- Volume 40, Issue 5, 1994

The intra-species differentiation of Pseudomonas aeruginosa was analysed by comparing the polymorphism of esterases by conventional polyacrylamide-agarose gel electrophoresis, the physicochemical properties of the variants of the major esterase P3 and the restriction fragment length polymorphism of ribosomal RNA gene regions (ribotyping) to O-serotyping for several panels of strains selected from among a series of 257 clinical isolates and two references strains, (ATCC nos. 10145 and 27853). The electrophoretic variation of four main kinds of esterase (P1–P4) and 11 additional esterases distinguished by their spectra of hydrolytic activity with synthetic substrates and by their sensitivity to di-isopropyl-fluorophosphate, allowed the discrimination of 67 zymotypes. Thirty-two esterase P3 variants were characterised by their pi, electrophoretic mobilities and titration curve analyses. They were distributed into two groups which, by these molecular criteria, seem to be distantly related. Combination of the patterns resulting from HindII, EcoRI and BelI restriction endonuclease digestions allowed the discrimination of 33 ribotypes among 134 strains. The strains exhibiting esterase P3 variants of group 2 presented a distinct ribotype and belonged to serotype O12. They could constitute a distinct group within the species. For the majority of the strains, the absence of correlation between zymotype, ribotype and serotype argues for a high level of heterogeneity within P. aeruginosa and indicates that the parallel use of the first two methods represent a potential tool for epidemiological study.

Three immunodominant antigens of Streptococcus equisimilis (Lancefield group C) with approximate mol. wts of 46, 66 and 105 kDa were recognised by human serum IgG and IgA immunoblotting. These antigens were identified consistently by various human sera but immunoblots with IgA (heavy chain) and secretory IgA (J chain) from human respiratory secretions gave more variable results. Antigens with similar migration rates were demonstrated in S.pyogenes, large colony human biotype group G streptococci, and streptococci of groups C and G from the “S. anginosus-milleri group”. Polyclonal antibody which was eluted from immunoblot substrates that contained the S. equisimilis 66-kDa antigen reacted with the 66-kDa antigen of S. pyogenes. Both polyclonal and monoclonal anti-vimentin antibodies identified the 46-kDa and 66-kDa antigens of S. equisimilis. The homology of these antigens among β-haemolytic streptococci has the potential to complicate both a strategy for the utilisation of immunoblotting for diagnostic purposes and the understanding of how such antigens may be involved in the pathogenesis of post-infectious sequelae.

Immunoblotting sera from cases of Streptococcus mutans or S. sobrinus endocarditis against an extract from S. sobrinus strain MUCOB 263 had identified three immunodominant antigenic bands at 190, 200 and 220 kDa. A lambda ZAPII DNA library was produced from the sheared genomic DNA of S. sobrinus MUCOB 263 and six identical positive clones were identified when this library was screened with serum from a patient with endocarditis caused by a bacterium from the mutans group of streptococci. On subcloning and sequencing, a protein containing 1548 amino acids was identified with a 99·2 % homology to the SpaA antigen of S. sobrinus and 68·4 % homology to the PAc antigen of S. mutans.

The prevalence and genotype of Shiga-like toxins (SLTs) in Escherichia coli O157 strains from patients in Germany with haemolytic uraemic syndrome (HUS) were investigated. This was done by PCR amplification of the B-subunit genes with two primer pairs—one complementary to slt-IB, and the other homologous to both slt-IIB and slt-IIvB sequences. To distinguish between slt-II and slt-IIv, the amplified DNA was digested with restriction endonucleases HaeIII and FokI. Of the 38 strains examined, 17 harboured sequences for slt-IIv; four contained only slt-IIv, three carried both slt-IIv and slt-I, and 10 strains had slt-IIv and slt-II. A further three genotypes (slt-I, slt-II, slt-I/slt-II) were found in the remaining 21 strains resulting in a total of six slt genotypes. To determine whether the slt genes were expressed, and whether genotypes correlated with phenotypes, all strains were subjected to cytotoxicity assays and colony ELISA. All 38 strains displayed cytotoxic activity to Vero cells in similar quantities. The SLT-I-specific monoclonal antibody (MAb)13C4 reacted with all 10 strains in which slt-I sequences were identified. Colony blot ELISA with the SLT-II specific MAb11E10 detected 27 of 28 strains with slt-II sequences, but did not react with any of the seven strains that carried slt-IIv, or slt-I and slt-IIv. The high SLT variability shown here has diagnostic implications and may well have consequences for the host response in infections associated with these pathogens.

The molecular basis of the non-expression of coagulase was investigated for 14 coagulase-negative isolates of Staphylococcus aureus obtained from different clinical samples. These isolates had typical S. aureus characteristics such as production of clumping factor, DNAase and protein A, but, with one exception, failed to produce detectable amounts of α-haemolysin. All 14 strains had DNA homologous to the coagulase gene (coa), but a coa-specific transcript was found in only seven of them. α-Haemolysin mRNA was detected in only eight strains without direct correlation to coa-mRNA expression. Thus, coagulase and α-haemolysin deficiencies in S. aureus may involve either transcriptional or post-transcriptional alterations although additional regulatory factors may influence the expression of both genes.

Respiratory secretions provide an efficient method for protecting the large surface area of the lower respiratory tract. To determine whether lung secretions contribute to antifungal defences, murine bronchoalveolar lavage fluid (BLF) was tested for anti-candidal activity against 49 oral and vaginal isolates belonging to six different Candida species. The yeasts were incubated in unconcentrated, cell-free lavage fluid from Sprague-Dawley rats and then cultured quantitatively to measure residual viability. Experiments with C. albicans indicated that sensitivity to BLF increased in a time- and dose-dependent manner. This activity was heat-stable (56°C) and consistent, irrespective of whether the BLF was derived from rats inoculated (orally) with candida or the uninoculated controls. Of the Candida spp. examined, C. albicans was the most susceptible followed by C. parapsilosis and C. tropicalis, whereas C. krusei, C. guilliermondii and C. glabrata were highly resistant. However, there were differences in susceptibility to BLF among different isolates within a given species. These results indicate that a heat-stable, soluble factor(s) in murine lavage fluid may suppress candidal colonisation of the lower respiratory tract and contribute to the defence mechanisms of the lungs.

A polymerase chain reaction (PCR) method was developed that was capable of detecting a wide range of medically important fungi from clinical specimens. The primer pair was designed in conserved sequences of 18S-ribosomal RNA genes shared by most fungi. The lower limit of detection of this PCR technique was 1 pg of Candida albicans genomic DNA by ethidium bromide staining and 100 fg after Southern analysis. A 687-bp product was amplified successfully by PCR from all 78 strains of 25 medically important fungal species studied, including Candida spp., Hansenula spp., Saccharomyces cerevisiae, Cryptococcus neoformans, Trichosporon beigelii, Malassezia furfur, Pneumocystis carinii, Aspergillus spp., and Penicillium spp., but not from any strains of Mucor spp., Escherichia coli, or methicillin-resistant Staphylococcus aureus (MRSA), calf thymus or human placenta. This specificity was subsequently confirmed by Southern analysis. PCR analysis of blood specimens collected from mice systemically infected with C. albicans and clinical samples including blood, cerebrospinal fluid and sputum appeared to be a more sensitive diagnostic method for invasive fungal infections than a conventional blood culture technique.

Multilocus enzyme electrophoresis was used to assess genetic relationships amongst 175 isolates of anaerobic intestinal spirochaetes, including 72 isolates from individuals living in different parts of the world, 102 from pigs and one from a dog. Amongst porcine isolates belonging to the genus Serpulina, a possible new species was identified. All but one of the isolates from man were clustered with the canine isolate and 59 porcine isolates in a distinct group that we have previously called “Anguillina coli”. The human and animal spirochaetes in this group had four-to-six axial flagella and most were recovered from individuals with diarrhoea. They included a strain of the so-called “Serpulina jonesii”, that was not a true serpulina. These 71 human isolates were distributed into 44 electrophoretic types and had a mean genetic diversity of 0·32. These were further divided into 26 clonal groups. Three of these clones also contained porcine isolates, one of which was strain P43/6/78, the agent of porcine intestinal spirochaetosis. Four of the clones contained human isolates from different sources. One included isolates from Western Australian Aboriginal children and from Italian adults, and the other three contained isolates from Western Australian Aboriginal children and from homosexual males in Sydney, New South Wales. There were no known connections between these human populations. The other spirochaete of human origin was Brachyspira aalborgi, which was distinct from isolates in the genera Serpulina and “Anguillina”. Both B. aalborgi and “A. coli” have been associated with human cases of intestinal spirochaetosis. Others have questioned the clinical significance of colonisation by B. aalborgi, but we suggest that isolates of “A. coli” may be the cause of some clinical cases of intestinal spirochaetosis.