Potential use of newly isolated bacteriophage as a biocontrol against Acidovorax citrulli.

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.

Emergence of Transferable mcr-9 Gene-Carrying Colistin-Resistant Salmonella enterica Dessau ST14 Isolated from Retail Chicken Meat in Korea.

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 blaTEM-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.

Characterization of Vancomycin-Resistant Enterococcus faecalis and Enterococcus faecium Isolated from Fresh Produces and Human Fecal Samples.

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.

Identification of a 12-gene Fusaric Acid Biosynthetic Gene Cluster in Fusarium Species Through Comparative and Functional Genomics.

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.

Monitoring of Fasciola Species Contamination in Water Dropwort by cox1 Mitochondrial and ITS-2 rDNA Sequencing Analysis.

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.

Twindemic Threats of Weeds Coinfected with Tomato Yellow Leaf Curl Virus and Tomato Spotted Wilt Virus as Viral Reservoirs in Tomato Greenhouses.

Tomato yellow leaf curl virus (TYLCV) and tomato spotted wilt virus (TSWV) are well-known examples of the begomovirus and orthotospovirus genera, respectively. These viruses cause significant economic damage to tomato crops worldwide. Weeds play an important role in the ongoing presence and spread of several plant viruses, such as TYLCV and TSWV, and are recognized as reservoirs for these infections. This work applies a comprehensive approach, encompassing field surveys and molecular techniques, to acquire an in-depth understanding of the interactions between viruses and their weed hosts. A total of 60 tomato samples exhibiting typical symptoms of TYLCV and TSWV were collected from a tomato greenhouse farm in Nonsan, South Korea. In addition, 130 samples of 16 different weed species in the immediate surroundings of the greenhouse were collected for viral detection. PCR and reverse transcription-PCR methodologies and specific primers for TYLCV and TSWV were used, which showed that 15 tomato samples were coinfected by both viruses. Interestingly, both viruses were also detected in perennial weeds, such as Rumex crispus, which highlights their function as viral reservoirs. Our study provides significant insights into the co-occurrence of TYLCV and TSWV in weed reservoirs, and their subsequent transmission under tomato greenhouse conditions. This project builds long-term strategies for integrated pest management to prevent and manage simultaneous virus outbreaks, known as twindemics, in agricultural systems.

Prevalence, distribution, enterotoxin profiles, antimicrobial resistance, and genetic diversity of Bacillus cereus group isolates from lettuce farms in Korea.

Lettuce wraps are popular in Korean cuisine for their high nutritional value and versatility as healthy additions to multiple dishes. Microbial contamination of lettuce is a major concern, as lettuce is consumed fresh without cooking. Among foodborne pathogens, the spore-forming, facultative anaerobic bacterium, Bacillus cereus is one of the frequently detected pathogen in lettuce in Korea. In this study, we investigated the prevalence and distribution of Bacillus cereus strains in lettuce production farms and further evaluated the enterotoxin gene profiles, antibiotic susceptibility, multidrug resistance pattern, and genetic differences among the B. cereus group isolates. Of the 140 samples isolated from 10 lettuce production farms, 30 samples (21.42%) were positive for B. cereus in which 19 (31.6%) and 10 (23.25%) were from soil and lettuce, respectively. The enterotoxin patterns A (hblCDA, nheABC, entFM, and cytK genes) and B (hblCDA, nheABC, and entFM genes) accounted for 50% and 20% of all the isolates, whereas the emetic gene cesB was not detected in any of the B. cereus group isolates. Antibiotic susceptibility testing of the B. cereus group isolates revealed that all the strains were predominantly resistant to ß-lactam antibiotics except imipenem and generally susceptible to most of the non ß-lactam antibiotics, including gentamycin, streptomycin, chloramphenicol, and tetracycline. ERIC-PCR and MLST analysis revealed high genetic diversity among the 30 B. cereus group isolates, which belonged to 26 different sequence types (STs) and seven new STs. Moreover, isolates with identical STs exhibited similar patterns of antibiotic resistance and enterotoxin profiles. Results of this study indicate a high prevalence of B. cereus group isolates in lettuce production farms in the Republic of Korea.

Microbiological assessment in strawberry production and recommendations to establish a good agricultural practice system.

This study conducted microbiological assessment in tunnel style strawberry greenhouses and packaging centers and suggested recommendations to establish a good agricultural practice for strawberry production. The samples from irrigation water, workers' gloves, harvest bins, soil, strawberry leaves and strawberries in greenhouses, packers' gloves, conveyor belts, packaging tables, and door knobs of entrances in packaging centers were collected. Bacterial cell counts of aerobic plate counts, coliforms, Escherichia coli, E. coli O157:H7, Salmonella, Staphylococcus aureus, and Bacillus cereus were then enumerated on appropriate selective media. In general, bacterial populations were similar (p ≥ 0.05) among strawberry greenhouses but not among packaging houses. E. coli and E. coli O157:H7 were negative in all samples, and low levels of Salmonella and B. cereus were detected. However, high bacterial cell counts of aerobic plate counts, coliforms, and S. aureus were found in most samples. These results suggest that food safety practice in strawberry greenhouses and packaging centers should be improved, and the results may be useful in the establishment of a good agricultural practice system for strawberry production.

Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolated from Fresh Produce and Agricultural Environments in Korea.

ABSTRACT: 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 ß-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.

Complete Genome Sequence of Biofilm-Forming Strain Staphylococcus haemolyticus S167.

Staphylococcus haemolyticus S167 has the ability to produce biofilms in large quantities. Genomic analyses revealed information on the biofilm-related genes of S. haemolyticus S167. Detailed studies of biofilm formation at the molecular level could provide a foundation for biofilm control research.

Mycobiota of ground red pepper and their aflatoxigenic potential.

To investigate contamination of ground red pepper with fungi and mycotoxin, we obtained 30 ground red pepper samples from 15 manufacturers in the main chili-pepper-producing areas in Korea. Fungal contamination was evaluated by spreading diluted samples on potato dextrose agar plates. The total fungi counts ranged from 0 to 7.3 × 103 CFU/g. In the samples, the genus Aspergillus had the highest incidence, while Paecilomyces was isolated most frequently. The next most frequent genera were Rhizopus, Penicillium, Cladosporium, and Alternaria. Within Aspergillus, A. ruber was predominant, followed by A. niger, A. amstelodami, A. ochraceus, A. terreus, A. versicolor, A. flavus, and A. fumigatus. The samples were analyzed for aflatoxins, ochratoxin A, and citrinin by ultra-perfomance liquid chromatography (UPLC) with a fluorescence detector. Ochratoxin A was detected from three samples at 1.03‒2.08 µg/kg, whereas no aflatoxins or citrinin were detected. To test the potential of fungal isolates to produce aflatoxin, we performed a PCR assay that screened for the norB-cypA gene for 64 Aspergillus isolates. As a result, a single 800-bp band was amplified from 10 A. flavus isolates, and one Aspergillus sp. isolate. UPLC analyses confirmed aflatoxin production by nine A. flavus isolates and one Aspergillus sp. isolate, which produced total aflatoxins at 146.88‒909.53 µg/kg. This indicates that continuous monitoring of ground red pepper for toxigenic fungi is necessary to minimize mycotoxin contamination.

Occurrence of Toxigenic Fusarium vorosii among Small Grain Cereals in Korea.

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.

Comparison of Trichothecene Biosynthetic Gene Expression between Fusarium graminearum and Fusarium asiaticum.

Nivalenol (NIV) and deoxynivalenol (DON) are predominant Fusarium-producing mycotoxins found in grains, which are mainly produced by Fusarium asiaticum and F. graminearum. NIV is found in most of cereals grown in Korea, but the genetic basis for NIV production by F. asiaticum has not been extensively explored. In this study, 12 genes belonging to the trichothecene biosynthetic gene cluster were compared at the transcriptional level between two NIV-producing F. asiaticum and four DON-producing F. graminearum strains. Chemical analysis revealed that time-course toxin production patterns over 14 days did not differ between NIV and DON strains, excluding F. asiaticum R308, which was a low NIV producer. Both quantitative real-time polymerase chain reaction and Northern analysis revealed that the majority of TRI gene transcripts peaked at day 2 in both NIV and DON producers, which is 2 days earlier than trichothecene accumulation in liquid medium. Comparison of the gene expression profiles identified an NIV-specific pattern in two transcription factor-encoding TRI genes (TRI6 and TRI10) and TRI101, which showed two gene expression peaks during both the early and late incubation periods. In addition, the amount of trichothecenes produced by both DON and NIV producers were correlated with the expression levels of TRI genes, regardless of the trichothecene chemotypes. Therefore, the reduced production of NIV by R308 compared to NIV or DON by the other strains may be attributable to the significantly lower expression levels of the TRI genes, which showed early expression patterns.

Gene-related strain variation of Staphylococcus aureus for homologous resistance response to acid stress.

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.

Occurrence of Fusarium mycotoxins in rice and its milling by-products in Korea.

A total of 201 samples of brown rice, polished rice, and two types of by-products, blue-tinged rice and discolored rice, were collected from rice stores maintained at 51 rice processing complexes in Korea. These samples were analyzed for the presence of Fusarium mycotoxins such as deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA). Contaminants (and their ranges) found in discolored rice samples were DON (59 to 1,355 ng g(-1)), NIV (66 to 4,180 ng g(-1)), and ZEA (25 to 3,305 ng g(-1)); those found in blue-tinged (less-ripe) rice were DON (86 to 630 ng g(-1)), NIV (50 to 3,607 ng g(-1)), and ZEA (26 to 3,156 ng g(-1)). Brown rice samples were contaminated mostly with NIV and ZEA (52 to 569 ng g(-1) and 47 to 235 ng g(-1), respectively). Polished rice samples were largely free from mycotoxins, although one sample was contaminated with NIV (77 ng g(-1)). When the fungal flora associated with each rice sample was investigated, blue-tinged rice was the most often contaminated with Fusarium graminearum (3.8%), followed by the discolored rice (2.4%) and brown rice (1.6%) samples. Using PCR, toxin genotyping of 266 isolates of F. graminearum revealed that most isolates (96%) were NIV producers. In conclusion, this survey is the first report of the cocontamination of Korean rice and its by-products with trichothecenes and ZEA. Importantly, it also provides new information on the natural contamination of rice by Fusarium mycotoxins.