We compared results of MIC and disk susceptibility tests on Haemophilus test medium (HTM) and those on comparative media. Ampicillin MICs were determined with seven ampicillin-resistant, non-f-lactamaseproducing (AmprNBLP) isolates by using HTM and supplemented brain heart infusion (sBHI) agar. Ampicilli and amoxicillin-clavulanate disk tests with 16 Amp'NBLP strains, 18 ampicillin-susceptible (Amps) isolates, and 17 ampicillin-resistant, .lactamase-producing (AmprBLP) strains were performed by using five media: laboratory-prepared HTM (PHTM), commercial HTM (CHTM), sBHI, enriched chocolate agar, and Mueller-Einton chocolate agar. We observed that five of seven and three of seven Amp'NBLP stains were misclassified as susceptible with PHTM (MIC, <2 ,ug/ml) with inocula of 103 and 1i@ CFU, respectively, but were resistant with sBHI (MIC,.2 ,ug/ml (.25 mm) and resistance (<21 mm) (1, 9). Presumably, these changes would allow identification of all ampicillin-resistant strains by using the standard 10-,ug ampicillin disk test. Previously, we showed with the former NCCLS criteria (7) and media that the 10-pLg disk test, in contrast to the 2-,ug ampicillin disk test, failed to detect 44% (8 of 18) of ampicillin-resistant, non-p-lactamase-producing (AmprNBLP) isolates; these isolates have an intermediate level of resistance compared with 3-lactamase-producing strains (median ampicillin MIC, 8 versus 32 p.g/ml, respectively, with an inoculum of 105 CFU) (4).Recently, we used HTM to compare the in vitro susceptibility of H. influenzae to three cephem antibiotics (6). In that study, we observed that the ampicillin MICs with HTM were lower for AmprNBLP strains compared with our previous data obtained with the same isolates in our laboratory when we used supplemented brain heart infusion medium (sBHI; Difco Laboratories, Detroit, Mich.); those data prompted this investigation. We compared the ampicillin MICs using HTM (2)

A 10-year-old boy presented with nuchal rigidity and cerebrospinal fluid (CSF) leukocytosis initially and again on day 6 of intravenous cefuroxime therapy (200 mg/kg/day). Both CSF specimens yielded nontypable beta-lactamase-negative Haemophilus influenzae that were susceptible by disk tests but relatively resistant to cefuroxime (MIC, 8- to 16-fold greater than that of control isolates). To define the mechanism of resistance, the cefuroxime resistance marker was transformed to a susceptible H. influenzae recipient; inactivation and permeability of beta-lactam substrate were tested and the penicillin-binding protein (PBP) profiles were examined. Inactivation of beta-lactam substrate was not detected and reduced permeability was not found. However, reduced beta-lactam binding to PBPs 4 and 5 was observed; 18- to 27-fold more penicillin and 2.5-to 4-fold more cefuroxime was required to saturate or block 50% of the binding sites of these PBPs, respectively. Thus, reduced affinity of PBPs 4 and 5 for beta-lactam substrate appears to be the mechanism of cefuroxime resistance in this strain. The reduced affinity of these targets appears to have contributed to the bacteriologic and clinical failure in this patient.

The antimicrobial activities of cefixime, cefpodoxime, and ceftibuten were determined with 18 ampicillinsusceptible (Amp'), 13 ampicillin-resistant P-lactamase-producing (AmprBLP), and 7 ampiciln-resistant non-j-lactamase-producing (AmprNBLP) strains of Haemophilus influenzae. An effect of inoculum density on apparent MIC, the bactericidal activity of these agents, and the targets of the three cephems were determined. The MICs of cefixime, cefpodoxime, and ceftibuten for 90% of the Amps and Amp%BLP isolates were 0.04, 0.08, and 0.08 ,ug/ml, respectively. In contrast, the MICs for 90% of the Amp'NBLP strains were 0.96, 1.92, and 7.68 ,ug/ml. No significant inoculum effect was observed for any group of strains comparing inocula of 103 and 105 CFU, whereas only the AmprNBLP isolates showed a marked effect at an inoculum of 106 CFU. Although bactericidal levels were achieved for the Amp' and AmnprBLP strains, tolerance to cefixime and ceftibuten was observed. The bactericidal activity for the AmprNBLP strains was limited, with cefixime showing the highest activity of the three cephems. Penicillin-binding proteins 2, 4, and 5 revealed high affinity, with 50% inhibitory concentration levels below the MIC for all three cephems, suggesting that these are important targets of these agents in H. influenzae. We conclude that the cephems are highly active in vitro against Amps and AmprBLP strains of H. influenzae, but less so against AmprNBLP isolates.