- Volume 30, Issue 1, 1989

Six isolates of Pseudomonas cepacia, representing various serotypes of the organism and possessing similar degrees of virulence in mice, were examined for their production of an extracellular toxic complex (ETC) in vitro. This compound is lethal for mice and produces extensive lung pathology in rats; it is composed of a surface carbohydrate antigen, lipopolysaccharide and protein. All six isolates produced the ETC. The LD50 values for the six ETC preparations ranged from 395 μg for strain 61g to 1750 μg for strain 90ee. Only two of the six ETC preparations contained ketodeoxyoctonate detectable by the methods used, and these two were the most toxic. Rabbit antiserum to the ETC of a serotype D strain could significantly protect mice only against serotype D strains. Examination of the various phases of growth of P. cepacia showed that there was extracellular release of the ETC beginning in the early logarithmic phase and continuing through the late stationary phase. The presence of the ETC in the supernatant fluids was due to release of this material rather than to cell lysis. In addition, at least one strain of P. cepacia was shown to produce an alginic acid-like compound.

Sera from 29 children and six adults were used to investigate the nature of antigenic cross-reactivity between Neisseria polysaccharea, N. lactamica and N. meningitidis B, 15P1.16 by immunoblotting. Major common antigens of 68-70 Kda, 60-65 Kda and 15-20 Kda were detected. Antibody directed against them uniformly decreased after absorption of the sera with the three different Neisseria species. Antigens of 55 Kda and 35 Kda specific to N. meningitidis, and one of 43 Kda specific to N. lactamica, were also demonstrated. Antibody against all antigens was more prevalent in bactericidal than in non-bactericidal sera, although these differences were statistically not significant. Differences in antibody prevalence between carriers of Neisseria spp. and non-carriers of these organisms were even less marked. Examination of sera by whole-cell enzyme-linked immunosorbent assay against N. meningitidis B, 15P1.16 and N. lactamica gave an absorbance ratio of 1:1. Only four sera from children showed no reactivity against the meningococcal strain. These common antigens are likely to be important in vaccine development.

Five strains of mice (C3H, CBA, BALB/c, TO and ICR) were inoculated intra-vaginally with Neisseria gonorrhoeae in an attempt to produce an animal model of gonorrhoea. Of a total of 68 mice inoculated, only three (4.4%) were culture-positive after 3 days. Histological examination of both the genital mucosa of inoculated animals, and the mucosa of genital tract organ cultures inoculated in vitro failed to show any evidence of gonococcal adherence or colonisation. Mice of these strains, therefore, appear resistant to gonococcal infection of the genital tract.

All smooth strains of Brucella bear two lipopolysaccharide (LPS) antigens in a ratio that defines the classification of strains in serovars, A (A≥M), M (M>A) and A.M (A = M). Anti-LPS-A monoclonal antibodies (MAb-A) were previously shown to convey protection to mice against B. abortus (A) strain 544, as shown by lower spleen counts than in controls at days 7 and 21 after challenge. Anti-LPS-M monoclonal antibodies (MAb-M) were obtained and tested for M-specificity with LPS from reference strains by ELISA, by agglutination of LPS-coated latex particles, and by inhibition of this agglutination. Antigens A and M of three strains were quantified by a homologous LPS-latex and MAb agglutination inhibition assay. Protection conferred by MAb-A and MAb-M against three strains, B. abortus 544 (A), B. abortus 292 (M) and B. melitensis H38 (M), was tested at equivalent challenge and MAb doses: intravenous challenge was adjusted to give similar infection at day 7;MAb doses were adjusted to the same specific ELISA titre. Under these conditions, MAb-A and MAb-M conferred both early and late protection, as shown at days 7 and 21, against the strains that bore the homologous major antigen, i.e., strain 544 on one hand and strains H38 and 292 on the other. In contrast, MAb directed against the minor antigen of the challenge strain conferred significant protection at day 7 only with strains 544 and H38 and no or inconsistent protection against strain 292, which expressed the lowest amount of minor antigen. Thus, early and late antibody-mediated immune mechanisms depend on amounts of surface LPS antigens accessible to specific antibodies. Therefore, to protect against the various strains of Brucella, an LPS-based vaccine should induce high litres of specific antibodies against both A and M antigens.

The novel lipopeptide antibiotic, daptomycin, at a concentration of 8 mg/L, caused gross morphological changes in both a methicillin-sensitive and a methicillin-resistant strain of Staphylococcus aureus and in a strain of Enterococcus faecalis. The earliest (after 1 h) surface lesion observed was the appearance of boss-like processes randomly distributed on the cell surface. Later, grossly deformed bacteria were seen and in two of the three bacteria prolonged exposure led to degeneration of the cells into an amorphous syncytial mass. Omission of calcium (which is known to potentiate the activity of daptomycin) from the culture medium did not affect the morphological response to an inhibitory concentration of the antibiotic.

Sixteen methicillin-resistant Staphylococcus aureus (MRSA) isolates, from a single nosocomial outbreak, were tested for molecular and phenotypic relationships. Two of the 16 outbreak strains were gentamicin resistant (Gmr) and the plasmids that they carried were characterised by reverse field electrophoresis, restriction endonuclease analysis and gene hybridisation. The gentamicin-resistant (Gmr) strains harboured two plasmids, a Gmr plasmid of 36.5 kb and a cryptic plasmid of 25.4 kb, whereas the other 14 isolates contained only the cryptic plasmid. Gentamicin resistance was encoded by a 2.5-kb HindIII fragment of the 32.8-kb plasmid and is similar to the 2.5-kb HindIII fragment also described for S. aureus Gmr plasmids from Australia and the USA. The Gmr plasmid was non-conjugal and was cured by ethidium bromide at a frequency of 4%. Two MRSA strains isolated subsequently from the same hospital were also Gmr and had identical plasmid and restriction endonuclease profiles to the two Gmr strains studied initially. Two other S. aureus isolates from the original carrier detected in this study and from his son were methicillin and gentamicin susceptible and had novel profiles. Since large plasmids show anomalous migration in agarose gels, more definitive analyses than simple plasmid identification should be considered when studying nosocomial outbreaks.

Strains of Salmonella of serotype Sendai, producing a mannose-resistant and eluting haemagglutinin (MREHA) when cultured at 37°C but not at 18°C, were examined by electronmicroscopy after negative staining. Production of this MREHA, previously thought to be nonfimbrial, was correlated with the presence of thick fimbriae with an external diameter of 13.6nm. These fimbriae were readily fragmented and, when purified, had an estimated Mr of 28 Kda. Production of fimbrial MREHA by Sendai strains was associated with the ability to adhere to a wide range of substrates and to form a fimbrial pellicle at the surface of liquid media incubated statically in air. The origin of this unusual Sendai fimbrial MREHA is unknown. Thin filamentous structures produced independently of fimbrial MREHA by Sendai strains were also described. Fimbrial MREHA was not produced by strains of the antigenically similar serotype Miami which, however, and unlike Sendai strains, formed mannose-sensitive haemagglutinin and type-1 fimbriae. The ability to differentiate strains of Miami and Sendai (serotype 1, 9, 12:a:1, 5) by means of their fimbriae is noted.

Highly purified toxin A of Clostridium difficile was obtained by bovine thyroglobulin affinity chromatography followed by two sequential anion-exchange chromatography steps on Q Sepharose FF and Mono Q. After Q Sepharose FF chromatography of a thyroglobulin affinity-purified toxin A preparation, two major peaks of cytotoxicity representing toxins A and B were detected. The homogeneity of the final toxin A preparation obtained from Mono Q anion-exchange fast protein liquid chromatography was ascertained by gel electrophoresis developed by silver stain. The mol. wt of toxin A in non-denaturing conditions was estimated to be 520-540 Kda by native polyacrylamide gel electrophoresis (PAGE) developed by silver stain. In contrast, with sodium dodecyl sulphate (SDS)-PAGE under reducing or non-reducing conditions, a major band of 240 Kda and 10 minor and 27 faint bands (non-reduced conditions), or four minor and 31 faint bands (reduced conditions) were detected after silver staining. In two-dimensional PAGE, the seven minor bands of >240 Kda obtained by non-reducing SDS-PAGE migrated to the 240-Kda position after reduction with β-mercaptoethanol.

Virulent Salmonella typhimurium, 2000 LD50, were injected intraperito-neally into inbred male A/J mice, genetically resistant to salmonella infection. Peritoneal exudate cells were harvested between 5 and 54 h after infection and examined by electronmicroscopy. Polymorphs were seen ingesting as well as digesting the pathogens as early as 5 h after infection. Macrophages were equally active in destroying the phagocytosed organisms throughout this period. In the meantime, the proliferation of salmonellae appeared to occur extracellularly in the peritoneal cavity as evidenced by their division.