Antibiotic resistance trends among Vibrio vulnificus and Vibrio parahaemolyticus isolated from the Chesapeake Bay, Maryland: a longitudinal study.

Antibiotics are often used to treat severe Vibrio infections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009-2012 and 2019-2022). Vibrio parahaemolyticus (n = 134) and Vibrio vulnificus (n = 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%-96%) of V. parahaemolyticus isolates from both sampling periods were resistant to ampicillin and only 2%-6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistant V. vulnificus isolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%-8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns among V. parahaemolyticus and V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibrio spp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, some Vibrio isolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed with Vibrio vulnificus infections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severe Vibrio spp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion.

A One-Step RPA-CRISPR Assay Using crRNA Based on Suboptimal Protospacer Adjacent Motif for Vibrio vulnificus Detection.

Vibrio vulnificus is a hazardous foodborne pathogen responsible for approximately 95% of seafood-related deaths. This highlights the urgent requirement for specialized detection tools to be developed and used by food enterprises and food safety authorities. The DETECTR (DNA endonuclease targeted CRISPR trans reporter) system that combines CRISPR/Cas and recombinase polymerase amplification (RPA) has been utilized to develop a molecular detection assay for V. vulnificus. However, because the incompatibility between RPA and Cas12a cleavage has not been addressed, it is a two-step assay that lacks convenience and presents contamination risk. Here, we developed a one-step RPA-CRISPR assay for V. vulnificus using a special crRNA targeting a sequence with a suboptimal protospacer adjacent motif (PAM). The entire assay, conducted at 37°C, takes only 40-60 min, yields results visualized under blue light, and exhibits exceptional specificity and sensitivity (detecting 4 pathogen genome copies per reaction). This study offers a valuable tool for detecting V. vulnificus, aiding in foodborne infection prevention, and exemplifies one-step RPA-CRISPR assays managing Cas-cleavage activity through PAM adjustments.

Characteristics of Vibrio vulnificus isolates from clinical and environmental sources.

Researchers have developed multiple methods to characterize clinical and environmental strains of Vibrio vulnificus. The aim of our study was to use four assays to detect virulence factors in strains from infected patients and those from surface waters/sediments/oysters of South Carolina and the Gulf of Mexico. Vibrio vulnificus strains from clinical (n = 81) and environmental (n = 171) sources were tested using three real-time PCR methods designed to detect polymorphisms in the 16S rRNA, vcg and pilF genes and a phenotypic method, the ability to ferment D-mannitol. Although none of the tests correctly categorized all isolates, the differentiation between clinical and environmental isolates was similar for the pilF, vcgC/E and 16S rRNA assays, with sensitivities of 74.1-79.2% and specificities of 77.4-82.7%. The pilF and vcgC/E assays are comparable in efficacy to the widely used 16S rRNA method, while the D-mannitol fermentation test is less discriminatory (sensitivity = 77.8%, specificity = 61.4%). Overall percent agreement for the D-mannitol fermentation method was also lower (66.7%) than overall percent agreement for the 3 molecular assays (78.0%-80.2%). This study demonstrated, using a large, diverse group of Vibrio vulnificus isolates, that three assays could be used to distinguish most clinical vs environmental isolates; however, additional assays are needed to increase accuracy.

An isothermal recombinase polymerase amplification and lateral flow strip combined method for rapid on-site detection of Vibrio vulnificus in raw seafood.

Vibrio vulnificus is an important foodborne pathogenic bacterium that mainly contaminates seafood. Rapid and accurate technologies that suitable for on-site detection are critical for effective control of its spreading. Conventional detection methods and polymerase chain reaction (PCR)-based and qPCR-based approaches have application limitations in on-site scenarios. Application of loop-mediated isothermal amplification (LAMP) technology was a good step towards the on-site detection. In this study, a recombinase polymerase amplification (RPA)-based detection method for V. vulnificus was developed combining with lateral flow strip (LFS) for visualized signal. The method targeted the conservative empV gene encoding the extracellular metalloproteinase, and finished detection in 35 min at a conveniently low temperature of 37 °C. It showed good specificity and an excellent sensitivity of 2 copies of the genome or 10-1 colony forming unit (CFU) per reaction, or 1 CFU/10 g in spiked food samples with enrichment. The method tolerated unpurified templates directly from sample boiling, which added the convenience of the overall procedure. Application of the RPA-LFS method for clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. This RPA-LFS combined method is well suited for on-site detection of V. vulnificus.

A comparison between farmed oysters using floating cages and oysters grown on-bottom reveals more potentially human pathogenic Vibrio in the on-bottom oysters.

Eating raw oysters can come with serious health risks, as oysters can potentially contain bacteria of the Vibrio genus that cause food-borne infections. Vibrio bacteria are concentrated by oysters and, when consumed, infections can result with severe symptoms such as diarrhoea, lesions on the extremities, or even death. Vibrio spp. concentrations are strongly affected by season, location, and other factors such as temperature and salinity. Previous research in North Carolina oysters has been conducted on wild and farmed oysters but not at the same time. Farmed, or aquaculture raised, oysters are considerably different from wild oysters and could possibly pose different health risks. Farmed oysters are handled, raised from seed, and often grown using suspended grow-out systems called 'floating cages'. Therefore, farmed oysters can be grown at the surface of the estuary, while wild oysters typically grow at the bottom of the water column. This project compared the concentrations of Vibrio spp. in suspended, farm-grown oysters and wild oysters at three sites, using a paired approach with farmed and wild oysters sampled in proximity. An important part of this comparison was identifying pathogenicity of the bacteria isolated from the samples. Distinction was made between off- and on-bottom farming. Interestingly, on-bottom oysters had more pathogenic V. vulnificus than off-bottom oysters.

Seasonal and Geographical Differences in Total and Pathogenic Vibrio parahaemolyticus and Vibrio vulnificus Levels in Seawater and Oysters from the Delaware and Chesapeake Bays Determined Using Several Methods.

Oyster and seawater samples were collected from five sites in the Chesapeake Bay, MD, and three sites in the Delaware Bay, DE, from May to October 2016 and 2017. Abundances and detection frequencies for total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus were compared using the standard most-probable-number-PCR (MPN-PCR) assay and a direct-plating (DP) method on CHROMagar Vibrio for total (tlh+ ) and pathogenic (tdh+ and trh+ ) V. parahaemolyticus genes and total (vvhA) and pathogenic (vcgC) V. vulnificus genes. The colony overlay procedure for peptidases (COPP) assay was evaluated for total Vibrionaceae DP had high false-negative rates (14 to 77%) for most PCR targets and was deemed unsatisfactory. Logistic regression models of the COPP assay showed high concordances with MPN-PCR for tdh+ and trh+V. parahaemolyticus and vvhA+V. vulnificus in oysters (85.7 to 90.9%) and seawater (81.1 to 92.7%) when seawater temperature and salinity were factored into the model, suggesting that the COPP assay could potentially serve as a more rapid method to detect vibrios in oysters and seawater. Differences in total Vibrionaceae and pathogenic Vibrio abundances between state sampling sites over different collection years were contrasted for oysters and seawater by MPN-PCR. Abundances of tdh+ and trh+V. parahaemolyticus were ∼8-fold higher in Delaware oysters than in Maryland oysters, whereas abundances of vcgC+V. vulnificus were nearly identical. For Delaware oysters, 93.5% were both tdh+ and trh+, compared to only 19.2% in Maryland. These results indicate that pathogenic V. parahaemolyticus was more prevalent in the Delaware Bay than in the Chesapeake Bay.IMPORTANCE While V. parahaemolyticus and V. vulnificus cause shellfish-associated morbidity and mortality among shellfish consumers, current regulatory assays for vibrios are complex, time-consuming, labor-intensive, and relatively expensive. In this study, the rapid, simple, and inexpensive COPP assay was identified as a possible alternative to MPN-PCR for shellfish monitoring. This paper shows differences in total Vibrionaceae and pathogenic vibrios found in seawater and oysters from the commercially important Delaware and Chesapeake Bays. Vibrio parahaemolyticus isolates from the Delaware Bay were more likely to contain commonly recognized pathogenicity genes than those from the Chesapeake Bay.

Septicemia, necrotizing fasciitis, and peritonitis due to Vibrio vulnificus treated with early use of polymyxin B hemoperfusion in a patient undergoing CAPD: a case report.

BACKGROUND: Vibrio vulnificus infection is a rare but fatal foodborne illness. Here, we report a case of Vibrio vulnificus peritonitis followed by severe septicemia in a patient undergoing continuous ambulatory peritoneal dialysis (CAPD) who was treated with hemoperfusion using polymyxin B immobilized fiber. CASE PRESENTATION: A 63-year-old man undergoing CAPD was admitted to the emergency room due to general weakness, fever, and abdominal pain with hazy dialysate. Two days before admission, he had eaten raw fish. Initial laboratory tests including peritoneal fluid analysis suggested peritonitis. Despite empirical intraperitoneal antibiotic treatment, his fever did not subside, and multiple vesicles on the extremities newly appeared. The result of initial peritoneal fluid culture and blood cultures reported Vibrio vulnificus as the most likely causative pathogen. Hemoperfusion with polymyxin B immobilized fiber was performed to control gram-negative bacterial septicemia with antibiotics targeting the pathogenic organism. The patient recovered completely and was discharged without complications. DISCUSSION AND CONCLUSION: Suspicion of Vibrio vulnificus infection in susceptible immunocompromised patients is important for early diagnosis and prompt management. Peritonitis should be noted as a clinical manifestation of Vibrio vulnificus infection in CAPD patients, and polymyxin B hemoperfusion along with proper antibiotics could be considered as a treatment option.

Prevalence and virulence of Vibrio species isolated from raw shrimp from retail markets in Reynosa, Mexico.

In this study, the prevalence of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio mimicus, Vibrio vulnificus and Vibrio spp. in shrimp from retail markets in Reynosa, Mexico was determined. A total of 765 isolates, identified as Vibrio spp. (59·1%), V. cholerae (17·8%), V. mimicus (6·7%) and V. parahaemolyticus (4·6%), were obtained; V. vulnificus was not detected. Most of the strains were isolated from supermarkets (48·1%), followed by street vendors (37·3%) and retail stores (14·6%). Moreover, several virulence genes were identified in V. cholerae: toxR (100%), OmpU (76·5%), hlyA (76·5%), VPI (19·9%) and tcpA (5·1%); in V. mimicus: vmh (100%), wzb (74·5%), pilF (54·9%), VPI (43·1%), OmpU (29·4%) and tdh (9·8%); and in V. parahaemolyticus: toxR (100%), tlh (100%), VP1680 (51·4%) and VPI (11·4%). These results show the low safety of this food and the potential risk to consumers' health, since this product in Mexican cuisine is sometimes served raw or semi-cooked. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the prevalence of pathogenic Vibrio cholerae, Vibrio mimicus and Vibrio parahaemolyticus isolated from shrimp that is commercialized in Reynosa city. This could represent a risk to consumers' health, since outbreaks related to shrimp contaminated with Vibrio have been previously reported. Additionally, shrimp fishing has a major role in Mexico's economy.

Faraday-Cage-Type Electrochemiluminescence Immunoassay: A Rise of Advanced Biosensing Strategy.

Electrochemiluminescence immunoassays are usually carried out through "on-electrode" strategy, i.e., sandwich-type immunoassay format, the sensitivity of which is restricted by two key bottlenecks: (1) the number of signal labels is limited and (2) only a part of signal labels could participate in the electrode reaction. In this Perspective, we discuss the development of an "in-electrode" Faraday-cage-type concept-based immunocomplex immobilization strategy. The biggest difference from the traditional sandwich-type one is that the designed "in-electrode" Faraday-cage-type immunoassay uses a conductive two-dimensional (2-D) nanomaterial simultaneously coated with signal labels and a recognition component as the detection unit, which could directly overlap on the electrode surface. In such a case, electrons could flow freely from the electrode to the detection unit, the outer Helmholtz plane (OHP) of the electrode is extended, and thousands of signal labels coated on the 2-D nanomaterial are all electrochemically "effective." Thus, then, the above-mentioned bottlenecks obstructing the improvement of the sensitivity in sandwich-type immunoassay are eliminated, and as a result a much higher sensitivity of the Faraday-cage-type immunoassay can be obtained. And, the applications of the proposed versatile "in-electrode" Faraday-cage-type immunoassay have been explored in the detection of target polypeptide, protein, pathogen, and microRNA, with the detection sensitivity improved tens to hundreds of times. Finally, the outlook and challenges in the field are summarized. The rise of Faraday-cage-type electrochemiluminescence immunoassay (FCT-ECLIA)-based biosensing strategies opens new horizons for a wide range of early clinical identification and diagnostic applications.

Vibrio vulnificus meningoencephalitis in a patient with thalassemia and a splenectomy.

Vibrio vulnificus usually causes wound infection, gastroenteritis, and septicemia. However, it is a rare conditional pathogen causing meningoencephalitis. We report a case of a young, immunocompromised man presenting with severe sepsis after exposure to sea water and consumption of seafood. The patient subsequently developed meningoencephalitis, and Vibrio vulnificus was isolated from his blood culture. The sequence was confirmed by Next-generation sequencing of a sample of cerebrospinal fluid, as well as from a bacteria culture. After the pathogen was detected, the patient was treated with ceftriaxone, doxycycline, and moxifloxacin for 6 weeks, which controlled his infection. In this case, we acquired his clinical and dynamic MRI presentations, which were never reported. Physicians should consider Vibrio vulnificus infections when they see a similar clinical course, brain CT and MRI findings, susceptibility factors and recent seafood ingestion or exposure to seawater. Due to high mortality, the early diagnosis and treatment of Vibrio vulnificus infections are crucial. Next-generation sequencing was found to be useful for diagnosis.

Virulence properties of Vibrio vulnificus isolated from diseased zoea of freshness shrimp Macrobrachium rosenbergii.

Macrobrachium rosenbergii is one of the most economically important freshwater shimp, with fast growth and high nutrient content in the agricultural development of China. However, it had been suffering diseases infection, causing mass death and great economic losses. In the present study, a bacteria strain was isolated from the diseased zoea of M. rosenbergii and was identified as Vibrio vulnificus by biochemical characteristics and 16S rRNA homologous analysis. The infection test showed that the strain GXFL1-3 was pathogenic to zoea and postlarva of M. rosenbergii, and the half lethal dose (LD50) were 1.16 × 106 CFU/mL and 1.45 × 106 CFU/mL, respectively. Detection of virulence-associated genes by PCR indicated that GXFL1-3 was positive for fur, OmpU, acfA, flaA, vvhA, vvp and tcp, the detection of extracellular enzymes and hemolysin showed that GXFL1-3 was positive for protease, amylase, lecithin, urease and hemolysin activity, further supporting its pathogenicity. A duplex PCR for rapid detection of V. vulnificus was established. Only V. vulnificus could amplify two specific bands of flaA and fur, while the other six strains of Vibrio were negative. The minimum detectable amount of template was 2.4 × 103 CFU/mL through sensitivity test.

Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae, V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products.

Seafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872-1:2007 and TS 21872-2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood.

Quantitative PCR enumeration of vcgC and 16S rRNA type A and B genes as virulence indicators for environmental and clinical strains of Vibrio vulnificus in Galveston Bay oysters.

Oysters from a reef in Galveston Bay, Texas, USA, were screened for more virulent clinical strains versus less virulent environmental strains of Vibrio vulnificus using a combination of quantitative PCR assays for the virulence correlating gene (clinical variant, vcgC) and 16S rRNA types A and B (type A = environmental, type B = clinical). The combination of vcgC and 16S rRNA type B loci to determine clinical type strains was suitable, as indicated by the strong correlation (R2 = 0.98; p < 0.001) between these gene counts over time and their relative proportion (up to 93.8% and 94.3%, respectively) to vvhA genes used to quantify all strains of V. vulnificus. A strong seasonal shift of V. vulnificus strain types was observed. Environmental strains (16S rRNA type A) predominated from April to mid-June as salinities increased from 22 to 27 PSU (practical salinity unit) and temperatures rose 20 to 28 °C, with peak gene quantities of 16 812 ± 56 CFU/g. As temperatures increased to ≥30 °C from mid-June to September and salinities rose above 27 PSU, clinical strains (16S rRNA type B; vcgC) predominated with peak quantities 31 868 ± 287 and 32 360 ± 178 CFU/g, respectively.

Vibrio parahaemolyticus and Vibrio vulnificus Recovered from Oysters during an Oyster Relay Study.

Vibrio parahaemolyticus and Vibrio vulnificus are naturally occurring estuarine bacteria and are the leading causes of seafood-associated infections and mortality in the United States. Though multiple-antibiotic-resistant V. parahaemolyticus and V. vulnificus strains have been reported, resistance patterns in vibrios are not as well documented as those of other foodborne bacterial pathogens. Salinity relaying (SR) is a postharvest processing (PHP) treatment to reduce the abundances of these pathogens in shellfish harvested during the warmer months. The purpose of this study was to evaluate the antimicrobial susceptibility (AMS), pathogenicity, and genetic profiles of V. parahaemolyticus and V. vulnificus recovered from oysters during an oyster relay study. Isolates (V. parahaemolyticus [n = 296] and V. vulnificus [n = 94]) were recovered from oysters before and during the 21-day relaying study to detect virulence genes (tdh and trh) and genes correlated with virulence (vcgC) using multiplex quantitative PCR (qPCR). AMS to 20 different antibiotics was investigated using microbroth dilution, and pulsed-field gel electrophoresis (PFGE) was used to study the genetic profiles of the isolates. Twenty percent of V. vulnificus isolates were vcgC+, while 1 and 2% of V. parahaemolyticus were tdh+ and trh+, respectively. More than 77% of the V. vulnificus isolates and 30% of the V. parahaemolyticus isolates were resistant to at least one antimicrobial. Forty-eight percent of V. vulnificus and 8% of V. parahaemolyticus isolates were resistant to two or more antimicrobials. All isolates demonstrated a high genetic diversity, even among those isolated from the same site and having a similar AMS profile. No significant effects of the relaying process on AMS, virulence genes, or PFGE profiles of V. vulnificus and V. parahaemolyticus were observed.IMPORTANCE Analysis of the antibiotic resistance profiles of V. vulnificus and V. parahaemolyticus isolated from oysters during this study indicated that more than 48% of V. vulnificus isolates were resistant to two or more antimicrobials, including those recommended by the CDC for treating Vibrio infections. Also, the V. parahaemolyticus isolates showed high MICs for some of the Vibrio infection treatment antibiotics. Monitoring of AMS profiles of this bacterium is important to ensure optimal treatment of infections and improve food safety. Our study showed no significant differences in the AMS profiles of V. vulnificus (P = 0.26) and V. parahaemolyticus (P = 0.23) isolated from the oysters collected before versus after relaying. This suggests that the salinity of the relaying sites did not affect the AMS profiles of the Vibrio isolates, although it did reduce the numbers of these bacteria in oysters (S. Parveen et al., J Food Sci 82:484-491, 2017, https://doi.org/10.1111/1750-3841.13584).

Inactivation of Vibrio sp. in pure cultures and mussel homogenates using high hydrostatic pressure.

The objective of this study was to determine the effect of high hydrostatic pressure (HHP) on the inactivation of Vibrio sp. in pure cultures and mussel homogenates. Four Vibrio strains including V. alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnificus were used. HHP treatments were performed with both pure Vibrio sp. cultures in alkaline peptone water (2% NaCl) and artificially inoculated mussel homogenates at pressure levels of 250, 350 and 450 MPa for 1 and 3 min at 25°C. Counts of Vibrio were determined before and after treatment using drop plating method. The effect of high pressure on the reduction level significantly differed among the respective Vibrio species. Vibrio vulnificus was the most susceptible species to HHP. To achieve a >5 log reduction in mussel homogenates, pressure treatment needs to be (i) 350-450 MPa for ≥1 min at 25°C for both V. alginolyticus and V. cholerae, (ii) 250 MPa for ≥3 min or 350-450 MPa for ≥1 min for V. vulnificus and (iii) 350 MPa for ≥3 min or 450 MPa for ≥1 min for V. parahaemolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: High hydrostatic pressure (HHP) has been applied to inactivate spoilage and pathogenic micro-organisms in a variety of food products, including seafood. Vibrio sp. are frequently reported as the main cause of foodborne illness associated with consumption of raw or undercooked seafood particularly shellfish worldwide. To date, data on the inactivation of Vibrio sp. via HHP are still limited and most of the trials only investigated HHP application in oysters and clams. This study demonstrates the efficacy of HHP inactivating Vibrio sp. in both pure culture and mussel homogenates.

Identification of a highly specific DNA aptamer for Vibrio vulnificus using systematic evolution of ligands by exponential enrichment coupled with asymmetric PCR.

Vibrio vulnificus is an important bacterial pathogen that causes serious infections in fish and is also highly pathogenic to humans. Many effective detection methods targeting this pathogen have previously been designed, but many of these methods are time-consuming, complicated and expensive. Thus, these approaches cannot be widely used by small aqacultural concerns. Although DNA aptamers have been used to detect pathogenic bacteria, these have not been applied to marine bacteria, including V. vulnificus. Therefore, we developed a highly specific DNA aptamer for V. vulnificus detection using systematic evolution of ligands by exponential enrichment (SELEX), coupled with asymmetric PCR. After 13 rounds of cross-selection, we identified a novel DNA aptamer (Vapt2). We evaluated the affinity, specificity and limit of detection (LOD) of this aptamer for V. vulnificus. We found that Vapt2 had a high affinity for V. vulnificus (Kd  = 26.8 ± 5.3 nM) and detected this pathogen at a wide range of concentrations (8-2.0 × 108  cfu/ml). Vapt2 bound to V. vulnificus with high selectivity in the presence of other pathogenic bacteria. Our study increases our knowledge of the possible applications of aptamers with respect to marine bacteria. Moreover, our work might provide a framework for the rapid detection of pathogenic bacteria and water pollution.

Differences in Abundances of Total Vibrio spp., V. vulnificus, and V. parahaemolyticus in Clams and Oysters in North Carolina.

Filter feeding shellfish can concentrate pathogenic bacteria, including Vibrio vulnificus and Vibrio parahaemolyticus, as much as 100-fold from the overlying water. These shellfish, especially clams and oysters, are often consumed raw, providing a route of entry for concentrated doses of pathogenic bacteria into the human body. The numbers of foodborne infections with these microbes are increasing, and a better understanding of the conditions that might trigger elevated concentrations of these bacteria in seafood is needed. In addition, if bacterial concentrations in water are correlated with those in shellfish, then sampling regimens could be simplified, as water samples can be more rapidly and easily obtained. After sampling of oysters and clams, either simultaneously or separately, for over 2 years, it was concluded that while Vibrio concentrations in oysters and water were related, this was not the case for levels in clams and water. When clams and oysters were collected simultaneously from the same site, the clams were found to have lower Vibrio levels than the oysters. Furthermore, the environmental parameters that were correlated with levels of Vibrio spp. in oysters and water were found to be quite different from those that were correlated with levels of Vibrio spp. in clams. IMPORTANCE: This study shows that clams are a potential source of infection in North Carolina, especially for V. parahaemolyticus These findings also highlight the need for clam-specific environmental research to develop accurate Vibrio abundance models and to broaden the ecological understanding of clam-Vibrio interactions. This is especially relevant as foodborne Vibrio infections from clams are being reported.

A double-quadratic model for predicting Vibrio species in water environments of Japan.

Vibrio spp. are natural inhabitants of marine and estuarine environments. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the major infectious agents for humans. Their densities are affected by environmental factors such as water temperature and salinity. The detailed contribution of each factor still remains to be elucidated. Here we conducted multi-coastal study in a 21-month period to examine relationships between environmental factors and V. cholerae, V. parahaemolyticus and V. vulnificus densities in sea surface water in eight coastal sites of four prefectures in Japan. Vibrio densities were measured by a most-probable-number with PCR method which is highly sensitive and quantitative (3/100 ml of detection limit). Vibrio densities were analyzed with environmental factors including water temperature, salinity, total dissolved substance, and pH, and their quadratics. A linear regression model suited best for prediction of V. cholerae density. A novel double-quadratic model suited best for the prediction of V. parahaemolyticus and V. vulnificus densities.

Raw ready-to-eat seafood safety: microbiological quality of the various seafood species available in fishery, hyper and online markets.

Microbiological quality of 206 raw ready-to-eat seafood samples was investigated according to species (gizzard shad, halibut, rockfish, tuna, oyster and squid) and distribution channels (fishery, hyper and online market). Enumeration of aerobic plate count and total coliforms (TC) and pathogenic bacteria (Bacillus cereus, Staphylococcus aureus and Vibrio parahaemolyticus) was performed, and level of microbiological quality was classified into four groups: satisfactory, acceptable, unsatisfactory and unacceptable. Qualitative analysis was also performed for Escherichia coli and eight foodborne pathogens (B. cereus, E. coli O157:H7, Listeria monocytogenes, Salmonella spp., S. aureus, Vibrio cholerae, V. parahaemolyticus, and Vibrio vulnificus). Raw ready-to-eat seafood products revealed 0·5% at an unsatisfactory level and 4·9% at an unacceptable level due to ≥4 log CFU g-1 of TC in squid and ≥3 log CFU g-1 of V. parahaemolyticus in gizzard shad respectively. Gizzard shad was shown to be potentially hazardous, as its sashimi is eaten with its skin attached. Bacillus cereus, E. coli, S. aureus, V. parahaemolyticus and V. vulnificus were qualitatively detected. Samples from the fishery market showed higher detection rate especially in V. parahaemolyticus (21·6%) and V. vulnificus (1·7%) which indicates the need to improve microbiological safety of raw ready-to-eat seafood products in fishery market. SIGNIFICANCE AND IMPACT OF THE STUDY: Raw ready-to-eat seafood products like sashimi can be easily contaminated with various bacteria from aquatic environments and human reservoirs, which subsequently bring about a risk in food poisoning due to no heating process before consumption. The results of this study provide comprehensive microbiological data on various species of raw ready-to-eat seafood from various distribution channels. It may contribute to establish reasonable standard and effective strategies to ensure a good microbiological quality of raw ready-to-eat seafood for the safety of meals, like sashimi and sushi.