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.     

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.     

Comparison of agar dilution, broth dilution, and disk diffusion testing of ampicillin against Haemophilus species by using in-house and commercially prepared media.

An evaluation to determine the optimal method for the in vitro susceptibility testing of Haemophilus strains to ampicillin was undertaken. In our hands, in-house-prepared Haemophilus Test Medium used by either the broth macrodilution or agar dilution method produced the most consistent results, especially for beta-lactamase-negative, ampicillin-resistant H. influenzae strains.     

Ampicillin disk diffusion susceptibility testing of Haemophilus influenzae.

A total of 114 strains of Haemophilus influenza were characterized with respect to beta-lactamase production and ampicillin MIC. Of this total, 41 strains produced a TEM-type beta-lactamase, and ampicillin MICs for these strains were greater than or equal to 2.0 microgram/ml. It was found that 54 strains lacked TEM-type beta-lactamase activity, and ampicillin MICs for them were less than or equal to 0.5 microgram/ml. The remaining 19 strains were beta-lactamase negative, but ampicillin MICs were greater than or equal to 2.0 micrograms/ml. Disk diffusion susceptibility tests were performed with two media, i.e., Mueller-Hinton agar containing 1.0% hemoglobin and 1.0% IsoVitaleX supplement (CHOC-MHA) and enriched chocolate agar (CHOC), by using disks containing 10 and 2 micrograms of ampicillin. If strains of H. influenzae for which ampicillin MICs were greater than or equal to 2.0 micrograms/ml were considered resistant, while strains for which MICs were less than or equal to 0.5 microgram/ml were considered susceptible, the following zone diameter interpretive criteria were identified as indicating ampicillin susceptibility: CHOC-MHA (10-micrograms disks), greater than or equal to 20 mm; CHOC-MHA (2-micrograms disks), greater than or equal to 17 mm; CHOC (10-micrograms disks), greater than or equal to 25 mm; and CHOC (2-micrograms disks), greater than or equal to 20 mm. In all cases, zones of inhibition less than those listed above would be interpreted as indicating resistance. Each of these four combinations was found to be essentially equivalent in identifying susceptible and resistant strains of H. influenzae, irrespective of beta-lactamase production.     

Reevaluation of interpretive criteria for Haemophilus influenzae by using meropenem (10-microgram), imipenem (10-microgram), and ampicillin (2- and 10-microgram) disks.

A collection of 300 Haemophilus influenzae clinical strains was used to assess in vitro susceptibility to carbapenems (meropenem, imipenem) by MIC and disk diffusion methods and to compare disk diffusion test results with two potencies of ampicillin disks (2 and 10 micrograms). The isolates included ampicillin-susceptible or- intermediate (167 strains), beta-lactamase-positive (117 strains), and beta-lactamase-negative ampicillin-resistant (BLNAR; 16 strains) organisms. Disk diffusion testing was performed with 10-micrograms meropenem disks from two manufacturers. Meropenem was highly active against H. influenzae strains (MIC50, 0.06 microgram/ml; MIC90, 0.25 microgram/ml; MIC50 and MIC90, MICs at which 50 and 90%, respectively, of strains are inhibited) and was 8- to 16-fold more potent than imipenem (MIC50, 1 microgram/ml; MIC90, 2 micrograms/ml). Five non-imipenem-susceptible strains were identified (MIC, 8 micrograms/ml), but the disk diffusion test indicated susceptibility (zone diameters, 18 to 21 mm). MIC values of meropenem, doxycycline, ceftazidime, and ceftriaxone for BLNAR strains were two- to fourfold greater than those for other strains. The performance of both meropenem disks was comparable and considered acceptable. A single susceptible interpretive zone diameter of > or = 17 mm (MIC, < = or 4 micrograms/ml) was proposed for meropenem. Testing with the 2-micrograms ampicillin disk was preferred because of an excellent correlation between MIC values and zone diameters (r = 0.94) and superior interpretive accuracy with the susceptible criteria at > or = 17 mm (MIC, < or = 1 microgram/ml) and the resistant criteria at < or = 13 mm (MIC, > or = 4 micrograms/ml). Among the BLNAR strains tested, 81.3% were miscategorized as susceptible or intermediate when the 10-micrograms ampicillin disk was used, while the 2-micrograms disk produced only minor interpretive errors (12.5%). Use of these criteria for testing H. influenzae against meropenem and ampicillin should maximize reference test and standardized disk diffusion test performance with the Haemophilus Test Medium. The imipenem disk diffusion test appears compromised and should be used with caution for detecting strains for which imipenem MICs are elevated.     

Simple screening method for beta-lactamase-positive and -negative ampicillin-resistant Haemophilus influenzae isolates.

A simple, inexpensive method for screening for beta-lactamase-positive and beta-lactamase-negative ampicillin-resistant Haemophilus influenzae isolates was developed. Disks containing 10 micrograms of cloxacillin yielded no zone of inhibition when placed on chocolate agar plates inoculated with beta-lactamase-positive (16 strains) or ampicillin-resistant (greater than or equal to 1 microgram/ml) beta-lactamase-negative (10 strains) H. influenzae, whereas ampicillin-susceptible (less than or equal to 0.5 microgram/ml; 36 strains) H. influenzae almost always (92%) showed a zone of inhibition.     

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.     

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.     

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.     

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.     

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).     

Rapid method for determining antimicrobial susceptibility of Haemophilus influenzae.

A method was developed to determine the susceptibility of Haemophilus influenzae to ampicillin, cefamandole, and chloramphenicol by using the MS-2 system (Abbott Laboratories) for determining minimum inhibitory concentrations (MIC). The MS-2 results for 132 strains of H. influenzae were compared with the results of agar disk diffusion, agar dilution, and beta-lactamase tests. Twenty-four strains (18.2%) of H. influenzae were resistant to ampicillin by the agar dilution method, as opposed to 25 strains by the MS-2 method. For a beta-lactamase-negative strain, the agar dilution MIC was 4 micrograms/ml, and the MS-2 MIC was 16 micrograms/ml. Twenty-one strains produced beta-lactamase; two beta-lactamase-negative strains were resistant by MS-2, agar dilution, and agar disk diffusion. In addition, one beta-lactamase-negative strain, for which the agar dilution MIC was 32 micrograms/ml and the MS-2 MIC was 16 micrograms/ml, was sensitive by agar disk diffusion. Overall, the MS-2 method compared favorably with the agar dilution method for determining the MIC of ampicillin, cefamandole, and chloramphenicol for H. influenzae.     

Tentative criteria for confirming the in vitro susceptibilities of Haemophilus influenzae and Neisseria gonorrhoeae to two fluoroquinolones (sparfloxacin and levofloxacin), including quality control parameters.

Sparfloxacin and levofloxacin were evaluated against 150 Haemophilus influenzae isolates and 149 Neisseria gonorrhoeae isolates in order to define susceptibility testing parameters. Sparfloxacin-susceptible H. influenzae strains were defined as those for which the MICs were < or = 0.25 microgram/ml and the zones were > or = 30 mm, and N. gonorrhoeae susceptible strains were those for which the MICs were < or = 0.03 microgram/ml and the zones were > or = 39 mm (5-micrograms disks). Levofloxacin-susceptible strains of H. influenzae included those for which the MICs were < or = 0.12 microgram/ml and the zones were > or = 32 mm and N. gonorrhoeae susceptible strains were those for which the MICs were < or = 0.12 microgram/ml and the zones were > or = 37 mm (5-micrograms disks). Criteria for a resistant category cannot yet be defined for either quinolone. In multilaboratory studies with different lots of Haemophilus Test Medium, replicate tests with the standard control strain of H. influenzae (ATCC 49247) were evaluated. For sparfloxacin disk tests, the proposed zone size limits were 33 to 42 mm and broth microdilution MIC limits were 0.004 to 0.016 microgram/ml, whereas for levofloxacin tests, zone size limits were 32 to 41 mm and broth microdilution MIC limits were 0.008 to 0.03 microgram/ml. Other multilaboratory studies evaluated tests with supplemented GC agar and N. gonorrhoeae ATCC 49226; for both drugs, zone size limits were 44 to 52 mm and agar dilution MIC limits were 0.004 to 0.016 microgram/ml.     

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.     

Relationship between in vitro susceptibility test results for chloramphenicol and production of chloramphenicol acetyltransferase by Haemophilus influenzae, Streptococcus pneumoniae, and Aerococcus species.

Haemophilus influenzae, Streptococcus pneumoniae, and Aerococcus species were tested for susceptibility to chloramphenicol by standard broth microdilution and disk-diffusion methods. MICs and zone diameter breakpoints were correlated with production of chloramphenicol acetyltransferase (CAT). A comparison of MICs and zone diameters indicated that the interpretative criteria for H. influenzae and S. pneumoniae should be an MIC of less than or equal to 4 micrograms/ml or a zone diameter greater than or equal to 25 mm for susceptible strains and an MIC of greater than or equal to 8 micrograms/ml or a zone diameter of less than or equal to 20 mm for resistant strains; for Aerococcus species, interpretative criteria should be an MIC of less than or equal to 8 micrograms/ml or a zone diameter of greater than or equal to 20 mm for susceptible strains and an MIC of greater than or equal to 32 micrograms/ml or a zone diameter of less than or equal to 12 mm for resistant strains. All but four strains of H. influenzae and one strain of S. pneumoniae that were resistant to chloramphenicol by these criteria produced CAT. For Aerococcus species, however, chloramphenicol-resistant strains were negative for CAT as determined by a commercially available disk test. When comparing susceptibility results with CAT production, thiamphenicol was a better indicator of the presence of the enzyme than chloramphenicol and may be useful in assaying resistance to chloramphenicol.     

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.     

Comparison of agar dilution, broth dilution, disk diffusion, and the E-test for susceptibility testing of penicillin-susceptible and penicillin-resistantStreptococcus pneumoniae

An evaluation to determine the optimal methods for the in vitro susceptibility testing of 41 clinical isolates and the ATCC 49619 strain ofStreptococcus pneumoniae to penicillin was undertaken. No very major or major interpretive errors were observed with the following test methods and media: agar dilution using either Mueller-Hinton medium with lysed horse blood or Haemophilus test medium; broth dilution using cation-adjusted Mueller-Hinton medium with lysed horse blood, Haemophilus test medium, or Todd-Hewitt medium; and the epsilometer test (E-test) using agar containing Mueller-Hinton medium and 5% sheep blood. The disk diffusion method using agar containing Mueller-Hinton medium and 5% sheep blood agar was an effective screening method, requiring confirmation by a dilution susceptibility test method.     

Influence of growth medium and supplement on growth of Haemophilus influenzae and on antibacterial activity of several antibiotics.

In the present study, five non-beta-lactamase- and five beta-lactamase-producing strains of Haemophilus influenzae were used to determine whether three different growth media, Mueller-Hinton broth and agar, brain heart infusion broth and agar, and tryptic soy broth and agar, and their added supplements (0.2% hemin-0.1% IsoVitaleX, 1% hemin-1% IsoVitaleX, 2% sheep blood, 10% Fildes enrichment, 5% Fildes enrichment, 1% supplement B, 5% horse erythrocytes, and 2% hemoglobin-1% IsoVitaleX) would influence the growth rate of this microorganism and the antibacterial activity of eight antibiotics, including ampicillin, tetracycline, chloramphenicol, gentamicin, cefamandole, erythromycin, trimethoprim-sulfamethoxazole (TMP-SMX), and cefoperazone. The growth curve studies were carried out with an initial inoculum of 10(4) bacteria per ml, and MICs were determined with an inoculum of 5 X 10(5) microorganisms. Mueller-Hinton broth, brain heart infusion broth, and tryptic soy broth enriched with 5% Fildes resulted in a maximal growth of more than 10(8) CFU/ml at 24 h. When 10% Fildes or 2% sheep blood was added as enrichment to Mueller-Hinton broth, a considerable reduction in the growth rate of H. influenzae strains resulted (P less than 0.01). Significant variations in MICs (P less than 0.01) were observed with chloramphenicol, TMP-SMX, erythromycin, and cefoperazone when brain heart infusion agar, Mueller-Hinton agar, or tryptic soy agar was used. Chloramphenicol, gentamicin, erythromycin, and TMP-SMX were all affected by the different enrichments added to Mueller-Hinton agar. MICs were in general higher with 5% Fildes enrichment and lower with 1% supplement B. Cefoperazone was the only drug which exhibited a lower MIC in 5% Fildes enrichment for ampicillin-resistant H. influenzae strains.     

Detection of ampicillin resistant Haemophilus influenzae in United Kingdom laboratories.

Susceptibility of Haemophilus influenzae clinical isolates to ampicillin reported by 23 laboratories, using a variety of methods, was compared with results obtained following retesting at The London Hospital Medical College. Beta lactamase production was not detected on initial isolation in 25 of 157 isolates (16%) found to be positive on retest. One hundred beta lactamase negative isolates, which gave reduced zone diameters (less than 20 mm) around 2 micrograms discs and required 1-64 mg/l ampicillin for inhibition, were detected at The London Hospital. Eighty five of these had been reported as sensitive to ampicillin by the laboratories of origin. Many of these 100 isolates showed reduced susceptibility to other beta lactam antibiotics. Accurate detection of non-enzymic reduced susceptibility to ampicillin may emerge as an important guide to the likely sensitivity of H influenzae isolates to the enzyme stable beta lactams.     

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