Characterization of bacteriophage VVP001 and its application for the inhibition of Vibrio vulnificus causing seafood-borne diseases.

Vibrio vulnificus is a major food-borne pathogen that causes septicemia and cellulitis with a mortality rate of >50%. However, there are no efficient natural food preservatives or biocontrol agents to control V. vulnificus in seafood. In this study, we isolated and characterized a novel bacteriophage VVP001. Host range and transmission electron microscopy morphology observations revealed that VVP001 belongs to the family Siphoviridae and specifically infects V. vulnificus. Phage stability tests showed that VVP001 is stable at a broad temperature range of -20 °C to 65 °C and a pH range from 3 to 11, which are conditions for food applications (processing, distribution, and storage). In vitro challenge assays revealed that VVP001 inhibited V. vulnificus MO6-24/O (a clinical isolate) growth up to a 3.87 log reduction. In addition, complete genome analysis revealed that the 76 kb VVP001 contains 102 open reading frames with 49.64% G + C content and no gene encoding toxins or other virulence factors, which is essential for food applications. Application of VVP001 to fresh abalone samples contaminated with V. vulnificus demonstrated its ability to inhibit V. vulnificus growth, and an in vivo mouse survival test showed that VVP001 protects mice against high mortality (survival rate >70% at a multiplicity of infection of 1000 for up to 7 days). Therefore, the bacteriophage VVP001 can be used as a good natural food preservative and biocontrol agent for food applications.

Molecular interaction between methicillin-resistant Staphylococcus aureus (MRSA) and chicken breast reveals enhancement of pathogenesis and toxicity for food-borne outbreak.

To study pathogenesis and toxicity of Staphylococcus aureus in foods, FORC_062 was isolated from a human blood sample and complete genome sequence has a type II SCCmec gene cluster and a type II toxin-antitoxin system, indicating an MRSA strain. Its mobile gene elements has many pathogenic genes involved in host infection, biofilm formation, and various enterotoxin and hemolysin genes. Clinical MRSA is often found in animal foods and ingestion of MRSA-contaminated foods causes human infection. Therefore, it is very important to understand the role of contaminated foods. To elucidate the interaction between clinical MRSA FORC_062 and raw chicken breast, transcriptome analysis was conducted, showing that gene expressions of amino acid biosynthesis and metabolism were specifically down-regulated, suggesting that the strain may import and utilize amino acids from the chicken breast, but not able to synthesize them. However, toxin gene expressions were up-regulated, suggesting that human infection of S. aureus via contaminated food may be more fatal. In addition, the contaminated foods enhance multiple-antibiotic resistance activities and virulence factors in this clinical MRSA. Consequently, MRSA-contaminated food may play a role as a nutritional reservoir as well as in enhancing factor for pathogenesis and toxicity of clinical MRSA for severe food-borne outbreaks.

Lelliottia jeotgali sp. nov., isolated from a traditional Korean fermented clam.

Strain PFL01T was isolated from traditional Korean fermented clam, jogae-jeotgal, and characterized. The strain was a facultative anaerobic, Gram-stain-negative bacterium that was rod-shaped, motile and beige-pigmented. The phylogenetic sequence analysis based on the 16S rRNA gene from PFL01T revealed that it was closely related to Lelliottia nimipressuralis LMG 10245T and Lelliottia amnigena LMG 2784T with 99.3 and 99.3 % sequence identities, respectively. Multilocus sequence type analysis of concatenated partial aptD, gyrB, infB and rpoB gene sequences showed a clear distinction of strain PFL01T from its closest related type strains. The discrimination was also supported by unique repetitive extragenic palindromic PCR (Rep-PCR, ERIC-PCR) fingerprint patterns. In addition, results from average nucleotide identity analyses with other species were less than 85 %. vitek and API analyses revealed distinct characteristics from other species of Lelliottia. The cellular fatty acid profile of the strain consisted of C16 : 0, cyclo-C17 : 0, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c as major components. The whole genome of PFL01T was 4.6 Mb with a G+C content of 55.3 mol%. Based on these results, strain PFL01T was classified as a novel species of the genus Lelliottia, for which the name Lelliottia jeotgali sp. nov. is proposed. The type strain in PFL01T (=KCCM 43247T=JCM 31901T).

Characterization and genome analysis of novel bacteriophages infecting the opportunistic human pathogens Klebsiella oxytoca and K. pneumoniae.

Klebsiella is a genus of well-known opportunistic human pathogens that are associated with diabetes mellitus and chronic pulmonary obstruction; however, this pathogen is often resistant to multiple drugs. To control this pathogen, two Klebsiella-infecting phages, K. oxytoca phage PKO111 and K. pneumoniae phage PKP126, were isolated from a sewage sample. Analysis of their host range revealed that they infect K. pneumoniae and K. oxytoca, suggesting host specificity for members of the genus Klebsiella. Stability tests confirmed that the phages are stable under various temperature (4 to 60 °C) and pH (3 to 11) conditions. A challenge assay showed that PKO111 and PKP126 inhibit growth of their host strains by 2 log and 4 log, respectively. Complete genome sequencing of the phages revealed that their genome sizes are quite different (168,758 bp for PKO111 and 50,934 bp for PKP126). Their genome annotation results showed that they have no human virulence-related genes, an important safety consideration. In addition, no lysogen-formation gene cluster was detected in either phage genome, suggesting that they are both virulent phages in their bacterial hosts. Based on these results, PKO111 and PKP126 may be good candidates for development of biocontrol agents against members of the genus Klebsiella for therapeutic purposes. A comparative analysis of tail-associated gene clusters of PKO111 and PKP126 revealed relatively low homology, suggesting that they might differ in the way they recognize and infect their specific hosts.

Methanocaldococcus bathoardescens sp. nov., a hyperthermophilic methanogen isolated from a volcanically active deep-sea hydrothermal vent.

A hyperthermophilic methanogen, strain JH146(T), was isolated from 26 °C hydrothermal vent fluid emanating from a crack in basaltic rock at Marker 113 vent, Axial Seamount in the northeastern Pacific Ocean. It was identified as an obligate anaerobe that uses only H2 and CO2 for growth. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain is more than 97% similar to other species of the genus Methanocaldococcus . Therefore, overall genome relatedness index analyses were performed to establish that strain JH146(T) represents a novel species. For each analysis, strain JH146(T) was most similar to Methanocaldococcus sp. FS406-22, which can fix N2 and also comes from Marker 113 vent. However, strain JH146(T) differs from strain FS406-22 in that it cannot fix N2. The average nucleotide identity score for strain JH146(T) was 87%, the genome-to-genome direct comparison score was 33-55% and the species identification score was 93%. For each analysis, strain JH146(T) was below the species delineation cut-off. Full-genome gene synteny analysis showed that strain JH146(T) and strain FS406-22 have 97% genome synteny, but strain JH146(T) was missing the operons necessary for N2 fixation and assimilatory nitrate reduction that are present in strain FS406-22. Based on its whole genome sequence, strain JH146(T) is suggested to represent a novel species of the genus Methanocaldococcus for which the name Methanocaldococcus bathoardescens is proposed. The type strain is JH146(T) ( = DSM 27223(T) = KACC 18232(T)).

In vivo Trial of Bifidobacterium longum Revealed the Complex Network Correlations Between Gut Microbiota and Health Promotional Effects.

Complete genome sequence analysis of Bifidobacterium longum subsp. longum BCBL-583 isolated from a Korean female fecal sample showed no virulence factor or antibiotic resistance gene, suggesting human safety. In addition, this strain has oxygen and heat tolerance genes for food processing, and cholesterol reduction and mucin adhesion-related genes were also found. For in vivo evaluations, a high fat diet (HFD) mouse model was used, showing that BCBL-583 administration to the model (HFD-583) reduced the total cholesterol and LDL-cholesterol in the blood and decreased pro-inflammatory cytokines but increased anti-inflammatory cytokines, substantiating its cholesterol reduction and anti-inflammation activities. Subsequent microbiome analysis of the fecal samples from the HFD mouse model revealed that BCBL-583 administration changed the composition of gut microbiota. After 9 weeks feeding of bifidobacteria, Firmicutes, Actinobacteria, and Bacteroidetes increased, but Proteobacteria maintained in the HFD mouse models. Further comparative species-level compositional analysis revealed the inhibitions of cholesterol reduction-related Eubacterium coprostanoligenes and obesity-related Lactococcus by the supplementation of B. longum BCBL-583, suggesting its possible cholesterol reduction and anti-obesity activities. The correlation analysis of HFD-583 between the gut microbiota compositional change and cholesterol/immune response showed that Verrucomicrobia, Firmicutes, Actinobacteria, and Bacteroidetes may play an important role in cholesterol reduction and anti-inflammation. However, correlation analysis of Proteobacteria showed the reverse correlation in HFD-583. Interestingly, the correlation analysis of B. longum ATCC 15707 administration to HFD model showed similar patterns of cholesterol but different in immune response patterns. Therefore, this correlation analysis suggests that the microbial composition and inflammatory cytokine/total-cholesterol may be closely related in the administration of BCBL-583 in the HFD mice group. Consequently, BCBL-583 could be a good probiotic strain for gut health promotion through gut microbiota modulation.

Microbiome Study of Initial Gut Microbiota from Newborn Infants to Children Reveals that Diet Determines Its Compositional Development.

To understand the formation of initial gut microbiota, three initial fecal samples were collected from two groups of two breast milk-fed (BM1) and seven formula milk-fed (FM1) infants, and the compositional changes in gut microbiota were determined using metagenomics. Compositional change analysis during week one showed that Bifidobacterium increased from the first to the third fecal samples in the BM1 group (1.3% to 35.1%), while Klebsiella and Serratia were detected in the third fecal sample of the FM1 group (4.4% and 34.2%, respectively), suggesting the beneficial effect of breast milk intake. To further understand the compositional changes during progression from infancy to childhood (i.e., from three weeks to five years of age), additional fecal samples were collected from four groups of two breast milk-fed infants (BM2), one formula milk-fed toddler (FM2), three weaning food-fed toddlers (WF), and three solid food-fed children (SF). Subsequent compositional change analysis and principal coordinates analysis (PCoA) revealed that the composition of the gut microbiota changed from an infant-like composition to an adult-like one in conjunction with dietary changes. Interestingly, overall gut microbiota composition analyses during the period of progression from infancy to childhood suggested increasing complexity of gut microbiota as well as emergence of a new species of bacteria capable of digesting complex carbohydrates in WF and SF groups, substantiating that diet type is a key factor in determining the composition of gut microbiota. Consequently, this study may be useful as a guide to understanding the development of initial gut microbiota based on diet.

Cancer-protective effect of a synbiotic combination between Lactobacillus gasseri 505 and a Cudrania tricuspidata leaf extract on colitis-associated colorectal cancer.

Previously, a synbiotic combination of probiotic Lactobacillus gasseri 505 (LG) and a new prebiotic, Cudrania tricuspidata leaf extract (CT) in fermented milk, designated FCT, showed an in vitro immunomodulatory effect and antioxidant activity. Although synbiotic combination might have cancer-protective effects, these activities have not been fully validated in vivo. Ten-week treatment of LG, CT, or FCT to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated colorectal cancer (CAC) mouse model reduced both the incidence of colonic tumors and damage to the colonic mucosa effectively, suggesting a cancer-protective effect. To understand these, biomarkers associated with inflammation, colon barrier, apoptosis, and cancer cell proliferation were monitored in AOM/DSS group versus LG/CT/FCT groups. A synbiotic combination (FCT) down-regulated pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1ß, and IL-6) and inflammation-associated enzymes (iNOS and COX-2), and up-regulated anti-inflammatory cytokines (IL-4 and IL-10). In addition, colon barrier experiment revealed that biomarkers of mucus layer (MUC-2 and TFF3) and tight junction (occludin and ZO-1) were up-regulated. Subsequent apoptosis experiment showed that pro-apoptotic factors (p53, p21, and Bax) were up-regulated and anti-apoptotic factors (Bcl-2 and Bcl-xL) were down-regulated. Furthermore, comparative metagenome analysis of gut microbiota revealed that Staphylococcus decreased but Lactobacillus, Bifidobacterium, and Akkermansia increased, supporting their protective effects, accompanied by increased short-chain fatty acids (SCFAs). Taken together, the FCT administration showed cancer-protective effects by reducing the risk of colitis-associated colon cancer via regulation of inflammation, carcinogenesis, and compositional change of gut microbiota. Consequently, the synbiotic combination (FCT) could be a novel potential health-protective natural agent against CAC.

Effects of PrObiotics on the Symptoms and Surgical ouTComes after Anterior REsection of Colon Cancer (POSTCARE): A Randomized, Double-Blind, Placebo-Controlled Trial.

We investigated microbiota changes following surgical colon cancer resection and evaluate effects of probiotics on microbiota and surgical recovery. This randomized double-blind trial was performed at four medical centers in South Korea. Of 68 patients expected to undergo anterior sigmoid colon cancer resection, 60 were eligible, of whom 29 and 31 received probiotics and placebo, respectively, for four weeks, starting at one week preoperatively. Third- and/or fourth-week information on anterior resection syndrome (ARS), inflammatory markers, and quality of life was obtained. Stool sample analysis was conducted after randomization and bowel preparation and at three and four postoperative weeks. Bacteria were categorized into Set I (with probiotic effects) and II (colon cancer-associated). The probiotic group's ARS score showed an improving trend (p = 0.063), particularly for flatus control (p = 0.030). Serum zonulin levels significantly decreased with probiotics. Probiotic ingestion resulted in compositional changes in gut microbiota; greater increases and decreases in Set I and II bacteria, respectively, occurred with probiotics. Compositional increase in Set I bacteria was associated with reduced white blood cells, neutrophils, neutrophil-lymphocyte ratio, and zonulin. Bifidobacterium composition was negatively correlated with zonulin levels in the probiotic group. Probiotics improved postoperative flatus control and modified postoperative changes in microbiota and inflammatory markers.

Role of Probiotics in Human Gut Microbiome-Associated Diseases.

Probiotics, including bacteria and yeast, are live microorganisms that have demonstrated beneficial effects on human health. Recently, probiotic bacteria are constantly being studied and their applications are also being considered in promising adjuvant treatments for various intestinal diseases. Clinical trials and in vivo experiments have extended our current understanding of the important roles that probiotics play in human gut microbiomeassociated diseases. It has been documented through many clinical trials that probiotics could shape the intestinal microbiota leading to potential control of multiple bowel diseases and promotion of overall wellness. In this review, we focused on the relationship between probiotics and the human gut microbiota and its roles in gut microbiome-associated diseases. Here, we also discuss future directions and research areas that need further elucidation in order to better understand the roles of probiotics in the treatment of intestinal diseases.

Isolation and Characterization of Bifidobacterium longum subsp. longum BCBL-583 for Probiotic Applications in Fermented Foods

Recent human gut microbiome studies have supported that the genus Bifidobacterium is one of the most beneficial bacteria for human intestinal health. To develop a new probiotic strain for functional food applications, fourteen fecal samples were collected from healthy Koreans and the strain BCBL-583 was newly selected and isolated from a 25-year-old Korean woman's fecal sample using the selective medium for Bifidobacterium. Subsequent fructose-6-phosphate phosphoketolase (F6PPK) test and 16S rRNA gene sequencing analysis of the strain BCBL-583 confirmed that it belongs to B. longum subsp. longum. The stress resistance tests showed that it has oxygen and heat tolerance activities (5- and 3.9-fold increase for 24 h at 60 and 120 rpm, respectively; 78.61 ± 6.67% survival rate at 45°C for 24 h). In addition, gut environment adaptation tests revealed that this strain may be well-adapted in the gut habitat, with gastric acid/bile salt resistance (85.79 ± 1.53%, survival rate under 6 h treatments of gastric acid and bile salt) and mucin adhesion (73.72 ± 7.36%). Furthermore, additional tests including cholesterol lowering assay showed that it can reduce 86.31 ± 1.85% of cholesterol. Based on these results, B. longum BCBL-583 has various stress resistance for survival during food processing and environmental adaptation activities for dominant survival in the gut, suggesting that it could be a good candidate for fermented food applications as a new probiotic strain.

Metagenomic Approach to Identifying Foodborne Pathogens on Chinese Cabbage.

Foodborne illness represents a major threat to public health and is frequently attributed to pathogenic microorganisms on fresh produce. Recurrent outbreaks often come from vegetables that are grown close to or within the ground. Therefore, the first step to understanding the public health risk of microorganisms on fresh vegetables is to identify and describe microbial communities. We investigated the phyllospheres on Chinese cabbage (Brassica rapa subsp. pekinensis, N = 54). 16S rRNA gene amplicon sequencing targeting the V5-V6 region of 16S rRNA genes was conducted by employing the Illumina MiSeq system. Sequence quality was assessed, and phylogenetic assessments were performed using the RDP classifier implemented in QIIME with a bootstrap cutoff of 80%. Principal coordinate analysis was performed using a weighted Fast UniFrac matrix. The average number of sequence reads generated per sample was 34,584. At the phylum level, bacterial communities were composed primarily of Proteobacteria and Bacteroidetes. The most abundant genera on Chinese cabbages were Chryseobacterium, Aurantimonadaceae_g, Sphingomonas, and Pseudomonas. Diverse potential pathogens, such as Pantoea, Erwinia, Klebsiella, Yersinia, Bacillus, Staphylococcus, Salmonella, and Clostridium were also detected from the samples. Although further epidemiological studies will be required to determine whether the detected potential pathogens are associated with foodborne illness, our results imply that a metagenomic approach can be used to detect pathogenic bacteria on fresh vegetables.

Analysis of Microbiota in Bellflower Root, Platycodon grandiflorum, Obtained from South Korea.

Bellflower root (Platycodon grandiflorum), which belongs to the Campanulaceae family, is a perennial grass that grows naturally in Korea, northeastern China, and Japan. Bellflower is widely consumed as both food and medicine owing to its high nutritional value and potential therapeutic effects. Since foodborne disease outbreaks often come from vegetables, understanding the public health risk of microorganisms on fresh vegetables is pivotal to predict and prevent foodborne disease outbreaks. We investigated the microbial communities on the bellflower root (n = 10). 16S rRNA gene amplicon sequencing targeting the V6-V9 regions of 16S rRNA genes was conducted via the 454-Titanium platform. The sequence quality was checked and phylogenetic assessments were performed using the RDP classifier implemented in QIIME with a bootstrap cutoff of 80%. Principal coordinate analysis was performed using the weighted Fast UniFrac distance. The average number of sequence reads generated per sample was 67,192 sequences. At the phylum level, bacterial communities from the bellflower root were composed primarily of Proteobacteria, Firmicutes, and Actinobacteria in March and September samples. Genera Serratia, Pseudomonas, and Pantoea comprised more than 54% of the total bellflower root bacteria. Principal coordinate analysis plots demonstrated that the microbial community of bellflower root in March samples was different from those in September samples. Potential pathogenic genera, such as Pantoea, were detected in bellflower root samples. Even though further studies will be required to determine if these species are associated with foodborne illness, our results indicate that the 16S rRNA gene-based sequencing approach can be used to detect pathogenic bacteria on fresh vegetables.

Genomic Insights of Weissella jogaejeotgali FOL01T Reveals Its Food Fermentation Ability and Human Gut Adaptive Potential for Probiotic Applications in Food Industries.

Although the genus Leuconostoc, generally found in various fermented foods, has often been suggested to be a novel probiotic for food fermentation and health promotion, the strains in this genus showed low acid tolerance and low osmotic stress resistance activities, which are required for survival during food fermentation events. Recently, a novel species of Weissella, W. jogaejeotgali FOL01T (= KCCM 43128 = JCM 30580), was isolated from Korean fermented clams. To determine the genomic features of this new species, its genome was completely sequenced and analyzed. The genome consists of a circular chromosome of 2,114,163 bp of DNA with a G+C content of 38.8%, and the plasmid pFOL01 consists of 35,382 bp of DNA with a G+C content of 39.1%. The genome analysis showed its potential for use in food fermentation and osmotic stress resistance abilities for processing in food industries. In addition, this strain was predicted to have acid tolerance and adhesion to the mucosal layer for survival and colonization in the gut. Subsequent experiments substantiated these abilities, suggesting that W. jogaejeotgali may have probiotic potential and a high survival rate during food fermentation. Therefore, it may be suitable as a novel probiotic strain for various applications in food industries.

The complete genome sequence of Cronobacter sakazakii ATCC 29544T, a food-borne pathogen, isolated from a child's throat.

BACKGROUND: Cronobacter sakazakii is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of severe diseases: meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. However, the pathogenesis mechanism of this pathogen remains largely unknown. To determine its pathogenesis at the genomic level, the genome of C. sakazakii ATCC 29544T was completely sequenced and analyzed. RESULTS: The genomic DNA, containing a circular chromosome and three plasmids, is composed of 4,511,265 bp with a GC content of 56.71%, containing 4380 predicted open reading frames (ORFs), 22 rRNA genes, and 83 tRNA genes. The plasmids, designated pCSK29544_p1, pCSK29544_p2, and pCSK29544_p3, were 93,905-bp, 4938-bp, and 53,457-bp with GC contents of 57.02, 54.88, and 50.07%, respectively. They were also predicted to have 72, 6, and 57 ORFs without RNA genes. CONCLUSIONS: The strain ATCC 29544T genome has ompA and ibeB-homologous cusC genes, probably associated with the invasion of human brain microvascular endothelial cells (BMECs). In addition, gene clusters for siderophore production (iucABCD/iutA) and the related transport system (eitCBAD) were detected in pCSK29544_p1 plasmid, indicating better iron uptake ability for survival. Furthermore, to survive under extremely dry condition like milk powder, this genome has gene clusters for biosynthesis of capsular proteins (CSK29544_00281-00284) and cellulose (CSK29544_01124-01127) for biofilm formation and a gene cluster for utilization of sialic acid in the milk (nanKTAR). The genome information of C. sakazakii ATCC 29544T would provide further understanding of its pathogenesis at the molecular level for the regulation of pathogenicity and the development of a rapid detection method using biomarkers.

Genomic Insights and Its Comparative Analysis with Yersinia enterocolitica Reveals the Potential Virulence Determinants and Further Pathogenicity for Foodborne Outbreaks.

Yersinia enterocolitica is a well-known foodborne pathogen causing gastrointestinal infections worldwide. The strain Y. enterocolitica FORC_002 was isolated from the gill of flatfish (plaice) and its genome was sequenced. The genomic DNA consists of 4,837,317 bp with a GC content of 47.1%, and is predicted to contain 4,221 open reading frames, 81 tRNA genes, and 26 rRNA genes. Interestingly, genomic analysis revealed pathogenesis and host immune evasion-associated genes encoding guanylate cyclase (Yst), invasin (Ail and Inv), outer membrane protein (Yops), autotransporter adhesin A (YadA), RTX-like toxins, and a type III secretion system. In particular, guanylate cyclase is a heat-stable enterotoxin causing Yersinia-associated diarrhea, and RTX-like toxins are responsible for attachment to integrin on the target cell for cytotoxic action. This genome can be used to identify virulence factors that can be applied for the development of novel biomarkers for the rapid detection of this pathogen in foods.

Biocontrol and Rapid Detection of Food-Borne Pathogens Using Bacteriophages and Endolysins.

Bacteriophages have been suggested as natural food preservatives as well as rapid detection materials for food-borne pathogens in various foods. Since Listeria monocytogenes-targeting phage cocktail (ListShield) was approved for applications in foods, numerous phages have been screened and experimentally characterized for phage applications in foods. A single phage and phage cocktail treatments to various foods contaminated with food-borne pathogens including E. coli O157:H7, Salmonella enterica, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Cronobacter sakazakii, and Vibrio spp. revealed that they have great potential to control various food-borne pathogens and may be alternative for conventional food preservatives. In addition, phage-derived endolysins with high host specificity and host lysis activities may be preferred to food applications rather than phages. For rapid detection of food-borne pathogens, cell-wall binding domains (CBDs) from endolysins have been suggested due to their high host-specific binding. Fluorescence-tagged CBDs have been successfully evaluated and suggested to be alternative materials of expensive antibodies for various detection applications. Most recently, reporter phage systems have been developed and tested to confirm their usability and accuracy for specific detection. These systems revealed some advantages like rapid detection of only viable pathogenic cells without interference by food components in a very short reaction time, suggesting that these systems may be suitable for monitoring of pathogens in foods. Consequently, phage is the next-generation biocontrol agent as well as rapid detection tool to confirm and even identify the food-borne pathogens present in various foods.

Characterization and Genomic Study of the Novel Bacteriophage HY01 Infecting Both Escherichia coli O157:H7 and Shigella flexneri: Potential as a Biocontrol Agent in Food.

BACKGROUND: Escherichia coli O157:H7 and Shigella flexneri are well-known food-borne pathogens causing severe food poisoning at low infectious doses. Bacteriophages have been approved for food applications by the US Food and Drug Administration (FDA) and have been suggested as natural food preservatives to control specific food-borne pathogens. To develop a novel natural food preservative against E. coli O157:H7 and S. flexneri, a new bacteriophage needs to be isolated and characterized. METHODOLOGY/PRINCIPAL FINDINGS: Bacteriophage HY01 infecting both E. coli O157:H7 and S. flexneri was isolated from a swine fecal sample. HY01 belongs to the family Myoviridae and is stable under various temperature and pH conditions. One-step growth curve analysis showed relatively short eclipse and latent periods as well as large burst size. The 167-kb genome sequence of HY01 was sequenced, and a comparative genome analysis with T4 for non-O157:H7 E. coli suggests that the receptor recognition protein of HY01 plays an important role in determination of host recognition and specificity. In addition, food applications using edible cabbage were conducted with two E. coli O157:H7 strains (ATCC 43890 and ATCC 43895), showing that treatment with HY01 inhibits these clinical and food isolates with >2 log reductions in bacterial load during the first 2 h of incubation. CONCLUSIONS/SIGNIFICANCE: HY01 can inhibit both E. coli O157:H7 and S. flexneri with large burst size and stability under stress conditions. The ability of HY01 to infect both E. coli O157:H7 and S. flexneri may be derived from the presence of two different host specificity-associated tail genes in the genome. Food applications revealed the specific ability of HY01 to inhibit both pathogens in food, suggesting its potential as a novel biocontrol agent or novel natural food preservative against E. coli O157:H7 and potentially S. flexneri.

Complete genome sequence of Vibrio parahaemolyticus strain FORC_008, a foodborne pathogen from a flounder fish in South Korea.

Vibrio parahaemolyticus is a Gram-negative, motile, nonspore-forming pathogen that causes foodborne illness associated with the consumption of contaminated seafoods. Although many cases of foodborne outbreaks caused by V. parahaemolyticus have been reported, the genomes of only five strains have been completely sequenced and analyzed using bioinformatics. In order to characterize overall virulence factors and pathogenesis of V. parahaemolyticus associated with foodborne outbreak in South Korea, a new strain FORC_008 was isolated from flounder fish and its genome was completely sequenced. The genomic analysis revealed that the genome of FORC_008 consists of two circular DNA chromosomes of 3266 132 bp (chromosome I) and 1772 036 bp (chromosome II) with a GC content of 45.36% and 45.53%, respectively. The entire genome contains 4494 predicted open reading frames, 129 tRNAs and 31 rRNA genes. While the strain FORC_008 does not have genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), its genome encodes many other virulence factors including hemolysins, pathogenesis-associated secretion systems and iron acquisition systems, suggesting that it may be a potential pathogen. This report provides an extended understanding on V. parahaemolyticus in genomic level and would be helpful for rapid detection, epidemiological investigation and prevention of foodborne outbreak in South Korea.

Complete genome sequence of Vibrio vulnificus FORC_017 isolated from a patient with a hemorrhagic rash after consuming raw dotted gizzard shad.

BACKGROUND: Vibrio vulnificus, a resident in the human gut, is frequently found in seafood, causing food-borne illnesses including gastroenteritis and severe septicemia. While V. vulnificus has been known to be one of the major food-borne pathogens, pathogenicity and virulence factors are not fully understood yet. To extend our understanding of the pathogenesis of V. vulnificus at the genomic level, the genome of V. vulnificus FORC_017 isolated from a female patient experiencing a hemorrhagic rash was completely sequenced and analyzed. RESULTS: Three discontinuous contigs were generated from a hybrid assembly using Illumina MiSeq and PacBio platforms, revealing that the genome of the FORC_017 consists of two circular chromosomes and a plasmid. Chromosome I consists of 3,253,417-bp (GC content 46.49 %) containing 2943 predicted open reading frames (ORFs) and chromosome II of 1,905,745-bp (GC content 46.90 %) containing 1638 ORFs. The plasmid pFORC17 consists of 70,069-bp (GC content 43.77 %) containing 84 ORFs. The average nucleotide identity (ANI) value of the FORC_017 and CMCP6 strains was 98.53, suggesting that they are closely related. CONCLUSIONS: Pathogenesis-associated genes including vvhA, rtx gene cluster, and various hemolysin genes were present in FORC_017. In addition, three complete secretion systems (Type I, II and VI) as well as iron uptake-related genes for virulence of the FORC_017 were detected, suggesting that this strain is pathogenic. Further comparative genome analysis revealed that FORC_017 and CMCP6 share major toxin genes including vvhA and rtx for pathogenesis activities. The genome information of the FORC_017 provides novel insights into pathogenicity and virulence factors of V. vulnificus.