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Colistin is an important antibiotic currently used to manage infections caused by multidrug-resistant pathogens in both humans and livestock animals. A new mobile colistin-resistance (mcr-9) gene w...

This study was conducted to characterize Escherichia coli strains and evaluate the spread of antimicrobial resistance among these strains from fresh produce and farm environments in Korea. We then conducted phenotypic and genetic studies on antimicrobial-resistant isolates. We determined the genetic epidemiological characteristics of isolates that produced extended-spectrum β-lactamase (ESBL) and confirmed plasmid transfer in isolates that carried blaCTX-M-type genes. E. coli strains were isolated from 8 samples of fresh produce and 152 samples from the farm environment collected from May 2014 to June 2016. Cephalosporin resistance was the most prevalent (61.8%) type of resistance among the isolates. Five ESBL-producing strains with high genetic homology with E. coli of human or livestock origin were identified. Lateral transfer of plasmids harboring blaCTX-M-type genes to transconjugants was successful. Two isolates from Chinese cabbage and from water samples collected from a nearby stream harbored the ISEcp1-blaCTX-M-55-orf477 operon and were confirmed as sequence type 1196 and the same type of plasmid replicon, suggesting that cross-contamination was highly likely. A high-risk clone of sequence type 69 (clonal complex 69) isolates was also recovered from the farm environment. This study provides genetic evidence that antimicrobial resistance factors in E. coli from farm environments originate in the clinic or in livestock, highlighting the fact that good agricultural practices in farming are important to inhibit the spread of antimicrobial resistance to bacteria on fresh produce. HIGHLIGHTS

Acidovorax citrulli, the gram-negative bacteria that causes bacterial fruit blotch (BFB), has been responsible for huge worldwide economic losses in watermelon and melon production since 1980. No commercial cultivar resistant to BFB has been reported. Of the two reported genotypes of A. citrulli, genotype I is the main causal agent of BFB in melon and genotype II causes disease in watermelon. After the isolation of the first bacteriophage against A. citrulli (ACP17), efforts have been made to isolate bacteriophages with wider host ranges by collecting samples from watermelon, pumpkin, and cucumber. The newly isolated phage ACPWH, belonging to the Siphoviridae family, has a head size of 60 ± 5 nm and tail size of 180 ± 5 nm, and can infect 39 out of 42 A. citrulli strains. ACPWH has genome size of 42,499 and GC content of 64.44%. Coating watermelon seeds with bacteriophage ACPWH before soil inoculation with A. citrulli resulted in 96% germination and survival, compared to 13% germination of uncoated control seeds. These results suggest that phage ACPWH may be an effective and low-cost biocontrol agent against BFB.

Colistin is an important antibiotic currently used to manage infections caused by multidrug-resistant pathogens in both humans and livestock animals. A new mobile colistin-resistance (mcr-9) gene was recently discovered; this discovery highlighted the need for rigorous monitoring of bacterial resistance against colistin. Salmonella is one of the major pathogens responsible for foodborne illnesses; however, there is minimal information regarding the presence of mcr genes in foodborne Salmonella strains. The aim of this study was to investigate the presence of mcr genes among 178 Salmonella strains isolated from chicken meat in Korea. Antimicrobial susceptibility was measured using the broth microdilution method. Bioinformatics characterization of colistin-resistant strains and genetic environment of the mcr-9 gene were analyzed using next-generation sequencing. Transferability of the mcr-9 carrying colistin-resistant Salmonella strain was tested using broth-mating conjugation. Thirteen of the 178 Salmonella isolates showed colistin resistance, but only one strain, Salmonella Dessau ST14 (KUFSE-SAL043) from a traditional chicken market in Korea, carried an mcr family gene, mcr-9. This strain also carried other acquired antimicrobial resistance genes such as bla(TEM-1B), qnrS1, and aac(6′)-Iaa. Only the IncX1 plasmid replicon type was detected in this strain. In the strain KUFSE-SAL043, the mcr-9 gene was located between two insertion sequences, IS903B and IS26, followed by the downstream regulatory genes qseB-like and qseC-like, which were located between IS1R and ΔIS1R. Conjugation tests revealed that the mcr-9 gene was successfully transferred to Escherichia coli J53 at a mean frequency of 2.03 × 10(−7). This is the first report of a transferable mcr-9 gene in Salmonella isolated from chicken meat in Korea, highlighting the possibility of transfer of colistin resistance. Therefore, the wide use of colistin should be reconsidered, and a One Health perspective should be adopted to monitor the antimicrobial resistance of Enterobacteriaceae strains in humans, livestock, and the environment.

Increased enterococcal infections in hospitals and multidrug-resistant and vancomycin-resistant enterococci (VRE) isolated from humans, animals, and food sources raised public health concern on the presence of VRE in multiple sources. We performed a comparative analysis of the antimicrobial resistance and genetics of VRE isolates derived from fresh produce and human fecal samples. Of 389 Enterococcus isolates, 8 fecal and 3 produce isolates were resistant to vancomycin and teicoplanin; all harbored vanA gene. The VRE isolates showed multidrug-resistant properties. The isolates from fresh produce in this study showed to have the common shared characteristics with the isolates from humans by the results of antimicrobial resistance, multilocus sequence typing, and Tn 1546 transposon analysis. Therefore, VRE isolates from fresh produce are likely related to VRE derived from humans. The results suggested that VRE may contaminate vegetables through the environment, and the contaminated vegetables could then act as a vehicle for human infections. Ongoing nationwide surveillance of antibiotic resistance and the promotion of the proper use of antibiotics are necessary.

Fusarium graminearum species complex (FGSC) causes Fusarium head blight in small grain cereals. To date, four species (F. graminearum, F. asiaticum, F. boothii, and F. meridionale ) belonging to FGSC frequently occur in Korean cereals. In addition, we first reported the occurrence of additional species (F. vorosii ) within FGSC, which was isolated from barley, corn, and rice in Korea. Phylogenetic analysis of the Fusarium isolates of this group using combined multi-gene sequences confirmed species identification. Moreover, the macroconidia produced by these isolates were morphologically similar to those of the F. vorosii holotype. Chemical analysis indicated that the F. vorosii isolates produced various trichothecenes such as nivalenol and deoxynivalenol with their acetyl derivatives along with zearalenone. Pathogenicity tests demonstrated that all of the F. vorosii isolates examined were pathogenic on barley, corn, and rice with variation in aggressiveness. This study is the first report of F. vorosii in Korean cereals, their pathogenicity towards barley and corn, and their ability to produce trichothecenes and zearalenone.

Fascioliasis, a food-borne trematode zoonosis, is a disease primarily in cattle and sheep and occasionally in humans. Water dropwort (Oenanthe javanica), an aquatic perennial herb, is a common second intermediate host of Fasciola, and the fresh stems and leaves are widely used as a seasoning in the Korean diet. However, no information regarding Fasciola species contamination in water dropwort is available. Here, we collected 500 samples of water dropwort in 3 areas in Korea during February and March 2015, and the water dropwort contamination of Fasciola species was monitored by DNA sequencing analysis of the Fasciola hepatica and Fasciola gigantica specific mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 500 samples assessed, the presence of F. hepatica cox1 and 1TS-2 markers were detected in 2 samples, and F. hepatica contamination was confirmed by sequencing analysis. The nucleotide sequences of cox1 PCR products from the 2 F. hepatica-contaminated samples were 96.5% identical to the F. hepatica cox1 sequences in GenBank, whereas F. gigantica cox1 sequences were 46.8% similar with the sequence detected from the cox1 positive samples. However, F. gigantica cox1 and ITS-2 markers were not detected by PCR in the 500 samples of water dropwort. Collectively, in this survey of the water dropwort contamination with Fasciola species, very low prevalence of F. hepatica contamination was detected in the samples.

The purposes of this study were to develop a small scale post-harvest facility, and consequently to evaluate the effects of applying the facility along with hygiene education on the level of microbial safety in Korean leeks production. A total of 135 samples were collected at three Korean leeks farms in Yangju, Gyeonggi province. Food safety indicators (Aerobic plate count (APC), coliform count, and Escherichia coli) and foodborne pathogens (E. coli O157:H7, Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus) on/in the samples were assessed. The microbial load measured as APC with harvesting tools such as comb, chopping board, and knife, at the farms where the small scale post-harvest facility had been operated (Farms A and B) was lower than that at another farm having no post-harvest facility (Farm C) by 1.44~2.33 log CFU / 100 cm. Moreover, the chopping board from Farm C was observed being contaminated with B. cereus at 6.03 log CFU / 100 cm. The coliform counts from the samples increased by 0.57~1.89 log CFU/g after leeks was submerged in ground water for washing. E. coli was recovered from leeks, soil, and the ground water used in the washing process, while no E. coli O157:H7, Salmonella spp., and L. monocytogenes was detected. Our results indicated that the small scale post-harvest facility developed in this study as well as the hygiene education played an important role in enhancing the level of microbial food safety in the leeks production environment. However, a disinfection technique could be needed during the washing step in order to prevent a potential contamination.

Antagonistic microbes were isolated from soils to control mycotoxin contamination of cereals by limiting the growth of mycotoxigenic Fusarium species. In total, 341 bacterial isolates were examined for antifungal activity against eight mycotoxigenic Fusarium species using dual culture assays. The screening identified 11 isolates that inhibited mycelial growth of all Fusarium species tested. The culture filtrates of 2 of the 11 isolates completely inhibited germination of conidia up to 21 days of incubation. These two isolates exhibited identical activity toward the fungi tested and were identified as Brevibacillus spp. based on 16S rRNA sequence homology. The most closely related species based on phylogenetic analysis was Brevibacillus reuszeri. Additional dual culturing using further fungal species showed that the antagonistic Brevibacillus inhibited the growth of most Fusarium species tested (39 of 46 species), two Epicoccum spp., one Alternaria sp., three Aspergillus spp. (3 of 11), and three Penicillium spp. (3 of 8). The in vivo assay was performed to test the efficacy of antagonistic Brevibacillus isolates on maize ears and revealed that the application of microbes suppressed ear rot (ANOVA, p = 0.0020). This Brevibacillus sp. may be an antagonist of the majority of Fusarium species, including mycotoxigenic species.

In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a polyketide-derived SM produced by multiple species of the fungal genus Fusarium. This SM is of concern because it is toxic to animals and, therefore, is considered a mycotoxin and may contribute to plant pathogenesis. Preliminary descriptions of the fusaric acid (FA) biosynthetic gene (FUB) cluster have been reported in two Fusarium species, the maize pathogen F. verticillioides and the rice pathogen F. fujikuroi. The cluster consisted of five genes and did not include a transcription factor or transporter gene. Here, analysis of the FUB region in F. verticillioides, F. fujikuroi, and F. oxysporum, a plant pathogen with multiple hosts, indicates the FUB cluster consists of at least 12 genes (FUB1 to FUB12). Deletion analysis confirmed that nine FUB genes, including two Zn(II)2Cys6 transcription factor genes, are required for production of wild-type levels of FA. Comparisons of FUB cluster homologs across multiple Fusarium isolates and species revealed insertion of non-FUB genes at one or two locations in some homologs. Although the ability to produce FA contributed to the phytotoxicity of F. oxysporum culture extracts, lack of production did not affect virulence of F. oxysporum on cactus or F. verticillioides on maize seedlings. These findings provide new insights into the genetic and biochemical processes required for FA production.

This study was conducted to investigate survival of Bacillus cereus (B. cereus) on stainless steel and polyvinyl chloride (PVC) and its transfer from two material to lettuce. The stainless steel and PVC were innoculated with B. cereus and stored at 6 combination conditions (temperature : 20oC and 30oC, relative humidity (RH) : 43%, 69%, and 100%). Although the total numbers of B. cereus at RH 43% and RH 69% were reduced by 3.53-4.00 log CFU/coupon within 24 h regardless of material type, the spore numbers of B. cereus was lasted at 3.0 log CFU/ coupon. When two materials were stored at 30oC, RH 100%, the spore numbers of B. cereus was rapidly increased by 3.0 log CFU/coupon. In addition, the reduction rate of B. cereus was decreased in the presence of organic matter. Transfer rate of B. cereus from surface of stainless steel and PVC to lettuce was increased by 10 times in the presence of water on the lettuce surface. As a result of this study, the presence of B. cereus on produce contact surfaces can increase the risk of cross-contamination. Thus, it is important that the packing table and conveyer belt in post harvest facility should be properly washed and sanitized after working to prevent cross-contamination.

Foodborne illness due to the consumption of contaminated raw strawberries is a continuing food safety concern. This study investigated and evaluated contamination levels of bacteria on strawberries at farms stage to evaluate potential hazards associated with fresh strawberries. A total of 315 samples, 105 samples from 5 sampling sites (A to E) of 21 farms and 210 samples from 1 sampling site of 6 farms, was collected every month for four months and analyzed to enumerate aerobic bacterial counts, Coliforms/E. coli, Bacillus cereus and Staphylococcus aureus. In addition, the prevalence study of five pathogens (S. aureus, E. coli, E. coli O157:H7, Salmonella spp. and Listeria monocytogenes) was performed on each sample. Aerobic bacterial counts ranged from 0.48 to 6.36 Log CFU/g, with the highest bacterial cell counts recorded for D and E sites. Coliforms were detected in 71 samples (22.5%) with a minimum of 0.48 cfu/g and a maximum of more than 4 Log CFU/g. B. cereus was detected in 98 samples (31.1%) among total samples analyzed. S. aureus was detected in 2 samples with a minimum of 0.48 Log CFU/g and a maximum of 1.38 Log CFU/g. E. coli, E. coli O157:H7, Salmonella spp. and L. monocytogenes were not isolated from any of the samples. The microbial contamination levels of strawberries determined in this study may be used as the fundamental data for microbiological risk assessment.

To evaluate microbiological and aflatoxin safety on traditional dried persimmon, a total of 315 samples were collected from 105 farms. The collected samples were assessed on aflatoxin and microorganisms (Aer- obic plate count, coliform count, Escherichia coli, Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus). The the APC of sliced dried persimmon, dried persimmon, and semi dried persimmon were 3.93 ± 0.96, 2.12 ± 0.93, and 1.50 ± 1.08 log CFU/g, respectively. S. aureus was detected in 40.0% of sliced dried persimmon, 29.5% of dried persimmon, and 23.5% of semi dried persimmon. E. coli recovered from dried persimmon and semi dried persimmon was 6.6%, and 2.9%, respectively. However, E. coli O157:H7, Sal- monella spp., and L. monocytogenes were not detected. According to the result of aflatoxin by ELISA and UPLC, aflatoxin was not detected in any sample. These data suggested that safety management system should be introduce to the farms producing traditional dried persimmon to enhance the safety of traditional dried persimmon.

This study assessed microbiological hazards at postharvest stage of dropwort farms (A, B, C, D, E, F, G, H, I) located in 4 different areas in Korea. The samples were assessed for sanitary indication bacteria (total aerobic bacteria, coliform, and Escherichia coli) and pathogenic bacteria (Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus). Total aerobic bacteria and coliform in 9 dropwort farms were detected at the levels of 0~7.00 and 0~4.25 log CFU/g, mL, of 100 cm. In particular, microbial contamination in worker’s hand showed higher than cultivation environment factors. Escherichia coli was detected in several farms of soil, irrigation water, washing water and worker’s hand and also, dropwort in these farms was contaminated with E. coli (positive reaction). In case of pathogenic bacteria, B. cereus was detected at the highest levels in soil. S. aureus was detected qualitatively from only one sample of dropwort washed by water. E. coli O157:H7 and L. monocytogenes were not detected. Although dropwort pass through 2 process (trimming and washing), the microbial contamination was not differ significantly before and after which indicates that current washing system was not effect on reduction of microorganism. From these results, the postharvest environment and workers have been considered as cross-contamination factors. Thus, processing equipments and personal hygiene should be managed to reduce the microbial contamination of dropwort. Accordingly management system such as good agricultural practices (GAP) criteria is needed for the safety of dropwort

While searching for healthier diets, people became more attentive to agricultural organic products. However, organic foods may be more susceptible to microbiological contamination because of the use of livestock manure compost and liquid manure, potential sources of pathogenic bacteria. This study was undertaken to investigate the persistence of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in soil, liquid manure amended soil, and liquid manure. Loamy soil, liquid manure amended soil, and liquid manure were inoculated with S. enterica, E. coli O157:H7, and L. monocytogenes. Samples were incubated in consistent moisture content at 25°C. Samples had been periodically collected during 120 days depending on the given conditions. S. enterica and E. coli O157:H7 survived over 120 days in loamy soil and over 60 days in liquid manure amended soil, respectively. L. monocytogenes decreased faster than other pathogens in soil. S. enterica, E. coli O157:H7, and L. monocytogenes survived for up to 5 days in liquid manure. S. enterica and E. coli O157:H7 in soil decreased by 2 to 2.5 log CFU g -1 for 120 days. S. enterica and E. coli O157:H7 in liquid manure amended soil decreased slowly for 21 days. However, S. enterica, E. coli O157:H7, and L. monocytogenes sharply decreased after 21 days. S. enterica, E. coli O157:H7, and L. monocytogenes in soil increased by 0.5 to 1.0 log CFU g -1 for 7 days. Foodborne pathogens in soil and liquid manure amended soil gradually decreased over time.

This study investigated the effect of adaptation of Staphylococcus aureus strains to the acidic condition of tomato in response to environmental stresses, such as heat and acid. S. aureus ATCC 13565, ATCC 14458, ATCC 23235, ATCC 27664, and NCCP10826 habituated in tomato extract at 35°C for 24 h were inoculated in tryptic soy broth. The culture suspensions were then subjected to heat challenge or acid challenge at 60°C and pH 3.0, respectively, for 60 min. In addition, transcriptional analysis using quantitative real-time PCR was performed to evaluate the expression level of acid-shock genes, such as clpB, zwf, nuoF, and gnd, from five S. aureus strains after the acid habituation of strains in tomato at 35°C for 15 min and 60 min in comparison with that of the nonhabituated strains. In comparison with the nonhabituated strains, the five tomato-habituated S. aureus strains did not show cross protection to heat, but tomato-habituated S. aureus ATCC 23235 showed acid resistance. In quantitative real-time-PCR analysis, the relative expression levels of acid-shock genes (clpB, zwf, nuoF, and gnd) were increased the most in S. aureus ATCC 23235 after 60 min of tomato habituation, but there was little difference in the expression levels among the five S. aureus strains after 15 min of tomato habituation. These results indicate that the variation of acid resistance of S. aureus is related to the expression of acid-shock genes during acid habituation.

The objective of this study was to analyze the microbiological hazards of Astragalus membranaceus Bunge on the post-harvest processing. Samples from processing equipments (cleaner, water, cart, table, tray and pack- aging machine), personal hygiene (hand) and harvested crops (before washing, after washing, after sorting, and after drying) were collected from four farms (A, B, C, and D) located in Chungchengbuk-do, Korea. The samples were analyzed for sanitary indication bacteria and pathogenic bacteria. First, total aerobic bacteria and coliform in process- ing facilities were detected at the levels of 0.93~4.86 and 0.33~2.28 log CFU/100 cm 2 and /mL respectively. In partic- ular, microbial contamination in hand (5.43~6.11 and 2.52~4.12 log CFU/Hand) showed higher than processing equipments. Among the pathogenic bacteria, Bacillus cereus was detected at the levels of 0.33~2.41 log CFU/100 cm 2 , 1.48~3.27 log CFU/Hand and 0.67~3.65 log CFU/g in equipments, hands, and plants and Staphylococcus aureus were detected in cleaner, table, hand and harvested crops (before washing and after sorting) by qualitative test. Escher- ichia coli O157:H7, Listeria monocytogenes, and Salmonella spp. were not detected. These results indicated that per- sonal hygiene and processing equipments should be managed to reduce the microbial contamination of A. membranaceus Bunge. Therefore, management system such as good agricultural practices (GAP) criteria is needed for hygienic agricultural products.

BACKGROUND: Azadirachta Indica extract(AIE) and Sophorae radix extract(SRE) are widely used as environment-friendly organic materials of plant origin in South Korea. METHODS AND RESULTS: In this study, the in vitro micronucleus(vitMN) tests of two samples of AIE and SRE were conducted to evaluate their genotoxicity using the Chinese hamster lung(CHL) cell. This study was composed of two parts; cytochalasin B(cyto B) test and non-cyto B test. Mitomycin C and colchicine were used as positive controls. As a result, the incidence of micronucleus(MN) in all AIE and SRE treated groups increased in dose- dependent manner, but were less than 2.2% in 1,000 binucleated cells. In addition, there were no significant increases of MN incidence in all AIE and SRE treated groups, compared with the negative control group. CONCLUSION: Therefore, we suggest that AIE samples and SRE samples used in this study may have no genotoxicity in the in vitro micronucleus test using the CHL cells. In our previous study, we reported that AIE and SRE did not cause genotoxicity in Ames test. According to the genotoxicity battery system, we concluded that AIE and SRE used in this study have no genotoxic effects to humans.

To analyze the hazards associated with cucumber and hot pepper cultivation areas, a total of 72 samples were obtained and tested to detect the presence of biological (sanitary indicative, pathogenic bacteria and fungi) and chemical hazards (heavy metals and pesticide residues). The levels of sanitary indicative bacteria (aerobic plate counts and coliforms) and fungi were ND-7.2 and ND-4.8 log CFU/(g, mL, hand, or 100 cm 2 ) in cucumber cultivation areas, and ND- 6.8 and 0.4-5.3 log CFU/(g, mL, hand, or 100 cm 2 ) in hot pepper cultivation areas. More specifically, the soil of hot pepper cultivation areas was contaminated with coliforms at a maximum level of 5.6 log CFU/g. Staphylococcus aureus was detected only in glove samples at a level of 1.4 log CFU/100 cm 2 and Bacillus cereus was detected in the majority of samples at a level of ND-4.8 log CFU/(g, mL, hand, or 100 cm 2 ). Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were not detected. Heavy metal (Zn, Cu, Ni, Pb, and Hg) chemical hazards were detected at levels lower than the regulation limit. Residual insecticides were not detected in cucumbers; however, hexaconazole was detected at a level of 0.016 mg/kg (maximum residue limit: 0.3 mg/kg) in hot peppers.

Livestock manure is a valuable source of nutrients and organic matter for plant. Thus, livestock manure compost is commonly used fertilizer in organic vegetable and fruit production in many countries. However, contaminated or inadequate manure compost can give negative effect to soil microorganisms. This study was conducted to investigate the survival difference of Salmonella enterica and Listeria monocytogenes in chicken and pig manure compost under the selected environmental conditions. Commercially available manure compost (pig, chicken) was inoculated with S. enterica and L. monocytogenes. Manure compost was incubated at 25℃ and consistent moisture content. Samples had been collected during 200 days depending on the given conditions. S. enterica survived for 130 days in pig manure compost and over 200 days in chicken manure compost, respectively. L. monocytogenes persisted for 120 days in pig manure compost and over 200 days in chicken manure compost, respectively. It is noted that the number of S. enterica and L. monocytogenes gradually decreased over time. The results indicate that S. enterica survived longer than L. monocytogenes in manure compost at 25℃. S. enterica and L. monocytogenes survived longer in chicken manure compost than in pig manure compost. Increased knowledge of pathogen behavior in agricultural environments is a valuable part of future work on improving risk evaluations and, in a longer perspective, in providing data for guidelines regarding safe handling of pathogen-contaminated manure compost and soil.