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.     

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.     

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.     

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.     

Standardized disk-diffusion susceptibility test for Haemophilus influenzae.

The emergence of ampicillin-resistant strains of Haemophilus influenzae has emphasized the need for an improved practical method for routine susceptibility testing of clinical isolates. We have previously described a simplified medium for quantitative dilution susceptibility testing that is composed of Mueller-Hinton medium plus Supplement C (Difco). In the present study, paired broth-dilution and disk-diffusion susceptibility tests with ampicillin and chloramphenicol were performed on 100 strains of Haemophilus (95 H. influenzae and five H. parainfluenzae), including 30 strains with previously documented ampicillin resistance. Disk-diffusion tests were performed in exactly the same manner as the standardized Kirby-Bauer procedure used for less fastidious organisms, except that supplemented Mueller-Hinton agar plates were incubated in an increased-CO2 atmosphere. Using this method, ampicillin-susceptible strains of Haemophilus produced zone diameters of 22 mm or more, while ampicillin-resistant strains produced zones of 18 mm or less. All strains were chloramphenicol-susceptible and produced zone diameters of 30 mm or more. This method would allow routine disk-diffusion testing of isolates of H. influenzae by hospital diagnostic laboratories, using a clear medium that closely resembles unsupplemented Mueller-Hinton agar.     

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.     

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.     

Evaluation of the capillary beta-lactamase test and antimicrobial susceptibility of Haemophilus influenzae.

The capillary beta-lactamase test for the detection of Haemophilus influenzae resistance to ampicillin was evaluated against 132 strains of H. influenzae recently isolated from clinical materials and four reference strains. Nineteen strains, including two of serotype b, were beta-lactamase-positive. The minimal inhibitory concentrations (MIC) of ampicillin for the 117 beta-lactamase-negative strains ranged from less than or equal to 0.125 to 2 microgram/ml (only one strain had a MIC of 2 microgram/ml). The range of MIC's of ampicillin was 4 to 64 microgram/ml for the 19 beta-lactamase-positive strains; all but two strains required 8 microgram/ml or more for inhibition. The capillary beta-lactamase test is an easy, rapid and reliable test for the detection of H. influenzae resistance to ampicillin. It is suitable for routine use in the clinical microbiology laboratory. The MIC of carbenicillin was higher for ampicillin-resistant than for ampicillin-susceptible strains, but the highest MIC (32 microgram/ml) was within achievable serum concentrations. Both cefamandole and chloramphenicol were active against all 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.     

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.     

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.     

Antimicrobial surveillance of Haemophilus influenzae in the United States during 2000-2001 leads to detection of clonal dissemination of a beta-lactamase-negative and ampicillin-resistant strain

A 2000-2001 U.S. Haemophilus influenzae surveillance study (n = 1,434) detected nine (0.6%) beta-lactamase-negative and ampicillin-resistant (BLNAR) isolates collected from two different hospitals. The MICs of ampicillin for all nine isolates were 4 microg/ml, with results being reproducible; and all nine isolates were susceptible to amoxicillin-clavulanate, cefuroxime, cefprozil, macrolides, and fluoroquinolones. Pulsed-field gel electrophoresis of genomic DNA following SmaI digestion demonstrated identical patterns for each of the nine isolates, suggesting intra- and interhospital dissemination of a BLNAR clone.     

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.     

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.     

Dynamics of long-term colonization of respiratory tract by Haemophilus influenzae in cystic fibrosis patients shows a marked increase in hypermutable strains

The persistence and variability of 188 Haemophilus influenzae isolates in respiratory tract of 30 cystic fibrosis (CF) patients over the course of 7 years was studied. Antibiotic susceptibility testing, DNA fingerprinting, and analysis of outer membrane protein profiles were performed on all isolates. A total of 115 distinct pulsed-field gel electrophoresis profiles were identified. Ninety percent of patients were cocolonized with two or more clones over the studied period. A third of the patients were cross-colonized with one or two H. influenzae strains; 11% of the clones persisted for 3 or more months. Biotype, outer membrane protein profiles, and resistance profiles showed variation along the studied period, even in persisting clones. Four isolates (2.1%) recovered from 3 patients were type f capsulate, with three of them belonging to the same clone. beta-Lactamase production was detected in 23.9% of isolates while 7% of the beta-lactamase-negative isolates presented diminished susceptibility to ampicillin (beta-lactamase-negative ampicillin resistance phenotype). Remarkably, 21.3% of the H. influenzae isolates presented decreased susceptibility to ciprofloxacin, which was mainly observed in persisting clones. Of the H. influenzae isolates from CF patients, 18 (14.5%) were found to be hypermutable in comparison with 1 (1.4%) from non-CF patients (P < 0.0001). Ten patients (33.3%) were colonized by hypermutable strains over the study period. A multiresistance phenotype and long-term clonal persistence were significantly associated in some cases for up to 7 years. These results suggest that H. influenzae bronchial colonization in CF patients is a dynamic process, but better-adapted clones can persist for long periods of time.     

Comparative molecular analysis of Haemophilus influenzae isolates from young children with acute lower respiratory tract infections and meningitis in Hanoi, Vietnam

Thirty-seven Haemophilus influenzae strains from nasopharyngeal swabs (NP) and 44 H. influenzae strains from cerebrospinal fluid (CSF) were investigated. Of the 37 H. influenzae isolates from NP, the serotypes of 30 isolates were nontypeable, 4 were type b, 2 were type c, and 1 was type a, whereas all of the 44 isolates from CSF were type b. The MICs of 16 antibiotics for the H. influenzae isolates from NP and CSF were similar, and no beta-lactamase-negative ampicillin-resistant strain was found. Molecular typing by pulsed-field gel electrophoresis (PFGE) showed that the 37 H. influenzae strains from NP had 22 PFGE patterns, with none predominating, and the 44 H. influenzae strains from CSF had 9 PFGE patterns, with patterns alpha (22 isolates) and beta (12 isolates) predominating. Our results indicate that two predominant types of H. influenzae type b strains have the potential to spread among children with meningitis in Hanoi, Vietnam.     

A simple disk diffusion test to identify beta-lactamase-negative, ampicillin-resistant Haemophilus influenzae--application of cephalexin, cefsulodin and cefaclor disks

Currently, beta-lactamase-negative (BLN) ampicillin-resistant (AR) strains of Haemophilus influenzae are prevalent in Japan. BLNAR strains are defined by the presence of specific mutation(s) in the ftsI gene but are not phenotypically distinguishable by ampicillin (ABPC) susceptibility. In the present study, we showed that cephalexin (CEX), cefsulodin (CFS), and cefaclor (CCL) disk diffusion tests can be effectively used to identify BLNAR strains. A total of 169 clinical isolates of BLN H. influenzae, consisting of 113 of BLNAR and 56 of BLN, ampicillin-susceptible (AS), were included. All the isolates were genetically defined by detection of the TEM gene and partial sequencing of the ftsI gene. The Clinical and Laboratory Standards Institute (CLSI) standard broth microdilution and disk diffusion tests for ABPC provided 20% and 19% false susceptible rates, respectively. Alternatively, 34 cephem agents were tested using disk diffusion. Of the agents tested, CEX, CFS, and CCL disks could effectively discriminate between BLNAR and BLNAS isolates. All the BLNAS isolates showed visible growth inhibitory zones around CEX and CFS disks, but 108 (95.6%) and 106 (93.8%) BLNAR isolates did not. The results indicated 100% predictive values (PVs) for BLNAR and PVs for BLNAS were 91.8% for CEX and 88.9% for CFS. The CLSI-based interpretations for CCL (> or =20 mm) also highly correlated with BLNAR and BLNAS, PVs for BLNAR and for BLNAS being 100% and 93.3%, respectively. With simplicity and discriminability of the test method, we recommend a CEX disk diffusion test in combination with a rapid beta-lactamase test to identify BLNAR isolates in clinical laboratories.     

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.     

Plasmid-linked ampicillin resistance in haempohilus influenza type b.

Four ampicillin-resistant, beta-lactamase-producing strains of Haempohilus influenzae type b were examined for the presence of plasmid deoxyribonucleic acid (DNA). Three resistant strains contained a 30 x 10-6-dalton (30Mdal) plasmid and one resitant strain contained a 3-Mdal plasmid. The ampicillin-sensitive Haemophilus strains examined did not contain plasmid DNA. Transformation of a sensitive H. influenzae strain to ampicillin resistance with isolated plasmid DNA preparations revealed that the structural gene for beta-lactamase resided on both plasmid species. DNA-DNA hybridization studies showed that the 30-Mdal Haemophilus plasmid contained the ampicillin translocation DNA segment (TnA) found on some R-factors of enteric origin of the H. influenzae plasmids.     

Rapid ampicillin susceptibility testing for Haemophilus influenzae.

Recent isolations of strains of Haemophilus influenzae resistant to ampicillin necessitate the development of a rapid, dependable, reproducible method of determining their antibiotic susceptibility. An agar-dilution method permitting susceptibility determinations on clinical specimens within 6-18 hours of specimen collection was designed. Chocolate agar biplates were made with one side having no additive and the other containing 2 mug/ml ampicillin. Seventy clinical specimens (cerebrospinal fluid, joint fluid, ear fluid, pleural fluid, blood culture broth) were streaked directly onto both sides of the plates when received in the laboratory and incubated at 35-37 C in 10% CO2. Reliable, readable results were usually available within 6-18 hours of receipt of the specimen and correlated completely with minimum inhibitory concentrations (MIC) determined by the agar-dilution plate method, although standard disk susceptibilities occasionally indicated false resistance. Susceptible strains (MIC less than 2 mug/ml) grew on the antibiotic-free side of the biplate only. The rapid determination of ampicillin susceptibility allows optimal antibiotic selection for the treatment of Haemophilus influenzae infections with early discontinuation of potentially toxic supplementary drugs.