicillin, clindamycin, metronidazole, imipenem and piperacillin-tazobactam was examined. Metronidazole, imipenem, and piperacillin-tazobactam showed the highest activity, with 98.5% of the isolates being fully susceptible to either agent. A high rate of reduced susceptibility to clindamycin among both Bacteroides spp. (37%) and Clostridium spp. (28%) was found. Almost all Bacteroides isolates were resistant to penicillin, and only 60% of Prevotella spp. were susceptible to this agent. The antibiotic susceptibility of anaerobic bacteria in Norway should be surveyed regularly.     

Mycobacterial testing in hospital laboratories: results from a questionnaire survey in Italy

Between 1999 and 2001, 355 hospital laboratories in Italy were asked to complete a questionnaire addressing mycobacterial test methods, 1-year workloads and laboratory safety features. Analysis of the data showed that rapid methods for mycobacterial testing were being used by most larger laboratories; however, sub-optimal methods were still in use in small and medium-size laboratories. In a country such as Italy, which has a low prevalence of tuberculosis cases, implementation of rapid technologies, combined with regionalisation of mycobacterial diagnostic services, seems to be the most reasonable and cost-effective strategy.     

Quality control criteria for testing the susceptibility of anaerobic bacteria to meropenem.

Reference values for quality control of in vitro susceptibility tests with meropenem against anaerobic bacteria were determined in a multilaboratory study by the approved National Committee for Clinical Laboratory Standards agar dilution method for the four quality control strains. The study protocol also included the evaluation of microdilution testing, medium additives, and multiple lots of media. The recommended MIC control ranges for three of the control organisms are as follows: Bacteroides fragilis ATCC 25285, 0.06 to 0.125 micrograms/ml; Bacteroides thetaiotaomicron ATCC 29741, 0.125 to 0.5 micrograms/ml; and Eubacterium lentum ATCC 43055, 0.125 to 0.5 micrograms/ml. The modal MIC for Clostridium perfringens ATCC 13124 was at or below the lowest concentration of meropenem tested, and no values are recommended.     

Current status of antifungal susceptibility testing methods.

Antifungal susceptibility testing is a very dynamic field of medical mycology. Standardization of in vitro susceptibility tests by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST), and current availability of reference methods constituted the major remarkable steps in the field. Based on the established minimum inhibitory concentration (MIC) breakpoints, it is now possible to determine the susceptibilities of Candida strains to fluconazole, itraconazole, voriconazole, and flucytosine. Moreover, utility of fluconazole antifungal susceptibility tests as an adjunct in optimizing treatment of candidiasis has now been validated. While the MIC breakpoints and clinical significance of susceptibility testing for the remaining fungi and antifungal drugs remain yet unclear, modifications of the available methods as well as other methodologies are being intensively studied to overcome the present drawbacks and limitations. Among the other methods under investigation are Etest, colorimetric microdilution, agar dilution, determination of fungicidal activity, flow cytometry, and ergosterol quantitation. Etest offers the advantage of practical application and favorable agreement rates with the reference methods that are frequently above acceptable limits. However, MIC breakpoints for Etest remain to be evaluated and established. Development of commercially available, standardized colorimetric panels that are based on CLSI method parameters has added more to the antifungal susceptibility testing armamentarium. Flow cytometry, on the other hand, appears to offer rapid susceptibility testing but requires specified equipment and further evaluation for reproducibility and standardization. Ergosterol quantitation is another novel approach, which appears potentially beneficial particularly in discrimination of azole-resistant isolates from heavy trailers. The method is yet investigational and requires to be further studied. Developments in methodology and applications of antifungal susceptibility testing will hopefully provide enhanced utility in clinical guidance of antifungal therapy. However, and particularly in immunosuppressed host, in vitro susceptibility is and will remain only one of several factors that influence clinical outcome.     

Antimicrobial susceptibility testing of Streptococcus pneumoniae: quality assessment results.

Six strains of Streptococcus pneumoniae were distributed to 405 United Kingdom laboratories who were asked to test the susceptibility of the strains to penicillin, tetracycline, chloramphenicol and erythromycin and to provide details of methodology to test the standards of susceptibility testing. High error rates were seen only in failure to detect moderate resistance to penicillin (12%) and resistance to chloramphenicol (16%). Increased error rates were associated with several methods or practices. These included the use of certain culture media; failure to standardise the inoculum; inoculation by loop rather than by swab; failure to use control organisms; failure to measure zone sizes; the use of discs containing a high content of penicillin to test susceptibility to penicillin, and the use of high content discs for testing erythromycin, tetracycline, and chloramphenicol.     

Assessment of trimethoprim-sulfamethoxazole susceptibility testing methods for fastidious Haemophilus spp.

ObjectivesTo compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement.MethodsWe collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller–Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates.ResultsStrains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm.ConclusionsGiven the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution.     

Review of methods for susceptibility testing of anaerobes

In the USA, the National Committee for Clinical Laboratory Standards has studied and published a reference agar dilution method for the susceptibility testing of anaerobic bacteria. While numerous investigators both in Europe and the USA have evaluated a variety of methods with a variety of modifications, only the broth microdilution method appears to be appropriate for routine use. The problems of the choice of breakpoint, inoculum size, media, media additives, endpoint recognition and other parameters affecting test performance and interpretation, while troublesome for anaerobes, are not unique to this group of organisms. The increasing resistance of anaerobes and the ever existing need to provide therapeutic guidance, surveillance for resistance and susceptibility data on new drugs make the need for an accurate and reliable susceptibility test for anaerobes critical. The newer methods, while showing promise, need further evaluation with all agents that have a therapeutic indication for anaerobic infections.     

Quality control guidelines for testing cefotetan in the reference agar dilution procedure for susceptibility testing of anaerobic bacteria.

Reference values for quality control of in vitro susceptibility tests with cefotetan against anaerobic bacteria were determined in two independent multilaboratory studies with the approved National Committee for Clinical Laboratory Standards agar dilution method and three control strains (Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Clostridium perfringens ATCC 13124). The results of the two studies were in agreement. The recommended MIC control limits for B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 are 4.0 to 16 micrograms/ml and 32 to 128 micrograms/ml, respectively. MICs for C. perfringens ATCC 13124 were too variable to be useful for controlling tests with cefotetan.