- Volume 43, Issue 3, 1995

The effects of exotoxin A (EXA) from Pseudomonas aeruginosa on polymorphonuclear leucocytes (PMNLs) were studied in a mouse model and in vitro. P. aeruginosa PA103, which produced EXA, was 20 times more virulent for normal mice than was its EXA-deficient mutant, PA103-29. EXA was detected in the plasma of mice infected with P. aeruginosa PA103, and its presence correlated with increasing numbers of bacteria in the blood and internal organs. A monoclonal antibody (MAb) against EXA prevented the death of the mice if it was given simultaneously with, or 2 h before, infection with strain PA 103. The number of PMNLs in murine blood decreased by 50% within 30 min of intravenous injection of EXA, but this decrease was prevented by simultaneous or prior injection of MAb to the toxin. EXA inhibited in-vitro phagocytosis and killing of P. aeruginosa by human and murine PMNLs and decreased the number of the PMNLs by between 60 and 68%. Collectively, these results not only confirm that EXA. is toxic in vivo, but also suggest that this toxin accelerates the growth of virulent P. aeruginosa in mice.

An analysis by ELISA of 100 rheumatoid factor (RF)-positive sera selected at random from a collection of sera from patients with various auto-immune diseases and joint pains, and 100 RF-negative sera from the same collection matched by patient age and gender, showed that the RF-positive sera had highly significantly (p ≪ 0.0001) raised levels of IgM antibody, but not IgG antibody, to Proteus mirabilis over those of the RF-negative sera. This response was subsequently found to be associated with sera from patients who clinically had rheumatoid arthritis (RA). Sera from the RA patients had significantly greater amounts (p = 0.026) of IgM antibody to P. mirabilis than to the other organisms tested and these values were also highly significantly different (p ≪ 0.0001) from P. mirabilis IgM antibody levels in matched RF-negative sera. Sera from RA patients also had significantly greater amounts of IgA to P. mirabilis (p ≪ 0.0001) and greater amounts of IgM to Escherichia coli (p ≪ 0.0001) and Klebsiella pneumoniae (p ≪ 0.0001) than those in matched RF-negative sera. Other classes of antibody to these organisms and all classes of antibody to Pseudomonas aeruginosa were not raised in the sera of RA patients over those of RF-negative controls. The IgM response in RA patients was not specific for only one O serotype of P. mirabilis but was associated with all 11 different O serotypes of P. mirabilis tested and those of other Proteus spp. Moreover, the IgM antibodies to Proteus spp. appeared to be independent from C-reactive protein and RF. Fourteen of 27 sera from RA patients had an IgM antibody that reacted with an internal 90-kDa protein of P. mirabilis. This antibody was not found in RF-positive sera of patients with Sjogren’s disease or systemic lupus erythematosus, nor in other RF-negative sera, nor in those of healthy people, nor in those with osteoarthritis or ankylosing spondylitis. IgM antibodies capable of reacting with the haemolysin protein Hpm B of P. mirabilis were not found in sera of RA patients of unknown HLA status.

The cell wall and outer structures of Haemophilus ducreyi bacteria were investigated. The 24-kDa outer protein from two strains was purified with an SDS–PAGE preparative continuous-elution electrophoresis cell. The protein was further characterised by SDS–PAGE and immunoblotting, and the immunological properties were investigated by ELISA. Localisation on the bacterial surface was investigated by immuno-electron-microscopy with a polyclonal antiserum raised against the purified protein. A triple-laminar cell wall typical of gram-negative bacteria, close cellular contact between bacterial cells and outer blebs were seen on thin sections. An additional high mol. wt band of c. 165 kDa was seen when not treated by heating to 100°C. A high density fibrilla-like material was detected on the bacterial cell and in the environment by negative staining and immuno-electron-microscopy with antisera specific for the 24-kDa protein. The surface localisation of the 24-kDa protein was confirmed by an ELISA technique with the specific antiserum and whole bacterial cells as antigen. The presence of antibodies to the 24-kDa protein was demonstrated in antisera to 13 strains of H. ducreyi, indicating antigenic identity or within-species cross-reactivity. Low titres of antibodies to this protein were also detected in 19 antisera raised against different strains of gram-negative bacteria, indicating cross-reactivity with other species. Antibody response to the 24-kDa protein in rabbits immunised subcutaneously with live bacteria resulted in a secondary IgG response. Of 28 sera from patients with culture-verified chancroid, 26 manifested high titres of IgG antibodies to the 24-kDa protein, thus indicating the involvement of this antigen in the disease process in man.

Serotyping of Neisseria gonorrhoeae based on the differential recognition by a panel of six monoclonal antibodies (MAbs) against Protein IB (PIB) is a valuable tool for studying the epidemiology of gonorrhoea. However, the predominance of certain serovars in specific geographic regions necessitates the development of new MAbs or new genotyping approaches. Nucleotide and amino-acid sequence analysis of PIB from strains within the IB-3 and IB-7 serovars revealed strain differences within the same serovar. Based on the generation of PIB nucleotide and deduced amino-acid sequences that centred on amino-acid residues 196 and 237, eight serovar IB-3 strains and nine serovar IB-7 strains were separately subdivided into five groups. Intra-serovar differences were also established by pulsed-field gel electrophoresis (PFGE) of macro-restriction fragments generated by SpeI- and NheI-cleavage of genomic DNA. There was good correlation between the results based on PIB gene PCR-sequencing and those based on PFGE analysis of macro-restriction fragment patterns. These data demonstrate the high precision of PFGE analysis and indicate that this approach can be used as a rapid epidemiological subtyping system for major serovars of N. gonorrhoeae.

Neisseria gonorrhoeae isolates that require arginine—i.e., either citrulline (C), or ornithine (O)—uracil (U) and hypoxanthine (H) have generally been considered to be similar when characterised by auxotype, serovar and plasmid content. The MICs of penicillin, tetracycline, erythromycin, spectinomycin, cefoxitin and ceftriaxone for 552 isolates belonging to serovar IA-2 with these phenotypes were found to be similar. Therefore, restriction fragment length polymorphism analysis of rRNA genes (ribotyping), restriction enzyme (RE) analysis of chromosomal DNA, and pulsed-field gel electrophoresis (PFGE) were evaluated to determine whether these isolates could be distinguished by molecular methods. A subset of 27 isolates of N. gonorrhoeae that were OUH-requiring, CUH-requiring or OH-requiring, belonged to serovar IA-2 and carried a 2.6-MDa plasmid, were selected for further study. Based on the RE analysis of SwaI-digested genomic DNA, the 27 isolates fell into a single RE pattern, five ribotypes and 17 PFGE profiles which did not correlate with the specific arginine-requiring subtypes of these isolates. Each ribotype varied by the presence of only a single fragment, which was of a different size in each pattern, and 17 (63%) of the 27 isolates belonged to ribotype I. PFGE yielded the highest level of discrimination with 17 different profiles.

A total of 148 Vibrio cholerae isolates from a major shrimp production area in Southern Thailand were examined by colony hybridisation for genes encoding heat-stable enterotoxin (NAG-ST) and cholera toxin (CT). Only non-O1 V. cholerae strains were found to harbour NAG-ST (14 of 146) whereas no strains hybridised with the CT probe. NAG-ST-positive V. cholerae non-O1 strains were isolated from shrimp farms situated close to urban areas. Five different O serogroups were found among NAG-ST positive non-O1 strains. Southern blot and restriction endonuclease analysis of NAG-ST-positive strains revealed a high degree of genetic divergence. A total of seven classes of enterotoxin gene patterns were found with HindIII and EcoRI restriction endonucleases. Enterotoxin gene patterns correlated with O-antigen expression in 84% of isolates tested. In combination with other molecular techniques Southern blot analysis with an NAG-ST oligonucleotide probe could be useful for studying the molecular epidemiology of V. cholerae non-O1 strains.

The 4-methylumbelliferyl-N-acetyl-β-d-galactosaminide (UAG) test was evaluated in parallel with chlamydospore (CHL) and germ-tube (GT) tests for the identification of Candida spp. The UAG test gave 86.6% correct identification of C. albicans and C. tropicalis. Non-C albicans and non-C. tropicalis yeasts gave correct negative results in UAG tests. Only one isolate of Trichosporon beigelii gave a “false” positive reaction. The UAG test, which can be completed within 30 min, is a reliable test for screening non-C. albicans and non-C. tropicalis yeasts.