Genomovar status, virulence markers and genotyping of Burkholderia cepacia complex strains isolated from Brazilian cystic fibrosis patients

Burkholderia cepacia complex isolates obtained by microbiological culture of respiratory samples from Brazilian CF patients were studied by recA based PCR, screened by specific PCR for virulence markers and genotyped by RAPD. Forty-one isolates of B. cepacia complex were identified by culture and confirmation of identity and genomovar determination obtained in 32 isolates, with predominance of B. cenocepacia (53.1%). Virulence markers were not consistently found among isolates. Genotyping did not identify identical patterns among different patients. B. cenocepacia was the most prevalent B. cepacia complex member among our patients, and cross-infection does not seem to occur among them. V 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Distribution of virulence markers in clinical and environmental Vibrio cholerae non-O1/non-O139 strains isolated in Brazil from 1991 to 2000

One hundred seventy nine Vibrio cholerae non-O1/non-O139 strains from clinical and different environmental sources isolated in Brazil from 1991 to 2000 were serogrouped and screened for the presence of four different virulence factors. The Random Amplification of Polymorphic DNA (RAPD) technique was used to evaluate the genetic relatedness among strains. Fifty-four different serogroups were identified and V. cholerae O26 was the most common (7.8%). PCR analysis for three genes (ctxA, zot, ace) located of the CTX genetic element and one gene (tcpA) located on the VPI pathogenicity island showed that 27 strains harbored one or more of these genes. Eight (4.5%) strains possessed the complete set of CTX element genes and all but one of these belonged to the O26 serogroup suggesting that V. cholerae O26 has the potential to be an epidemic strain. The RAPD profiles revealed a wide variability among strains and no genetic correlation was observed.

Differences in virulence markers between Helicobacter pylori strains from the Brazilian Amazon region

Introduction This study compares virulence markers of Helicobacter pylori isolated from patients in 2 cities in the Brazilian Amazon. Methods The study analyzed 168 patients with chronic gastritis from Belém and 151 from Bragança, State of Pará, Brazil. Levels of bacterial DNA associated with cagA and vacA alleles were checked by PCR, and hematoxylin-eosin staining was used for histologic diagnosis. Results In Bragança 87% of patients were genotype s1m1 cagA-positive (s1m1 cagA+), compared with 76% in Belém. In samples from patients in both cities, there was an association between s1m1 cagA+ strains and gastric mucosal damage. Conclusions Both cities have a high frequency of s1m1 cagA+ strains of H. pylori.

Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in São Paulo, Brazil

Bacteroides fragilis has been isolated from several human and non-human monomicrobial and mixed infections. In this study, some virulence markers and the antimicrobial susceptibility of bacteria of the B. fragilis group isolated from children's stools were evaluated. All the 64 isolates showed the following characteristics: capsulated, beta-hemolytic, hydrophilic, and serum-resistant. Only, 24 (37.5%) strains were resistant at 60ºC, for 30 min, and among them, 12 (18.75%) were resistant at 60ºC, for 60 min. Also, none strain was resistant at 100ºC. Four strains were able to hemagglutinate erythrocytes and D-mannose, D-galactose, D-arabinose, and D-xylose inhibited hemagglutination in 2 B. fragilis strains (p76a, p76b). The hemagglutination in the strain B. uniformis p3-2 was inhibited by D-xylose and D-galactose. The bft gene detection and the enterotoxin production were observed only in 13 EF-enterotoxigenic species. Fragilysin activity was confirmed on HT-29 cells. The antimicrobial determination confirmed that both imipenem and metronidazole were efficient against B. fragilis species; all the strains were resistant to lead and nickel. Plasmids of 2.9, 4.4, 4.8, and 8.9 kb were observed in 6 tested strains. These results show the values of the species identification from clinical infections, as well as of the periodic evaluation of the resistance patterns of the B. fragilis group at Brazilian medical institutions.

Ribotyping and virulence markers of Yersinia pseudotuberculosis strains isolated from animals in Brazil

Ribotyping and virulence markers has been used to investigate 68 Yersinia pseudotuberculosis strains of serogroups O:1a and O:3. The strains were isolated from clinical material obtained from healthy and sick animals in the Southern region of Brazil. Ribotypes were identified by double digestion of extracted DNA with the restriction endonucleases SmaI and PstI, separation by electrophoresis and hybridization with a digoxigenin-labeled cDNA probe. The presence of the chromosomal virulence marker genes inv, irp1, irp2, psn, ybtE, ybtP-ybtQ, and ybtX-ybtS, of the IS100 insertion sequence, and of the plasmid gene lcrF was detected by polymerase chain reaction. The strains were grouped into four distinct ribotypes, all of them comprising several strains. Ribotypes 1 and 4 presented distinct profiles, with 57.3% genetic similarity, ribotypes 2 and 3 presented 52.5% genetic similarity, and genetic similarity was 45% between these two groups (1/4 and 2/3). All strains possessed the inv, irp1, and irp2 genes. Additionally, strains of serogroup O:1a carried psn, ybtE, ybtP-ybtQ, ybtX-ybtS, and IS100. As expected lcrF was only detected in strains harboring the virulence plasmid. These data demonstrate the presence of Y. pseudotuberculosis strains harboring genotypic virulence markers in the livestock from Southern Brazil and that the dissemination of these bacteria may occur between herds.

Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheria boydii

The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a saprophytic filamentous fungus recognized as a potent etiologic agent of a wide variety of infections in immunocompromised as well as in immunocompetent patients. Very little is known about the virulence factors expressed by this fungal pathogen. The present review provides an overview of recent discoveries related to the identification and biochemical characterization of potential virulence attributes produced by P. boydii, with special emphasis on surface and released molecules. These structures include polysaccharides (glucans), glycopeptides (peptidorhamnomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases, phosphatases and superoxide dismutase), which have been implicated in some fundamental cellular processes in P. boydii including growth, differentiation and interaction with host molecules. Elucidation of the structure of cell surface components as well as the secreted molecules, especially those that function as virulence determinants, is of great relevance to understand the pathogenic mechanisms of P. boydii.

Molecular typing and virulence markers of Yersinia enterocolitica strains from human, animal and food origins isolated between 1968 and 2000 in Brazil

Molecular typing and virulence markers were used to evaluate the genetic profiles and virulence potential of 106 Yersinia enterocolitica strains. of these strains, 71 were bio-serotype 4/O: 3, isolated from human and animal clinical material, and 35 were of biotype 1 A or 2 and of diverse serotypes, isolated from food in Brazil between 1968 and 2000. Drug resistance was also investigated. All the strains were resistant to three or more drugs. The isolates showed a virulence-related phenotype in the aesculin, pyrazinamidase and salicin tests, except for the food isolates, only two of which were positive for these tests. For the other phenotypic virulence determinants (autoagglutination, Ca++ dependence and Congo red absorption), the strains showed a diverse behaviour. The inv, ail and ystA genes were detected in all human and animal strains, while all the food isolates were positive for inv, and 3% of them positive for ail and ystA. The presence of virF was variable in the three groups of strains. The strains were better discriminated by PFGE than by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). A higher genomic similarity was observed among the 4/O: 3 strains, isolated from human and animal isolates, than among the food strains, with the exception of two food strains possessing the virulence genes and grouped close to the 4/O: 3 strains by ERIC-PCR. Unusually, the results revealed the virulence potential of a bio-serotype 1 A/O: 10 strain, suggesting that food contaminated with Y. enterocolitica biotype 1 A may cause infection. This also suggests that ERIC-PCR may be used as a tool to reveal clues about the virulence potential of Y. enterocolitica strains. Furthermore, the results also support the hypothesis that animals may act as reservoirs of Y. enterocolitica for human infections in Brazil, an epidemiological aspect that has not been investigated in this country, confirming data from other parts of the world.

Ribotyping and virulence markers of Yersinia pseudotuberculosis strains isolated from animals in Brazil

Ribotyping and virulence markers has been used to investigate 68 Yersinia pseudotuberculosis strains of serogroups O:1a and O:3. The strains were isolated from clinical material obtained from healthy and sick animals in the Southern region of Brazil. Ribotypes were identified by double digestion of extracted DNA with the restriction endonucleases SmaI and PstI, separation by electrophoresis and hybridization with a digoxigenin-labeled cDNA probe. The presence of the chromosomal virulence marker genes inv, irp1, irp2, psn, ybtE, ybtP-ybtQ, and ybtX-ybtS, of the IS100 insertion sequence, and of the plasmid gene lcrF was detected by polymerase chain reaction. The strains were grouped into four distinct ribotypes, all of them comprising several strains. Ribotypes 1 and 4 presented distinct profiles, with 57.3% genetic similarity, ribotypes 2 and 3 presented 52.5% genetic similarity, and genetic similarity was 45% between these two groups (1/4 and 2/3). All strains possessed the inv, irp1, and irp2 genes. Additionally, strains of serogroup O:1a carried psn, ybtE, ybtP-ybtQ, ybtX-ybtS, and IS100. As expected lcrF was only detected in strains harboring the virulence plasmid. These data demonstrate the presence of Y. pseudotuberculosis strains harboring genotypic virulence markers in the livestock from Southern Brazil and that the dissemination of these bacteria may occur between herds.

Production of cytolethal distending toxin and other virulence characteristics of Escherichia coli strains of serogroup O86

Genetic and phenotypic virulence markers of different categories of diarrhoeagenic Escherichia coli were investigated in 106 strains of enteropathogenic E. coli (EPEC) serogroup O86. The most frequent serotype found was O86:H34 (86%). Strains of this serotype and the non motile ones behaved as EPEC i.e., carried eae, bfpA and EAF DNA sequences and presented localised adherence to HeLa cells. Serotypes O86:H2, O86:H6, O86:H10, O86:H18, O86:H27 and O86:H non determined, belonged to other categories. The majority of the strains of serotype O86:H34 and non motile strains produced cytolethal-distending toxin (CDT). The ribotyping analysis showed a correlation among ribotypes, virulence markers and serotypes, thus suggesting that CDT production might be a property associated with a universal clone represented by the O86:H34 serotype.

Shigella in Brazilian children with acute diarrhoea: prevalence, antimicrobial resistance and virulence genes

Diarrhoeal disease is still considered a major cause of morbidity and mortality among children. Among diarrhoeagenic agents, Shigella should be highlighted due to its prevalence and the severity of the associated disease. Here, we assessed Shigella prevalence, drug susceptibility and virulence factors. Faeces from 157 children with diarrhoea who sought treatment at the Children's Hospital João Paulo II, a reference children´s hospital in Belo Horizonte, state of Minas Gerais, Brazil, were cultured and drug susceptibility of the Shigella isolates was determined by the disk diffusion technique. Shigella virulence markers were identified by polymerase chain reaction. The bacterium was recovered from 10.8% of the children (88.2% Shigella sonnei). The ipaH, iuc, sen and ial genes were detected in strains isolated from all shigellosis patients; set1A was only detected in Shigella flexneri. Additionally, patients were infected by Shigella strains of different ial, sat, sen and set1A genotypes. Compared to previous studies, we observed a marked shift in the distribution of species from S. flexneri to S. sonnei and high rates of trimethoprim/sulfamethoxazole resistance.

Virulence properties and antimicrobial susceptibility of Shiga toxin-producing Escherichia coli strains isolated from healthy cattle from Parana State, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Virulence characteristics and epidemiology of Yersinia enterocolitica and Yersiniae other than Y-pseudotuberculosis and Y-pestis isolated from water and sewage

Aims: To determine the species, bio-sero-phagetypes, antimicrobial drug resistance and also the pathogenic potential of 144 strains of Yersinia spp. isolated from water sources and sewage in Brazil.Methods and Results: the 144 Yersinia strains were characterized biochemically, serologically and had their antibiotic resistance and phenotypic virulence markers determined by microbiological and serological standard techniques. The Y. enterocolitica strains related to human diseases were also tested for the presence of virulence genes, by the PCR technique. The isolates were classified as Y. enterocolitica, Y. intermedia, Y. frederiksenii, Y. kristensenii and Yersinia biochemically atypical. The 144 isolates belonged to various bio-serogroups. Half of the strains showed resistance to three or more drugs. The Y. enterocolitica strains related to human diseases exhibited phenotypic virulence characteristics and virulence genes.Conclusions: Water from various sources and sewage are contaminated with Yersinia spp. in Brasil. Among these bacteria, virulent strains of Y. enterocolitica were found, with biotypes and serogroups related to human diseases.Significance and Impact of the Study: This is the first documented description of the occurrence of pathogenic Y. enterocolitica in water sources and sewage in Brazil. The occurrence of virulence strains of Y. enterocolitica shows that the environment is a potential source of human infection by this species in this country.

Prevalence and virulence properties of Vibrio cholerae non-O1, Aeromonas spp. and Plesiomonas shigelloides isolated from Cambe Stream (State of Parana, Brazil)

The incidence of Vibrio cholerae, Aeromonas spp, and Plesiomonas shigelloides was determined in Rater samples from Cambe Stream. The samples were collected from seven different sites. The serogroups, virulence markers and drug resistance profiles were also evaluated. Twelve. Aer. hydrophila, 12 Aer. caviae, eight Aer. sobria, seven Ple. shigelloides and two V. cholerae non-O1 were isolated. They belonged to different serogroups and all produced haemolysis in different assays. Five of the Aeromonas strains and one of V, cholerae non-O1 were positive for enterotoxin activity. Haemagglutination and its inhibition, using erythrocytes of different origins, was variable for Aeromonas spp and V. cholerae, while none of the Plt. shigelloides haemagglutinated in association with any type of erythrocyte. All isolates exhibited multiple drug resistance. These results indicate that the occurrence of V. cholerae non-O1, Aeromonas spp, and Ple. shigelloides, in water used for vegetable irrigation, human recreation and animal consumption, among others, represents a potential risk for humans.

Survey of Minas frescal cheese from Southwest Minas Gerais for virulence factors and antimicrobial resistance in Escherichia coli isolates

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)