Inactivation of Human Norovirus GII.4 and Vibrio parahaemolyticus in the Sea Squirt (Halocynthia roretzi) by Floating Electrode-Dielectric Barrier Discharge Plasma.

Human norovirus (HNoV) GII.4 and Vibrio parahaemolyticus may be found in sea squirts. Antimicrobial effects of floating electrode-dielectric barrier discharge (FE-DBD) plasma (5-75 min, N2 1.5 m/s, 1.1 kV, 43 kHz) treatment were examined. HNoV GII.4 decreased by 0.11-1.29 log copy/µL with increasing duration of treatment time, and further by 0.34 log copy/µL when propidium monoazide (PMA) treatment was added to distinguish infectious viruses. The decimal reduction time (D1) of non-PMA and PMA-treated HNoV GII.4 by first-order kinetics were 61.7 (R2 = 0.97) and 58.8 (R2 = 0.92) min, respectively. V. parahaemolyticus decreased by 0.16-1.5 log CFU/g as treatment duration increased. The D1 for V. parahaemolyticus by first-order kinetics was 65.36 (R2 = 0.90) min. Volatile basic nitrogen showed no significant difference from the control until 15 min of FE-DBD plasma treatment, increasing after 30 min. The pH did not differ significantly from the control by 45-60 min, and Hunter color in "L" (lightness), "a" (redness), and "b" (yellowness) values reduced significantly as treatment duration increased. Textures appeared to be individual differences but were not changed by treatment. Therefore, this study suggests that FE-DBD plasma has the potential to serve as a new antimicrobial to foster safer consumption of raw sea squirts.

Colorimetric detection of norovirus by helicase-dependent amplification method based on specific primers integrated with HRPzyme.

Noroviruses (NoVs) are the most common causes of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and significant causes of foodborne illness. In the USA, approximately 21 million illnesses attributable to NoVs have annually occurred. Therefore, there is a great demand to develop a rapid, low-cost, and accurate detection method for NoVs. This study first reported colorimetric helicase-dependent amplification (HDA) methods based on specific primers integrated with HRPzyme for the rapid and sensitive detection of NoV GI and GII. The colorimetric HDA methods exhibited a detection limit of 10 copies mL-1 of each NoV GI and GII and were confirmed to be specific to each NoV GI and GII. The period required to complete the HDA method was 2 h, including a step of RNA extraction and cDNA synthesis without expensive instruments such as a thermal cycler and detector. The cutoff value of the method for the oyster artificially inoculated with a known amount of NoV was all 102 copies g-1 for NoV GI and GII. Therefore, the HDA method developed in this study can be useful tool for the on-site detection of NoVs in food samples.

Distribution and antimicrobial resistance of Vibrio parahaemolyticus isolated from fish and shrimp aquaculture farms along the Korean coast.

The present study investigated the distribution and antimicrobial susceptibility patterns of Vibrio parahaemolyticus in water samples and aquatic animals (fish and shrimp) from major aquaculture farms along the Korean coast in 2018. V. parahaemolyticus is the most common pathogen causing seafood-borne illness. The strain was detected in 34.7% of all samples tested, and was detected at higher levels during summer to autumn when the water temperature is higher. Although more than 90.0% of V. parahaemolyticus isolates were sensitive to 13 of the 15 antimicrobials tested, which is useful for treating V. parahaemolyticus infectious disease, the isolates exhibited higher resistance to two antibiotics (colistin and ampicillin), which should be excluded as treatment options for these infections. Koreans typically enjoy consuming raw seafood. To reduce the potential human health risk of raw seafood consumption, the prevalence and antimicrobial resistance of V. parahaemolyticus in aquaculture environments should be continuously valuated.

Antibiotic resistance in shellfish and major inland pollution sources in the drainage basin of Kamak Bay, Republic of Korea.

Shellfish-growing areas in marine environments are affected by pollutants that mainly originate from land, including streams, domestic wastewater, and the effluents of wastewater treatment plants (WWTPs), which may function as reservoirs of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs). The objective of this study was to identify the occurrence and distribution of antibiotic resistance at five oyster sampling sites and 11 major inland pollution sources in the drainage basin of Kamak Bay, Republic of Korea. Culture-based methods were used to estimate the diversity and abundance of antibiotic-resistant Escherichia coli strains isolated from oysters and major inland pollution sources. The percentages of ARB and multiple antibiotic resistance index values were significantly high in discharge water from small fishing villages without WWTPs. However, the percentages of antibiotic-resistant E. coli isolates from oysters were low, as there was no impact from major inland pollutants. Fourteen ARGs were also quantified from oysters and major inland pollution sources. Although most ARGs except for quinolones were widely distributed in domestic wastewater discharge and effluent from WWTPs, macrolide resistance genes (ermB and msrA) were detected mainly from oysters in Kamak Bay. This study will aid in tracking the sources of antibiotic contamination in shellfish to determine the correlation between shellfish and inland pollution sources.

Assessment of potential infectivity of human norovirus in the traditional Korean salted clam product "Jogaejeotgal" by floating electrode-dielectric barrier discharge plasma.

This study investigated the antiviral effects of floating electrode-dielectric barrier discharge (FE-DBD) plasma treatment (1.1 kV, 43 kHz, N2 1.5 m/s, 5-30 min) against human norovirus (HuNoV) GII.4 in Jogaejeotgal Infectivity was assessed using real-time quantitative-PCR (RT-qPCR) following treatment of samples with propidium monoazide (PMA) and sodium lauroyl sarcosinate (Sarkosyl). This study also investigated the effects of FE-DBD plasma treatment on Jogaejeotgal quality (assessed using pH value and Hunter colors). Following inoculation, the average titers of HuNoV GII.4 in Jogaejeotgal significantly (P < 0.05) decreased with increases in the FE-DBD plasma treatment time in both the non-PMA-treated and PMA + Sarkosyl-treated samples; in the non-PMA and PMA + Sarkosyl treated Jogaejeotgal, HuNoV GII.4 titers (log10 copy number/µL) were to: 3.16 and 2.95 (5 min), 2.90 and 2.48 (10 min), 2.82 and 2.40 (15 min), 2.58 and 2.26 (20 min), 2.48 and 2.06 (25 min), and 2.23 and 1.91 (30 min), respectively. The average titers of HuNoV demonstrated significant (P < 0.05) reductions of 0.35 log10 (55.3%) in PMA + Sarkosyl-treated samples compared with the non-PMA treated samples following exposure to 5-30 min of FE-DBD plasma. Reductions of >1-log for HuNoV in PMA + Sarkosyl- treated Jogaejeotgal required treatments of FE-DBD of 5-30 min. Using the first order kinetic model (R2 = 0.95), GII.4 decimal reduction time (D-value) resulting from FE-DBD plasma was 23.75 min. The pH and Hunter colors ("L", "a", and "b") were not significantly different (P > 0.05) between the untreated and FE-DBD plasma-treated Jogaejeotgal. Based on these results, the PMA + Sarkosyl/RT-qPCR method could be assessing HuNoV viability following 5-30 min treatment of FE-DBD plasma. Furthermore, may be an optimal treatment for Jogaejeotgal without altering the food quality (color and pH).

Virucidal Effects of Dielectric Barrier Discharge Plasma on Human Norovirus Infectivity in Fresh Oysters (Crassostrea gigas).

This study investigates the effects of dielectric barrier discharge (DBD) plasma treatment (1.1 kV, 43 kHz, N2 1.5 L/min, 10~60 min) on human norovirus (HuNoV) GII.4 infectivity in fresh oysters. HuNoV viability in oysters was assessed by using propidium monoazide (PMA) as a nucleic acid intercalating dye before performing a real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, the impact of the DBD plasma treatment on pH and Hunter colors was assessed. When DBD plasma was treated for 60 min, the HuNoV genomic titer reduction without PMA pretreatment was negligible (<1 log copy number/µL), whereas when PMA treatment was used, HuNoV titer was reduced to >1 log copy number/µL in just 30 min. D1 and D2-value of HuNoV infectivity were calculated as 36.5 and 73.0 min of the DBD plasma treatment, respectively, using the first-order kinetics model (R2 = 0.98). The pH and Hunter colors were not significantly different (p > 0.05) between the untreated and DBD-plasma-treated oysters. The results suggest that PMA/RT-qPCR could help distinguish HuNoV infectivity without negatively affecting oyster quality following >30 min treatment with DBD plasma. Moreover, the inactivation kinetics of nonthermal DBD plasma against HuNoV in fresh oysters might provide basic information for oyster processing and distribution.

Level of contamination in the feces of several species at major inland pollution sources in the drainage basin of Yeoja Bay, Republic of Korea.

We assessed the levels of fecal contamination and the originating species of 12 major inland pollutants in the drainage basin of Yeoja Bay. The presence of the human-specific (HF183), ruminant-specific (BacR and Rum-2-Bac), pig-specific (Pig-Bac-2 and Pig-2-Bac), avian-specific (GFD), and gull-specific (Gull2) markers in water samples (n = 34) from 12 inland pollution sources around Yeoja Bay was analyzed. HF183 was detected in 97% of the water samples, and all major inland pollution sources were contaminated with human feces. BacR and Rum-2-Bac were detected in 94% and 11%, respectively, of the water samples. Pig-2-Bac was not detected in the inland pollution sources, but site L5 might be contaminated with swine feces. Gull2 was not detected, whereas GFD was detected in 26% of the water samples. This study highlights the utility of a MST toolbox approach for characterizing the water quality of inland pollution sources and identifying the feces producing species.

Bacteriological quality evaluation of seawater and oysters from the Hansan-Geojeman area in Korea, 2011-2013: impact of inland pollution sources.

From 2011 to 2013, we conducted a full sanitary survey of pollution sources in proximity to a shellfish growing area in the Hansan-Geojeman region in Korea, which includes a designated shellfish growing area. In the sea area, 1152 seawater and 209 oyster samples were collected and examined to evaluate their bacteriological quality. There were 758 potential pollution sources in the drainage area, including 40 sources discharging water in 2013. Fecal coliform (FC) concentrations and impact radii of discharges ranged from 1.8 to 700,000 MPN/100 mL and from 3 to 600 m, respectively; however, the pollutants did not reach the designated area. This demonstrates that the dilution of waste was sufficient such that no significant impact occurred within the designated shellfish growing area. The variation in the FC levels of seawater was closely related to season and rainfall. The FC levels of seawater and oysters from the designated area met the regulation limits set by various countries. No pathogens were found in any oysters. The results of the survey indicate that the oysters produced in this area are apparently safe for raw consumption based on their bacterial quality.

Bioaccumulation of heavy metals in oysters from the southern coast of Korea: assessment of potential risk to human health.

From 2009 to 2013, 80 oyster and 16 seawater samples were collected from the southern coast of Korea, including designated shellfish growing areas for export. The concentrations and bioaccumulation of heavy metals were determined, and a potential risk assessment was conducted to evaluate their hazards towards human consumption. The cadmium (Cd) concentration in oysters was the highest of three hazardous metals, including Cd, lead (Pb), and mercury (Hg), however, below the standards set by various countries. The metal bioaccumulation ratio in oysters was relatively high for zinc and Cd but low for Hg, Pb, arsenic, and chromium. The estimated dietary intakes of all heavy metals for oysters accounted for 0.02%-17.75% of provisional tolerable daily intake. The hazard index for all samples was far <1.0, which indicates that the oysters do not pose an appreciable hazard to humans for the metal pollutants of study.

Production of monoclonal antibody against Listeria monocytogenes and its application to immunochromatography strip test.

An immunochromatography (ICG) strip test based on a monoclonal antibody for the rapid detection of L. monocytogenes in meat and processed-meat samples was developed in this study. A monoclonal antibody (MAb) specific to L. monocytogenes was produced from cloned hybridoma cells (FKLM-3B12-37) and used to develop an ICG strip test. The antibody showed a stronger binding to L. monocytogenes than other Listeria species, and a weak cross-reaction to S. aureus based on an ELISA. The detection limit of the ICG strip test was 10(5) cell/ml. In total, 116 meat and processed-meat samples were collected and analyzed using both the ICG strip test and a PCR. The ICG strip test and PCR indicated L. monocytogenes contamination in 34 and 27 meat samples, respectively. The 7 meat samples not identified as L. monocytogenes positive by the PCR were also tested using an API kit and found to be contaminated by Listeria species. In conclusion, the ICG strip test results agreed well with those obtained using the PCR and API kit. Thus, the developed ICG has potential use as a primary screening tool for L. monocytogenes in various foods and agricultural products, generating results within 20 min without complicated steps.

Incidence and polymerase chain reaction assay of Listeria monocytogenes from raw milk in Gyeongnam Province of Korea.

A total of 50 raw milk samples from Gyeongnam Province of Korea were examined for the incidence of Listeria monocytogenes between July 1998 and August 1998. L. monocytogenes isolated by biochemical test was confirmed by polymerase chain reaction (PCR) with two sets of primers designed from the invasion-associated protein (iap) gene. After standard PCR with external primers, the amplified DNA was confirmed by a second round of PCR with internal primers (nested PCR). Both the external and internal primers generated 468-bp and 287-bp products. respectively. Only one (G9 strain) of the three suspect samples that tested positive in biochemical tests for L. monocytogenes from 50 raw milk samples was also PCR positive. Following this procedure. PCR-positive G9 strain was confirmed by Southern blot using the 287-bp internal iap probe again. The detection limit of G9 strain by standard PCR assay was as few as 102 cells, equivalent to approximately I pg of L. monocytogenes DNA. These PCR assays may be useful for novel detection as well as rapid confirmation for L. monocytogenes from food samples and the field.