- Volume 46, Issue 12, 1997

Bacteroides spp., particularly B. fragilis, are well-recognised bacterial pathogens. Production of the typical β-lactamases of Bacteroides restricts the therapeutic use of β-lactam agents mainly to the β-lactamase inhibitor combinations and carbapenems. These compounds have the advantage of broad-spectrum activity and the ability to combat polymicrobial infections. Resistance of Bacteroides spp. to β-lactam antibiotics appears to be increasing, largely because of an overall increase in β-lactamase activity. There has been a rise in the prevalence of isolates showing high-level production of typical Bacteroides β-lactamases and an increase in reports other potent β-lactamase types. In the case of B. fragilis, metallo-enzymes are a particular threat to current therapeutic practice, as they are not inhibited by common β-lactamase inhibitors and are able to hydrolyse carbapenems. The presence of permeability barriers may confer low-level β-lactam resistance and supplement the effect of β-lactamase activity. There are also sporadic reports of loss of β-lactam activity because of reduced affinity of the penicillin-binding proteins.

Serratia marcescens isolates from 164 patients with suspected nosocomial infection in several hospitals in the greater Paris region were investigated by analysis of the electrophoretically demonstrable allelic variations of gene loci coding for five esterases and five other enzymes. All the loci were polymorphic and the mean number of alleles per locus was 6.1. A total of 72 distinctive electrophoretic types (ETs) representing multilocus genotypes was distinguished. the isolates were divided into two groups according to their resistance to antibiotics: 82 multiresistant isolates (MRI) and 82 relatively susceptible isolates (RSI). Seventy-two MRI (88%) were in four genetically related ETs: ET1, ET2, ET8 and ET9; ET1 was found in 48 isolates, whereas the remaining MRI were in 10 ETs, and all RSI in 61 ETs. Three ETs contained both MRI and RSI. the mean coefficients of genetic diversity for the 10 enzyme loci among ETs and isolates were smaller for MRI than for RSI, while the modal ET of MRI resembled that of RSI. the epidemiological significance of isolates varied according to their ET. Thus, isolates belonging to ET1, ET2 and ET8 were responsible for outbreaks or for sporadic infections, whereas isolates of other ETs were responsible for only sporadic infections. the temporal distribution of ET1 isolates among hospitals identified seven outbreaks in seven clinical departments.

A new serogroup of Vibrio cholerae non-O1, designated as O139, has emerged causing cholera-like disease among adults. Laboratory and field studies clearly show that there is no cross-protection between O1 and O139 pathogenic strains. Since colonisation of the intestine is a most important step in the pathogenesis of cholera caused by O1 strains and colonising antigens are known to be protective, investigation of the colonising antigens of O139 strain was initiated. By TnphoA mutagenesis, mutants were generated with insertions in the genome encoding membrane spanning or secretory proteins. Screening of the mutants for adherence to rabbit intestinal surface and colonisation in 5-day-old mice resulted in the identification of mutant clones, which were less adhesive than was the wild-type parent strain and which could not efficiently colonise the gut. Such non-colonising strains were attenuated in virulence. Analysis of the proteins by SDS-PAGE revealed that the non-colonising mutants did not express a 40-kDa outer-membrane protein.

It has been shown that cytokines have potential as therapeutic adjuvants in vaccination. Interleukin-5 (IL-5) is a cytokine that regulates antibody production, in particular enhancing IgA production by activated mucosal B cells. This study examined the expression of a cloned cytokine gene encoding murine IL-5 (mIL-5) by an attenuated aroA strain (SL5631) of Salmonella serotype Dublin. the resulting strain, SL5631(pTRXFUS-mIL-5), expressed mIL-5 as a fusion with thioredoxin as demonstrated by immunological and biological assays. When strain SL5631(pTRXFUS-mIL-5) was used as a live vaccine in BALB/c mice, it colonised and multiplied at higher levels in spleens and livers than the strain carrying the empty plasmid. A reduction in invasiveness of SL5631(pTRXFUS-mIL-5) was observed in in-vitro invasion assays. Enhanced IgA response against salmonella LPS in mucosal secretions and enhanced IgA and IgG responses were detected by ELISA and ELISPOT methods in sera of mice immunised with the strain expressing mIL-5. Results with IL-5-deficient mice showed that the enhanced IgA response was due to Salmonella-expressed mIL-5 rather than endogenous mIL-5.

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 ISP8 (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 pTZ19U. 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 ISP8. 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 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.

Counter-current immuno-electrophoresis was evaluated as a diagnostic test for the serodiagnosis of typhoid fever with somatic (O), flagellar (H) and capsular polysaccharide (Vi) antigens of Salmonella typhi on the sera of patients who were blood culture positive (confirmed typhoid cases) or had high Widal agglutination titres, ≥ 320, (presumptive typhoid cases). Of the 37 sera from confirmed cases, 30% showed positivity with O antigen, 24% with H antigens and 51% with Vi antigen. In patients with a presumptive diagnosis, 45% were positive for O antibody, 27% for flagellar antibody and 52% for Vi antibody. When all three antigens were combined the reactivity to any of the antigens was found to be 59% in confirmed typhoid cases, 79% in presumptive typhoid cases and 93% in patients who were simultaneously positive by blood culture and Widal agglutination. However, none of the sera from 45 controls gave a positive precipitation reaction with any of the antigens. It is concluded that counter-current immuno-electrophoresis is a rapid test with low sensitivity and high specificity with Vi antigen, a panel of antigens being most effective, and is, therefore, recommended for rapid diagnosis of typhoid fever.