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Article Title 1 Pandemic Vibrio parahaemolyticus O3:K6 present in the Coastal 2 Environment of Northwest Mexico is associated with recurrent 3

diarrheal cases from 2004 to 2010 4 Jorge Velazquez-Roman1,2, Nidia León-Sicairos2,3, Héctor Flores-Villaseñor4, 5 Santiago Villafaña-Rauda1 and Adrian Canizalez-Roman2,5* 6 7 Sección de posgrado, Escuela Superior de Medicina IPN1, 14330 Mexico D.F.; 8 School of Medicine, Autonomous University of Sinaloa, 80246 Culiacan Sinaloa, 9 Mexico2; Pediatric Hospital of Sinaloa, 80200 Culiacan Sinaloa, Mexico3; Programa 10 Regional Para el Doctorado en Biotecnología, FCQB-UAS, 80040 Culiacán Sinaloa, 11 México4; The Sinaloa State Public Health Laboratory, Secretariat of Health, 80020 12 Culiacan Sinaloa, Mexico5. 13 14 Running title: Pandemic V. parahaemolyticus in the Northwest Mexico 15 Key words: Serologic, Isolation, Vibrio parahaemolyticus, biosurveillance, Public 16 Health. 17 *Corresponding author 18 Correspondent footnote: Autonomous University of Sinaloa, School of Medicine, 19 80246 Culiacan Sinaloa, Mexico. Phone: (52) 6677538802, Fax: (52) 6677538801. E-20 mail: [email protected] 21

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.06953-11 AEM Accepts, published online ahead of print on 13 January 2012

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Abstract 22 In 2004, more than 1230 cases of gastroenteritis due to pandemic O3:K6 strains of 23 Vibrio parahaemolyticus were reported in southern Sinaloa, a state in Northwestern 24 Mexico. Recurrent sporadic cases arose from 2004 to 2010, spreading from the south 25 to the north. In the present study, Vibrio parahaemolyticus was detected in both 26 environmental samples and clinical cases along the Pacific Coast of Sinaloa during 27 2004 to 2010. An evaluation was made of the serotypes, distribution of virulence 28 genes, and presence of pandemic O3:K6 strains. A total of 144 strains were isolated 29 from environmental samples (from sediment, seawater and shrimp), and 154 clinical 30 strains were isolated. A total of 10 O serogroups and 30 serovars were identified in all 31 strains. Environmental strains (n=144) belonged to 10 O serogroups and 28 serovars 32 while clinical strains (n=154) belonged to 8 O serogroups and 14 serovars. Ten 33 serovars were shared by both environmental and clinical strains. Among 144 34 environmental isolates, 4.1% (6/144) belonged to the pandemic clone, which 83.3% 35 with the orf8 gene and O3:K6 accounted for 67%. On the other hand, pathogenic 36 strains (tdh and/or trh) accounted for 52% (75/144) of the environmental isolates. 37 Interestingly, among 154 clinical isolates, 80.5% (124/154) were pandemic strains, 38 with O3:K6 (tdh, toxRS/New and orf8) representing the predominant serovar (99.2%, 39 123/124). Overall, our results indicate that in spite of a high serodiversity and 40 prevalence of pathogenic Vibrio parahaemolyticus in the environment, pandemic 41 strain O3:K6 caused >79% of reported cases from 2004 to 2010 in Sinaloa Mexico. 42


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Introduction 43 Vibrio parahaemolyticus (V. parahaemolyticus) is a marine bacterium, which is also 44 responsible for acute diarrheal illness in human beings. Eating raw seafish or 45 contaminated seafood is responsible for acute gastroenteritis (49). Although the 46 presence of V. parahaemolyticus is extensive in estuarine environments, not all strains 47 of V. parahaemolyticus are considered pathogenic. For example, virulent strains are 48 essentially limited to those that are positive for the tdh gene (encoding the 49 thermostable direct hemolysin, TDH), because it has been associated with most 50 clinical strains, but with very few environmental strains (27, 44). Some clinical 51 isolates also carry the trh gene, encoding a TDH-related hemolysin (TRH). 52

Until 1996, infections caused by V. parahaemolyticus were usually associated 53 with diverse serovars (for example O1:K38, O3:K29, O4:K8, O3:K6, O2:K3, O4:K8 54 and other serotypes) (45, 56) and had shown a local distribution, emerging in different 55 areas of the world during the warmer months of the year. In February 1996, however, 56 a unique serovar (O3:K6) of V. parahaemolyticus with genetic markers specific, 57 abruptly appeared in Calcutta, India (45). In subsequent years, O3:K6 isolates similar 58 to those isolated in Calcutta were reported from food-borne outbreaks and from 59 sporadic cases in Southeast Asia, as well as Atlantic and Gulf coasts of the USA (16, 60 36, 45), and more recently there have been such reports in Europe (35), Africa (3) and 61 South America (24). This widespread occurrence of a single serotype of V. 62 parahaemolyticus had not previously been reported and it became evident that a 63 pandemic strain had emerged. 64

Several studies reported that the toxRS operon encoded by these newly 65 identified pandemic strains, contains a unique sequence (toxRS/new) encoding 66 transmembrane proteins involved in the regulation of virulence associated genes (36). 67


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Besides, the open reading frame (ORF8), derived from a filamentous phage (f237), 68 has been associated with pandemic V. parahaemolyticus strains. However, some 69 pandemic strains lacking ORF8 were recently reported (8, 33, 46). Then, the main 70 features of the O3:K6 pandemic clone isolates are, a distinctive toxRS sequence 71 (toxRS/new) (36), with orf8 (42) and tdh genes in the absence of the trh gene, the 72 latter of which is found in some other pathogenic strains. In general, an isolate 73 possessing both tdh and toxRS/new can be considered as a pandemic strain (46). 74 Recent studies have shown that since 1996 at least 21 serovariants have emerged (40) 75 that share genetic markers specific for the pandemic serovar O3:K6 (8, 15, 36, 40, 51, 76 55). It has been reported that these serotypes probably originated from the same clone 77 as O3:K6 (8, 15, 36). On the other hand, non-O3:K6 serovars with pandemic traits are 78 increasingly isolated worldwide, and therefore they could possibly have a pandemic 79 potential (2). 80

In Mexico, there had been few reports of environmental tdh positive strains 81 isolated from water and fish (11), and recently, between September and October of 82 2004, more than 1230 cases of gastroenteritis were reported in a relatively small 83 geographic area, the southern part of Sinaloa, a state located in Northwest Mexico. 84 These cases were associated with consumption of raw or undercooked shrimp 85 collected in a lagoonal system. In Mexico, this was the first outbreak of gastroenteritis 86 caused by the pandemic strain of O3:K6 V. parahaemolyticus (9). In subsequent 87 years, recurrent sporadic cases were present in this state; however, the cases occurred 88 from South to North areas in the state. The present study focuses on the dissemination 89 of this pandemic strain in the state of Sinaloa from 2004 to 2010, and within this 90 context identifies the serotypes, serodiversity and virulence genes of Vibrio 91 parahaemolyticus isolated from environmental (from shrimp, seawater and sediment) 92


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and clinical samples. To our knowledge, this is the first report on the prevalence of V. 93 parahaemolyticus strains (toxigenic and pandemic O3:K6) isolated in clinical and 94 environmental samples over an extended period of time in Mexico. 95 96


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Materials and Methods 97 Area of study and sample collection. This study was performed in the state of 98 Sinaloa, located in the Northwest of Mexico. Sinaloa has over 650 km of coastline, 99 most (75%) of it facing the Sea of Cortez and some (25%) bordering the Pacific 100 Ocean. Sampling collection was performed into three regions along the Sinaloa state 101 divided in nine sites (three sites for each region) from September 2004 to December 102 2010. The sites represent leading shrimp producers in the State and clinical cases 103 were also detected near of these regions. A total of 2,266 environmental samples 104 (shrimp, 380; sediment, 65; and seawater, 1,821) were collected in the northern region 105 (Gua=Guasave, Ang=Angostura, Moc=Mocorito), the centre region (Nav=Navolato, 106 Cln=Culiacán, Elo=Elota) and the southern region (Maz=Mazatlán, Ros=Rosario, 107 Esc=Escuinapa) of Sinaloa (Fig. 1). Stool and environmental samples were not 108 collected in 2005. Stool specimens or rectal swabs (n=3023) were collected from 109 persons with gastroenteritis who had eaten seafood and requested attention in public-110 sector health-care agencies from September 2004 to December 2010 (Table 1). 111 Written informed consent was obtained and donors of human samples were selected 112 as recommended by the ethics committee of the Faculty of Medicine-UAS and the 113 Sinaloa State Public Health Laboratory. 114 115 Bacteriological analyses. All samples were processed as described in the 116 Bacteriological Analytical Manual of the Food and Drug Administration (32) and also 117 as described by Canizalez-Roman et al. 2011 (13). Briefly, 50 grams of Shrimp and 118 Sediment samples and 50 mL of Seawater were weighed and add into 450 mL of 119 sterile alkaline peptone water (APW) pH 8.6 each and homogenized in a Stomacher-120 400 circulator. The APW homogenate was incubated at 37ºC for 6-8 hours. The non-121


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bloody stool samples were collected in Cary–Blair transport medium and transported 122 at room temperature (RT) to the laboratory within 2 h. These specimens were also 123 enriched in APW (pH 8.6) for 6-8 h at 37°C. After incubation, the enrichment broths 124 (APW) were streaked onto thiosulfate–citrate–bile salts–sucrose (TCBS) agar plates 125 and (or) CV medium (CHROMagar, Paris, France) and incubated at 37°C for 18–24 126 h. At least three typical colonies (of V. parahaemolyticus) from each plate were 127 isolated and subjected to identification by Biochemical test and PCR as mentioned 128 below. After identification of Vibrio parahaemolyticus, a single colony from each 129 sample was used to continue the analysis (serotyping and virulence genes). 130 131 Extraction and purification of chromosomal DNA. Chromosomal DNA was 132 extracted using the Wizard genomic DNA purification kit (Promega Corp.), according 133 to the manufacturer’s instructions. Briefly, 3 ml of 16 to 18h culture in LB broth 134 containing 3% NaCl was harvested by centrifugation at 13,000 X g to 16,000 X g for 135 5 min. Cells were lysed at 80°C in nuclei lysis solution (Promega Corp.). RNase 136 solution was added to the cell lysate, followed by incubation at 37°C for 1 h and 137 cooling at room temperature. Protein precipitation solution (Promega Corp.) was 138 added to the RNase-treated cell lysate and vortexed vigorously. After incubation and 139 centrifugation at 13,000 X g to 16,000 X g for 3 min, the DNA was precipitated by 140 adding 0.6 volume isopropanol at room temperature. The DNA pellet was washed 141 with 70% ethanol, air dried, and dissolved in DNA rehydration solution (Promega 142 Corp.). Prehydrated DNA was stored at 2 to 8°C until use. 143 144 PCR assays. PCR amplification was performed in a 25 µL volume consisting of 1x 145 GoTaq Green Master Mix (Promega) primer targeting genes tl (7), R72H (34, 50), tdh 146


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and trh (7), and 0.5 µg of purified genomic DNA template, with the remaining 147 volume consisting of molecular biology grade water. PCR was routinely carried out in 148 a Thermal Cycler C1000 (BIO-RAD Laboratories, Hercules, California) under the 149 following cycling conditions: an initial period of DNA denaturation at 94 °C for 3 150 min, followed by 35 cycles of 1 min at 94 °C, 1 min at 58 °C, and 1 min at 72 °C, and 151 a final extension of 5 min at 72 °C. PCR assays for the amplification of the toxRS 152 (GS) and orf8 pandemic marker genes were performed using specific primers 153 previously reported to detect toxRS/New (36) sequences unique to the pandemic clone 154 of V. parahaemolyticus and the orf8 (39) sequence of phage f237, respectively. 155 Briefly, the PCR conditions were as follows: toxRS/New gene, initial denaturation at 156 94°C for 3 min, followed by 25 cycles of 30 s at 94°C, 30 s at 45°C and at 1 min at 157 72°C, with a final extension of 5 min at 72°C and orf8 gene denaturation at 94°C for 3 158 min, followed by 30 cycles at 94°C for 30 s, 30 s at 55°C, 30 s at 72°C, and a final 159 extension of 5 min at 72°C. Negative control reactions were performed 160 simultaneously by replacing the template DNA with sterilized water in the PCR 161 mixture. Ten microlitre aliquots of each amplification product were separated by 162 electrophoresis in a 2% agarose gel. Ethidium bromide staining (0.5 mg/mL) allowed 163 for the visualization of DNA fragments with a digital imaging system (Kodak, Model 164 E1 logia 100 imaging system). The sizes of the PCR fragment were compared against 165 a 50-bp DNA ladder (Promega DNA step ladder). 166 167 Serotyping. Serotyping of the V. parahaemolyticus isolates was done using a 168 commercially available V. parahaemolyticus antiserum test kit (Denka Seiken, Tokyo, 169 Japan) that has O1 to O11 antisera and 71 K antisera and by following the 170 manufacturer's instructions. Briefly, overnight strains were first grown on LB agar 171


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containing 3% NaCl and incubated at 37°C; a pool of colonies were collected and re-172 suspended in (1 ml) normal saline, an aliquot (0.5 ml) was boiled for 2 hours and 173 these lysates were used for serotyping based on the O antigen. The remaining cell 174 suspension (that not boiled) was used for serotyping based on the K antigen. 175 176 177


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Results 178 Detection of V. parahaemolyticus 179 A total of 2,266 environmental samples (380 samples from shrimp, 1,821 from 180 seawater and 65 from sediment) collected from nine sites in northern, central and 181 southern Sinaloa, from September 2004 to December 2010 (Fig. 1), were analyzed for 182 the presence of V. parahaemolyticus strains. These strains were isolated from 6.3% 183 (144) of the samples. Of the 144 strains isolated, 70 (49%) were obtained from shrimp 184 samples, 50 (35%) from seawater, and 24 (16%) from sediment (Table 1). In clinical 185 samples taken during the same period, Vibrio parahaemolyticus was detected in 154 186 of 3023 (5.09%) stool specimens or rectal swabs from persons with gastroenteritis 187 who had eaten seafood (Table 1). Regarding environmental samples, the presence of 188 V. parahaemolyticus was mainly detected in southern sites, whereas in respect to 189 cases of diarrhea, this bacterium was commonly isolated from patients from both 190 southern and northern Sinaloa (Table 1 and Fig. 1). 191 192 Serovars of V. parahaemolyticus isolates 193 V. parahaemolyticus serotyping was performed for epidemiological purposes and 194 serves as an important marker for both diarrheagenic and pandemic strains. As shown 195 in Table 2, 30 serovars were identified among 200 isolates that were serotyped and 196 recognized with antisera O and K from the 298 isolates (144 environmental isolates 197 and 154 clinical strains). Approximately 38% (55/144) of the environmental isolates 198 could be typed by both O and K antisera, resulting in 10 different O groups and 23 199 different K types and 28 serovars. 19.4% (28/144) of the strains were not recognized 200 by O antisera, while 61% (88/144) were not recognized by K antisera, and 27 strains 201 of the latter did not react to O:K antisera (OUT:KUT). The serogroups O1, O2, O3, 202


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O4, O5, O6, O7, O8, O10, and O11 accounted for 26% (37/144), 6.25% (9/144), 203 15.3% (22/144), 6.25% (9/144), 10.42% (15/144), 5.6% (8/144), 0.69% (1/144), 3.5% 204 (5/144), 6.25% (9/144), and 0.69% (1/144) of all the environmental isolates, 205 respectively. Hence, the most prevalent O serogroups were O1, O3 and O5. Isolates 206 that could be serotyped by O antisera, but not by any of the available K antisera, 207 represented 42.3% (61/144) of the samples. Twenty three were serogroup O1:KUT 208 (untyped), one was O2:KUT, eleven were O3:KUT, five O4:KUT, eight O5:KUT, 209 two O6:KUT, one was O7:KUT, eight were O10:KUT, and one was O11:KUT). The 210 most frequent serovars were O1:KUT, O5:KUT and O3:KUT, comprising 23, 8 and 211 11 strains, respectively. Among serotypes recognized by the antisera O and K, the 212 most frequent were O2:K28, O3:K6 and O5:K17, comprising 7, 4 and 3 strains, 213 respectively. One (1/144) strain was recognized by K antisera but not by O antisera 214 (OUT:K8, Table 2). 215

In clinical isolates, there were 8 O serogroups and 14 different K types, and 14 216 serovars that could be identified in the 146 strains recognized by both currently 217 available O and K antisera. These 146 strains accounted for 94.8% of 154 clinical 218 strains found with V. parahaemolyticus. Six clinical strains (3.9%) could be 219 recognized only by O antisera but not by K antisera (one was O1:KUT, one O4:KUT, 220 one O5:KUT, one O8:KUT and two were O11:KUT). Serogroups O1 and O3 were 221 the most commonly recognized, accounting for 89% (137/154) of all clinical strains. 222 Importantly, O3:K6 was the predominant serovar of clinical strains throughout the 223 period of the study, accounting for 79.8% (123/154). Besides O3:K6, other serotypes 224 were found, including O1:K9, O1:K33, O3:K58, O3:K68, O4:K8, O4:K10, O4:K12, 225 O4:K63, O5:K15, O5:K17, O6:K46, O8:K20 and O10:K52 (Table 2). 226 227


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Detection of virulence and pandemic marker genes by PCR 228 V. parahaemolyticus strains isolated from environmental and clinical samples were 229 screened for virulence and pandemic marker genes orf8, toxRS/New, tdh and trh, by 230 PCR. Based on the presence or absence of virulence genes, we classified the isolates 231 into three groups: pandemic, pathogenic (non-pandemic) and nonpathogenic strains. 232 The features of pandemic strains include tdh positive, toxRS/New positive, orf8 233 positive (or negative), and trh negative. Pathogenic strains are tdh and/or trh positive. 234 On the contrary, nonpathogenic strains are characteristically both tdh and trh 235 negative. 236 Among environmental V. parahaemolyticus strains, 4.1% (6/144) of the 237 isolates were identified as pandemic: four were pandemic O3:K6 strains containing 238 the orf8 gene (2 detected in seawater, 1 in shrimp and 1 in sediment samples), one 239 pandemic O3:KUT strain (detected in seawater), and one pandemic O10:KUT strain 240 without orf8 (detected in sediment). A total of 52% (75/144) of environmental isolates 241 were found to carry the virulence genes (tdh and/or trh). Of these, 68% (51/75) had 242 the tdh gene and belonged to 22 different serotypes, most of which were OUT:KUT 243 (n=22). Others included O1:K25, O1:K26, O1:K33, O1:K56, O2:K28, O3:K59, 244 O4:K68, O8:K21 and O10:K52. The 30.6% (23/75) of environmental isolates with 245 both tdh-trh genes included O1:K32, O1:K41, O3:K58, O5:K15 and OUT:KUT 246 serotypes. We detected only 1 isolate with the trh gene that had the O1:KUT serotype. 247 Nonpathogenic strains were found in 43.8 % (63/144) of environmental isolates, 248 belonging to serotypes O1:K26, O1:K32, O1:K33, O1:K56, O2:K28, O4:K10, 249 O4:K12, O4:K53, O5:K15, O5:K17, O8:K20 and O10:K52, with a high frequency of 250 O1:KUT (11) serovar (Table 2). 251

Among clinical V. parahaemolyticus strains, 80.5% (124/154) of isolates were 252


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identified as belonging to the pandemic serotype with orf8 gene. Of these 123 253 belonged to serovar O3:K6 and one isolate belonged to serovar O4:K12. 12.9% 254 (20/154) of the clinical isolates encode tdh and/or trh. Of these 4 isolates encoded tdh 255 only and were serotypes O1:K33, O3:K58, O10:K52 and OUT:KUT. On the other 256 hand, 5 isolates, with serotypes O3:K6 (3), O8:K20 and OUT:KUT, were positive for 257 both tdh and orf8 genes. We also detected seven isolates that were tdh and trh 258 positive, with serotypes O3:K58, O3K68, O4:K8, O4:K63, O4:K10 and O4:KUT, as 259 well as 3 isolates that were tdh, trh and orf8 positive, with serotypes O1:K9, O1:KUT 260 and O6:K46. One of the clinical isolates was only trh positive (tdh negative), with the 261 O5:K17 serotype (Table 2). Interestingly, 6.5% (10/154) of clinical isolates belong to 262 the nonpathogenic group, this strains did not encoded neither the tdh nor trh gene, 263 with serotypes O5:K15 (2), O5:KUT (1), O8:KUT (1), O10:K52 (4) and O11:KUT 264 (2). Unlike those serovars detected in environmental isolates, in clinical samples the 265 most prevalent serotype was O3:K6 and the following serotypes were detected not 266 only in environmental but also in clinical strains: O1:K33, O3:K6, O3:K58, O4:K10, 267 O4:K12, O5:K15, O5:K17, O6:K46, O8:K20 and O10:K52. 268 269 Yearly presence of the O3:K6 pandemic clone and pathogenic strains 270 During the present study, the pandemic clone O3:K6 serotype was the most prevalent 271 one isolated from clinical samples in every year. V. parahaemolyticus was present 272 with high proportions of these clinical strains (70.5% to 100%) in most years. Only in 273 two years (2007 and 2009) gastroenteritis cases by V. parahaemolyticus were not 274 detected. On the contrary, in environmental strains the O3:K6 pandemic clone was 275 detected only in 2008 and 2010, at a proportion of 7.5% and 4%, respectively (Table 276 3). 277


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It is noteworthy that in clinical V. parahaemolyticus strains, pathogenic 278 versions (tdh and/or trh) were detected in low proportions, from 2.98% to 21.3% 279 through the years (except in 2007 and 2010, when pathogenic strains were not 280 detected). However, the pathogenic strains in the environmental were detected every 281 year in high proportions, from 20% to 92.5% (except in 2004, when pathogenic 282 strains were not detected) (Table 3). 283 284


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Discussion 285 V. parahaemolyticus, a halophilic bacterium, has in recent years emerged as a 286 worldwide pandemic pathogen causing gastroenteritis related to consumption of 287 seafood (5, 18, 55). Before 2004 in Mexico, there had been no reports of outbreaks 288 caused by V. parahaemolyticus (9), and few reports of environmental tdh positive 289 strains had been isolated from water and fish (11). The first outbreak in Mexico (1230 290 cases), was caused by the pandemic strain of O3:K6 and associated with the 291 consumption of raw or undercooked shrimp in southern Sinaloa, a state in the 292 Northwest of Mexico (9). Since 2004, recurrent sporadic cases have shown up in this 293 state. In recent years, new cases have arisen in different areas of Sinaloa (including 294 southern and northern areas). The current contribution is a study developed during 6 295 years (from September 2004 to December 2010) along the Pacific Coast of Sinaloa. In 296 both clinical cases and environmental samples, we detected the presence of pandemic 297 and pathogenic strains belonging to different serotypes. It turns out that the pandemic 298 clone O3:K6 (tdh, toxRS/New, and/or orf8 genes) was the most prevalent serotype 299 isolated from clinical samples in every year of the study. It was also detected in 300 environmental samples (seawater, sediment and shrimp), suggesting that it is endemic 301 to this setting. To our knowledge, this is the first report of the prevalence of V. 302 parahaemolyticus strains (toxigenic and pandemic) isolated in Mexico. 303

Our data show that in Sinaloa, as of 2004 the infections caused by O3:K6 304 pandemic clone spread rapidly from the southern to northern region, over more than 305 650 km of coastline. This spreading of V. parahaemolyticus is possibly due to the 306 movement of oceanic waters or to human activities, such as ballast water discharges 307 (4). This spreading of pandemic V. parahaemolyticus is still a speculative question 308 that requires further investigation since the pandemic clone can potentially migrate to 309


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south Mexico and the USA. 310 Notably, it has been shown that in different parts of the world the distribution 311

of V. parahaemolyticus in the marine environment (seawater, sediment and marine 312 flora and fauna), is highly related with a combination of environmental parameters 313 including sea surface temperature, salinity, turbidity and chlorophyll concentration 314 (12, 28-31). Kaneko and Colwell (29-31) reported the seasonal cycle of V. 315 parahaemolyticus in the sediment, water and plankton of Chesapeake Bay (USA), 316 determining that the bacterium might survive in sediments during the winter and be 317 released into the water column in late spring or early summer when the temperature 318 increase. In tropical countries the seasonal cycle of the organism is correlated with the 319 rainy and dry seasons; the lowest numbers are found in rainy months, and the highest 320 numbers are found in the dry season (43). Beside, in tropical waters the temperature 321 fluctuates less throughout the year, while the salinity fluctuates more (17). In the 322 present study, the isolation rate of V. parahaemolyticus was low (6.3%) in the marine 323 environment. However, we found a moderate prevalence in sediment of 36% (24/65) 324 and in shrimp samples of 18% (70/380). The prevalence of V. parahaemolyticus in 325 seawater samples was only 2.7% (50/1821), despite high number of seawater samples 326 (1821) collected in this study. Thus, we speculate that the observed prevalence may 327 also be produced by seasonal variation of the V. parahaemolyticus population (due to 328 salinity, turbidity and chlorophyll concentration) by "hibernating" in the sediment or 329 in partnership with the marine fauna, during the sampling period (mainly from 330 November to January). But we cannot prove these hypotheses due to insufficient or 331 absent data in this work (sea surface temperature, salinity and chlorophyll). 332 Additional research is needed to determine the possibility of changes in the relative 333 abundance of pathogenic V. parahaemolyticus during different seasons of the year on 334


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the Pacific Coast of Mexico, particularly in the Sinaloa State, as well as the 335 identification of associated environmental factors (e.g. temperature or salinity 336 effects). 337

Serotyping analysis demonstrates that there were fewer serogroups and 338 serovars in clinical than in environmental isolates, clearly demonstrating that clinical 339 isolates belong to a specific group of strains, in agreement with other studies (14). 340 Whether these strains will contain others or unknown virulence determinants remain 341 to be investigated. Among the diversified environmental samples, O1 was the 342 predominant O serogroup found, accounting for 26% of all isolates. This study on the 343 Pacific Coast of Mexico differs with the unique serogroup study developed on the 344 Gulf coast of this same country, where O3 was the most frequently encountered 345 serogroup (30%) among the environmental strains (11). Several researchers have 346 pointed out that certain serogroups are constant in some specific geographical areas 347 (1, 48). 348

On the other hand, throughout the present study O3:K6 was the predominant 349 serovar of the clinical strains recognized, accounting for 79.8% (123/154) of the total 350 number of strains isolated. The regional predominance of a specific serotype of V. 351 parahaemolyticus has occasionally been reported in distinct geographic areas. Along 352 the United States Pacific Coast, V. parahaemolyticus O4:K12 was the dominant 353 serotype causing infections (1). Similarly, O3:K6 was the predominant serovar in 354 Peru in 2007 (23). In Chile the O3:K6 pandemic clone was also the predominant 355 serovar, causing outbreaks in Puerto Montt that began in 2004 and reached a peak in 356 2005, with 3,600 clinical cases. Up to 2006, every analyzed case in Chile was caused 357 by the serovar O3:K6 pandemic strain. Among the 475 cases reported in the summer 358 of 2007, 73% corresponded to the pandemic strain (10, 21, 24). In 2009, Hart et al. 359


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(25) reported that the decrease of outbreaks caused by O3:K6 was associated with a 360 change in serotype of many pandemic isolates to O3:K59, and to the emergence of 361 new clinical strains. Contrarily, no change in the prevalence of the O3:K6 pandemic 362 clone was observed in the present study. We identified 8 serovars that were not listed 363 by the Japanese committee on the serological typing of V. parahaemolyticus, two in 364 clinical (O1:K9, O1:K33) and six in environmental strains (O3:K32, O3:K68, 365 O4:K68, O6:K6, O8:K8, O10:K52). These serovars have also been found in several 366 other studies (14, 38, 40, 41, 53, 57). 367

It has been demonstrated that the existence of the tdh and/or trh genes in a 368 strain is associated with its ability to cause gastroenteritis (44). Therefore, the 369 presence of these genes is routinely used to determine the pathogenicity of V. 370 parahaemolyticus strains (30). In molecular epidemiological studies, it has been 371 shown that clinical isolates possess the tdh and/or trh genes in up to 90% of clinical 372 strains (serotype O3:K6 strain has increasing prominence) (14, 22, 45), whereas their 373 presence in environmental isolated strains is rare (14, 17, 26, 40, 44, 52, 58). Data on 374 the current study are in accordance with these previous observations, as 93% of all 375 clinical strains carried tdh and/or trh genes (80.5% pandemic group and 12.9% 376 pathogenic group). Interestingly, we detected a high proportion of strains (52%) with 377 pathogenic markers (tdh and/or trh), but a low proportion (4.1%) of strains belonging 378 to the pandemic group, in the environmental isolates represented by many different 379 serotypes. The high rate of detection of pathogenic V. parahaemolyticus in the marine 380 environment may be due to a decreased prevalence of total V. parahaemolyticus and 381 not to a greater abundance of pathogenic strains. However, data related to the 382 prevalence of pathogenic Vibrio parahaemolyticus has not previously been 383


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investigated at the Pacific coasts of Mexico. This study provides novel information on 384 the abundance and characteristics of pathogenic V. parahaemolyticus in this setting. 385

We found that most of the clinical isolates belong to pandemic strains (tdh, 386 toxRS/New positive) with orf8, and that O3:K6 was the dominant pandemic serovar 387 during the six-year period of study. Previous studies have shown that isolates 388 belonging to the new clone O3:K6 (tdh, toxRS/New positive) are associated with 389 outbreaks of human gastroenteritis (36, 45, 46); however, similar to our study, these 390 strains are rarely isolated from environmental samples (14, 19, 54). In the current 391 contribution, the few environmental isolates belonging to the pandemic serotype were 392 O3:K6 (in seawater, shrimp and sediment), O3:KUT (in seawater) and O10:KUT (in 393 sediment). Moreover, the discovery of these strains in seawater and sediment suggests 394 a complete seasonal cycle of V. parahaemolyticus, as has been reported by Kaneko 395 and Colwell in the Chesapeake Bay (USA) (30, 31). Thus, this is the first report about 396 the presence of the pandemic group in the environment in Mexico. 397

Along the approximately 650 km of the Sinaloa Pacific coastal area, seawater 398 temperatures range from 23 to 30.8°C (47), with an average humidity of 68%. It has 399 been reported that elevated temperatures (>30 °C) are appropriate for the proliferation 400 of this pathogen (19). The importance of water temperature in the epidemiology of 401 infections is reflected by the fact that most outbreaks occur during the warmer 402 months. The progressive spread of V. parahaemolyticus and its colonization of new 403 areas has been related to an unusual increase in seawater temperatures in coastal 404 zones (16, 27, 45). This might account for the outbreaks of diarrhea caused by V. 405 parahaemolyticus during the summer of 2004 in Alaska (38), and also in southern 406 Chile at Puerto Montt during 2004-2007, where seawater temperatures are normally 407 low and V. parahaemolyticus infection is very rare. In the latter case, ≈7,000 cases 408


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were reported, perhaps due to the climatic change, such as the warm ocean currents 409 commonly referred to as the El Niño phenomenon (10, 20, 21, 24). The arrival of El 410 Niño waters to South America causes a general disruption of the environmental 411 conditions of coastal areas, displacing the native species southwards (4). Besides 412 weather changes, the spread of non-indigenous microorganisms has been attributed to 413 the water ballast of ships (37, 39). Ballast water-mediated invasions have been 414 proposed for the entrance of O3:K6 strains into Texas in 1998 (16) as well as into 415 Coruña, Spain, in 2004 (35). According to Cabanillas et al, the O3:K6 pandemic may 416 have been transported to Mexico via the port of Mazatlan (in southern Sinaloa) (9). 417 However, the ecology and seasonality of V. parahaemolyticus in the marine 418 environment of northwest Mexico is still unclear. 419

Recently, the analysis of the arrival of the O3:K6 clone at the Pacific coasts of 420 South America has provided novel insights linking the origin of the invasion in 1997 421 from the Asian (India) populations and describing the successful establishment of the 422 O3:K6 populations, first in Peru and subsequently in the South of Chile (4). Thus, we 423 speculate that this Peruvian foothold of the O3:K6 clone could possibly be the origin 424 of the arrival of this strain to the coasts of Northwestern Mexico (Sinaloa) in 2003-425 2004. However, the dynamics of dissemination of the environmental human pathogen 426 Vibrio parahaemolyticus are uncertain. 427

Results obtained within the present study revealed the presence and 428 persistence of V. parahaemolyticus (O3:K6) in marine environments in Sinaloa. 429 Records of the Sinaloa State Public Health Laboratory indicate that from 1999 to 430 2003, diarrhea cases related to seafood consumption (<0.5%) were not widespread in 431 Sinaloa state in spite of the large consumption of raw shellfish. From arrival of the 432 pandemic strain O3:K6, possibly between 2003 and 2004, cases of seafood-related 433


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diarrhea have now increased (to 5%). 434 Noteworthy in this study was the prevalence of a few similar serotypes of V. 435

parahaemolyticus between heterogeneous environmental (especially from shrimp) 436 and clinical strains from stools (O1:K33, O3:K6, O3:K58, O4:K10, O4:K12, O5:K15, 437 O5:K17, O6:K46, O8:K20, O10:K52). The fact that we isolated similar serotypes and 438 virulence genes from patients and incriminated environmental sources during the 439 outbreaks of several epidemics also suggests a certain association between serotypes 440 found in the environment and those linked to gastroenteritis (6). Although serotyping 441 technique is good for differentiating clinical and environmental isolates of V. 442 parahaemolyticus further molecular methods for the analysis of genetic variability 443 among strains isolated should be performed. For this purpose, in future studies, we 444 would like to compare the phenotypic and genotypic properties, including DNA 445 fingerprints by pulsed-field gel electrophoresis (PFGE) or Multilocus sequence typing 446 (MLST), of the Mexican V. parahaemolyticus (O3:K6) strains isolated from clinical 447 sources with those from Mexican environments (shrimp, seawater and sediment). 448

Data from present study is in accordance with other reports, in which V. 449 parahaemolyticus environmental strains show a high serological variability. We 450 demonstrate that the pandemic clone O3:K6 harboring toxRS/New and orf8, along 451 with tdh, is predominant in infections all along coastal areas of Sinaloa. In this same 452 area, serotyping and virulence factors showed some similar distributions of pandemic 453 and pathogenic V. parahaemolyticus, isolated from clinical and environmental 454 sources. The fact that certain serotypes and virulence factors were found in both 455 clinical and environmental isolates, while many others were only observed in 456 environmental isolates, implies that certain serotypes with virulence factors are more 457 relevant for human disease. The current results provide the first available data on the 458


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distribution, prevalence and detection of virulence factors and pandemic genes of V. 459 parahaemolyticus in clinical and environmental strains in Mexico. The presence and 460 persistence of pandemic and pathogenic environmental V. parahaemolyticus strains in 461 Sinaloa is matter of concern for public health authorities, as the potential of outbreaks 462 along the northern Mexican coastline of the Pacific Ocean is now well established. 463 Therefore, this information is important for preventing public health problems that not 464 only are likely to affect the populations in Sinaloa, but also might spread to other 465 states in Mexico and to the USA. 466 467


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Acknowledgments 468 This work was supported by a grant from Consejo Nacional de Ciencia y Tecnología 469 Mexico (CB-2007-01-84405), PROFAPI 2010/025 and CECyT 2009 to ACR. JVR 470 was supported by a scholarship from CONACYT No. 202157 and a “Doctores 471 Jovenes” scholarship from the UAS and a DIM scholarship from the ESM-IPN. We 472 would like to thank the Department of Microbiology and Epidemiology, the Sinaloa 473 State Public Health Laboratory, and Elizandra Quiñonez for their technical help. We 474 would also like to thank Bruce Allan Larsen for reviewing the English in this 475 manuscript and Dr. Jorge E. Vidal from the Rollins School of Public Health, Emory 476 University for critical reading. 477 478


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Figure legends 708 Figure 1. Map showing the geographical location of sampling sites. clinical cases 709 (stools specimens or rectal swabs, n=3023) and environmental (seawater, sediment 710 and shrimp, n=2,266) samples during 2004 to 2010 in the coast region of northern 711 (Gua=Guasave, Ang=Angostura, Moc=Mocorito), central (Nav=Navolato, 712 Cln=Culiacán, Elo=Elota) and southern (Maz=Mazatlán, Ros=Rosario, 713 Esc=Escuinapa) Sinaloa. 714 715 716 717


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TABLE I. Distribution of Vibrio parahaemolyticus strains isolated from clinical (stools) and environmental (seawater, sediment and shrimp) samples during 2004 to 2010.

No. of isolates

YEAR CLINICAL

ENVIRONMENTAL

Total

SHRIMP SEAWATER SEDIMENT

S C N S C N S C N S C N Total

2004 67 67 8 3 11 2006 17 34 10 61 15 4 20 10 49 2007 2 3 5 2008 9 1 10 32 4 4 40 2009 6 1 6 1 14 2010 12 4 16 3 1 13 2 6 25

TOTAL 154 144 S, southern region; C, central region; N, northern region of the state of Sinaloa. Total of clinical samples n=3023, and environmental samples n=2,266 (shrimp 380,sediment 65, and seawater 1,821).


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TABLE II. Serovar, virulence and pandemic characteristics of 298 Isolates during 2004 - 2010

Virulence genes CLINICAL ENVIRONMENTAL

O serogroup Serovars No. of isolates tdh trh toxRS/New Orf-8 F Sh SW S

O1 O1:KUT 1 + - - + 1 1 + + + - 1 4 + + - - 4 7 + - - - 4 3 1 - + - - 1 11 - - - - 3 3 5

O1:K9 1 + + + - 1 O1:K25 1 + - - - 1 O1:K26 1 + - - - 1

1 - - - - 1 O1:K32 1 + + - - 1

1 - - + - 1 1 - - - - 1

O1:K33 2 + - - - 1 1 1 - - - - 1

O1:K41 2 + + - - 1 1 O1:K56 1 + - - - 1

1 - - - - 1 O2 O2:KUT 1 - - - - 1

O2:K28 1 - - - + 1 1 - - + - 1 4 + - - - 1 3 2 - - - - 2

O3 O3:KUT 1 + - + + 1 3 + + - - 1 2 1 - - + - 1 5 + - - - 3 1 1 1 - - - - 1

O3:K4 1 + + - - 1 O3:K6 3 + - - + 3

127 + - + + 123 1 2 1 O3:K29 1 + - - - 1 O3:K32 1 + - - - 1 O3:K33 1 - - - + 1 O3:K58 2 + + - - 1 1

1 + - - - 1 O3:K59 1 + - - - 1 O3:K68 2 + + - - 1 1

1 + - - - 1


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O4 O4:KUT 1 - - - + 1 3 + + - - 1 2 1 + - - - 1 1 - - - - 1

O4:K8 1 + + - - 1 O4:K10 2 + + - - 2

1 - - - - 1 O4:K12 1 + - + + 1

1 - - - - 1 O4:K53 1 - - - - 1 O4:K63 1 + + - - 1 O4:K68 1 + - - + 1

O5 O5:KUT 1 - - - + 1 1 + + - - 1 2 + - - - 2 5 - - - - 1 2 2

O5:K15 1 + + - - 1 3 - - - - 2 1

O5:K17 3 - - - - 1 1 1 1 - + - - 1

O5:K68 1 - - - + 1 1 - - - - 1

O6 O6:KUT 2 - - - - 2

O6:K6 1 - - - + 1 1 + - - - 1

O6:K46 1 + - - - 1 1 + + - + 1 3 - - - - 2 1

O7 O7:KUT 1 + - - - 1 O8 O8:KUT 1 - - - - 1

O8:K8 1 + - - - 1 1 - - - - 1

O8:K20 1 + - - + 1 1 - - - + 1

O8:K21 2 + - - - 1 1 O10 O10:KUT 1 + - + - 1

2 + + - - 1 1 3 + - - - 1 1 1 2 - - - - 1 1

O10:K52 2 + - - - 1 1 4 - - - - 4

O11 O11:KUT 1 + + - - 1


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2 - - - - 2 OUT OUT:K8 1 - - - - 1

OUT:KUT 1 + - - + 1 1 - - - + 1 2 - - + - 2 3 + + - - 1 2 13 + - - - 1 10 1 1 9 - - - - 3 4 2

TOTAL 298 154

Sh: Shrimp; Sw: Seawater; S: Sediment; F: Feces


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TABLE III. O3:K6 Pandemic clone and pathogenic strains in isolates of Vibrio parahaemolyticus from environmental and clinical samples per year

YEAR O3:K6 Pandemic Clone Pathogenic strains

Clinical Environmental Clinical Environmental

2004 88% (59/67) 0% (0/11) 2.98% (2/67) 0% (0/11)

2006 70.5% (43/61) 0% (0/49) 21.3% (13/61) 36.7% (18/49)

2007 - 0% (0/5) - 60% (3/5)

2008 80% (8/10) 7.5% (3/40) 20% (2/10) 92.5% (37/40)

2009 - 0% (0/14) - 85.7% (12/14)

2010 100% (16/16) 4% (1/25) 0% (0/16) 20% (5/25)

Total 81.8% (126/154) 2.7% (4/144) 11% (17/154) 52% (75/144)

Pandemic Clone: Serotype O3:K6 (tdh+, toxRS/New + and/or Orf-8+) Pathogenic strains: tdh+ and or trh+ or both genes. -: Denotes absence of Vibrio parahaemolyticus isolates.


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