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.     

Multiply-resistant Haemophilus influenzae type b causing systemic disease in children in Australia

Until 1984 strains of Haemophilus influenzae type b (Hib) isolated from children with systemic infections in Australia had been uniformly sensitive to chloramphenicol. In July 1984 a child with bacteremia in Adelaide, South Australia, yielded a strain of Hib resistant to ampicillin, chloramphenicol and tetracycline. In October and November 1984 similar isolates were obtained on cerebrospinal fluid culture from 2 children with meningitis. The 3 isolates showed an identical resistance pattern and inactivated ampicillin and chloramphenicol. Each isolate was fully sensitive to moxalactam (latamoxef), and to cefotaxime. Both children with meningitis were treated initially with ampicillin and chloramphenicol given intravenously. One of these, a girl aged 16 mth, showed no improvement and a sixth cranial nerve palsy developed. When ampicillin and chloramphenicol were ceased and moxalactam was substituted there was a rapid clinical improvement. As initial therapy in children with bacterial meningitis we advocate using either moxalactam and ampicillin in combination or a suitable third generation cephalosporin such as cefotaxime.     

Haemophilus influenzae: antibiotic susceptibility.

Ampicillin resistance was first reported among clinical isolates of Haemophilus influenzae in 1972. Reports of chloramphenicol resistance followed shortly thereafter. The principal mechanism of resistance to these two antibiotics is enzymatic. Although other mechanisms have been described, they are found in comparatively few strains. The genetic information for the inactivating enzymes is plasmid mediated and therefore readily transmissible to susceptible strains. Consequently, effective therapy for invasive disease caused by this pathogen has been seriously compromised. As antibiotic susceptibility became less predictable, in vitro testing became increasingly important. Unfortunately, the standardization of methods for laboratory testing has been slow and complicated by the fastidious nature of the organisms. This review traces the development of antibiotic resistance in H. influenzae, discusses the mechanisms which appear to be important in mediating resistance, explores newer antimicrobial agents which might be useful in the treatment of infection, and analyzes the various approaches to in vitro testing.     

Course of ampicillin sensitivity in Haemophilus influenzae responsible for purulent meningitis in children. Therapeutic consequences

Bacterial meningitis caused by Haemophilus influenzae observed from 1976 to 1980 (10 cases) are compared with those observed in 1981 (9 cases). From 1976 to 1980, 9 strains (90%) are ampicillin-sensitive. In 1981, 5 strains (55,5%) are ampicillin-sensitive and 4 strains (45,5%) are ampicillin-resistant and betalactamase positive. These 9 strains are serotype b. Therefore, since 1981, initial treatment of bacterial meningitis in infancy is a new cephalosporin, in this study is cefotaxime.     

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.     

Inhibitory activity of ampicillin and mecillinam on Haemophilus influenzae

Mecillinam, an antibiotic of the beta-lactam group, is active against Gram negative bacilli, with the exception of a few species such as Haemophilus influenzae. The activities of ampicillin, mecillinam, and ampicillin-mecillinam combinations at ratios of 1/1, 2/1, 4/1, 1/2 and 1/4, on 21 Haemophilus influenzae strains, were studied by agar dilution and determination of minimal inhibitory concentrations (MIC). Ampicillin at concentrations of 0.12 to 0.25 mg/l was active on susceptible strains. Beta-lactamase-producing strains were inhibited by 4 and 8 mg/l ampicillin. MICs of mecillinam were above 128 mg/l for 19 strains; two strains were susceptible to 2 mg/l. In every case, antibacterial activity was greater with the ampicillin-mecillinam combination than with either one of the antibiotics alone. The maximum antibacterial effect was obtained at ratios of 1/1 and 1/2; in this case, susceptible strains were inhibited by 0.06 mg/l ampicillin. Kinetic evaluation (killing curves) showed that ampicillin has a bactericidal effect at concentrations under the MIC when it is combined with mecillinam at a 1/1 ratio.     

Quantitative antibiotic susceptibility testing: Haemophilus influenzae type B.

Twelve strains of Haemophilus influenzae were tested for susceptibility to gentamicin, ampicillin, chloramphenicol and cephalothin using two methods, agar dilution and microdilution (broth). Although inocula and incubation conditions were standardized, significant differences in the minimal inhibitory concentration (MIC) were seen as a result of growth media. Method dependent differences were also observed for some antibiotics. Notwithstanding such variation, high level resistance of H. influenzae to ampicillin was readily detected by either broth or agar dilution tests.     

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.     

Epidemiology and antimicrobial susceptibility of non-typeable Haemophilus influenzae in otitis media in Taiwanese children

Background

Concerns about non-typeable Haemophilus influenzae (NTHi) in otitis media (OM) have grown after the introduction of pneumococcal conjugate vaccine (PCV). We aim to better understand the clinical role of NTHi in pediatric OM.

Haemophilus influenzae type b antigenuria in children.

Detection of Haemophilus influenzae type b (HIb) antigenuria by latex agglutination has been shown to be sensitive, specific, and rapid. In children, antigenuria persisted for a mean duration of 10 days and a maximum of 18 days. Antigenuria was demonstrated in 25 of 30 patients with HIb infection but not in 62 with other types of infection. In five children, antigenuria confirmed the diagnosis in the absence of bacteriological confirmation. In five other children, antigenuria was not found, but in this group the antigen was detected in another body fluid or HIb was recovered.     

Haemophilus influenzae infections in the H. influenzae type b conjugate vaccine era

The widespread use of Haemophilus influenzae type b (Hib) conjugate vaccines has nearly eradicated invasive Hib disease where the vaccines are used. This success was accompanied by a shift in capsular serotypes of invasive H. influenzae disease, with nontypeable strains replacing type b strains as the most common bloodstream isolate, but there is no convincing evidence of a true increase in the incidence of non-serotype b invasive infections. H. influenzae causes predominantly mucosal infections. The introduction of vaccines for otitis media and global shifts in antimicrobial susceptibility emphasize the importance of continued surveillance of H. influenzae colonization and disease patterns.     

Low rate of nasopharyngeal carriage and high rate of ampicillin resistance for Haemophilus influenzae among healthy children younger than 5 years old in northern Taiwan.

BACKGROUND AND PURPOSE: Surveillance data of colonization by Haemophilus influenzae in Taiwan are lacking. This study aimed to define the nasopharyngeal carriage rate of H. influenzae among children younger than 5 years in northern Taiwan, and to determine the antibiotic susceptibility, serotype and the clonal relationship of these isolates. METHODS: Nasopharyngeal specimens were obtained from 511 healthy children younger than 5 years. All H. influenzae isolates were serotyped. The minimal inhibitory concentrations for various antibiotics were determined. Pulsed-field gel electrophoresis (PFGE) was used for clonal analysis. RESULTS: Among 511 children, 269 (52.6%) had been vaccinated with at least one dose of H. influenzae type b (Hib) conjugate vaccine, 236 (46.2%) were unvaccinated and 6 (1.2%) had no vaccination records available. Twenty six H. influenzae strains were isolated. There were three Hib isolates and the others were nontypeable H. influenzae (NTHi). The carriage rate for Hib was 0.6% (3/511) and of NTHi was 5% (23/511). Three (1.27%) of the 236 unvaccinated children were carriers of Hib, whereas none of the 269 vaccinated children carried Hib. Two out of the three Hib isolates and 14 (60.9%) of 23 NTHi isolates were ampicillin-resistant. Multidrug resistance was found in 7 (26.9%) of the isolates. Among the isolates, 61.5% were beta-lactamase producers; there were no beta-lactamase-negative ampicillin-resistant isolates. The PFGE restriction patterns showed a wide diversity of genotypes. CONCLUSIONS: There is very low nasopharyngeal carriage of Hib among children younger than 5 years in northern Taiwan. This may explain why the incidence of invasive Hib disease is also low in Taiwan. In addition, we found a high prevalence of beta-lactamase-positive ampicillin-resistant nasopharyngeal H. influenzae isolates.     

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.     

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.     

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.     

Rapid methods of detecting enzymatic resistance to ampicillin and chloramphenicol in Haemophilus influenzae

Several methods used for the detection of beta-lactamase activity in Haemophilus influenzae are described. The rapid iodemetric, acidimetric, and chromogenic cephalosporin techniques are specific tests for the presence of beta-lactamase. The Gots test can also be used for the detection of enzymatic resistance to ampicillin and chloramphenicol.     

A comparison of chloramphenicol and ampicillin as bactericidal agents for Haemophilus influenzae type B.

In tests of bactericidal action against H. influenzae type b strains isolated from patients with meningitis, chloramphenicol was found to be far more reliable than ampicillin in dealing with large inocula, and more rapidly effective against both large and relatively small inocula. These findings provide a laboratory explanation for the somewhat better record of chloramphenicol as an agent for treatment of haemophilus meningitis.