- Volume 49, Issue 6, 2000

Patterns of invasiveness of Salmonella serotypes Typhimurium, Choleraesuis and Dublin in Caco-2 cells (without centrifugation) were compared with previously published studies of the rabbit ileal invasion assay (RIIA) and (where relevant) a HEp-2 cell invasion assay. Optimal conditions for the use of Caco-2 cell monolayers in bacterial invasion assays were defined. Centrifuge-assisted attachment of bacteria to cells was not used routinely as this increased the invasiveness of known hypo-invasive strains and detachment of Caco-2 cells. Inocula with too high bacterial numbers resulted in rapid acidification of media and detachment of the monolayers. The invasiveness of Typhimurium strains TML, WAKE, WII8, LT7, SL1027 and M206 in Caco-2 cells reflected that seen in the RIIA. The invasiveness of Choleraesuis strain A50 was similar to that in the RIIA except that bacteria grown at 37°C and used without storage at 4°C were slightly more invasive than those grown at 37°C and stored at 4°C before use. Dublin strain 3246 showed no apparent temperature-regulated invasiveness in Caco-2 cells, in contrast to the results observed in the RIIA. Dublin strain 3246 did not cleave tight junctions in the Caco-2 cell monolayer as it did in rabbit ileal epithelia both in vitro and in vivo . Three Tn phoA insertion LPS mutants of Typhimurium TML were uniformly hypo-invasive in both Caco-2 cells and the RIIA; in contrast, they were differentially invasive in HEp-2 cells. Three smooth Tn phoA insertion mutants of Typhimurium TML ( invH, invG and pagC ) were hypo-invasive in both the Caco-2 and HEp-2 cell invasion assays but not in the RIIA.

Helicobacter pylori is known to transform to coccoid forms which might be involved in faecal–oral transmission. When the bacteria enter the intestine, they encounter anaerobiosis that is unfavourable for growth. The effect of anaerobiosis was investigated to determine whether H. pylori is viable under such conditions. H. pylori in the late logarithmic growth phase transformed from spiral to coccoid forms when transferred to and incubated anaerobically in fresh medium. Acridine orange staining indicated that the viability of coccoid forms was significantly reduced, but still measurable even at day 5 or 7 of anaerobic culture. The cells retained low but significant levels of the major sigma factor RpoD at day 5 or 7 of anaerobic culture. The cellular structures of coccoid forms contained polyphosphate granules at day 1 and even at day 7 when incubated anaerobically, whereas only a few granules were observed under micro-aerobic conditions. Poor formation of polyphosphate granules in micro-aerobic cultures correlated particularly well with lower levels of acridine orange staining. These results suggest that acridine orange-positive anaerobic coccoid forms are viable to a certain extent and that polyphosphate may support this viability.

Both polymorphonuclear cell infiltration and increased epithelial apoptosis are seen in gastric mucosa in the presence of Helicobacter pylori infection. This study examined the association between bacterial ability to stimulate an oxidative burst in neutrophils and epithelial apoptosis. Biopsy specimens were obtained from 15 patients to detect apoptotic cells by the TUNEL method. H. pylori strains isolated from corresponding stomach biopsy samples were tested for the ability to stimulate an oxidative burst in human neutrophils. Neutrophils were isolated from healthy subjects without H. pylori infection and the oxidative burst was measured by flow cytometry with dichlorofluorescein diacetate. Stimulation with H. pylori increased both the percentage of activated cells and fluorescence intensity. There was a significant positive correlation between the number of epithelial apoptotic cells and fluorescence intensity. Increased neutrophil oxidative burst stimulated by H. pylori may play a role in enhanced gastric mucosal DNA damage and consequent atrophic gastritis and gastric cancer.

A bacterial toxin that causes progressive distension and death of Chinese hamster ovary (CHO) cells and HeLa cells, termed cytolethal distending toxin (Cdt), has been identified in several diarrhoeagenic bacteria, including Campylobacter spp. ( C. jejuni and C. coli ), some pathogenic strains of Escherichia coli and Shigella spp. Genes encoding this toxin were identified as a cluster of three adjacent genes cdtA , cdtB and cdtC . Homologues of cdtB from five species of enterohepatic helicobacters ( Helicobacter hepaticus , H. bilis , H. canis and two novel Helicobacter spp. isolated from mice and woodchuck, respectively) were identified by means of degenerative PCR primers, cloned and sequenced. The similarities of these partial cdtB nucleotide sequences from these Helicobacter spp. to those of cdtB genes known to be present in other bacteria were: C. jejuni , 58.3–64.8%; E. coli , 52.3–57.8%, Haemophilus ducreyi , 53.4–58.4% and Actinobacillus actinomycetemcomitans , 52.7–58.1%. Bacterial lysates from four of these helicobacters caused characteristic cytolethal distension of HeLa cells. Cdt caused cell cycle arrest at G2/M phase as measured by flow cytometry. The results demonstrated the presence of a toxin in these Helicobacter spp. belonging to the family of Cdt.

A set of 46 epidemiologically related or unrelated Candida ( Torulopsis ) glabrata isolates from four different medical centres in Germany and Hungary, and the type strain of this species, were genetically typed by arbitrarily primed PCR (AP-PCR). The resulting band patterns of C. glabrata strains were compared with those of other species of the genus Candida including C. albicans , C. guilliermondii , C. kefyr , C. parapsilosis , C. tropicalis and C. krusei . After preliminary trials of various reaction parameters and control experiments to test the reproducibility of this method, it was found that consistently reproducible amplification patterns were obtained only when rigorously optimised and standardised reaction conditions were employed. Discriminatory abilities were studied with 29 generated 10-mer oligonucleotides of different G+C content. Typing of clinical isolates with the optimised AP-PCR protocol was then performed with the primer 50-1, with a G+C content of 50%. Sufficiently discriminatory polymorphisms were observed among the band patterns of the Candida species included. The gel electrophoresis patterns of each species showed an adequate similarity. Variations in minor bands were characteristic for comparison at the isolate level. Only three AP-PCR genotypes were identified among the clinical isolates of C. glabrata tested. Two of these genotypes were closely related and appeared to be widespread within German and Hungarian isolates. The third genotype of C. glabrata showed a distinct band pattern. With optimised, validated and standardised assay conditions, the feasibility, sensitivity and rapidity of AP-PCR may offer a discriminatory method for genotyping of yeasts in epidemiological studies, as well as in the control of nosocomial infections.

This paper reports the results of characterising and selecting a strain of Lactobacillus for potential use as a probiotic in regenerating the vaginal flora of women with recurrent episodes of bacterial vaginosis (BV). BV is a condition characterised by a depletion of vaginal lactobacilli accompanied by an overgrowth of a mixed vaginal flora of aerobic, anaerobic and micro-aerophilic species in very large numbers. BV has been associated with various gynaecological and obstetric complications and has an extremely high recurrence rate, due in part to the failure to establish a normal vaginal flora after antimicrobial therapy. A total of 60 vaginal isolates of lactobacilli was assessed for characteristics considered important for vaginal re-colonisation. The characteristics studied were the in-vitro inhibitory activity of the lactobacilli against bacterial species isolated from women with recurrent BV, acid production after growth of the lactobacilli in liquid culture, production of hydrogen peroxide (H2O2) and adhesiveness of the lactobacilli to exfoliated vaginal epithelial cells (VEC). Four strains of lactobacilli, L. acidophilus (61701 and 61880), L. crispatus (55730) and L. delbrueckii subsp. delbrueckii (65407), demonstrated the greatest inhibitory activity against the BV-associated bacterial species. Two of these isolates (55730 and 61880) produced H2O2. All four isolates produced a highly acidic environment after growth in liquid medium (pH < 4). Only one of these (strain 61701) was strongly adherent to VEC (>100 bacteria/VEC). A further isolate ( L. acidophilus 48101) did not demonstrate maximum inhibitory activity against BV-associated bacteria, but was found to be a strong producer of H2O2 and was also highly adherent to VEC. Isolates 61701 and 48101 could be candidates for use as probiotics for vaginal re-colonisation.

The effect of influenza vaccination on the occurrence and severity of influenza virus infection in a population residing in nursing homes for the elderly was studied during an influenza A (H3N2) epidemic in Japan. Of 22 462 individuals living in 301 welfare nursing homes, 10 739 received either one dose (2027 subjects) or two doses (8712 subjects) of inactivated, subunit trivalent influenza vaccine. During the period Nov. 1998 to March 1999, there were 950 cases of influenza infection diagnosed clinically, with virus isolation or serology. There were statistically significantly fewer cases of influenza, hospital admissions due to severe infection and deaths due to influenza in the vaccinated cohort (256 cases, 32 hospital admissions, 1 death) than in the unvaccinated controls (694 cases, 150 hospital admissions, 5 deaths; reduction rates 59.8%, 76.9% and 79.1% respectively). Vaccination was almost equally effective in those who received one dose of vaccine and those who received two doses. No serious adverse reactions to vaccination were recorded. Thus influenza vaccination is safe and effective in this population, and should be an integral part of the routine care of persons aged ≥65 years residing in nursing homes.