An ELISA-on-a-chip biosensor system coupled with immunomagnetic separation for the detection of Vibrio parahaemolyticus within a single working day.

In this study, we constructed a rapid detection system for a foodborne pathogen, Vibrio parahaemolyticus, by using enzyme-linked immunosorbent assay (ELISA)-on-a-chip (EOC) biosensor technology to minimize the risk of infection by the microorganism. The EOC results showed a detection capability of approximately 6.2x10(5) cells per ml, which was significantly higher than that of the conventional rapid test kit. However, this high level of sensitivity required cultivation of the pathogen prior to analysis, which typically exceeded a day. To shorten the test period, we combined the EOC technology with immunomagnetic separation (IMS), which could enhance the sensitivity of the biosensor. IMS was carried out with magnetic particles coated with a monoclonal antibody specific to the microbe. To test the performance of the IMS-EOC method, fish intestine samples were prepared by artificially inoculating less than 1 or 5 CFU/10 g, allowing for enrichment over predetermined times, and analyzing the sample by using the EOC sensor after concentrating the culture 86-fold via IMS. Using this approach, the bacterium was detected after (at most) 9 h, which approximately corresponds to standard working hours. Thus, the IMS-EOC method allowed for the rapid detection of V. parahaemolyticus, which is responsible for foodborne diseases, and this method could be used for early isolation of contaminated foods before distribution.

Comparison of bioaccumulation and elimination of Escherichia coli and male-specific bacteriophages by ascidians and bivalves.

Levels of Escherichia coli and male-specific bacteriophages (MSBs) were determined in the filter feeders obtained from retail markets, commercial farms, and wild beds in Korea. The accumulation and elimination of E. coli and MSBs were compared between ascidians and bivalves (oysters and mussels) during relaying and depuration. E. coli concentrations in ascidians from retail markets ranged between < 20 and 460 most probable number/100 g while MSBs were not detected. E. coli levels in bivalves from commercial farms and wild beds were not significantly different but bacterial levels in ascidians were consistently lower. Ascidians exhibited much lower ability than bivalves to accumulate E. coli and MSBs during relaying in a polluted coastal area. This study also shows that an equilibrium was developed between levels of microbes in water and ascidians and shellfish during relaying. E. coli and MSBs in ascidians decreased quickly during depuration in a clean seawater tank. However, after 1 day, E. coli in bivalves decreased by only 1.1-1.6 logs, and the elimination of MSBs was negligible. Therefore, depuration is an effective means to reduce the health risk of contaminated ascidians.

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from oysters in Korea.

Vibrio parahaemolyticus is the most prevalent gastroenteritis-causing pathogen in Korea and in some other Asian countries. It is frequently found in oysters and other seafood. This study monitored changes in the prevalence of V. parahaemolyticus and environmental parameters in oyster aquaculture environments in Korea. From June to October 2014, we tested oysters (Crassostrea gigas) from shellfish-harvesting areas off the west coast of Korea. These 71 isolates were the sum of 16 (22.5%), 19 (26.8%), 23 (32.4%), and 13 (18.3%) isolates collected in July, August, September, and October, respectively. These 71 isolates had the following profiles of resistance against 16 antibiotics: all isolates were resistant to ampicillin and vancomycin, and 52.2, 50.7, and 50.7% of isolates exhibited resistance to cephalothin, rifampin, and streptomycin, respectively. PCR analysis for the presence of the species-specific toxR gene confirmed that 38 (53.5%) of the total 71 isolated strains were positive for V. parahaemolyticus. In PCR analysis for virulence of V. parahaemolyticus, of the 71 isolates tested in the present study, only 38 (53.5%) were positive for the trh virulence gene and 71 (100%) was negative for the tdh virulence gene.

Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from farmed fish in Korea from 2005 through 2007.

The antimicrobial resistance patterns to 15 antimicrobial agents of Vibrio parahaemolyticus and Vibrio alginolyticus isolated from farmed fishes, including olive flounder (Paralichthys olivaceus), black rockfish (Sebastes schlegeli), red sea bream (Pagrus major), and sea bass (Lateolabrax japonicus), were investigated from 2005 through 2007. A total of 218 V. parahaemolyticus isolates and 153 V. alginolyticus isolates were obtained from the 180 fish samples collected from fish farms located along the southern coast of Korea. We found that 65.1% of V. parahaemolyticus and 85.6% of V. alginolyticus isolates showed antimicrobial resistance against more than one antimicrobial agent. The prevalence of resistance in V. parahaemolyticus isolates to ampicillin was highest (57.8%), followed by resistance to rifampin (11.9%), streptomycin (8.7%), and trimethoprim (6.4%). V. alginolyticus isolates were also most resistant to ampicillin (75.2%), followed by tetracycline (15.0%), trimethoprim (12.4%), and rifampin (9.8%). The prevalence of multiresistance to four or more antimicrobials was higher in V. alginolyticus (11.1%) than in V. parahaemolyticus (5%). Antimicrobial resistance rates per isolate of V. parahaemolyticus and V. alginolyticus possessing virulence genes were not different from those of the rest of the isolates.

Evaluation of the sensitivity and specificity of primer pairs and the efficiency of RNA extraction procedures to improve noroviral detection from oysters by nested reverse transcription-polymerase chain reaction.

Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoV-polluted sea areas are considered as the significant vectors of viral transmission. To improve NoV detection from oyster using nested reverse transcription-polymerase chain reaction (RT-PCR), we evaluated the sensitivity and specificity of previously published primer pairs and the efficiency of different RNA extraction procedures. Among the primer pairs used for RT-PCR, the sensitivity of GIF1/GIR1-GIF2/GIR1 and GIIF1/GIIR1-GIIF2/GIIR1 was higher than that of other primer pairs used in nested RT-PCR for the detection of NoV genogroup I (NoV GI) and NoV GII from both NoV-positive stool suspension and NoV-seeded oyster concentrates, respectively; the resulting products showed neither unspecific bands in the positive samples nor false-positive bands in the negative controls. The extraction of NoV RNA from oyster samples using a QIAamp® Viral RNA Mini kit with a QIAshredder™ Homogenizer pretreatment afforded more efficient recovery (mean recovery for NoV GI and GII, 6.4%) and the procedure was less time consuming (<30 min) than most other RNA extraction procedures. The results of RNA extraction procedure and primer pairs evaluated by nested RT-PCR assay in this study can be useful for monitoring NoV contamination in oysters, which is an indicator of possible public health risks.