Antimicrobial Resistance, Virulence Properties and Genetic Diversity of Salmonella Typhimurium Recovered from Domestic and Imported Seafood.

The quantity of seafood imported and produced by domestic aquaculture farming has increased. Recently, it has been reported that multidrug-resistant (MDR) Salmonella Typhimurium may be associated with seafood. However, information is limited to the antimicrobial resistance, virulence properties, and genetic diversity of S. Typhimurium recovered from imported and domestic seafood. This study investigated the antimicrobial resistance, virulence properties, and genetic diversity of S. Typhimurium isolated from domestic and imported catfish, shrimp, and tilapia. A total of 127 isolates were tested for the presence of multidrug-resistance (MDR), virulence genes (invA, pagC, spvC, spvR), and genetic diversity using the Sensititre micro-broth dilution method, PCR, and pulsed-field gel electrophoresis (PFGE), respectively. All isolates were uniformly susceptible to six (amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, imipenem, nitrofurantoin, and trimethoprim/sulfamethoxazole) of the 17 tested antimicrobials and genetically diverse. Fifty-three percent of the Salmonella isolates were resistant to at least one antimicrobial and 49% were multidrug resistant. Ninety-five percent of the isolates possessed the invA gene, 67% pagC, and 43% for both spvC, and spvR. The results suggest that S. Typhimurium recovered from seafood is frequently MDR, virulent, and have the ability to cause salmonellosis.

Prevalence and Abundance of Bacterial Pathogens of Concern in Shrimp, Catfish and Tilapia Obtained at Retail Stores in Maryland, USA.

Outbreaks of human gastroenteritis have been linked to the consumption of contaminated domestic and imported seafood. This study investigated the microbiological quality of seafood obtained from retail stores on the Eastern Shore of Maryland. A total of 440 samples of domestic and imported frozen shrimp, catfish and tilapia samples were analyzed for aerobic plate count (APC), total coliforms, Escherichia coli and seafood-borne-pathogens (Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Campylobacter jejuni). The prevalence of APC, coliforms and E. coli positive samples was 100%, 43% and 9.3%, respectively. Approximately 3.2%, 1.4%, 28.9% and 3.6% of the samples were positive for V. parahaemolyticus, V. vulnificus, Salmonella and Campylobacter jejuni, respectively. The MPN/g ranges were 150-1100 MPN/g for vibrios, 10-1100 MPN/g for Salmonella and 93-460 MPN/g for C. jejuni in seafood, respectively. Comparing bacterial prevalence by type or source of seafood, the only significant difference identified was Salmonella-positive imported tilapia (33.3%) versus domestic tilapia (19.4%). The quantitative data on pathogen levels in the present study provide additional information for quantitative risk assessment not available in previous surveys. The findings of this study suggest the association of potential food safety hazards with domestic and imported seafood and warrant further large-scale studies and risk assessment.

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).

High Salinity Relaying to Reduce Vibrio parahaemolyticus and Vibrio vulnificus in Chesapeake Bay Oysters (Crassostrea virginica).

Cases of Vibrio infections in the United States have tripled from 1996 to 2009 and these infections are most often associated with the consumption of seafood, particularly oysters (Crassostrea virginica). Information is needed on how to reduce numbers of Vibrio parahaemolyticus and Vibrio vulnificus in bi-valve molluscan shellfish (for example, oysters). The purpose of this study was to evaluate the effectiveness of high salinity relaying or treatment in recirculating aquaculture systems (RASs) as methods to reduce the abundance of V. parahaemolyticus and V. vulnificus in oysters. For relaying field trials, oysters were collected from approved harvest waters, temperature abused outside under a tarp for 4 h, and then transferred to high (29 to 33 ppt.) and moderate (12 to 19 ppt.) salinities. For RAS treatment trial, oysters were transferred to 32 to 34 ppt. salinity at 15 °C. After 7, 14, 21, and in some instances 28 d, oysters were collected and analyzed for V. parahaemolyticus and V. vulnificus levels using multiplex real-time PCR. Initial levels of V. parahaemolyticus and V. vulnificus ranged from 3.70 to 5.64 log10 MPN/g, and were reduced by 2 to 5 logs after 21 to 28 d in high salinity water (29 to 34 ppt.). Oyster mortalities averaged 4% or less, and did not exceed 7%. Relaying of oysters to high salinity field sites or transfer to high salinity RAS tanks was more effective in reducing V. vulnificus compared with V. parahaemolyticus. These results suggest that high salinity relaying of oysters is more effective in reducing V. vulnificus than V. parahaemolyticus in the oyster species used in this study.

Seafood Processing and Safety.

Food microorganisms are found on all surfaces (skin and gills) and in the intestines of fishery products.[...].

Effects of modified atmosphere packaging on toxin production by Clostridium botulinum in raw aquacultured summer flounder fillets (Paralichthys dentatus).

Packaging fishery products under vacuum atmosphere packaging (VAC) and modified atmosphere packaging (MAP) conditions can significantly extend the shelf life of raw, refrigerated fish products. There is considerable commercial interest in marketing VAC and MAP refrigerated (never frozen) raw fish fillets. The objective of this study was to determine if Clostridium botulinum toxin development precedes microbiological spoilage in raw, refrigerated flounder fillets. Aquacultured flounder (Paralichthys dentatus) individual fish fillets either were packed with a film having an oxygen transmission rate (OTR) of 3000 cm3 m(-2) 24 h(-1) at 22.8 degrees C or were vacuum packaged or packaged under 100% CO2 with a film having an OTR of 7.8 cm3 m(-2) 24 h(-1) at 21.1 degrees C and were stored at 4 and 10 degrees C. Samples were analyzed by aerobic plate count (APC) for spoilage and qualitatively for botulinum toxin with a mouse bioassay. The results demonstrate that flounder fillets (4 degrees C) packaged with a film having an OTR of 3,000 were microbiologically spoiled (APC, > 10(7) CFU/g) on day 15, but there was no toxin formation, even after 35 days of storage. However, at 10 degrees C, toxin production occurred (day 8), but it was after microbial spoilage and absolute sensory rejection (day 5). Vacuum-packaged fillets and 100% CO2 fillets (4 degrees C) packaged with a film having an OTR of 7.8 were toxic on days 20 and 25, respectively, with microbial spoilage (APC, >10(7) CFU/g) not occurring during the tested storage period (i.e., >35 days). At 10 degrees C, in vacuum-packaged flounder, toxin formation coincided with microbiological spoilage (days 8 to 9). In the 100% CO2-packaged fillets, toxin formation occurred on day 9, with microbial spoilage occurring on day 15. This study indicates that films with an OTR of 3,000 can be used for refrigerated fish fillets and still maintain the safety of the product.

Inactivation of vibrio parahaemolyticus and vibrio vulnificus in phosphate-buffered saline and in inoculated whole oysters by high-pressure processing.

Inactivation studies for Vibrio parahaemolyticus TX-2103 (serotype O3:K6) and Vibrio vulnificus MO-624 (clinical isolate) were conducted in phosphate-buffered saline (PBS) and in inoculated oysters under high-pressure processing conditions. V. parahaemolyticus was more resistant than V. vulnificus in PBS at all pressures and times. A 6-log reduction of V. parahaemolyticus and V. vulnificus in PBS at 241 MPa required 11 and 5 min, respectively, which included a 3-min pressure come-up time. A 4.5-log reduction of V. parahaemolyticus in oysters at 345 MPa required 7.7 min, which included a 6.7-min pressure come-up time. More than a 5.4-log reduction of V. vulnificus in oysters at 345 MPa occurred during the 6-min pressure come-up time. Both V. parahaemolyticus and V. vulnificus in PBS and in oysters were reduced to nondetectable numbers at 586 MPa during the 8- and 7-min pressure come-up times, respectively.

Comparison of kinetic models to describe high pressure and gamma irradiation used to inactivate Vibrio vulnificus and Vibrio parahaemolyticus prepared in buffer solution and in whole oysters.

Comparisons of different models in inactivation kinetics were conducted on data obtained from high-pressure and gamma-irradiation processing. Vibrio vulnificus (MO-624) and Vibrio parahaemolyticus (O3:K6 TX-2103) suspended in phosphate-buffered saline (pH 7.4, 10(7) CFU/ml) were exposed to pressures from 207 to 379 MPa for 1 to 20 min. Inoculated whole oysters (106 CFU/g) were exposed to pressure from 276 to 379 MPa for 1 to 15 min. Pure cultures and inoculated oysters (10(6) CFU/g) also were irradiated (gamma irradiation) at doses of less than 3 kGy. Four mathematical models, the Bigelow model, Arrhenius equation, Fermi equation, and Weibull frequency distributions, were applied to microbial survival data, and performances of the different kinetic models were compared. Weibull frequency distributions can predict the high-pressure inactivation of Vibrio spp. with more accuracy in both pure cultures and inoculated oyster samples. The Fermi model provided a better description of gamma-irradiation inactivation kinetics compared with the traditional Bigelow model.

Nutritional composition and microflora of the fresh and fermented skate (Raja Kenojei) skins.

The proximate compositions of fresh and fermented skate skin were each 75.95% and 74.5% moisture, 22.7% and 21.8% protein, 0.5% and 0.7% lipid and 0.6% and 0.9% ash, respectively. The predominant minerals were potassium and phosphorus (i.e. 53.5 and 33.0 mg/100 g in fresh skin, and 10.46 and 10.51 mg/100 g in fermented skin, respectively). Amino acid concentrations were lower in the fermented skin compared with the fresh skin. Histidine, glycine, alanine and glutamic acid were the major free amino acids in both skins. Palmitic acid (C16:0) was the major fatty acid in both fresh (16.68%) and fermented (20.38%) skate skin. Omega-3 polyunsaturated fatty acids were higher in fresh skin (22.17%) and fermented skin (24.54%) compared with omega-6 polyunsaturated fatty acids. The predominant microflora present in the both fresh and fermented skin were Photobacterium sp. and Vibrio sp. Total plate counts for the fresh and fermented skin were 2.4x10(4) CFU/g and 7.7x10(7) CFU/g, respectively.

Microbiological Hazards and Emerging Food-Safety Issues Associated with Seafoods †.

The United States is entering into a new era in which dwindling natural fisheries resources are forcing regulatory agencies to develop a more holistic approach to seafood safety and natural marine resource issues. Public health issues associated with seafoods can be grouped as (i) environmentally induced (i.e., natural or anthropogenic), (ii) process-induced, (iii) distribution-induced, or (iv) consumer-induced hazards. Similarly, loss of habitat and ecosystem degradation threaten the future viability of fisheries and have important ramifications for seafood safety. In the United States, large-scale legistlative efforts are underway to reexamine regulatory food control systems. The driving forces behind these efforts are the discovery of new emerging pathogens for which little information is available and dramatic improvements in analytic technology that allow for detection of low levels of microbial and chemical contaminants in foods. The global nature of seafood trading issues and the worldwide implementation of new preventative food safety programs such as hazard analysis for critical control points are driving some of the efforts to build new scientific bridges that will reevaluate current risk analysis strategies. New scientific bridges are needed to close the gaps between the scientific community and society concerning the effects of anthropogenic impacts on seafood safety and the heatlh of coastal habitats and associated fishery resources. The driving force behind this latter issue is the realization that the United States has lost over half of its original coastal wetlands areas. Protecting, conserving, and restoring the health and safety of our fisheries resources will require an integrated approach of food science and fishery science.