- Volume 24, Issue 3, 1987

A quantitative assay for Clostridium difficile cytotoxin has been developed, based on the observation that suspended fibroblasts exposed to cytotoxin fail to adhere to plastic. A dye-binding technique was used to quantitate adherent cells, in order to obviate microscopy. Adherent BHK cells were fixed with glutaraldehyde and cell protein was stained with Coomassie blue R-250. Cell-bound dye was eluted and estimated spectrophotometrically. The amount of eluted dye was proportional to the number of adherent cells and cell staining was time dependent. Cytotoxin was purified by gel-permeation and ion-exchange chromatography and migrated as a single band on SDS-PAGE. After exposure of suspended BHK cells to purified cytotoxin, their adhesion to plastic was inhibited in a manner which depended on concentration of cytotoxin and on time and temperature of exposure. This study provides the basis for a C. difficile cytotoxin assay that is quantitative, rapid and reproducible and may have wider applicability in the study of other toxins or agents that inhibit cell adhesion.

A procedure was developed for enumeration of total associated, attached and intracellular bacteria after interaction of Yersinia spp. with epithelial cells in vitro. Isogenic cultures of Y. enterocolitica grown at 25°C had greater affinity for epithelial cells (Henle, HeLa and Vero) than for polystyrene, and they invaded the cells. Y. kristenseni and Y. intermedia showed less attachment to either surface and were non-invasive. The degree of attachment to cells and invasion by Y. enterocolitica was related to number of bacteria added and interaction time, whereas attachment to polystyrene occurred rapidly and did not change. Y. enterocolitica was more hydrophobic when grown at 35°C than at 25°C according to partitioning in a biphasic dextran-polyethylene glycol system, and attached strongly to both polystyrene and epithelial-cell monolayers. Y. kristenseni grown at 25°C was also hydrophobic but did not have the same attachment properties. Y. kristenseni and Y. intermedia showed slightly reduced electrostatic interactions with the anion exchangers DEAE-Sepharose and DEAE-Trisacryl. Attachment of Y. enterocolitica to epithelial cells probably involves non-specific surface properties that are not entirely explicable by hydrophobic and electrostatic interactions, whereas invasion of epithelial cells appears to resemble “receptor-mediated endocytosis”.

Rabbits were given, by the intra-gastric route, two isogenic strains of Yersinia enterocolitica that differed only in the presence or absence of the virulence plasmid. Clinical illness and characteristic morphological lesions of Y. enterocolitica infection were seen only in rabbits infected with the plasmid-bearing strain (MCH700S). Although rabbits infected with a strain lacking the plasmid (MCH700L) remained healthy, mild histological changes in the small intestine, consisting of epithelial-cell damage, dilatation of lymphatics and a slight increase in neutrophil polymorphonuclear leukocytes in lamina propria, were seen in the first 12 h after inoculation. Bacteria, which were identified as Y. enterocolitica by indirect fluorescent antibody staining, were seen in dilated lymphatics. These early lesions tended to abate quickly and were no longer detectable at 24 h. Strain MCH700L was recovered from the mesenteric lymph nodes in increasing numbers until 24 h after inoculation; the number then began to decrease rapidly. In contrast, the early lesions in rabbits given strain MCH700S progressed to micro-abscesses, focal destruction of villi, and ulcerations beginning 24 h after inoculation; the number of bacteria recovered from the lymph nodes continued to increase beyond 24 h after inoculation. Bacteria were also recovered from the liver and spleen. These results suggest that both plasmid-bearing and non-bearing strains of Y. enterocolitica are capable of penetrating the intestinal mucosa. However, the virulence plasmid is required for invading bacteria to proliferate in the host tissue and to establish infection.

The presence of R-plasmid RP1 increased the adhesion of chemostat-grown iron- and carbon-limited Proteus mirabilis to the surfaces of various medical prostheses and to glass. Similar results were obtained with iron-limited Pseudomonas aeruginosa and anaerobically-grown Escherichia coli. Changes in the surface properties of P. mirabilis indicated that the R-plasmid-mediated increase in negative charge was one of the factors that promoted adhesion.

The bactericidal action of non-immune whole blood on Proteus mirabilis was increased when the bacteria contained the R-plasmid RP1. This effect was due mainly to increased phagocytosis. Iron-depleted stationary-phase cells were more sensitive than carbon-depleted cells. The contribution of serum was usually negligible but was increased during a minor non-specific infection. Most plasmid-containing phenotypes were more sensitive than were those without plasmids but there were considerable differences between stationary and exponentially-growing cells. The R-plasmid-mediated increase in sensitivity to phagocytosis may be due in part to the presence of additional glycosylated proteins in the outer membrane.

The vaginal bacteriology of 10 healthy asymptomatic women was assessed during the menstrual cycle. Samples were taken from the posterior vaginal fornix for quantitative analysis. There were no significant alterations in the total vaginal flora at different stages of the menstrual cycle. The mean number of species isolated per specimen declined from 4·6 in week 1 to 2·9 in week 4. This decline was not caused by a decrease in the occurrence or concentration of any one organism or group of organisms. The vaginal pH decreased from a mean of 6·6 in week 1 to 4·3 in week 4, this increased acidity could not be attributed to the action of lactobacilli as their total incidence or concentration did not change during the menstrual cycle.

Amongst 58 isolates of motile aeromonads evaluated for the ability to produce β haemolysin, haemolytic activity was significantly associated with strains belonging to the Aeromonas hydrophila and A. sobria groups. Of erythrocytes from nine animal species tested, mouse red blood cells provided the best indicator system for detection of β-haemolysin activity. Furthermore, differences in the stability of the β haemolysins of selected A. sobria and A. hydrophila isolates at different temperatures, and in the presence of urea or dithiothreitol were observed.

The genital carriage of Ureaplasma urealyticum, Mycoplasma hominis and Chlamydia trachomatis was assessed in 72 women admitted to hospital in spontaneous preterm labour and in 26 women requiring preterm delivery for other reasons who formed a control group. Women in preterm labour significantly more often carried ureaplasmas, had large numbers of M. hominis and subsequently developed chorioamnionitis than women in the control group. M. hominis, in particular, occurred more frequently and in large numbers in women who had chorioamnionitis associated with ruptured membranes. Genital carriage of the various micro-organisms appeared not to be associated with fetal growth retardation, although subsequent isolation of ureaplasmas from infants was common. It is suggested that mid-second-trimester vaginal specimens should be cultured on a research basis to establish whether these various micro-organisms identify women at risk of labouring preterm.

Small numbers of bacteria capable of growing on agar supplemented with amoxycillin 40 mg/L were isolated from the saliva of 9 out of 20 adult volunteers in a previous study. All the bacteria were identified as Streptococcus sanguis although no strains produced dextran in conventional tests. However, using a specific assay, all the antibiotic-resistant strains were found to secrete glucosyltransferases (GTF), the enzymes that synthesise these extracellular polysaccharides; the production of GTF-S, the enzyme that synthesises dextran, was 22–43% less than that of an antibiotic-sensitive control strain. Enzyme production by both antibiotic-resistant and sensitive bacteria was markedly inhibited by dextran primer. The amoxycillin-resistant bacteria were resistant to other penicillins; their resistance to erythromycin was variable but they were uniformly sensitive to cephalothin and clindamycin. As dextran production has been proposed as a key factor in the colonisation of damaged heart valves by bacteria such as S. sanguis, these highly resistant bacteria may not pose a threat to the susceptible individual.

A strain of Pseudomonas aeruginosa (3-Post) was resistant to cefsulodin and ceftazidime at 37°C but sensitive at 20°C. Resistance was mediated by chromosomally-encoded β lactamase which was synthesised at a high level during growth above 30°C but at a low, inducible level during growth below 27°C.

During growth in magnesium (Mg++)-deficient mineral media, Pseudomonas aeruginosa cells synthesise large amounts of H1 outer-membrane protein and are resistant to polymyxins and EDTA. It has been suggested that H1 protein replaces Mg++ as an outer-membrane-stabilising component in Mg++ -deprived cells, thereby removing the EDTA target and blocking an adsorption site for polymyxins. Induction of H1 protein synthesis also occurred in P. aeruginosa cells grown in Antibiotic No. 3 Broth (Ab3B), although this medium is not Mg++ -deficient. Generally, significant induction of H1 protein did not occur in P. aeruginosa cultures grown in other complex media such as Proteose Peptone and Nutrient Broth, which contained less Mg++ than Ab3B, nor in Isosensitest Broth or Mueller Hinton Broth, which contained higher Mg++ concentrations. H1-protein-induced P. aeruginosa cells from Ab3B cultures, unlike those from Mg++ -deficient mineral-broth culture, remained fully sensitive to polymyxin B and, with one exception, to EDTA. It is concluded that induction of H1 protein does not itself confer resistance to polymyxin B, and has no more than a minor role in EDTA resistance. Other cell-wall changes, such as phospholipid modifications and the absence of Mg++, probably account for the resistance of Mg++ -deprived cells.

Methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus from diverse geographic origins were analysed by polyacrylamide-agarose gel electrophoresis for esterase polymorphism. Three kinds of esterase bands, designated A, B and C, were defined by their ranges of activity toward five synthetic substrates and their resistance to di-isopropyl fluorophosphate. There were five allozymes of esterase A, four of esterase B and four of esterase C. Eighteen distinct combinations of allozymes (zymotypes) were distinguished amongst 105 strains analysed. Two major zymotypes were represented by 35 and 19 strains respectively, whereas other zymotypes were represented by one or, at most, seven strains. The coefficient of genetic diversity was lower for methicillin-resistant strains than for methicillin-sensitive strains. Most of the methicillin-resistant strains are represented by the two major zymotypes which differed from each other by the electrophoretic behaviour of the three esterases. These results indicate that, on the basis of esterase electrophoretic polymorphism, methicillin resistance is expressed in genetically different strains.