[Show abstract][Hide abstract] ABSTRACT: Plague outbreaks are occasionally reported in Brazil. Unfortunately, due to great genetic similarity, molecular subtyping of Yersinia pestis strains is difficult. Analysis of multiple-locus variable number of tandem repeats (VNTR), also known as MLVA, has been found to be a valuable tool to discriminate among strains. To check for genetic differences, strains obtained from two different ecological complexes in Brazil collected during two different epidemiological events, an epizootic in Sítio Alagoinha in 1967 and an outbreak in Planalto da Borborema in 1986, were subtyped through MLVA using 12 VNTR loci. Three clusters (A, B and C) were observed. Of the 20 strains from the epizootic, 18 fit into cluster A. Cluster A was divided into two subgroups: A(1) (15 strains) and A(2) (3 strains). Of the 17 strains from the outbreak, 15 fit into cluster B. Cluster B was divided into three subgroups: B(1) (4 strains), B(2) (4 strains) and B(3) (7 strains). Cluster C is a singleton with one epizootic strain. The external standards, Y. pestis CO92 and Y. pseudotuberculosis IP32953, formed two clusters of singletons. The stability of 12 VNTR loci of three unrelated cultures included in this study was assessed. The 12 VNTR loci were stable through multiple serial subcultures in the laboratory. MLVA revealed that Y. pestis populations in Brazil are not monomorphic, and that there is intraspecific genetic diversity among Brazilian plague strains. We conclude that there is some correlation among genetic groups of this species, related to the temporal and geographic origin of isolates.
[Show abstract][Hide abstract] ABSTRACT: Traditional methods of typing Vibrio cholerae define virulent strains according to their recognition by sera directed against the known epidemic serogroups O1 and O139, overlooking potentially virulent non-O1/non-O139 strains. Here, we have undertaken the characterization of eight clinical isolates of non-O1/non-O139 V. cholerae, collected during cholera outbreaks in Brazil. Seven of these were typed as O26 and one, 17155, was defined as non-typable. A PCR-based approach has previously detected in these strains several virulence genes derived from the CTXvarphi prophage and generally associated with pathogenic strains. Here, the presence of the O1-specific wbeN gene was investigated through PCR and found to be restricted to strain 17155, as well as one of the O26 strains, 4756, although neither strain was recognized by O1-specific antisera. The same two isolates were the only strains able to express the cholera toxin in culture, assayed by western blotting. They also possessed four repeats of the heptanucleotide TTTTGAT upstream of the ctxAB genes encoding the cholera toxin. The remaining strains possessed only two intact repeats, whereas pathogenic O1 possessed four to six repeats. To define their evolutionary relationships, selected 16S-23S intergenic rRNA spacer regions were sequenced from the various strains and the resulting sequences used to build phylogenetic trees. Strains 4756 and 17155 always clustered with control O1 strains, whereas the remaining O26 strains clustered separately. These results confirm that, despite their serological phenotype, these two strains are genotypically related to O1 strains and potentially able to produce epidemic cholera.
Clinical Microbiology and Infection 06/2009; 16(1):62-7. · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To examine the virulence factors and the genetic relationship isolates of the serogroup O3 of Vibrio parahaemolyticus in outbreaks of diarrhoea in the northeast region of Brazil.
Eighteen samples of the O3:K6 and O3:KUT serotypes of V. parahaemolyticus were analysed by multiplex polymerase chain reaction (m-PCR) for detection of the tl, tdh and trh genes, by random-amplified polymorphic DNA (RAPD) using two primers, and by amplification of the rDNA 16S-23S region. The gene tl was amplified in all the samples, tdh in 16 while trh in none; amplification of rDNA 16S-23S generated only one profile; each RAPD primer produced two amplification patterns allowing grouping two tdh(-) Kanagawa-negative isolates.
V. parahaemolyticus with characteristics of the pandemic clone appears to be widely disseminated in the studied region. Because of the genetic uniformity of the isolates, elucidation of outbreaks or tracking the source of contamination by the present molecular techniques seems useless.
Detection of V. parahaemolyticus with virulence potential of pandemic clone from two outbreaks and from several isolated gastroenteritis cases points out the need for inclusion of this micro-organism in the Brazilian routine monitoring of the diarrhoeas for elucidation of their aetiology.
Journal of Applied Microbiology 04/2008; 105(3):691-7. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The performance of a single-tube nested-PCR (STNPCR) technique was evaluated for plague diagnosis in comparison to conventional (one step) and two step nested PCR (NPCR). Assays were carried out with primers targeting the gene caf1 that encodes the Yersinia pestis F1 antigen. For STNPCR inner primers were immobilized onto the inside of the microtube caps and after the first amplification they were eluted by inversion of the tube. This procedure avoids opening the tube, reducing the risks of false-positive results by cross-contamination. The immobilized primers are stable for several months at -20 degrees C, thus, the tubes can be prepared beforehand and stored until use. STNPCR was more sensitive than conventional PCR, and less sensitive than NPCR. This drawback is compensated by a lower risk of cross-contamination. The experiments with infected animals showed that NPCR and STNPCR were able to produce positive results in all samples tested, despite contamination with other organisms. In contrast, conventional PCR yielded positive results in a smaller number of samples. Three out of 62 culture-negative rodents from plague areas, were positive by STNPCR. In conclusion, the PCR approaches evaluated, particularly NPCR and STNPCR have potential to be used as alternative tools in epidemiological surveys of plague. Furthermore, as the results can be obtained quickly (less than 24 hour), these techniques could be useful in emergency situations in which the rapidity in diagnosis is essential for adoption of immediate measures of control.
Advances in experimental medicine and biology 02/2007; 603:351-9. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.
Memórias do Instituto Oswaldo Cruz 12/2004; 99(7):727-32. · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the utility of random amplification of polymorphic DNA (RAPD) technique for routine practice in public health laboratories for epidemiological studies of Vibrio cholerae O1 isolates.
Seventy-nine strains were examined by PCR for the toxin genes (ctx A, zot and ace), virulence-associated genes (tcp A and tox T) and RAPD sequences. Except for one strain (no. 1123) from the Amazonas State, all the strains analysed carried the genes ctx A, zot, ace, tcp A and tox T. RAPD fingerprinting revealed variability but no correlation with serotype, biotype or geographical origin of the isolates was found.
A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V. cholerae O1 strains.
The simplicity and discriminative capacity of this technique make it useful for detecting genetic diversity among micro-organisms from a defined group or for outbreak investigation.
Journal of Applied Microbiology 02/2004; 96(3):447-54. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Most Brazilian Yersinia pestis isolates display a typical plasmid profile composed of the three classical plasmids: pYV, pPst and pFra. However, some cultures lack at least one of these plasmids, while a few of them harbour atypical DNA bands of molecular weight ranging from 147 to 11.5 kb. To investigate whether Y. pestis displaying atypical plasmid content could be propagated among rodents in nature through flea bites, we carried out studies with fleas ( Xenopsylla cheopis) and rodents ( Calomys callosus) reared in the laboratory and five Y. pestis cultures differing in plasmid content. The results suggest that: (1) the single presence of pYV is not sufficient for the transmission of Y. pestis by fleas, (2) pPst is not essential for transmission, (3) two atypical DNA bands of molecular weight of 30 kb and >90 kb have no biological role, and (4) pFra is required for the transmission of Y. pestis by flea bites. Other studies are needed to determine whether this plasmid alone is sufficient for transmission.
Parasitology Research 02/2003; 89(3):159-62. · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the effectiveness of multiplex-PCR in Yersinia pestis identification in samples preserved in Cary & Blair medium and to evaluate if this technique would uncover Y. pestis-positives among culture-negative samples.
Multiplex-PCR was used to detect Y. pestis in Cary & Blair preserved bubo aspirates from experimentally infected guinea pigs and to re-analyze samples from a plague outbreak after prolonged storage in Cary & Blair. Variation in the target genes amplification was observed over time.
Multiplex-PCR proved to be more effective than culture for plague diagnosis, both for old and recent samples. This technique would be a valuable tool for the plague control programme.
The multiplex-PCR technique can be useful for the detection and characterization of Y. pestis even when the bacteria are no longer viable and when culture diagnosis has been hampered by the growth of contaminants.
Letters in Applied Microbiology 01/2003; 37(5):361-4. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aims: To investigate whether modifications in Yersinia pestis isolates from three plague foci from the state of Ceará, Brazil, had occurred over the years as a consequence of genetic adaptation to the environment.Methods and Results: The isolates were studied with respect to susceptibility to antimicrobial drugs, plasmid and protein profiling, pigmentation on Congo red-agar plates, and the presence of some pathogenicity genes using PCR. Most of the expected virulence markers were detected in the cultures examined. There was no evidence of any alteration that could be associated with their origin (patients, rodents and fleas) or period of isolation (1971–1997).Conclusions, Significance and Impact of the Study: Phenotypic or genotypic changes were not detected in the cultures examined. However, the results obtained will serve as a reference to follow the evolution of Y. pestis in these foci.
[Show abstract][Hide abstract] ABSTRACT: To investigate whether modifications in Yersinia pestis isolates from three plague foci from the state of Ceará, Brazil, had occurred over the years as a consequence of genetic adaptation to the environment.
The isolates were studied with respect to susceptibility to antimicrobial drugs, plasmid and protein profiling, pigmentation on Congo red-agar plates, and the presence of some pathogenicity genes using PCR. Most of the expected virulence markers were detected in the cultures examined. There was no evidence of any alteration that could be associated with their origin (patients, rodents and fleas) or period of isolation (1971-1997).
Phenotypic or genotypic changes were not detected in the cultures examined. However, the results obtained will serve as a reference to follow the evolution of Y. pestis in these foci.
Letters in Applied Microbiology 02/2002; 35(6):543-7. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AIMS: To investigate genetic diversity among Staphylococcus aureus and to delineate the geographical distribution of the strains found. METHODS AND RESULTS: RAPD-PCR and ribotyping-PCR were employed for the characterization of Staph. aureus isolates from bovine and nosocomial origin. Among the strains, five to nine groups were distinguished by RAPD-PCR, depending on which primer was used, while ribotyping-PCR distinguished seven ribotypes. CONCLUSIONS, AND SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate the genetic heterogeneity of the strains studied, and the large dissemination of some clones throughout different regions and hosts, findings that may allow the monitoring of Staph. aureus infections.
Letters in Applied Microbiology 02/2002; 35(1):32-6. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an attempt to elucidate the virulence factors and the pathogenic mechanisms of Providencia alcalifaciens, 36 isolates identified in 1994-1995 in Recife city, Brazil were analysed by PCR to investigate the presence of DNA sequences homologous to virulence genes described in other invasive enterobacteria, as well as their ability to invade HeLa cells, their plasmid profiles and antibiotic resistance patterns. The genetic diversity of the isolates was also analysed by RAPD-PCR. No homologous sequences of virulence genes were observed with any of the P. alcalifaciens isolates studied. Ten isolates had no plasmid and 26 harboured one-to-five plasmids of 147-<6.9 kb. Invasion of HeLa cells was observed in only 10 isolates. No correlation between the plasmid content of the strains, their invasion of HeLa cells or their resistance to antimicrobial drugs could be established. The isolates could be distributed into 10 genotypic groups by RAPD-PCR. Considering the genotypic profile and ability to invade HeLa cells, 7 of the 10 invasive isolates belonged to the same genotypic group. The presence of invasive isolates in the same or a related genotypic group suggests the existence of a clonal lineage responsible for the invasiveness.
Journal of Medical Microbiology 01/2001; 50(1):29-34. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Revista do Instituto de Medicina Tropical de São Paulo 01/1999; 41(6):339-42. · 0.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Considering the possibility that invasiveness could be a neglected factor of virulence in Vibrio fluvialis-linked enteritis, since a dysenteric form of the disease was seen in Bangladesh, we studied 12 Brazilian strains of the organism, six clinical and six environmental, to determine whether they might be able to enter into HeLa cell monolayers or would carry plasmids incidentally involved in invasiveness. Four human and two environmental isolates attached to but did not enter into the cells. Though five strains harbored plasmids, no relationship was found between the carriage of these genetic elements and adhesiveness.
Memórias do Instituto Oswaldo Cruz 01/1994; 89(2):221-3. · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pathogenic Yersinia pestis isolates were collected during a plague outbreak at the Paraiba State in 1986. The Y. pestis isolates were investigated for the presence of virulence-associated factors and plasmid content. All strains analysed were proficient in the expression of the VW and fraction 1 antigens, pigment adsorption and pesticin-fibrinolysin-coagulase production. A similar plasmid profile composed by four plasmid with molecular weight of 60, 44, 14.9, and 6.4 Megadaltons (MD) was found in all strains. DNA cleavage with EcoRI restriction enzyme further demonstrated the uniform plasmid content of the Y. pestis isolates. Seven additional Y. pestis strains, previously isolated in the same region but in an endemic state, showed the same plasmid fingerprint. The lack of any detectable difference between epidemic and endemic isolates as well as the value of plasmid fingerprints in epidemiology of Y. pestis is discussed.
Revista do Instituto de Medicina Tropical de São Paulo 01/1989; 31(5):295-300. · 0.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During a plaque outbreak in the Borborema Plateau focus (Paraiba), bacteriological and serological studies were carried out in material from 452 patients (48 positives), 1,938 rodents and other small mammals (75 positives), 4,756 dogs (141 positives) and 2,047 cats (57 positives) obtained from 41 counties (out of which, 21 produced positive samples). Twenty Yersinia pestis strains isolated from material from 3 patients and 17 rodents, displayed biochemical reactions, virulence factors, antibiotic susceptibility and animal experimental pathogenicity similar to those observed in strains previously isolated. According to our findings this recent plague outbreak did not exhibit different factors from those observed during prior outbreaks in other plague foci in the northeast of Brazil.
Memórias do Instituto Oswaldo Cruz 01/1989; 84(2):249-56. · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 326 samples of diarrheal feces obtained from children whose ages ranged from zero to 5 years, admitted in two rehydration hospitals in the city of Recife, Pernambuco, were analyzed. Feces were placed in Cary-Blair medium (4 degrees C) for shipment to the laboratory. There was no difference in the rate of bacteria isolation if the samples were analyzed within the period from 3 to 7 days of collection. 19.02% of the analyzed samples were positives for at least one of the searched bacteria, 26 Salmonella belonging to 3 species, 21 classic enteropathogenic E. coli, 1 invasive E. coli, 10 Shigella belonging to 3 serotypes and 1 Yersinia enterocolitica were found.
Memórias do Instituto Oswaldo Cruz 01/1988; 83(4):475-9. · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Supernatant of boiled spleen saline-suspensions of Yersinia pestis experimentally infected animals were used as template for PCR amplification without DNA extraction. PCR sensitivity was enhanced by a second round of amplification (Nested). No amplification was observed from non-infected animals.
Revista do Instituto de Medicina Tropical de São Paulo 38(5):371-3. · 0.96 Impact Factor