Subtractive hybridisation was used to screen for and identify conserved DNA sequences associated with clinical, clonal populations of . DNA from strain ISP8 (a clinical isolate) was digested with I and hybridised with randomly fragmented pooled DNA from 10 non-clinical (community) isolates of . The mixture of DNA fragments was then ligated to I-digested and dephosphorylated pTZ19U. One recombinant plasmid (pWASA) was identified as containing specific DNA from strain ISP8; DNA sequencing revealed a 40-bp I DNA fragment (WASA). PCR amplification and hybridisation analysis, with pWASA as a probe, showed that 84% of clinical isolates from a clonal line present in hospitals in major eastern Australian cities carried sequences homologous to WASA, compared with only 10% of community isolates. The isolates that hybridised were closely related by RFLP analysis to strain ISP8. the plasmid pWASA was used to identify, isolate and clone a 3.5-kb I fragment (DSA) from strain ISP8 total DNA. Sequence analysis of the DSA fragment identified two open reading frames (ORFs) of 2475 and 576 bp, respectively; the larger ORF1 contained a series of six tandem repeats, each consisting of 384 nucleotides. the nucleotide sequence of the repeats was 96% identical, and no significant sequence homologies to previously described protein sequences were identified. However, tandem repeats of amino acids are structural motifs characteristic of a number of gram-positive surface proteins, and are thought to play a role in generating genetic and phenotypic diversity in these and other bacteria.


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