Microbial dynamics of South Korean beef and surroundings along the supply chain based on high-throughput sequencing.

Microbiological safety and quality of beef is crucial as beef can serve as a reservoir for a variety of bacteria, including spoilage-related and foodborne pathogens. Controlling microbial contamination is a critical aspect of food quality and safety, but it is difficult to prevent as there are several potential sources of contamination from production to distribution. In this study, the microbiological ecology of cattle/beef and associated environmental samples (n = 69) were trace-investigated to reveal microbiome shifts in cattle/beef and possible cross-contaminants throughout the entire supply chain using 16S rRNA gene sequencing. Pseudomonas, Psychrobacter, and Acinetobacter, known as spoilage bacteria, opportunistic pathogens, or antibiotic-resistant bacteria, were the main microorganisms present in cattle/beef, and Staphylococcus became abundant in the final products. The dominance of Acinetobacter and Pseudomonas was noticeable in the slaughtered carcasses and slaughterhouse environment, indicating that the slaughterhouse is a critical site where hygienic practices are required to prevent further contamination. Taxonomic similarities between cattle/beef and several environmental samples, as well as diversity analysis, presented a high potential for microbial transmission. Source tracking identified environmental samples that primarily contributed to the microbiota of cattle/beef. Farm floor (48%), workers' gloves (73%), and carcass splitters (20%) in the slaughterhouse were found to be major sources influencing the microbiome of cattle/beef at the farm, slaughterhouse, and processing plant, respectively. These findings demonstrated the dynamics of bacterial communities in cattle/beef according to stage and detected potential contamination sources, which may aid in a better understanding and control of microbial transmission in beef production.

Bacterial profile of pork from production to retail based on high-throughput sequencing.

Pork is a common vehicle for foodborne pathogens, including Salmonella spp. and Yersinia enterocolitica. Cross-contamination can occur at any stage of the pork production chain, from farm to market. In the present study, high-throughput sequencing was used to characterize bacterial profiles and track their changes along the whole supply chain. Tracked meat samples (pig on the farm, carcass in the slaughterhouse, unprocessed carcass and processed meat in the processing plant, and fresh pork at the local retail stores) and their associated environmental samples (e.g., water, floor, feed, feces, and workers' gloves) were collected from sequential stages (n = 96) and subjected to 16S rRNA metataxonomic analyses. At the farm, a total of 652 genera and 146 exclusive genera were identified in animal and environmental samples (pig, drain, floor, fan, and feces). Based on beta diversity analysis, it was demonstrated that the microbial composition of animal samples collected at the same processing step is similar to that of environmental samples (e.g., drain, fan, feces, feed, floor, gloves, knives, tables, and water). All animal and environmental samples from the slaughterhouse were dominated by Acinetobacter (55.37 %). At the processing plant, belly meat and neck meat samples were dominated by Psychrobacter (55.49 %). At the retail level, key bacterial players, which are potential problematic bacteria and important members with a high relative abundance in the samples, included Acinetobacter (8.13 %), Pseudomonas (6.27 %), and Staphylococcus (2.13 %). In addition, the number of confirmed genera varied by more than twice that identified in the processing plant. Source tracking was performed to identify bacterial contamination routes in pork processing. Animal samples, including the processing plant's carcass, the pig from the farm, and the unwashed carcass from the slaughterhouse (77.45 %), along with the processing plant's gloves (5.71 %), were the primary bacterial sources in the final product. The present study provides in-depth knowledge about the bacterial players and contamination points within the pork production chain. Effective control measures are needed to control pathogens and major pollutants at each stage of pork production to improve food safety.

Microbial trace investigation throughout the entire chicken supply chain based on metagenomic high-throughput sequencing.

As poultry possesses a high risk of contamination by various pathogens and has repeatedly been linked to foodborne outbreaks, ensuring microbiological safety throughout the chicken production chain is essential. In this study, bacterial communities in chickens and associated environments (n = 72), including feces, floors, gloves, and worktables, were trace investigated from the broiler farm, slaughterhouse, meat processing plant, and the market by amplicon sequencing of the V4 region of the 16S rRNA. The bacterial composition in live chickens along the production chain significantly changed across the stages, with distinct microbiota noted at each step. Pseudomonas, Shewanella, Acinetobacter, and Psychrobacter were dominant in the final products. Staphylococcus was abundant in live birds originally (36.83 %) but dramatically decreased after slaughter (3.07 %, 0.06 %, and 0.42 % in slaughtered, processed, and market carcasses, respectively), which may be attributed to defeathering. The proportion of Enterobacteriaceae, Acinetobacter, and Pseudomonas increased from 0.95 %, 0.03 %, and 0.04 % before slaughter to 13.57 %, 34.19 %, and 21.90 %, respectively, after slaughter, highlighting the importance of hygiene management in the succeeding steps. Diversity analysis revealed the possibility of bacterial transmission between samples from the processing plant and the market. Source tracking was performed to identify microbial contamination routes in the chicken microbiome; the major bacterial sources in the final products were the samples from the processing plant (such as processed carcasses, gloves, and worktables), accounting for 93.53 % of the total microbial sources. These results suggest that in-depth knowledge of microbial transmission between chickens and their surroundings can facilitate a precise understanding of microbiological concerns across the poultry production system and help establish safety management measures for the poultry industry.

Microbial investigation of aquacultured olive flounder (Paralichthys olivaceus) from farm to table based on high-throughput sequencing.

The microbial ecologies of fish, such as the olive flounder (Paralichthys olivaceus), one of the most widely consumed fish in East Asia, remain to be elucidated. The microbiome of olive flounder and related environmental samples (i.e., feed, water, workers' aprons and gloves) were collected from six different sources (i.e., a fish farm, a transporting truck, a Wando market and restaurant, and a Seoul market and restaurant). These samples (n = 102) were investigated at various farm-to-distribution stages based on their 16S rRNA sequences. The microbial communities of fish from the farms and trucks were dominated by Photobacterium (>86 %) and showed distinct differences from fish from the Wando and Seoul markets and restaurants. There was also a significant difference in fish microbiomes according to geographical location. The relative abundances of Shewanella, Acinetobacter, Enterobacteriaceae, and Pseudomonas increased as the distribution and consumption stages of the supply chain advanced. The percentages of Shewanella (24.74 %), Acinetobacter (18.32 %), and Enterobacteriaceae (11.24 %) in Wando, and Pseudomonas (42.98 %) in Seoul markets and restaurants implied the importance of sanitation control in these areas. Alpha and beta diversity results corresponded to taxonomic analyses and showed the division of two groups (i.e., fish from the production and transporting stage (farm and truck fish) and fish from the distribution and consumption stages (market and restaurant fish)). The present study provides an in-depth understanding of olive flounder and its environmental microbiomes and suggests control measures to improve food safety.

Identification and phylogenetic analysis of Enterococcus isolates using MALDI-TOF MS and VITEK 2.

The bacterial genus Enterococcus encompasses 38 species. Two of the most common species are E. faecalis and E. faecium. Recently, however, there has been an increase in clinical reports concerning less prevalent Enterococcus species, such as E. durans, E. hirae, and E. gallinarum. Rapid and accurate laboratory methods are needed to facilitate the identification of all these bacterial species. In the present study, we compared the relative accuracy of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), VITEK 2, and 16S rRNA gene sequencing using 39 enterococci isolates from dairy samples, and compared the resultant phylogenetic trees. We found that MALDI-TOF MS correctly identified all isolates at the species level except for one, whereas the VITEK 2 system, which is an automated identification system using biochemical characteristics of species, misidentified ten isolates. However, phylogenetic trees constructed from both methods showed all isolates in similar positions. Our results clearly showed that MALDI-TOF MS is a reliable and rapid tool for identifying Enterococcus species with greater discriminatory power than the biochemical assay method of VITEK 2.

A Combined In Vitro and In Vivo Assessment of the Safety of the Yeast Strains Kluyveromyces marxianus A4 and A5 Isolated from Korean Kefir.

Kefir is a traditional fermented milk containing beneficial bacteria and yeasts. Despite Kluyveromyces marxianus, isolated from kefir, gaining increasing attention as a potential probiotic yeast owing to its biological function, Saccharomyces boulardii is the only species considered as a probiotic yeast. We evaluated the safety of K. marxianus strains A4 and A5, isolated from Korean kefir, in comparison with that of S. boulardii. Virulence attributes were preliminarily assessed in vitro including their ability of gelatin hydrolysis, pseudohyphae formation, and hemolysis. To evaluate in vivo safety, the strains were challenged in a healthy animal model, four-week-old female BALB/c mice. Mice were orally administered 0.2 mL of 0.9% sterilized saline (NC_S; n = 6), S. boulardii ATCC MYA-796 (high concentration, S.b_H; low concentration, S.b_L; n = 6 for each), K. marxianus A4 (high concentration, A4_H; low concentration, A4_L; n = 6 for each), or K. marxianus A5 (high concentration, A5_H; low concentration, A5_L; n = 6 for each) for 2 weeks. At study end, body weight, spleen and liver weights, and blood parameters were assessed. K. marxianus A4 and A5 were tested negative for gelatinase and hemolysis. Overall, hematological, plasma biochemical, and cytokine (interleukin-1ß and tumor necrosis factor-α) parameters were comparable between the experimental and negative control (NC) groups. Notably, the interleukin-6 level of the A5_H group was significantly lower than that of the NC group (p < 0.05), suggesting anti-inflammatory potential of K. marxianus A5.

Red seaweed extracts reduce methane production by altering rumen fermentation and microbial composition in vitro.

A series of in vitro batch culture incubations were carried out to investigate changes in rumen fermentation characteristics, methane (CH4) production, and microbial composition in response to supplementation with five different red seaweed species (Amphiroa anceps, AANC; Asparagopsis taxiformis, ATAX; Chondracanthus tenellus, CTEN; Grateloupia elliptica, GELL; and Gracilaria parvispora, GPAR). Prior to the incubations, the total flavonoid and polyphenol content of the red seaweed extracts was quantified. The incubated substrate consisted of timothy hay and corn grain [60:40 dry matter (DM) basis]. Treatments were substrate mixtures without seaweed extract (CON) or substrate mixtures supplemented with 0.25 mg/mL of red seaweed extract. Samples were incubated for 6, 12, 24, 36, and 48 h. Each sample was incubated in triplicates in three separate runs. In vitro DM degradability, fermentation parameters (i.e., pH, volatile fatty acids, and ammonia nitrogen), total gas production, and CH4 production were analyzed for all time points. Microbial composition was analyzed using 16S rRNA amplicon sequencing after 24 h of incubation. The highest CH4 reduction (mL/g DM, mL/g digested DM, and % of total gas production) was observed in ATAX (51.3, 50.1, and 51.5%, respectively, compared to CON; P < 0.001) after 12 h of incubation. The other red seaweed extracts reduced the CH4 production (mL/g DM; P < 0.001) in the range of 4.6-35.0% compared to CON after 24 h of incubation. After 24 h of incubation, supplementation with red seaweed extracts tended to increase the molar proportion of propionate (P = 0.057) and decreased the acetate to propionate ratio (P = 0.033) compared to the CON. Abundances of the genus Methanobrevibacter and total methanogens were reduced (P = 0.050 and P = 0.016) by red seaweed extract supplementation. The linear discriminant analysis effect size (P < 0.05, LDA ≥ 2.0) showed that UG Succinivibrionaceae, Anaeroplasma, and UG Ruminococcaceae, which are associated with higher propionate production, starch degradation, and amylase activity were relatively more abundant in red seaweed extracts than in the CON. Our results suggest that supplementation with red seaweed extracts altered the microbiota, leading to the acceleration of propionate production and reduction in CH4 production.

Distribution and Characterization of Antimicrobial Resistant Pathogens in a Pig Farm, Slaughterhouse, Meat Processing Plant, and in Retail Stores.

The emergence of antibiotic resistance in foodborne pathogens isolated from meat pro-ducts and their producing environment has been an increasing and leading threat to public health. The aim of the study was to identify pathogens and their antimicrobial resistance isolated from pig production to pork meat distribution phases. Through this study, food spoilage and foodborne or clinical pathogenic bacteria were isolated and identified from pork (belly and neck) meat product and its related environmental samples that include pig swabs, diets, feces, liquid manure, workers' gloves, dust fan swabs, carcass swabs, floor swabs, and drain water in the affiliated farm, slaughterhouse, meat processing plant, and in retail stores. All carcasses at the slaughterhouse and meat products at the meat processing plant were tracked from pigs at a targeted farm. Nine different selective media agars were used to effectively isolate various pathogenic bacteria. A total of 283 presumptive pathogenic bacteria isolated from 126 samples were selected and identified using MALDI-ToF MS. Twenty-three important foodborne pathogens were identified, and some of them, Shiga-toxin-producing E. coli (STEC), Listeria monocytogenes, Staphylococcus aureus, and Yersinia enterocolitica, were further confirmed using PCR. The PFGE patterns of 12 STEC isolates were grouped by sample source or site. All the foodborne pathogens used in the study were not resistant to amoxicillin/clavulanate, ciprofloxacin, and gentamicin, whereas some of the STEC, L. monocytogenes, and S. aureus isolates were resistant to various antibiotics, including ampicillin, erythromycin, tetracycline, and vancomycin. The most common antimicrobial resistance pattern in the pathogenic STEC isolates was AMP-KAN-STR-SXT-TET. Consequently, this study provides valuable information for the distribution of antimicrobial-resistant pathogens along the pork meat production chain and can assist farmers and stakeholders to develop a systematic strategy for reducing the current emergence and spread of antimicrobial resistance in the different phases of pig production and distribution.

Inactivation of Airborne Avian Pathogenic E. coli (APEC) via Application of a Novel High-Pressure Spraying System.

Infectious diseases of livestock caused by novel pathogenic viruses and bacteria are a major threat to global animal health and welfare and their effective control is crucial for agronomic health and for securing global food supply. It has been widely recognized that the transmission of infectious agents can occur between people and/or animals in indoor spaces. Therefore, infection control practices are critical to reduce the transmission of the airborne pathogens. ViKiller®-high-pressure sprayer and Deger®-disinfectant are newly developed spraying systems that can produce an optimal size of disinfectants to reduce airborne microbes. The system was evaluated to reduce the infection caused by avian pathogenic Escherichia coli (APEC), an airborne bacterium which survives in indoor spaces. pH-neutral electrolyzed water (NEW) containing 100 ppm of free chlorine, laboratory-scale chambers, a recently developed sprayer, and a conventional sprayer were used in the study. A total of 123 day-of-hatch male layer chicks (Hy-Line W-36) were randomly classified into five groups (negative control (NC): no treatment; treatment 1 (Trt 1): spraying only NEW without APEC; treatment 2 (Trt 2): spraying NEW + APEC using a high-pressure sprayer; treatment 3 (Trt 3): spraying NEW + APEC using a conventional sprayer; positive control (PC): spraying only APEC). Experimental chicks in the chambers were daily exposed to 50 mL of NEW and/or APEC (1.0 × 106 cfu/mL) until the end of the experiment (day 35). APEC strains were sprayed by ViKiller®. At least four chicks in each group were evaluated weekly to monitor APEC infection and determine the lesion. Data showed that our spraying system significantly reduced airborne APEC concentrations, mortality rate, respiratory infection, and APEC lesions in birds in the chamber space (p < 0.05). The results demonstrate that the antibacterial effect of the novel spraying sprayer with NEW on APEC was far superior compared to the conventional sprayer. This study provides a new insight for preventive measures against airborne microorganisms in indoor spaces.

Survivability of Kluyveromyces marxianus Isolated From Korean Kefir in a Simulated Gastrointestinal Environment.

Kluyveromyces marxianus accounts for > 90% of the yeast population of kefir, and recently, its probiotic potential has been actively explored with a focus on its health benefits and safety. Herein, the survivability of five kefir-isolated K. marxianus strains (Km A1-A5) in a simulated gastrointestinal (GI) environment was evaluated and compared with those of commercial probiotic yeast, Saccharomyces boulardii MYA-796. To further explore the potential to survive in the host GI tract, biochemical activities, hydrophobicity assay, biofilm formation, auto-aggregation analysis, and phenol tolerance of the strains were assessed. K. marxianus A4 exhibited the best survivability among all tested strains, including the clinically proven probiotic yeast strain S. boulardii MYA-796 (p = 0.014) in the artificial GI tract ranging from pH 2.0 to 7.5. In addition, the five K. marxianus strains and S. boulardii MYA-796 displayed different assimilation of lactose, xylitol, D-sorbitol, and DL-lactate, indicating that K. marxianus metabolized a wide range of substances and, thus, might be more feasible to nourish themselves in the host GI tract for survival. K. marxianus strains showed a greater hydrophobicity of cell surface, abilities to biofilm formation and auto-aggregation, and phenol tolerance than S. boulardii MYA-796, suggesting greater potential for survival in the host GI tract.

Properties of broiler breast meat with pale color and a new approach for evaluating meat freshness in poultry processing plants.

The current trend in monitoring meat quality is to move the quality measurements from the laboratory to the processing line. To provide better meat quality control in the commercial poultry processing plants, we evaluated the quality of broiler breast meat samples, observing different colors, and assessed their freshness using a Torrymeter. Different colors were classified based on the mean ± standard deviation of lightness (L*) values in 1,499 broiler breast fillets: Dark (L* < 56), normal (56 ≤ L* ≤ 62), and pale (L* > 62). To characterize the differences between the pale and normal color groups, we evaluated additional fillets for meat quality traits. Changes in meat quality during storage were also evaluated. The L* and Torrymeter values (freshness values) allowed us to distinguish between the pale and normal meat samples. Normal and pale fillets showed a significant difference in pH, Torrymeter values, and water-holding capacity (P < 0.001). The L* values were significantly correlated with cook and drip loss (P < 0.01) and were higher (paler, +1.2 L* unit) at 72-h postmortem than at 4-h postmortem. Torrymeter values were correlated with cook loss (P < 0.05) and pH (P < 0.001), and significantly decreased with the increase in storage period (P < 0.001). These results suggest the applicability of the Torrymeter, a fast and non-destructive device, in distinguishing stale and fresh breast fillets. With its portability and simplicity, the Torrymeter is expected to be a valuable tool to estimate meat freshness. Especially, the use of Torrymeter for evaluating pale breast fillets may allow easy identification and separation of fillets according to their pale, soft, and exudative properties in commercial poultry processing lines.

Effect of folic acid supplementation on proliferation and apoptosis in bovine mammary epithelial (MAC-T) cells.

Folic acid (FA) is known to be an important micronutrient in humans; however, information regarding the effect of FA supplementation on bovine mammary epithelial (BME) cells is insufficient. FA supplementation is reported to increase milk production in dairy cows, but the underlying molecular mechanisms are unknown. This study examined the effects of FA supplementation on the proliferation and apoptosis of a BME cell line (MAC-T). MAC-T cells were treated with various concentrations (deficient in FA (DF) < 0.01 ng/mL; low-level FA (LF) 3.1 ng/mL; normal FA (NF) 15.4 ng/mL; and high-level FA (HF) 30.8 ng/mL) based on serum folate (10-20 ng/mL) in milking cows. HF treatment significantly increased the proliferation of MAC-T cells. Cellular apoptosis was observed mainly in the DF group. The number of apoptotic cells in DF media was significantly higher than that in NF media. The bcl-2/bax mRNA expression ratio was significantly increased in the HF group compared to that in the DF group. FA supplementation significantly increased the ratio of Bcl-2/Bax protein levels in MAC-T cells. FA supplementation increases proliferation and decreases apoptosis in these cells. This study might provide information regarding the molecular mechanism through which FA supplementation is associated with increased milk yield.

Prevalence, Characterization, and Antimicrobial Susceptibility of Listeria monocytogenes from Raw Beef and Slaughterhouse Environments in Korea.

The prevalence of Listeria monocytogenes in raw beef and in slaughterhouse environments was investigated from April 2019 to February 2020. Three hundred raw beef samples were purchased from 50 retailers and 10 restaurants (5 samples per source). One hundred and thirty-four samples from slaughterhouse environments were collected by swabbing (10 × 10 cm) the surfaces, gloves, splitting saw, and drains. L. monocytogenes was detected and identified according to the method described in ISO 11290-1, and confirmed by 16S rRNA sequencing. L. monocytogenes was detected in raw beef (2/300, 0.7%), gloves used in carcass splitting (6/21, 28.6%), the splitting saw (1/18, 5.6%), and the drain zone (1/15, 6.7%). All isolates were serotype 1/2a or 1/2c, based on screening using multiplex PCR-based serogrouping assay and serotyping kit for O-H antigens. Pulsed-field gel electrophoresis (PFGE) following ApaI digestion of eight PFGE pulsotypes and four PFGE groups were identified. Biofilm formation analysis using Crystal Violet staining revealed the highest biofilm formation in strain LM-16, followed by D190613. Although L. monocytogenes isolates were susceptible to most antimicrobials, some resistance to penicillin (8/15, 53.3%) and tetracycline (2/15, 13.3%) was observed. Through PFGE, G190426, G190829, and G200210 isolated from the same location in this study were genetically homologous similar to the LM-16 strain, previously isolated from beef carcass in 2006. These results suggest that LM-16 has been continuously present in biofilms in the slaughterhouse environments since 2006. Our study indicates that L. monocytogenes contamination in raw beef could consistently occur during beef processing in slaughterhouse environments through contact with gloves, splitting saws, and drains.

Detection of Campylobacter jejuni from Fresh Produce: Comparison of Culture- and PCR-based Techniques, and Metagenomic Approach for Analyses of the Microbiome before and after Enrichment.

ABSTRACT: In this study, we compared the efficiency of culture-based methods with or without membrane filtration, real-time PCR, and digital droplet PCR (ddPCR) for the detection of Campylobacter in fresh produce. Alfalfa sprouts, clover sprouts, coleslaw, and lettuce salad spiked with Campylobacter jejuni were enriched in Bolton broth for 48 h, and enrichment cultures were either directly inoculated onto modified charcoal-cefoperazone-deoxycholate agar or applied on membrane filters placed on the surface of plating media. In parallel, 2-mL Bolton broth cultures were taken to extract DNA for real-time PCR and ddPCR assays and bacterial community analysis. A developed primer set for ddPCR and real-time PCR was evaluated for its inclusivity and exclusivity using pure culture of C. jejuni and non-C. jejuni strains, respectively. In pure culture, the primer set reacted only with C. jejuni strains and showed negative reaction to non-C. jejuni strains. There was no significant difference (P > 0.05) in the detection efficiency of positive Campylobacter isolates from coleslaw and lettuce salad using four detection methods. However, for sprout samples, the detection efficiency of the culture method was significantly (P < 0.05) lower than those of the two PCR assays and the filtration method. The analysis also revealed the presence of Pseudomonas and Acinetobacter as the most prevalent competing microbiota in enriched culture and only Acinetobacter on agar plates in the selective culture step.

Effectiveness of calcium hypochlorite, quaternary ammonium compounds, and sodium hypochlorite in eliminating vegetative cells and spores of Bacillus anthracis surrogate.

BACKGROUND: The spore-forming bacterium Bacillus anthracis causes anthrax, an often-fatal infection in animals. Therefore, a rapid and reliable strategy to decontaminate areas, humans, and livestock from B. anthracis is very critical. OBJECTIVES: The aim of this study was performed to evaluate the efficacy of sodium hypochlorite, calcium hypochlorite, and quaternary ammonium compound (QAC) sanitizers, which are commonly used in the food industry, to inhibit spores and vegetative cells of B. anthracis surrogate. METHODS: We evaluated the efficacy of sodium hypochlorite, calcium hypochlorite, and a QAC in inhibiting vegetative cells and spores of a B. anthracis surrogate. We treated a 0.1-mL vegetative cell culture or spore solution with 10 mL sanitizer. The samples were serially diluted and cultured. RESULTS: We found that 50 ppm sodium hypochlorite (pH 7), 1 ppm calcium hypochlorite, and 1 ppm QAC completely eliminated the cells in vegetative state. Exposure to 3,000 ppm sodium hypochlorite (pH 7) and 300 ppm calcium hypochlorite significantly eliminated the bacterial spores; however, 50,000 ppm QAC could not eliminate all spores. CONCLUSIONS: Calcium hypochlorite and QAC showed better performance than sodium hypochlorite in completely eliminating vegetative cells of B. anthracis surrogate. QAC was ineffective against spores of the B. anthracis surrogate. Among the three commercial disinfectants tested, calcium hypochlorite most effectively eliminated both B. anthracis vegetative cells and spores.

Characterization of a novel bacteriophage φCJ22 and its prophylactic and inhibitory effects on necrotic enteritis and Clostridium perfringens in broilers.

High necrotic enteritis (NE) incidence and mortality rates in poultry can be caused by Clostridium perfringens (CP) coinfected with Eimeria spp., a causative agent of coccidiosis. Banning of prophylactic use of antibiotics in feed has been accompanied by increased NE outbreaks, resulting in economically devastating losses to the broiler industry. To determine alternatives for controlling NE, we isolated CP-specific bacteriophages (BP), characterized their properties, evaluated their inhibitory effects on pathogenic CP, selected a highly effective phage (φCJ22), and used φCJ22 as a dietary supplement in experimental NE-afflicted broiler chickens. Male broilers (n = 780) were randomly assigned to 60 pens (n = 13 broilers/pen) and into 5 groups [CP-uninfected negative control (NC), basal diet (BD) without CP and BP; CP-infected positive control (PC), BD + CP; and 3 BP groups receiving low- (LP; BD + CP+105 BP), medium- (MP; BD + CP+106 BP), and high-phage (HP; BD + CP+107 BP plaque-forming units/kg) concentrations]. The results showed that MP and HP groups presented an antimicrobial activity toward clinical CP isolate strains, and the groups decreased NE lesions and mortality rates without changes in chicken performance at the end of the experimental period. After CP-challenge body weight gain and feed efficiency were significantly lower in phage-fed groups than that in the PC group (P < 0.05), and NE-associated mortality was the lowest in the HP group (P < 0.001). Moreover, histopathology revealed lesser gastrointestinal mucosal damage in the NC and BP-treated (LP, MP, and HP) groups than that in the PC group, and MP and HP significantly lowered viable CP number in the cecum content by up to 1.24log10 relative to only CP-infected PC group (P < 0.05). These findings suggest that addition of φCJ22 to chicken feed might effectively ameliorate NE, which is accompanied by reduced CP strains in the gut and compensate the performance of NE-afflicted broilers.

Synergistic effects of the early administration of Lactobacillus kefiranofaciens DN1 and Kluyveromyces marxianus KU140723-05 on the inhibition of Salmonella Enteritidis colonization in young chickens.

In this study, we aimed to assess the feasibility of the lactic acid bacterium Lactobacillus kefiranofaciens DN1 (LKF_DN1) and the yeast Kluyveromyces marxianus KU140723-05 (KMA5), recently isolated from kefir, as probiotics. Specifically, we evaluated the effect of early administration of these 2 microbes on the inhibition of Salmonella Enteritidis (SE) colonization in neonatal chicks. We also examined the effects of exposure of chicks to probiotics before SE exposure on the reduction in the number of gut SE. A total of 108 1-day-old specific-pathogen-free male layer chicks were used for 3 independent experiments. The experimental chicks were randomly divided into 6 groups (negative control: basal diet [BD] without probiotics and SE; positive control: BD; probiotic group [PG] 1: BD + LKF_DN1; PG2: BD + KMA5; PG3: BD + LKF_DN1 + KMA5; and PG4: BD+ a commercial product IDF-7), all of which, except negative control, were coadministered with SE strain resistant to rifampicin (SERR). We found that the administration of LKF_DN1 and/or KMA5 reduced the number of viable cells of the SERR strain in chicks by up to 1.90 log10, relative to positive control chicks. Compared with late administration (day [D] 10 and D11), early administration (D1 and D2) of the probiotics was more effective in reducing SERR cell numbers in the gut. Furthermore, we detected no significant difference in the reduction of gut SERR cell numbers in chicks from the same groups exposed to the probiotics at D10 and D11 before and after administration with SERR. Collectively, our findings indicate that, as dietary additives, LKF_DN1 and KMA5 showed potential probiotic activity in chicks. Moreover, the combination of the lactic acid bacteria and/or yeast strain was found to rapidly reduce SE numbers in the chick gut and showed a prolonged inhibitory effect against SE colonization. We, thus, propose that the administration of these 2 probiotics, as early as possible after hatching, would be considerably effective in controlling SE colonization in the guts of chicks.

Prevalence, toxin-typing, and antimicrobial susceptibility of Clostridium perfringens from retail meats in Seoul, Korea.

Clostridium perfringens is a ubiquitous, Gram-positive, spore-forming bacterium. It can contaminate many types of retail meat products and cause food poisoning by producing enterotoxins in the small intestines of humans and domestic animals. We investigated the prevalence, toxin-encoding gene profile, and antimicrobial resistance of C. perfringens in beef, chicken, and pork meat purchased from retail markets in Seoul, Korea. C. perfringens was detected according to the International Organization for Standardization 7937, with some modifications, and confirmed using the Vitek 2 system. In total, 38 C. perfringens strains were isolated from 200 meat samples (38/200, 19%; thirty-three from chicken, and five from beef). Among the six toxins evaluated, including alpha, beta, epsilon, iota, enterotoxin (encoded in the cpe gene), and netB, only the cpa gene was detected in all isolates by polymerase chain reaction (PCR) amplification. The antimicrobial resistance of the isolates was evaluated using the agar dilution method and resistance to ampicillin (12/38, 31.6%), tetracycline (38/38, 100%), chloramphenicol (26/38, 68.4%), metronidazole (13/38, 34.2%), and imipenem (27/38, 71%) was observed. Interestingly, 30 of the 38 isolates (78.9%) were multiple-drug resistant, showing resistance to more than three different antimicrobial classes.

CYPminer: an automated cytochrome P450 identification, classification, and data analysis tool for genome data sets across kingdoms.

BACKGROUND: Cytochrome P450 monooxygenases (termed CYPs or P450s) are hemoproteins ubiquitously found across all kingdoms, playing a central role in intracellular metabolism, especially in metabolism of drugs and xenobiotics. The explosive growth of genome sequencing brings a new set of challenges and issues for researchers, such as a systematic investigation of CYPs across all kingdoms in terms of identification, classification, and pan-CYPome analyses. Such investigation requires an automated tool that can handle an enormous amount of sequencing data in a timely manner. RESULTS: CYPminer was developed in the Python language to facilitate rapid, comprehensive analysis of CYPs from genomes of all kingdoms. CYPminer consists of two procedures i) to generate the Genome-CYP Matrix (GCM) that lists all occurrences of CYPs across the genomes, and ii) to perform analyses and visualization of the GCM, including pan-CYPomes (pan- and core-CYPome), CYP co-occurrence networks, CYP clouds, and genome clustering data. The performance of CYPminer was evaluated with three datasets from fungal and bacterial genome sequences. CONCLUSIONS: CYPminer completes CYP analyses for large-scale genomes from all kingdoms, which allows systematic genome annotation and comparative insights for CYPs. CYPminer also can be extended and adapted easily for broader usage.

Prevalence, Antibiotic-Resistance, and Virulence Characteristics of Vibrio parahaemolyticus in Restaurant Fish Tanks in Seoul, South Korea.

Vibrio parahaemolyticus is a marine bacterium that causes foodborne diarrhea. Many seafood restaurants keep live fish and shellfish in fish tanks for use in raw seafood dishes; thus, the present study aimed to investigate the prevalence, antibiotic-resistance, and virulence characteristics exhibited by V. parahaemolyticus detected in restaurant fish-tank water samples collected in Seoul, South Korea. Fish-tank water samples were collected from 69 restaurants in Seoul, and screened for the presence of V. parahaemolyticus via both a commercial detection kit, and a real-time polymerase chain reaction (RT-PCR) to detect the toxR gene. Antibiotic susceptibility and virulence determinants of V. parahaemolyticus isolates were evaluated and identified using standard disk-diffusion and RT-PCR methods, respectively. Thirty-five (50.7%) of the 69 analyzed water samples were found to be contaminated with V. parahaemolyticus. Those isolates were most often resistant to ampicillin (51.4% of isolates), followed by amikacin and tetracycline (11.4%), and ceftazidime (8.6%). Thirty (85.7%) out of the 35 isolates carried all four cytotoxicity-inducing type III secretion system 1 (T3SS1) genes [specifically, 34 (97.1%), 33 (94.3%), 35 (100%), and 32 (91.4%) isolates carried genes encoding the VP1670, VP1686, VP1689, and VP1694 T3SS1 proteins, respectively]. The type VI secretion systems (T6SS1 and T6SS2) genes were also detected in 11 (31.4%) and 27 (77.1%) isolates, respectively. However, virulence determinants such as the hemolysin (tdh and trh), urease (ureC), T3SS2α, or T3SS2ß genes that are known to be associated with enterotoxicity were not detected in all isolates. Although some known major virulence genes were not detected in the V. parahaemolyticus isolates, the results of this study indicate that restaurant fish tanks are a potential source of antibiotic-resistant V. parahaemolyticus. The presented data support the need for strict guidelines to regulate the maintenance of restaurant fish tanks to prevent antibiotic-resistant foodborne vibriosis.