Viability of Yersinia pestis subcultures in agar stabs.

UNLABELLED: Since its identification as the causative agent of plague in 1894, thousands of Yersinia pestis strains have been isolated and stored. Here, we report the ability of Y. pestis to survive up to 47 years in agar stabs, in rubber-stoppered tubes, under refrigeration (+4 to +10°C), although overall subculture recovery rates were poor and inversely related to the length of time stored. Genetic characterization of virulence gene presence among these subcultures was suggestive of significant variation in the genomic stability of Y. pestis subcultures stored under these conditions. Specifically, we found variation in the presence of plasmid and chromosomal virulence markers (genes pla, lcrV, caf1 and irp2) among multiple subcultures of Y. pestis strains in the 'Collection of Yersinia pestis' (Fiocruz-CYP) maintained by the SRP of FIOCRUZ-PE in Brazil. This variation, together with all of the inherent temporal, geographic and other genetic variation represented by all of the recoverable strains in this historical collection was preserved in new frozen culture stocks stored at -70°C as a result of this study. These frozen culture stocks represent a valuable resource for future comparative studies of Y. pestis. SIGNIFICANCE AND IMPACT OF THE STUDY: We report the ability of Yersinia pestis to survive up to 47 years in agar stabs, in rubber-stoppered tubes, under refrigeration (+4 to +10°C), although overall subculture recovery rates were poor and inversely related to the length of time stored. Genetic characterization of virulence gene presence among these subcultures was suggestive of significant variation in the genomic stability of Y. pestis subcultures stored under these conditions. This variation, together with all of the inherent temporal, geographic and other genetic variation represented by all of the recoverable strains in the historical 'Collection of Yersinia pestis' (Fiocruz-CYP) maintained by the SRP of FIOCRUZ-PE in Brazil was preserved in new frozen culture stocks stored at -70°C as a result of this study. These frozen culture stocks represent a valuable resource for future comparative studies of Y. pestis.

Genetic diversity of Yersinia pestis in Brazil.

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.

Characterization of potentially virulent non-O1/non-O139 Vibrio cholerae strains isolated from human patients.

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.

Vibrio parahaemolyticus serovar O3:K6 gastroenteritis in northeast Brazil.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

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.

Evaluation of a RAPD-based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil.

AIMS: 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. MATERIALS AND RESULTS: 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. CONCLUSION: A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V. cholerae O1 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence.

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.

Retrospective study of a plague outbreak by multiplex-PCR.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Transmission of Yersinia pestis cultures with different plasmid content from Xenopsylla cheopis to Calomys callosus.

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.

Typing of Yersinia pestis isolates from the state of Ceará, Brazil.

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.

Typing of human and bovine Staphylococcus aureus by RAPD-PCR and ribotyping-PCR.

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.

[Isolation of pathogenic bacteria in pasteurized type C milk sold in Recife City, Pernambuco, Brazil]. / Pesquisa de bactérias patogênicas em leite pasteurizado tipo C comercializado na cidade do Recife, Pernambuco, Brasil.

In order to improve information about the microbiological quality of the milk commercially available in the city of Recife, 250 samples of pasteurized type-C milk and 50 samples of raw milk were analyzed for Yersinia enterocolitica and Listeria monocytogenes and verify the possible occurrence of Yersinia enterocolitica and Listeria monocytogenes. These bacteria can develop in refrigeration temperatures and are responsible for food-born diseases. Neither Y. enterocolitica nor L. monocytogenes were found in the samples analyzed. However, the presence of Y. intermedia and Y. frederiksenii was detected, these environmental species behave as opportunist pathogens. Through the methodology used for Listeria isolation, one isolate of Salmonella Montevideo was obtained from a sample of pasteurized milk and another isolated from one sample of raw milk. Besides these, several other bacteria species were found. It is likely that the large microbiota present in the samples and the procedures employed to destroy it could have hindered the isolation of Y. enterocolitica and L. monocytogenes.

Pesquisa de bactérias patogênicas em leite pasteurizado tipo C comercializado na cidade do Recife, Pernambuco, Brasil / Isolation of pathogenic bacteria in pasteurized type C milk sold in Recife City, Pernambuco, Brazil

In order to improve information about the microbiological quality of the milk commercially available in the city of Recife, 250 samples of pasteurized type-C milk and 50 samples of raw milk were analyzed for Yersinia enterocolitica and Listeria monocytogenes and verify the possible occurrence of Yersinia enterocolitica and Listeria monocytogenes. These bacteria can develop in refrigeration temperatures and are responsible for food-born diseases. Neither Y. enterocolitica nor L. monocytogenes were found in the samples analyzed. However, the presence of Y. intermedia and Y. frederiksenii was detected, these environmental species behave as opportunist pathogens. Through the methodology used for Listeria isolation, one isolate of Salmonella Montevideo was obtained from a sample of pasteurized milk and another isolated from one sample of raw milk. Besides these, several other bacteria species were found. It is likely that the large microbiota present in the samples and the procedures employed to destroy it could have hindered the isolation of Y. enterocolitica and L. monocytogenes.

Visando complementar as informações sobre a qualidade microbiológica do leite comercializado na cidade do Recife, foram analisadas 250 amostras de leite pasteurizado tipo C e 50 amostras de leite cru para a pesquisa de Yersinia enterocolitica e Listeria monocytogenes, bactérias patogênicas capazes de se desenvolverem em temperatura de refrigeração. Y. enterocolitica não foi encontrada em nenhuma das amostras analisadas, entretanto foi detectada a presença de Y. intermedia e Y. frederiksenii, espécies ambientais que se comportam como patógenos oportunistas. L. monocytogenes também não foi encontrada, mas, através da metodologia empregada para seu isolamento foi obtido um isolamento de Salmonella Montevideo em uma amostra de leite pasteurizado e outro em leite cru. Além dessas, várias outras bactérias foram encontradas, supondo-se que a ampla microbiota crescida nos meios empregados pode ter interferido no isolamento da Y. enterocolitica e L. monocytogenes.

Acute attacks in the extremities of persons living in an area endemic for bancroftian filariasis: differentiation of two syndromes.

The natural history of lymphatic disease in human filariasis remains unclear, but recurrent episodes of acute lymphangitis are believed to constitute a major risk factor for the development of chronic lymphoedema and elephantiasis. Prospective analysis of 600 patients referred to the filariasis clinic of the Centro de Pesquisas Aggeu Magalhães/FIOCRUZ in Recife, Brazil, indicated that 2 distinct acute syndromes accompanied by lymphangitis occur in residents of this filariasis-endemic area. One syndrome, which we call acute filarial lymphangitis (AFL), is caused by the death of adult worms. It is relatively uncommon in untreated persons, usually is asymptomatic or has a mild clinical course, and rarely causes residual lymphoedema. The second syndrome, of acute dermatolymphangioadenitis (ADLA), is not caused by filarial worms per se, but probably results from secondary bacterial infections. ADLA is a common cause of chronic lymphoedema and elephantiasis in Recife as well as in other areas of Brazil where lymphatic filariasis is not present. The syndromes of AFL and ADLA can be readily distinguished from each other by simple clinical criteria.

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.

Ausência de Yersinia enterocolitica em alimentos, e reservatórios animais, em áreas do Estado de Pernambuco, Brasil / The absence of Yersinia enterocolitica in foods and animal reservoirs in areas of the state of Pernambuco, Brazil

Através das análises efetuadas, em 96 amostras de hortaliças cruas, coletadas em 5 restaurantes da cidade do Recife, que servem almoço no peso, não foram encontradas Yersinia enterocolitica nem outras enterobactérias patogênicas. As análises realizadas a partir dos "swabs" orais e retais, obtidos em 15 suínos aparentemente sadios do município de Bonito, no Estado de Pernambuco, também não evidenciaram a presença de Y. enterocolitica. Foram obtidas amostras para análises em 22 roedores e um espécimen de marsupial, entre os quais também não foram encontrados nem Y. enterocolitica nem outros enteropatógenos.

A search for the presence of enteropathogenic bacteria in fresh vegetables obtained in 5 restaurants from the city of Recife, revealed neither Yersinia enterocolitica nor other pathogenic bacteria in 96 samples analyzed. Furthermore, Y. enterocolitica was not found in the oral and rectal swabs taken from 15 apparently healthy pigs at an abattoir in the municipality of Bonito in the Pernambuco State. Another search in which twenty one rodents from four species and one marsupial specimen were examined did not detect the presence of Yersinia and other enteropathogenic bacteria.

[The absence of Yersinia enterocolitica in foods and animal reservoirs in areas of the state of Pernambuco, Brazil]. / Ausência de Yersinia enterocolitica em alimentos, e reservatórios animais, em áreas do Estado de Pernambuco, Brasil.

A search for the presence of enteropathogenic bacteria in fresh vegetables obtained in 5 restaurants from the city of Recife, revealed neither Yersinia enterocolitica nor other pathogenic bacteria in 96 samples analyzed. Furthermore, Y. enterocolitica was not found in the oral and rectal swabs taken from 15 apparently healthy pigs at an abattoir in the municipality of Bonito in the Pernambuco State. Another search in which twenty one rodents from four species and one marsupial specimen were examined did not detect the presence of Yersinia and other enteropathogenic bacteria.

A simple PCR-based procedure for plague diagnosis.

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.

Plague surveillance in Brazil: 1983-1992.

Plague caused by Yersinia pestis, has persisted in Brazil in several natural foci spread throughout rural areas in the States of Ceara, Paraiba, Pernambuco, Piaui, Rio Grande do Norte, Alagoas, Bahia, Minas Gerais and Rio de Janeiro. Nationwide surveillance of plague in Brazil based on serological testing started in 1983. We now present an update report of the examinations carried out in our laboratory from 1983 to 1992. The passive hemagglutination test for antibodies against fraction 1A antigen of Y. pestis and the passive hemagglutination inhibition control were employed for testing a total of 220,769 sera. Samples analyzed included 2,856 sera from clinically diagnosed plague cases or suspects, 49,848 sera from rodents of 24 species and 2 species of small wild carnivores (marsupials), 122,890 sera from dogs, and 45,175 sera from cats. Specific antibodies were found in 92 (3.22%) human sera; 143 (0.29%) sera from rodents of 8 species and from the two species of marsupials, 1,105 (0.90%) sera from dogs and 290 (0.64%) sera from cats. The presence of significant levels of specific anti-F1A antibodies among rodents and wild or domestic carnivores (dogs and cats) indicates that all the Brazilian plague foci remain active in spite of the absence of human cases in some of them.