- Volume 32, Issue 1, 1990

A diverse range of heterotrophic bacteria was screened for the presence of carbonic anhydrase (CA) activity, sensitivity to inhibition of growth by acetazolamide (CA inhibitor), and the presence of protein binding monospecific antibody prepared against purified Neisseria sicca CA. CA activity was demonstrated only in strains of N. sicca and N. gonorrhoeae. However, all Neisseria strains, including various isolates of N. meningitidis and N. lactamica, were sensitive to acetazolamide, when grown in air, and showed serological cross-reaction with N. sicca CA. Strains of other genera were resistant to acetazolamide. A number of strains including members of the genera Pseudomonas, Staphylococcus, Streptococcus, Serratia and Proteus also strongly expressed a gene product(s) immunologically related to CA. The presence of CA cross-reacting proteins, which lack hydrase activity, is discussed in relation to the function of the various mammalian CA isoenzymes.

The adherence of clinical isolates of Staphylococcus aureus to cultured mouse epidermal cells was studied. Adherence of the isolates to the cells varied from strain to strain. When epidermal cell differentiation was induced by raising the calcium concentration in the medium, three out of 10 strains tested adhered better to calcium-induced differentiated cells than to undifferentiated cells, and one strain demonstrated higher adherence to undifferentiated cells than to differentiated cells. No significant difference between the adherences to both types of epidermal cells was observed with the other six strains. No relationship was observed between adherence and surface hydrophobicity with bacterial cells. Lipoteichoic acid and N-acetyl sugars caused limited inhibition of adherence. The adherence assay method employed in this study is useful for investigating the effects of epidermal cell differentiation on bacterial adherence in vitro.

The effect of the O2-dependent antimicrobial systems of the human neutrophil on the intraphagocytic activity of difloxacin, ciprofloxacin, pefloxacin and fleroxacin was determined by use of a radioassay with Staphylococcus aureus as the test organism. The fluoroquinolones exhibited good intraphagocytic activity with normal neutrophils. However the intracellular bioactivities of the four antimicrobial agents were substantially less in tests with neutrophils from two patients with chronic granulomatous disease. These observations suggest a synergic interaction between fluoroquinolones and the O2-dependent antimicrobial systems of phagocytes in the eradication of intracellular microbial pathogens.

The opsonic requirement for phagocytosis and killing and cell-surface hydrophobicity of five strains of Staphylococcus saprophyticus isolated from clinical sources were studied. Phagocytosis and killing of bacteria by human granulocytes were measured in suspension. Bacterial aggregating cell-surface hydrophobicity was determined by salt aggregation, and the absorptive hydrophobicity was measured by hydrophobic interaction chromatography. All strains were well opsonised by pooled normal human serum 10%. Ingestion of these bacteria could be detected to a variable extent in the absence of extracellular opsonins; heat-inactivated serum 10% or intravenous IgG concentrate 1 mg/ml improved phagocytosis of all strains. Significantly increased rates of both the ingestion and killing of one of the five strains occurred in the presence of IgG or in the absence of opsonins, compared to those found with each of the other four. This particular strain had significantly stronger adsorptive surface hydrophobicity than the other four strains, and with all strains there was a correlation between hydrophobicity and phagocytosis by granulocytes in the absence of opsonins.

A DNA-based assay has been developed for the detection of Toxoplasma gondii. The assay makes use of the polymerase chain reaction (PCR) to amplify part of the P30 gene on the parasite’s DNA. Following gel electrophoresis, the amplified DNA can be detected either directly on the gel or by Southern hybridisation with radioactive or non-radioactive DNA probes. The assay has been used to detect the DNA from different isolates of T. gondii in a background of human or mouse DNA. Together with other information such as clinical data, CT scans and serology, the PCR assay should improve the diagnosis of toxoplasmosis in immunosuppressed and immunocompromised patients as well as in fetal tissues.

The optimum rabbit ileal loop (RIL) reacting doses of the new cholera toxin (NCT) produced by cholera toxin gene-negative (CT-) strain X-392 and of the enterotoxin produced by cholera toxin gene-positive (CT+) strain 569B of Vibrio cholerae Ol were found to be 32 μg and 22 μg respectively. Production of NCT by the CT+ strain, in addition to CT, was confirmed by in-vivo neutralisation tests. Anti-569B-enterotoxin neutralised the optimum RIL reacting activity of NCT completely at 1 in 16 dilution, whereas the activity of 569B enterotoxin was only partially neutralised (44%) by anti-NCT. Similarly, partial neutralisation (66%) was observed when purified anti-CT was mixed with 569B enterotoxin. Therefore, the fluid accumulation produced in the RIL by 569B enterotoxin was the combined effect of both CT and NCT. No antigenic relationship between NCT and CT could be demonstrated in gel-diffusion tests.

The mode of action of Vibrio vulnificus haemolysin (VVH) on mast cells from the peritoneal cavity of the rat was examined. VVH induced histamine release, and damage to the mast cells, in a dose-dependent fashion. When 1 μg of VVH was added to c. 105 mast cells at 37°C, histamine release was observed after a lag period of 5-10 s, and was complete within 5 min. The action was temperature-dependent, and was not induced at 4°C. Disodium cromoglycate, a membrane stabiliser for mast cells, inhibited the histamine release significantly, but the effect was not dose-dependent. Moreover, leakage of lactate dehydrogenase from VVH-treated mast cells was observed. These results suggest that VVH acts on the cell membrane of mast cells and is cytolytic.

The prevalence of Aeromonas spp. and Plesiomonas shigelloides was determined in patients attending the enteric laboratory of the Department of Medical Microbiology and Parasitology, Lagos University Teaching Hospital, Nigeria. During the 12-month study (October 1986-September 1987), Aeromonas spp. were isolated from 53 (2.26%) of 2350 patients with diarrhoea and only 2 (0.4%) of 500 patients without diarrhoea (p < 0.01). Similarly, P. shigelloides was isolated from 16 (0.68%) patients with diarrhoea and none of the controls (p > 0.05). The seasonality, age and sex distribution of diarrhoea associated with Aeromonas spp. and P. shigelloides in this study were similar to those of diarrhoea associated with other recognised enteropathogens in Nigeria. Both species may play a role in the aetiology of acute diarrhoeal disease in that environment.

The effects of incorporating ampicillin, some bile salts and sugars into media on the primary recognition and selection of aeromonads from faeces were examined. Most (88%) of the 101 Aeromonas strains examined had an ampicillin MIC of ≥40 mg/L, and would be isolated on blood agar containing ampicillin 30 mg/L. The strains with an ampicillin MIC of < 40mg/L were all of human origin and predominantly A. caviae. Although ampicillin at 10, 20 or 30 mg/L in blood agar inhibited faecal bacteria, the ability to detect Aeromonas strains with a high ampicillin MIC was less when the lower concentrations of ampicillin were used, without any improvement in the isolation of those strains with a low ampicillin MIC. Thirty-seven strains were tested for sensitivity to several different bile salts and found to be at least as resistant to them as Escherichia coli NCTC 10418. Bile salt sensitivity was not related to the species or source of a strain. There were minor differences in sensitivity to bile salts between some strains which related to whether strains had been isolated originally in the presence of bile salt or not. The effects of the presence of E. coli, Klebsiella spp. and Enterococcus faecalis on the growth of Aeromonas strains in mixed culture on media with and without carbohydrate were examined. The colony size of some Aeromonas strains was reduced in mixed culture but colony counts were not affected with any Aeromonas strains. The effect of carbohydrate in the medium on Aeromonas colonies was apparent in the presence of other bacteria that fermented carbohydrate, when the non-fermenting Aeromonas colonies were often indistinguishable from those of the fermenters. When xylose was added to the medium the recognition of colonies of the non-fermenting aeromonads among those of other fermenting bacteria was difficult because their colonies were often of the same colour. This may explain the difficulty of isolating aeromonads from mixed cultures on some carbohydrate-containing media. It is recommended that if aeromonads are to be recognised in selective culture from faeces by their failure to ferment carbohydrate, a medium be used which inhibits the growth of non-aeromonad bacteria as much as possible.

Outer membranes were prepared by the Sarkosy1 method from 30 strains of Pasteurella multocida and the closely related Taxon 13, which had been isolated from cattle. The patterns of the outer membrane proteins (OMPs) on SDS-PAGE were generally similar to one another, though the four major proteins (a-d) varied somewhat in molecular mass; these patterns allowed the strains to be arranged into 12 groups. Taxon 13 strains and typical P. multocida strains were indistinguishable, both types being found within the same group. Mice were vaccinated with heat-killed bacteria of three strains and challenged with 10 LD50 of homologous and heterologous live bacteria, representing groups based on OMP patterns; the best protection was afforded by strain W674, which protected against nine of the 17 challenge strains; but there was no correlation between protection and PAGE pattern. Pre-vaccination and pre-challenge sera were used in immunoblotting to probe OMPs from protective and non-protective strains. All three vaccines produced antibody to proteins a and d; these proteins appeared to be common to all strains, varying in molecular mass but not in overall antigenic expression. The antibody response to the other two major OMPs appeared to be PAGE-group specific. There was no correlation between protection and the antigen pattern seen by immunoblotting.

When the three serotypes of Bordetella pertussis (types 1,2,3; 1,2 and 1,3) were labelled with agglutinins and protein-A gold, agglutinogen 1 was found on fimbriae and on the cell surface of types 1,2,3 and 1,2 but on the cell surface only of non-fimbriate type 1,3 organisms. In contrast, agglutinogen 2 was located on fimbriae only. Agglutinogen 3 was not labelled. When protein-A gold was replaced by immunoglobulin-G gold, agglutinogen 3 was found on the cell surface only, even of fimbriate bacteria of type 1,2,3. The implications of these findings for acellular vaccines are discussed.