Diversity of Campylobacter jejuni and Campylobacter coli genotypes from human and animal sources from Rio de Janeiro, Brazil.

To compare the genotypes of Campylobacter jejuni and Campylobacter coli isolates of human and animal origin collected in Rio de Janeiro City, 30 C. jejuni and 35 C. coli isolates from animal sources (n=45) and human patients with gastroenteritis (n=20) were genotyped by PCR-based techniques, namely random amplified polymorphic DNA (RAPD-PCR) and enterobacterial repetitive intergenic consensus sequence (ERIC-PCR). RAPD-PCR identified 50 types and ERIC-PCR identified 22 genotypes, among the 65 Campylobacter isolates. Both PCR methods discriminated the C. jejuni and C. coli groups of isolates. Combining the results of both methods, no single genotype was shared between isolates from human and animal sources. Two groups of two C. coli isolates each with identical genotypes were found among poultry and pig isolates. A high level of genetic diversity observed among the Campylobacter isolates suggests lack of overlap between isolates from different sources.

Non-effect of p22-phox -930A/G polymorphism on end-organ damage in Brazilian hypertensive patients.

The p22-phox subunit is an essential component of NAD(P)H oxidase enzymatic complex, which is considered the major source of oxidative stress products in the cardiovascular system. The -930G allele of p22-phox has been associated with higher promoter activity, increased NAD(P)H oxidase-mediated oxidative stress and hypertension. We recently reported that left ventricular hypertrophy is accompanied by increased myocardial p22-phox expression in aortic-banded rats, suggesting that this protein might be involved in hypertensive cardiac hypertrophy.

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.

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.

Antimicrobial resistance and plasmid profiles of Campylobacter jejuni and Campylobacter coli from human and animal sources.

AIMS: The purpose of this study was to determine the susceptibility of Campylobacter jejuni and Campylobacter coli isolates to antimicrobial agents and to investigate the presence of plasmid DNA. METHODS AND RESULTS: A total of 15 clinical isolates from children faeces, and 29 animal isolates of Campylobacter jejuni (n=22) and Campylobacter coli (n=22) were tested for susceptibility to 9 antimicrobial agents using a disc diffusion method, and screened for the presence of plasmid DNA by agarose gel electrophoresis. Of the 44 isolates, 56.8% were resistant to sulphonamide, 25% to norfloxacin, 18.2% to erythromicin, ciprofloxacin and ampicillin, and 13.6% to tetracycline. All isolates were susceptible to gentamicin, chloramphenicol and cefotaxime. Plasmids were detected in one Camp. jejuni (4.54%) strain isolated from sheep and in six (27.27%) Camp. coli strains isolated from rhesus monkey(3), swine(2), and poultry(1) with sizes ranging from 3.4 to 50 kb. CONCLUSIONS: The majority of the human isolates were susceptible to antibiotics commonly used for the treatment of campylobacteriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The origin and spread of Campylobacter resistance to antibiotics are discussed, with particular respect to the current situation in Brazil.

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.

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.

Otite média crônica: perfil de sensibilidade de espécies facultativas e anaeróbicas prevalentes: estudo de 40 casos / Chronic otitis media: sensitivity profile of prevalent facultative and anaerobic species: study of 40 cases

Este trabalho apresenta um traçado do perfil de sensibilidade a antimicrobianos das principais bactérias isoladas de exudatos purulentos de 40 pacientes com Otite Média Crônica. Oitenta e duas bactérias facultativas e aeróbias e sessenta e três bactérias anaeróbias estritas foram isoladas, tendo a infecçäo polimicrobiana predominado em 70 por cento dos casos estudados. Em ordem decrescente de freqüência, as bactérias mais comumente isoladas foram: Peptostreptococcus (13,1 por cento), Pseudomonas aeruginosa (12,4 por cento), Bacteroides sp (8,2 por cento), Proteus mirabilis (6,9 por cento), Staphylococcus aureus (6,9 por cento). Prevotella melaninogenica (6,9 por cento) e Bacteroides do grupo fragilis (6,9 por cento), onde a espécie fragilis foi prevalente. Múltipla resistência aos antimicrobianos testados foi uma constante entre as bactérias aeróbicas e facultativas a resistência à oxacilina em 20 por cento das cepas de S. aureus estudadas foi constatado. Resistência à penicilina (100 por cento), tetraciclina (80 por cento) e cefoxitina (20 por cento) foi observado entre as espécies de Bacteroides do grupo fragilis