Disk diffusion susceptibility testing ofHaemophilus influenzae using Haemophilus test medium

A three-center collaborative study was performed with the aim of determining the reproducibility of disk diffusion and broth microdilution susceptibility tests ofHaemophilus influenzae with 19 different antimicrobial agents using a newly described medium, Haemophilus Test Medium. The results of both methods were found to be highly reproducible. Using Haemophilus Test Medium, inhibitory zone diameters and MICs were then determined for 150 test strains ofHaemophilus influenzae, selected with an emphasis on known mechanisms of resistance. Regression analysis of zone diameter-MIC comparisons was used to define zone diameter interpretive criteria and MIC correlates for Haemophilus Test Medium disk diffusion tests performed withHaemophilus spp. for the following antimicrobial agents: ampicillin, amoxicillin/clavulanate, ampicillin/sulbactam, cefaclor, cefuroxime, cefamandole, ceftriaxone, cefotaxime, ceftizoxime, ceftazidime, cefixime, trimethoprim/sulfamethoxazole, rifampin, chloramphenicol, tetracycline, imipenem, aztreonam and ciprofloxacin.     

Effects of various test media on the activities of 21 antimicrobial agents against Haemophilus influenzae

As considerable variation in the antimicrobial susceptibility of Haemophilus influenzae has been reported, the effects of various test media on the susceptibility of H. influenzae were studied. MICs were determined by three laboratories for 21 antimicrobial agents against a panel of 100 selected isolates. Testing was performed using a reference NCCLS frozen broth microdilution method with Haemophilus test medium (HTM) broth and dried commercial MIC trays rehydrated with the following media: in-house and commercially prepared HTM broth, Mueller-Hinton broth with 2% lysed horse blood and NAD, IsoSensitest broth with 2% lysed horse blood and NAD, and IsoSensitest broth-based HTM. Overall, all results were very reproducible, with the MIC at which 50% of the isolates tested are inhibited (MIC(50)), MIC(90), and geometric mean MIC being within one doubling dilution by all six methods and at all three testing centers for 15 of the 21 agents tested. Interlaboratory differences were more marked than intralaboratory differences or differences among media. Cefprozil, cefaclor, and trimethoprim-sulfamethoxazole results differed the most, while results for ampicillin, amoxicillin-clavulanic acid, cefdinir, cefixime, ceftriaxone, and clarithromycin were the most reproducible. However, these variations in results caused considerable differences in susceptibility rates for agents for which NCCLS susceptible breakpoints were close to the geometric mean MIC, particularly for cefaclor and cefprozil. This was much less of a problem when pharmacokinetic-pharmacodynamic breakpoints were used. Reproducible susceptibility results were obtained for a wide range of agents against H. influenzae in three laboratories using a variety of media that support the growth of this fastidious species.     

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).     

Evaluation of in vitro methods for testing susceptibility of anaerobes to ampicillin-sulbactam and amoxicillin-clavulanic acid.

A total of 97 anaerobic bacteria were tested for susceptibility to ampicillin, ampicillin-sulbactam, and amoxicillin-clavulanic acid by broth microdilution and disk elution methods, the results of which were compared with those of the reference agar dilution method. With the broth microdilution method, approximately 95% of MICs were within 1 dilution of those of the reference agar method, with a definite (0.6 to 0.7 dilution) trend toward lower MICs. The disk elution test performed satisfactorily, but additional anaerobic isolates resistant to ampicillin-sulbactam and/or amoxicillin-clavulanic acid (currently rare) are needed to assure the predictability of resistance by the disk elution test.     

Antimicrobial susceptibilities of clinical isolates of Haemophilus influenzae and Moraxella catarrhalis collected during 1999–2000 from 13 countries

Objective   To determine antimicrobial activity against Haemophilus influenzae and Moraxella catarrhalis .
Methods   A central laboratory performed NCCLS susceptibility testing for all isolates and β -lactamase and capsular serotype determinations for H. influenzae .
Results   A total of 2712 H. influenzae and 1079 M. catarrhalis were collected . H. influenzae susceptibilities were >90% for amoxicillin/clavulanate, cefaclor, loracarbef, cefprozil, cefuroxime, ciprofloxacin, azithromycin and clarithromycin and were <80% for trimethoprim/sulfamethoxazole and ampicillin. 19.3% were β -lactamase positive. The most common serotype was type-b (5.6%); 86.1% were nontypeable. M. catarrhalis had MIC₉₀ within therapeutic range for all antimicrobials except ampicillin.
Conclusion   The conclusion of the study is that antimicrobials, except ampicillin and trimethoprim/sulfamethoxazole, remain good empiric choices against H. influenzae and M. catarrhalis .     

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.     

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.     

Revised disk diffusion interpretive criteria for cefaclor,loracarbef, cefprozil and cefixime when testingHaemophilus influenzae on Haemophilus Test Medium

The aim of the current five-center collaborative study was to reassess the interpretive criteria for cefaclor, loracarbef, cefprozil and cefixime previously adopted or proposed by the National Committee for Clinical Laboratory Standards (NCCLS) for disk diffusion susceptibility tests withHaemophilus influenzae on Haemophilus Test Medium (HTM) agar. MICs and zones of inhibition were determined using NCCLS methods, HTM and two collections of strains ofHaemophilus influenzae. One group of strains consisted of 118 stock organisms taken largely from various recent U.S. antibiotic resistance surveillance studies. The emphasis in this selected group of organisms was on strains that were beta-lactamase negative but ampicillin resistant (BLNAR) by some other mechanism. The second collection of test organisms consisted of 50 recent clinical isolates ofHaemophilus influenzae obtained from each of the five participating study centers. This group was considered representative of the type ofHaemophilus influenzae currently recovered from clinical sources in the USA. Frequency distribution assessment and error-rate bounded analysis of scattergram comparisons of MICs and zone sizes were used to develop the following zone diameters interpretive for disk diffusion susceptibility tests withHaemophilus influenzae on HTM agar: cefaclor, 20 mm (susceptible, S) and 16 mm (resistant, R); loracarbef, 19 mm (S) and 15 mm (R); and cefprozil, >18 mm (S) and <14 mm (R). The respective MIC correlates for all three antimicrobial agents were 8 µg/ml (S) and 32 µg/ml (R). For cefixime, a zone diameter size of 21 mm was recommended to define the susceptible category, with an MIC correlate of 1 µg/ml. Only a susceptible category was defined for cefixime testing. These new interpretive criteria have recently been adopted by the NCCLS.     

Proposed changes in interpretive criteria and potency of ampicillin and ampicillin-sulbactam disks for susceptibility tests.

The accuracy of disk susceptibility tests with ampicillin and ampicillin-sulbactam was not improved when the amount of ampicillin was increased from 10 to 20 or 30 micrograms per disk. For testing members of the family Enterobacteriaceae, ampicillin disk tests correlated better with broth microdilution tests when the zone size standards were altered from greater than or equal to 14 greater than or equal to 17 mm for susceptible and from less than or equal to 11 to less than or equal to 13 mm for resistant. The same zone size breakpoints apply to tests with ampicillin-sulbactam disks (10/10 micrograms). When Staphylococcus, Branhamella, and Haemophilus species are tested against ampicillin, interpretive breakpoints are those separating beta-lactamase-producing strains from nonproducing strains. However, when ampicillin-sulbactam is tested, beta-lactamase enzymes are efficiently inhibited by the sulbactam component, and thus zone size standards for ampicillin do not apply: zone size standards for the Enterobacteriaceae can be used for testing the combination against Staphylococcus, Branhamella, and Haemophilus species. For Streptococcus, Enterococcus, and Listeria species, only ampicillin disks need be tested, since ampicillin-sulbactam disks give essentially identical results.     

Interpretive accuracy of the disk diffusion method for testing newer orally administered cephalosporins against Morganella morganii.

Eight newer orally administered cephems (cefdinir, cefetamet, cefixime, cefpodoxime, cefprozil, ceftibuten, cefuroxime, and loracarbef) were tested against 100 clinical strains of Morganella morganii to determine the extent of serious interpretive very major (false-susceptible) errors when current criteria for the disk diffusion test are applied. Agar dilution MICs and disk diffusion tests were performed as recommended by the National Committee for Clinical Laboratory Standards (Villanova, Pa.) (NCCLS), and the methods were compared by regression analysis using the method of least squares and by error rate bounding. The following results are listed in the order of increasing error rates: cefdinir, loracarbef, and cefprozil, < or = 1% very major error; ceftibuten, 8% minor errors; cefuroxime, 21% minor errors; cefixime, cefpodoxime, and cefetamet, very major errors of 15, 24, and 36%, respectively. M. morganii produces unacceptable rates of test error with cefuroxime, cefixime, cefpodoxime, and cefetamet. The latter two cephalosporins currently have NCCLS table footnote warnings covering the problem observed with this organism. The inclusion of cefuroxime and cefixime in the NCCLS table footnote is strongly recommended.     

Serotypes and antimicrobial susceptibility in clinical isolates of Haemophilus influenzae from Korean children in prevaccination era

Fifty-five strains of Haemophilus influenzae recovered at a children's hospital in Korea from 1992 through 1997, were analyzed for serotype and antibiotic resistance. Antimicrobial susceptibility was tested by broth dilution method. Among the 55 strains, 26 were from normally sterile body fluids, of which 17 were from the immunocompetent children. Spectrum in the immunocompetent included meningitis (47%), bacteremic pneumonia (18%), and bacteremia without focus (35%). Three (12%) of 26 invasive infections were caused by non-type b: one type d and two type f. Nine of 29 non-sterile body fluid isolates belonged to one of encapsulted serotypes: four a, two c, one of each of b, d and e. Thirty two (58%) strains were resistant to ampicillin, and all of which produced beta-lactamase. All of the strains were highly susceptible to amoxicillin/clavulanate, cefixime, cefuroxime, azithromycin and ciprofloxacin, while 1 (2%), 7 (13%), 4 (7%) and 4 (7%) strains were intermediate to cefprozil, cefaclor, loracarbef, and clarithromycin, respectively. The serotype distribution of H. influenzae in Korean children is similar to those in developed countries before the introduction of Hib conjugate vaccine, and ampicillin resistance rate is among the highest published to date.     

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.     

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 in-house and commercially prepared haemophilus test media for disk diffusion testing of ampicillin against Haemophilus species.

The performance characteristics of two lots of Haemophilus Test Medium (HTM) prepared in-house at Mayo Clinic, one lot obtained from BBL (Cockeysville, Md.) and one lot obtained from Remel (Lenexa, Kans.), for disk diffusion susceptibility testing of ampicillin against 81 Haemophilus strains were assessed. Insufficient growth occurred in 2.5 to 13.6% of strains, depending on the HTM used, and was most frequent for beta-lactamase-negative, ampicillin-resistant strains when they were tested with one lot of HTM prepared in-house. Results for all 14 beta-lactamase-positive Haemophilus strains were in complete agreement for all lots of HTM when either 1990 or 1993 National Committee for Clinical Laboratory Standards (NCCLS) interpretive guidelines were followed. However, these beta-lactamase-positive Haemophilus strains could be as precisely, but more rapidly, identified by the cefinase disk method. Results for 56 beta-lactamase-negative, ampicillin-susceptible strains and 11 beta-lactamase-negative, ampicillin-resistant strains varied significantly among all HTM lots tested and as to whether 1990 or 1993 NCCLS interpretive guidelines were followed. We conclude that in our hands disk diffusion testing with HTM prepared in-house or obtained from two commercial sources and following either 1990 or 1993 NCCLS guidelines is an unacceptable method for determining the susceptibilities of Haemophilus strains to ampicillin.     

Cefixime disk susceptibility test criteria.

A total of 583 bacterial isolates was tested for susceptibility to cefixime by broth microdilution and by disk agar diffusion with 5-, 10-, and 30-microgram disks. At MIC breakpoints of less than or equal to 1.0 and greater than or equal to 4 micrograms/ml for susceptible and resistant, respectively, the 5-microgram disk showed slightly better discrimination. The 5-microgram cefixime disk is recommended with proposed interpretive breakpoint criteria of: less than or equal to 17 mm, resistant; 18 to 20 mm, intermediate; and greater than or equal to 21 mm, susceptible.     

Comparison of broth microdilution and disk diffusion test for antimicrobial resistance testing in Yersinia enterocolitica 4/O:3 strains

The aim of this study was to compare the results obtained from two methods for the determination of antimicrobial resistance in 110 Yersinia enterocolitica 4/O:3 strains. Ten antimicrobial agents were tested using broth microdilution and disk diffusion. Similar results were determined for six antimicrobials. Very major errors (false-susceptible by disk diffusion test) were detected for ampicillin (at a rate of 1.8%). Major errors (false-resistant by disk diffusion test) were found for streptomycin (0.9%) and sulfamethoxazole (1.8%). Minor errors (intermediate susceptible by disk diffusion and resistant or susceptible by microdilution) were obtained for ampicillin (2.7%) and sulfamethoxazole (13.6%). All Y. enterocolitica were resistant to at least one antimicrobial agent. Resistances to three classes of antimicrobial agents were obtained by 3% of the strains included in the study. A slightly higher frequency of multiresistance was obtained by disk diffusion (3%) compared with broth microdilution (1%). Resistance to streptomycin was found frequently (13%); in contrast, resistance to tetracycline was rare (1%). The disk diffusion test produced unacceptably high rates of very major errors for ampicillin and a high frequency of minor errors for sulfamethoxazole. Susceptibility tests should thus be carried out by the more reliable method of microdilution. Most of the antimicrobials that can be used for therapy were very effective when tested against Y. enterocolitica. In order to identify changes in susceptibilities as early as possible, antimicrobial resistance in Y. enterocolitica should be regularly surveyed.     

Proposed quality control guidelines for antimicrobial susceptibility tests using tilmicosin.

Quality control guidelines for tilmicosin, a novel veterinary-use-only macrolide, were developed in a multi-laboratory study according to established National Committee for Clinical Laboratory Standards (NCCLS) procedures (M23-T2). Tilmicosin was incorporated into Sensititre plates for broth microdilution endpoint testing and into two lots of 15-micrograms disks for Kirby-Bauer agar disk diffusion testing. One common lot and five unique lots of Mueller-Hinton media were used. (Broth was cation adjusted, and agar was supplemented with 5% defibrinated sheep blood.) Bacteria used for reference strains included Pasteurella haemolytica 128K, Pasteurella multocida ATCC 43137, and Staphylococcus aureus ATCC 29213 (microdilution) and ATCC 25923 (disk). Replicate tests were conducted. Disk diffusion and broth microdilution quality control ranges are proposed.     

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.     

In vitro activity of new antibiotics againstHaemophilus influenzae

The activity of sulbactam/ampicillin, aztreonam, cefetamet, cefixime, ciprofloxacin, enoxacin, fleroxacin and ofloxacin was determined against 160Haemophilus influenzae strains using a standardized microdilution method. The strains were categorized into four groups according to beta-lactamase production and their resistance to ampicillin, chloramphenicol, erythromycin, tetracycline and cotrimoxazole. All isolates tested, regardless of their beta-lactamase production or resistance to the above mentioned drugs, were susceptible to the nine antibiotics. The most active antimicrobial agents were aztreonam, cefixime, and the two quinolone derivatives ciprofloxacin and ofloxacin.