Toxigenic V. cholerae, V. parahaemolyticus, and V. vulnificus in oysters from the Gulf of Mexico and sold in Mexico City.

Oysters can accumulate potentially pathogenic bacteria, such as Vibrio cholerae, V. parahaemolyticus, and V. vulnificus. The aim of this study was to detect the presence of these Vibrio species and their toxigenic variants in oysters from the Gulf of Mexico sold in Mexico City. Oyster samples were studied using traditional culture and molecular polymerase chain reaction analysis. V. cholerae was present in 30.4% of the samples and its toxigenic variant chxA+ in 26.1%. It was isolated only in deshelled oysters, mainly in the dry season. V. parahaemolyticus was present in 95.7% of the samples and the toxigenic variant was found in 17.4%. V. vulnificus was identified in 60.9% of the samples, 38% of which corresponded to the environmental genotype and 21.7% to the clinical genotype, mainly in the cold season. Consumption of the oysters analyzed poses health risks due to the presence of Vibrio species, especially in deshelled oysters.

Occurrence in Mexico, 1998-2008, of Vibrio cholerae CTX+ El Tor carrying an additional truncated CTX prophage.

The seventh cholera pandemic caused by Vibrio cholerae O1 El Tor (ET) has been superseded in Asia and Africa by altered ET possessing the cholera toxin (CTX) gene of classical (CL) biotype. The CL biotype of V. cholerae was isolated, along with prototypic and altered ET, during the 1991 cholera epidemic in Mexico and subsequently remained endemic until 1997. Microbiological, molecular, and phylogenetic analyses of clinical and environmental V. cholerae isolated in Mexico between 1998 and 2008 revealed important genetic events favoring predominance of ET over CL and altered ET. V. cholerae altered ET was predominant after 1991 but not after 2000. V. cholerae strains isolated between 2001 and 2003 and a majority isolated in 2004 lacked CTX prophage (Φ) genes encoding CTX subunits A and B and repeat sequence transcriptional regulators of ET and CL biotypes: i.e., CTXΦ(-). Most CTXΦ(-) V. cholerae isolated in Mexico between 2001 and 2003 also lacked toxin coregulated pili tcpA whereas some carried either tcpA(ET) or a variant tcpA with noticeable sequence dissimilarity from tcpA(CL). The tcpA variants were not detected in 2005 after CTXΦ(+) ET became dominant. All clinical and environmental V. cholerae O1 strains isolated during 2005-2008 in Mexico were CTXΦ(+) ET, carrying an additional truncated CTXΦ instead of RS1 satellite phage. Despite V. cholerae CTXΦ(-) ET exhibiting heterogeneity in pulsed-field gel electrophoresis patterns, CTXΦ(+) ET isolated during 2004-2008 displayed homogeneity and clonal relationship with V. cholerae ET N16961 and V. cholerae ET isolated in Peru.

Quantification of Vibrio species in oysters from the Gulf of Mexico with two procedures based on MPN and PCR.

Oysters can accumulate potentially pathogenic water bacteria. The objective of this study was to compare two procedures to quantify Vibrio species present in oysters to determine the most sensitive method. We analyzed oyster samples from the Gulf of Mexico, commercialized in Mexico City. The samples were inoculated in tubes with alkaline peptone water (APW), based on three tubes and four dilutions (10-1 to 10-4). From these tubes, the first quantification of Vibrio species was performed (most probable number (MPN) from tubes) and bacteria were inoculated by streaking on thiosulfate-citrate-bile salts-sucrose (TCBS) petri dishes. Colonies were isolated for a second quantification (MPN from dishes). Polymerase chain reaction (PCR) was used to determine species with specific primers: ompW for Vibrio cholerae, tlh for Vibrio parahaemolyticus, and VvhA for Vibrio vulnificus. Simultaneously, the sanitary quality of oysters was determined. The quantification of V. parahaemolyticus was significantly higher in APW tubes than in TCBS dishes. Regarding V. vulnificus counts, the differences among both approaches were not significant. In contrast, the MPNs of V. cholerae obtained from dishes were higher than from tubes. The quantification of MPNs through PCR of V. parahaemolyticus and V. vulnificus obtained from APW was sensitive and recommendable for the detection of both species. In contrast, to quantify V. cholerae, it was necessary to isolate colonies on TCBS prior PCR. Culturing in APW at 42 °C could be an alternative to avoid colony isolation. The MPNs of V. cholerae from dishes was associated with the bad sanitary quality of the samples.

Genetic characterization of Vibrio vulnificus strains isolated from oyster samples in Mexico.

Vibrio vulnificus strains were isolated from oysters that were collected at the main seafood market in Mexico City. Strains were characterized with regard to vvhA, vcg genotype, PFGE, multilocus sequence typing (MLST), and rtxA1. Analyses included a comparison with rtxA1 reference sequences. Environmental (vcgE) and clinical (vcgC) genotypes were isolated at nearly equal percentages. PFGE had high heterogeneity, but the strains clustered by vcgE or vcgC genotype. Select housekeeping genes for MLST and primers that were designed for rtxA1 domains divided the strains into two clusters according to the E or C genotype. Reference rtxA1 sequences and those from this study were also clustered according to genotype. These results confirm that this genetic dimorphism is not limited to vcg genotyping, as other studies have reported. Some environmental C genotype strains had high similarity to reference strains, which have been reported to be virulent, indicating a potential risk for oyster consumers in Mexico City.

Genomic stability among O3:K6 V. parahaemolyticus pandemic strains isolated between 1996 to 2012 in American countries.

BACKGROUND: The V. parahaemolyticus pandemic clone, results in the development of gastrointestinal illness in humans. Toxigenic strains of this species are frequently isolated from aquatic habitats and organisms such as mollusks and crustaceans. Reports on the isolation of the pandemic clone started in 1996, when a new O3:K6 clone was identified in Asia, that rapidly spread worldwide, becoming the predominant clone isolated from clinical cases. In this study whole genome sequencing was accomplished with an Illumina MiniSeq platform, upon six novel V. parahaemolyticus strains, that have been isolated in Mexico since 1998 and three representative genomes of strains that were isolated from reported outbreaks in other American countries, and were deposited in the GenBank. These nine genomes were compared against the reference sequence of the O3:K6 pandemic strain (RIMD 2210633), which was isolated in 1996, to determine sequence differences within American isolates and between years of isolation. RESULTS: The results indicated that strains that were isolated at different times and from different countries, were highly genetically similar, among them as well as to the reference strain RIMD 2210633, indicating a high level of genetic stability among the strains from American countries between 1996 to 2012, without significant genetic changes relative to the reference strain RIMD 2210633, which was isolated in 1996 and was considered to be representative of a novel O3:K6 pandemic strain. CONCLUSIONS: The genomes of V. parahaemolyticus strains isolated from clinical and environmental sources in Mexico and other American countries, presented common characteristics that have been reported for RIMD 2210633 O3:K6 pandemic strain. The major variations that were registered in this study corresponded to genes non associated to virulence factors, which could be the result of adaptations to different environmental conditions. Nevertheless, results do not show a clear pattern with the year or locality where the strains were isolated, which is an indication of a genomic stability of the studied strains.

Molecular analyses of Vibrio cholerae O1 clinical strains, including new nontoxigenic variants isolated in Mexico during the Cholera epidemic years between 1991 and 2000.

We studied the evolution of Vibrio cholerae O1 during the 1991 to 2000 cholera epidemic in Mexico by biochemical, serological, and molecular characterization of strains collected during this period. Strains were divided into toxigenic and nontoxigenic groups according to the presence or absence of genes encoding cholera toxin. As previously reported, we characterized two populations among toxigenic strains, which were present from the first year of the epidemic. BglI rRNA analysis revealed that these strains had ribotype profiles, denoted M5 and M6 in our study, that were identical to those previously designated Koblavi B5 or Popovic 5 and Popovic 6a or Tamayo B21a, respectively. Ribotype M5 was isolated between 1991 and 1993. This ribotype had a low level of genetic variation as detected by pulsed-field gel electrophoresis (PFGE). Ribotype M6 persisted from 1991 to 2000. However, PFGE profiles suggested that two epidemiologically unrelated strains coexisted within this single ribotype from 1995 until the end of the epidemic. We identified three new BglI ribotypes, Mx1, Mx2, and Mx3, from nontoxigenic V. cholerae O1 strains isolated between 1998 and 2000; one of them grouped strains positive for the toxin-coregulated pilus island. They differed from nontoxigenic clones isolated in Latin America and on the U.S. Gulf Coast and are probably autochthonous Mexican V. cholerae O1 variants. Most of these new variants were isolated from states surrounding the Gulf of Mexico, where the highest incidence of cholera in the country was recorded. Thus, the Mexican Gulf Coast, like the U.S. Gulf Coast, may act as an environmental reservoir of V. cholerae O1.

Whole-genome comparison between reference sequences and oyster Vibrio vulnificus C-genotype strains.

Whole-genome sequences of Vibrio vulnificus clinical genotype (C-genotype) from the CICESE Culture Collection, isolated from oysters, were compared with reference sequences of CMCP6 and YJ016 V. vulnificus C-genotype strains of clinical origin. The RAST web server estimated the whole genome to be ~4.8 Mb in CICESE strain 316 and ~4.7 Mb in CICESE strain 325. No plasmids were detected in the CICESE strains. Based on a phylogenetic tree that was constructed with the whole-genome results, we observed high similarity between the reference sequences and oyster C-genotype isolates and a sharp contrast with environmental genotype (E-genotype) reference sequences, indicating that the differences between the C- and E-genotypes do not necessarily correspond to their isolation origin. The CICESE strains share 3488 genes (63.2%) with the YJ016 strain and 3500 genes (63.9%) with the CMCP6 strain. A total of 237 pathogenicity associated genes were selected from reference clinical strains, where-92 genes were from CMCP6, 126 genes from YJ016, and 19 from MO6-24/O; the presence or absence of these genes was recorded for the CICESE strains. Of the 92 genes that were selected for CMCP6, 67 were present in both CICESE strains, as were as 86 of the 126 YJ016 genes and 13 of the 19 MO6-24/O genes. The detection of elements that are related to virulence in CICESE strains-such as the RTX gene cluster, vvhA and vvpE, the type IV pili cluster, the XII genomic island, and the viuB genes, suggests that environmental isolates with the C-genotype, have significant potential for infection.

Occurrence and Abundance of Pathogenic Vibrio Species in Raw Oysters at Retail Seafood Markets in Northwestern Mexico.

Seafood has frequently been associated with foodborne illness because pathogens are easily introduced during seafood cultivation, handling, and processing. Vibrio parahaemolyticus and Vibrio cholerae are human pathogens that cause gastroenteritis and cholera, respectively, and Vibrio vulnificus can cause fatal wound infections and septicemia. However, information about the occurrence of these pathogens in oysters from the Pacific coast of Mexico is limited to V. parahaemolyticus. In the present study, we evaluated the presence and abundance of these three Vibrio species in 68 raw oysters (Crassostrea corteziensis) obtained from retail seafood markets in Sinaloa, Mexico. The most probable number (MPN)-PCR assay was used for amplification of the tlh (thermolabile hemolysin), ompW (outer membrane protein), and vvhA (hemolytic cytolysin) genes that are specific to V. parahaemolyticus, V. cholerae, and V. vulnificus, respectively. All oyster samples were positive for at least one Vibrio species. V. parahaemolyticus, V. cholerae, and V. vulnificus prevalences were 77.9, 8.8, and 32.3% overall, respectively, and most species were present in all sample periods with increased prevalence in period 3. The tdh (thermostable direct hemolysin) gene was detected in 30.1%, trh (TDH-related hemolysin) was detected in 3.7%, and tdh/trh was detected in 7.5% of the total tlh-positive samples (53 of 68), whereas the pandemic serotype O3:K6 (orf8 positive) was detected in only 1 sample (1.8%). The total prevalence of tdh and/or trh was 41.5%. In none of the samples positive for V. cholerae were the cholera toxin (ctxA) and cholix (chxA) toxigenic genes or the rfb gene encoding the O1 and O139 antigens amplified, suggesting the presence of non-O1 non-O139 V. cholerae strains. Our results clearly indicated a high prevalence of pathogenic Vibrio species in raw oysters from retail seafood markets in Mexico. Consumption of these raw oysters carries the potential risk of foodborne illness, which can be limited by cooking.

Detection of Alcanivorax spp., Cycloclasticus spp., and Methanomicrobiales in water column and sediment samples in the Gulf of Mexico by qPCR.

Water column and sediment samples were collected in the southern Gulf of Mexico (GoMex) during 3 oceanographic cruises: XIXIMI-04 (September 2015), XIXIMI-05 (June 2016), and XIXIMI-06 (August 2017). DNA that was extracted from the samples was analyzed by qPCR to detect and quantify bacterial groups that have been reported to metabolize alkanes (Alcanivorax) and aromatic hydrocarbons (Cycloclasticus) and are involved in methane production (Methanomicrobiales). The results were then analyzed with regard to the water masses that are currently detected in the GoMex. Generally, we observed a decrease in the proportion of Alcanivorax and a rise in those of Cycloclasticus and Methanomicrobiales in samples from the surface to deep waters and in sediment samples. Scatterplots of the results showed that the relative abundance of the 3 groups was higher primarily from the surface to 1000 m, but the levels of Cycloclasticus and Methanomicrobiales were high in certain water samples below 1000 m and in sediments. In conclusion, oil-degrading bacteria are distributed widely from the surface to deep waters and sediments throughout the southern GoMex, representing a potential inoculum of bacteria for various hydrocarbon fractions that are ready for proliferation and degradation in the event of an oil spill from the seafloor or along the water column.

Genetic Analysis of Vibrio parahaemolyticus O3:K6 Strains That Have Been Isolated in Mexico Since 1998.

Vibrio parahaemolyticus is an important human pathogen that has been isolated worldwide from clinical cases, most of which have been associated with seafood consumption. Environmental and clinical toxigenic strains of V. parahaemolyticus that were isolated in Mexico from 1998 to 2012, including those from the only outbreak that has been reported in this country, were characterized genetically to assess the presence of the O3:K6 pandemic clone, and their genetic relationship to strains that are related to the pandemic clonal complex (CC3). Pathogenic tdh+ and tdh+/trh+ strains were analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Also, the entire genome of a Mexican O3:K6 strain was sequenced. Most of the strains were tdh/ORF8-positive and corresponded to the O3:K6 serotype. By PFGE and MLST, there was very close genetic relationship between ORF8/O3:K6 strains, and very high genetic diversities from non-pandemic strains. The genetic relationship is very close among O3:K6 strains that were isolated in Mexico and sequences that were available for strains in the CC3, based on the PubMLST database. The whole-genome sequence of CICESE-170 strain had high similarity with that of the reference RIMD 2210633 strain, and harbored 7 pathogenicity islands, including the 4 that denote O3:K6 pandemic strains. These results indicate that pandemic strains that have been isolated in Mexico show very close genetic relationship among them and with those isolated worldwide.

Integrated view of Vibrio cholerae in the Americas.

Latin America has experienced two of the largest cholera epidemics in modern history; one in 1991 and the other in 2010. However, confusion still surrounds the relationships between globally circulating pandemic Vibrio cholerae clones and local bacterial populations. We used whole-genome sequencing to characterize cholera across the Americas over a 40-year time span. We found that both epidemics were the result of intercontinental introductions of seventh pandemic El Tor V. cholerae and that at least seven lineages local to the Americas are associated with disease that differs epidemiologically from epidemic cholera. Our results consolidate historical accounts of pandemic cholera with data to show the importance of local lineages, presenting an integrated view of cholera that is important to the design of future disease control strategies.

Effect of Vibrio alginolyticus on larval survival of the blue mussel Mytilus galloprovincialis.

The effect of increasing concentrations of Vibrio alginolyticus on survival of Mytilus galloprovincialis larvae was studied in a 48 h static bioassay in 1 l glass bottles. Five bacterial densities were tested ranging from 10(2) to 10(6) bacteria ml(-1). Larval survival and normality (veliger larvae with the typical D-shape) were evaluated after 48 h. An inverse relationship between bacterial concentration and larval survival and normality was observed. In spite of high larval survival (79%) under conditions of high bacterial density (10(5) bacteria ml(-1)), the percent of normal larvae was 11%. Besides an irregular shape, abnormal larvae also presented velum reduction. Results from this study suggest that concentrations of V. alginolyticus lower than 10(3) bacteria ml(-1) should be maintained during M. galloprovincialis larval culture.

Diversity and distribution of cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae.

Non-toxigenic non-O1, non-O139 Vibrio cholerae strains isolated from both environmental and clinical settings carry a suite of virulence factors aside from cholera toxin. Among V. cholerae strains isolated from coastal waters of southern California, this includes cholix toxin, an ADP-ribosylating factor that is capable of halting protein synthesis in eukaryotic cells. The prevalence of the gene encoding cholix toxin, chxA, was assessed among a collection of 155 diverse V. cholerae strains originating from both clinical and environmental settings in Bangladesh and Mexico and other countries around the globe. The chxA gene was present in 47% of 83 non-O1, non-O139 strains and 16% of 72 O1/O139 strains screened as part of this study. A total of 86 chxA gene sequences were obtained, and phylogenetic analysis revealed that they fall into two distinct clades. These two clades were also observed in the phylogenies of several housekeeping genes, suggesting that the divergence observed in chxA extends to other regions of the V. cholerae genome, and most likely has arisen from vertical descent rather than horizontal transfer. Our results clearly indicate that ChxA is a major toxin of V. cholerae with a worldwide distribution that is preferentially associated with non-pandemic strains.