- Volume 37, Issue 1, 1992

Seventy-three clinical isolates of “Campylobacter upsaliensis” were screened for the presence of plasmids. Plasmid bands were found in 68 (93%) isolates, from which 14 plasmid types were identified. Type 5 was found only in blood-culture isolates, whereas types 7–14 were found only in faecal isolates. Plasmid-free isolates and the other plasmid profiles were present in both faecal and blood isolates. The reproducibility of these profiles was largely dependent on the method of plasmid isolation. The chloroform-phenol lysis method was the most efficient and reliable method of preparing plasmid DNA for plasmid profile analysis. The success of this method may be largely attributable to two factors: (1) the efficacy of cell lysis was independent of either an alkaline agent or of heat; (2) the inactivation of nucleases by the utilisation of chloroform-phenol to lyse the cells. Furthermore, this method of plasmid DNA preparation is ideally suited for use on clinical isolates, especially when rapid plasmid profile analysis may be required.

The iron uptake mechanisms of enteropathogenic Escherichia coli (EPEC) were examined and compared with those of control E. coli strains. The incidence of aerobactin production was similar (39% and 37% respectively) in the two groups. The quantities of enterochelin produced by aerobactin-negative EPEC and control strains were similar, as were the quantities of enterochelin produced by aerobactin-positive EPEC and control strains. The ability to use haem or haemoglobin as an iron source in an iron-restricted environment was found in 80.4% and 60.8% of EPEC strains respectively, and in 76.6% and 56.6% of control E. coli strains. The ability of E. coli strains to use these compounds was not related to the production of enterochelin or aerobactin or to the production of haemolysins, and may be an important characteristic of bowel organisms. When growing in an iron-limited environment, the iron contained in haemoglobin was used in preference to ovotransferrin-bound iron. During periods of haemoglobin-stimulated growth, the enterochelin uptake system was shown to be fully expressed and may be involved in transport of haemoglobin-derived iron into the cell. Uptake of ovotransferrin-bound iron took place immediately upon exhaustion of haemoglobin-derived iron. The ability to use iron derived from haem compounds represents an alternative iron uptake mechanism for organisms growing in an iron-limited environment and allows greater flexibility during growth in vivo.

The production of toxins by 79 strains of Campylobacter jejuni isolated in Costa Rica from children with compylobacter-induced diarrhoea (44 strains) and from chickens (35 strains) was studied. An enterotoxic effect giving a rounding of mouse adrenocortical tumour (Y1) cells, which could be neutralised with antitoxin against Escherichia coli heat-labile enterotoxin, was detected in supernates from 16 (62%) of 26 strains from children with watery diarrhoea, in 5 (28%) of 18 strains from children with bloody or inflammatory diarrhoea, and in 12 (34%) of the 35 strains from chickens. Cytotoxic effects in human lung fibroblasts (MRC-5), African Green monkey kidney (Vero) cells and human cervical carcinoma (HeLa) cells were observed in none of the 26 strains from children with watery diarrhoea, in 2 (11%) of the 18 strains from children with bloody or inflammatory diarrhoea, and in 6 (17%) of the 35 strains from chickens. The simultaneous production of enterotoxin and cytotoxin was detected in four strains. The cytotoxic effect, which was most prominent in cells freshly seeded at a low density, appeared as a lethal rounding of the cells. Fibroblasts were more sensitive than epithelial cells. The effects of the supernates were inactivated by heating at 100° for 10 min and decreased after 1 week at 4 °. The production of toxins was lost after storage of the strains for one year at −70°C.

Although Campylobacter jejuni is now recognised as a common enteric pathogen, the mechanisms by which this organism produces enteritis remain ill-defined. It has been proposed that its abilities to adhere to and enter epithelial cells represent properties essential to virulence. However, the characteristics of these interactions and factors that may influence the association of C. jejuni with epithelial cells are incompletely described. We have determined that the ability of C. jejuni to bind to epithelial cell lines in vitro is significantly affected by the growth temperature and growth stage of the bacteria, but not by growth-medium composition. Binding of C. jejuni to cultured cells is not affected by temperature or phylogenetic origin of the target cell, and exhibits a non-uniform or patchy distribution. In contrast, internalisation is markedly diminished at low temperature, appears to involve active invagination of the target cell membrane via pseudopod formation, and is maximal when cells of human origin are employed.

Lactobacilli isolated from the vaginas of healthy women (39 strains) and from the vaginal discharge of women with bacterial vaginosis (15 strains) were investigated for their binding to 125I-fibronectin. Nine of the 54 strains bound fibronectin at pH 7.2. The binding capacity of these nine strains was about the same as that observed with Staphylococcus aureas Cowan 1. The binding was specific; an excess of unlabelled fibronectin or its amino-terminal 29-kDa fragment effectively competed for binding, whereas bovine serum albumin, human IgG and orosomucoid did not. Incubation of lactobacilli with fibronectin for different periods revealed a time-dependent increase in binding. Lowering the pH to 4•0 increased the binding capacity of all of the lactobacilli tested; binding occurred with strains that had previously failed to bind at pH 7•2. The increased binding of lactobacilli to fibronectin at a low pH may play a role in the maintenance of the ecological balance of the vagina.

An explant adult foreskin cell culture (FS2–3) was compared with human lung carcinoma cell culture (A549) with regard to the ability of Haemophilus ducreyi to produce a cytopathic effect. The survival of H. ducreyi for up to 26 days in FS2–3 cells was far greater than in any previously described in-vitro culture system. H. ducreyi survived for up to 7 days in A549 cells. The H. ducreyi cells grew and formed “fungal-like” microcolonies on the eukaryotic monolayer. Portions of the microcolonies remained attached despite extensive washing. Transmission electronmicroscopy indicated that, at 48 h after infection, the H. ducreyi cells did not penetrate the FS2–3 cells but they were closely associated with them; there was only a 2–5 nm gap between the H. ducreyi cell wall and the FS2–3 membrane. The virulent H. ducreyi strains RO18 and 35000 produced a cytopathic effect on FS2–3 cells that did not appear to be due to a soluble toxin. These strains did not produce any CPE on A549 cells. H. influenzae and the avirulent H. ducreyi strain CIP542, inoculated in the same concentration and incubated for the same length of time, did not produce CPE on FS2–3 cells. This study demonstrated that the use of FS2–3 foreskin cell culture provided an in-vitro approach for evaluating the cytopathic effect of virulent H. ducreyi whereby, unlike in other in-vitro systems, viability of the micro-organism could be readily maintained.

Sixteen lectins were examined for their ability to agglutinate 298 strains of Neisseria gonorrhoeae. Seven lectins failed to agglutinate any of the strains; the remaining nine lectins gave 22 different agglutination patterns. The 298 strains were divided into 14 serovars with a single panel of monoclonal antibody typing reagents; lectin agglutination subdivided these into 57 serovar/lectin patterns. A combination of two monoclonal antibody serotyping panels divided the strains into 32 serovar combinations; lectin agglutination further subdivided these into 79 serovar/lectin patterns. There was no correlation between lectin pattern and serovar. Lectin agglutination is a simple supplementary typing method and could be particularly useful in micro-epidemiological studies.

Clinical (66) and reference (5) strains of pigmented gram-negative anaerobic bacilli, identified as Prevotella intermedia (47), Pr. melaninogenica (1), Pr. corpora (8), Porphyromonas asaccharolyticus (12), P. endodontalis (1) and P. gingivalis (2), were examined by pyrolysis mass spectrometry (PMS) and in conventional tests. Numerical classification based on conventional test reaction patterns (CTRPs) resolved five clusters, four comprising strains identified as Pr. intermedia, Pr. corpora, Pr. melaninogenica, and P. gingivalis respectively, and one comprising strains identified as P. asaccharolyticus and P. endodontalis. Numerical classification based on PMS showed a similar division, with decreasing homogeneity of chemical composition in the order Pr. intermedia, Pr. corpora, P. asaccharolyticus, which agreed with the order of homogeneity in CTRPs. PMS clusters corresponding to the genus Porphyromonas were clearly distinct from those of the genus Prevotella. PMS and CTRP classification disagreed on cluster membership for six strains. PMS identification from blind challenge sets was in agreement with conventional identification for 64 of 67 strains.

A firmly adherent mass of slime plus organisms (biofilm) accumulates on the sides of culture tubes when some strains of coagulase-negative staphylococci are grown in a chemically-defined medium containing [14C]glucose. This mass was washed (to remove labelled medium) and then counted after adding scintillation fluid. Organisms from the liquid culture were also washed and counted to check that [14C]glucose had been utilised to label the bacteria. Nine strains were examined in this way, and the results were compared with those obtained with four older techniques for recognising slime production or adherent bacteria. The new method is quick, and has advantages of reproducibility and good discrimination between strains; there was a 15-fold difference in counts in the biofilm between slime-producing and non-producing strains respectively. With the new radiolabel assay, the effects of several antibacterial compounds on the build-up of the biofilm were investigated with four slime-producing strains. Tunicamycin, chloramphenicol and 5-fluorouracil, at levels below their minimum growth-inhibitory concentrations, each greatly diminished biofilm formation; several other drugs had less effect.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins (WCP), immunoblot analysis and DNA restriction-endonuclease analysis (REA) were applied as potential typing methods to 31 clinically significant strains of Moraxella (Branhamella) catarrhalis, five of which came from a suspected outbreak of nosocomial infection in a respiratory-diseases ward. Twelve of 31 isolates were placed in four groups, each of which contained strains indistinguishable by the three typing techniques used. Each of a further two groups contained two strains, and they were similar by at least one technique; the remaining 15 strains were unique by all three methods. Four of five strains from the suspected outbreak were indistinguishable by SDS-PAGE of WCP, immunoblotting and REA. Results show that SDS-PAGE of WCP, immunoblotting and REA are suitable techniques for characterising M. catarrhalis and that there is a considerable degree of strain heterogeneity. Nosocomial infection with M. catarrhalis may be relatively common and further epidemiological studies with a combination of typing techniques are indicated.

Seven isolates of B. piliformis, the agent of Tyzzer’s disease, obtained from various host species, were examined for cytotoxic activity by incubating culture filtrates on BRL 3A rat-hepatocyte and 3T3 mouse-fibroblast cell lines. One isolate exhibited cytopathic effects on BRL 3A cells, but not on 3T3 cells. Three other isolates were strongly cytotoxic for 3T3 cells but only slightly so for BRL 3A cells. The remaining three isolates showed no cytotoxicity for either cell line. The cytotoxic products were > 100 kDa in mol. wt, thermolabile, and partly destroyed by trypsin treatment. The data show that some B. piliformis isolates produce cytotoxic proteins, which may contribute to the pathogenesis of Tyzzer’s disease.