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.