Extractable surface proteins of indigenous probiotic strains confer anti-adhesion knack and protect against methicillin-resistant Staphylococcus aureus induced epithelial hyperpermeability in HT-29 cell line.

Probiotic intervention has been long believed to have beneficial effects on human health by curbing the intestinal colonization of pathogens. However, the application of live probiotics therapy may not be an ideal approach to circumvent the infections of superbug origin due to the risk of horizontal antibiotic resistance genes transfer. In this study, the anti-adhesion ability of extractable cell surface proteins from two indigenous potential probiotic strains (Lactiplantibacillus plantarum A5 and Limosilactobacillus fermentum Lf1) and two standard reference strains (Lactobacillus acidophilus NCFM and Lacticaseibacillus rhamnosus LGG) was evaluated against clinical isolates of Methicillin-Resistant Staphylococcus aureus (MRSA) on porcine gastric mucin and HT-29 cells. The surface proteins from the probiotic strains were extracted by treatment with 5 M lithium chloride. The surface protein quantification and SDS-PAGE profiling indicated that the yield and protein patterns were strain-specific. Surface proteins significantly hampered the mucoadhesion of MRSA isolates via protective, competitive, and displacement. Similarly, the treatment with surface proteins probiotic strains displayed anti-adhesion against MRSA isolates on HT-29 cells without affecting the viability of the cell line. Surface proteins treatment to the confluent monolayer of HT-29 cells maintained the epithelial integrity; however, MRSA isolates (109 cells/mL) showed considerable alteration in the epithelial integrity by exacerbating the FITC-dextran transflux. Contrarily, the co-treatment with surface proteins with MRSA isolates significantly lowered the FITC-dextran transflux across the differentiated HT-29 monolayer. Overall, the findings of this study suggest that probiotic-derived surface proteins could be the novel biotherapeutics to combat the MRSA colonization and their concomitant intestinal infections.

Gut bacterial profile in Indian children of varying nutritional status: a comparative pilot study.

PURPOSE: Childhood malnutrition is a multifactorial disease, responsible for nearly half of all deaths in children under five. Lately, the probable association of a dysbiotic gut to malnutrition is also being eagerly investigated. The current study is an attempt to investigate this purported association through assessing the abundance of major gut bacterial phyla (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria), probionts (Bifidobacteria and Lactobacillus), butyrogens (Faecalibacterium and Roseburia) and pathogens (Escherichia and Klebsiella). METHODS: The study was conducted in the suburbs of Chandigarh, India in the year 2017. The children enrolled in the study were part of Anganwadis (Rural Child Care Centres) set up under Integrated Child Development Scheme (ICDS) of Government of India where community-based management approach is being widely used for treatment of malnutrition. We used qPCR based absolute quantification as well as the 16S rRNA amplicon sequencing approach for our study. The study population included 30 children in the age group of 2-5 years who were categorized into three groups Healthy, Moderate Acute Malnutrition (MAM) and Severe Acute Malnutrition (SAM), with 10 children in each group. The selection of participants was made based on Z scores. Further, statistical tools like the One-way ANOVA, PCA and PLSDA were employed to analyze and compare the gut bacterial profile. RESULTS: Our investigation through the qPCR (Absolute quantification) approach revealed a significantly higher abundance of Actinobacteria in healthy, in comparison to children suffering from Severe Acute Malnutrition (SAM). Consequently, the same trend was also reflected with respect to Bifidobacterium, a prominent member of the Actinobacteria phylum. Conversely, a significant higher abundance of Lactobacillus with the diminishing nutritional status was recorded. Escherichia showed a significant higher abundance in healthy subjects compared to the malnourished; however, no such difference in abundance of Klebsiella was observed. The other target phyla [Bacteroidetes, Firmicutes and Proteobacteria] and genera (Faecalibacterium and Roseburia) showed differences in abundance; however, these were non-significant. Similarly, the bacterial taxonomy analysis of 16S rRNA gene amplicon sequencing data revealed the higher abundance of phylum Actinobacteria and its member Bifidobacterium with lower prevalence of Lactobacillus in healthy children. CONCLUSION: The pattern of gut microbiota profile in malnourished subjects suggests a dysbiotic gut depleted in Bifidobacteria, a core member of the consortia of beneficial anaerobes of the healthy child gut.

Characterization of Antibiotic Resistance and Virulence Traits Present in Clinical Methicillin-Resistant Staphylococcus aureus Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) is a notorious superbug which poses serious health threats to humanity. The severity of the infections depends on the prevalence of virulence factors and antibiotic resistance. In this study, attempts have been made to nominate the two most virulent and multidrug-resistant MRSA isolates demonstrating the preliminary features of intestinal adhesion for the futuristic applications of probiotics and postbiotics as antagonists to combat MRSA infections. In this context, six clinical isolates of MRSA were polyphasically characterized for their identity, multidrug resistance, and few selected virulence determinates such as hemolytic activity and production of coagulase, nuclease, and capsule. The gut colonizing ability of MRSA isolates was assessed by mucoadhesion, auto-aggregation, and cell surface hydrophobicity. An antibiogram of MRSA isolates suggested the resistance towards several antibiotics with multiple antibiotic resistance (MAR) index >0.5 (12/241, 12/206, and 5/255) as well as their genome portraying mecA mediated methicillin resistance. Besides exhibiting strong biofilm formation ability, all the isolates exhibited positive responses towards tested virulence assays coupled with their genome displaying Coa, NucA, and CapE genes. On the other hand, isolates exhibited different levels of auto-aggregation (37.90 ± 1.8 to 51.53 ± 3.1%) and mucin adhesion ability (68.93 ± 0.61% to 86.62 ± 1.96%) with a significant (P ≤ 0.05) variation in adhesion to different hydrocarbons. Finally, multivariate Principal Component Analysis and Hierarchical Cluster Analysis (HCA) heatmap using Euclidean distance measurement indicated MRSA 12/206 and 5/255 as most resistant and virulent isolates with the potential to adhere to the hydrophobic gut niche.

Evaluation of casein & whey protein hydrolysates as well as milk fermentates from Lactobacillus helveticus for expression of gut hormones.

BACKGROUND & OBJECTIVES: Milk proteins play a beneficial role in the regulation of food intake, postprandial glycaemia and enteroendocrine hormone secretions and thus are receiving considerable attention for the management of metabolic inflammatory disorders such as type 2 diabetes mellitus (T2DM). The objective of this study was to evaluate the efficacy of peptide/s obtained from milk proteins (casein and whey) as well as from the milk fermented with Lactobacillus helveticus as secretagogues for gut hormones and to purify and characterize the active peptides. METHODS: Effect of hydrolysates of casein protein (CP) and whey protein (WP) and L. helveticus fermented milk on the expression of proglucagon, pro-gastric inhibitory peptide (GIP) and cholecystokinin (CCK) genes was monitored by real-time quantitative polymerase chain reaction. The active glucagon-like peptide-1 (GLP-1) secretion was also quantitatively measured using ELISA. RESULTS: Hydrolysates of CP and WP as well as fermentates of L. helveticus induced the proglucagon, pro-GIP and CCK expression and secretion of GLP-1 in STC-1 (pGIP/Neo) cells. However, intact casein exhibited maximum GLP-1 secretion and proglucagon expression. Two active peptides (F5 and F7) derived from CP1 and WP3 hydrolysates having the ability to upregulate the GLP-1 secretion by 1.6 and 1.8 folds were obtained, and the mass was found to be 786 and 824 Da, respectively, as determined by electrospray ionization-mass spectrometry. However, no single active peptide from L. helveticus fermented milk could be obtained. INTERPRETATION & CONCLUSIONS: Casein as well as fermentates obtained from L. helveticus fermented milk showed higher potential for GLP-1 induction. These can be explored as novel therapeutics to T2DM effectively after demonstrating their in vivo efficacy in appropriate animal models.

Ameliorative Effect of Surface Proteins of Probiotic Lactobacilli in Colitis Mouse Models.

The increase in concern from viable cells of probiotics specifically in acute inflammatory conditions has led to the emergence of the concept of postbiotics as a safer alternative therapy in the field of health and wellness. The aim of the present study was to evaluate the efficacy of surface proteins from three probiotic strains in dextran sodium sulfate and trinitrobenzenesulphonic acid = induced colitis mouse models. The molecular weight of total surface proteins extracted from the three probiotic strains ranged from ∼25 to ∼250 kDa with the presence of negligible levels of endotoxins. Surface layer proteins (SLPs) (∼45 kDa) were found to be present only in the Lactobacillus acidophilus NCFM strain. In the in vivo study, significant differences were not observed in the weight loss and general appetite, however, the decrease in colon length was apparent in TNBS colitis control mice. Further, the administration of these surface proteins significantly reversed the histopathological damages induced by the colitogens and improved the overall histological score. The oral ingestion of these surface proteins also led to a decrease in myeloperoxidase activity and TNF-α expression while the IL-10 levels significantly increased for the strain NCFM followed by MTCC 5690 and MTCC 5689. Overall, the present study signifies the ameliorative role of probiotic surface proteins in colitis mice, thereby, offering a potential and safer alternative for the management of inflammatory bowel disorders.

Modulation of intestinal barrier function to ameliorate Salmonella infection in mice by oral administration of fermented milks produced with Lactobacillus plantarum MTCC 5690 - a probiotic strain of Indian gut origin.

Probiotic Lactobacillus plantarum MTCC 5690, a probiotic strain of Indian gut origin, and milk formulations produced with the same were explored in this study as biotherapeutics by evaluating their functional efficacy against Salmonella infection in mice. The efficacy of milk formulations (fermented/unfermented) of MTCC 5690 for enhancement of intestinal barrier function was determined by monitoring the permeability and histopathology of the intestine. Infected mice fed with probiotic Dahi, fermented probiotic drink and sweetened fermented probiotic drink maintained the health and integrity of the intestinal epithelium as compared to those fed with PBS, milk, unfermented probiotic milk and Dahi. Our relative expression data revealed that the changes caused by MTCC 5690 in intestinal barrier function components were established through modulation of the key regulatory receptors Toll-like receptor 2 and Toll-like receptor 4. The results suggest that fermented milks of MTCC 5690 could enhance the defences of the intestinal barrier in enteric infection condition and, therefore, can be explored as a dietary-based strategy to reduce Salmonella infection in the human gut.