- Volume 23, Issue 1, 1987

Alkali-treated lipopolysaccharides (LPS) from Ogawa and Inaba serotypes of Vibrio cholerae were chemically coupied to cell-surface proteins of V. cholerae. The reaction product was eluted in the void volume when fractionated on a column of Sephacryl S-300. The material did not enter the gel when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). The bivalent protein-polysaccharide conjugate was nonpyrogenic, as determined by the Limulus lysate assay. It was immunogenic and elicited, in rabbits, antibodies against both intact LPS and cell surface proteins, as determined by enzyme linked immunosorbent assay. LPS from Ogawa serotype was resolved into two major bands by SDS-PAGE and that from the Inaba serotype into one major band. Immunoblotting studies indicated that antisera to the protein-polysaccharide conjugate contained antibodies to the major LPS fractions from both serotypes. Antisera to the protein-polysaccharide conjugate tested by crossed-immunoelectrophoresis produced immunoprecipitation with whole-cell sonicates of both biotypes and serotypes of V. cholerae. Such antisera also possessed agglutinating and complement-mediated bactericidal activities towards V. cholerae strains of both biotypes and serotypes. These results suggest that a bivalent cell-surface protein-polysaccharide conjugate of V. cholerae could be developed as a nonpyrogenic vaccine against cholera.

Oral inoculation of neonatal MFI mice with enterotoxigenic strains of Escherichia coli that possessed the K99 or F41 antigen or both resulted in severe diarrhoea with high mortality. The diarrhoea was associated with increased fluid in the gut, greatly increased numbers of E. coli in gut homogenates and reduced weight gain compared to control animals. Further studies with strain B44 demonstrated greatly increased numbers of E. coli on the surface of the intestinal mucosa and haemo-concentration. The infection was transmissible between litter-mates. There was no evidence of invasion of the intestinal tissue of infected animals. Gnotobiotic Balb C mice and endotoxin-resistant mice were susceptible to oral inoculation with bovine enterotoxigenic E. coli strains, but neonatal rats were not susceptible to infection with enterotoxigenic E. coli strains B44 or 431.

Porcine strains of E. coli that possessed K88 or 987P antigen did not infect neonatal MFI mice but an “atypical” porcine strain (431) which possessed both K99 and F41 antigens caused diarrhoea and a high mortality. The disease in neonatal mice resembled acute diarrhoea caused by these bacteria in other species, particularly the calf, and the model should be of value in assessing the efficacy of therapeutic agents.

The faecal microbial flora of a patient with severe irritable bowel syndrome related to multiple food intolerances was very variable and contained a high proportion of facultative bacteria and an unusual incidence of Clostridium species.

A typing scheme for Morganella morgani based on bacteriocin (morganocin) production and sensitivity is described. These characteristics were determined by testing 160 strains in all combinations and permitted their differentiation into 90 types. Morganocin production was induced with mitomycin C and morganocin sensitivity determined with a diluted inoculum on Lab-lemco agar at 30°C. Most strains (82.5%) produced morganocins and 49 different types were defined. Most strains (97.5%) were sensitive to morganocins and usually to several different types. The scheme is more discriminating than other reported methods. The finding in an epidemiological survey of the carriage of certain strains in the bowel for several weeks suggests that in practice the method is stable and reproducible.

Whole-cell-polypeptide profiles obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were used in conjunction with the API-Staph® technique to identify different strains of Staphylococcus aureus, S. epidermidis, S. saprophyticus and S. capitis. Complete concordance of results from both techniques was achieved with all strains examined. Visual analysis of the polypeptide patterns and comparison by use of the coefficient of Dice showed minor differences in band pattern between strains of the same species but each species produced a pattern distinguishable from that of any other. The results suggest that although SDS-PAGE can be used to identify staphylococcal species, this type of analysis will not readily provide the basis for a typing method.

Thirty Streptococcus faecalis isolates from mixed dental plaque samples were classified into four groups on the basis of biotype, tetracycline susceptibility, phage type and serotype combinations. The organisms were from patients on haemodialysis, from staff of the dialysis unit, and from controls. Three biotypes were distinguished by seven biochemical tests: production of acid from inositol, sucrose and xylose; rapid or delayed production of acid from sorbitol; gelatin liquefaction; and production of alkaline phosphatase and β-galactosidase. With a set of eight typing antisera for S. faecalis, 15 strains were non-typable, 12 were serotype 1 and three were serotype 19. With a set of 17 bacteriophages specific for S. faecalis, all of the oral isolates were typable; 40% were lysotype I1 and the remainder lysotype V6b. On the basis of biotype-serotype-phage-type combinations, indications of possible spread of strains between haemodialysis patients and dialysis unit staff were obtained. Biotyping and serotyping of 13 German isolates of S. faecalis of phage type I1 from four clinical sources and tripartite typing of three control strains provided additional evidence for the potential of biotyping in distinguishing between strains of identical serotype and phage type. One oral isolate of S. faecium was of phage type XX. None of the oral isolates of S. faecalis, of which 14 exhibited delayed sorbitol fermentation, reacted with group-G streptococcal grouping reagents or antiserum. Slow sorbitol fermentation does not appear to be a definitive phenotypic marker for S. faecalis strains possessing antigens that react with both group-D and group-G grouping reagents.

Chewing sticks are widely used in Nigeria for dental and oral hygiene. Invitro susceptibility tests were done with crude extracts from nine popular sticks on four species of Bacteroides. Serindeia warneckei chewing stick had the greatest and most consistent inhibitory effect on the four species; extracts from bark and pulp were bactericidal at concentrations of ≤1%. Extracts of other sticks, when inhibitory, were only so at higher concentrations—in the range 2–30%. All the black-pigmented oral anaerobes were very susceptible to eight of the nine chewing-stick extracts but non-pigmented anaerobes showed variable susceptibilities.

The effect of capsulation on the ability of Bacteroides fragilis, B. asaccharolyticus and anaerobic gram-positive cocci to induce bacteraemia and seeding to various organs was investigated. The test species were injected into mice subcutaneously alone, or mixed with other aerobic or facultative organisms. Capsulate anaerobes were isolated more frequently from the blood, spleen, liver, and kidneys of infected animals than were non-capsulate organisms. After injection of single anaerobic strains, capsulate organisms were recovered from 163 (39%) of 420 animals; non-capsulate anaerobes were recovered from only 14 (3%) of 420 animals. After injection of B. fragilis mixed with aerobic or facultative organisms, the capsulate B. fragilis strain was isolated more often and for longer periods than the non-capsulate strain. Capsulate B. fragilis was also recovered more often 5 days after injection with other organisms, than when injected alone. These data demonstrate that capsulate Bacteroides spp. and anaerobic gram-positive cocci are more virulent than non-capsulate strains in single and mixed infections.

The adherence of Fusobacterium necrophorum to the surface of bovine ruminal epithelial cells was paralleled by the organism’s haemagglutinating ability. Treatment of the bacterial cells with haemagglutinin antiserum caused a reduction in the degree of attachment. The purified haemagglutinin became bound to the membranes of ruminal epithelial cells but lost its adherence when pre-incubated with haemagglutinin antiserum. These findings suggest that the adherence of F. necrophorum to the membrane of the ruminal cells is mediated by haemagglutinin.

Under uniform experimental conditions, different degrees of selective attachment of Staphylococcus aureus and Candida albicans to epithelial cells of the labium majus, the labium minus, and the vagina were compared and contrasted with those found in studies with cells of the buccal and nasal mucosa and forearm skin by a novel analysis of adherence density. For both micro-organisms, the larger, rougher cells of the labium majus gave the highest adherence scores matched only by the interaction of S. aureus with fully keratinised nasal epithelial cells. Increasing acidity to pH 3.5 enhanced microbial adherence to vaginal cells. Menstruation also influenced attachment; highest densities were reached between the third and fourth weeks of the cycle. Autogenous ribitol teichoic acid was found to block the attachment of S. aureus to labium majus and labium minus cells by 76% and 81% respectively, and to vaginal cells by 66%. Adherence is considered to be an important attribute of vulvar ecology and may be a determinant of infectious disease.

The action of ciprofloxacin and norfloxacin on two strains of Escherichia coli was studied by diverse methods including electronmicroscopy, viable counting and continuous turbidimetric monitoring. During the first few hours of exposure to inhibitory concentrations of the drugs, the opacity of bacterial cultures continued to increase for a period that was inversely proportional to the drug concentration. This change corresponded to the appearance of filamentous bacteria, swollen forms and some lysis. There was subsequently a gradual drop in opacity during which extensive lysis occurred. As judged by viable counts of bacteria washed free of drug, cell death occurred within 30 min of first exposure to the drugs and continued over a 3-h period. Ultrastructure studies demonstrated that lysis was preceded by the formation of vacuoles, predominantly at the poles of the cells. At these sites, breaks in the cell walls eventually occurred, resulting in extrusion of the cytoplasmic contents.