Hong-Sik Yu

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.

Geographical and annual variation in lipophilic shellfish toxins from oysters and mussels along the south coast of Korea.

To better understand critical aspects of diarrhetic shellfish poisoning (DSP) occurrence in a chief producing region of bivalves in Korea, the geographical and annual variation of DSP toxins and other lipophilic toxins in mussels (Mytilus galloprovincialis) and oysters (Crassostrea gigas) were investigated by liquid chromatography-tandem mass spectrometry in an area on the south coast of Korea from 2007 to 2009. The total lipophilic shellfish toxin (LST) levels in bivalves showed geographical and annual variations. LSTs were detected mostly in the hepatopancreas of mussels from Jinhae Bay throughout the entire year, except in November and December of 2007, but were almost undetectable in all samples during the entire year in 2009. The peak DSP toxin (okadaic acid plus dinophysistoxin 1) levels in the hepatopancreas of mussels from Jinhae Bay and the Tongyeong region were 945.3 and 37.6 ng/g, respectively. The DSP toxin content was about 10 times higher in mussels than in oysters collected from the same region. The major toxins in bivalves were okadaic acid and dinophysistoxin 1; however, pectenotoxin 2 or yessotoxin was occasionally detected as a major component. The results of a quantitative analysis of phytoplankton showed that Dinophysis acuminata was the most probable source of the LSTs, with the exception of yessotoxin. When the highest DSP toxin level was measured (945.3 ng/g in the hepatopancreas of mussels from Jinhae Bay), the toxin concentration in whole mussel tissue was calculated to be 114.0 ng/g. The calculated highest DSP toxin level in whole oyster tissue from both regions was 15.0 ng/g. The calculated maximum toxicities in whole mussel and oyster tissues were lower than the regulatory limit (160 to 200 ng/g) in Korea, the European Union, and the United States. Korean oysters (242 samples) and mussels (214 samples) were thus deemed safe for consumption. But because such variation was detected in a relatively small area of the coast, it is possible that at some locations or during a specific period LST levels could exceed the standard and a few consumers could be at risk of experiencing DSP.

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.

First detection and seasonal variation of lipophilic toxins okadaic acid, dinophysistoxin-1, and yessotoxin in Korean gastropods.

Okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2, and yessotoxin (YTX) are classes of lipophilic toxins found in marine animals. OA and DTX1 accumulation causes diarrhetic shellfish poisoning, a worldwide public health problem. Diarrhetic shellfish poisoning has not previously been reported in gastropods, which are widely consumed in Korea. Seasonal variation in marine lipophilic toxins in gastropods was investigated using liquid chromatography-tandem mass spectrometry. Eighty specimens of Neptunea cumingii, 65 specimens of Rapana venosa, and 95 specimens of Batillus cornutus were collected at the Tongyeong fish market on the southern coast of Korea between May 2009 and December 2010. OA, DTX1, and YTX were detected in meat and digestive glands in all gastropod species studied. Pectenotoxin-2 was not found in any sample tested. Lipophilic toxins were detected in the digestive glands of gastropods; no lipophilic toxin was detected in the salivary glands of the carnivorous gastropods, N. cumingii and R. venosa. The highest concentrations of OA (21.5 ng/g) and DTX1 (8.4 ng/g) were detected in the digestive glands of R. venosa, and the maximum concentration of YTX (13.7 ng/g) was found in the digestive glands of N. cumingii. The maximum toxicities in gastropod tissues were lower than the European standard for acceptable levels. The concentrations of lipophilic toxins in carnivorous gastropods showed a high degree of seasonal variation; lipophilic toxins in carnivorous gastropods were found predominantly in spring and summer. This is the first report of the occurrence of lipophilic toxins in Korean gastropods.

Simultaneous Determination of Amoxicillin and Ampicillin in Fish Meat Using High-Performance Liquid Chromatography

A simultaneous high-performance liquid chromatography assay method for amoxicillin and ampicillin in fish products was developed, evaluated, and validated by monitoring these antibiotics in fish samples obtained from aquaculture and distribution. The recovery rate of this method was higher than those of conventional methods and was 95.3-106.6% for amoxicillin and 81.4-92.4% for ampicillin. Our pretreatment procedure sufficiently removed or reduced materials affecting HPLC analysis, such as low-molecular-weight substances. The performance limit of this method was evaluated as 0.01 ppm of amoxicillin and ampicillin in fish muscle. Finally, 171 fish samples, including olive flounder (Paralichthys olivaceus), common sea bass (Lateolabrax japonicus), and black rock fish (Sebastes schlegeli) collected from fish farms in the coastal area between April and September 2005 were analyzed to evaluate the overall efficiency of the method and to monitor the actual of amoxicillin and ampicillin usage in fish farms. The results indicated that the developed method was suitable for analyzing amoxicillin and ampicillin in fish muscle, and determined that those antibiotics were being used for fish farming but were not detected in fish samples during the shipping and distribution stages.

Genetic diversity of noroviruses detected in oysters in Jinhae Bay, Korea

Norovirus (NoV) is considered the major causative agent of food poisoning outbreaks in Korea. Most such outbreaks originate from vegetables or drinking water, but bivalves are known to be an important NoV vector. Environmental contamination of NoV in ground and river water has been reported in Korea, but the NoV contamination status of shellfish-growing areas based on regular surveys is not available. In this study, we investigated the NoV contamination status of oyster-growing areas in the Jinhae Bay of Korea to evaluate the circulation of various NoV genotypes. A total of 39 NoV-positive samples detected from February, 2010 to February, 2011 were sequenced and evaluated. Based on the sequencing results, five genotypes (GI.1, GI.2, GI.5, GI.6, and GI.7) were identified in NoV GI, and seven genotypes (GII.2, GII.3, GII.4, GII.7, GII.12, GII.13, and GII.17) in NoV GII. GI.1 (52.6%; 10 of 19). Both GII.3 and GII.4 (30.0%; 6 of 20, respectively) were identified as the most prevalent GI and GII strains in oyster during the investigation period.

Characterization of Vibrio parahaemolyticus isolated from oysters in Korea: Resistance to various antibiotics and prevalence of virulence genes

Vibrio parahaemolyticus, found frequently in oysters, is the most prevalent gastroenteritis-causing pathogen in Korea and in several other Asian countries. This study monitored changes in the environmental parameters and occurrence of V. parahaemolyticus in oyster aquaculture sites. Of the 44 presumed V. parahaemolyticus isolates obtained, when tested against 16 antibiotics, 90.9, 86.4, and 75.0% of the 44 isolates exhibited resistance to vancomycin, ampicillin, and streptomycin, respectively. PCR analysis for the presence of the toxR gene confirmed 31 of the 44 isolates as being positive V. parahaemolyticus strains. The toxR positive isolates were tested for the presence of thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) virulence genes. Only 9.1% toxR positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The occurrence of multi drug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields.

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.

Distribution and Antimicrobial Resistance of Vibrio parahaemolyticus Isolated from Korean Shellfish

The contamination status of Vibrio parahaemolyticus in commercially valuable shellfish from the south and west coasts of Korea and the antimicrobial resistance patterns of isolated V. parahaemolyticus were investigated from July through October, 2011. The range of V. parahaemolyticus concentrations in oysters Crassostrea gigas and short neck clams Venerupis philippinarum was

Defecation of Norovirus from the Oyster Crassostrea gigas by Depuration Following Translocation of the Growing Area

이매패류(이하 패류)는 분변-경구 감염 환을 통한 세균성 및 바이러스성 장관계 질환의 주요 전파 매개체로 간주되고 있다 (Butt et al., 2004). 이는 지역사회에서 장관계 질환이 유행하게 되면 해당 병원체가 하수 및 오수를 통하여 담수 및 기수 환경 으로 배출되게 되고(Da Silva et al., 2007; Iwai et al., 2009), 패 류는 여과섭식 작용을 통해 서식해역에 존재하는 미생물을 체 내에 농축하여 사람이 섭취하는 경우 재감염을 유발하기 때문 이다(Lees, 2000). 또한 전통적으로 패류를 생식하거나 살짝 익 혀먹는 식습관 때문에 오염된 패류에 의한 재감염의 위험성이 사라지지 않고 있다(Lees, 2000). 더욱이 70년대 이후 패류 냉 동가공품이 국제적으로 유통되면서 오염된 제품에 의한 감염 의 시공간적 범위가 확대되고 있다(Pinto et al., 2009; Polo et al., 2010). 이러한 패류에 의한 감염증 발생을 예방하고자 유럽 및 북미 대부분 국가와 남미 및 아시아 일부 국가에서는 엄격한 패류 위생 규정을 마련하였으며 유럽과 북미의 경우 그 역사가 백 여년에 이르고 있다(Lees, 2000). 동 규정에는 패류 생산해역 의 분변계 대장균 (fecal coliform) 농도 또는 패육 중의 대장균 (Escherichia coli) 농도를 기준으로 패류 생산해역의 등급을 구 분하고 그 등급에 따라 패류의 수확 전후 관리조건을 명시하고 있다(European Commission, 2004; US FDA, 2014). 그러나 이러한 노력에도 불구하고 패류 생식과 관련된 감염증이 지속 적으로 발생하고 있으며(Butt et al., 2004), 특히 노로바이러스 에 오염된 굴 섭취에 따른 장관계 질환이 세계적으로 유행하고 있다(Kroneman et al., 2008; Scallan et al., 2011). 오염된 활 패류 중 미생물의 농도를 저감화할 수 있는 방법 중 시설 내 정화 및 자연정화가 현재 상업적 이용되고 있는 대표적 인 것들이다(Lees, 2000; Dore et al., 2010). 시설 내 정화는 패 자연정화에 의한 양식굴(Crassostrea gigas) 중 노로바이러스 저감화 유홍식*·박용수·안세라·박큰바위·심길보·송기철·이태식