A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic Vibrio cholerae O1 and O139 Postexposure to Simulated Gastric Fluid.

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic Vibrio cholerae O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a "stressed state" due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI.

Recent Vibrio cholerae O1 Epidemic Strains Are Unable To Replicate CTXΦ Prophage Genome.

Cholera, an acute diarrheal disease, is caused by pathogenic strains of Vibrio cholerae generated by the lysogenization of the filamentous cholera toxin phage CTXΦ. Although CTXΦ phage in the classical biotype are usually integrated solitarily or with a truncated copy, those in El Tor biotypes are generally found in tandem and/or with related genetic elements. Due to this structural difference in the CTXΦ prophage array, the prophage in the classical biotype strains does not yield extrachromosomal CTXΦ DNA and does not produce virions, whereas the El Tor biotype strains can replicate the CTXΦ genome and secrete infectious CTXΦ phage particles. However, information on the CTXΦ prophage array structure of pathogenic V. cholerae is limited. Therefore, we investigated the complete genomic sequences of five clinical V. cholerae isolates obtained in Kolkata (India) during 2007 to 2011. The analysis revealed that recent isolates possessed an altered CTXΦ prophage array of the prototype El Tor strain. These strains were defective in replicating the CTXΦ genome. All recent isolates possessed identical rstA and intergenic sequence 1 (Ig-1) sequences and comparable rstA expression in the prototype El Tor strain, suggesting that the altered CTXΦ array was responsible for the defective replication of the prophage. Therefore, CTXΦ structures available in the database and literatures can be classified as replicative and nonreplicative. Furthermore, V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a significant change in the dissemination pattern of the current cholera pandemic occurred. IMPORTANCE Cholera is an acute diarrheal disease caused by pathogenic strains of V. cholerae generated by lysogenization of the filamentous cholera toxin phage CTXΦ. The analysis revealed that recent isolates possessed altered CTXΦ prophage array of prototype El Tor strain and were defective in replicating the CTXΦ genome. Classification of CTXΦ structures in isolated years suggested that V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a critical change had occurred in the dissemination pattern of the current cholera pandemic.

Investigation of an imported cholera case in China with whole genome sequencing.

Determining the source and genetic characteristics of the imported pathogen is critical in the control of infectious diseases. Here, we reported the investigation of an imported cholera case in China in 2018 with a recent travel history in Nepal and India. Stool culture from the patient was identified as Vibrio cholerae serogroup O1, biotype El Tor, serotype Ogawa. The strain 2018HL24 possessed intact Vibrio seventh pandemic island I (VSP-I), Vibrio pathogenicity Island 1 and 2 (VPI-1, VPI-2). A VSP-II variant with a 13 kb deletion was also detected, which was identical to those observed in V. cholerae in cluster "Nepal-4". Phylogenetic analysis based on the core genome SNPs showed that the isolate was most closely related to the V. cholerae isolated in northern India not far from the border of Nepal in 2012 (16 SNPs). Combining the epidemiological data with phylogenetic analysis results, we speculate that the patient may got infected in Nepal-India region.

Toxigenic Vibrio cholerae evolution and establishment of reservoirs in aquatic ecosystems.

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.

Safety and immunogenicity of a killed bivalent (O1 and O139) whole-cell oral cholera vaccine in adults and children in Vellore, South India.

This open-label study assessed the safety and immunogenicity of two doses (14 days apart) of an indigenously manufactured, killed, bivalent (Vibrio cholerae O1 and O139), whole-cell oral cholera vaccine (SHANCHOL; Shantha Biotechnics) in healthy adults (n = 100) and children (n = 100) in a cholera endemic area (Vellore, South India) to fulfill post-licensure regulatory requirements and post-World Health Organization (WHO) prequalification commitments. Safety and reactogenicity were assessed, and seroconversion rates (i.e. proportion of participants with a ≥ 4-fold rise from baseline in serum vibriocidal antibody titers against V. cholerae O1 Inaba, O1 Ogawa and O139, respectively) were determined 14 days after each vaccine dose. No serious adverse events were reported during the study. Commonly reported solicited adverse events were headache and general ill feeling. Seroconversion rates after the first and second dose in adults were 67.7% and 55.2%, respectively, against O1 Inaba; 47.9% and 45.8% against O1 Ogawa; and 19.8% and 20.8% against O139. In children, seroconversion rates after the first and second dose were 80.2% and 68.8%, respectively, against O1 Inaba; 72.9% and 67.7% against O1 Ogawa; and 26.0% and 18.8% against O139. The geometric mean titers against O1 Inaba, O1 Ogawa, and O139 in both adults and children were significantly higher after each vaccine dose compared to baseline titers (P < 0.001; for both age groups after each dose versus baseline). The seroconversion rates for O1 Inaba, O1 Ogawa, and O139 in both age groups were similar to those in previous studies with the vaccine. In conclusion, the killed, bivalent, whole-cell oral cholera vaccine has a good safety and reactogenicity profile, and is immunogenic in healthy adults and children. Trial Registration: ClinicalTrials.gov NCT00760825; CTRI/2012/01/002354.

Genomic plasticity associated with antimicrobial resistance in Vibrio cholerae.

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.

Investigating the virulence genes and antibiotic susceptibility patterns of Vibrio cholerae O1 in environmental and clinical isolates in Accra, Ghana.

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.

Comparative genomics for non-O1/O139 Vibrio cholerae isolates recovered from the Yangtze River Estuary versus V. cholerae representative isolates from serogroup O1.

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.

Glucose Metabolism by Escherichia coli Inhibits Vibrio cholerae Intestinal Colonization of Zebrafish.

The Vibrio cholerae O1 serogroup is responsible for pandemic cholera and is divided into the classical and El Tor biotypes. Classical V. cholerae produces acid when using glucose as a carbon source, whereas El Tor V. cholerae produces the neutral product acetoin when using glucose as a carbon source. An earlier study demonstrated that Escherichia coli strains that metabolize glucose to acidic by-products drastically reduced the survival of V. cholerae strains in vitro In the present study, zebrafish were fed 1% glucose and either inoculated with single V. cholerae or E. coli strains or coinfected with both V. cholerae and E. coli A significant decrease in classical biotype colonization was observed after glucose feeding due to acid production in the zebrafish intestine. El Tor colonization was unaffected by glucose alone. However, the El Tor strain exhibited significantly lower colonization of the zebrafish when either of the acid-producing E. coli strains was coinoculated in the presence of glucose. An E. coli sugar transport mutant had no effect on V. cholerae colonization even in presence of glucose. Glucose and E. coli produced a prophylactic effect on El Tor colonization in zebrafish when E. coli was inoculated before V. cholerae infection. Thus, the probiotic feeding of E. coli inhibits V. cholerae colonization in a natural host. This suggests that a similar inhibitory effect could be seen in cholera patients, especially if a glucose-based oral rehydration solution (ORS) is administered in combination with probiotic E. coli during cholera treatment.

Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia.

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.

Vibrio cholerae accessory colonisation factor AcfC: a chemotactic protein with a role in hyperinfectivity.

Vibrio cholerae O1 El Tor is an aquatic Gram-negative bacterium responsible for the current seventh pandemic of the diarrheal disease, cholera. A previous whole-genome analysis on V. cholerae O1 El Tor strains from the 2010 epidemic in Pakistan showed that all strains contained the V. cholerae pathogenicity island-1 and the accessory colonisation gene acfC (VC_0841). Here we show that acfC possess an open reading frame of 770 bp encoding a protein with a predicted size of 28 kDa, which shares high amino acid similarity with two adhesion proteins found in other enteropathogens, including Paa in serotype O45 porcine enteropathogenic Escherichia coli and PEB3 in Campylobacter jejuni. Using a defined acfC deletion mutant, we studied the specific role of AcfC in V. cholerae O1 El Tor environmental survival, colonisation and virulence in two infection model systems (Galleria mellonella and infant rabbits). Our results indicate that AcfC might be a periplasmic sulfate-binding protein that affects chemotaxis towards mucin and bacterial infectivity in the infant rabbit model of cholera. Overall, our findings suggest that AcfC contributes to the chemotactic response of WT V. cholerae and plays an important role in defining the overall distribution of the organism within the intestine.

Deletion of gene encoding the nucleoid-associated protein H-NS unmasks hidden regulatory connections in El Tor biotype Vibrio cholerae.

Hypervirulent atypical El Tor biotype Vibrio cholerae O1 isolates harbour mutations in the DNA-binding domain of the nucleoid-associated protein H-NS and the receiver domain of the response regulator VieA. Here, we provide two examples in which inactivation of H-NS in El Tor biotype vibrios unmasks hidden regulatory connections. First, deletion of the helix-turn-helix domain of VieA in an hns mutant background diminished biofilm formation and exopolysaccharide gene expression, a function that phenotypically opposes its phosphodiesterase activity. Second, deletion of vieA in an hns mutant diminished the expression of σE, a virulence determinant that mediates the envelope stress response. hns mutants were highly sensitive to envelope stressors compared to wild-type. However, deletion of vieA in the hns mutant restored or exceeded wild-type resistance. These findings suggest an evolutionary path for the emergence of hypervirulent strains starting from nucleotide sequence diversification affecting the interaction of H-NS with DNA.

Changing facades of Vibrio cholerae: An enigma in the epidemiology of cholera.

Cholera, caused by the Gram-negative bacterium Vibrio cholerae, has ravaged humanity from time immemorial. Although the disease can be treated using antibiotics along with administration of oral rehydration salts and controlled by good sanitation, cholera is known to have produced mayhems in ancient times when little was known about the pathogen. By the 21st century, ample information about the pathogen, its epidemiology, genetics, treatment and control strategies was revealed. However, there is still fear of cholera outbreaks in developing countries, especially in the wake of natural calamities. Studies have proved that the bacterium is mutating and evolving, out-competing all our efforts to treat the disease with previously used antibiotics and control with existing vaccines. In this review, the major scientific insights of cholera research are discussed. Considering the important role of biofilm formation in the V. cholerae life cycle, the vast availability of next-generation sequencing data of the pathogen and multi-omic approach, the review thrusts on the identification of suitable biofilm-inhibiting targets and the discovery of anti-biofilm drugs from nature to control the disease.

Characterization of highly virulent multidrug resistant Vibrio cholerae isolated from a large cholera outbreak in Ghana.

OBJECTIVE: The purpose of this study was to investigate the virulent factors of Vibrio cholerae which caused an unprecedented large cholera outbreak in Ghana in 2014 and progressed into 2015, affected 28,975 people with 243 deaths. RESULTS: The V. cholerae isolates were identified to be the classical V. cholerae 01 biotype El Tor, serotype Ogawa, responsible for the large cholera outbreak in Ghana. These El Tor strains bear CtxAB and Tcp virulent genes, making the strains highly virulent. The strains also bear SXT transmissible element coding their resistance to antibiotics, causing high proportions of the strains to be multidrug resistant, with resistant proportions of 95, 90 and 75% to trimethoprim/sulfamethoxazole, ampicillin and ceftriaxone respectively. PFGE patterns indicated that the isolates clustered together with the same pattern and showed clusters similar to strains circulating in DR Congo, Cameroun, Ivory Coast and Togo. The strains carried virulence genes which facilitated the disease causation and spread. This is the first time these virulent genes were determined on the Ghanaian Vibrio strains.

Analysis of Vibrio seventh pandemic island II and novel genomic islands in relation to attachment sequences among a wide variety of Vibrio cholerae strains.

Vibrio cholerae O1 El Tor, the pathogen responsible for the current cholera pandemic, became pathogenic by acquiring virulent factors including Vibrio seventh pandemic islands (VSP)-I and -II. Diversity of VSP-II is well recognized; however, studies addressing attachment sequence left (attL) sequences of VSP-II are few. In this report, a wide variety of V. cholerae strains were analyzed for the structure and distribution of VSP-II in relation to their attachment sequences. Of 188 V. cholerae strains analyzed, 81% (153/188) strains carried VSP-II; of these, typical VSP-II, and a short variant was found in 36% (55/153), and 63% (96/153), respectively. A novel VSP-II was found in two V. cholerae non-O1/non-O139 strains. In addition to the typical 14-bp attL, six new attL-like sequences were identified. The 14-bp attL was associated with VSP-II in 91% (139/153), whereas the remaining six types were found in 9.2% (14/153) of V. cholerae strains. Of note, six distinct types of the attL-like sequence were found in the seventh pandemic wave 1 strains; however, only one or two types were found in the wave 2 or 3 strains. Interestingly, 86% (24/28) of V. cholerae seventh pandemic strains harboring a 13-bp attL-like sequence were devoid of VSP-II. Six novel genomic islands using two unique insertion sites to those of VSP-II were identified in 11 V. cholerae strains in this study. Four of those shared similar gene clusters with VSP-II, except integrase gene.

Selection of affinity peptides for interference-free detection of cholera toxin.

Cholera toxin is a major virulent agent of Vibrio cholerae, and it can rapidly lead to severe dehydration, shock, causing death within hours without appropriate clinical treatments. In this study, we present a method wherein unique and short peptides that bind to cholera toxin subunit B (CTX-B) were selected through M13 phage display. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides. Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB5.

Genome Sequence Analysis of Vibrio cholerae clinical isolates from 2013 in Mexico reveals the presence of the strain responsible for the 2010 Haiti outbreak. / El análisis molecular de la secuencia del genoma completo de aislados clínicos de Vibrio cholerae en México en 2013 revela la presencia de la cepa causante de la epidemia de Haití en 2010.

The first week of September 2013, the National Epidemiological Surveillance System identified two cases of cholera in Mexico City. The cultures of both samples were confirmed as Vibrio cholerae serogroup O1, serotype Ogawa, biotype El Tor. Initial analyses by pulsed-field gel electrophoresis and by polymerase chain reaction-amplification of the virulence genes, suggested that both strains were similar, but different from those previously reported in Mexico. The following week, four more cases were identified in a community in the state of Hidalgo, located 121 km northeast of Mexico City. Thereafter a cholera outbreak started in the region of La Huasteca. Genomic analyses of the strains obtained in this study confirmed the presence of pathogenicity islands VPI-1 and VPI-2, VSP-1 and VSP-2, and of the integrative element SXT. The genomic structure of the 4 isolates was similar to that of V. cholerae strain 2010 EL-1786, identified during the epidemic in Haiti in 2010. This study shows that molecular epidemiology is a very powerful tool to monitor, prevent and control diseases of public health importance in Mexico.

La primera semana de septiembre de 2013, el Sistema Nacional de Vigilancia Epidemiológica identificó dos casos de cólera en Ciudad de México. Los cultivos de ambas muestras se confirmaron como Vibrio cholerae serogrupo O1, serotipo Ogawa, biotipo El Tor. Los análisis iniciales por electroforesis por campos pulsados y por reacción en cadena de la polimerasa indicaron que ambas cepas eran similares, pero diferentes de las previamente reportadas en México. La semana siguiente se identificaron cuatro casos más en una comunidad del Estado de Hidalgo, ubicada a 121 kilómetros al noreste de Ciudad de México. Posteriormente se inició un brote de cólera en la región de La Huasteca. Los análisis genómicos de cuatro cepas obtenidas en este estudio confirmaron la presencia de las islas de patogenicidad VPI -1 y VPI-2, VSP-1 y VSP-2, y del elemento integrador SXT. La estructura genómica de los cuatro aislamientos fue similar a la de V. cholerae cepa 2010 EL-1786, identificada durante la epidemia en Haití en 2010. Este estudio pone de manifiesto que la epidemiología molecular es una herramienta muy poderosa para vigilar, prevenir y controlar enfermedades de importancia en salud pública en México.

Large cholera outbreak in Brong Ahafo Region, Ghana.

BACKGROUND: A nationwide outbreak of Vibrio cholerae occurred in Ghana in 2014 with Accra, the nation's capital as the epi-center. The outbreak spread to the Brong Ahafo Region (BAR) which is geographically located in the middle of the country. In this region a review of data collected during the outbreak was carried out and analyzed descriptively to determine the hot spots and make recommendations for effective response to future outbreaks. METHODS: A review of patient records and line lists of cases of cholera reported in all hospitals during the period of the outbreak (July-December 2014) was conducted. Hospitals used IDSR (Integrated Disease Surveillance and Response system) standard case definitions to detect and report cases for management. The GPS coordinates of all districts and health facilities were collected and utilized in the construction of spot maps. We also obtained populations (denominators) from the BAR Health surveillance unit of the Ghana Health Service. All the data thus collected was analyzed descriptively and expressed as frequencies and rates. RESULTS: A total of 1035 cases were reported, 550 (53.4%) were males and the rest females. Their ages ranged from 1 to 95 years; (mean age of 28.2 ± 19.6 years). The most affected (23.5%) was the 20-29 year old age group. On the 30th July, 2014, a 26 year old male (recorded as the index case of the cholera outbreak in the Brong Ahafo region) with a history of travel from Accra reported to the Nkoranza district hospital with a history of symptoms suggestive of cholera. The reporting of cholera cases reached their peak (17.3%) in week 15 of the outbreak (this lasted 25 weeks). An overall attack rate of 71/100,000 population, and a case fatality rate of 2.4% was recorded in the region. Asutifi South district however recorded a case fatality of 9.1%, the highest amongst all the districts which recorded outbreaks. The majority of the cases reported in the region were from Atebubu-Amanten, Sene West, Pru, and Asunafo North districts with 31.1, 26.0, 18.2 and 9.9% respectively. Vibrio cholerae serotype O1 was isolated from rectal swabs/stool samples tested. CONCLUSION: Vibrio cholerae serotype O1 caused the cholera-outbreak in the Brong Ahafo Region and mainly affected young adult-males. The most affected districts were Atebubu-Amanten, Sene west, Pru (located in the eastern part of the region), and Asunafo North districts (located in the south west of the region). Case Fatality Rate was higher (2.4%) than the WHO recommended rate (<1%). Active district level public health education is recommended on prevention and effective response for future outbreaks of cholera.

Genetic diversity of environmental Vibrio cholerae O1 strains isolated in Northern Vietnam.

Cholera epidemics have been recorded periodically in Vietnam during the seventh cholera pandemic. Since cholera is a water-borne disease, systematic monitoring of environmental waters for Vibrio cholerae presence is important for predicting and preventing cholera epidemics. We conducted monitoring, isolation, and genetic characterization of V. cholerae strains in Nam Dinh province of Northern Vietnam from Jul 2013 to Feb 2015. In this study, four V. cholerae O1 strains were detected and isolated from 110 analyzed water samples (3.6%); however, none of them carried the cholera toxin gene, ctxA, in their genomes. Whole genome sequencing and phylogenetic analysis revealed that the four O1 isolates were separated into two independent clusters, and one of them diverged from a common ancestor with pandemic strains. The analysis of pathogenicity islands (CTX prophage, VPI-I, VPI-II, VSP-I, and VSP-II) indicated that one strain (VNND_2014Jun_6SS) harbored an unknown prophage-like sequence with high homology to vibriophage KSF-1 phi and VCY phi, identified from Bangladesh and the USA, respectively, while the other three strains carried tcpA gene with a distinct sequence demonstrating a separate clonal lineage. These results suggest that the aquatic environment can harbor highly divergent V. cholera strains and serve as a reservoir for multiple V. cholerae virulence-associated genes which may be exchanged via mobile genetic elements. Therefore, continuous monitoring and genetic characterization of V. cholerae strains in the environment should contribute to the early detection of the sources of infection and prevention of cholera outbreaks as well as to understanding the natural ecology and evolution of V. cholerae.