Moo-Sang Kim

Antimicrobial resistance and resistance genes in Escherichia coli strains isolated from commercial fish and seafood.

The purpose of this study was to investigate the antimicrobial resistance and to characterize the implicated genes in Escherichia coli isolated from commercial fish and seafood. Fish and seafood samples (n=2663) were collected from wholesale and retail markets in Seoul, Korea between 2005 and 2008. A total of 179 E. coli isolates (6.7%) from those samples were tested for resistance to a range of antimicrobial agents. High rates of resistance to the following drugs were observed: tetracycline (30.7%), streptomycin (12.8%), cephalothin (11.7%), ampicillin (6.7%) and ticarcillin (6.1%). No resistances to amikacin, amoxicillin/clavulanic acid and cefoxitin were observed. Seventy out of 179 isolates which were resistant to one or more drugs were investigated by PCR for the presence of 3 classes of antimicrobial resistance genes (tetracycline, aminoglycosides and beta-lactams), class 1, 2 and 3 integrons. Gene cassettes of classes 1 and 2 integrons were further characterized by amplicon sequencing. The tetracycline resistance genes tetB and tetD were found in 29 (41.4%) isolates and 14 (20%) isolates, respectively. The beta-lactam resistance gene, bla(TEM) was found in 15 (21.4%) isolates. The aminoglycoside resistance gene, aadA was found in 18 (25.7%) isolates. Class 1 integron was detected in 41.4% (n=29) of the isolates, while only 2.9% (n=2) of the isolates were positive for the presence of class 2 integron. Two different gene cassettes arrangements were identified in class 1 integron-positive isolates: dfrA12-aadA2 (1.8 kb, five isolates) and aadB-aadA2 (1.6 kb, four isolates). One isolate containing class 2 integron presented the dfrA1-sat-aadA1 gene cassette array. These data suggest that commercial fish and seafood may act as the reservoir for multi-resistant bacteria and facilitate the dissemination of the resistance genes.

Prevalence, antibiotic resistance, and molecular characterization of Salmonella serovars in retail meat products.

The prevalence of Salmonella was determined in chicken meat (n = 26), beef (n = 49), and pork (n = 56) collected from wholesale markets, retail stores, and traditional markets in Seoul, South Korea, in 2009. Antibiotic resistance was assessed, and the molecular subtypes of Salmonella isolates were ascertained using an automated repetitive sequence-based PCR (rep-PCR) system (DiversiLab). A total of 18 Salmonella strains were isolated from 17 of 131 samples: 16 strains from each of 16 samples and 2 strains from the same pork sample. The prevalence of Salmonella from the retail meats was 2.0% in beef, 8.9% in pork, and 42.3% in chicken meat. Among 10 different serotypes, Salmonella enterica Panama was recovered from a beef sample, and Salmonella London and Salmonella Montevideo were the predominant serotypes from pork and chicken meat, respectively. The highest antibiotic resistance observed was to erythromycin (100%) followed by streptomycin (22.2%) and tetracycline and chloramphenicol (16.7%). Of the 18 isolates, 5 (27.8%) were resistant to two or more antibiotics, and 1 isolate from chicken meat was resistant to eight antibiotics, including cephalosporins. Differentiation between all of the Salmonella isolates except between Salmonella Montevideo and Salmonella London was successfully performed with the automated rep-PCR system, indicating that it can be added to the toolbox for source tracking of foodborne pathogens associated with outbreaks.

Antimicrobial resistance profiles among Escherichia coli strains isolated from commercial and cooked foods.

A total of 4330 food samples of which microbiological standard for Escherichia coli is negative in Korea were determined for the frequency of E. coli. Ninety six samples (2.2%) were positive for E. coli. Detection rate of E. coli varied significantly by food type and ranged from 0.3% to 10.9%. Seasoned raw meat (yukhoe) and cold bean-soup had the highest prevalence for E. coli (10.9%) followed by gimbap (5.2%), meat broth for cold noodle (2.9%) and sprout (2.1%). E. coli isolates (n=96) were investigated for their phenotypic and genotypic antimicrobial resistance patterns. Seventeen E. coli isolates (17.7%) were resistant to one or more antimicrobial agents tested. High rates of resistance to the following drugs were observed: tetracycline (15.6%), streptomycin (12.5%), ampicillin (10.4%), nalidixic acid (9.4%) and ticarcillin (9.4%). All ampicillin resistant isolates were screened for extended-spectrum β-lactamase (ESBL) production by the combination disk test. None of the E. coli isolates produced ESBLs. Seventeen out of 96 E. coli isolates which were resistant to at least one antibiotic were investigated by PCR for the presence of 3 classes of antimicrobial resistance genes (tetracycline, aminoglycosides and beta-lactams). The tetracycline resistance genes tetA and tetB were found in 7 and 5 isolates, respectively. The aminoglycoside resistance genes, strA/B, aphA1, aadA and aac(3)-IV were found in 9, 5, 2 and 2 isolates, respectively. The beta-lactam resistance gene, bla(TEM) was found in 7 isolates. Results of this study show that 13 E. coli isolates were multidrug resistant (to three or more antibiotics) and 12 isolates carried at least one antimicrobial resistance gene. These isolates can act as the reservoir for antimicrobial resistance genes and facilitate the dissemination of these genes to other pathogenic and commensal bacteria. Adequate intervention to reduce microbial contamination of these foods is strongly recommended.

Microbial Quality of Fresh Vegetables and Fruits in Seoul, Korea

ABSTRACT - A total of 187 samples of leafy vegetables and fruits were acquired at traditional markets anddepartment stores in Seoul, Korea. Samples were tested for microorganism distributions and for the presence of patho-genic bacteria. The aerobic mesophilic counts ranged between 2.5 and 9.4 log CFU/g, with the highest count recordedfrom the dropwort. Counts of psychrotrophic microorganisms were as high as those of the mesophilic microorgan-isms. Total coliform populations between 1.0 and 7.8 log CFU/g were found in 90.9% of the samples. Microbiologicalcounts for fruits were very low. Escherichia coli was isolated in 24 (12.8%) samples. Staphylococcus aureus andClostridium perfringens contamination were found in 15 (8.0%) and 20 (10.7%) samples. Salmonella species andListeria monocytogenes were detected in 2.7 and 0.5% of samples, respectively. Among the total 187 samples, 8 sam-ples were contaminated by more than two pathogens. E. coli O157:H7 was not detected in any of the samples. Themicrobial contamination levels determined in the present study may be used as the primary data to execute microbialrisk assessment of fresh vegetables and fruits. Key words : Vegetables, microorganism, foodborne pathogen, safety

Human Enteric Viruses in Groundwater

Waterborne outbreaks of enteric viruses are a major public health concern. The present study has been carried out to assess the presence of enteric viruses responsible for human acute gastroenteritis (AGE) in groundwater intended for drinking and produce washing. In total, 62 samples from groundwater for drinking and produce washing collected from Dec 2007 to Dec 2008 in Seoul were tested for enteric viruses using conventional RT–PCR, ELISA, and real-time RT–PCR. Our results showed that enteric viruses were detected in 7 (8.8%) groundwater samples. Rotaviruses were detected in 3 (4.8%) of the samples by ELISA; human adenoviruses were detected in 2 (3.2%) of the samples by ELISA; and nested RT–PCR detected noroviruses in 2 (3.2%) of the samples. In one of the groundwater sample, the norovirus RNA was detected by conventional RT–PCR which was confirmed positive by real-time RT–PCR. Additionally, real-time RT–PCR successfully detected norovirus RNA in five out of 62 water samples (8.1%). The data demonstrate that real-time RT–PCR will be useful as a rapid and sensitive method for detecting norovirus in water samples. Phylogenetic analysis revealed that the noroviruses detected in two of the groundwater samples belonged to GII-4. These studies can provide important information for the prevalence of enteric viruses in Korean groundwater.

Outbreak of rotavirus variant P[8] in Seoul, South Korea

An epidemiologic study was performed to determine the genetic variability of rotaviruses in Seoul, South Korea. In 3,174 stool specimens from children with acute diarrhea at five referral hospitals, 571 (18%) possessed the antigen of group A rotavirus detectable by ELISA—10.8% in 2004 and 28.1% in 2005. VP7 genotyping revealed that the G3 type was found in 25.6% of all typed isolates, G4 in 23.8%, G2 in 21.6%, and G1 in 17.6%. VP4 genotyping showed that the P[8] type was detected in 66.7%, P[6] in 15.6%, P[4] in 13.0%, and P[9] in 0.2%. Because the variant P[8] type could not be amplified initially by conventional P typing primers (1T‐1), PCR were performed using newly designed 1T‐1S primer, which revealed that 307 specimens were the variant P[8] type. Uncommon combinations such as G4P[6] and G2P[8] were also found with relatively high prevalence, 14.6% and 12.8%, respectively. Variant P[8] types were associated with an outbreak of rotavirus in 2005. J. Med. Virol. 80:1661–1665, 2008.

Development of a selective enrichment broth supplemented with bacteriological charcoal and a high concentration of polymyxin B for the detection of Campylobacter jejuni and Campylobacter coli in chicken carcass rinses.

A new Campylobacter-selective enrichment broth supplemented with bacteriological charcoal and a high concentration of polymyxin B was developed (charcoal-cefoperazone-polymyxin B-deoxycholate broth; CCPD broth). We compared the ability of CCPD broth to detect Campylobacter jejuni and Campylobacter coli in chicken carcass rinses to that of modified Bolton (mBolton) broth. Eighty whole chickens were purchased from retailers and rinsed with 400 mL buffered peptone water. The rinsed samples were enriched with 2× blood-free mBolton enrichment broth or 2× CCPD broth at 42 °C for 48 h and then streaked onto modified charcoal-cefoperazone-deoxycholate agar (mCCDA). The Campylobacter isolation rate was significantly higher in CCPD broth than in mBolton broth (CCPD broth, 61 out of 80; mBolton broth, 34 out of 80; p<0.05). Moreover, the selectivity of CCPD broth agar was also superior to that of mBolton broth when comparing the number of contaminated mCCDA plates (CCPD broth, 16 out of 80; mBolton broth, 58 out of 80; p<0.05) and the growth index of competing flora (CCPD broth, 1.4; mBolton broth, 2.9; p<0.05).

Rapid detection method for hepatitis A virus from lettuce by a combination of filtration and integrated cell culture-real-time reverse transcription PCR.

We have developed a rapid and simple method for filtration using a positively charged membrane to concentrate hepatitis A virus (HAV) from lettuce and an integrated cell culture-real-time reverse transcription PCR (ICC-real-time RT-PCR) to detect infectious HAV. The most suitable buffer for HAV concentration by filtration was 100 mM Tris-HCl, 50 mM glycine (pH 9.5). Filtration using the NanoCeram matrix was compared with polyethylene glycol precipitation for viral concentration from lettuce inoculated with 6 log RNA copies of HAV. The recovery rate of filtration was statistically higher than that of polyethylene glycol precipitation (47.3 versus 24.9%, respectively). The sensitivity of ICC-real-time RT-PCR for detection of infectious HAV was determined by inoculation of FRhK-4 cells with HAV (4 log to 0 log RNA copies). ICC-real-time RT-PCR detected infectious HAV on average 5 days earlier than cytopathic effects at all inoculation levels. HAV recovered from lettuce (approximately 3 log RNA copies) was also analyzed with ICC-real-time RT-PCR. Infectious HAV was detected within 2 days postinfection by ICC-real-time RT-PCR, whereas cytopathic effects were not observed until 7 days postinfection. Coupled with a virus concentration and purification system using a positively charged membrane, ICC-real-time RT-PCR has the potential to become a novel and rapid method for the detection of infectious HAV in vegetables.

Determination of Post-harvest Fungicide in Citrus Fruits Using LC-MS

Post-harvest fungicide residue was measured in citrus fruits. Samples were collected from local markets in Seoul and analyzed using liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS results were validated for the assay of pesticides by using linearity, accuracy, precision, and limits of detection and quantification. The linearity in the concentration ranged from 0.005 to 2.0 mg/kg (R 2 >0.999). Sample recoveries ranged from 80.2 to 98.3% with relative standard deviations below 4.0% for spiking levels from 0.01 to 1.0 mg/kg. The limits of detection ranged between 0.002 and 0.008 mg/kg, and the limits of quantification ranged between 0.006 and 0.027 mg/kg. The highest residue levels for carbendazim, thiabendazole, imazalil, and azoxystrobin in citrus fruits were 0.541, 0.958, 0.721, and 0.052 mg/kg, respectively. The pesticide residues found in citrus fruits were blow maximum residue limits (MRLs) and are not a serious public health problem.

Comparison of Real-Time PCR and Culture Methods for Detection of Campylobacter jejuni in Various Foods

In this study, performances of culture methods using two selective media and real-time PCR were evaluated for detection of Campylobacter jejuni (C. jejuni) in various food samples. Sausage, ground beef, and radish sprouts inoculated with C. jejuni were enriched in Hunt broth and then streaked onto modified cefoperazone charcoal deoxycholate agar and Preston agar, followed by incubation under microaerobic conditions. The enriched Hunt broth (1 mL) was used in real-time PCR assay. No statistical differences were observed in sensitivity among the two selective media and real-time PCR for sausage and ground beef. However, the number of positives by real-time PCR in radish sprouts was much higher than the two selective media (p