Seongbeom Cho

Prevalence and characterization of Escherichia coli O157 isolates from Minnesota dairy farms and county fairs.

Samples were collected from 26 organic and conventional farms and 12 county fairs in Minnesota during 2001 and 2002 to identify the presence of Escherichia coli O157. Immunomagnetic separation was used for isolation of E. coli O157. Isolates were further characterized by the presence of virulence marker genes (stx1, stx2, eaeA, E-hly, katP, etpD, and espP), antimicrobial susceptibility profiles, and genotypes. During 2001, E. coli O157 was isolated from 16 (5.2%) of 305 fecal samples and from 7 (36.8%) of 19 farms. During 2002, E. coli O157 was isolated from 6 (4.5%) of 132 fecal samples from weaned calves at 4 (23.5%) of 17 farms. During 2001 and 2002, cattle manure samples were collected from 12 county fairs, and E. coli O157 was isolated from 19 (11%) of 178 samples and 9 (75%) of 12 county fairs. Among 40 E. coli O157 isolates, 17 isolates (43%) had both the stx1 and stx2 genes, and 21 strains (53%) had the stx2 gene only. Thirteen percent of O157 isolates were resistant to tetracycline, and 25% were resistant to sulfadimethoxine. Heterogeneity of E. coli O157 strains was demonstrated by the presence of 22 different pulsed-field gel electrophoresis (PFGE) patterns. Four PFGE patterns matched those of isolates previously found in humans. The presence of E. coli O157 at county fairs suggests the potential for transmission to the public, who may have contact with cattle or their environment.

Instant coffee consumption may be associated with higher risk of metabolic syndrome in Korean adults.

AIMS Cumulative evidence suggests that coffee consumption may have beneficial effects on metabolic diseases; however, few previous studies have considered the types of coffee consumed and the additives used. We investigated the relationship between coffee consumption and metabolic syndrome (MetSyn) and its components. METHODS We analyzed 17,953 Korean adults, aged 19-65 years, using cross-sectional data from the Korean National Health and Nutrition Examination Survey (KNHANES, 2007-2011). Coffee consumption level, types of coffee consumed, and the additives used were assessed based on a food frequency questionnaire and 24-h recall. Demographic and lifestyle factors were assessed using self-administered questionnaires. Data on metabolic biomarkers were obtained from a health examination. Multivariable logistic regression was used to determine the odds ratios of prevalent metabolic syndrome and its components according to frequency and type of coffee consumption. RESULTS We found that 76% of the subjects were habitual coffee drinkers, most of whom consumed instant coffee mix containing sugar and powder creamer. After multivariable adjustment, the odds ratios (95% CI) comparing those who consumed coffee ≥3 times/day with those who consumed coffee <1 time/week were 1.37 (1.15-1.63) for obesity, 1.33 (1.11-1.59) for abdominal obesity, 1.28 (1.09-1.51) for hypo-HDL cholesterolemia, and 1.37 (1.10-1.72) for metabolic syndrome. Instant-coffee drinkers were observed to have elevated risks of these metabolic conditions. CONCLUSIONS Consumption of coffee, particularly instant coffee mix, may have harmful effects on MetSyn, perhaps partly deriving from excessive intake of sugar and powder creamer.

Development of a loop-mediated isothermal amplification assay for rapid, sensitive detection of Campylobacter jejuni in cattle farm samples.

Campylobacter jejuni is a leading cause of bacterial foodborne disease worldwide. The detection of this organism in cattle and their environment is important for the control of C. jejuni transmission and the prevention of campylobacteriosis. Here, we describe the development of a rapid and sensitive method for the detection of C. jejuni in naturally contaminated cattle farm samples, based on real-time loop-mediated isothermal amplification (LAMP) of the hipO gene. The LAMP assay was specific (100% inclusivity and exclusivity for 84 C. jejuni and 41 non-C. jejuni strains, respectively), sensitive (detection limit of 100 fg/μl), and quantifiable (R(2) = 0.9133). The sensitivity of the LAMP assay was then evaluated for its application to the naturally contaminated cattle farm samples. C. jejuni strains were isolated from 51 (20.7%) of 246 cattle farm samples, and the presence of the hipO gene was tested using the LAMP assay. Amplification of the hipO gene by LAMP within 30 min (mean ~10.8 min) in all C. jejuni isolates (n = 51) demonstrated its rapidity and accuracy. Next, template DNA was prepared from a total of 186 enrichment broth cultures of cattle farm samples either by boiling or using a commercial kit, and the sensitivity of detection of C. jejuni was compared between the LAMP and PCR assays. In DNA samples prepared by boiling, the higher sensitivity of the LAMP assay (84.4%) compared with the PCR assay (35.5%) indicates that it is less susceptible to the existence of inhibitors in sample material. In DNA samples prepared using a commercial kit, both the LAMP and PCR assays showed 100% sensitivity. We anticipate that the use of this rapid, sensitive, and simple LAMP assay, which is the first of its kind for the identification and screening of C. jejuni in cattle farm samples, may play an important role in the prevention of C. jejuni contamination in the food chain, thereby reducing the risk of human campylobacteriosis.

Development of a loop-mediated isothermal amplification assay for detecting Listeria monocytogenes prfA in milk

A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed for detecting Listeria monocytogenes prfA in milk. The inclusivity of 23 L. monocytogenes and the exclusivity of 16 non-L. monocytogenes strains were both 100% in the assay. The limit of detection (LoD) of the LAMP assay in Listeria enrichment broth (LEB) was 2.22 CFU/mL after 12 h and 24 h of incubation. The LoDs of the LAMP assay in LEB with artificially contaminated milk (LEB-M) incubated for 12 h (2.22×101 CFU/mL) and 24 h (2.22 CFU/mL) were lower than those of the PCR and real-time PCR assays. Comparison of the LoDs in LEB with those in LEB-M showed that the LAMP assay was less influenced by the milk compounds than the real-time PCR assay. Our results indicate that the LAMP assay can be utilized as a potential screening tool for L. monocytogenes in milk.

Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA

Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be 85.56±0.07℃ for cattle, 84.96±0.08℃ for pig, and 85.99±0.05℃ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); 84.91±0.11℃ for goat and 83.90±0.11℃ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and 86.31±0.23℃ for chicken, 88.66±0.12℃ for duck, and 84.49±0.08℃ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from 10 pg/μL to 100 fg/μL levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species.

Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle.

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on Tm values of 85.03 ± 0.54℃ for stx1 and 87.47 ± 0.35℃ for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/µL), and quantifiable (R2 = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.

Helicobacter apodemus sp. nov., a new Helicobacter species identified from the gastrointestinal tract of striped field mice in Korea

A novel Helicobacter species was identified from the gastrointestinal tract of the Korean striped field mouse (Apodemus agrarius). Biochemical testing, ultrastructure characterization, and 16S rRNA gene sequence analysis suggested that this bacterium represents a distinct taxon. The bacterium was positive for urease activity, susceptible to cephalothin and nalidixic acid, and weakly positive for oxidase and catalase activity. Electron microscopy revealed that the bacterium has spirally curved rod morphology with singular bipolar nonsheathed flagella. Genotypically, the isolated bacterial strains (YMRC 000215, YMRC 000216, and YMRC 000419) were most closely related to a reference strain of Helicobacter mesocricetorum (97.25%, 97.32%, and 97.03% 16S rRNA sequence similarities, respectively). The 16S rRNA sequences of these strains were deposited into GenBank under accession numbers AF284754, AY009129, and AY009130, respectively. We propose the name Helicobacter apodemus for this novel species.

Development and Application of a Method for Rapid and Simultaneous Determination of Three β-agonists (Clenbuterol, Ractopamine, and Zilpaterol) using Liquid Chromatography-tandem Mass Spectrometry

β-agonists are anabolic compounds that promote fat loss and muscle gain, and their administration to livestock may provide economic benefits by increasing growth rate and feed efficiency. For these reasons, β-agonists are also commonly added to livestock feed as growth promoters. This can introduce a significant risk of secondary human poisoning through intake of contaminated meat. A new method for the simultaneous determination of three β-agonists (clenbuterol, ractopamine, and zilpaterol) was developed in this study and applied to various meat samples. The limits of quantification, derived through a validation test following Codex guidelines, were 0.2 μg/kg for clenbuterol and zilpaterol, and 0.4 μg/kg for ractopamine. The average recoveries for clenbuterol, ractopamine, and zilpaterol ranged from 109.1% to 118.3%, 95.3% to 109.0%, and 94.1% to 120.0%, respectively. The recovery and coefficient of variation (CV) values fell within the acceptable range according to the Codex guidelines. This method reduced the analysis time without decreasing detection efficiency by modifying the pretreatment steps. This method could be utilized to manage the safety of imported meat products from countries where zilpaterol use is still permitted, thereby improving public health and preventing β-agonist poisoning due to secondary contamination.

Inhibitory effects of resveratrol on hepatitis B virus X protein-induced hepatocellular carcinoma

Liver cancer occurs very frequently worldwide and hepatocellular carcinoma (HCC) accounts for more than 80% of total primary liver cancer cases. In this study, the anticarcinogenic effects of resveratrol against hepatitis B virus (HBV)-induced HCC were investigated by using HBV X-protein-overexpressing Huh7 (Huh7-HBx) human hepatoma cells. MTT assay showed that resveratrol decreased cell viability. Fluorescence-activated cell-sorter analysis showed that resveratrol induced G1 cell cycle arrest without increasing the sub-G1 phase cell population. Therefore, we evaluated its effect on regulation of cyclin D1, which is critically involved in G1/S transition. Resveratrol lowered cyclin D1 transcription. Western blot analysis of the effects of resveratrol on upstream cyclin D1 transcriptional signaling, extracellular signal-related kinase (ERK), p90RSK, Akt, and p70S6K revealed inhibition of Akt but not the ERK signaling pathway. Collectively, the results indicate that resveratrol inhibits Huh7-HBx proliferation by decreasing cyclin D1 expression through blockade of Akt signaling. We investigated the anticarcinogenic effect and mechanism of resveratrol in xenograft model mice implanted with Huh7-HBx cells. Intraperitoneal resveratrol injection reduced tumor size in the mice. Expression of survivin was reduced, but cyclin D1 was not affected. The results demonstrate that resveratrol treatment may help manage HBV-induced HCC by regulating survivin.

Antibiotic resistance patterns and genetic relatedness of Enterococcus faecalis and Enterococcus faecium isolated from military working dogs in Korea

Enterococcus spp. are normally present in the gastrointestinal tracts of animals and humans, but can cause opportunistic infections that can be transmitted to other animals or humans with integrated antibiotic resistance. To investigate if this is a potential risk in military working dogs (MWDs), we analyzed antibiotic resistance patterns and genetic relatedness of Enterococcus spp. isolated from fecal samples of MWDs of four different age groups. Isolation rates of Enterococcus spp., Enterococcus (E.) faecalis, and E. faecium, were 87.7% (57/65), 59.6% (34/57), and 56.1% (32/57), respectively, as determined by bacterial culture and multiplex PCR. The isolation rate of E. faecalis gradually decreased with age (puppy, 100%; adolescent, 91.7%; adult, 36.4%; and senior, 14.3%). Rates of resistance to the antibiotics ciprofloxacin, gentamicin, streptomycin, sulfamethoxazole/trimethoprim, imipenem, and kanamycin among Enterococcus spp. increased in adolescents and adults and decreased in senior dogs, with some isolates having three different antibiotic resistance patterns. There were indistinguishable pulsed-field gel electrophoresis patterns among the age groups. The results suggest that Enterococcus is horizontally transferred, regardless of age. As such, periodic surveillance studies should be undertaken to monitor changes in antibiotic resistance, which may necessitate modification of antibiotic regimens to manage antibiotic resistance transmission.