- Volume 39, Issue 5, 1993

A range of solid and liquid media was evaluated for the ability to maintain survival of Helicobacter pylori strains under different conditions. Chocolate agar slopes maintained survival of most strains for longer than 3 days, some strains surviving for up to 9 days, despite a decreased number of viable cells. Temperature and atmosphere did not significantly influence the performance of these slopes. The BBL Campy Pouch system also achieved a considerable recovery rate of H. pylori after storage for 3 days at the same range of temperatures. Brain-heart infusion broth with horse serum was superior among the liquid media tested, maintaining the viability of H. pylori for c. 3 days at temperatures ranging from –4°C to 21°C. Chocolate agar slopes are recommended as suitable for transport of H. pylori strains.

A polymerase chain reaction (PCR) assay with oligonucleotide primers homologous to a portion of the urease C gene of Helicobacter pylori was evaluated for specificity with pure DNA and biopsy material. The assay was used to test for the presence of the organism in dental plaque. The species specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from H. cinaedi, H. felis, H. fennelliae, H. mustelae and H. nemestrinae. Sixty-two gastric biopsy samples collected from 14 patients (antrum, body and duodenal sites) were cultured and PCR was performed on the samples after culture. Primer sites were conserved in genomically diverse strains. Samples prepared by single-step heat lysis of bacterial cells and biopsy material did not inhibit PCR. The overall specificity was 96% irrespective of genotype. H. pylori was not cultured from dental plaque (15 patients), neither was H. pylori DNA detected by PCR in either urea breath test-positive or -negative individuals. The results showed that primer pair sequences within the urease C gene are conserved in most strains and provide an accurate basis for detecting H. pylori. As the PCR assay was not inhibited and did not yield false positive results with crude extracts from organisms or in the presence of biopsy material, its value as a diagnostic test was confirmed.

Isolates from a presumptive nosocomial outbreak of Clostridium difficile infection at a large teaching hospital were typed by pyrolysis mass spectrometry (PMS) and antibiograms. One isolate, from the putative index case, was dissimilar from the outbreak strain, but 24 isolates from 16 patients were indistinguishable by both methods. The outbreak centred on two wards for the acute care of the elderly, with a few cases elsewhere. Transfer of patients appeared to be the route of transmission between wards. There was a significant fall in the incidence of cases following intervention by the Infection Control Unit. This included ward inspection, advice on antibiotic usage and advice on prevention of faecal-oral transfer, particularly by proper handwashing. Subsequent monitoring of C. difficile infection showed a background of sporadic, dissimilar isolates with occasional apparent cross-infection incidents limited to a few patients. In suspected outbreaks, patterns of antibiotic susceptibility may be useful in initial screening, before referral for more sophisticated typing. There was excellent correlation between PMS results, antibiograms and epidemiological information.

A bacteriological study of isolates from the oral cavity of patients with Kawasaki disease (KD), age-matched non-KD patients and healthy children, showed that over half the KD and control isolates had gram-positive, catalase-negative cocci. About 50% of these organisms were identified as viridans streptococci by means of an API Strep 20 kit. Further identification by fluorometric DNA-DNA hybridisation demonstrated that the predominant species were S oralis and S. mitis, each of which accounted for 25% of the isolates of viridans streptococci; 40% of viridans strains were unidentifiable; and S. sanguis and S. parasanguis were minor components. Studies in vivo showed that insertion of culture supernates of most of the viridans streptococci increased capillary permeability and induced redness with swelling and occasional bleeding in rabbit skin. One-third of S. mitis strains and one-fifth of the unidentified strains caused aggregation of human blood platelets, whereas S. oralis and other strains had no such effect. The distribution of extracellular lipoteichoic acids and glucan produced in the presence of sucrose was also examined. There were no significant differences in the recovery rate of viridans streptococci forming these biologically active extracellular products between KD and control groups.

Brucella strains exhibit either a rough (R) or a smooth (S) colonial phase identifiable by bacteriological methods. This depends on the biosynthesis and translocation to the surface in S but not in R strains, of the O-polysaccharide chain of the lipopolysaccharide (LPS) molecule. B. melitensis biovar 1 strain EP exhibited simultaneously both S and R characteristics in relation to colonial morphology, agglutination by monospecific anti-M and anti-R sera, activity of bacteriophages lytic for rough Brucella spp. (phage R/C) and for smooth B. melitensis (phage Iz). B. melitensis strain EP expressed fewer O-chains with a similar distribution of molecular weights than B. melitensis reference strain 16M by SDS-PAGE and immunoblotting, but higher amounts of R-LPS. Quantitative determination of S-LPS by a turbidimetric latex inhibition immunoassay with monoclonal antibodies confirmed the limited expression of S-LPS in strain EP. As with other gram-negative bacteria, the phenomenon could be attributed to a deficiency in one step of the biosynthetic assembly of the O-chains.

Seventeen strains of Escherichia coli serotype O157:H7 producing Shiga-like toxin were examined for the presence of plasmids and for the ability to adhere to HEp-2 and Intestine 407 cells. All of the strains possessed a common 60-MDa plasmid. To determine the role of the 60-MDa plasmid, plasmid-cured strains were compared with the parent strains for their ability to produce pili and to adhere to epithelial cells in culture. The total cell lysate protein and outer-membrane protein (OMP) profiles were also compared. Both the parent strains and their plasmid-cured derivatives produced pili. Immunofluorescence assay results indicated that the plasmid-cured and parent strains adhered equally well to HEp-2 and Intestine 407 cells; overall adherence was greater with intestinal cells than HEp-2 cells. SDS-PAGE of polypeptides synthesised in an E. coli system in vitro showed that plasmid DNA encodes c. 35 proteins. SDS-PAGE of OMP preparations demonstrated that the 60-MDa plasmid appears to be involved in the synthesis of a 33-kDa OMP. Two strains cured of the 60-MDa plasmid, one that possessed no plasmids and one that still contained a 2.2-MDa plasmid, produced exopolysaccharide (EPS) when cultured on solid medium at 25°C. Two other strains, which were cured of the 60-MDa plasmid but contained a 4.5-MDa plasmid, did not produce visible amounts of EPS. Gas chromatography analysis showed that the EPS consisted of fucose, glucose and galactose in an approximate molar ratio of 2.0:0.9:1.1 and also had 7% of a uronic acid sugar as part of its structure.

Cysteine-dependent (cys−) Escherichia coli and Klebsiella spp., defective in sulphate assimilation, were isolated from urine and stool samples of infected patients. These isolates reverted to prototrophy under conditions of cysteine deprivation but the revertant strains and a prototrophic wild-type E. coli strain became auxotrophic for cysteine in a cysteine-enriched medium. This suggested that excess cysteine acts as a repressor of the cys HIJ operon known to control aspects of cysteine biosynthesis. A group of mostly elderly patients infected with cys− strains suffered a disproportionate amount of renal impairment as compared with a control group. In renal impairment, sulphur compounds, including cysteine, are retained. This raises the possibility that these raised levels of cysteine and related compounds may enhance the selection of cys− strains in vivo.

A surface protein of Bordetella bronchiseptica was purified in one step by affinity chromatography with bovine submaxillary mucin coupled to agarose. The purified protein, with a mol. wt of 200 kDa and an iso-electric point of pI 6.5, showed haemagglutinating activity for bovine erythrocytes. The haemagglutinin (HA) inhibited the adherence of B. bronchiseptica to a rat lung cell line (L2) and was able to bind to N-acetylneuraminic acid. These findings suggest that the HA of B. bronchiseptica is an adhesin.

Aspergillus fumigatus produced a 33-kDa serine protease (ALP) in vitro and in vivo. In vitro, this alkaline protease was secreted when the fungus was cultivated in the absence of protein, if the pH of the medium remained close to neutrality. Western blotting and immuno-electronmicroscopy studies showed that ALP was localised in the wall of the fungus and was degraded after secretion in the culture medium under conditions of low pH. Although present in the lung during infection, ALP did not appear to be diagnostically useful and was different from the precipitating chymotrypsin antigen used in the diagnosis of aspergilloma.