Molecular Characterization of the β-Lactamases from Clinical Isolates of Moraxella ( Branhamella ) catarrhalis Obtained from 24 U.S. Medical Centers during 1994–1995 and 1997–1998

Abstract

The ␤-lactamases from 403 Moraxella (Branhamella) catarrhalis clinical isolates obtained during 1994-1995 and 1997-1998 U.S. multicenter surveillance studies were characterized by isoelectric focusing. The overall prevalences of the BRO-1 and BRO-2 enzymes among ␤-lactamase-positive isolates were estimated to be 97.5 and 2.5%, respectively. The minimum inhibitory concentrations (MICs) of ampicillin for all BRO-2-producing isolates were <1 g/ml; however, numerous ␤-lactamase-positive isolates for which the ampicillin MICs were <1 g/ml produced the BRO-1 enzyme (88.1%).Isoelectric focusing (IEF) of ␤-lactamases extracted from Moraxella (Branhamella) catarrhalis typically reveals one of two different banding patterns; these have been referred to as Ravasio (BRO-1) and 1908 (BRO-2) (7, 15). The BRO-1 pattern has been the most prevalent, accounting for 87 to 95% of M. catarrhalis ␤-lactamases characterized in the United States, France, Denmark, Sweden, England, and Scotland, with the remaining 5 to 13% identified as 3,5,6,8,11,13).Although substrate hydrolysis rates of BRO-1 and BRO-2 enzymes are similar, BRO-1-producing isolates are more resistant to penicillins than BRO-2 strains (8, 10, 15). The higher ␤-lactam minimum inhibitory concentrations (MICs) associated with BRO-1 strains have been attributed to larger amounts of enzyme produced (15).The purpose of this study was to characterize the ␤-lactamases of M. catarrhalis isolates collected during 1994-1995 and 1997-1998 U.S. multicenter surveillance studies. Enzyme type was determined by IEF, and the relationship to ampicillin susceptibility was examined.Collection and identification of isolates. Between 1 November 1994 and 30 April 1995, 606 clinical isolates of M. catarrhalis were obtained from 24 U.S. health care institutions. Between 1 November 1997 and 30 April 1998, 513 additional clinical isolates of M. catarrhalis were obtained from the same 24 centers. Stock cultures of all isolates were prepared by using an absorbent bead system (Prolab Diagnostics, Austin, Tex.), and organisms were stored at Ϫ70°C until further use. The identity of the organisms was confirmed using conventional criteria (9). The details of these two studies and antimicrobial susceptibility patterns of the isolates collected have been described previously (4, 14).␤-Lactamase production and susceptibility testing. ␤-Lactamase production was assessed with a nitrocefin disk assay (Cefinase; Becton-Dickinson Microbiology Systems, Cockeysville, Md.). MICs of ampicillin were determined by a broth microdilution procedure (total volume per well, 100 l; final inoculum concentration, ca. 5 ϫ 10 5 CFU/ml) in cation-adjusted Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines (12). Microdilution trays were incubated at 35°C in ambient air for 22 to 24 h prior to the determination of the results. Ampicillin was tested at 12 different concentrations (from 0.015 to 32 g/ml) to limit the number of off-scale...

Contemporary Prevalence of BRO β-Lactamases in Moraxella catarrhalis : Report from the SENTRY Antimicrobial Surveillance Program (North America, 1997 to 2004)

“…Isolates carrying BRO-1 are usually more resistant to ampicillin than those carrying BRO-2 and have become more widespread and predominant (1). BRO-2 has been reported among M. catarrhalis isolates at rates less than 15% in the 1980s (8, 11), 4.8% from 1984 to 1994 (11,15,16), 2.1% from 1994 to 1995, and 3.1% in 1997 and 1998 (13). Although the rate of ␤-lactamase-mediated penicillin resistance in M. catarrhalis has been generally stable at Ͼ95% (9), the differential prevalence of the BRO-1 and the BRO-2 enzymes has not been determined in North America since 1998.…”

Production of BRO β-Lactamases and Resistance to Complement in European Moraxella catarrhalis Isolates

“…Bootsma et al (5) reported a 21-bp deletion in the promoter region of the ␤-lactamase gene from a BRO-2-producing strain. In addition, Richter et al (22) suggested that additional mutations or deletions or insertions in the promoter sequence might explain the variations seen in antibiotic susceptibilities within the populations of BRO-1-and BRO-2-producing M. catarrhalis isolates.…”

“…The 21-bp deletion in the BRO-2 promoter region, for example, is the one most likely to have a notable effect on promoter activity, affecting both the potential Ϫ10 and Ϫ35 sequences of BRO-1. However, additional, asyet-unknown mutations or deletions and/or insertions in the regulatory sequence might also explain the variations seen in the antibiotic susceptibilities of the BRO-1-and BRO-2-producing clinical isolates (22). Because of this, we sequenced the putative promoter region and a part of the gene of all of the ␤-lactamase-positive isolates (5).…”

“…All of the ␤-lactamase-positive isolates were selected for ␤-lactamase extraction and isoelectric focusing (IEF) of the enzymes as described by Richter et al (22). IEF of ␤-lactamase enzymes was performed with commercially prepared polyacrylamide gels (Ampholine PAGplate; Pharmacia Biotech, Uppsala, Sweden) with a pH range of 5.5 to 8.5 (22).…”

“…The incidence of these ␤-lactamase-producing isolates varied from 83 to 98% in the 13 participating countries, and no increase was observed during the study period. All of the ␤-lactamase-producing isolates were screened for the presence of BRO-1 and BRO-2 ␤-lactamase by IEF (22) and for the bro1 and bro2 genes by PCR and sequencing methods (5). IEF and sequencing displayed corresponding results for all ␤-lactamase-producing isolates.…”