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.

Serotypes of Vibrio cholerae Non-O1 Isolated from Water Supplies for Human Consumption in Campeche, México and their Antibiotic Susceptibility Pattern

The presence of Vibrio cholerae non-O1 in water supplies for human consumption in the city of Campeche and rural locality of Bécal was investigated. V. cholerae non-O1 was detected in 5.9% of the samples obtained in deep pools of Campeche. Studies conducted in Bécal and neighbourhood of Morelos in Campeche indicated that collected samples harbored V. cholerae non-O1 in 31.5% and 8.7% respectively. There was a particular pattern of distribution of V. cholerae non-O1 serotypes among different studied regions. Accordingly, V. cholerae non-O1 serotype O14 predominated in the deep pools of Campeche and together with V. cholerae non-O1, O155 were preferentially founds in samples taken from intradomiciliary faucets in the neighbourhood of Morelos. Samples from Bécal predominantly presented the serotype O112. 60% and 53.8% of all studied strains of V. cholerae non-O1 proved to be resistant to ampicillin and carbenicillin. 3.1%, 7.7% and 6.2% presented resistant to doxycycline, trimethoprim-sulfamethoxazole and erythromycin respectively. The study showed the necessity of performing a strong epidemiologic surveillance for emergence and distribution of V. cholerae non-O1

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.

Detection of viable and viable nonculturable Vibrio cholerae O1 through cultures and immunofluorescence in the Tucumán rivers, Argentina

Vibrio cholerae has been sporadically isolated from rivers in Tucumán, Argentina, since the outbreak in 1991. The aim of this study was to determine the environmental reservoir of the bacterium in these rivers, assessing the presence of Vibrio cholerae non-O1 and O1 (the latter both in its viable culturable and non culturable state) and its relationship to environmental physicochemical variables. 18 water samplings were collected in the Salí River (in Canal Norte and Banda) and the Lules River between 2003 and 2005. Physical-chemical measurements (pH, water temperature, electrical conductivity and dissolved oxygen) were examined. Vibrio cholerae was investigated with conventional culture methods and with Direct Immunofluorescence (DFA-VNC) in order to detect viable non culturable organisms. All isolated microorganisms corresponded to Vibrio cholerae non-O1 and non-O139 (Lules 26%, Canal Norte 33% and Banda 41%). The majority was found during spring and summer and correlated with temperature and pH. Non culturable Vibrio cholerae O1 was detected year round in 38 of the 54 water samples analyzed. Application of the Pearson correlation coefficient revealed that there was no relationship between positive immunofluorescence results and environmental physicochemical parameters. Genes coding for somatic antigen O1 were confirmed in all DFA-VNC-positive samples, whereas the virulence-associated ctxA and tcpA genes were confirmed in 24 samples.

Estudo molecular de Vibrio cholerae não-O1 isolado de zooplâncton da Baía de São Marcos/São Luis - MA, Brasil

O estudo foi desenvolvido com o objetivo de analisar o perfil plasmidial, pesquisar genes de virulência e identificar os perfis genéticos de 31 cepas de Vibrio cholerae não O1 isoladas de zooplâncton dos estuários dos rios Anil e Bacanga em São Luis MA. O estudo do DNA plasmidial revelou a presença de 2 a 3 plasmídeos em 10 cepas, com pesos moleculares variando de 5,5 a 40 kilobases. A ribotipagem revelou um perfil comum a todas as cepas. A amplificação do DNA genômico por PCR não revelou os genes ctxA, ace e zot, mostrando tratar-se de cepas não patogênicas, enquanto a RAPD-PCR identificou múltiplos perfis genéticos, achado compatível com o grande potencial de variabilidade desta espécie.

Occurrence and composition of class 1 and class 2 integrons in clinical and environmental O1 and non-O1/non-O139 Vibrio cholerae strains from the Brazilian Amazon

This study identified and characterised class 1 and 2 integrons in clinical and environmental Vibrio cholerae O1 and non-O1/non-O139 strains isolated from the Brazilian Amazon. The aadA2 and aadA7 gene cassettes were found in class 1 integrons in two genotypes of environmental V. cholerae non-O1/non-O139. Empty integrons were found in strains from the Brazilian cholera epidemic. A class 2 integron was detected in one strain from the V. cholerae Amazonia lineage harbouring sat1 and aadA1 genes. All isolates were resistant to aminoglycosides, indicating aadA functionality. These findings suggest that environmental bacteria act as cassette reservoirs that favour the emergence of resistant pathogens.

SURVIVAL, INDUCTION AND RESUSCITATION OF Vibrio cholerae FROM THE VIABLE BUT NON-CULTURABLE STATE IN THE SOUTHERN CARIBBEAN SEA

The causative agent of cholera, Vibrio cholerae, can enter into a viable but non-culturable (VBNC) state in response to unfavorable conditions. The aim of this study was to evaluate the in situ survival of V. cholerae in an aquatic environment of the Southern Caribbean Sea, and its induction and resuscitation from the VBNC state. V. cholerae non-O1, non-O139 was inoculated into diffusion chambers placed at the Cuare Wildlife Refuge, Venezuela, and monitored for plate, total and viable cells counts. At 119 days of exposure to the environment, the colony count was < 10 CFU/mL and a portion of the bacterial population entered the VBNC state. Additionally, the viability decreased two orders of magnitude and morphological changes occurred from rod to coccoid cells. Among the aquatic environmental variables, the salinity had negative correlation with the colony counts in the dry season. Resuscitation studies showed significant recovery of cell cultivability with spent media addition (p < 0.05). These results suggest that V. cholerae can persist in the VBNC state in this Caribbean environment and revert to a cultivable form under favorable conditions. The VBNC state might represent a critical step in cholera transmission in susceptible areas.

Caracterização molecular de Vibrio cholerae O1 sacarose negativa de isolados clínicos e ambiente na Amazônia brasileira

O V.cholerae é um microorganismo autóctone do ambiente aquático e os sorogrupos O1 e O 139 estão ligados a pandemia e epidemia de cólera. Os V.cholerae não O1 e não O139 ou vibrios não aglutinantes (NAGs) estão envolvidos em casos isolados e surtos de diarréia semelhantes à cólera. No decorrer da sétima pandemia houve o surgimento de diversos isolados “El Tor atípicos”. Entre estes se encontra a variante bioquímica do V.cholerae O1 que não fermenta a sacarose no TCBS em 18 a 24 horas que é o tempo de incubação convencional. Neste trabalho foram estudados 138 isolados de V.cholerae O1 e não O1 não fermentador da sacarose no TCBS de procedência clínica e ambiental, obtidos entre 1994 e 1995 na Amazônia Brasileira (Estados do Pará, Amapá e Amazonas). Avaliou-se a fermentação da sacarose no TCBS e em caldo; o perfil de suscetibilidade a oito diferentes antimicrobianos em ágar difusão; a relação clonal entre os V.cholerae O1 e NAG clínicos e ambientais pelo PFGE e a presença de genes de virulência ctxAB e tcpA pela reação em cadeia da polimerase. Observou-se que as amostras de V.cholerae não fermentaram a sacarose em 24 de incubação no ágar TCBS e em caldo, 43% utilizaram a sacarose em 24 horas e 57% a fermentavam tardiamente (tempo superior a 24 horas). Os isolados apresentaram baixo percentual de resistência a antimicrobianos (8,7%) e nenhum caso de multiresistência. Em relação aos genes de virulência, de um modo geral, os isolados de V.cholerae O1 apresentavam o tcpA e o ctxAB. Nos não O1 estes estavam ausentes, com exceção de um isolado clínico não O1 (gene tcpA+). A análise do PFGE revelou pulsotipos distintos entre os O1 e NAGs, embora dois destes últimos tenham apresentado relação clonal com os O1 clínicos. Todos os O1 clínicos apresentaram relação clonal com isolados de referência da sétima pandemia.

Diversidade genética de isolados ambientais de Vibrio cholerae da Amazônia brasileira

Vibrio cholerae, agente etiológico da cólera, é uma bactéria nativa de ambientes aquáticos de regiões temperadas e tropicais em todo o mundo. A cólera é endemica e epidemica em países da África, Ásia e Americas Central e do Sul. Neste trabalho o objetivo foi estudar a diversidade genética de isolados desta espécie, de ambientes aquáticos da Amazônia brasileira. Um total de 148 isolados de V.cholerae não-O1 e não-O139 (NAGs) e O1 ambientais da Amazônia, obtidos entre 1977 e 2007, foram caracterizados e comparados a linhagens clínicas de V.cholerae O1 da sexta e sétima pandemias. Utilizou-se os perfis de macrorestrição definidos em eletroforese em gel de agarose em campo pulsado (PFGE), para determinar a relação clonal entre V.cholerae non-O1 e O1 ambientais e clínicos. A presença de genes de virulência (hlyA/hem, hlyB, hlyC, rtxA, rtxC, tcp, ctx, zot, ace, stn/sto) e integrons de classe 1, 2 e 3 (intI 1, 2 e 3), foi analisada utilizando-se a reação em cadeia da polimerase. A análise dos perfis de macrorestrição revelou que os NAGs apresentaram uma grande diversidade genética comparada aos V.cholerae O1. Isolados de NAGs e O1 segregaram em distintos grupos e a maioria dos O1 ambientais apresentou relação clonal com isolados clínicos da sétima pandemia de cólera. A distribuição dos genes de virulência entre os NAGs é diferente a dos O1, os quais, em geral, foram positivos para todos os genes de virulência estudados exceto stn/sto e integrons de classe 1, 2 e 3. Alguns V.cholerae O1 ambientais pertencentes a linhagem da sétima pandemia, apresentaram uma extensiva perda de genes. Diferentes NAGs foram stn/sto+ e intI 1+. Dois alelos do gene aadA foram encontrados: aadA2 e aadA7. De modo interessante os V.cholerae O1 ambientais pertencentes à linhagem pandêmica, só foram isolados durante o período da última epidemia de cólera na região Amazônica brasileira (1991-1996).

Associação de Vibrio cholerae com o zooplâncton de águas estuárias da Baía de São Marcos/São Luis - MA, Brasil

Foi investigado, no período de outubro de 1997 a outubro de 1998, a possível associação de Vibrio cholerae com o zooplâncton dos estuários dos rios Anil e Bacanga, em São Luis - MA, Brasil, a presença da forma viável, mas não cultivável de Vibrio cholerae O1 e a correlação entre pH, salinidade e temperatura da água com a sobrevivência da bactéria. Amostras de zooplâncton foram coletadas em dois pontos fixos em cada estuário. O método clássico de isolamento e imunofluorescência direta foram empregados na detecção da bactéria. Nas 52 amostras obtidas de zooplâncton houve predomínio de Copepodes. O cultivo permitiu a obtenção de 55 isolados de Vibrio cholerae não O1. Os sorogrupos O1 e O139 foram demonstrados, respectivamente em 37 (71,1%) e 17 (32,7%) na imunofluorescência. Formas viáveis, mas não cultiváveis de Vibrio cholerae O1 foram detectadas em 70,8% das amostras estudadas. Correlação significativa foi constatada entre salinidade e pH da água e isolamento de Vibrio cholerae.

OCCURRENCE OF PATHOGENIC MICROORGANISMS IN FISH SOLD IN SAO PAULO, BRAZIL

This study investigated the presence of potentially human pathogenic strains of Vibrio spp., Aeromonas spp., Escherichia coli, Salmonella spp. and Staphylococcus aureus in fish commercialized in street markets of Sao Paulo city, Brazil. Twenty fish of different species were analyzed for foodborne pathogens using conventional methods. High levels of fecal contamination were detected in 25% of samples. S. aureus was isolated from 10% of samples. All were negative for Salmonella. Vibrio species, including Vibrio cholerae non-O1/non-O139, were observed in 85% of samples although Vibrio parahaemolyticus was not found in this study. Aeromonas spp., including A. hydrophila, was isolated from 50% of fish samples. The occurrence of these pathogens suggests that the fish commercialized in Sao Paulo may represent a health risk to the consumers.

Ballast water: a review of the impact on the world public health

Since the nineteenth century ships have been using ballast water (BW) for safety, stability, propulsion and maneuverability, as well as to redress loss of fuel weight and water consumption, and to maintain structural stress at acceptable levels. Ballast water has been spreading many non-native species around the globe, but little is known about the extent and potential significance of ship-mediated transfer of microorganisms. The global movements of ballast water by ships create a long-distance dispersal mechanism for human pathogens that may be important in the worldwide distribution of microorganisms, as well as for the epidemiology of waterborne diseases. Only a few studies have been carried out on this subject, most of them involving ballast water containing crustacean larvae and phytoplankton. Specialized microbiological studies on these waters are necessary to avoid a repeat of what happened in 1991, when epidemic cholera was reported in Peru and rapidly spread through Latin America and Mexico. In July of 1992, Vibrio cholerae was found in the USA and the Food and Drug Administration (FDA) determined that it came from ballast water of ships whose last port of call was in South America. In Brazil, just a few studies about the subject have been performed. An exploratory study by the Brazilian National Health Surveillance Agency (Agencia Nacional de Vigilancia Sanitaria - ANVISA) found in ballast water different microorganisms, such as fecal coliforms, Escherichia coli, Enterococcus faecalis, Clostridium perfringens, coliphages, Vibrio cholerae O1 and Vibrio cholerae non-O1. Until now, Brazil has been focusing only on organisms transported to its territory from other countries by ballast water, to avoid their establishment and dissemination in Brazilian areas. Studies that can assess the probability that water ballast carries pathogenic microorganisms are extremely important, as is the examination of ships that arrive in the country. Treatment of the human infections caused by BW exists but none is completely safe and efficient.

The neuraminidase gene is present in the non-toxigenic Vibrio cholerae Amazonia strain: a different allele in comparison to the pandemic strains

The neuraminidase gene, nanH, is present in the O1, non-toxigenic Vibrio cholerae Amazonia strain. Its location has been assigned to a 150 kb NotI DNA fragment, with the use of pulsed-field gel electrophoresis and DNA hybridization. This NotI fragment is positioned inside 630 kb SfiI and 1900 kb I-CeuI fragments of chromosome 1. Association of the pathogenicity island VPI-2, carrying nanH and other genes, with toxigenic strains has been described by other authors. The presence of nanH in a non-toxigenic strain is an exception to this rule. The Amazonia strain nanH was sequenced (Genbank accession No. AY825932) and compared to available V. cholerae sequences. The sequence is different from those of pandemic strains, with 72 nucleotide substitutions. This is the first description of an O1 strain with a different nanH allele. The most variable domain of the Amazonia NanH is the second lectin wing, comprising 13 out of 17 amino acid substitutions. Based on the presence of nanH in the same region of the genome, and similarity of the adjacent sequences to VPI-2 sequences, it is proposed that the pathogenicity island VPI-2 is present in this strain.

Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139.

The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.