- Volume 32, Issue 4, 1990

Of 39 strains of Escherichia coli isolated from foodstuffs, all were resistant to at least one of a panel of four metallic ions tested. The most common resistance (94.9%) was against cadmium, followed by arsenate (76.9%), silver (71.8%) and mercury (61.5%). Multiple resistance to three (35.9%) or four (38.5%) metals was seen more often than resistance to two (18%) or one (7.7%) metal only. The opposite trend was seen in antibiotic resistance; resistance to one (30%) or two (49%) antibiotics was more common than to three or more antibiotics (13%). Resistance to kanamycin correlated with resistance to silver and cadmium ions and resistance to ampicillin or cephalothin was, with one exception, associated with resistance to cadmium ions.

It has been shown previously that Trichomonas vaginalis, a common protozoan pathogen of the female genito-urinary tract, has binding sites for oestrogen and that its growth, adherence and chemotaxis are altered after exposure to physiological concentrations of oestrogens. Two other species of trichomonad showed no physiological response to oestrogens but did have high affinity oestrogen binding with low binding capacity. To determine whether oestrogen binding sites occur in other pathogenic micro-organisms, these studies with eukaryotic pathogens were extended to prokaryotic bacteria. Numerous bacteria previously considered to be oestrogen responsive, other species of the same genera, and control bacteria (not considered oestrogen responsive) were examined. High affinity protein-containing oestrogen binding sites were demonstrated in several bacterial genera. Direct oestrogenic effects on micro-organisms, as well as the traditional oestrogenic effects on host cells, may explain why certain infections are more common or more severe after exposure to oestrogen.

An enzyme immunoassay (EIA) with a monoclonal antibody to cryptosporidium oocysts was developed and evaluated for the examination of faecal and environmental samples. The EIA was as sensitive as microscopy for detecting Cryptosporidium but also produced some positive results which could not be confirmed by a modified Ziehl-Neelsen technique or by immunofluorescence microscopy. These specimens reacted also with heterologous antibodies in EIA, suggesting false positive results.

There was a marked peak in human cases of cryptosporidiosis in the Sheffield area in May and June 1986. Extensive epidemiological investigations failed to find a common source of food or a consistent history of animal contact, but did suggest that a waterborne outbreak of cryptosporidiosis may have occurred. Cryptosporidium oocysts were found in untreated water and in fish from a reservoir complex implicated by epidemiological analysis. Laboratory investigations confirmed that cattle on a farm adjacent to the reservoir complex were a possible source of contamination.

Purified oocysts of Cryptosporidium were applied to the top of a sand filter which had been constructed in the laboratory. The filter was eluted with distilled water; fractions were collected and examined for Cryptosporidium by modified Ziehl-Neelsen technique and immunoflourescence microscopy and by an enzyme immunoassay. The results indicate that oocysts of Cryptosporidium do not easily pass through the sand filter, and that some disintegration of oocysts may occur during filtration.

Monoclonal antibody (MAb) to diphtheria toxin was produced in mouse hybridomas, and shown by ELISA to be of sub-class IgG1. Hybridomas were inoculated into mice to produce ascitic fluid from which MAb was purified by caprylic acid. The MAb was shown by immunoblotting to be directed against the A fragment of the toxin and also against the intact toxin molecule. After conjugation with fluorescein isothiocyanate, it was used in an immunoassay to detect toxin in culture supernates of Corynebacterium diphtheriae and C. ulcerans. The assay correlated well with the Elek test and with virulence in guinea-pigs; but it gave occasional false positive results, probably by binding of MAb to defective toxin.

Shiga toxin and Shiga-like toxins (SLTs, syn. Verotoxins) are currently detected by tissue culture assays that are expensive, time-consuming and require specialised facilities and experienced personnel. We have developed a rapid method to detect Shiga toxin and SLT-I (Verotoxin 1) based on their binding to globotriosyl ceramide (Gb3). Bound toxin was then detected by an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The direct detection of cytotoxins from pure culture plates and from a mixed bacterial culture was studied. Using polymyxin extraction (0.1 g/L, 30 min, 37°C) and Gb3-based ELISA we detected toxin from reference strains Shigella dysenteriae 1 strain 60R (Shiga toxin) and Escherichia coli 026: H11 strain H30 (SLT-I), and from clinical isolates of E. coli O157: H7 and O26: H11 (both SLT-I) from 11 patients with diarrhoea, haemorrhagic colitis or haemolytic uraemic syndrome. Toxin production by these strains was confirmed by a radiolabelled HeLa cell assay and the structural genes were detected by DNA hybridisation. The Gb3-based ELISA could detect SLT-I in extracts of a mixed culture even when the toxin-positive strains represented only 1% of the mixture. No cross-reactivity was found with bacteria that produce other cytotoxins, such as other E. coli and Shigella, Salmonella, Aeromonas and Campylobacter spp.

Cell walls from different species of Mycobacterium were purified on a sucrose step gradient. The components derived from these preparations were characterised by sodium dodecyl sulphate—polyacrylamide gel electrophoresis, followed by staining or by Western blotting. Surface-exposed polypeptide molecules were also identified by biotinylation. Many protein and glycoprotein molecules were identified in the cell walls. Some of these molecules were immunogenic in man and experimental animals and showed wide variability from species to species. The data suggest that these molecules could be of significance in the diagnosis and pathophysiology of mycobacterial diseases.

A combination of radiometric broth culture (Bactec 13A) and the Genprobe nucleic acid hybridisation system was used to detect and identify Mycobacterium tuberculosis and M. avium from the blood of patients with acquired immune deficiency syndrome (AIDS).

The immune response to different serogroups and serotypes of N. meningitidis has been examined in acute and convalescent sera from patients with meningococcal diseases. The focus of the study was the c. 70-Kda iron-regulated outer-membrane protein (FeRP-70). FeRP-70 was demonstrated on all strains of different serogroups and serotypes examined by sodium dodecylsulphate-polyacrylamide gel electrophoresis or Western blots of outer-membrane proteins (OMPs). Immunoblotting experiments demonstrated the presence of considerable amounts of anti-FeRP-70 IgG antibodies in the acute and convalescent sera of six patients; the antibodies reacted with homologous and heterologous strains. However, sera from two patients who died of severe meningococcal septicaemia had no antibodies against FeRP-70 or any other OMPs demonstrable by immunoblotting. Absorbed rabbit hyperimmune sera reacted with FeRP-70 of their homologous strains, but, unlike human sera, with only a few of the heterologous strains. We believe that FeRP-70 is strongly immunogenic in vivo, cross-reactive amongst different strains, and that man and animals differ considerably in their response to similar meningococcal antigens. The functional attribution of human antibody response against this protein requires further exploration.

Fresh clinical isolates of Salmonella spp. and Streptococcus pyogenes were analysed by pyrolysis-mass spectrometry (Py-MS). The results formed the basis of mathematically derived characterisations of individual strains and these were compared with the results of phage typing for the salmonellas and M protein typing for the streptococci. Py-MS was shown to be a rapid and reproducible method for inter-strain comparisons, giving evidence of identity and non-identity between strains that agreed well with the results of conventional tests. Py-MS has potential value as a rapid, relatively inexpensive and highly discriminatory method of epidemiological analysis in bacterial disease.