Use of Haemophilus test medium for broth microdilution antimicrobial susceptibility testing of Streptococcus pneumoniae.

A recently described medium (Haemophilus test medium [HTM]) for antimicrobial susceptibility testing of Haemophilus influenzae was evaluated in this study for broth microdilution testing of Streptococcus pneumoniae. A total of 137 clinical isolates was tested against 11 antimicrobial agents, using Mueller-Hinton broth supplemented with 3% lysed horse blood in parallel with HTM. Inocula of 5 X 10(5) CFU/ml and incubation for 20 to 24 h were used with both media. All isolates of S. pneumoniae produced acceptable growth in both media, and MICs determined in HTM agreed closely with those determined in lysed horse blood. Drugs which provided a MIC within 1 log2 concentration difference in both media included penicillin (100%), ampicillin (98.0%), amoxicillin-clavulanate (100%), ampicillin-sulbactam (100%), cephalexin (98.9%), cefaclor (96.8%), cefuroxime (99.0%), chloramphenicol (96.2%), tetracycline (96.2%), and erythromycin (100%). HTM MICs with trimethoprim-sulfamethoxazole were 1 to 2 log2 concentration increments higher in 92.0% of isolates than MICs determined in lysed horse blood. Based on the results of this study, HTM appears to represent a promising alternative medium for broth microdilution susceptibility testing of S. pneumoniae.     

Haemophilus influenzae ATCC 49766, an alternative quality control strain for monitoring broth microdilution susceptibility tests with selected beta-lactams.

A beta-lactamase-negative, ampicillin-resistant strain of Haemophilus influenzae is currently used for quality control of broth microdilution tests performed with Haemophilus test medium (HTM). Studies with eight lots of HTM broth documented the fact that MIC limits for some antimicrobial agents are unrealistically stringent; i.e., only three of eight lots of HTM broth were satisfactory for testing cefaclor. An alternative, ampicillin-susceptible strain of H. influenzae (ATCC 49766) was found to provide much more reproducible results with five problematic drugs (cefaclor, cefuroxime, cefamandole, loracarbef, and cefonicid). Multilaboratory studies defined MIC control limits for both control strains tested against 12 antimicrobial agents.     

Interpretive criteria for susceptibilities of Haemophilus influenzae to ampicillin, amoxicillin, and amoxicillin-clavulanic acid.

One hundred fifty isolates of Haemophilus influenzae (including 30 beta-lactamase-positive strains and 23 beta-lactamase-negative, ampicillin-resistant strains) were tested for susceptibilities to ampicillin, amoxicillin, and amoxicillin-clavulanic acid (A/C) by the broth microdilution method in Haemophilus test medium (HTM) and in Mueller-Hinton medium with lysed horse blood and by the disk diffusion method on HTM agar. Our results support the use of HTM for susceptibility testing of H. influenzae but raise a number of questions regarding the interpretive criteria currently in use, particularly with respect to the fourfold difference in MIC susceptibility breakpoints for ampicillin and A/C and a resulting high proportion of A/C-susceptible beta-lactamase-negative, ampicillin-resistant strains.     

Multicenter evaluation of the use of Haemophilus test medium for broth microdilution antimicrobial susceptibility testing of Streptococcus pneumoniae and development of quality control limits.

A five-laboratory collaborative study was undertaken to determine the precision and accuracy of broth microdilution susceptibility tests of Streptococcus pneumoniae isolates performed with Haemophilus test medium (HTM) compared with tests performed with lysed horse blood-supplemented Mueller-Hinton broth (LHB). The intra-and interlaboratory reproducibilities of MICs of 10 antimicrobial agents determined with the two media were found to be quite similar and highly reproducible in both media. On the basis of favorable performance in this study, S. pneumoniae ATCC 49619 is recommended as a quality control strain to assess the performance of HTM when this medium is used for testing of pneumococci. Testing of 293 unique clinical isolates of S. pneumoniae with both media in the respective participant laboratories allowed a direct comparison of MIC results and a calculation of interpretive error rates. Although there were some slight differences between MICs determined with HTM and MICs determined with LHB, few very major or major errors resulted from testing the clinical isolates against the 10 antimicrobial agents. However, MIC-interpretive criteria specific for S. pneumoniae should be developed and promulgated through a national consensus mechanism.     

Haemophilus test medium versus Mueller-Hinton broth with lysed horse blood for antimicrobial susceptibility testing of four bacterial species

Studies were undertaken to determine whether broth microdilution susceptibility tests could be standardized by using a single medium for testing fastidious respiratory pathogens. Mueller-Hinton broth with lysed horse blood and the broth version of Haemophilus Test Medium (HTM) were directly compared. Ten orally administered agents were found to give essentially identical results in both media but minor differences were noted. Because the tests are easier to read when HTM broth is used, that medium is to be preferred for routine testing ofHaemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes andMoraxella catarrhalis isolates by the microdilution procedure.     

Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae.

The need for complex growth media has complicated routine susceptibility testing of Haemophilus influenzae because of antagonism of certain antimicrobial agents by the medium or because of difficulties in interpretation of growth endpoints. Haemophilus test medium (HTM) is a simple, transparent medium for broth- or agar-based tests with H. influenzae. HTM incorporates Mueller-Hinton medium with additions of 15 micrograms of hematin per ml, 15 micrograms of NAD per ml, and 5 mg of yeast extract per ml as growth-promoting additives. Agar or broth microdilution MICs of 10 antimicrobial agents for a collection of 179 H. influenzae isolates determined by using HTM compared favorably with MICs determined by the conventional agar or broth dilution methods recommended by the National Committee for Clinical Laboratory Standards. Disk diffusion tests performed with HTM allowed accurate categorization of susceptible and resistant strains and were easier to interpret than tests performed with Mueller-Hinton chocolate agar. A particular advantage of HTM was the reliability of broth- or agar-based test results with trimethoprim-sulfamethoxazole. The results of the study suggest modification of current National Committee for Clinical Laboratory Standards MIC-interpretive criteria for H. influenzae with amoxicillin-clavulanate, chloramphenicol, and trimethoprim-sulfamethoxazole. Error rate-bounded analysis of MICs and disk diffusion zone sizes also suggest modified zone-interpretive criteria for ampicillin, amoxicillin-clavulanate, chloramphenicol, and tetracycline with HTM or conventional media. Interpretive zone sizes are newly proposed for cefaclor and rifampin disk diffusion tests.     

Broth microdilution testing of Haemophilus influenzae with haemophilus test medium versus lysed horse blood broth. Canadian Haemophilus Study Group.

Broth microdilution testing of 702 community-acquired isolates of Haemophilus influenzae from across Canada was performed with both Mueller-Hinton broth supplemented with 3% lysed horse blood broth (LHB) (BBL Microbiology Systems, Cockeysville, Md.) and haemophilus test medium (HTM). The prevalence of beta-lactamase production was found to be 26% with no regional variation. MICs determined with LHB tended to be higher than those with HTM, but interpretive errors due to these differences were observed only rarely with trimethoprim-sulfamethoxazole (n = 5), cefaclor (n = 8), and cefamandole (n = 3). The interobserver variability in MIC determinations was found to be greater when LHB was used than when HTM was used. There was no difference in intraobserver variability between the two medium formulations. beta-Lactamase-positive isolates developed false resistance to amoxicillin-clavulanate 2 weeks after microdilution panels of both types of medium were stored at -20 degrees C but not when panels were stored at -70 degrees C. In conclusion, this study supports the use of HTM rather than LHB for sensitivity testing of H. influenzae because of its lower rate of interobserver variability and its ability to support the growth of these organisms, which is comparable to that of LHB.     

Quantitative antimicrobial susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae by using the E-test.

The E-test (PDM Epsilometer; AB Biodisk, Solna, Sweden) is an antimicrobial agent gradient-coated plastic test strip which allows MIC determinations on agar media. The test is performed in a manner similar to the agar disk diffusion procedure. A collection of Haemophilus influenzae and Streptococcus pneumoniae strains possessing various resistance mechanisms was used to evaluate the E-test method. H. influenzae strains were tested with both Haemophilus test medium (HTM) and PDM ASM II chocolate agar, while the S. pneumoniae strains were tested on Mueller-Hinton sheep blood agar. E-test MICs for a total of 10 antimicrobial agents were compared with broth microdilution MICs determined according to National Committee for Clinical Laboratory Standards methods. In general, E-test MICs for both species were quickly and easily interpreted and agreed within one log2 MIC increment in 89.8% of tests with H. influenzae and in 80.4% of pneumococcal tests. The majority of disagreements between the E-test and conventional MICs occurred with trimethoprim-sulfamethoxazole because of trailing and diffuse E-test MIC endpoints with both species. Ampicillin MICs for beta-lactamase-producing H. influenzae determined by the E-test differed at times from those determined by conventional testing because of the vagaries of interpreting colonies growing within the E-test inhibition ellipses. E-test penicillin MICs for pneumococci tended to be 1 to 2 log2 dilutions lower than those determined by using Mueller-Hinton broth supplemented with lysed horse blood. Nevertheless, strains of both species with documented resistance to the study drugs were detected by E-tests, i.e., 0.7% of the tests had very major errors with H. influenzae and 0.8% had very major errors with S. pneumoniae. Thus, the E-test represents a potential alternative method for antimicrobial susceptibility testing of these two fastidious bacterial species.     

Variability of clarithromycin and erythromycin susceptibility tests with Haemophilus influenzae in four different broth media and correlation with the standard disk diffusion test.

Four separate laboratories performed antimicrobial susceptibility tests with 40 Haemophilus influenzae isolates, each tested in triplicate. Erythromycin and a new macrolide, clarithromycin (A-56268; TE-031), were tested by the disk diffusion method, by the agar dilution procedure in two different media, and by broth microdilution tests in four different media. Erythromycin MICs for 90% of the strains were 16 micrograms/ml in Mueller-Hinton broth with 3% lysed horse blood and NAD, 4.0 micrograms/ml in hemophilus test medium, and 2.0 micrograms/ml in supplemented Schaedler broth or in the fastidious broth medium from Beckman Instruments, Inc. Clarithromycin MICs were generally 1 doubling dilution greater than erythromycin MICs in each of the media. Erythromycin disk tests corresponded best with MICs determined in the fastidious broth medium. In that same medium, clarithromycin MICs were about 1 doubling dilution greater than what would be expected from the results of disk tests. Because there were fewer growth failures, hemophilus test medium is recommended for microdilution tests with H. influenzae. Incubation of all tests for a full 24 h without an increased CO2 atmosphere was needed to achieve maximal precision of the tests. Interlaboratory and intralaboratory reproducibility of all tests was satisfactory.     

Antimicrobial susceptibility testing of less commonly isolated Haemophilus species using Haemophilus test medium.

Haemophilus test medium (HTM) was developed recently for dilution and disk diffusion antimicrobial agent susceptibility testing of Haemophilus influenzae. The application of HTM to the testing of other, less frequently encountered Haemophilus species recovered from humans was evaluated in this study by using commercially prepared HTM (BBL Microbiology Systems, Cockeysville, Md.) in broth microdilution and agar disk diffusion susceptibility tests with 18 antimicrobial agents. A total of 93.3% of 90 isolates belonging to six Haemophilus species provided acceptable growth in HTM agar disk diffusion tests, while only 63.3% (57 of 90) provided acceptable growth in the broth microdilution tests. However, HTM agar dilution testing provided an alternative method for those strains (primarily H. haemolyticus) which failed to grow adequately in broth. Based on the latest National Committee for Clinical Laboratory Standards guidelines (standard M2-T4) for interpretation of HTM disk tests of H. influenzae, the overall very major, major, and minor errors for all 18 drugs and six species tested were 0.2, 0.7, and 3.4%, respectively. Thus, the use of HTM in agar or broth susceptibility tests can be recommended for testing the less commonly encountered Haemophilus species by using the same test conditions and interpretive guidelines developed for H. influenzae.     

Development of interpretive criteria and quality control limits for broth microdilution and disk diffusion antimicrobial susceptibility testing of Streptococcus pneumoniae.

A five-center collaborative study was undertaken to develop quality control and specific interpretive criteria for susceptibility testing of Streptococcus pneumoniae against 12 antimicrobial agents. MICs were determined for 248 pneumococcal clinical isolates (with an emphasis on resistant strains) by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure incorporating lysed horse blood-supplemented Mueller-Hinton broth. NCCLS disk diffusion testing was also performed for each isolate by using Mueller-Hinton sheep blood agar incubated in 5% CO2. Repetitive testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of quality control ranges which encompassed approximately 95% of MIC and zone size values observed in the study. Good intra- and interlaboratory reproducibilities were seen with these testing methods and all of the drugs examined. On the basis of the results of this study, MIC interpretive criteria are proposed for 11 agents. Comparisons of MICs and disk diffusion zone sizes allowed disk diffusion zone size interpretive criteria to be proposed for five drugs and confirmed the use of the oxacillin disk test for prediction of penicillin susceptibility among pneumococci. Excessive numbers of minor-category interpretive errors precludes recommendation at this time of the disk diffusion method for testing of pneumococci against five of the drugs. Use of these proposed quality control and interpretive criteria should provide for reproducible test results and allow recognition of recently emerging resistance among pneumococcal clinical isolates.     

Influence of variations in test methods on susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin

The National Committee for Clinical Laboratory Standards standard broth microdilution method for testing the susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin was evaluated by altering one variable at a time. Variables that were tested included age of colony for inoculum preparation, inoculum density, test medium, incubation atmosphere, and incubation time. For the macrolide, azalide, and ketolide agents, incubation in 5 to 7% CO(2) most significantly affected the MICs, producing nearly twofold increases for clarithromycin and telithromycin and a greater than threefold increase for azithromycin. For ampicillin, a 10-fold increase in inoculum density increased the geometric mean MICs for beta-lactamase-negative strains from 1. 50 to 2.45 microg/ml. In addition, 206 H. influenzae strains were tested for their susceptibilities to the same drugs by the broth microdilution tests in two media, as well as by agar dilution tests, disk diffusion tests, and Etests, on six different agar media. The three standard methods with Haemophilus test medium (HTM) compared favorably with each other except for a high minor discrepancy rate (27%) by the disk diffusion test with ampicillin and clarithromycin. Agar dilution test MICs on the five comparative media were generally higher than those on HTM agar but were only rarely more than one twofold concentration higher. Etest MICs of azithromycin and telithromycin were more than twofold higher than agar dilution and broth microdilution MICs on HTM; ampicillin Etest MICs were nearly twofold lower. The use of media other than HTM agar appears to have a minimal effect on susceptibility test results for the ketolide, azalide, or macrolide drugs that we tested against H. influenzae.     

Evaluation of the Dade MicroScan MICroSTREP Antimicrobial Susceptibility Testing Panel with Selected Streptococcus pneumoniae Challenge Strains and Recent Clinical Isolates

The MicroScan MICroSTREP panel is a recently marketed frozen broth microdilution panel for susceptibility testing of various streptococci, including Streptococcus pneumoniae. The panel contains 10 antimicrobial agents in cation-adjusted Mueller-Hinton broth supplemented with 3% lysed horse blood, similar in concept to the National Committee for Clinical Laboratory Standards (NCCLS) reference broth microdilution method for testing streptococci. A group of 210 isolates of S. pneumoniae were selected to include isolates with previously documented resistance to agents incorporated in the MICroSTREP panel, as well as recent invasive clinical isolates. All isolates were tested simultaneously with the MICroSTREP panel and an NCCLS reference panel whose drug concentrations were prepared to coincide with those of the MICroSTREP panel. Of the 210 isolates, 5 failed to grow in the MICroSTREP panel; 3 of those also did not grow in the reference panel. Essential agreement of MICs determined by the two methods (test MIC ± one dilution of the reference MIC) was 99.6% overall and ranged from 98.0% with chloramphenicol to 100% with penicillin, ceftriaxone, erythromycin, tetracycline, and vancomycin. There were no very major or major interpretive category errors resulting from the MICroSTREP panel tests. Minor interpretive category errors ranged from 12.2% with cefotaxime and 9.8% with ceftriaxone (due mainly to clustering of MICs for the selected strains near the breakpoints) to 0% with chloramphenicol and vancomycin. These results indicate that the MicroScan MICroSTREP frozen panels provide susceptibility results with pneumococci that are essentially equivalent to results derived by the NCCLS reference broth microdilution procedure.     

Investigation of ampicillin-intermediate strains of Haemophilus influenzae by using the disk diffusion procedure and current National Committee for Clinical Laboratory Standards guidelines.

It was noted in our laboratory that certain strains of Haemophilus influenzae yielded zone sizes interpreted as resistant to the ampicillin (AMP) disk on chocolate-Mueller-Hinton agar (CMH) but showed no evidence of beta-lactamase (beta-Lac) activity. Although it is known that a second mechanism of AMP resistance exists, strains with this mechanism are uncommon. To investigate this apparent discrepancy, a study of 100 consecutive clinical isolates of H. influenzae collected over a 6-month period was performed. Isolates were simultaneously tested against five antibiotics (AMP, chloramphenicol, cefotaxime, ciprofloxacin, and AMP-sulbactam) on CMH and on two brands of Haemophilus test medium (HTM) by using the disk diffusion procedure and National Committee for Clinical Laboratory Standards (NCCLS) standards. By using CMH and NCCLS standard M2-A3-S2, strains of H. influenzae showing zone sizes of greater than or equal to 20 mm with AMP were considered sensitive. By using HTM and NCCLS standard M2-A4, strains showing zone sizes of greater than or equal to 25 mm to AMP on HTM were considered sensitive. Intermediate strains had zone sizes of 22 to 24 mm. The majority of isolates (68%) were sensitive to all antibiotics. Two percent of the isolates were resistant to chloramphenicol. Seventeen percent of the isolates were AMP-resistant, beta-Lac-producing strains of H. influenzae. Thirteen percent of the isolates gave at least one intermediate or resistant zone for AMP but were beta-Lac negative. MIC determinations with NCCLS standard M7-A2 were performed with resistant and intermediate strains. MICs for beta-Lac-producing strains of H. influenzae were >/= 8.0 microgram/ml. MICs for beta-Lac-negative strains were 相似文献   

Activity of Gatifloxacin against Haemophilus influenzae and Moraxella catarrhalis, Including Susceptibility Test Development, E-Test Comparisons, and Quality Control Guidelines for H. influenzae

In vitro antimicrobial activity and susceptibility testing interpretation criteria and quality control were studied for gatifloxacin, a new 8-methoxy fluoroquinolone, tested against Haemophilus influenzae. Moraxella catarrhalis (600 strains) and H. influenzae (1,400 strains) from the SENTRY Antimicrobial Surveillance Program in North America (Canada and the United States) were also tested against gatifloxacin and 12 other antimicrobial agents. Gatifloxacin (MIC at which 90% of the isolates are inhibited [MIC90], /=18 mm) was also suggested for H. influenzae testing. No interpretive errors were observed. Quality control guidelines for H. influenzae ATCC 49247 were determined by using the NCCLS M23-T3 (1998) study design. The results from the nine-laboratory protocol suggested the following control ranges: for broth microdilution tests, 0.004 to 0.03 microg/ml; for disk diffusion testing, 33 to 41 mm. Gatifloxacin appears to be a potent anti-Haemophilus fluoroquinolone compound with in vitro testing interpretive criteria that will produce accurate results (disk diffusion, broth microdilution, and E-test).     

Surveillance of susceptibility testing methodologies for Haemophilus influenzae in Canada, including evaluation of disk diffusion test.

We surveyed 75 clinical laboratories to determine if National Committee for Clinical Laboratory Standards (NCCLS) were being used for the susceptibility testing of Haemophilus influenzae. Of the 66 laboratories that performed susceptibility testing, all claimed to follow current NCCLS guidelines. However, upon further questioning, only 23, all of which used disk diffusion testing, accurately interpreted and followed the guidelines. Proficiency testing of 22 of these laboratories found that an unacceptable number of interpretive errors (> 10%) occurred. These results query the merit of routine disk diffusion susceptibility testing of H. influenzae to beta-lactam agents.     

Evaluation of disk diffusion methods to detect low-level β-lactamase-negative ampicillin-resistant Haemophilus influenzae

We evaluated the efficacy of disk diffusion methods for detection of low-level β-lactamase-negative ampicillin-resistant (low-BLNAR) Haemophilus influenzae. Four hundred and seventy unselected, recent clinical isolates were tested with ampicillin (10 μg), cefaclor (30 μg) and cefuroxime (30 μg) on iso-Sensitest agar enriched with nicotinamide adenine dinucleotide (NAD) and horse blood [ST agar; Swedish Reference Group for Antibiotics (SRGA) guidelines], and on chocolate agar (in-house guidelines). Selected isolates (n = 147) were subjected to partial sequencing of the ftsI gene. Forty-seven strains (10.0%) were genotypically identified as low-BLNAR, which was confirmed by determination of minimal inhibitory concentration (MIC) using microbroth dilution method: only low level resistance to ampicillin was detected [MIC ≤1 μg/mL; MIC(50) = 0.5 μg/mL, implying susceptibility by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antibiotic Susceptibility Testing (EUCAST) interpretative criteria]. The MIC of cefuroxime varied between 1 and 4 μg/mL (MIC(50) = 2 μg/mL), indicating susceptibility to cefuroxime by CLSI but not by EUCAST guidelines. Disk diffusion methods were able to discriminate low-BLNAR H. influenzae from the wild-type population with sensitivities ranging from 87% to 98% and specificities from 96% to 99%. Cefaclor was found to be superior to cefuroxime and ampicillin. Cefaclor zone diameter breakpoints of 30/29 and 23/22 mm are suggested for ST agar and chocolate agar, respectively.     

Development of interpretive criteria and quality control limits for macrolide and clindamycin susceptibility testing of Streptococcus pneumoniae.

A six-laboratory collaborative study was conducted to develop MIC and zone diameter quality control limits and interpretive criteria for antimicrobial susceptibility testing of Streptococcus pneumoniae with azithromycin, clarithromycin, dirithromycin, and clindamycin. The MICs of all of the agents plus erythromycin for 302 clinical isolates of pneumococci that had been selected with an emphasis on resistant strains were determined by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure. The zone diameters of the isolates were also determined for the same agents except erythromycin by the NCCLS disk diffusion test procedure. Repeated testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of MIC and zone diameter quality control ranges for these agents. Interpretive criteria for the MIC of azithromycin were established and were as follows: susceptible, < or = 0.5 microgram/ml; intermediate, 1 microgram/ml; and resistant, > or = 2 micrograms/ml. The interpretive criteria advocated for the MICs of clarithromycin and clindamycin were as follows: susceptible, < or = 0.25 microgram/ml; intermediate, 0.5 microgram/ml; and resistant, > or = 1 microgram/ml. Comparison of MICs and disk diffusion zone diameters led to the development of interpretive criteria for the zone diameters for azithromycin, clarithromycin, and clindamycin that correlated well with these MIC breakpoints. Testing of this organism collection also led to the reestablishment of the erythromycin MIC breakpoints as being identical to those of clarithromycin, which resulted in equivalent cross-susceptibility and cross-resistance for the three macrolides that are currently marketed in the United States. Thus, the susceptibility of pneumococci to azithromycin and clarithromycin can be predicted accurately by testing only erythromycin in clinical laboratories. This recommendation, as well as the interpretive and quality control criteria that are described, have been accepted by NCCLS and are included in the latest NCCLS susceptibility testing guidelines.     

Performance of Haemophilus Test Media prepared with 12 different lots of Mueller-Hinton agar from four manufacturers.

Haemophilus Test Media (HTM) were prepared from 12 different lots of Mueller-Hinton agar. When tested with Haemophilus influenzae ATCC 49247, most lots were initially rejected because of small zones of inhibition for cefaclor, cefuroxime, and cefamandole disks, whereas five other drugs performed satisfactorily on the 11 lots that supported growth of the control strain. At the same time, tests of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 documented the acceptability of these agar media, with or without HTM supplements. The current control limits for cefaclor, cefuroxime, and cefamandole appear to be unrealistic. Because the beta-lactamase-negative, ampicillin-resistant control strain of H. influenzae (ATCC 49247) has altered penicillin-binding proteins, it is resistant to ampicillin and is probably also resistant to cefaclor, cefuroxime, and cefamandole. Consequently, media that produce no or very small zones of inhibition with those three drugs might be clinically correct and should not be rejected. One manufacturer provided three lots of Mueller-Hinton agar that gave unusually large zones of inhibition with all beta-lactams. The three other manufacturers provided eight Mueller-Hinton agars that were satisfactory for the preparation of HTM agar, provided that small zones of inhibition with cefaclor, cefuroxime, and cefamandole disks are accepted as the preferred result.     

Interpretive criteria and quality control parameters for testing of susceptibilities of Haemophilus influenzae and Streptococcus pneumoniae to trimethoprim and trimethoprim-sulfamethoxazole. The Antimicrobial Susceptibility Testing OC Group.

Two hundred twenty-eight strains of Haemophilus influenzae and 234 strains of Streptococcus pneumoniae were tested by broth microdilution and disk diffusion methods for susceptibility to trimethoprim (TMP) and TMP-sulfamethoxazole (SMX) to evaluate proposed criteria. Data are presented to support the proposed TMP MIC breakpoints of < or = 2.0 micrograms/ml for susceptibility and > or = 4.0 micrograms/ml for resistance for both species and TMP-SMX MIC breakpoints of < or = 2.0-38 micrograms/ml for susceptibility and > or = 4.0-76 micrograms/ml for resistance. Corresponding zone diameter breakpoints for H. influenzae for both drugs are proposed: < or = 10 mm = resistant; > or = 16 mm = susceptible. A 10-laboratory study documented reproducibility of such tests with standard control strains. The following control limits are proposed for tests of H. influenzae ATCC 49247 against TMP; MIC, 0.12 to 0.5 microgram/ml; zone diameter, 27 to 33 mm. The current limits for TMP-SMX were confirmed. For tests of S. pneumoniae ATCC 49619, MICs of TMP were 1.0 to 4.0 micrograms/ml and the current TMP-SMX MIC range was confirmed. Disk susceptibility tests of either drug against pneumococci were not reproducible, and consequently neither quality control limits nor interpretive criteria could be established. Endpoint interpretation and lot-to-lot variability in Mueller-Hinton agars were significant factors leading to interlaboratory variability.