RESUMO
V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no-clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and “in vivo” studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.
En este trabajo se analizó la presencia y expresión de genes de virulencia en V. cholerae no-O1 no-O139 de origen clínico y ambiental, aislados en Córdoba, Argentina. La mayoría de las cepas estudiadas contiene los genes toxR y hlyA, pero no ctxA, zot, ace, tcpA y stn. Se analizó la actividad hemolítica y citotóxica de estas cepas en los sobrenadantes de cultivo, así como su potencial enterotóxico en ensayos de asa ileal ligada de conejo. Además, los aislamientos fueron comparados por sus perfiles genéticos en PFGE. Las cepas del medio ambiente mostraron variación en su fenotipo de virulencia y no mostraron relación clonal. Las cepas clínicas fueron muy enterotóxicas, hemolíticas, proteolíticas y mostraron perfiles indistinguibles de PFGE, aunque mostraron diferencias en su actividad citotóxica. En este trabajo se describen por primera vez, utilizando ensayos de cultivo celular e “in vivo”, propiedades de virulencia de V. cholerae no-O1 no-O139 aislados en Argentina.
Assuntos
Animais , Humanos , Coelhos , Vibrio cholerae não O1/patogenicidade , Argentina/epidemiologia , Técnicas de Tipagem Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/fisiologia , Chlorocebus aethiops , Células COS/microbiologia , Toxina da Cólera/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Diarreia/epidemiologia , Diarreia/microbiologia , Eletroforese em Gel de Campo Pulsado , Enterotoxinas/genética , Enterotoxinas/isolamento & purificação , Enterotoxinas/fisiologia , Deleção de Genes , Genes Bacterianos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/fisiologia , Metaloendopeptidases/genética , Metaloendopeptidases/isolamento & purificação , Metaloendopeptidases/fisiologia , Filogenia , Vibrioses/epidemiologia , Vibrioses/microbiologia , Vibrio cholerae não O1/efeitos dos fármacos , Vibrio cholerae não O1/genética , Vibrio cholerae não O1/isolamento & purificação , Virulência/genética , Microbiologia da ÁguaRESUMO
V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and "in vivo" studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.
Assuntos
Vibrio cholerae não O1/patogenicidade , Animais , Argentina/epidemiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/fisiologia , Técnicas de Tipagem Bacteriana , Células COS/microbiologia , Chlorocebus aethiops , Toxina da Cólera/genética , DNA Bacteriano/genética , Diarreia/epidemiologia , Diarreia/microbiologia , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Enterotoxinas/genética , Enterotoxinas/isolamento & purificação , Enterotoxinas/fisiologia , Deleção de Genes , Genes Bacterianos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/fisiologia , Humanos , Metaloendopeptidases/genética , Metaloendopeptidases/isolamento & purificação , Metaloendopeptidases/fisiologia , Filogenia , Coelhos , Vibrioses/epidemiologia , Vibrioses/microbiologia , Vibrio cholerae não O1/efeitos dos fármacos , Vibrio cholerae não O1/genética , Vibrio cholerae não O1/isolamento & purificação , Virulência/genética , Microbiologia da ÁguaRESUMO
V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and [quot ]in vivo[quot ] studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.
RESUMO
Vibrio ordalii is a major cause of vibriosis in wild and cultured marine salmonids and carries pMJ101, a 30-kb cryptic plasmid that replicates in the absence of DNA polymerase I without producing single-stranded intermediates. A recombinant derivative harboring the pMJ101 replication region proved to be compatible with pJM1, a plasmid containing the iron acquisition system required for the virulence of V. anguillarum 775, another important pathogen that causes vibriosis. Sequence analysis of a 1.56-kb fragment harboring the pMJ101 replication region revealed the presence of typical features found in DNA origins including an AT-rich region, 11 dam-methylation sites of which 5 are within the putative ori region, and five copies of the 9-bp consensus sequence for DnaA binding. Gel retardation assays demonstrated that the latter replication element indeed binds DnaA purified from Escherichia coli. A potential open reading frame encoding a hydrophilic protein with a predicted pI of 10.3 and an M(r) of 33,826 was found adjacent to the ori region. Although these properties are typical of DNA-binding proteins, no significant homology was found between this predicted protein, named RepM, and other previously characterized proteins. Reverse transcriptase-polymerase chain reaction analysis of total RNA demonstrated the presence of repM mRNA in V. ordalii. The major initiation site of this mRNA was located 187 nucleotides upstream of the GTG initiation codon as determined by nuclease S1 protection assays. This transcription initiation site is preceded by putative -10 and -35 promoter sequences that control the expression of the repM replication gene. These results demonstrate that the replication region of pMJ101 shares some structural and sequence similarities with other DNA replication regions, which include DnaA binding and methylation sites and an open reading frame encoding a distinct protein required for its replication.
Assuntos
Plasmídeos/genética , Replicon , Vibrio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Primers do DNA/genética , Replicação do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Origem de Replicação , Salmonidae/microbiologia , Vibrio/metabolismo , Vibrio/patogenicidadeRESUMO
The 30-kb pMJ101 plasmid is found as a high-copy-number pool in all the pathogenic strains of Vibrio ordalii examined so far. The replication functions of pMJ101 were localized within a 2.4-kb EcoRV-HindIII restriction fragment by using different subclones in combination with Bal31 exonuclease deletions and Tn5 insertion mutants. Recombinant clones carrying this fragment were able to replicate in Escherichia coli cells deficient in either DNA Polymerase I (PolA-) or integration host factor functions. However, the viability of recombinant plasmids containing the pMJ101 origin of replication was dependent on the expression of the gene encoding the DnaA protein. Electrophoretic analysis of plasmid-encoded proteins in an in vitro transcription-translation coupled system revealed that the replication region of pMJ101 encodes a 36-kDa protein. The expression of this protein was correlated with the ability of different recombinant plasmids harboring this pMJ101 DNA region to replicate in the PolA- E. coli strain. Replication typing showed that pMJ101 is not related to any of the plasmid incompatibility groups contained in the bank of rep probes described by M. Couturier et al. (Microbiol. Rev. 52, 375-395, 1988).
