Considerable biochemical diversity and polynucleotide sequence variation have been reported amongst strains of spp. However, sequence heterogeneity has not been investigated at gene loci of clinical relevance. In this study, sequence heterogeneity in the β-lactamase structural gene, , amongst 91 clinical isolates of spp. that showed resistance to various third-generation cephalosporins was investigated. Variation was examined by high-stringency polymerase chain reactions (PCR) with primers homologous to the known sequences of strains OS60 and I113, and NF85. If an isolate contained an gene homologous to one of these three characterised genes, a single PCR band of a predictable size was generated with the appropriate primer set; 50 (60%) of isolates gave a PCR product of the expected size with the OS60 primer set and nine (10%) gave a product with the I113 primer set. All these 59 isolates were identified as by API-20E strips. Six isolates (7%) gave a product with the NF85 primer set but only four of these were identified as in API-20E tests; the other two isolates were identified as . Of the 91 isolates, 28 (31%), were identified as either or , but gave no PCR product with any primer set tested. Five of these showed no homology to any of the reference strain PCR products in hybridisation tests. Nevertheless, all showed β-lactamase activity. Overall, this method allowed the identification of novel gene loci, which may serve as a basis for the identification of spp. rapidly at a molecular level.


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