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.

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.

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.

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.

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.

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.

Influence of sodium reduction and storage temperature on the growth of total microbes and Bacillus cereus in naturally contaminated hamburger patty and loaf bread.

This study aimed to investigate the influence of sodium reduction and storage temperature on the growth of total microbes and Bacillus cereus in naturally contaminated hamburger patty and loaf bread, respectively. The sodium reduction rate of hamburger patty and loaf bread was 20% and 30%, respectively, and experimental samples were kept at 4 °C, 25 °C, and 40 °C for 60 h. The microbiological analysis included the colony count of total microbes and B. cereus. The water activity (Aw), titratable acidity (TA), and pH were assessed as factors that inhibit microbial growth. In this study, Aw, TA, and pH of all samples were affected by the growth of total microbes and B. cereus during the storage period. Hence, these results suggested that sodium reduction in processed foods should be preferentially applied as a potent inhibition strategy after accurate assessment of inhibitors for different food types.

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.

Comparison of Direct Syringe Filtration and Membrane Filtration for the Selective Isolation of Campylobacter jejuni from Ready-to-Eat Sprouts.

Culture method using enrichment broth and selective agar is one of the most common isolation methods for detecting Campylobacter jejuni from food. However, the overgrowth of competing bacteria in enrichment culture complicates the selective isolation of C. jejuni. In this study, we compared an enrichment/plating method for the isolation of C. jejuni from sprout samples with an enrichment/plating method with syringe or membrane filtration when transferring enriched broths to plates. Four types of sprout samples were artificially contaminated with various levels of C. jejuni and incubated in 100 mL of Bolton broth for 48 h. Enrichment broths were either directly transferred onto modified charcoal-cefoperazone-deoxycholate agar or filtered through membrane or with a syringe. A significantly higher (p < 0.05) isolation rate of Campylobacter positives was obtained with both filtration methods (58-61%) than with the method without filtration (10%). Membrane filtrations yielded 61%, whereas syringe yielded 58% positives. In most cases of unfiltered samples (98%), high competing flora covered most of the plate, making differentiation and picking of suspicious colonies difficult. However, less plates were contaminated with competing flora in both filtration methods. Only 5% of plates were contaminated in the syringe filtration method, whereas no competing flora was observed in membrane filtration (0%).

Supplementation of Modified Mannitol-Yolk-Polymyxin B Agar with Cefuroxime for Quantitative Detection of Bacillus cereus in Food.

The presence of unwanted competing flora has been the most common confounding factor in the enumeration of Bacillus cereus (B. cereus) using selective media such as mannitol-yolk-polymyxin B agar (MYPA). The objective of this study was to improve MYPA selectivity for B. cereus by supplementation with a second-generation cephalosporin, cefuroxime. The performance of cefuroxime-supplemented MYPA (cefu-MYPA) was evaluated by comparison with original MYPA in 60 food products with established microbiological standards for B. cereus contamination. Cefu-MYPA demonstrated superior recoverability and selectivity for B. cereus compared with original MYPA in most tested foods. B. cereus numbers on MYPA and cefu-MYPA were 363.5 and 462.0 CFU/g, respectively. Competing flora on cefu-MYPA was detected in significantly less samples (70%) compared to original MYPA (93%). In addition, the detection and isolation of suspected colonies were significantly improved in cefu-MYPA because of the reduction or elimination of competing flora in all tested foods except fruit juice, indicating superior selectivity of the modified medium. Our findings suggest that cefuroxime supplementation of MYPA would markedly improve the detection rate of B. cereus, particularly in foods with high levels of indigenous flora.

Risk Assessment for Salmonellosis in Chicken in South Korea: The Effect of Salmonella Concentration in Chicken at Retail.

Salmonellosis caused by chicken consumption has been a critical issue in food safety worldwide, including in Korea. The probability of illness from consumption of chicken was simulated in study, based on the recipe of Dakgalbi, a commonly eaten chicken dish in Korea. Additionally, the processing stage at slaughterhouses to decrease Salmonella concentration in broilers was modeled to explore its effect on the likelihood of illness. A Monte Carlo simulation model was created using @RISK. Prevalence of Salmonella in chickens at the retail stage was found to be predominantly important in determining the probability of illness. Other than the prevalence, cooking temperature was found to have the largest impact on the probability of illness. The results also demonstrated that, although chlorination is a powerful tool for decreasing the Salmonella concentration in chicken, this effect did not last long and was negated by the following stages. This study analyzes the effects of variables of the retail-to-table pathway on the likelihood of salmonellosis in broiler consumption, and also evaluates the processing step used to decrease the contamination level of Salmonella in broilers at slaughterhouses. According to the results, we suggest that methods to decrease the contamination level of Salmonella such as chlorination had little effect on decreasing the probability of illness. Overall, these results suggest that preventing contamination of broiler with Salmonella must be a top priority and that methods to reduce the concentration of Salmonella in broilers at slaughterhouses hardly contribute to safe consumption of Salmonella-contaminated chicken.

Antimicrobial and anti-biofilm activities of Lactobacillus kefiranofaciens DD2 against oral pathogens.

Background: Streptococcus mutans and Streptococcus sobrinus are major causative bacterial pathogens of dental caries. Objective: We investigated the applicability of three Lactobacillus strains (L. kefiranofaciens DD2, DD5, and DD6) isolated from kefir and three commercial Lactobacillus strains (L. plantarum ATCC 10,012, L. johnsonii JCM 1022, and L. rhamnosus ATCC 7469) as potential oral probiotics with respect to their survivability in an experimental oral environment, antimicrobial activity, and anti-biofilm formation activity against S. mutans and S. sobrinus. Results: Strains DD2, ATCC 10012, ATCC 7469, and JCM 1022 had the best oral survivability, including aerotolerance and enzymatic resistance, and inhibited the growth and biofilm formation of S. mutans and S. sobrinus. In particular, DD2 suppressed all three classes of biofilm formation-associated genes: those associated with carbohydrate metabolism and those encoding regulatory biofilm and adhesion proteins. Conclusions: These results indicate that the novel kefir isolate L. kefiranofaciens DD2 effectively and directly inhibits S. mutans and S. sobrinus.

Dual function of Lactobacillus kefiri DH5 in preventing high-fat-diet-induced obesity: direct reduction of cholesterol and upregulation of PPAR-α in adipose tissue.

SCOPE: Kefir consumption inhibits the development of obesity and non-alcoholic fatty liver disease (NALFD) in mice fed 60% high-fat diet (HFD). To identify the key contributor of this effect, we isolated lactic acid bacteria (LAB) from kefir and examined their anti-obesity properties from in vitro screening and in vivo validation. METHODS AND RESULTS: Thirteen kefir LAB isolates were subjected to survivability test using artificial gastrointestinal environment and cholesterol-reducing assay. Lactobacillus kefiri DH5 showed 100% survivability in gastrointestinal environments and reduced 51.6% of cholesterol; thus, this strain was selected for in vivo experiment. Compared to the HFD-saline group, the HFD-DH5 group showed significantly lower body weight (34.68 versus 31.10 g; p < 0.001), epididymal adipose tissue weight (1.39 versus 1.05 g; p < 0.001), blood triglyceride (38.2 versus 31.0 mg/dL; p < 0.01) and LDL-cholesterol levels (19.4 versus 15.7 mg/dL; p < 0.01). In addition, L. kefiri DH5 administration significantly modulated gut microbiota of HFD-fed mice. The hepatic steatosis was significantly milder (Lesion score, 2.1 versus 1.2; p < 0.001) and adipocyte diameter was significantly smaller (65.1 versus 42.2 µm; p < 0.001) in the HFD-DH5 group. L. kefiri DH5 upregulated PPAR-α, FABP4, and CPT1 expression in the epididymal adipose tissues (2.29-, 1.77-, and 2.05-fold change, respectively), suggesting a reduction in adiposity by stimulating fatty acid oxidation. CONCLUSION: L. kefiri DH5 exerts anti-obesity effects by direct reduction of cholesterol in the lumen and upregulation of PPAR-α gene in adipose tissues.

Kefir alleviates obesity and hepatic steatosis in high-fat diet-fed mice by modulation of gut microbiota and mycobiota: targeted and untargeted community analysis with correlation of biomarkers.

Kefir is a probiotic beverage containing over 50 species of lactic acid bacteria and yeast. In this study, the anti-obesity and anti-non-alcoholic fatty liver disease (NAFLD) effects of kefir were comprehensively addressed along with targeted and untargeted community analysis of the fecal microbiota in a high-fat diet (HFD)-induced obese mouse model. HFD-fed C57BL/6 mice were orally administrated either kefir or milk (control) once a day for 12 weeks, and body and organ weight, fecal microbiota and mycobiota, histopathology, blood cholesterol and cytokines and gene expressions were analyzed. Compared to the control, mice in the kefir group exhibited a significantly lower body weight (34.18 g vs. 40.24 g; p=0.00004) and histopathological liver lesion score (1.13 vs. 3.25; p=0.002). Remarkably, the kefir-fed mice also harbored more Lactobacillus/Lactococcus (7.01 vs. 6.32 log CFU/g), total yeast (6.07 vs. 5.01 log CFU/g) and Candida (5.56 vs. 3.88 log CFU/g). Kefir administration also up-regulated genes related to fatty acid oxidation, PPARα and AOX, in both the liver and adipose tissue (PPARα, 2.95- and 2.15-fold; AOX, 1.89- and 1.9-fold, respectively). The plasma concentration of IL-6, a proinflammatory marker, was significantly reduced following kefir consumption (50.39 pg/ml vs. 111.78 pg/ml; p=0.03). Strikingly, the populations of Lactobacillus/Lactococcus, total yeast and Candida were strongly correlated with PPARα gene expression in adipose and hepatic tissue (r=0.599, 0.580 and 0.562, respectively). These data suggest that kefir consumption modulates gut microbiota and mycobiota in HFD-fed mice, which prevents obesity and NAFLD via promoting fatty acid oxidation.

Evaluation of Selective-Enrichment and Chromogenic Media for Salmonella Detection in Raw Shell Egg Contents with a Low Microbial Load.

The current study was conducted to evaluate the ability to recover Salmonella from shell egg contents by culture methods. A total of 4,000 eggs were obtained from a grading and packing center located in the Gyeonggi Province of South Korea, and 200 samples were created by pooling 20 broken eggs. The pooled samples were held at room temperature for 4 d before a 25-mL aliquot of each pool was added to 225 mL of modified trypticase soy broth (mTSB) and incubated at 35°C for 24 ± 2 h. A loopful of the culture was streaked onto chromogenic Druggan-Forsythe-Iversen (DFI) agar and incubated at 36 ± 1°C for 18-24 h. In addition, 1 mL and/or 0.1 mL of the mTSB cultures were added to 10 mL of Muller-Kauffmann tetrathionate with novobiocin (MKTTn) or Rappaport-Vassiliadis (RV) broth, and they were incubated for 24 ± 2 h at 35 ± 2°C or 42 ± 0.2°C, respectively. A loopful from these cultures was streaked onto Brilliant Green (BG), xylose lysine deoxycholate (XLD), and bismuth sulfite (BS) agar plates, respectively. Directly streaking onto DFI agar revealed the presence of Salmonella in 14 out of the 200 pooled samples (7%); whereas the combination of RV medium and BG, XLD, and BS agar detected the pathogen in only 9 (4.5%), 7 (3.5%), and 3 (1.5%) of the pooled samples, respectively. When MKTTn broth was used, Salmonella was detected in 7 (3.5%), 2 (1%), and 0 (0%) of the samples when streaked onto BG, XLD, and BS agar, respectively. The results indicate that direct plating onto DFI agar without enrichment was the most suitable among the methods evaluated in this study for detecting Salmonella in raw shell egg contents with a low microbial load.

Modulation of gut microbiota and increase in fecal water content in mice induced by administration of Lactobacillus kefiranofaciens DN1.

Lactobacillus kefiranofaciens is the key probiotic bacterium in kefir. In this study, we investigated the effects of oral consumption of L. kefiranofaciens on the fecal quality and intestinal microbiota of mice. Four-week-old Balb/c mice were divided into two groups (n = 8 each) and administered 0.2 mL of saline (control group) or saline containing 2 × 108 cfu L. kefiranofaciens DN1 (LKF_DN1 group) for two weeks. At the end of the experiment, their fecal samples were collected and the fecal quality and microbiota were assessed. The LKF_DN1 group exhibited higher total fecal weight and fecal weight per stool sample than the control group (p < 0.05). Interestingly, the fecal water content was significantly higher in the fecal samples of the LKF_DN1 group than in those of the control group (p < 0.05). The numbers of total bacteria, Firmicutes, Bacteroidetes, Lactobacillus, and Prevotella were significantly higher in the LKF_DN1 group than in the control group (p < 0.05). In contrast, the number of opportunistic pathogens, including Proteobacteria and Enterobacteriaceae, and the percentage of genus Clostridium among the total bacteria were significantly reduced in the LKF_DN1 group (p < 0.05). Our data suggest that regular L. kefiranofaciens DN1 administration could alleviate constipation and improve gut microbiota.

Establishing Quantitative Standards for Residual Alkaline Phosphatase in Pasteurized Milk.

The alkaline phosphatase (ALP) assay is a rapid and convenient method for verifying milk pasteurization. Since colorimetric ALP assays rely on subjective visual assessments, their results are especially unreliable near the detection limits. In this study, we attempted to establish quantitative criteria for residual ALP in milk by using a more objective method based on spectrophotometric measurements. Raw milk was heat-treated for 0, 10, 20, 30, and 40 min and then subjected to ALP assays. The quantitative criteria for residual ALP in the milk was determined as 2 µg phenol/mL of milk, which is just above the ALP value of milk samples heat-treated for 30 min. These newly proposed methodology and criteria could facilitate the microbiological quality control of milk.