Molecular typing and long-term comparison of clostridium difficile strains by pulsed-field gel electrophoresis and PCR-ribotyping.

Thirty-two related and 68 unrelated isolates of Clostridium difficile, isolated in different Italian hospitals since 1987, were analysed by PFGE and PCR-ribotyping to investigate their genetic relatedness. The isolates were classified into 28 groups by PFGE and 20 ribotypes by PCR-ribotyping. A single clone of C. difficile was recognised as the cause of three geographically and chronologically distant outbreaks. The correlation between PFGE and PCR-ribotyping results was good, with agreement for 77 (84%) of the 92 isolates typed by both methods. However, among sporadic isolates the discriminatory power of PFGE was more evident. Eight isolates that were untypable by PFGE could be analysed by PCR-ribotyping. The dendrograms generated showed that the genetic relatedness of the C. difficile isolates obtained by both techniques was comparable. The majority of the isolates in recent years appeared to be genetically unrelated to isolates from past infections. However, two clonal groups identified in all time periods had a common origin and this seems to indicate that they share some advantageous biological characteristics. The constant monitoring of C. difficile epidemiology will allow acquisition of further important data on this nosocomial pathogen.

Bacteroides fragilis enterotoxin induces the expression of IL-8 and transforming growth factor-beta (TGF-beta) by human colonic epithelial cells.

Bacteroides fragilis toxin (BFT) has been shown to be capable of inducing intestinal mucosal inflammation in animals. Such inflammation may be responsible for diarrhoea, which occurs in some, but not all human carriers of enterotoxigenic strains of B. fragilis (ETBF). We have studied responses to BFT by different human intestinal epithelial cell lines and subsequently investigated the expression of IL-8 and TGF-beta by T84 cells. The latter were selected because their responses to BFT, characterized by morphological changes and cell death by apoptosis, were similar to those we have recently observed in primary human colonocytes. We show that BFT dose-dependently increased the expression of transcripts and protein of the polymorphonuclear cell chemoattractant IL-8. BFT also dose-dependently induced the release of TGF-beta, which has been shown to enhance the repair of the injured intestinal epithelium. However, the secreted TGF-beta was almost exclusively in the biologically inactive form, as determined by Mv1Lu bioassay. Our studies therefore suggest that exposure of colonic epithelial cells in vivo to high concentrations of BFT can initiate an inflammatory response via secreted IL-8. BFT-induced release of latent TGF-beta may facilitate the subsequent repair of the injured epithelium, following its activation by proteases from neighbouring cells. Variation in cytokine responses by colonic epithelial cells in vivo could be an important determinant in the development of mucosal disease and symptoms in response to ETBF.

Heterogeneity in responses by primary adult human colonic epithelial cells to purified enterotoxin of Bacteroides fragilis.

BACKGROUND: Enterotoxigenic strains of Bacteroides fragilis (ETBF) have been implicated in diarrhoeal illness in livestock and children, but their role in adult human colonic disease is unknown. AIMS: To investigate responses by primary adult human colonic epithelial cells to purified B fragilis toxin (BFT). METHODS: BFT was purified from culture supernatant of a highly toxigenic strain of ETBF. Morphological changes to primary colonic epithelial cells, in response to purified BFT, were studied in organ culture of colonic biopsy specimens from 15 adults. RESULTS: BFT induced epithelial cell cytotoxicity in colonic biopsy specimens from 12/15 subjects. The BFT induced morphological changes were characterised by epithelial cell rounding, separation from adjacent cells, and detachment from the basement membrane. In severely affected specimens, almost all the epithelial cells were affected. There was heterogeneity between subjects in the rate at which BFT induced epithelial cell cytotoxicity occurred. Furthermore, in colonic biopsy specimens from three subjects, exposure to BFT did not induce any significant morphological changes to epithelial cells. CONCLUSION: BFT is capable of inducing cytotoxicity in primary adult human colonic epithelial cells. Such an effect of ETBF derived BFT on epithelial cells in the colon in vivo would be expected to lead to mucosal inflammation and diarrhoea. Heterogeneity in responses by primary colonocytes probably reflects the outcome of host-BFT interactions. Such interactions in vivo could determine the occurrence of colonic disease in some individuals but not others.

Detection of enterotoxigenic Bacteroides fragilis by PCR.

Strains of enterotoxigenic Bacteroides fragilis (ETBF) are associated with diarrhea in young farm animals and, at least in particular settings, in children. Enterotoxin production by ETBF is currently detected by a tissue culture assay with HT-29 cells. We have developed a PCR assay based on the detection of the enterotoxin gene to identify ETBF in culture and in stool samples. Overall, 113 bacterial strains were examined, including 3 B. fragilis reference strains, 75 B. fragilis isolates (comprising 40 ETBF isolates), 20 Bacteroides spp. other than B. fragilis, and 15 strains belonging to other genera. Complete agreement was found between the results of the tissue culture assay and those of the PCR for our strains. PCR was also used to detect ETBF directly in fecal samples. Stools from two healthy volunteers were spiked with known numbers of ETBF and were processed by three different methods. A culture method, which required inoculation of the stools on selective plates and the collection of the whole bacterial growth ("sweeps"), was found to be the most sensitive. PCR performed with the plate sweeps yielded amplification products with a detection limit of 10(5) to 10(4) CFU/g of feces. By this method 18 samples of diarrheic stools (10 positive and 8 negative for ETBF) were examined. The results of the PCR were in accordance with the culture results in all cases. The proposed PCR assay represents a diagnostic tool for the rapid identification of ETBF in culture as well as in fecal samples.

Recovery of Bilophila wadsworthiafrom Clinical Specimens in Italy.

This report is the first survey in Italy to evaluate the incidence of recovery of Bilophila wadsworthia in clinical situations. The survey was carried out at the departments of Microbiology in two Northern Italian hospitals over a one-year period. Tests for B. wadsworthia were carried out on a range of specimens from different body sites, when etiology by anaerobes was suspected. Out of a total of 350 samples examined, 67% were positive in bacteriological tests. Mixed anaerobic infections were detected in 53 specimens, corresponding to 23% of all cases. Strains of B. wadsworthia were isolated from 12 samples, equivalent to 5% and 22% of total and mixed/anaerobic infections, respectively. Bilophila wadsworthia was always isolated in mixed infections, mainly from the large intestine (67% of cases). The infectious process of B. wadsworthia was often complicated by abscess formation, regardless of body site. Interestingly, a strain was isolated from one case of bacteremia. The microorganisms most frequently isolated with B. wadsworthia were Escherichia coli for facultative species (38%), and Bacteroides fragilis, from anaerobic isolates (25%). Production of beta-lactamases by B. wadsworthia isolates was found in ten strains (83%), which appeared to be penicillin G resistant at concentration equal to or greater than the break-point (4 microg/mL). Epidemiological and clinical data from this and previous studies point to the involvement of B. wadsworthia in mixed infections. To assess the specific contribution of the species to the disease, studies of pathogenetic factors are to be considered in parallel. Nonetheless, production of beta-lactamases by most B. wadsworthia isolates could easily interfere with the therapeutical approach to infections involving the new species. The addition of a selective medium to culture specimens from the abdominal cavity should be considered in order to detect the presence of B. wadsworthia.

Detection of enterotoxigenic Bacteroides fragilis and its toxin in stool samples from adults and children in Italy.

Stool samples from children and adults with and without diarrhea were examined for the presence of enterotoxigenic Bacteroides fragilis (ETBF) and its enterotoxin. A cytotoxic assay with HT-29 cells followed by neutralization with a hyperimmune antiserum were used to detect B. fragilis enterotoxin. ETBF isolates were recovered from 12% of healthy children and 17% of children with diarrhea (P = .42) and from 15% of healthy adults and 9.4% of adults with diarrhea (P = .31). Fecal B. fragilis enterotoxin was detected in four children (two with diarrhea and two without diarrhea) and in four adults with diarrhea. This study shows that in Italy, the rate of ETBF carriage is high, regardless if diarrhea is present. In some instances, the presence of ETBF is associated with detectable levels of fecal enterotoxin, but the significance of this finding deserves further evaluation.

Anaerobic yeast killer systems.

The influence of anaerobic conditions on the expression of the killer phenomenon of several yeast isolates belonging to recognized killer systems coded by different genetic determinants (Pichia spp., Kluyveromyces lactis, Saccharomyces cerevisiae) was studied. Anaerobiosis influenced the activity of killer toxins from some individual isolates of the genera Pichia and Saccharomyces on sensitive strains of P. anomala, K. lactis and Candida albicans. However, no influence was detectable on a S. cerevisiae sensitive isolate. Thus, anaerobic conditions seem to interfere more with the metabolic process of sensitive strains than with toxin production by killer yeasts. The selection of a panel of killer yeasts, able to display their activity against reference sensitive yeast isolates under anaerobic conditions in a medium that favored the growth of anaerobes, allowed the use of the killer system to type Bacteroides fragilis isolates for epidemiological purposes.

Diagnostic potential of IgA coated Candida cells in mucous membrane candidiasis.

The significance of in vivo IgA coated yeast cells for the diagnosis of candidiasis of the oral and vaginal mucosal membranes was evaluated by direct immunofluorescence in 70 patients with or without clinical symptoms, shown to be positive for yeast growth in the cultural test. Most of the patients with clinically suspected candidiasis of the mucosal membranes gave positive results by serologic assays in contrast to the majority of symptomless patients. The diagnostic approach proved to be essentially consistent with the clinical signs, persistence of infection, response to antifungal therapy and quantitative cultural data.

[Diagnostic significance of secretory IgA coated yeast cells in oral candidiasis]. / Significato diagnostico di IgA secretorie adese a cellule lievitiformi nelle candidosi orali.

The significance of in vivo IgA coated yeast cells for the diagnosis of candidiasis of the oral mucosal membranes was evaluated by direct immunofluorescence in 42 patients with or without clinical symptoms, shown to be positive for yeast growth in the cultural test. Most of the patients with clinically suspected candidiasis of the mucosal membranes gave positive results by serologic assays in contrast to the majority of symptomless patients. The diagnostic approach proved to be essentially consistent with the clinical signs, persistance of infection, response to antifungal therapy and quantitative cultural data.

A multicentre study to evaluate the effect of sulbactam/ampicillin combination on anaerobic micro-organisms.

Ampicillin combined with the beta-lactamase inhibitor sulbactam was compared with ampicillin alone, cefoxitin and metronidazole against 569 clinical strains of anaerobic organisms. The strains included 289 species of Bacteroides, 160 strains of Clostridium and 120 strains of various species of Streptococcus/Peptostreptococus, Fusobacterium, Veillonella, Eubacterium, Bifidobacterium, Actinomyces and Propionibacterium. Sulbactam/ampicillin was as effective as cefoxitin and metronidazole against all anaerobic species tested, inhibiting more than 90% of strains at the breakpoints (16, 32 and 32 mg/l, respectively). Sulbactam/ampicillin was also significantly more active than ampicillin against strains of Bacteroides, the minimal inhibitory concentration being at least four-fold lower. In contrast, the activity of the combination did not differ from that of ampicillin alone against Fusobacterium species and Gram-positive rods and cocci.

The diagnosis of small bowel bacterial overgrowth. Reliability of jejunal culture and inadequacy of breath hydrogen testing.

The reliability of a single jejunal culture in the diagnosis of small bowel bacterial overgrowth has recently been questioned. Seventy-seven patients thought to have bacterial overgrowth, defined as a jejunal culture yielding at least 10(6) organisms per milliliter of aspirate, took part in the study. Bacterial overgrowth was found in 74% of the patients with predisposing conditions and in 32% of those with no clear causes of bacterial colonization. The intestinal juice of some patients was taken at two different levels of the proximal jejunum, using both the closed- and open-tube systems. Highly significant correlations (rs = 0.90, p less than 0.001) were found between the numbers of bacteria per milliliter at the 2 jejunal levels and between the numbers of bacteria per milliliter of jejunal aspirate obtained from the closed and open tubes (rs = 0.84, p less than 0.001). Compared with the jejunal culture, the gas chromatography of volatile fatty acids in jejunal aspirate and the glucose- and lactulose-hydrogen breath tests showed sensitivities of 56%, 62%, and 68% and specificities of 100%, 83%, and 44%, respectively. This work demonstrates the reliability of jejunal cultures and the inadequacy of breath hydrogen testing in the prediction of positive jejunal cultures. When results of testing for volatile fatty acids in jejunal aspirates are positive, this always indicates the presence of bacterial overgrowth; thus, this procedure would avoid the more complicated, time-consuming, and costly bacteriological analysis of jejunal samples.

In-vitro activity of meropenem against clinical isolates in a multicentre study in Italy.

A multicentre in-vitro study was undertaken to evaluate the susceptibility of bacterial pathogens isolated in different Italian hospitals to meropenem. A total of 1399 aerobic and 452 anaerobic strains was analysed. Comparative agents were imipenem, cefotaxime, ceftazidime, ceftriaxone, piperacillin, ciprofloxacin, gentamicin, amikacin, plus vancomycin when appropriate. The MIC ranges (mg/l) of meropenem were: 0.015-2 for Klebsiella spp., Proteus spp., Morganella morganii and Providencia spp.; less than 0.008-1 for Escherichia coli; 0.016-32 for Serratia spp.; 0.03-2 for Enterobacter spp. and Citrobacter spp.; 0.03- greater than 128 for Acinetobacter anitratus; 0.03-32 for Pseudomonas spp.; less than 0.008-0.5 for Haemophilus spp. and Neisseria spp.; 0.015-64 for Staphylococcus spp.; 0.06- greater than 128 for Enterococcus spp.; less than 0.008-0.25 for Streptococcus spp.; 0.016-8 for Fusobacterium spp.; 0.03-8 for Bacteroides spp.; less than 0.06-0.5 for anaerobic Gram-positive cocci; 0.08-2 for Clostridium spp. Meropenem exhibited superior antibacterial activity against the aerobic and anaerobic strains tested when compared to the other beta-lactam drugs. The new carbapenem was as active as ciprofloxacin and more active than imipenem and the aminoglycosides against Enterobacteriaceae and Ps. aeruginosa. It was also more active than ciprofloxacin against most strains of Gram-positive cocci. Meropenem was slightly less potent than imipenem against staphylococci and enterococci, with the exception of oxacillin-susceptible Staph. aureus against which meropenem and imipenem exhibited similar antibacterial activity.

High homology of membrane proteins electrophoretic pattern in Bacteroides fragilis as confirmed by various solubilizing methods.

Membrane proteins from 25 strains of B. fragilis isolated in different laboratories in Northern Italy were examined by SDS-PAGE and isoelectrofocusing. The electrophoretic patterns of inner and outer membrane after Sarkosyl and SDS solubilization of all the isolates were consistently similar to that of the reference strain. The protein profiles of the different species belonging to the B. fragilis group are clearly distinguishable with negligible similarities. Our data clearly show that this approach is extremely helpful and reliable in providing additional verification of the identity of strains recognized by conventional tests. In this connection PAGIF analysis of triton solubilized isolated envelopes reduces technical time and difficulties, thus improving analytical accuracy.

Isolation of Chlamydia trachomatis from the prostatic cells in patients affected by nonacute abacterial prostatitis.

To assess the presence of Chlamydia trachomatis in nonacute abacterial prostatitis 30 patients with urethral cultures positive for Chlamydia trachomatis underwent microbiological studies, including cultures of transrectal aspiration biopsies of the prostate. Chlamydia trachomatis was isolated from 10 of the prostatic specimens (33 per cent). In 3 cases a nonspecific cytopathogenic effect caused the destruction of the tissue cultures. Our findings demonstrate that Chlamydia trachomatis may cause ascending infections of the prostate and that this microorganism may have an etiological role in the pathogenesis of nonacute abacterial prostatitis.