A comparative evaluation of the kefir yeast Kluyveromyces marxianus A4 and sulfasalazine in ulcerative colitis: anti-inflammatory impact and gut microbiota modulation.

Although only Saccharomyces boulardii has been studied for ulcerative colitis (UC), probiotic yeasts have immense therapeutic potential. Herein, we evaluated the kefir yeast Kluyveromyces marxianus A4 (Km A4) and its anti-inflammatory effect with sulfasalazine in BALB/c mice with dextran sulfate sodium (DSS)-induced colitis. Oral administration continued for 7 days after the mice were randomly divided into seven groups: control (CON, normal mice administered with saline), DSS-induced colitis mice administered saline (DSS), and DSS-induced colitis mice administered sulfasalazine only (S), Km A4 only (A4), Km A4 plus sulfasalazine (A4 + S), S. boulardii ATCC MYA-796 (Sb MYA-796) only (Sb), and Sb MYA-796 plus sulfasalazine (Sb + S). The ß-glucan content of Km A4 was significantly higher than that of Sb MYA-796 (P < 0.05). Body weight gain (BWG) significantly correlated with colon length, cyclooxygenase-2 (Cox-2) levels, and Bacteroides abundance (P < 0.05). In colitis-induced mice, the A4 + S group had the lowest histological score (6.00) compared to the DSS group (12.67), indicating the anti-inflammatory effects of this combination. The A4 + S group showed significantly downregulated expression of interleukin (Il)-6, tumor necrosis factor-α (Tnf-α), and Cox-2 and upregulated expression of Il-10 and occludin (Ocln) compared to the DSS group. Mice treated with A4 + S had enhanced Bacteroides abundance in their gut microbiota compared with the DSS group (P < 0.05). Bacteroides were significantly correlated with all colitis biomarkers (BWG, colon length, Il-6, Tnf-α, Il-10, Cox-2, and Ocln; P < 0.05). The anti-inflammatory effects of Km A4 could be attributed to high ß-glucan content and gut microbiota modulation. Thus, treatment with Km A4 and sulfasalazine could alleviate UC.

Effect of Cucumis melo L. peel extract supplemented postbiotics on reprograming gut microbiota and sarcopenia in hindlimb-immobilized mice.

Postbiotics made from lactic acid bacteria may ameliorate sarcopenia via the metabolic reprogramming of gut dysbiosis. This study investigated the anti-sarcopenic effect of postbiotics (WDK) produced from polyphenol-rich melon peel extract (Cucumis melo L. var. makuwa, KEE) and whey with Lentilactobacillus kefiri DH5 (DH5) in C2C12 skeletal muscle cells and hindlimb-immobilized mice. WDK significantly ameliorated palmitate-induced atrophy of C2C12 cells, restoring myotube length and diameter. It also upregulated the expression of myogenic genes including Atrogin-1, Igf-1, and MyoD. Hindlimb-immobilized C57BL/6J mice were randomly divided and orally administered 10 mL/kg body weight of saline (CON), Whey, Whey + DH5 (WD), DH5 + KEE, Whey + DH5 + KEE postbiotic (WDK) for three weeks (n = 10/group). Interestingly, WDK significantly improved muscle function in hindlimb-immobilized mice by restoring both the grip strength and the mass of the soleus muscle, which was closely related to the upregulation of the myoD gene. WDK increased microbial diversity and modulated the distribution of intestinal bacteria, particularly those involved in protein synthesis and the production of butyrate. There was a significant correlation between myogenic biomarkers and butyrate producing gut microbiota. Restoration of muscle mass and function following postbiotic WDK is strongly related to the regulation of myogenic genes by in part remodulating gut microbiota. In conclusion, these findings suggest that polyphenol- and whey-based postbiotics WDK may have potential as an effective manner to combat the progression of sarcopenia.

Gut microbiota modulation via short-term administration of potential probiotic kefir yeast Kluyveromyces marxianus A4 and A5 in BALB/c mice.

Kefir yeast, Kluyveromyces marxianus, has been evaluated for its potential probiotic properties-survivability, non-pathogenicity, and antioxidant and anti-microbial activities. However, host gut microbiota modulation of kefir yeasts remains unclear. Here, we compared kefir yeast strains K. marxianus A4 (Km A4) and K. marxianus A5 (Km A5) with Saccharomyces boulardii ATCC MYA-796 (Sb MYA-796) by investigating their adherence to colorectal adenocarcinoma (Caco-2) cells and gut microbiota modulation in BALB/c mice. The kefir yeast strains exhibited higher intestinal cell adhesion than Sb MYA-796 (p < 0.05). Bacteroidetes, Bacteroidales, and Bacteroides were more abundant in the 1 × 108 CFU/mL of Km A4 treatment group than in the control group (p < 0.05). Moreover, 1 × 108 CFU/mL of Km A5 increased Corynebacteriales and Corynebacterium compared to the 1 × 108 CFU/mL of Km A4 treatment group (p < 0.01). The results showed that Km A4 and Km A5 had good Caco-2 cell adhesion ability and modulated gut microbiota upon short-term administration in healthy mice. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01268-3.

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.

Gold Nanoparticle and Polymerase Chain Reaction (PCR)-Based Colorimetric Assay for the Identification of Campylobacter spp. in Chicken Carcass.

Campylobacteriosis is a common cause of gastrointestinal disease. In this study, we suggest a general strategy of applying gold nanoparticles (AuNPs) in colorimetric biosensors to detect Campylobacter in chicken carcass. Polymerase chain reaction (PCR) was utilized for the amplification of the target genes, and the thiolated PCR products were collected. Following the blending of colloid AuNPs with PCR products, the thiol bound to the surface of AuNPs, forming AuNP-PCR products. The PCR products had a sufficient negative charge, which enabled AuNPs to maintain a dispersed formation under electrostatic repulsion. This platform presented a color change as AuNPs aggregate. It did not need additional time and optimization of pH for PCR amplicons to adhere to the AuNPs. The specificity of AuNPs of modified primer pairs for mapA from Campylobacter jejuni and ceuE from Campylobacter coli was activated perfectly (C. jejuni, p-value: 0.0085; C. coli, p-value: 0.0239) when compared to Salmonella Enteritidis and Escherichia coli as non-Campylobacter species. Likewise, C. jejuni was successfully detected from artificially contaminated chicken carcass samples. According to the sensitivity test, at least 15 ng/µL of Campylobacter PCR products or 1×103 CFU/mL of cells in the broth was needed for the detection using the optical method.

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.

Effect of postbiotics derived from kefir lactic acid bacteria-mediated bioconversion of citrus pomace extract and whey on high-fat diet-induced obesity and gut dysbiosis.

The objective of this study was to develop a highly bioactive postbiotic for weight management by bioconversion of whey (WHE) and polyphenol-rich citrus pomace extract (CPX) using kefir lactic acid bacteria (LAB). WHE and CPX bioconverted by kefir LAB (CPB) were fed to C57BL/6J mice on high-fat diets for five weeks and compared with oral administrations of saline (CON), WHE, CPX, and kefir LAB. Hesperetin, a potential therapeutic agent for obesity, was increased in the CPB after bioconversion from an inactive precursor. Compared with the CON group, the CPB group showed significantly reduced body weight gain, adipose tissue weight/body weight ratio, hypertriglyceridemia, and adipocyte diameter along with increased gene expression related to energy expenditure in adipose tissue (p < 0.05). Interestingly, the abundance of gut microbiota related to butyrate production was significantly altered in the CPB group compared with the CON group. There was a significant correlation between obesogenic biomarkers and the abundance of butyrate-producing and obesogenic gut microbiota. In conclusion, kefir LAB-derived bioconversion of WHE and CPX may be effective in combating obesity and obesity-related diseases.

Characterization of Cronobacter sakazakii Strains Originating from Plant-Origin Foods Using Comparative Genomic Analyses and Zebrafish Infectivity Studies.

Cronobacter sakazakii continues to be isolated from ready-to-eat fresh and frozen produce, flours, dairy powders, cereals, nuts, and spices, in addition to the conventional sources of powdered infant formulae (PIF) and PIF production environments. To understand the sequence diversity, phylogenetic relationship, and virulence of C. sakazakii originating from plant-origin foods, comparative molecular and genomic analyses, and zebrafish infection (ZI) studies were applied to 88 strains. Whole genome sequences of the strains were generated for detailed bioinformatic analysis. PCR analysis showed that all strains possessed a pESA3-like virulence plasmid similar to reference C. sakazakii clinical strain BAA-894. Core genome analysis confirmed a shared genomic backbone with other C. sakazakii strains from food, clinical and environmental strains. Emerging nucleotide diversity in these plant-origin strains was highlighted using single nucleotide polymorphic alleles in 2000 core genes. DNA hybridization analyses using a pan-genomic microarray showed that these strains clustered according to sequence types (STs) identified by multi-locus sequence typing (MLST). PHASTER analysis identified 185 intact prophage gene clusters encompassing 22 different prophages, including three intact Cronobacter prophages: ENT47670, ENT39118, and phiES15. AMRFinderPlus analysis identified the CSA family class C ß-lactamase gene in all strains and a plasmid-borne mcr-9.1 gene was identified in three strains. ZI studies showed that some plant-origin C. sakazakii display virulence comparable to clinical strains. Finding virulent plant-origin C. sakazakii possessing significant genomic features of clinically relevant STs suggests that these foods can serve as potential transmission vehicles and supports widening the scope of continued surveillance for this important foodborne pathogen.

Prevalence and Virulence Characteristics of Enterococcus faecalis and Enterococcus faecium in Bovine Mastitis Milk Compared to Bovine Normal Raw Milk in South Korea.

Enterococcus spp. are pathogens that cause environmental mastitis and are difficult to eliminate owing to their resistance to antibiotics. To compare the virulence characteristics of isolates from bovine mastitis milk (BMM) and bovine normal raw milk (NRM), we isolated Enterococcus spp. from 39 dairy farms in South Korea from 2015−2020. A total of 122 Enterococcus spp. were identified, with Enterococcus faecalis (73.8%) accounting for the majority, followed by Enterococcus faecium (26.2%). E. faecalis isolated from BMM harbored gelE, asa1, esp, and cylA genes with a prevalence of 85.7, 71.4, 54.3, and 30.0%, respectively. These genes were significantly more abundant in BMM than in NRM, except for asa1 (p < 0.0001). Interestingly, strong biofilm and gelatinase formation was predominately observed for BMM isolates and this was significantly correlated to the presence of esp and gelE genes (p < 0.05). BMM isolates demonstrated higher resistance to tetracycline (59.3%), followed by chloramphenicol (21.0%), rifampicin (18.5%), doxycycline (4.9%), ciprofloxacin (1.2%), and nitrofurantoin (1.2%), than those from NRM. E. faecalis harboring esp, gelE, and cylA may be causative agents for bovine mastitis and act as a reservoir for the transmission of virulence factors to humans.

Effects of kefir lactic acid bacteria-derived postbiotic components on high fat diet-induced gut microbiota and obesity.

Cellular components, surface layer protein (SLP) and exopolysaccharides (EPS) of postbiotic lactic bacteria (PLAB) can rehabilitate high-fat diet-induced dysbiosis and obese characteristic gut microbiome. However, it is not clear whether and how PLAB components affect gut microbiota and specifically adipocyte gene expression. Furthermore, SLP and EPS of PLAB in combination with polyphenolics of prebiotic wine grape seed flour (GSF) may have greater benefit on high-fat diet (HFD)-induced obesity and gut microbiota imbalance. To investigate interactions, C57BL/6 mice were fed a HFD and orally administered saline (CON), 250 mg/Kg EPS, or 120 mg/Kg SLP or saline with fed 2% GSF (GSF) or combination (42 mg/Kg EPS + 20 mg/Kg SLP + 0.5% GSF; ALL). There were significant reductions of HFD-induced body weight gain, adipose weight, serum triglyceride, and insulin resistance by the SLP and ALL diets compared to CON, with the most profound effect by ALL. ALL significantly affected the distribution of intestinal bacterial genus and species particularly those involved in production of short chain fatty acid (SCFA) and anti-obesogenic action. Microarray analysis from adipose tissue showed that ALL significantly affected expression of genes related to fatty acid biosynthesis, autophagy, inflammatory response, immune response, brown adipose tissue development and response to lipoteichoic acid and peptidoglycan (p < 0.05). Interestingly, expression of Akp13 (A-kinase anchoring protein 13) gene, which is related to body mass index and immune response, was negatively associated with the abundance of obesogenic and SCFAs producing gut bacteria. These data suggest that a combination of postbiotic kefir LAB cellular components and prebiotic GSF establishes a healthy intestinal microbiota that in part was associated with the prevention of obesity and obesity-related diseases.

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.

Isolation and Characterization of Halophilic Kocuria salsicia Strains from Cheese Brine.

Kocuria salsicia can survive in extreme environments and cause infections, including catheter-related bacteremia, in humans. Here, we investigated and evaluated the characteristics of nine K. salsicia strains (KS1-KS9) isolated from cheese brine from a farmstead cheese-manufacturing plant in Korea from June to December, 2020. Staphylococcus aureus American Type Culture Collection (ATCC) 29213 was used as a positive control in the growth curve analysis and biofilm-formation assays. All K. salsicia isolates showed growth at 15% salt concentration and temperatures of 15°C, 25°C, 30°C, 37°C, and 42°C. KS6 and KS8 showed growth at 5°C, suggesting that they are potential psychrotrophs. In the biofilm-formation analysis via crystal violet staining, KS6 exhibited the highest biofilm-forming ability at various temperatures and media [phosphate buffered saline, nutrient broth (NB), and NB containing 15% sodium chloride]. At 25°C and 30°C, KS3, KS6, and KS8 showed higher biofilm-forming ability than S. aureus ATCC 29213. The antimicrobial resistance of the isolates was evaluated using the VITEK® 2 system; most isolates were resistant to marbofloxacin and nitrofurantoin (both 9/9, 100%), followed by enrofloxacin (7/9, 77.8%). Five of the nine isolates (5/9, 55.6%) showed multidrug resistance. Our study reports the abilities of K. salsicia to grow in the presence of high salt concentrations and at relatively low temperatures, along with its multidrug resistance and tendency to form biofilms.

Phylogenetic Comparison and Characterization of an mcr-1-Harboring Complete Plasmid Genome Isolated from Enterobacteriaceae.

Global dissemination of mobilized colistin resistance (mcr)-1-carrying plasmids has been reported. This study aimed to investigate the global dissemination of these plasmids using whole genome sequencing to provide better understanding on genetic characteristics. Sixty-seven complete plasmid genomes harboring mcr-1 were obtained. Phylogeny was built against full plasmid genomes. Different replicon types of plasmid were compared in terms of antimicrobial resistance genes (ARGs), insertion sequence, and other functional genes. Five different replicon types of plasmid (IncX4, IncI2, IncP1, IncHIA, and IncFIB) were found to harbor mcr-1. IncX4 and IncI2 types of plasmid were well clustered in accordance with the country where they were isolated (and not as IncHIA and IncFIB). Three insertion sequences (ISApl1, ISKpn26, and IS1294) were identified in up- and/or downstream of mcr-1. Plasmids IncX4 and IncI2 were observed across the sample origin. Plasmids IncX4 showed high uniformity regardless of the origin of isolates and harbored H-NS coding genes, a facilitator for successful plasmid transfer. All three insertion sequences were observed in IncI2 plasmids. IncHI2 plasmids harbored various ARGs in addition to mcr-1. Our results elucidate the characteristics and phylogenetic relationships of complete mcr-1-harboring plasmids, indicating that global dissemination of mcr-1 is primarily owing to plasmid transfer rather than clonal spread.

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.

Effect of Surface Layer Proteins Derived from Paraprobiotic Kefir Lactic Acid Bacteria on Inflammation and High-Fat Diet-Induced Obesity.

The antiobesity action of nonviable probiotic lactic acid bacteria (PLAB) may be attributed to bacterial cellular components recognized by host cells. The anti-inflammation and antiobesity properties of surface layer proteins (SLPs) that are cellular components isolated from kefir PLAB were determined in macrophage RAW 264.7 cells and obese mice. Kefir SLPs significantly decreased secretion of IL-6 and production of NF-kB p65 protein by LPS-stimulated RAW 264.7 cells in a dose-response manner. C57BL/6J mice were fed a high-fat (HF) diet with oral administration of either saline (CON) or kefir SLPs for 6 weeks. SLPs significantly improved body weight gain and adipose tissue weight, plasma triglyceride concentrations, and insulin resistance. Profiling of adipocyte gene expression showed that the antiobesity effect was significantly related to the expression of genes associated with adipogenesis, autophagy, and inflammatory/immune response, and fatty acid oxidation. Taken together, SLPs are a novel bioactive component in kefir PLABs to target obesity and obesity-related disorders.

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.

Evaluation of Ceftazidime as an Antibiotic Supplement in Mannitol-Yolk-Polymyxin B Agar Used for Enumeration of Bacillus cereus in Ready-to-Eat Vegetables.

ABSTRACT: The Bacillus cereus group of bacteria, which causes foodborne diseases, can be detected by culture on selective media. However, the presence of competing flora is the most common factor preventing the accurate enumeration of B. cereus on selective agars. In this study, we improved the selectivity of mannitol-yolk-polymyxin B agar (MYPA) and its modified version containing trimethoprim (mMYPA) developed in our previous study by supplementation with ceftazidime (16 µg/mL). Ceftazidime-supplemented MYPA (C-MYPA16) and mMYPA (C-mMYPA16) were evaluated for bacteria recovery and selectivity with three types of ready-to-eat vegetables. Four B. cereus and one Bacillus thuringiensis strains were mixed and artificially inoculated into vegetable salad, radish sprouts, and sprout mix and then recovered on MYPA, mMYPA, C-MYPA16, and C-mMYPA16. In all tested vegetables, mMYPA, C-MYPA16, and C-mMYPA16 culture resulted in similar recovery of B. cereus and B. thuringiensis (P > 0.05), whereas radish sprout and sprout mix colonies grown on MYPA were undistinguishable. C-mMYPA16 was the most selective medium because it eliminated most of the competing flora, especially that in sprouts, without negatively affecting the recovery of B. cereus and B. thuringiensis. Our results indicate that supplementation of mMYPA with ceftazidime may improve the selectivity of this medium for B. cereus and B. thuringiensis in food testing.

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.

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.