The genomic DNA of 58 isolates of methicillin-resistant Staphylococcus aureus (MRSA) obtained during an infection outbreak at two major Canberra hospitals was analysed for restriction fragment length polymorphism (RFLP) by digestion with the endonuclease SmaI and resolution of the fragments by pulsed-field gel electrophoresis. Based on the fraction of common fragments generated by the endonuclease, DNA similarities among the isolates were estimated. Distance matrix analysis showed that the MRSA isolates could be divided into two major clusters (RFLP types I and 11) and one minor one (type 46). A fourth group of miscellaneous isolates was found to be heterogeneous in terms of DNA sequence similarity. The epidemiological data indicated that RFLP type I was most common in the intensive care units in the two hospitals, with particular subtypes of RFLP type I concentrated in individual units. RFLP type I1 and the miscellaneous group were more generally distributed. Type 46 isolates appear to be related to a group which was present in epidemics in Melbourne hospitals in the early 1980s. Using the standard phage set, the RFLP type I group was largely untypable. However, type I1 isolates were all phage typable, with a shared susceptibility to phages 29/85/95/90; type 46 isolates had a shared susceptibility to phages 85/90. The miscellaneous isolates were of variable phage types.

Clinical isolates of Staphylococcus aureus collected from hospitals in Australia were analyzed for genetic similarities using restriction fragment length polymorphisms. Methicillin-resistant (Mcr) isolates from Melbourne (1982) and from Hobart (1986) were closely related to a methicillin-multiresistant S. aureus (MRSA) strain, ANS46, originally isolated in Melbourne in 1982 and studied extensively since. Methicillin-sensitive (Mcs) isolates were isolated concurrently with the Melbourne and Hobart Mcr isolates. These were found to be similar to induced Mcs variants of ANS46; these laboratory variants have lost approximately 40-70 kb of DNA carrying multiple resistance (R) determinants clustered around the mec gene. The Melbourne and Hobart Mcs isolates appear to be natural variants lacking this region of their chromosome. A clinical Mcr and Mcs pair of isolates differing only in the presence of an R-cluster near the mec gene were also isolated in Melbourne in 1990; these are not of the same clonal line as the earlier types from Melbourne and Hobart. These data suggest that insertion or deletion (or both) similar to that produced by known mutagens occurs in the mec region of the chromosome of MRSA in clinical populations under natural selective pressures; such processes may be important in the balance of resistant and sensitive staphylococci in hospitals and other clinical environments.

A 26-kDa protein (OMP26) isolated and purified from nontypeable Haemophilus influenzae (NTHI) strain 289 has been shown to enhance clearance of infection following pulmonary challenge with NTHI in rats. DNA sequence analysis revealed that it was 99% identical to a gene encoding a cell envelope protein of the H. influenzae Rd strain (TIGR accession no. HI0916). The deduced amino acid sequence revealed a hydrophilic polypeptide rich in basic amino acids. Restriction fragment length polymorphism analysis suggested that the OMP26 gene was relatively conserved among isolates of NTHI. Analysis of the deduced amino acid sequence of the OMP26 gene from 20 different isolates showed that similarity with NTHI-289 ranged from 96.5% (1 isolate) to 99.5% (14 isolates). Two recombinant forms of OMP26, a full length 28-kDa protein (equivalent to preprotein) and a 26-kDa protein lacking a 23-amino-acid leader peptide (equivalent to processed protein), were assessed in immunization studies for the ability to induce an immune response that would be as effective as the native protein in enhancing the clearance of NTHI following pulmonary challenge in rats. Immunization with the recombinant protein that included the leader peptide was more effective in enhancing pulmonary clearance, and it induced a better cell-mediated response and higher titers of systemic and mucosal antibody. This study has characterized a 26-kDa protein from NTHI that shows significant potential as a vaccine candidate.

Subtractive hybridisation was used to screen for and identify conserved DNA sequences associated with clinical, clonal populations of Staphylococcus aureus. DNA from S. aureus strain ISPS (a clinical isolate) was digested with TaqI and hybridised with randomly fragmented pooled DNA from 10 non-clinical (community) isolates of S. aureus. The mixture of DNA fragments was then ligated to AccI-digested and dephosphorylated pTZl9U. One recombinant plasmid (pWASA) was identified as containing specific DNA from strain ISP8; DNA sequencing revealed a 40-bp TaqI 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 ISPS. The plasmid pWASA was used to identify, isolate and clone a 3.5-kb DraI fragment (DSA) from strain ISP8 total DNA. Sequence analysis of the DSA fragment identified two open reading frames (ORFs) of 2475 and 576bp, respectively; the larger O W 1 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.

The effects of UV-B radiation generated in the laboratory and as a component of sunlight on the viability and particular biochemical activities of the bacterium Staphylococcus aureus have been examined. UV-B radiation progressively inhibits protein synthesis (assayed as 3H-alanine incorporation) and kills cells. Cell respiration, and RNA and DNA synthesis (3H-uridine and 3H-thymidine incorporation) were not greatly affected by UV-B irradiation. The OH. and 1O2-free radical scavengers protected cells against killing and inhibition of protein synthesis by UV-B, suggesting that such radicals mediate the effects of UV-B on this organism. A similar protective effect using a ferric ion chelator suggests an important role for metallic ions in UV-B lethality.