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Determination of clonality and relatedness of Vibrio cholerae isolates by genomic fingerprinting, using long-range repetitive element sequence-based PCR.

ABSTRACT: A high-throughput method which is applicable for rapid screening, identification, and delineation of isolates of Vibrio cholerae, sensitive to genome variation, and capable of providing phylogenetic inferences enhances environmental monitoring of this bacterium. We have developed and optimized a method for genomic fingerprinting of V. cholerae based on long-range PCR. The method uses a primer set directed to enterobacterial repetitive intergenic consensus sequences, a high-fidelity DNA polymerase, and analysis via conventional agarose gel electrophoresis. Long ( approximately 10 kb), highly reproducible amplicons were generated from V. cholerae isolates, including those from different geographical locations and historical strains isolated during the period 1931-2000. The amplicons yielded reduced variability in their densitometric band patterns to /=90% similarity, discriminating O serotypes and biotypes (classical versus El Tor) as well as pathogenic and nonpathogenic strains. Compared to genome similarity measured by DNA-DNA hybridization, the results showed good correlation (r = 0.7; P < 0.001), with five times less measurement error and without bias. The method permits both phylogenetic inference and clonal differentiation of individual V. cholerae strains, enables robust, high-throughput analysis, and does not require specialized equipment to perform. With access to a curated public database furnished with appropriate analytical software applications, the method should prove useful in large-scale multilaboratory surveys, especially those designed to detect specific pathogens in the natural environment.

SUBMITTER: Chokesajjawatee N

PROVIDER: S-EPMC2546650 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Determination of clonality and relatedness of Vibrio cholerae isolates by genomic fingerprinting, using long-range repetitive element sequence-based PCR.

Chokesajjawatee Nipa N Zo Young-Gun YG Colwell Rita R RR

Applied and environmental microbiology 20080707 17

A high-throughput method which is applicable for rapid screening, identification, and delineation of isolates of Vibrio cholerae, sensitive to genome variation, and capable of providing phylogenetic inferences enhances environmental monitoring of this bacterium. We have developed and optimized a method for genomic fingerprinting of V. cholerae based on long-range PCR. The method uses a primer set directed to enterobacterial repetitive intergenic consensus sequences, a high-fidelity DNA polymeras ...[more]

PMID: 18606790

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Project description:Evidence suggests that a repertoire of Vibrio cholerae genes are differentially expressed in vivo, and regulation of virulence factors in vivo may follow a different pathway. Our work was aimed at characterization of in vivo-grown bacteria and identification of genes that are differentially expressed following infection by RNA arbitrarily primed (RAP)-PCR fingerprinting. The ligated rabbit ileal loop model was used. The motility of in vivo-grown bacteria increased by 350% over that of in vitro-grown bacteria. Also, the in vivo-grown cells were more resistant to killing by human serum. By using the RAP-PCR strategy, five differentially expressed transcripts were identified. Two in vitro-induced transcripts encoded polypeptides for the leucine tRNA synthatase and the 50S ribosomal protein, and the three in vivo-induced transcripts encoded the SucA and MurE proteins and a polypeptide of unknown function. MurE is a protein involved in the peptidoglycan biosynthetic pathway. The lytic profiles of in vivo- and in vitro-grown cells suspended in distilled water were compared; the former was found to be slightly less sensitive to lysis. Ultrathin sections of both cells observed under the transmission electron microscope revealed that in contrast to the usual wavy discontinuous membrane structure of the in vitro-grown cells, in vivo-grown cells had a more rigid, clearly visible double-layered structure. The V. cholerae murE gene was cloned and sequenced. The sequence contained an open reading frame of 1,488 nucleotides with its own ribosome-binding site. A plasmid containing the murE gene of V. cholerae was transformed into V. cholerae 569B, and a transformed strain, 569BME, containing the plasmid was obtained. Ultrathin sections of 569BME viewed under a transmission electron microscope revealed a slightly more rigid cell wall than that of wild-type 569B. When V. cholerae 569B and 569BME cells were injected separately into ligated rabbit ileal loops, the transformed cells had a preference for growth in the ileal loops versus laboratory conditions.

Project description:BACKGROUND:Household contacts of cholera patients have a 100 times higher risk of developing a cholera infection than the general population. To compare the genetic relatedness of clinical and water source Vibrio cholerae isolates from cholera patients' households across three outbreaks, we analyzed these isolates using whole-genome-sequencing (WGS) and multilocus variable-number tandem-repeat analysis (MLVA). RESULTS:The WGS analyses revealed that 80% of households had source water isolates that were more closely related to clinical isolates from the same household than to any other isolates. While in another 20% of households an isolate from a person was more closely related to clinical isolates from another household than to source water isolates from their own household. The mean pairwise differences in single nucleotide-variant (SNV) counts for isolates from the same household were significantly lower than those for different households (2.4 vs. 7.7 p < 0.0001), and isolates from the same outbreak had significantly fewer mean pairwise differences compared to isolates from different outbreaks (mean: 6.2 vs. 8.0, p < 0.0001). Based on MLVA in outbreak 1, we observed that the majority of households had clinical isolates with MLVA genotypes related to other clinical isolates and unrelated to water source isolates from the same household. While in outbreak 3, there were different MLVA genotypes between households, however within the majority of households, the clinical and water source isolates had the same MLVA genotypes. The beginning of outbreak 2 resembled outbreak 1 and the latter part resembled outbreak 3. We validated our use of MLVA by comparing it to WGS. Isolates with the identical MLVA genotype had significantly fewer mean pairwise SNV differences than those isolates with different MLVA genotypes (mean: 4.8 vs. 7.7, p < 0.0001). Furthermore, consistent with WGS results, the number of pairwise differences in the five MLVA loci for isolates within the same household was significantly lower than isolates from different households (mean: 1.6 vs. 3.0, p < 0.0001). CONCLUSION:These results suggest that transmission patterns for cholera are a combination of person-to-person and water-to-person cholera transmission with the proportions of the two modes varying within and between outbreaks.

Project description:BackgroundSome microorganisms can produce pigments such as melanin, which has been associated with virulence in the host and with a survival advantage in the environment. In Vibrio cholerae, studies have shown that pigment-producing mutants are more virulent than the parental strain in terms of increased UV resistance, production of major virulence factors, and colonization. To date, almost all of the pigmented V. cholerae strains investigated have been induced by chemicals, culture stress, or transposon mutagenesis. However, during our cholera surveillance, some nontoxigenic serogroup O139 strains and one toxigenic O1 strain, which can produce pigment steadily under the commonly used experimental growth conditions, were obtained in different years and from different areas. The genes VC1344 to VC1347, which correspond to the El Tor strain N16961 genome and which comprise an operon in the tyrosine catabolic pathway, have been confirmed to be associated with a pigmented phenotype. In the present study, we investigated the mechanism of pigment production in these strains.ResultsSequencing of the VC1344, VC1345, VC1346, and VC1347 genes in these pigmented strains suggested that a deletion mutation in the homogentisate oxygenase gene (VC1345) may be associated with the pigmented phenotype, and gene complementation confirmed the role of this gene in pigment production. An identical 15-bp deletion was found in the VC1345 gene of all six O139 pigment-producing strains examined, and a 10-bp deletion was found in the VC1345 gene of the O1 strain. Strict sequence conservation in the VC1344 gene but higher variance in the other three genes of this operon were observed, indicating the different stress response functions of these genes in environmental adaption and selection. On the basis of pulsed-field gel electrophoresis typing, the pigment-producing O139 strains showed high clonality, even though they were isolated in different years and from different regions. Additionally all these O139 strains belong to the rb4 ribotype, which contains the O139 strains isolated from diarrheal patients, although these strains are cholera toxin negative.ConclusionDysfunction of homogentisate oxygenase (VC1345) causes homogentisate accumulation and pigment formation in naturally pigmented strains of V. cholerae. The high clonality of these strains may correlate to an environmental survival advantage in the V. cholerae community due to their pigment production, and may imply a potential protective function of melanin in environmental survival of such strains.

Dataset Information

Determination of clonality and relatedness of Vibrio cholerae isolates by genomic fingerprinting, using long-range repetitive element sequence-based PCR.

Publications

Determination of clonality and relatedness of Vibrio cholerae isolates by genomic fingerprinting, using long-range repetitive element sequence-based PCR.

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