- Volume 25, Issue 3, 1988

Yersinia pseudotuberculosis was examined for its haemagglutinating activity and adherence to cultured epithelial cells (HEp-2) in relation to possession of a virulence (VW) plasmid and to growth conditions. VW-lacking (VW–) bacteria were isolated from ten VW+ strains of each serovar which, after they were grown on CFA plates at 37°C, agglutinated the erythrocytes from five different species. In contrast to the bacteria possessing the plasmid (VW+) half of the VW– bacteria, grown on CFA plates at 37°C, did not agglutinate any of the erythrocytes used and the other half agglutinated only human erythrocytes. Furthermore, when grown on CFA plates at 25°C, neither VW+ nor VW– bacteria showed a haemagglutinating activity. When the bacteria were grown in CFA broth, only two strains grown at 25°C did not agglutinate any of the erythrocytes tested. The VW+ and VW– bacteria of the remaining strains, grown either at 25°C or 37°C, showed relatively high haemagglutinating activity. Adherence to HEp-2 cells did not correlate with haemagglutinating activity in Y. pseudotuberculosis; the VW+ bacteria grown at 37°C adhered to HEp-2 cells more efficiently than either the VW+ derivatives or the VW+ bacteria grown at 25°C, regardless of the growth medium. These results indicate that some of the haemagglutinins detected on Y. pseudotuberculosis are not involved in the adherence to HEp-2 cells.

Because of the central rôle postulated for Pertussis Toxin in the pathogenesis of whooping cough, and the well-established ability of this toxin to alter insulin and glucose levels in animal blood, a study of insulin and glucose levels in hospitalised pertussis patients and in controls was made. With blood specimens collected in heparin-fluoride anticoagulant, the geometric mean plasma-insulin level (13·3 μU/ml) in a series of 24 pertussis patients was slightly, but statistically significantly, higher than that (8·9 μU/ml) in a series of 27 non-pertussis controls with other infectious diseases (p < 0·02). Portions of the same blood specimens collected in lithium-heparin anticoagulant yielded higher mean plasma-insulin values of 15·5 and 11·4 μU/ml respectively, with no significant difference between them (p > 0.05). Mean plasma glucose concentrations were not significantly different between the two groups, and hypoglycaemia was not detected in any pertussis patient. There were no statistically significant differences between pertussis and control children in the mean levels of plasma calcium, magnesium or phosphate.

Two hybridoma cell lines that produce monoclonal antibodies against Aeromonas hydrophila haemolysin were established by fusion of myeloma and spleen cells obtained from a mouse immunised with haemolysin detoxified with tetranitro-methane. Enzyme-linked immunosorbent assay (ELISA) showed that the two purified monoclonal antibodies, B7 and Bl 1, recognised the same epitope on the haemolysin molecule. Antibody B7 neutralised the haemolytic and enterotoxic activities of the haemolysin. It is concluded that the same site on the haemolysin molecule is responsible for both haemolytic and enterotoxic activities.

For many organisms, mucosal association is an important virulence determinant. Although studied in detail for other intestinal pathogens, this aspect of pathogenicity has not been studied for Clostridium difficile. We compared the ability of an avirulent non-toxigenic strain (M-1), a highly virulent toxigenic strain (B-1), and a poorly virulent toxigenic strain (BAT) of C. difficile to adhere to different regions of the gastrointestinal tract of hamsters pre-treated with clindamycin. Strain B-1 associated with the gut mucosa significantly better than strain M-1 (p <0·001) for all sites other than the caecum, and achieved significantly higher levels in the caecal contents (p<0·001). The same was true when strain B-1 was compared with strain BAT except that there was no significant difference for the large bowel mucosa. To assess the possible role of toxin in promoting mucosal association, e.g., by compromising host defences or exposing masked adherence sites, strain M-1 was given to animals after intra-caecal administration of crude toxin preparations from strain B-1, which were heat-inactivated in control experiments. The addition of this toxin increased significantly the mucosal association of M-1 for the small bowel only, whereas the inactivated toxin had no significant effect. These results imply that there may be intrinsic differences between strains in their ability to colonise and associate with the gut mucosa, which may partly depend on their ability to produce toxin. These differences do not correlate with cell-surface hydrophobicity or the presence of plasmids, flagella or fimbriae.

A high degree of non-specific resistance to a lethal systemic Escherichia coli infection was induced in mice by pretreatment with a small dose (< 5 μg/mouse) of the homologous lipopolysaccharide (LPS) or with heterologous rough-type LPS from E. coli K-12. The route of LPS administration, intraperitoneally or subcutaneously, did not influence the development of resistance, suggesting that a systemic cell activation was responsible for the effect. The enhanced elimination of bacteria was similar to that in mice recovering from a sublethal E. coli infection. In the LPS-treated mice, elimination of the challenge bacteria from the peritoneal cavity and the blood started 3–4 h after challenge whereas, in controls, the bacterial numbers continued to increase until the mice died. The detoxified LPS derivative, monophosphoryl lipid A (MPL), also increased the survival of mice infected with E. coli O18 :K1. However, the dose of MPL required for optimal infection resistance was 100-fold greater than that of native, E. coli K-12 LPS, corresponding to the 100-fold reduced toxicity of MPL for mice and rabbits in lethality and pyrogenicity assays.

Mice surviving a sublethal E. coli O18:K1 infection possess a greatly increased resistance to a subsequent lethal E. coli O18:K1 peritonitis. A similar increase in resistance can also be achieved by LPS pretreatment. The early course of the infection in the convalescent mice at day 1 was identical to that in LPS-pretreated mice. At this time, the convalescent mice were also able to restrict the growth of the heterologous E. coli O78(ColV) strain, suggesting that non-specific phagocyte activation was responsible for the increased destruction of the bacteria. At day 4, the kinetics of infection in convalescent mice were identical to those in mice passively immunised with specific anti-K1 capsule antiserum. A rapid decline in the numbers of viable homologous, but not heterologous, bacteria took place in the peritoneal cavity suggesting effective antibody-mediated opsonisation followed by phagocytosis and killing of the bacteria.

Normal human plasma and serum were found to inhibit the growth of Torulopsis glabrata and, to a lesser extent, other yeasts. The factor responsible for the inhibition of T. glabrata was not dialysable, was heat stable at 56°C for up to 4 h and could be partly removed by absorption with viable T. glabrata but not Candida albicans. It was fungistatic at low concentrations and fungicidal at high concentrations, stable up to 4 years between –20°C and –70°C, but for only a few weeks at 4°C. Studies with Cohn fractions of serum showed that the inhibitory components were in either the alpha or beta globulin fraction or both. The combined effects of transferrin and IgM accounted for about 70% of the total inhibition observed. We were unable to identify the component responsible for the residual inhibition of growth. The inhibitory effect was totally neutralised by tetracyclines, quinolones, sulphamethoxazole and by very low concentrations of polyenes, imidazoles and 5-fluorocytosine.

Cultures of Escherichia coli carrying Coll plasmids received in conjugation from strains of Salmonella typhimurium and S. agona were examined for abortive infection (Abi) of phage BF23 and for enhanced resistance to the lethal action of UV-irradiation (Uvr). The Abi character of stored cultures of E. coli was also compared with the reaction of the same stock culture tested 5 years before. Seven of the eight potential types differentiated by three characters were represented among 160 Coll plasmids: Colla Abi+ Uvr+ (3 plasmids), Colla Abi– Uvr+ (1), Colla Abi– Uvr– (2), Collb Abi+ Uvr+ (85), Collb Abi+ Uvr– (5), Collb Abi– Uvr+ (4), Collb Abi– Uvr– (60). Recognition that different plasmid types could be carried by strains of a clone proved useful in the interpretation of the epidemic spread of strains of S. typhimurium of phage type/biotype 141/9f in Scotland and in tracing the ancestry of a recently emerged rhamnose non-fermenting mutant strain of S. agona.

The activities of β-lactam antibiotics were compared against Enterobacter cloacae clinical isolates and mutants which had inducible, stably-derepressed, and basal expression of a pI 8·4 subtype of the Ia chromosomal β-lactamase. These activities were correlated with the results of studies of the β-lactamase-lability and β-lactamase-inducer-power of the antibiotics. Cefoxitin and ampicillin were labile, and induced β-lactamase production strongly at concentrations below their MIC values. Consequently, β-lactamase-inducible and β-lactamase-stably-derepressed organisms were highly resistant (MIC <256 mg/L) to these antibiotics, whereas enzyme-basal strains and mutants were much more susceptible (MIC 1–16 mg/L). Imipenem also induced β-lactamase production strongly at concentrations below its MIC, but was more stable than ampicillin and cefoxitin. It was active against enzyme-inducible and stably-derepressed organisms at 0·25–0·5 mg/L and against β-lactamase-basal organisms at 0·06–0·25 mg/L. Thus the β-lactamase afforded only very low-level protection against imipenem ; this appeared to be by a non-hydrolytic mechanism, with the enzyme binding to the antibiotic in a relatively stable complex. This complex, which probably was an intermediate in a hydrolytic pathway, was isolated by gelfiltration chromatography and shown to have a breakdown half-life of 47 ± 2 min. Cefotaxime, ceftriaxone and mezlocillin were labile to the pI 8·4 β-lactamase but induced β-lactamase production weakly at concentrations below their MIC values. Consequently, β-lactamase-inducible and β-lactamase-basal organisms remained equally susceptible (MIC 0·06–4 mg/L), but stably-derepressed organisms were considerably more resistant (MIC>64 mg/L) to these antibiotics.