Detection of plasmid-mediated beta-lactamases with DNA probes

Abstract

P-Lactamase identffication by colony hybridization with 32P-labeled DNA probes for TEM-1, SHV-1, OXA-1, OXA-2, PSE-1, PSE-2, and PSE-4 was compared with isoelectric focusing in 122 clinical isolates making a variety of enzyme types. All strains producing a probe-type enzyme gave a positive hybridization reaction. Cross-hybridization was observed between TEM-1 and TEM-2 or TLE-1, between SHV-1 and SHV-2, between OXA-1 and OXA-4, between OXA-2 and OXA-3 (weak), between PSE-2 and OXA-6 or OXA-5 (weak), and among PSE-1, PSE-4, and CARB-3. With allowance for such cross-hybridization, only six strains gave false-positive reactions, and the procedure was 99% specific.Biochemical studies have established that plasmids in gram-negative bacteria determine a variety of P-lactamase types. The enzymes can be differentiated by substrate profile, reactions with inhibitors, molecular weight, and particularly isoelectric point (pl) into at least 25 kinds (16,19,32). Some, such as TEM-1 and SHV-1, have broad-spectrum but relatively unspecialized activity. As their name implies, the OXA set hydrolyzes oxacillin and related P-lactams with particular efficiency, whereas CARB enzymes, also given PSE designations because they were first found in Pseudomonas aeruginosa, are notably effective in carbenicillin hydrolysis. Distinguishing between the enzymes is of value for epidemiological studies where the presence of a less common P-lactamase type may allow a particular strain or plasmid to be traced (20). The distribution of P-lactamase types in different organisms may also help elucidate where these enzymes originated and how they have spread in response to the use and elaboration of P-lactam antibiotics.Studies relying on isoelectric focusing have demonstrated TEM-1 as the predominant P-lactamase in ampicillin-resistant Escherichia coli and Salmonella spp., whereas SHV-1 is preponderant in Klebsiella spp. (29). In P. aeruginosa carbenicillin or ticarcillin resistance due to ,-lactamase production is the result mainly of PSE-1 or PSE-4 (24, 33).If P-lactamase genes are as diverse as the enzymes they encode, it should be possible to develop DNA probes for P-lactamase identification that would allow more rapid studies with large numbers of strains. DNA fragments suitable as probes for TEM-1 (3, 12, 22), OXA-1 (12, 23), OXA-2 (2), and PSE-1 (12) have been described and have been studied for specificity by utilizing purified plasmid DNA. The purpose of this investigation was to apply such probes to clinical isolates to evaluate their sensitivity and specificity. MATERIALS AND METHODSBacterial strains. strains were demonstrated to grow on plates containing 25 ,ug of ampicillin per ml and to produce ,-lactamase as determined by the nitrocefin test (21). Because no probetype P-lactamases were detected among P. aeruginosa isolates from this source, 20 P. aeruginosa clinical isolates with known plasmid-mediated P-lactamases collected by A. Philippon in France before 1981 were also included in hybridization studies.Analytical isoelectric focusin...

Extended-Spectrum β-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat

“…Early detection of ␤-lactamase genes was performed using DNA probes that were specific for TEM and SHV enzymes (7,55,70). However, using DNA probes can sometimes be rather labor intensive.…”

Molecular Detection of Antimicrobial Resistance

“…Although no explanation was offered for this discrepancy, the method was considered convenient for the rapid screening of isolates. Radiolabeled fragment probes were also used to detect the genes for TEM-1, SHV-1, OXA-1, OXA-2, PSE-1, PSE-2, and PSE-4 (127). Some of the probes cross-hybridized with related ␤-lactamase genes.…”

Identification and Minisequencing-Based Discrimination of SHV β-Lactamases in Nosocomial Infection-AssociatedKlebsiella pneumoniaein Brisbane, Australia

“…Previously used methods include DNA hybridization (5,11,29), oligotyping (20), restriction fragment length polymorphism analysis (27), an immunoassay system (6), PCRsingle stranded conformation polymorphism (2,25,26), and the ligase chain reaction (17). The clinically significant effect on phenotype conferred by single-nucleotide changes in SHV ESBLs makes them ideal candidates for the development of a minisequencing protocol.…”