Colitis caused by Clostridioides difficile infection in a domestic dog: A case report.

Clostridioides difficile has been identified as one of the primary etiologic agents of nosocomial diarrhea and pseudomembranous colitis in humans and other mammals associated following broad-spectrum antibiotics use. In Rio de Janeiro, Brazil we describe a case of C. difficile infection (CDI) in a 13-year-old male dog.

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays.

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

Use of rep-PCR to define genetic relatedness among Bacteroides fragilis strains.

Bacteroides fragilis, a component of the normal flora and an important anaerobic pathogen in non-intestinal endogenous infections, has recently been associated with enteric diseases. In this study, 41 B. fragilis strains were analysed in relation to their genetic diversity. This collection included two reference strains (ATCC 23745 and 25285), 20 isolates from non-intestinal infections, six from intestinal infections, five from intestinal microflora and eight from an aquatic environment. The fingerprints were generated by using two repetitive sequences (REP and ERIC) as primers to PCR (rep-PCR). A dendrogram was obtained with the Taxotron Program. Three clusters (threshold genotypes I, II and III) were observed when the genetic distance was 0.30. These results confirm previous data found regarding the genotypical diversity of B. fragilis.

Expression of Bacteroides fragilis virulence markers in vitro.

Bacteroides fragilis isolates from intestinal and non-intestinal infections, normal flora and the environment were examined for properties linked with interactions among cells in vitro. Different adhesion molecules were detected in agglutination assays with human erythrocytes and tests for auto-agglutination and adherence to human colon carcinoma cells (HT29). There was no correlation between these properties, indicating that independent molecules are involved. Treatment with trypsin, heat or EDTA inhibited agglutination and adherence, suggesting that these molecules are proteins. The lack of correlation with the origin of the strains did not permit any of these activities to be recognised as virulence markers. The expression of fragilysin, a protease associated with damage to intestinal cells and bacterial translocation, was examined. Only those strains from patients with diarrhoea expressed this protease activity in assays with HT29 cells and this was confirmed by specific PCR for the bft gene. The activity of fragilysin as an enterotoxin was confirmed in the rabbit intestinal ligated loop assay. The association of this property only with strains from intestinal infections indicates that it is too early to suggest this protease as a determinant factor of B. fragilis invasiveness.

Determinants of resistance in Bacteroides fragilis strains according to recent Brazilian profiles of antimicrobial susceptibility.

Susceptibility profiles of 99 Bacteroides fragilis strains for 9 antimicrobial agents were defined by using an agar dilution method. The isolates were uniformly susceptible to imipenen and metronidazole. All isolates were resistant to ampicillin. The resistance rates to amoxicillin/clavulanate, cefoxitin, cefotaxime, chloramphenicol, clindamycin and tetracycline were 3.0, 12.1, 15.1, 1.0, 18.2 and 75.7%, respectively. Sixteen strains showed reduced susceptibility to metronidazole (MIC 2-4 mg/L) but none had nim genes using PCR. All strains were also investigated for the presence of cepA, cfiA, cfxA, ermF and tetQ genes by PCR methodology and 92.9, 4.9, 24.2, 2 and 64.6% of the strains were respectively found positive. These results reflect the importance of surveys of susceptibility profiles and the relevance of detecting major genetic determinants to monitor the dissemination of these genes.

Clostridium difficile: a problem of concern in developed countries and still a mystery in Latin America.

Clostridium difficile-associated disease (CDAD) is caused by a spore-forming bacterium and can result in highly variable disease, ranging from mild diarrhoea to severe clinical manifestations. Infections are most commonly seen in hospital settings and are often associated with on-going antibiotic therapy. Incidences of CDAD have shown a sustained increase worldwide over the last ten years and a hypervirulent C. difficile strain, PCR ribotype 027/REA type BI/North American pulsed-field (NAP) type 1 (027/BI/NAP-1), has caused outbreaks in North America and Europe. In contrast, only a few reports of cases in Latin America have been published and the hypervirulent strain 027/BI/NAP-1 has, so far, only been reported in Costa Rica. The potential worldwide spread of this infection calls for epidemiological studies to characterize currently circulating strains and also highlights the need for increased awareness and vigilance among healthcare professionals in currently unaffected areas, such as Latin America. This review attempts to summarize reports of C. difficile infection worldwide, especially in Latin America, and aims to provide an introduction to the problems associated with this pathogen for those countries that might face outbreaks of epidemic strains of C. difficile for the first time in the near future.

Clonality of Fusobacterium nucleatum in root canal infections.

Fusobacterium nucleatum is a gram-negative non-spore-forming, non-motile, obligate anaerobic rod that is normally isolated from the oral cavity. Several studies have reported a significant heterogeneity within the F. nucleatum species. The aim of the present study was to analyze the clonal diversity of F. nucleatum strains isolated from intracanal infections and to evaluate the presence of Enterobacterial Repetitive Intergenic Consensus (ERIC)-like sequences in the genome of F. nucleatum. Samples were collected from 13 single-root teeth from adult patients, all having carious lesions, necrotic pulps and radiographic evidence of periradicular bone loss. F. nucleatum was isolated from two different patients (subjects 5 and 7) by culture. Amplification of 19 colonies from subject 5 and 15 colonies from subject 7 using ERIC primers resulted in four clonal types, two per subject. An intense amplicon of approximately 700 bp was generated by ERIC-PCR for all F. nucleatum isolates and F. nucleatum ssp. polymorphum ATCC 10953. The amplification reaction using primer 1254 confirmed the results obtained with the ERIC primer. Our findings indicate that DNA fingerprints provided by ERIC- and Arbitrarily Primed (AP)-PCR may constitute a powerful tool for investigating F. nucleatum clonal diversity.

Bacteroides fragilis adherence to Caco-2 cells.

The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76-100%) to non- or weakly adherent (0-25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.

Teste alternativo de sensibilidade de bactérias anaeróbias estritas a agentes antimicrobianos / Alternative susceptibility test of anaerobic bacteria to antimicrobial agents

Um total de 25 amostras de bactérias anaeróbias, Bacteroides fragilis, Clostridium perfringens e Clostridium difficile foram avaliadas quanto a sensibilidade a vários antimicrobi anos pelo método de eluiçäo do disco em caldo BHI pré-reduzido esterilizado em anaerobiose (PRAS) e os resultados foram comparados com aqueles obtidos por uma metodologia de BHI näo-PRAS. Uma grande correlaçäo entre os dois métodos justifica a indicaçäo desta alternativa como procedimento satisfatório em rotina bacteriológica