The Role of OmpR in Bile Tolerance and Pathogenesis of Adherent-Invasive Escherichia coli

Gut microbiota dysbiosis toward adherent-invasive Escherichia coli (AIEC) plays an important role in Crohn’s disease (CD). The OmpR transcriptional regulator is required for the AIEC LF82 prototype strain to adhere and invade intestinal epithelial cells. In this study, we explored the role of OmpR in AIEC pathogenesis using a panel of eight Escherichia coli strains isolated from CD patients and identified as AIEC. The deletion of ompR together with the implementation of two cell-based assays revealed that the role of OmpR in adhesion in vitro was not conserved in AIEC clinical strains. Nevertheless, we showed that OmpR was required for robust gut colonization of transgenic mice expressing human CEACAM receptors, suggesting that OmpR is involved in alternative virulence mechanisms in AIEC strains. We found that deletion of ompR compromised the ability of AIEC strains to cope with the stress induced by bile salts, which may be key for AIEC pathogenesis. More specifically, we demonstrated that OmpR was involved in a tolerance mechanism toward sodium deoxycholate (DOC), one of bile salts main component. We showed that the misregulation of OmpF or the loss of outer membrane integrity are not the drivers of OmpR-mediated DOC tolerance, suggesting that OmpR regulates a specific mechanism enhancing AIEC survival in the presence of DOC. In conclusion, the newly discovered role of OmpR in AIEC bile tolerance suggests that OmpR inhibition would interfere with different aspects of AIEC virulence arsenal and could be an alternative strategy for CD-treatment.

In Vitro Evaluation of Probiotic Properties of Indigenous Yeasts Isolated from Nigerian Fermented Food Products

Probiotics are living microbial food supplements which beneficially affect the host by improving the intestinal microbial balance. Before an organism can be designated as probiotic there are certain criteria that must be fulfilled. These include acid and bile tolerance, antimicrobial activity, ability to co-aggregate, hydrophobicity etc. One hundred and eighty one indigenous yeast isolates recovered from various fermented food products of Nigeria were characterized and grouped using phenotypic methods. Forty two selected yeast isolates were identified using molecular method which involved sequencing of D1 and D2 domain of the large subunit of ribosomal DNA. Then nine indigenous Saccharomyces cerevisiae were evaluated for their probiotic characteristics such as acid and bile tolerance, transit in simulated gastric and intestinal juices, autoaggregation and hydrophobicity. Saccharomyces cerevisiae SC10 was included as a positive control. The S. cerevisiae were able to grow in the presence of acidic medium with pH as low as 2 and 3. In the minimum inhibitory concentration test with 0-1% ox bile, all the S. cerevisiae tested were able to grow. The growth for 3% bile tolerance test ranged from 4.81 to 5.35 log cfu/ml. These isolates were able to survive in simulated gastro-intestinal transit. All the yeast isolates exhibited bile salt deconjugation activity against sodium glycodeoxycholate and were able to grow in the presence of all other bile salts investigated. Autoaggregation ability (an adhesive property) of the indigenous yeast isolates ranged from 89.80% for S. cerevisiae BK19 to 99.91% for S. cerevisiae OB03. The native yeast isolates also exhibited high percentage hydrophobicity, another adhesive property of probiotics. The values obtained ranged from 31.62 to 83.45% for isolates AG23A and OB 17. These observations indicate that the native yeast isolates from Nigerian fermented foods have the potential of being use as probiotics for making functional foods.

Enhancing bile tolerance of Lactobacilli is involved in the hypolipidemic effects of liraglutide

Liraglutide is an analogue of human glucagon-like peptide-1 (GLP-1), an endogenous intestinal hormone which play essential roles in the regulation of glycolipid metabolism. To investigate the role of lactic acid bacteria (LAB) in the lipid-lowering effect of liraglutide, forty mice were divided into normal saline-treated basal diet (NFD), normal saline-treated high-fat food (HFD), 10.0 mg/kg/d simvastatin-treated HFD (SIM + HFD), 200 and 400 μg/kg/d liraglutide-treated HFD (LL + HFD and HL + HFD) groups for 5 weeks. 16S rDNA sequencing, real-time quantitative PCR and western blot were used to detected changes of intestinal flora, cholesterol 7α-hydroxylase (CYP7A1), LDL-receptor (LDLR) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Results showed that liraglutide could up-regulate CYP7A1 and LDLR, whereas down-regulate HMGCR. Besides, liraglutide enhance the abundance of lactobacillaceae in gut of hyperlipidemic mice and increase the bile tolerance ability of LAB by up-regulating bile salt hydrolases, and the lysate of liraglutide-sensitive LAB could also directly down-regulate HMGCR, the key enzyme in cholesterol synthesis, and inhibit hepatocyte steatosis. These findings might provide new theoretical guidance for clinical application of liraglutide and threw a light on research and development of anti-obesity, hypolipidemic and cholesterol-lowering drugs or functional foods.

Quantitative assessment of available probiotic products in community pharmacies in Benin City, Nigeria

Purpose: To assess four probiotic products available in community pharmacies in Benin City, Nigeria for accuracy of information on product labels with regard to the quantity and type of microorganisms, pH and bile tolerance, and antimicrobial activity.Methods: Percent label compliance of products was determined, in addition to isolation, identification and enumeration of microorganisms. Determination of pH and bile tolerance was conducted using turbidity studies in MRS broth. Antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Klebsiella pneumonia, Staphylococcus aureus and Candida albicans pathogens was investigated using agar overlay technique.Results: In each product, there was 100 % label compliance with regard to name of probiotic organism, storage condition, dose, expiration date, contact details and batch number. Three-quarters (75 %) of the probiotic products indicated product net quantity, National Agency for Food and Drug Administration and Control (NAFDAC) number, and microbial count; 50 % of products indicated the excipients used, while only 25 % of the products showed their indications. None of the products indicated strain designation. In species identification, Enterococcus faecium was absent in a multi-species product PB1, while PB3 contained Saccharomyces cerevisiae instead of Saccharomyces boulardii. Enumeration showed comparatively low quantities of probiotic organisms. Tolerance to pH 3 and pH 7, and bile levels of 0.3 and 2 % were within acceptable range. The probiotic organisms demonstrated antimicrobial effect specifically against P. aeruginosa, E. coli, B. subtilis, K. pneumonia, S. aureus and C. albicans.Conclusion: Antimicrobial effect and tolerance to pH and bile salts were consistent with acceptable properties of probiotics. However, there is need for total compliance with the indications, strain designation, excipients, and actual quantity of the individual probiotic organisms in the formulations. Keywords: Probiotics, Strain designation, Agar overlay technique, Saccharomyces cerevisiae, Saccharomyces boulardii

The effect of bile acids on the growth and global gene expression profiles in Akkermansia muciniphila

Akkermansia muciniphila is a prominent member of the gut microbiota and the organism gets exposed to bile acids within this niche. Several gut bacteria have bile response genes to metabolize bile acids or an ability to change their membrane structure to prevent membrane damage from bile acids. To understand the response to bile acids and how A. muciniphila can persist in the gut, we studied the effect of bile acids and individual bile salts on growth. In addition, the change in gene expression under ox-bile condition was studied. The growth of A. muciniphila was inhibited by ox-bile and the bile salts mixture. Individual bile salts have differential effects on the growth. Although most bile salts inhibited the growth of A. muciniphila, an increased growth was observed under culture conditions with sodium deoxycholate. Zaragozic acid A, which is a squalene synthase inhibitor leading to changes in the membrane structure, increased the susceptibility of A. muciniphila to bile acids. Transcriptome analysis showed that gene clusters associated with an ABC transporter and RND transporter were upregulated in the presence of ox-bile. In contrast, a gene cluster containing a potassium transporter was downregulated. Membrane transporter inhibitors also decreased the tolerance to bile acids of A. muciniphila. Our results indicated that membrane transporters and the squalene-associated membrane structure could be major bile response systems required for bile tolerance in A. muciniphila. Key points • The growth of Akkermansia muciniphila was inhibited by most bile salts. • Sodium deoxycholate increased the growth of A. muciniphila. • The genes encoding transporters and hopanoid synthesis were upregulated by ox-bile. • The inhibitors of transporters and hopanoid synthesis reduced ox-bile tolerance.

Microencapsulation of Probiotics by Exopolysaccharides-sodium Alginate and Evaluation of their Survival in Simulated GI Conditions

Background: The viability of the probiotics reduces because of the high acid and bile salt concentrations present in the stomach. Probiotic cultures can be protected during processing, storage and passage of the probiotic product through the gastrointestinal tract by the technique of microencapsulation. Objective: In the present study, Exopolysaccharides (EPS) and sodium alginate are incorporated in a different ratio to form the encapsulation matrix. They have also been checked for improving the viability of lactic acid bacteria in simulated GI conditions. Methods: EPS were isolated from a reference strain of L. acidophillus and the two strains P8 and Cu3 isolated from pumpkin and cucumber. Microencapsulated beads were formed using a different combination of EPS and sodium alginate and were analysed in terms of viability, bile tolerance, acid tolerance, survival in gastrointestinal conditions, and antibiotic susceptibility conditions. Results: The results indicated that the increasing concentration of EPS has significantly increased the survival of microencapsulated probiotic in simulated gastrointestinal conditions. The treatment T4 was found to be the best for all the tested conditions. Conclusion: This study has shown the potential application of EPS in microencapsulation to protect probiotics against the adverse condition of the gastrointestinal tract.

Probiotic Potential of Traditional Indian Fermented Drinks

Probiotics are living microorganisms, which when taken in adequate amount, provide various health benefits by maintaining the balance of bacteria in the intestine. Probiotics are purported to have countless health benefits, some of which include improved digestion, enhanced immunity, prevention of cancer and diabetes. The most common group of probiotics include species of Lactobacillus, Bifidobacterium and Enterococcus. In order to work as an effective probiotic, the microbial strain is expected to exhibit certain desirable characteristics like acid and bile tolerance, antimicrobial activity, adhesion to intestinal epithelium, etc. The fermented products contain a myriad of bacteria, some of which are characterized as probiotics and are responsible for various health benefits associated with the product. The fermented foods and drinks have been consumed in India since time immemorial. The art of fermentation has been a part of the traditional knowledge of India for thousands of years. The use of fermented products is strongly linked to the culture and tradition of India. Some traditional fermented drinks of India having probiotic potential include Koozh, Toddy, Kanji, Hamei and Handia. Further research on the probiotic potential of traditional fermented drinks may pave a path for their medical usage and commercial development.

Anti-microbial activity of potential probiotic lactic acid bacteria against Methicillin-Resistant Staphylococcus Aureus (MRSA)

Lactic acid bacteria (LAB)are the essential ingredients in probiotic foods, intestinal microflora, and dairy products able to cope up and exist in diverse environmental ranges. Samples were collected using sterile test tubes and transported to a laboratory in the icebox for further biochemical characterization. Gram test and catalase activity were examined after microscopically distinct colonies were sub-cultured to pure colonies based on standard gram and catalase test protocols. Subsequently, these bacteria were characterized for their ability to grow at various salt concentrations (5%,10%, and15%) and temperature gradients (15°C, 30°C, 45°C). Acid-tolerance was analyzed by growing the colonies in MRS broth adjusted to acidic pH (pH 3) and pH 7.2 (control). Bile tolerance of LAB isolates was assayed by growing in 0.3% bile-supplemented MRS agar. Bile salt hydrolase (BSH) activity was studied by growing 10 μl of the prepared overnight culture on BSH screening media containing MRS agar plate supplemented with bile salts. The LAB isolates were checked for antimicrobial activity by agar well diffusion assay. All isolates found gram-positive, catalase-negative and non-motile, convex elevation and entire margin. All LAB isolates were able to grow at 5-10% Nacl concentration, whereas moderately grow at 10% concentration but rarely grow at 15% Nacl concentration. BCM2, BBM3 and BGM1 record the highest acidic resistance viability percentage 94.9%, 92.7%, and 91.8% respectively. BCM3 has the lowest acidic resistance with a viable percentage of 87.4%. BBM1 records the highest bile tolerance activity whereas BCM2 has the lowest bile tolerance. All isolates were found BSH positive. The study reveals LAB isolates showed a putative probiotic potential.Article highlightsLAB are the main ingredients of probiotic products commercially available in the market nowadays. To effectively functioning the host gastrointestinal tract probiotics, need to have certain criteria like acid and bile tolerance this study revealsAcid tolerance activity of LAB isolated from bovine milkBile tolerance activity of LAB isolated from bovine milkBile salt hydrolase (BSH) activity of LAB isolatesAntimicrobial activity of LAB against MRSA

Investigation of bile tolerance and deconjugation ability of various Lactobacillus Casei group strains

Lactobacillus casei group is commonly used as probiotics. Various factors affect their viability in gastrointestinal tract. Twenty two strains of Lactobacillus casei group were assessed for bile tolerance and deconjugation attribute. All strains had shown moderate activity at 0% bile concentration. At 1% bile concentration CSCC 2607 has shown highest growth while remaining Lactobacillus strains have shown fair growth. At 1.5% bile concentration only seven Lactobacillus strains showed growth and ASCC 1521 showed highest activity. All seven strains were able to deconjugate both sodium glycocholate and sodium taurocholate at varying levels. ASCC 1520 showed lowest deconjugation activity for sodium glycocholate. All seven strains showed lower deconjugation of sodium taurocholate compared to sodium glycocholate. ASCC 290 was least capable of deconjugation of sodium taurocholate. When mixture of sodium glycocholate and sodium taurocholate having the similar molar ratio in human bile, was taken, all seven strains showed consistent cholic acid secretion. ASCC 1521 also showed highest cholic acid liberation while ASCC 1520 showed lowest deconjugation ability. This study suggests the importance to identify all the useful Lactobacillus strains which are able to tolerate and deconjugate, high bile concentration in order to study further for their therapeutic benefits.

Preliminary Studies on Isolation, Bile Tolerance and Antibiogram of Potential Probiotics (Probionts) from Locally Fermented Food Products at Beach Market, Nsukka Metropolis, Enugu State, Nigeria

Aims: The study aims to isolate, evaluate bile tolerance and antibiogram studies of potential probiotics (Lactobacillus spp) from locally fermented Food Products (Akamu, Aqua Rafa® Yoghurt, Ogiri, Okpeye) and Kunu at Beach Market, Nsukka. Study Design: A ten - fold serial dilution and spread plate method using De Man, Rogosa and Sharpe (MRS) medium was adopted for isolation of potential Probionts. Place and Duration of Study: Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, University of Nigeria Nsukka, between March - September, 2018. Methodology: Only catalase negative and Gram positive isolates characteristic of lactobacilli were used. Bile tolerance test was performed by monitoring the bacterial growth at different Bile salt concentrations (0.2%, 0.3% and 2%). The antibiogram of the isolates was assessed using the Kirby-Bauer disc diffusion method against commercial antibiotic discs of ampicillin, vancomycin, gentamycin, ciprofloxacin, methicillin and erythromycin. Results: All the 18 screened isolates were tolerant to bile salt at 0.2 % and 0.3 % with inhibition of growth at 2 % bile concentration. All isolates were observed to be resistant to methicillin (100 %) but very sensitive to gentamycin (11%) and ciprofloxacin (22%) respectively. The isolates showed intermediate resistance to other antibiotics: vancomycin (33%), erythromycin (33%) and ampicillin (44%). The decreasing pattern of resistance was thus: methicillin > ampicillin > vancomycin and erythromycin > ciprofloxacin > gentamycin. Isolates from Yoghurt (66.67%) and Ogiri (53.33%) provided most of the resistant isolates. Methicillin would provide best antagonist potential as all the isolates exhibited very high level of resistance (100 %). Conclusion: These results suggest that all the eighteen potential Lactobacillus spp strain show potential for probiotic applications and the locally fermented food products are rich sources of probionts.