- Volume 40, Issue 1, 1994

Sixty-two selected strains of Salmonella serotype Enteritidis of 33 phage types (PTs), and one strain classified as RDNC, were characterised by four different chromosomally based typing methods to elucidate genetic relationships among strains of different phage types. Based on IS200-hybridisation patterns, two major groups, containing strains of the most commonly encountered phage types, and six minor groups (seven with the RDNC strain included) were observed. IS200 pattern was a stable epidemiological marker in strains of all phage types except PT 6a and 14b. Ribotyping separated strains of the phage types into one major and five minor groups; the pattern of the RDNC strain was not seen with other strains. More than one ribotype was observed among strains of Enteritidis PTs 6, 7, 14b and 21. By pulsed-field gel electrophoresis, strains of 21 of the 33 phage types formed one large cluster when bands > 125 kb were used as the criterion for separation. Among strains belonging to PTs 1, 6, 7 and 14b, more than one pattern was observed by this method. By probing with five random cloned fragments of the Enteritidis chromosome, strains from 27 of 31 phage types examined showed the same hybridisation pattern. With the combined use of four genotypic methods, two groups of strains, representing eight and seven of 33 Enteritidis phage types, were formed; these two groups may be considered as the main evolutionary lines of Enteritidis. Strains of the remaining phage types, and the RDNC strain, belonged to separate groups.

The ability of cefotaxime, ciprofloxacin, piperacillin and tobramycin to cause release of endotoxin was examined in vitro with cultures of Enterobacter cloacae and Escherichia coli. Endotoxin was measured by a quantitative limulus amoebocyte lysate assay and its presence was confirmed by silver staining of the lipopolysaccharide moiety following SDS-PAGE. The morphology of the bacteria during antibiotic exposure was examined by scanning electronmicroscopy. Cefotaxime, ciprofloxacin and piperacillin caused significant endotoxin release, correlating with their ability to affect cell-wall morphology, causing filamentation, wall breakage and cell lysis. In contrast, little endotoxin was released when bacteria were exposed to tobramycin and no morphological changes were observed when bacteria were exposed to bactericidal concentrations of this aminoglycoside. Its antimicrobial spectrum and bactericidal activity make tobramycin an appropriate agent for treatment of sepsis caused by gram-negative bacteria and its lack of propensity to elicit excessive release of endotoxin may avoid exacerbation of endotoxin-related shock in sepsis.

Vibrio cholerae non-O1 strains were screened for the presence of cholera enterotoxin (CT) genes by means of digoxigenin-labelled polynucleotide CTA and CTB probes. In-vitro production of CT was investigated by the Y1 mouse adrenal cell assay, enzyme-linked immunosorbent assay (ELISA) and a commercial, reversed passive latex agglutination (RPLA) kit. Only two (0·25%) of 790 strains tested gave positive results with the CTA and CTB probes. The production of other bacterial cytotoxin(s) made it impossible to use the characteristic cell-rounding effect on Y1 cells for the detection of CT. CT production by the probe-positive strains was confirmed by the immunoassays. Two hundred and fifty-two of the 788 probe-negative strains were tested by both cell assay and immunoassays. Of these, 90% produced cytotoxin(s) in the cell assay. In addition, 37% gave positive results in CT-ELISA, but negative results with LT-ELISA and VET-RPLA. These results indicate the presumed presence of a toxin in V. cholerae non-O1 that is able to bind GM1 and react with antisera to CT, but which is not identical to CT.

Adherence of 18 staphylococcal strains to 13 types of uncoated plastic tubes made from 10 different plastic materials were investigated by binding of radiolabelled bacteria in phosphate-buffered saline for 2 h at 37°C. The different materials could be divided into five groups based on their ability to bind staphylococci. Lowest adhesion was found for plasticised polyvinylchloride. Simple assays for the relative binding of peroxidase-labelled human IgG or fibrinogen did not predict the result of adhesion studies. Neither bacterial surface hydrophobicity measured in a two-phase partitioning assay, nor hydrophobicity of materials (wettability) as measured by their contact angles in water correlated with bacterial adhesion. Adhesion of staphylococci to certain plastic materials was greatly influenced by the method used for sterilisation of the material.

The presence of a Teflon catheter had no effect on the in-vitro activity of a range of antibacterial agents against slime producing and non-producing Staphylococcus epidermidis strains as determined by a microdilution assay. The susceptibility of S. epidermidis attached to Teflon catheters for 6,24 and 48 h was also evaluated. MICs for planktonic and attached bacteria were similar. When bacteria attached to Teflon for 6 h were used as inocula, MBC values increased 32–8192-fold for the antibacterial agents tested. Similar results were observed when bacteria attached for 24 and 48 h were used as inocula. The activity of a high concentration (16 × MBC) of these antimicrobial agents against S. epidermidis biofilms in Teflon catheters was evaluated ; for five slime non-producing strains, the highest reduction (around 99 %) in bacterial viability was produced by cloxacillin and teicoplanin; for the slime producers, the highest effect (99·5 % reduction) was shown by amikacin, clindamycin cloxacillin and ciprofloxacin but all cases still showed bacterial counts higher than 103 cfu/catheter segment. It is concluded that adherence of S. epidermidis to Teflon catheters decreases the bactericidal activity of the antibacterial agents tested in vitro.

Herpes simplex virus type-1 (HSV-1) induces Fc- and C3b(i)-receptors on infected cells. The role of these receptors in baceterial superinfection was studied by comparing the adherence of non-opsonised and opsonised bacteria to HSV-infected and non-infected HEp-2 cells. A flow cytometric adherence assay, based on the fluorescent quantitation of FITC-labelled bacteria, was developed. Opsonisation of Staphylococcus epidermidis with human serum, resulted in a marked increase in adherence to HSV-infected cells and revealed a role for C3b(i)R- and FcR-mediated adhesion. However, the enhanced adherence never exceeded the level of attachment to non-infected cells. Increased adherence of other pathogenic bacteria, including Escherichia coli, Streptococcus pneumoniae, Haemophilus influenzae and Pseudomonas aeruginosa was not observed, indicating that the HSV-receptors play a minor role in secondary infections. Bacterial adhesion factors such as the fimbriae of E. coli played a more dominant role in the adherence of bacteria to HSV-infected cells.

The enteropathogenicity of Aeromonas strains that showed mannose-resistant adhesion to INT407 cells was evaluated by infecting Caco-2 cells and observing them by light and electronmicroscopy. Five of six strains adhered in large numbers to Caco-2 cells in the presence of mannose and caused cytopathic effects. Two strains of Aeromonas spp. seemed to invade Caco-2 cells, as membrane-bound bacteria were seen within the cytoplasm of these cells; however, staining by acridine orange-crystal violet appeared to show intracellular fluorescent bacteria in three strains. Fimbriae did not appear to play an important role in adhesion because fimbrial structures were not seen by transmission electronmicroscopy. Adhesion of four strains was inhibited by the addition of l-fucose. The strains were negative in the fluorescence actin staining test, which in enteropathogenic Escherichia coli strains correlates with the ability to attach and efface intestinal microvilli. The DNA of the Aeromonas strains did not hybridise with the E. coli eae and ipaB probes, associated with attaching and effacing ability and invasion, respectively. These results give support to the enteropathogenicity of adhesive strains of Aeromonas spp., although the mechanisms of adhesion, and possibly invasion, remain to be elucidated.

Outer membrane (OM) antigens of several oral treponemes were studied. SDS-PAGE revealed a 56–58-kDa protein as a major component of isolated OM vesicles. Immunoblot analysis with eluted antibodies prepared from Treponema denticola ATCC 33520 recognised the 56–58-kDa protein, which was highly conserved in the 17 T. denticola strains tested. The protein was also a component of a T. denticola desoxycholate-extractable, ethanol-soluble antigen (DES-Ag) but was not present in T. pectinovorum or T. vincentii ATCC 35580 and strain N9 whole-cell lysates. Electronmicroscopy of OM vesicles showed typical treponemal ultrastructure. Immunogold labelling of T. denticola ATCC 33520 with T. denticola ATCC 33520-specific eluted antibodies recognised only the OM surface of the cell. These results suggest that the 56–58-kDa antigen comprises surface-orientated epitopes and that this antigen may be specific for T. denticola.

Polyclonal rabbit anti-idiotypic (anti-Id) antibodies against two monoclonal antibodies (MAbs) specific for the flagella of Clostridium chauvoei were produced, purified and characterised. Lack of cross-reactivity with heterologous MAbs indicated that the anti-Id antibodies were highly specific. The surface-exposed epitopes of the flagellar filament recognised with protective MAb were further distinguished by the anti-Id antibodies. Moreover, each anti-Id antibody inhibited the binding of its related MAb to flagellar antigens in a competitive enzyme-linked immunosorbent assay, suggesting that the anti-Id antibodies bore an internal image of the flagellar antigens. The survival rate of mice was increased to nearly twice that of controls by immunisation with anti-Id 41, which had been produced with a protective MAb; in contrast, anti-Id 114, produced with a non-protective MAb, failed to immunise. The results suggest that an anti-Id antibody containing an internal image of C. chauvoei flagella might be used as a vaccine.