Jaime Martinez-Urtaza

Determination of Molecular Phylogenetics of Vibrio parahaemolyticus Strains by Multilocus Sequence Typing

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.

Environmental occurrence and clinical impact of Vibrio vulnificus and Vibrio parahaemolyticus: a European perspective.

Vibrio vulnificus and Vibrio parahaemolyticus are ubiquitous Gram-negative bacterial pathogens found naturally in marine and estuarine waters, and are a leading cause of seafood-associated bacterial illness. These pathogens are commonly reported in the USA and in many Asian countries, including China, Japan and Taiwan; however, there is growing concern that V. vulnificus and V. parahaemolyticus may represent an important and increasing clinical problem in Europe. Several factors underlie the need for a greater understanding of these non-cholera vibrios within a European context. First, there is a growing body of evidence to suggest that V. vulnificus and V. parahaemolyticus infections are increasing, and tend to follow regional climatic trends, with outbreaks typically following episodes of unusually warm weather. Such findings are especially alarming given current predictions regarding warming of marine waters as a result of global climatic change. Second, a myriad of epidemiological factors may greatly increase the incidence as well as clinical burden of these pathogens - including increasing global consumption and trade of seafood produce coupled to an increase in the number of susceptible individuals consuming seafood produce. Finally, there is currently a lack of detailed surveillance information regarding non-cholerae Vibrio infections in Europe, as these pathogens are not notifiable in many countries, which probably masks the true clinical burden of many human infections. This review will present a pertinent overview of both the environmental occurrence and clinical impact of V. vulnificus and V. parahaemolyticus in Europe.

Characterization of Pathogenic Vibrio parahaemolyticus Isolates from Clinical Sources in Spain and Comparison with Asian and North American Pandemic Isolates

ABSTRACT In spite of the potential risk involved with contamination of seafood with Vibrio parahaemolyticus, there is a lack of information on the occurrence of pathogenic V. parahaemolyticus in Europe. This organism was isolated in 1999 from a large outbreak (64 cases admitted to a single hospital) associated with raw oyster consumption in Galicia, Spain, one of the most important regions in shellfish production worldwide. Two V. parahaemolyticus isolates from the 1999 Galicia outbreak, three additional clinical isolates obtained in the same period from hospitals in Spain, two reference strains from clinical sources, and five Spanish environmental isolates were examined. Seventeen isolates belonging to the pandemic clone isolated in Asia and North America were included in the study for comparison. All isolates were characterized by serotyping, PCR for virulence-related genes, pulsed-field gel electrophoresis (PFGE), and plasmid analysis. Four of the five clinical isolates from hospitals in Spain belonged to serotype O4:K11; the remaining isolate was O4:K untypeable (KUT). All five isolates were positive for V. parahaemolyticus toxR and tlh (species-specific genes) and tdh and negative for trh and group-specific PCR (a PCR method for detection of the pandemic clone). PFGE analysis with NotI and SfiI discriminated the European isolates in two closely related PFGE types included in a homogeneous cluster, clearly differentiated from the Asian and North American isolates. The five environmental isolates belonged to serotypes O2:K28, O2:KUT, O3:K53, O4:KUT, and O8:K22 and were negative for all virulence genes. The five isolates were discriminated into five different PFGE types unrelated to any other isolate included in the study. While the virulence characteristics (tdh positive, trh negative) of the Spanish clinical isolates matched those of the O3:K6 clone from Asia and North America, they were clearly excluded from this clone by group-specific PCR, PFGE, and serotyping. The results of this study suggest that a unique and specific clone could be related to the V. parahaemolyticus infections in Europe.

Pandemic Vibrio parahaemolyticus O3:K6, Europe

To the Editor: Vibrio parahaemolyticus is a halophilic member of the genus Vibrio that inhabits temperate and tropical marine environments worldwide. Strains that produce the thermostable direct hemolysin or the thermostable direct hemolysin-related hemolysin, which are encoded by tdh and trh genes, respectively, are considered pathogenic. While almost all clinical strains have these virulence factors, these strains represent <1% of all environmental strains.

Influence of Environmental Factors and Human Activity on the Presence of Salmonella Serovars in a Marine Environment

ABSTRACT The temporal and spatial distribution of Salmonella contamination in the coastal waters of Galicia (northwestern Spain) relative to contamination events with different environmental factors (temperature, wind, hours of sunlight, rainfall, and river flow) were investigated over a 4-year period. Salmonellae were isolated from 127 of 5,384 samples of molluscs and seawater (2.4%), and no significant differences (P < 0.05) between isolates obtained in different years were observed. The incidence of salmonellae was significantly higher in water column samples (2.9%) than in those taken from the marine benthos (0.7%). Of the 127 strains of Salmonella isolated, 20 different serovars were identified. Salmonella enterica serovar Senftenberg was the predominant serovar, being represented by 54 isolates (42.5%), followed by serovar Typhimurium (19 isolates [15%]) and serovar Agona (12 isolates [9.4%]). Serovar Senftenberg was detected at specific points on the coast and could not be related to any of the environmental parameters analyzed. All serovars except Salmonella serovar Senftenberg were found principally in the southern coastal areas close to the mouths of rivers, and their incidence was associated with high southwestern wind and rainfall. Using multiple logistic regression analysis models, the prevalence of salmonellae was best explained by environmental parameters on the day prior to sampling. Understanding this relationship may be useful for the control of molluscan shellfish harvests, with wind and rainfall serving as triggers for closure.

Environmental Determinants of the Occurrence and Distribution of Vibrio parahaemolyticus in the Rias of Galicia, Spain

ABSTRACT Infections associated with Vibrio parahaemolyticus on the coast of Galicia (in northwestern Spain) were reported to be linked to large outbreaks of illness during 1999 and 2000. Little information is available about the ecological factors that influence the emergence of V. parahaemolyticus infections in this temperate region. We carried out a 3-year study to investigate the occurrence and distribution of V. parahaemolyticus at 26 sites located in the four main rias of Galicia in association with environmental and oceanographic variables. V. parahaemolyticus was detected in all the areas investigated and throughout the complete period of study with an overall incidence of 12.5%. Salinity was the primary factor governing the temporal and spatial distribution of V. parahaemolyticus, whereas seawater temperature had a secondary effect and only modulated the abundance in periods and areas of reduced salinities. Higher occurrence of V. parahaemolyticus was observed during periods of lower salinity in autumn, with a total of 61 positive samples (18%) and a mean density of 1,234 most probable number/100 g. V. parahaemolyticus was primarily detected in areas of reduced salinity close to freshwater discharge points, where it was found in up to 45% of the samples. Characterization of the isolates obtained from the study resulted in the first identification of two pathogenic tdh-positive strains of V. parahaemolyticus recovered from the marine environment in Galicia. These isolates showed serotypes identical to and DNA profiles indistinguishable from those of the clinical clone of V. parahaemolyticus dominant in infections in Spain in the last 10 years.

Identification of tdh-positive Vibrio parahaemolyticus from an outbreak associated with raw oyster consumption in Spain

Between August and September 1999, a total of 64 cases of illness were identified in three episodes of acute gastroenteritis associated with the consumption of live oysters from a typical outdoor street market in Galicia (northwest Spain). Nine case patients were hospitalized and analysis of their stool samples revealed the presence of Vibrio parahaemolyticus. The strains isolated from two stool samples were studied for antibiotic susceptibility, biochemical characteristics and presence of virulence factors. Both isolates were Kanagawa phenomenon positive and produced thermostable direct hemolysin, which is related to pathogenicity in humans. These results show the presence of pathogenic V. parahaemolyticus in mollusks harvested in Europe and reveal the risk of illness associated with their consumption, suggesting the revision of V. parahaemolyticus risk assessment associated with consumption of raw live shellfish.

Epidemiology of Vibrio parahaemolyticus Outbreaks, Southern Chile

One-sentence summary for table of contents: Outbreaks are decreasing and the O3:K6 pandemic strain is being replaced by a new serotype and new strains.

Emergence of asiatic vibrio diseases in south america in phase with El Niño

Background: The seventh pandemic of Vibrio cholerae unexpectedly reached the coast of Peru in 1991, causing an explosive emergence of infections throughout the American continents. The origin and routes of dissemination are as yet unknown. A new Vibrio epidemic arose in 1997 in South America (northern Chile) when the pandemic clone of Vibrio parahaemolyticus was for the fist time detected outside of Asia. These 2 cases were concurrent with 2 episodes of El Niño. Methods: We carried out a survey of records of V. parahaemolyticus infection and of strains existing in the Instituto Nacional de Salud of Peru between 1994 and 2005. Association between the El Niño event and the V. parahaemolyticus disease was analyzed through generalized additive models applied to time-series data with negative binomial response, selecting some oceanographic factors distinctive of the movement of the El Niño waters. Results: Epidemiologic data and laboratory investigations of the strains showed that V. parahaemolyticus infections caused by the pandemic clone emerged in the coasts of Peru linked to the 1997 El Niño episode. The epidemic dissemination of this clone matched the expansion and dynamics of the poleward propagation and the receding of the El Niño waters. This pattern was similar to previously reported onset of cholera epidemic in 1991. Conclusions: These findings identify the El Niño episodes as a reliable vehicle for the introduction and propagation of Vibrio pathogens in South America. The movement of oceanic waters seems to be one of the driving forces of the spread of Vibrio diseases.

Evaluation of different procedures for the optimized detection of Vibrio parahaemolyticus in mussels and environmental samples.

Vibrio parahaemolyticus is a marine bacterium with a worldwide distribution and is frequently associated with human outbreaks of infection. Detection and isolation of V. parahaemolyticus from natural sources is often problematical because of limitations in the analytical procedures. We evaluated a combination of conventional and molecular protocols previously described for the investigation of V. parahaemolyticus, with the aim of identifying the best procedures for improved detection of this organism in environmental matrixes. A total of 259 samples of zooplankton (103), mussels (48) and seawater (108) were investigated by an Absence-Presence method (A/P), whereas 118 samples of zooplankton (70) and mussels (48) were analyzed by the Most Probable Number (MPN) method. All samples were processed by a two-step enrichment procedure, firstly with APW broth and then with SPB as selective secondary broth. Detection of V. parahaemolyticus was by direct-PCR and by plate culture on TCBS and CHROMagar Vibrio, after sample enrichment in APW and SPB. With the A/P method, V. parahaemolyticus was detected in 23.6% samples by direct-PCR, whereas only 11.2% samples were positive with the plate culture method. With the MPN method, V. parahaemolyticus was detected in 54.2% and 27.1% of the samples by direct-PCR and plate culture respectively; this indicated the existence of 31% false negative results with the A/P method. No significant differences between the use of a single (APW) or two-step enrichment (APW+SPB) were observed by direct-PCR with A/P or MPN, although a significant higher presence of V. parahaemolyticus was detected by plate culture in both protocols with the two-step enrichment procedure. In conclusion, direct-PCR after sample enrichment in APW broth was the most successful method for detection of V. parahaemolyticus with the A/P procedure and enumeration by MPN. Better detection was obtained with MPN than with the A/P protocol. Conversely, the plate culture procedure showed better results with the two-step enrichment protocol in which CHROMagar Vibrio was used as the selective agar.