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1.
PLoS Negl Trop Dis ; 14(4): e0008128, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32236098

RESUMEN

Cholera continues to be an important public health concern in developing countries where proper hygiene and sanitation are compromised. This severe diarrheal disease is caused by the Gram-negative pathogen Vibrio cholerae belonging to serogroups O1 and O139. Cholera toxin (CT) is the prime virulence factor and is directly responsible for the disease manifestation. The ctxB gene encodes cholera toxin B subunit (CTB) whereas the A subunit (CTA) is the product of ctxA gene. Enzymatic action of CT depends on binding of B pentamers to the lipid-based receptor ganglioside GM1. In recent years, emergence of V. cholerae Haitian variant strains with ctxB7 allele and their rapid spread throughout the globe has been linked to various cholera outbreaks in Africa and Asia. These strains produce classical type (WT) CTB except for an additional mutation in the signal sequence region where an asparagine (N) residue replaces a histidine (H) at the 20th amino acid position (H20N) of CTB precursor (pre-CTB). Here we report that Haitian variant V. cholerae O1 strains isolated in Kolkata produced higher amount of CT compared to contemporary O1 El Tor variant strains under in vitro virulence inducing conditions. We observed that the ctxB7 allele, itself plays a pivotal role in higher CT production. Based on our in silico analysis, we hypothesized that higher accumulation of toxin subunits from ctxB7 allele might be attributed to the structural alteration at the CTB signal peptide region of pre-H20N CTB. Overall, this study provides plausible explanation regarding the hypertoxigenic phenotype of the Haitian variant strains which have spread globally, possibly through positive selection for increased pathogenic traits.


Asunto(s)
Alelos , Toxina del Cólera/genética , Cólera/microbiología , Genes Bacterianos/genética , Vibrio cholerae O1/genética , Técnicas de Tipificación Bacteriana , Cólera/epidemiología , Toxina del Cólera/química , Toxina del Cólera/metabolismo , Brotes de Enfermedades , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Haití/epidemiología , Humanos , ARN Bacteriano , Serogrupo , Virulencia/genética , Factores de Virulencia/genética
2.
Proc Natl Acad Sci U S A ; 117(14): 7897-7904, 2020 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-32229557

RESUMEN

The spread of cholera in the midst of an epidemic is largely driven by direct transmission from person to person, although it is well-recognized that Vibrio cholerae is also capable of growth and long-term survival in aquatic ecosystems. While prior studies have shown that aquatic reservoirs are important in the persistence of the disease on the Indian subcontinent, an epidemiological view postulating that locally evolving environmental V. cholerae contributes to outbreaks outside Asia remains debated. The single-source introduction of toxigenic V. cholerae O1 in Haiti, one of the largest outbreaks occurring this century, with 812,586 suspected cases and 9,606 deaths reported through July 2018, provided a unique opportunity to evaluate the role of aquatic reservoirs and assess bacterial transmission dynamics across environmental boundaries. To this end, we investigated the phylogeography of both clinical and aquatic toxigenic V. cholerae O1 isolates and show robust evidence of the establishment of aquatic reservoirs as well as ongoing evolution of V. cholerae isolates from aquatic sites. Novel environmental lineages emerged from sequential population bottlenecks, carrying mutations potentially involved in adaptation to the aquatic ecosystem. Based on such empirical data, we developed a mixed-transmission dynamic model of V. cholerae, where aquatic reservoirs actively contribute to genetic diversification and epidemic emergence, which underscores the complexity of transmission pathways in epidemics and endemic settings and the need for long-term investments in cholera control at both human and environmental levels.


Asunto(s)
Cólera/microbiología , Ecosistema , Filogenia , Vibrio cholerae O1/clasificación , Asia/epidemiología , Cólera/epidemiología , Cólera/genética , Cólera/patología , Brotes de Enfermedades , Genoma Bacteriano/genética , Haití/epidemiología , Humanos , Vibrio cholerae O1/genética , Vibrio cholerae O1/patogenicidad , Microbiología del Agua
3.
Zhonghua Yu Fang Yi Xue Za Zhi ; 54(3): 301-305, 2020 Mar 06.
Artículo en Chino | MEDLINE | ID: mdl-32187936

RESUMEN

Objective: To analyze the genomic recombination of the vibrio cholerae serogroup O1 El Tor pandemic strains. Methods: A total of 292 complete or draft genome sequences of Vibrio cholerae O1 serogroup El Tor strains isolated from 1937 to 2015 were selected from National Biotechnology Information Center database. The genome alignment of strains was computed by snippy software by using N16961 as reference sequence. Then ClonalFrameML software was used to do the recombinant analysis. The wilcox.test function in agricolae package was used to compare the number recombinant segments and the total length of recombinant regions between small and large chromosomes. The kruskal function was used to compare the number recombinant segments and the total length of recombinant regions among different isolation continents. The KOBAS tool was used to do the gene ontology enrichment analysis of recombinant hotspot genes. Results: Of all 292 strains of Vibrio cholerae, 163 strains (55.8%) were recombined. The median of normalized recombinant segment number of small chromosome was 4.7×10(-6) (9.3×10(-7), 2.0×10(-5)), which was significantly larger than that of large chromosome [2.4×10(-6) (3.4×10(-7), 5.7×10(-6))] (P<0.001). The median (P(25),P(75)) of recombinant segment number of strains isolated from Africa, Asia, Europe, North America and South America were 23(1.0,33.0), 1.0(0.0,34.0), 6.0(2.0,13.0), 0.0(0.0,1.0) and 29.5(6.8,56.8), respectively, and the difference was statistically significant (P<0.001). The median (P(25),P(75)) of total length of recombinant regions of strains isolated from Africa, Asia, Europe, North America and South America were 233.0(4.0, 461.0), 11.0(0.0, 695.5), 56.0(4.0,111.0), 0.0(0.0,9.0) and 347.5(132.8,1 323.5) bp, respectively, and the difference was statistically significant (P<0.001). Gene ontology Enrichment analysis showed that the functions of 62 recombinant hotspot genes were mainly enrichment in chemotaxis, taxis, response to external stimulus, receptor activity and molecular transducer activity. Conclustion: In this study, we found that there were significant differences in the number of recombinant fragments and the length of recombinant regions between large and small chromosomes of Vibrio cholerae El Tor. We also found significant differences in the number of recombinant fragments and the total length of recombinant regions among different continents.


Asunto(s)
Cólera/epidemiología , Genoma Viral , Pandemias , Recombinación Genética , Vibrio cholerae O1/genética , Cólera/diagnóstico , Cólera/microbiología , Variación Genética , Genómica , Humanos , Análisis de Secuencia de ADN , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/virología
4.
J Med Microbiol ; 69(3): 372-378, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31961790

RESUMEN

Introduction. The emergence of novel strains of Vibrio cholerae O1 El Tor biotype has gained attention due to causing several epidemics around the world. Variant strains have evolved as a result of the acquisition of genes that confer extended virulence and pathogenicity.Aim. This study aimed to determine the presence of the most recently emerging Haitian-like genetic traits among the isolates from Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, Southern India. We also wanted to detect the prevalence of the sulfamethoxazole and trimethoprim (SXT) element, which is an integrating conjugative element (ICE) and the antimicrobial resistance genes present in our isolates.Methodology. Identification of Haitian-specific alleles was done by mismatched amplification mutation assay PCR (MAMA-PCR). The presence of SXT elements was carried out by PCR by detecting int, eex, att-prfC and setR genes. Detection of antibiotic resistance determinant, sul(1,2,3); dfr(A1,18,5) for trimethoprim resistance, tet(A,B,C,D,E,Y,G,M), tet34 for tetracycline resistance and erm(A,B,C), mph(A,B), ere(A,B), msr(A,D) for azithromycin resistance were targeted by PCR. The MIC of tetracycline, ciprofloxacin and azithromycin was determined by the E-test method.Results. Of the 95 isolates, 60 % of the isolates were found to carry Haitian-specific alleles of ctxB, tcpA and rtxA gene, 100 % of the isolates were found to carry SXT elements. All the isolates harboured the four conserved genes of the SXT element, except one which had only eex, att-prfC, setR genes. About 99 % harboured sul2 and dfrA1 genes. No tet and macrolide genes were detected. We observed a progressive increase in the MIC of azithromycin ranging from 0.75 µg ml-1 to 2 µg ml-1.Conclusion. None of the isolates were the prototype El Tor biotype. All the isolates were a Haitian variant. The presence of SXT elements across all our isolates and their creeping MIC of azithromycin is a matter of concern. Further testing for other genetic determinants of resistance will be carried out in our future studies.


Asunto(s)
Antibacterianos/farmacología , Azitromicina/farmacología , Cólera/epidemiología , Farmacorresistencia Bacteriana/genética , Vibrio cholerae O1/genética , Alelos , Cólera/microbiología , Ciprofloxacino/farmacología , Heces/microbiología , Transferencia de Gen Horizontal , Genotipo , Haití , Humanos , India/epidemiología , Pruebas de Sensibilidad Microbiana , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Tetraciclina/farmacología , Vibrio cholerae O1/aislamiento & purificación
5.
Int J Antimicrob Agents ; 55(3): 105853, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31770631

RESUMEN

It is evident from previous cholera epidemics/outbreaks in India, Africa and America that isolates of the seventh pandemic Vibrio cholerae El Tor (7PET) with Haitian cholera toxin (HCT) genotype were associated with increased mortality. The present study highlights the emergence of 7PET-HCT isolates causing two cholera outbreaks in Walsang and Wagdari (Solapur, India) in 2016. Molecular analyses revealed that 7PET strains from earlier outbreaks (2010 and 2012) were progenitors of the current 7PET-HCT isolates. Isolates from the 2016 outbreaks carried qnrVC and floR genes and showed reduced susceptibility to tetracycline, ciprofloxacin and azithromycin, drugs recommended by the World Health Organization (WHO) for the treatment of cholera. Remarkably, protein profiling and mass spectrometry revealed disappearance of the outer membrane protein U (OmpU) porin in 7PET-HCT isolates from the second outbreak in 2016. Downregulation of ompU gene expression was also confirmed at the transcriptional level. Strains with downregulated OmpU showed reduced minimum inhibitory concentrations (MICs) for polymyxin B, which is a pore-forming antimicrobial agent. A multipronged approach is of utmost importance to prevent further spread of circulating 7PET-HCT strains. There is a pressing need for the formulation and implementation of international policies to closely monitor the effective use of antibiotics in order to prevent the further rise and spread of antimicrobial resistance.


Asunto(s)
Antibacterianos/farmacología , Cólera , Vibrio cholerae O1 , Antibacterianos/uso terapéutico , Cólera/tratamiento farmacológico , Cólera/epidemiología , Cólera/microbiología , Toxina del Cólera/genética , Brotes de Enfermedades , Haití , Humanos , Pruebas de Sensibilidad Microbiana , Vibrio cholerae O1/efectos de los fármacos , Vibrio cholerae O1/genética
6.
PLoS One ; 14(12): e0225848, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31825986

RESUMEN

BACKGROUND: Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is widely used by laboratory-based surveillance networks for subtyping pathogens causing foodborne and water-borne disease outbreaks. However, Whole Genome Sequencing (WGS) has recently emerged as the new more powerful reference for pathogen subtyping, making a data conversion method necessary which enables the users to compare the MLVA identified by either method. The MLVAType shiny application was designed to extract MLVA profiles of Vibrio cholerae isolates from WGS data while ensuring backward compatibility with traditional MLVA typing methods. METHODS: To test and validate the MLVAType algorithm, WGS-derived MLVA profiles of nineteen Vibrio cholerae isolates from Democratic Republic of the Congo (n = 9) and Uganda (n = 10) were compared to MLVA profiles generated by an in silico PCR approach and Sanger sequencing, the latter being used as the reference method. RESULTS: Results obtained by Sanger sequencing and MLVAType were totally concordant. However, the latter were affected by censored estimations whose percentage was inversely proportional to the k-mer parameter used during genome assembly. With a k-mer of 127, less than 15% estimation of V. cholerae VNTR was censored. Preventing censored estimation was only achievable when using a longer k-mer size (i.e. 175), which is not proposed in the SPAdes v.3.13.0 software. CONCLUSION: As NGS read lengths and qualities tend to increase with time, one may expect the increase of k-mer size in a near future. Using MLVAType application with a longer k-mer size will then efficiently retrieve MLVA profiles from WGS data while avoiding censored estimation.


Asunto(s)
Repeticiones de Minisatélite/genética , Vibrio cholerae O1/genética , Secuenciación Completa del Genoma , Algoritmos , Sitios Genéticos , Genoma Bacteriano , Uganda
7.
PLoS Negl Trop Dis ; 13(12): e0007934, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31869327

RESUMEN

BACKGROUND: Tanzania is one of seven countries with the highest disease burden caused by cholera in Africa. We studied the evolution of Vibrio cholerae O1 isolated in Tanzania during the past three decades. METHODOLOGY/PRINCIPAL FINDINGS: Genome-wide analysis was performed to characterize V. cholerae O1 responsible for the Tanzanian 2015-2017 outbreak along with strains causing outbreaks in the country for the past three decades. The genomes were further analyzed in a global context of 590 strains of the seventh cholera pandemic (7PET), as well as environmental isolates from Lake Victoria. All Tanzanian cholera outbreaks were caused by the 7PET lineage. The T5 sub-lineage (ctxB3) dominated outbreaks until 1997, followed by the T10 atypical El Tor (ctxB1) up to 2015, which were replaced by the T13 atypical El Tor of the current third wave (ctxB7) causing most cholera outbreaks until 2017 with T13 being phylogenetically related to strains from East African countries, Yemen and Lake Victoria. The strains were less drug resistant with approximate 10-kb deletions found in the SXT element, which encodes resistance to sulfamethoxazole and trimethoprim. Nucleotide deletions were observed in the CTX prophage of some strains, which warrants further virulence studies. Outbreak strains share 90% of core genes with V. cholerae O1 from Lake Victoria with as low as three SNPs difference and a significantly similar accessory genome, composed of genomic islands namely the CTX prophage, Vibrio Pathogenicity Islands; toxin co-regulated pilus biosynthesis proteins and the SXT-ICE element. CONCLUSION/SIGNIFICANCE: Characterization of V. cholerae O1 from Tanzania reveals genetic diversity of the 7PET lineage composed of T5, T10 and T13 sub-lineages with introductions of new sequence types from neighboring countries. The presence of these sub-lineages in environmental isolates suggests that the African Great Lakes may serve as aquatic reservoirs for survival of V. cholerae O1 favoring continuous human exposure.


Asunto(s)
Cólera/epidemiología , Cólera/microbiología , Epidemias , Genoma Bacteriano , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/genética , Evolución Molecular , Genotipo , Humanos , Epidemiología Molecular , Tanzanía/epidemiología , Vibrio cholerae O1/aislamiento & purificación
9.
Microbiol Immunol ; 63(9): 350-358, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31407393

RESUMEN

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975-1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.


Asunto(s)
Cólera/epidemiología , Cólera/microbiología , Brotes de Enfermedades , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/genética , Vibrio cholerae O1/aislamiento & purificación , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana/métodos , Toxina del Cólera/genética , ADN Bacteriano/genética , Genotipo , Técnicas de Genotipaje , Humanos , Kenia/epidemiología , Pruebas de Sensibilidad Microbiana , Fenotipo , Polimixina B/farmacología , Vibrio cholerae O1/efectos de los fármacos , Virulencia/genética , Factores de Virulencia/genética
10.
BMC Genomics ; 20(1): 360, 2019 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-31072300

RESUMEN

BACKGROUND: Toxigenic Vibrio cholerae serogroup O1 is the causative pathogen in the sixth and seventh cholera pandemics. Cholera toxin is the major virulent factor but other virulence and virulence-related factors play certain roles in the pathogenesis and survival in the host. Along with the evolution of the epidemic strains, the virulence-related genes also experience variation, gain and loss, and lead to genetic divergence in different strains. RESULTS: In this study, we analyzed the virulence-related gene profiles in the toxigenic serogroup O1 strains isolated from 1923 to 2015, the genomes of which were publicly available. The virulence-related genes of the V. cholerae O1 strains were annotated based on the Virulence Factors Database (VFDB). An average of 230.1 virulence-related genes per strain were identified; significant differences in the average numbers were found between the classical and El Tor biotypes, and increasing trends in the number of virulence-related genes along with the isolation years were observed in the El Tor biotype strains. A total of 176 homologs of virulence-related genes were found from these strains, of which 25 belonged to the core genes, suggesting their conservative and necessary roles in V. cholerae pathogenesis. We described the diversities of the homologs by defining gene sequence type, and illustrated its association with gene duplication; we found that gene duplication clearly increased the complexity of the gene sequence types in the core virulence-related genes. In addition, we provided virulence-related gene profiles whose genetic characteristic depend on the isolation years from the view of gene gain and loss, variation, gene duplication and gene sequence type number. CONCLUSIONS: Our study reveals the comprehensive variation dynamics of the virulence-related genes in toxigenic V. cholerae serogroup O1 during epidemics. The increasing trend for the virulence-related genes may suggest the evolutional advantage of strains by gaining virulence-related genes with diverse functional categories.


Asunto(s)
Proteínas Bacterianas/genética , Cólera/epidemiología , Variación Genética , Serogrupo , Vibrio cholerae O1/genética , Factores de Virulencia/genética , Cólera/microbiología , Humanos , Filogenia , Vibrio cholerae O1/aislamiento & purificación
11.
Mol Cell Probes ; 45: 65-67, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31082474

RESUMEN

A loop-mediated isothermal amplification assay was developed. It was designed for recognizing Vibrio cholerae O1/O139, where atpA, rfbN, and wfbR genes were adopted. The assay specifically detected the target with sensitivities of 5-67 copies per reaction in 1 h. The assay will aid rapid detection of the cholera bacterium.


Asunto(s)
Proteínas Bacterianas/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Vibrio cholerae O139/aislamiento & purificación , Vibrio cholerae O1/aislamiento & purificación , Cólera/diagnóstico , Monitoreo del Ambiente , Humanos , Límite de Detección , Vibrio cholerae O1/genética , Vibrio cholerae O139/genética
12.
Proc Natl Acad Sci U S A ; 116(13): 6226-6231, 2019 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-30867296

RESUMEN

The Bay of Bengal is known as the epicenter for seeding several devastating cholera outbreaks across the globe. Vibrio cholerae, the etiological agent of cholera, has extraordinary competency to acquire exogenous DNA by horizontal gene transfer (HGT) and adapt them into its genome for structuring metabolic processes, developing drug resistance, and colonizing the human intestine. Antimicrobial resistance (AMR) in V. cholerae has become a global concern. However, little is known about the identity of the resistance traits, source of AMR genes, acquisition process, and stability of the genetic elements linked with resistance genes in V. cholerae Here we present details of AMR profiles of 443 V. cholerae strains isolated from the stool samples of diarrheal patients from two regions of India. We sequenced the whole genome of multidrug-resistant (MDR) and extensively drug-resistant (XDR) V. cholerae to identify AMR genes and genomic elements that harbor the resistance traits. Our genomic findings were further confirmed by proteome analysis. We also engineered the genome of V. cholerae to monitor the importance of the autonomously replicating plasmid and core genome in the resistance profile. Our findings provided insights into the genomes of recent cholera isolates and identified several acquired traits including plasmids, transposons, integrative conjugative elements (ICEs), pathogenicity islands (PIs), prophages, and gene cassettes that confer fitness to the pathogen. The knowledge generated from this study would help in better understanding of V. cholerae evolution and management of cholera disease by providing clinical guidance on preferred treatment regimens.


Asunto(s)
Cólera/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Transferencia de Gen Horizontal , Genoma Bacteriano/genética , Vibrio cholerae/genética , Antibacterianos/farmacología , Conjugación Genética/genética , Elementos Transponibles de ADN/genética , Diarrea/microbiología , Evolución Molecular , Heces/microbiología , Variación Genética , Islas Genómicas/genética , Humanos , Imipenem/farmacología , India , Secuencias Repetitivas Esparcidas/genética , Fenotipo , Plásmidos/genética , Profagos/genética , Proteoma , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae/patogenicidad , Vibrio cholerae O1/genética , Vibrio cholerae O1/aislamiento & purificación , Vibrio cholerae O1/patogenicidad , Secuenciación Completa del Genoma
13.
BMC Infect Dis ; 19(1): 76, 2019 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-30665342

RESUMEN

BACKGROUND: Cholera has been endemic in Ghana since its detection in 1970. It has been shown that long-term survival of the bacteria may be attained in aquatic environments. Consequently, cholera outbreaks may be triggered predominantly in densely populated urban areas. We investigated clinical and environmental isolates of Vibrio cholerae O1 in Accra to determine their virulence genes, antibiotic susceptibility patterns and environmental factors maintaining their persistence in the environment. METHODS: Water samples from various sources were analyzed for the presence of V. cholerae O1 using culture methods. Forty clinical isolates from a previous cholera outbreak were included in the study for comparison. Antibiotic susceptibility patterns of the bacteria were determined by disc diffusion. Virulence genes were identified by analyzing genes for ctx, tcpA (tcpAEl Tor tcpACl), zot, ompW, rbfO1 and attRS using PCR. Physicochemical characteristics of water were investigated using standard methods. One-way ANOVA and student t - test were employed to analyze the relationship between physicochemical factors and the occurrence of V. cholerae O1. RESULTS: Eleven V. cholerae O1 strains were successfully isolated from streams, storage tanks and wells during the study period. All isolates were resistant to one or more of the eight antibiotics used. Multidrug resistance was observed in over 97% of the isolates. All isolates had genes for at least one virulence factor. Vibrio cholerae toxin gene was detected in 82.4% of the isolates. Approximately 81.8% of the isolates were positive for tcpAEl Tor gene, but also harbored the tcpAcl gene. Isolates were grouped into thirteen genotypes based on the genes analyzed. High temperature, salinity, total dissolved solids and conductivity was found to significantly correlate positively with isolation of V. cholerae O1. V. cholerae serotype Ogawa biotype El tor is the main biotype circulating in Ghana with the emergence of a hybrid strain. CONCLUSIONS: Multidrug resistant V. cholerae O1 with different genotypes and pathogenicity are present in water sources and co-exist with non O1/O139 in the study area.


Asunto(s)
Antibacterianos/farmacología , Cólera/microbiología , Agua Dulce/microbiología , Vibrio cholerae O1/efectos de los fármacos , Vibrio cholerae O1/patogenicidad , Toxina del Cólera/genética , Farmacorresistencia Bacteriana Múltiple/genética , Proteínas Fimbrias/genética , Genotipo , Ghana , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Ríos/microbiología , Vibrio cholerae O1/genética , Virulencia/genética , Factores de Virulencia/genética , Pozos de Agua
14.
Appl Environ Microbiol ; 85(3)2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30446560

RESUMEN

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. Vibrio mimicus occasionally produces CT, encoded by ctxAB on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in V. mimicus ctx-positive (ctx +) strains (i.e., V. mimicus strains containing ctx) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novel ctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized as an RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv array. Additionally, novel variants of tcpA and toxT, respectively, showing phylogenetic lineage to a clade of V. cholerae non-O1 and to a clade of V. cholerae non-O139, were identified. The V. mimicus strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked Vibrio seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region similar to those seen with some classical strains of V. cholerae O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the ctx + V. mimicus strains were clonally related. However, their in vitro CT production and in vivo toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental V. mimicus strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of V. cholerae hybrid strains.IMPORTANCE Natural diversification of CTXФ and ctxAB genes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains of V. cholerae This report, showing the occurrence of CTXET comprising a novel variant of ctxAB in V. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of V. cholerae Identification and cluster analysis of the newly discovered alleles of tcpA and toxT suggest their horizontal transfer from an uncommon clone of V. cholerae The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФEnv and of a CTXФET in V. mimicus probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.


Asunto(s)
Toxina del Cólera/metabolismo , Regulón , Vibrio cholerae O1/metabolismo , Vibrio mimicus/genética , Vibrio mimicus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cólera/microbiología , Toxina del Cólera/genética , Microbiología Ambiental , Evolución Molecular , Variación Genética , Humanos , Filogenia , Vibrio cholerae O1/genética , Vibrio mimicus/clasificación , Vibrio mimicus/aislamiento & purificación , Factores de Virulencia/genética , Factores de Virulencia/metabolismo
15.
Mol Genet Genomics ; 294(2): 417-430, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30488322

RESUMEN

Vibriocholerae, which is autochthonous to estuaries worldwide, can cause human cholera that is still pandemic in developing countries. A number of V. cholerae isolates of clinical and environmental origin worldwide have been subjected to genome sequencing to address their phylogenesis and bacterial pathogenesis, however, little genome information is available for V. cholerae isolates derived from estuaries, particularly in China. In this study, we determined the complete genome sequence of V. cholerae CHN108B (non-O1/O139 serogroup) isolated from the Yangtze River Estuary, China and performed comparative genome analysis between CHN108B and other eight representative V. cholerae isolates. The 4,168,545-bp V. cholerae CHN108B genome (47.2% G+C) consists of two circular chromosomes with 3,691 predicted protein-encoding genes. It has 110 strain-specific genes, the highest number among the eight representative V. cholerae whole genomes from serogroup O1: there are seven clinical isolates linked to cholera pandemics (1937-2010) and one environmental isolate from Brazil. Various mobile genetic elements (such as insertion sequences, prophages, integrative and conjugative elements, and super-integrons) were identified in the nine V. cholerae genomes of clinical and environmental origin, indicating that the bacterium undergoes extensive genetic recombination via lateral gene transfer. Comparative genomics also revealed different virulence and antimicrobial resistance gene patterns among the V. cholerae isolates, suggesting some potential virulence factors and the rising development of resistance among pathogenic V. cholerae. Additionally, draft genome sequences of multiple V. cholerae isolates recovered from the Yangtze River Estuary were also determined, and comparative genomics revealed many genes involved in specific metabolism pathways, which are likely shaped by the unique estuary environment. These results provide additional evidence of V. cholerae genome plasticity and will facilitate better understanding of the genome evolution and pathogenesis of this severe water-borne pathogen worldwide.


Asunto(s)
Cólera/genética , Genoma Bacteriano/genética , Vibrio cholerae O1/genética , Vibrio cholerae no O1/genética , Brasil , China , Cólera/microbiología , Elementos Transponibles de ADN/genética , Estuarios , Transferencia de Gen Horizontal/genética , Variación Genética , Genómica , Humanos , Anotación de Secuencia Molecular , Filogenia , Ríos , Serogrupo , Vibrio cholerae O1/patogenicidad , Vibrio cholerae no O1/patogenicidad , Virulencia/genética
16.
J Microbiol Immunol Infect ; 52(4): 563-570, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-29428381

RESUMEN

BACKGROUND: Cholera is an important health problem in Sabah, a Malaysian state in northern Borneo; however, Vibrio cholerae in Sabah have never been characterized. Since 2002, serogroup O1 strains having the traits of both classical and El Tor biotype, designated as atypical El Tor biotype, have been increasingly reported as the cause of cholera worldwide. These variants are believed to produce clinically more severe disease like classical strains. PURPOSE: The purpose of this study is to investigate the genetic diversity of V.cholerae in Sabah and whether V.cholerae in Sabah belong to atypical El Tor biotype. METHODS: ERIC-PCR, a DNA fingerprinting method for bacterial pathogens based on the enterobacterial repetitive intergenic consensus sequence, was used to study the genetic diversity of 65 clinical V.cholerae O1 isolates from 3 districts (Kudat, Beluran, Sandakan) in Sabah and one environmental isolate from coastal sea water in Kudat district. In addition, we studied the biotype-specific genetic traits in these isolates to establish their biotype. RESULTS: Different fingerprint patterns were seen in isolates from these three districts but one of the patterns was seen in more than one district. Clinical isolates and environmental isolate have different patterns. In addition, Sabah isolates harbor genetic traits specific to both classical biotype (ctxB-1, rstRCla) and El Tor biotype (rstRET, rstC, tcpAET, rtxC, VC2346). CONCLUSION: This study revealed that V.cholerae in Sabah were genetically diverse and were atypical El Tor strains. Fingerprint patterns of these isolates will be useful in tracing the origin of this pathogen in the future.


Asunto(s)
Cólera/microbiología , Variación Genética , Vibrio cholerae O1/genética , Dermatoglifia del ADN/métodos , ADN Bacteriano/aislamiento & purificación , Microbiología Ambiental , Genes Bacterianos/genética , Humanos , Malasia , Epidemiología Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Vibrio cholerae O1/aislamiento & purificación
17.
Microbiol Immunol ; 62(10): 635-650, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30211956

RESUMEN

Vibrio cholerae O1 causes cholera, and cholera toxin, the principal mediator of massive diarrhea, is encoded by ctxAB in the cholera toxin (CTX) prophage. In this study, the structures of the CTX prophage region of V. cholerae strains isolated during the seventh pandemic wave 1 in Asian countries were determined and compared. Eighteen strains were categorized into eight groups by CTX prophage region-specific restriction fragment length polymorphism and PCR profiles and the structure of the region of a representative strain from each group was determined by DNA sequencing. Eight representative strains revealed eight distinct CTX prophage regions with various combinations of CTX-1, RS1 and a novel genomic island on chromosome I. CTX prophage regions carried by the wave 1 strains were diverse in structure. V. cholerae strains with an area specific CTX prophage region are believed to circulate in South-East Asian countries; additionally, multiple strains with distinct types of CTX prophage region are co-circulating in the area. Analysis of a phylogenetic tree generated by single nucleotide polymorphism differences across 2483 core genes revealed that V. cholerae strains categorized in the same group based on CTX prophage region structure were segregated in closer clusters. CTX prophage region-specific recombination events or gain and loss of genomic elements within the region may have occurred at much higher frequencies and contributed to producing a panel of CTX prophage regions with distinct structures among V. cholerae pathogenic strains in lineages with close genetic backgrounds in the early wave 1 period of the seventh cholera pandemic.


Asunto(s)
Toxina del Cólera/genética , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción/genética , Polimorfismo de Nucleótido Simple/genética , Profagos/genética , Vibrio cholerae O1/genética , Asia/epidemiología , Cólera/epidemiología , Toxina del Cólera/clasificación , Cromosomas Bacterianos/genética , ADN Bacteriano , Genes Bacterianos/genética , Variación Genética , Islas Genómicas , Humanos , Pandemias , Análisis de Secuencia de ADN , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/aislamiento & purificación , Vibrio cholerae O1/patogenicidad
18.
Lett Appl Microbiol ; 67(4): 329-336, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29981154

RESUMEN

Whole genome sequencing was utilized to investigate the genomic profile of Vibrio cholerae O1 strains, isolated from symptomatic patients in a low-income urban area of Dhaka, Bangladesh. Comparative genomics using bioinformatics tools were applied to identify major virulence factors, biotype and antimicrobial resistance genes in three V. cholerae O1 strains (VC-1, 2 and 3) isolated from two case patients. A phylogenetic SNP (single nucleotide polymorphism)-based analysis was conducted to infer the relatedness to V. cholerae O1 strains isolated elsewhere. The V. cholerae strains were the El Tor variant carrying ctxB1 (standard classical genotype). SNP-based global phylogeny revealed that the three isolates were strictly clonal and the closest neighbouring genomes were epidemic clones of V. cholerae O1 isolated in 2010 from cholera patients in Pakistan. All strains harboured the integrase gene of the SXT element (intSXT ), antimicrobial resistance genes for aminoglycosides, phenicol, sulphonamide and trimethoprim except VC-1 that lacked sulphonamide resistance genes. The multilocus sequence typing (MLST) revealed that the strains belonged to sequence type, ST69. The study provides knowledge on current genetic traits of clinical V. cholerae O1 circulating in urban household clusters of Bangladesh which may help in predicting emergence of new pandemic strains in Bangladesh. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio cholerae has frequently experienced genetic changes with rapid evolution of pandemic clones in the Ganges Delta region. Whole genome sequencing can reveal genetic information of current pathogenic V. cholerae in Bangladesh which includes cefotaxime genotypes, virulence factors, altered antimicrobial resistance pattern as well as mobile genetic element compared to global pandemic strains. This study data could be used in planning future surveillance strategies in Ganges Delta region by informing new epidemiology of current outbreak strains.


Asunto(s)
Cólera/epidemiología , Cólera/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Genoma Bacteriano/genética , Vibrio cholerae O1 , Adulto , Aminoglicósidos/farmacología , Antibacterianos/farmacología , Bangladesh/epidemiología , Preescolar , Toxina del Cólera/genética , Brotes de Enfermedades , Femenino , Genómica/métodos , Genotipo , Humanos , Tipificación de Secuencias Multilocus , Filogenia , Polimorfismo de Nucleótido Simple/genética , Sulfonamidas/farmacología , Trimetoprim/farmacología , Vibrio cholerae O1/efectos de los fármacos , Vibrio cholerae O1/genética , Vibrio cholerae O1/aislamiento & purificación , Secuenciación Completa del Genoma/métodos , Adulto Joven
19.
Environ Microbiol Rep ; 10(5): 594-604, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30058121

RESUMEN

Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the acute diarrheal disease cholera. Entry into a viable but non-culturable (VBNC) state is a survival strategy by which V. cholerae withstands natural stresses and is important for the transition between the aquatic and host environments during the V. cholerae life cycle. In this study, the formation of VBNC V. cholerae induced by cold seawater exposure was investigated using RNA sequencing (RNA-seq). The analysis revealed that the expression of 1420 genes was changed on VBNC state formation. In the VBNC cells, genes related to biofilm formation, chitin utilization and stress responses were upregulated, whereas those related to cell division, morphology and ribosomal activity were mainly downregulated. The concurrent acquisition of a carbon source and the arrest of cell division in cells with low metabolic activity help bacteria increase their resistance to unfavourable environments. Moreover, two transcriptional regulators, SlmA and MetJ, were found to play roles in both VBNC formation and intestinal colonization, suggesting that some genes may function in both processes. This acquired knowledge will improve our understanding of the molecular mechanisms of stress tolerance and may help control future cholera infections and outbreaks.


Asunto(s)
Frío , Regulación Bacteriana de la Expresión Génica , Agua de Mar , Vibrio cholerae O1/crecimiento & desarrollo , Vibrio cholerae O1/genética , Animales , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Intestinos/microbiología , Ratones , Mutación , ARN Bacteriano/genética , Estrés Fisiológico/genética
20.
PLoS Negl Trop Dis ; 12(6): e0006492, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29864113

RESUMEN

BACKGROUND: For almost 50 years sub-Saharan Africa, including Uganda, has experienced several outbreaks due to Vibrio cholerae. Our aim was to determine the genetic relatedness and spread of strains responsible for cholera outbreaks in Uganda. METHODOLOGY/PRINCIPAL FINDINGS: Sixty-three V. cholerae isolates collected from outbreaks in Uganda between 2014 and 2016 were tested using multiplex polymerase chain reaction (PCR), multi-locus variable number of tandem repeat analysis (MLVA) and whole genome sequencing (WGS). Three closely related MLVA clonal complexes (CC) were identified: CC1, 32% (20/63); CC2, 40% (25/63) and CC3, 28% (18/63). Each CC contained isolates from a different WGS clade. These clades were contained in the third wave of the 7th cholera pandemic strain, two clades were contained in the transmission event (T)10 lineage and other in T13. Analysing the dates and genetic relatedness revealed that V. cholerae genetic lineages spread between districts within Uganda and across national borders. CONCLUSION: The V. cholerae strains showed local and regional transmission within Uganda and the East African region. To prevent, control and eliminate cholera, these countries should implement strong cross-border collaboration and regional coordination of preventive activities.


Asunto(s)
Cólera/epidemiología , Cólera/microbiología , Epidemias , Vibrio cholerae O1/genética , Cólera/transmisión , Estudios Transversales , Genotipo , Humanos , Repeticiones de Minisatélite , Tipificación de Secuencias Multilocus , Reacción en Cadena de la Polimerasa Multiplex , Filogenia , Uganda/epidemiología , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/aislamiento & purificación , Secuenciación Completa del Genoma/métodos
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