Host-microbe interaction and pathogen exclusion mediated by an aggregation-prone surface layer protein of Lactobacillus helveticus.

Probiotic surface layer proteins (Slps) have multiple functions and bacterial adhesion to host cells is one of them. The precise role of Slps in cellular adhesion is not well understood due to its low native protein yield and self-aggregative nature. Here, we report the recombinant expression and purification of biologically active Slp of Lactobacillus helveticus NCDC 288 (SlpH) in high yield. SlpH is a highly basic protein (pI = 9.4), having a molecular weight of 45 kDa. Circular Dichroism showed a prevalence of beta-strands in SlpH structure and resistance to low pH. SlpH showed binding to human intestinal tissue, enteric Caco-2 cell line, and porcine gastric mucin, but not with fibronectin, collagen type IV and laminin. SlpH inhibited the binding of the enterotoxigenic E. coli by 70 % and 76 % and that of Salmonella Typhimurium SL1344 by 71 % and 75 % to enteric Caco-2 cell line in the exclusion and competition assays, respectively. The pathogen exclusion and competition activity and tolerance to harsh gastrointestinal conditions show the potential for developing SlpH as a prophylactic or therapeutic agent against enteric pathogens.

Postbiotic Lipoteichoic acid of probiotic Lactobacillus origin ameliorates inflammation in HT-29 cells and colitis mice.

Lipoteichoic acid (LTA) is a key surface component of probiotic lactobacilli that is involved in important cellular functions including cross talk with the host immune cells. In this study, the anti-inflammatory and ameliorative properties of LTA from probiotic lactobacilli strains were assessed in in vitro HT-29 cells and in vivo colitis mice. The LTA was extracted with n-butanol and its safety was confirmed based on its endotoxin content and cytotoxicity in HT-29 cells. In the Lipopolysaccharide stimulated HT-29 cells, the LTA from the test probiotics evoked a visible but non-significant increase in IL-10 and decrease in TNF-α levels. During the colitis mice study, probiotic LTA treated mice showed substantial improvement in external colitis symptoms, disease activity score and weight gain. The treated mice also showed improvements in key inflammatory markers such as the gut permeability, myeloperoxidase activity and histopathological damages in colon, although non-significant improvements were recorded for the inflammatory cytokines. Furthermore, structural studies by NMR and FTIR revealed increased level of D-alanine substitution in the LTA of LGG strain over MTCC5690. The present study demonstrates the ameliorative effect of LTA as a postbiotic component from probiotics which can be helpful in building effective strategies for combating gut inflammatory disorders.

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.

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.

Human immunodeficiency virus infection amongst newly diagnosed tuberculosis patients and their clinico-radiological profile: A prospective study from Western India.

BACKGROUND: Since time immemorial tuberculosis (TB) has been and continues to be one of the most significant infections causing human disease. In tropical countries, TB remains a leading cause of death. Human immunodeficiency virus (HIV) epidemic continues to fuel this global TB epidemic. The rapid growth of the HIV epidemic in many countries has resulted in an equally dramatic rise in the estimated number of new TB cases, which present therefore the integration of HIV and TB testing at the primary level is need of the hour. METHODS: A prospective study was conducted on newly diagnosed, untreated TB patients aged 15-45 year and patients were screened for HIV infection. Clinico-radiological spectrum of TB among HIV seropositive and seronegative patients was evaluated. RESULTS: Out of a total of 307 patients screened, 17 (5.54%) were found to be HIV seropositive. Seroprevalence was found significantly (P < 0.01, χ2 9.301) more common in 26-35 year age group and higher in Extrapulmonary TB (EPTB) patients than that in pulmonary TB (PTB) patients (9.90% vs 3.4%). Fever was the most common presenting symptom for TB in HIV seropositive patients. On examination pallor (58.82% vs. 15.86%), oral ulcer (35.3% vs. 0.35%) was found more common and on chest X-ray mid-lower zone involvement and mediastinal lymphadenopathy were more common in HIV seropositive patients. CONCLUSION: HIV seropositivity rates among newly diagnosed TB patients aged 15-45 year was 5.54 percent. The presentation of TB was more often atypical among these patients. Thus, an integrated model of TB and HIV at primary healthcare service delivery is an efficient use of resources that would address the two very important co-epidemics and thereby result in better management.

Genome Sequences of Bacillus sporothermodurans Strains Isolated from Ultra-High-Temperature Milk.

Here, we report the draft genome sequences of 3 Bacillus sporothermodurans strains isolated from ultra-high-temperature milk products in South Africa and Brazil and the type strain MB 581 (DSM 10599). The genomes will provide valuable information on the molecular dynamics of heat resistance in B sporothermodurans.

Assessing the Safety and Efficacy of Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 in Colitis Mouse Model.

Probiotic lactobacilli have an unprecedented history of safe use, although some cases of infections have raised concerns about their safety, and hence, a rigorous screening of any new strain even of Lactobacillus is a must in order to study possible adverse interactions with the host, particularly under unhealthy conditions. The present study was, therefore, undertaken to investigate the safety as well as therapeutic efficacy of probiotic Lactobacillus plantarum MTCC 5690 and L. fermentum MTCC 5689 strains in dextran sodium sulfate (DSS)-induced colitis mouse model. Both MTCC 5690 and MTCC 5689 did not induce any detrimental effect on the colitic mice, as was reflected by normal colon and caecum length, blood biochemistry, hematology, and absence of inflammation. Although translocation of both the strains was observed in extraintestinal organs, probiotic-fed mice had significantly improved intestinal permeability and decreased myeloperoxidase (MPO) activity. Probiotic interventions also led to an improved health index and better growth of colitis mice compared to colitis animals with no probiotic intervention. These results point towards the safe use of L. plantarum MTCC 5690 and L. fermentum MTCC 5689 as biotherapeutics for amelioration of inflammatory conditions after establishing their efficacy in human clinical trials.

Assessing safety of Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 using in vitro approaches and an in vivo murine model.

Lactobacilli have a long history of safe use in human nutrition, however, inclusion of any new strain, despite its safe usage evidence, warrants proper analysis of its safety and toxicity under the purview of existing regulations. In the present investigation, Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 were evaluated for their safety and toxicity using both in vitro and in vivo approaches. The in vitro assays included mucin degradation, hemolytic activity, biogenic amine production and platelet aggregation assay. The safety was also assessed using acute, subacute and subchronic assays, bacterial translocation studies, intravenous and intravenous administration and genotoxicity assay in murine model. The outcome of this toxicological safety assessment indicated that both the test strains lacked any harmful metabolic activity or any genotoxic effects. Furthermore, the results of oral toxicity studies in mice revealed that short term administration of high cell mass concentration of 1012 cfu/animal as well as long term feeding of the probiotic strains did not alter any hematological, general health parameters or cause any organ specific disorder. Based upon these scientific assessments and supported by long history of safe use, both MTCC 5690 and MTCC 5689 may be considered safe for human consumption.

Mechanistic insights into the host-microbe interaction and pathogen exclusion mediated by the Mucus-binding protein of Lactobacillus plantarum.

Surface adhesins of pathogens and probiotics strains are implicated in mediating the binding of microbes to host. Mucus-binding protein (Mub) is unique to gut inhabiting lactic acid bacteria; however, the precise role of Mub proteins or its structural domains in host-microbial interaction is not well understood. Last two domains (Mubs5s6) of the six mucus-binding domains arranged in tandem at the C-terminus of the Lp_1643 protein of Lactobacillus plantarum was expressed in E. coli. Mubs5s6 showed binding with the rat intestinal mucus, pig gastric mucins and human intestinal tissues. Preincubation of Mubs5s6 with the Caco-2 and HT-29 cell lines inhibited the binding of pathogenic enterotoxigenic E. coli cells to the enterocytes by 68% and 81%, respectively. Pull-down assay suggested Mubs5s6 binding to the host mucosa components like cytokeratins, Hsp90 and Laminin. Mubs5s6 was predicted to possess calcium and glucose binding sites. Binding of Mubs5s6 with these ligands was also experimentally observed. These ligands are known to be associated with pathogenesis suggesting Mub might negotiate pathogens in multiple ways. To study the feasibility of Mubs5s6 delivery in the gut, it was encapsulated in chitosan-sodium tripolyphosphate microspheres with an efficiency of 65% and release up to 85% in near neutral pH zone over a period of 20 hours. Our results show that Mub plays an important role in the host-microbial cross-talk and possesses the potential for pathogen exclusion to a greater extent than mediated by L. plantarum cells. The functional and technological characteristics of Mubs5s6 make it suitable for breaking the host-pathogen interaction.

Improvement in glucose tolerance and insulin sensitivity by probiotic strains of Indian gut origin in high-fat diet-fed C57BL/6J mice.

PURPOSE: Diabetes and obesity are characterized by glucose intolerance, fat deposition, inflammation, and dyslipidemia. Recent reports postulated that distinct gut microbiota alterations were observed in obese/diabetic subjects and modulating gut microbiota beneficially through specific probiotics could be a potential therapeutic option for type 2 diabetes/obesity. Therefore, we attempted to study the efficacy of probiotics of Indian gut origin (Lactobacillus plantarum MTCC5690 and Lactobacillus fermentum MTCC5689) along with a positive control, Lactobacillus rhamnosus (LGG) on glucose/lipid homeostasis in high-fat-diet-induced diabetic animal model. METHODS: C57BL/6J male mice were divided into seven groups (n = 6 per group) comprising feeding on: (1) Normal Pellet Diet (NPD), (2) High-Fat Diet (HFD), (3) HFD with LGG, (4) HFD with MTCC5690, (5) HFD with MTCC5689, (6) HFD with metformin, and 7) HFD with vildagliptin for a period of 6 months. Biochemical markers, glucose tolerance, insulin resistance, and GLP-1 and LPS levels were assessed by standard protocols. Gut integrity was measured by intestinal permeability test. Transcriptional levels of tight junction proteins (TJPs) were probed in small intestinal tissues while inflammatory signals and other pathway specific genes were profiled in liver, visceral adipose tissue, and skeletal muscle. RESULTS: Mice fed with HFD became insulin resistant, glucose intolerant, hyperglycemic, and dyslipidemic. Diabetic mice were characterized to exhibit decreased levels of GLP-1, increased gut permeability, increased circulatory levels of LPS, decrease in the gene expression patterns of intestinal tight junction markers (occludin and ZO-1), and increased proinflammatory gene markers (TNFα and IL6) in visceral fat along with decreased mRNA expression of FIAF and adiponectin. Diabetic mice also exhibited increased mRNA expression of ER stress markers in skeletal muscle. In addition, liver from HFD-fed diabetic mice showed increased gene expressions of proinflammation, lipogenesis, and gluconeogenesis. Probiotic interventions (most prominently the MTCC5689) resisted insulin resistance and development of diabetes in mice under HFD feeding and beneficially modulated all the biochemical and molecular alterations in a mechanistic way in several tissues. The metabolic benefits offered by the probiotics were also more or less similar to that of standard drugs such as metformin and vildagliptin. CONCLUSION: Native probiotic strains MTCC 5690 and MTCC 5689 appear to have potential against insulin resistance and type 2 diabetes with mechanistic, multiple tissue-specific mode of actions.

Expression of fibronectin-binding protein of L. acidophilus NCFM and in vitro refolding to adhesion capable native-like protein from inclusion bodies.

The ability of Lactobacilli to adhere to host epithelial surface and intestinal tracts is important for colonization and persistence of bacteria in the host gut. Extracellular matrix components like fibronectin, mucin, collagen and other adhesion molecules serve as substratum for attachment of bacteria. However, the precise structure, function and mechanism of binding of microbial surface adhesion proteins such as Fibronectin-binding protein (FBP) with host molecules remains unclear. This is primarily due to limitations in high expression of these proteins in biologically active form. To study adhesion of its FBP (64 kDa), the fbp gene of L. acidophilus NCFM was cloned and expressed in E. coli. However, the fibronectin-binding protein expressed in soluble form could not be purified by Ni-NTA affinity chromatography possibly because of partially buried Histidine tag in the recombinant fusion protein. Therefore, the protein was expressed as inclusion bodies (IBs) at 37 °C and solubilized in urea followed by purification in denatured form by Ni-NTA affinity chromatography. The purified denatured protein was refolded in vitro to structurally stable and biologically active form. The conformational properties of the refolded protein were studied by circular dichroism, which showed prominence of α+ ß structural element. The refolded FBP also showed significant binding to human intestinal tissue sections. Our optimized refolding protocol from IBs of this recombinant probiotic FBP led into high amounts of biologically active protein. Our results help in increasing understanding of structure-function relation of surface adhesion proteins and host-microbial interactions.

Expression of recombinant truncated domains of mucus-binding (Mub) protein of Lactobacillus plantarum in soluble and biologically active form.

Mucins amount to 70% of total proteins present in mammalian mucus and serve as important substrata for bacterial adhesion. In probiotic bacteria such as Lactobacillus plantarum, surface adhesion proteins mediate its adhesion to mucus and adhesion is pivotal in bi-directional host-microbe interactions. Mucus binding (Mub) proteins are a group of bacterial surface adhesion proteins that bind to mucin proteins. The structural framework and functional role of these proteins needs immediate attention but is poorly understood because of their large size, low yield and lack of highly purified protein. The lp_1643 gene of L. plantarum encodes a large Mub protein of 240 kDa and has six mucus binding (Mub) domains in tandem. In this study, the fragment of lp_1643 containing the last two domains with their preceding spacers herein referred to as Mubs5s6 was cloned and expressed in E. coli for probing its functional role in the adhesion of L. plantarum. The protein was expressed with a solubility enhancing maltose binding protein (MBP) fusion tag, yet the MBP-Mubs5s6 protein expressed majorly (>90%) as biologically insoluble inclusion bodies. Thus, extensive optimization of culture conditions was carried out to achieve high level soluble expression (∼70%) of Mubs5s6 protein from its initial low level of solubility. The recombinant protein was purified up to homogeneity by affinity chromatography. Recombinant MBP-Mubs5s6 protein showed strong adhesion potential by binding with human intestinal tissue sections. Our results show a step-by-step hierarchical approach to improve the solubility of difficult-to-express extracellular surface proteins while retaining high functional viability.

Adhesion of Lactobacilli and their anti-infectivity potential.

The probiotic potential of lactic acid bacteria primarily point toward colonizing ability of Lactobacilli as the most important attribute for endowing all the known beneficial effects in a host. Lactobacillus species exert health-promoting function in the gastrointestinal tract through various mechanisms such as pathogen exclusion, maintenance of microbial balance, immunomodulation, and other crucial functions. It has been seen that many surface layer proteins are involved in host adhesion, and play significant role in the modification of some signaling pathways within the host cells. Interaction between different bacterial cell surface proteins and host receptor has been imperative for a better understanding of the mechanism through which Lactobacilli exert their health-promoting functions.

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.

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.

Live and heat-killed probiotic Lactobacillus casei Lbs2 protects from experimental colitis through Toll-like receptor 2-dependent induction of T-regulatory response.

Inflammatory bowel disease (IBD) is a group of inflammatory disorders of the intestine caused by dysregulated T-cell mediated immune response against commensal microflora. Probiotics are reported as therapeutically effective against IBD. However, variable efficacy of the live probiotic strains, difference in survival and persistence in the gut between the strains and the lack of insight into the mechanisms of probiotic action limit optimal therapeutic efficacy. Our aims were to evaluate the lactobacillus strains isolated from the North Indian population for the generation of regulatory cells and cytokines in the intestine, to study their effects on pro-inflammatory mediators in the mouse model of inflammatory bowel disease and to explore the underlying mechanisms of their actions. Among the selected lactobacillus strains, Lactobacillus casei Lbs2 (MTCC5953) significantly suppressed lipopolysaccharide-induced pro-inflammatory cytokine (TNF-alpha, IL-6) secretion. Both live and heat-killed Lbs2 polarized Th0 cells to T-regulatory (Treg) cells in vitro, increased the frequency of FoxP3(+) Treg cells in the mesenteric lymph nodes (MLNs) and alleviated macroscopic and histopathological features of colitis in probiotic-fed mice. Moreover, the levels of IL-12, TNF-alpha and IL-17A were suppressed, while IL-10 and TGF-beta levels were augmented in the colonic tissues of Lbs2-treated mice. The induced Treg (iTreg) cells secreted IL-10 and TGF-beta and exerted suppressive effects on the proliferation of effector T-cells. Adoptive transfer of iTreg cells ameliorated the disease manifestations of murine colitis and suppressed the levels of TNF-alpha and IL-17A. Finally, Lbs2 effects were mediated by Toll-like receptor 2 (TLR2) activation on the dendritic cells. This study identified live and heat-killed Lbs2 as putative therapeutic candidates against IBD and highlighted their Toll-like receptor 2-dependent immunomodulatory and regulatory function.

Synthetic Biology: Applications in the Food Sector.

Synthetic biology also termed as "genomic alchemy" represents a powerful area of science that is based on the convergence of biological sciences with systems engineering. It has been fittingly described as "moving from reading the genetic code to writing it" as it focuses on building, modeling, designing and fabricating novel biological systems using customized gene components that result in artificially created genetic circuitry. The scientifically compelling idea of the technological manipulation of life has been advocated since long time. Realization of this idea has gained momentum with development of high speed automation and the falling cost of gene sequencing and synthesis following the completion of the human genome project. Synthetic biology will certainly be instrumental in shaping the development of varying areas ranging from biomedicine, biopharmaceuticals, chemical production, food and dairy quality monitoring, packaging, and storage of food and dairy products, bioremediation and bioenergy production, etc. However, potential dangers of using synthetic life forms have to be acknowledged and adoption of policies by the scientific community to ensure safe practice while making important advancements in the ever expanding field of synthetic biology is to be fully supported and implemented.

Exploring specific primers targeted against different genes for a multiplex PCR for detection of Listeria monocytogenes.

The efficacy of six different sets of primers targeted against 16S rRNA and virulence genes such as 'iap', 'hly' and 'prf' was evaluated in separate PCR assays. The primer pairs targeted against 16S rRNA resulted into amplification of 1.2 kb PCR product. However, sets of primers targeted against different regions of 'iap' produced 371 and 660 bp PCR products, respectively. The primer pair targeted against 'prf' gene could produce 508 bp product. Three primer pairs targeted against different regions of 'hly', i.e., 'hly', 'hly A' and 'hly K9' were able to amplify 713, 276 and 384 bp products, respectively. The PCR conditions were also optimized in respect of two internal sets of primers falling within 'iap' and 'hly' genes that amplified 119 and 188 bp products to verify the PCR results obtained with respective external sets of primers. Three different combinations involving four sets of primers based on 16S rRNA, 'iap', 'hly' and 'prf' were explored in respective multiplex PCR assays in order to select a suitable combination. Combination 1 and 3 worked successfully as revealed by amplification of all the four bands of expected sizes on agarose gel. However, while optimizing the different parameters for developing a functional multiplex PCR, it was observed that in both these combinations, only two of the amplified products, i.e., 1.2 kb and 713 bp could be invariably detected. Hence, these two primers were combined in the multiplex PCR and the conditions were optimized for application in dairy foods for detection of Listeria monocytogenes.

Adhesion of indigenous Lactobacillus plantarum to gut extracellular matrix and its physicochemical characterization.

Adhesion to the human intestinal epithelial cell is considered as one of the important selection criteria of lactobacilli for probiotic attributes. Sixteen Lactobacillus plantarum strains from human origins were subjected for adhesion to extracellular matrix (ECM) components, and their physiochemical characterization, incubation time course and effect of different pH on bacterial adhesion in vitro were studied. Four strains showed significant binding to both fibronectin and mucin. After pretreatment with pepsin and trypsin, the bacterial adhesion to ECM reduced to the level of 50 % and with lysozyme significantly decreased by 65-70 %. Treatment with LiCl also strongly inhibited (90 %) the bacterial adhesion to ECM. Tested strains showed highest binding efficacy at time course of 120 and 180 min. Additionally, the binding of Lp91 to ECM was highest at pH 6 (155 ± 2.90 CFU/well). This study proved that surface layer components are proteinaceous in nature, which contributed in adhesion of lactobacillus strains. Further, the study can provide a better platform for introduction of new indigenous probiotic strains having strong adhesion potential for future use.

Draft Genome Sequence of Lactobacillus casei Lbs2.

We report here a 3.2-Mb draft assembled genome of Lactobacillus casei Lbs2. The bacterium shows probiotic and immunomodulatory activities. The genome assembly and annotation will help to identify molecules and pathways responsible for interaction between the host immune system and the microbe.