- Volume 39, Issue 6, 1993

Case of culture-confirmed clinically typical haemorrhagic colitis caused by verocytotoxin-producing (VT+) Escherichia coli O157 and age- and sex-matched control patients were examined for antibodies to E. coli O157. Serum samples from 28 cases and 34 patients in control group 1 were examined for VT1- and VT2-neutralising antibodies, E. coli O157 agglutinating antibodies, and by an enzyme immunoassay (EIA) technique for IgG antibodies against smooth lipopolysaccharide purified from E. coli O157 and for IgG antibodies against whole intact E. coli O157 cells. Differences between antibody tires were significant when compared by a Wilcoxon two-sample test for E. coli O157 agglutinating antibodies (p < 0.05) and IgG antibodies against whole cells (p < 0.001). The whole-cell EIA was used further to examine faecal samples from 93 cases and 47 patients in control group 2 for IgA antibodies. Elevated levels of faecal IgA specific for E. coli O157 were found in 59 (63.4%) of 93 cases but in only 10 (21.2%) of 47 control patients (p < 0.001); follow-up faecal samples from five cases all showed marked rises in levels of IgA that appeared to coincide with cessation of excretion of the organism. Detection of specific faecal IgA with a whole-cell EIA, although requiring further evaluation, may be a useful addition to tests currently available for the diagnosis of infection by VT+ E. coli O157.

Porphyromonas gingivalis is associated strongly with severe periodontitis, but little information is available on possible transmission routes of this species. This study evaluaated three DNA-based molecular typing methods for use in epidemiological surveys of P. gingivalis. In total, 32 isolates from eight married couples were investigated by: (i) restriction endonuclease analysis (REA) of whole chromosomal DNA; (ii) hybridisation of DNA fragments with ribosomal DNA (ribotyping); and (iii) amplification of DNA by the polymerase chain reaction with arbitrary primers (AP-PCR). The data obtained with the three methods were in broad agreement: In six of the eight couples, the isolates from husband and wife were indistinguishable, but isolates from unrelated individuals showed distinct types with all three methods. For some isolates, minor differences in REA pattern were obtained which could not be correlated with differences in ribotype or AP-PCR type. Ribotyping showed differences between isolates from one individual, which were indistinguishable with the other two methods. The patterns obtained with ribotyping or AP-PCR were simple in comparison to the relatively complex REA patterns. Although all three methods were concordant, AP-PCR was found to be the least time-consuming method. The data support the suggestion that P. gingivalis can be transmitted between spouses.

Phenotypic loss of protein A production was tested in six methicillin-resistant (McR) Staphylococcus aureus (MRSA) isolates and their isogenic methicillin-sensitive (McS) variants by a radiolabelled IgG-binding assay with washed cells and by Western blotting of supernates prepared from lysed washed cells. Genomic DNA was probed for homology with the protein A gene (spa) in EcoRI digests and for homology to the methicillin resistance gene (mec) in HindIII digests. The McS variants had lost homology with mec. An isogenic pair of McR and McS strains, and derivatives of S. aureus 8325-4 with site-specific mutations of the accessory gene regulator locus (agr) and spa, were tested for adherence to human peritoneal mesothelial cells in monolayer culture. The isogenic pair were also tested for adherence to HEp-2 and Vero cell monolayers in assays with 3H thymidine-labelled bacteria. McR isolates produced protein A which was absent from three strains that had become McS. This correlated with deletion of the spa locus. Spa homology, but reduced production of protein A, was retained in one McS strain which also showed reduced adherence to HEp-2, Vero and mesothelial cells (p < 0.05) compared with the parent McR strain. A spa mutation in strain 8325-4 did not significantly affect adherence to mesothelial cells but mutation in agr increased adherence significantly in both Spa+ and Spa- strains.

Ten Staphylococcus intermedius isolates from cases of canine pyoderma and 10 from healthy carriers were examined by SDS-PAGE of exoproteins, immunoblotting and restriction endonuclease digest analysis. Similarities between banding patterns of the isolates were calculated as Dice coefficients for all three methods. For SDS-PAGE and immunoblotting, no significant differentiation was found between the pyoderma and “healthy” groups. Analysis of DNA digested with BglII indicated that S. intermedius is genetically heterogeneous; Dice coefficients for the pyoderma group were distinct from those for the healthy group (p < 0.001), and cluster analysis confirmed that the pyoderma isolates (9) formed a group separate from the majority (6 of 9) of the normal isolates.

The capacity of 11 strains of oral streptococcal species (Streptococcus sanguis, S. oralis, S. mitis and S. sobrinus) to produce hydrogen peroxide (H2O2) was studied in vitro. Detection of this property in solid media, particularly with trypticase soy agar-benzidine-peroxidase, was more sensitive than in liquid media. The addition of carbohydrates (arabinose, xylose, mannose, sorbose and lactose), sorbitol and saccharine to buffered trypticase soy broth increased H2O2 production in S. oralis NCTC 11427, although the concentrations obtained with some substrates (glucose, galactose, mannitol and xylitol) were lower than those obtained in controls. In S. sanguis NCTC 7863, H2O2 production was detected only with galactose, sorbitol, lactose and saccharin.

The ability of 25 lectins, isolated from different plants and fungi, to agglutinate 95 clinical isolates of β-haemolytic streptococci was examined. Cell suspensions were untreated, trypsin-treated or boiled at pH 2.0. None of the 95 untreated cell suspensions gave a visible reaction with any of the lectins. When the cells were trypsinised, 42 strains were agglutinated with one or more lectin and after boiling at pH 2, all the strains were agglutinated. After treatment with trypsin, 20 different agglutination patterns were observed, and after boiling, 19 patterns, four of which were similar. A correlation was found between Lancefield group C and some of these patterns. Some lectins reacted specifically with group C streptococci; DBA and WFA, both specific for D-Ga1NAc, DSA, a G1cNAc-specific lectin, and RPA, which showed a complex specificity, reacted only with group C strains. Furthermore, the lectin of Maackia amurensis reacted with 50% of group B streptococci only. Agglutination assays with lectins were reproducible, easy to perform, relatively inexpensive and, therefore, applicable to studies of cell-wall structure and epidemiology of β-haemolytic streptococci.

Probes constructed from a 4.05-kb EcoR1 digest fragment of a mupirocin resistance plasmid and a 751-bp internal part of this fragment hybridised with DNA from all of 36 independent high-level mupirocin-resistant staphylococci tested from seven centres; most were Staphylococcus aureus. In most instances the probes detected an EcoR1 digest fragment of approximately 4 kb. Probes did not hybridise to DNA from low-level resistant strains, nor from strains sensitive to mupirocin.

High-level mupirocin resistance in coagulase-negative staphylococci isolated from patients undergoing peritoneal dialysis was investigated by transfer of the resistance determinants, usually in the form of a plasmid, to Staphylococcus aureus strains, cleavage of the plasmid by restriction endonuclease and hybridisation with a probe comprising a 4.05 kb EcoR1 fragment of a plasmid from a S. aureus strain. In most instances the mupirocin-resistant staphylococci isolated from each patient were different according to the species, antibiogram and plasmid profile data. The mupirocin resistance determinant was carried on various plasmids as judged by EcoR1 restriction fragment length polymorphisms. All hybridised at about 4 kb with the S. aureus probe.

The type strains of Vi-phage type E1, M1 and A of Salmonella typhi, together with drug-resistant and drug-sensitive strains of phage types E1 and M1 isolated in 1992 from patients associated with India or Pakistan, and a drug-resistant strain of phage type A isolated in South Africa in 1991, were characterised with respect to the presence of plasmids conferring resistance to antimicrobial drugs and their chromosomal insertion sequence IS200 profiles. The three type strains, the drug-sensitive strains of Vi-phage types E1 and M1, and a strain of phage type M1 resistant to ampicillin and trimethoprim but not to chloramphenicol, did not contain plasmids. In contrast, for strains of phage types E1 and M1 resistant to chloramphenicol, ampicillin and trimethoprim, and for the drug-resistant strain of phage type A, the complete spectrum of resistance was encoded by high molecular mass plasmids belonging to the H1 incompatibility group. Characterisation of IS200 profiles demonstrated that at least 13 IS200 copies were distributed on the chromosome of all strains tested. Although the IS200 profiles of the type strains of Vi-phage types A, E1 and M1 were identical, it was possible to distinguish between drug-sensitive and drug-resistant strains of Vi-phage types E1 and M1 isolated from patients infected in India and Pakistan by this method. It was concluded that although IS200 typing is not as discriminatory as phage typing for the primary subdivision of S. typhi, it may be useful for certain epidemiological investigations and, in particular, for investigating the origins of strains with multiple drug resistance.

A beneficial role of the antibody response to Pseudomonas aeruginosa seen in cystic fibrosis (CF) patients has not been established. We investigated a possible role for these antibodies as inhibitors of the adherence of P. aeruginosa to mammalian cells. An adhesion model system was used, employing buccal epithelial cells in an enzyme-labelled immunoassay procedure on microtitration plates. Total levels of IgG, IgA, IgM and the four IgG subclasses were estimated in 11 CF patients and 10 healthy controls. Most of the CF patients demonstrated increased levels of all these immunoglobulin types. Sera from seven patients with elevated serum IgG were observed to cause greater inhibition of the adherence of P. aeruginosa to buccal cells than were the sera from four CF patients with low serum IgG and from ten healthy controls. Nevertheless, the levels of individual anti-P. aeruginosa IgG subclass antibodies varied amongst the patients and did not correlate with the degree of inhibition of bacterial adherence. Negative affinity chromatography was used to obtain antibody fractions enriched for IgG1, IgG2 or IgG4 and protein A-sepharose chromatography was used to isolate IgG3 antibodies from CF patients. The IgG1-, IgG2-, or IgG4- enriched fractions similarly inhibited the adherence of P. aeruginosa in the test system, whereas three of five IgG3-enriched fractions from CF patients had no greater effect on adhesion than did IgG from control individuals.

The IgG subclass response to the major outer-membrane proteins (OMPs) of Pseudomonas aeruginosa was investigated in 11 cystic fibrosis (CF) patients and 10 healthy controls. Inhibition of adhesion of P. aeruginosa to buccal epithelial cells by the IgG serum fractions from the CF patients has been established previously. The CF patients demonstrated marked heterogeneity in their individual IgG subclass response to pseudomonal OMPs. The predominant IgG1 and IgG4 responses were directed towards OMPs F, H2 and, with IgG1 only, to protein I. Proteins of 42 and 46 kDa primarily elicited an IgG2 response but some patients produced IgG4 antibodies. The IgG3 response varied from very weak in some patients to a strong reaction with proteins D2, E, G and I in others. The range of antigen-specific IgG subclass responses was similar in CF patients whose IgG fractions strongly inhibited the adherence of P. aeruginosa to epithelial cells and in those whose fractions gave only weak inhibition of adherence. There was no indication that an antibody response towards any particular OMP was implicated in the inhibition of bacterial adherence. Thus, the IgG subclass response to OMPs did not exert a significant effect on adherence when investigated in isolation, but may possibly play some role in combination with other processes.

Developments in molecular biology have offered a wide range of nucleic acid probes to detect the genome of hepatitis B virus (HBV). We have tested the ability of two enzyme-linked (alkaline phosphatase) probes to detect HBV-DNA. These hybridise with the S and C regions of the genome of HBV and are used to determine the clinical significance of detecting the two regions. A total of 66 serum samples from patients at different stages of HBV infection was examined. HBV-DNA was detected with at least one of the probes in 17 (85%) patients with HBeAg-positive chronic hepatitis, five (26.3%) with anti-HBe-positive chronic hepatitis and six (66.6%) with acute hepatitis. Although both probes were able to detect as little as 10 pg/ml (2.86 × 106 g. E./ml) of a full length HBV-DNA standard, the C-region-directed probe did not react in one patient with acute hepatitis, two with HBeAg-positive and three with anti-HBe-positive chronic hepatitis. When C-region-directed probes are used for diagnostic purposes, results should always be accompanied by hybridisation with probes directed against other regions showing less variability (e.g. S region).