Genome mining of Bacillus licheniformis MCC2514 for the identification of lasso peptide biosynthetic gene cluster and its characterization.

The probiotic strain Bacillus licheniformis MCC2514 has been shown to produce a strong antibacterial peptide and the whole genome sequence of this strain is also reported in our previous study. The present study is focused on the genome level investigation of this peptide antibiotic and its characterization. Genome mining of the culture revealed the presence of three putative bacteriocin clusters, viz. lichenicidin, sonorensin and lasso peptide. Hence, the mode of action of the peptide was investigated by reporter assay, scanning electron microscopy, and Fourier Transform Infrared spectroscopy. Additionally, the peptide treated groups of Kocuria rhizophila showed a reduction in the fold expression for transcription-related genes. The gene expression studies, quantitative ß-galactosidase induction assay using the RNA stress reporter strain, yvgS along with the homology studies concluded that lasso peptide is responsible for the antibacterial activity of the peptide which acts as an inhibitor of RNA biosynthesis. Gene expression analysis showed a considerable increase in fold expression of lasso peptide genes at various fermentation hours. Also, the peptide was isolated, and its time-kill kinetics and minimum inhibitory concentration against the indicator pathogen K. rhizophila were examined. The peptide was also purified and the molecular weight was determined to be ~ 2 kDa. Our study suggests that this bacteriocin can function as an effective antibacterial agent in food products as well as in therapeutics as it contains lasso peptide, which inhibits the RNA biosynthesis.

Improved production of RNA-inhibiting antimicrobial peptide by Bacillus licheniformis MCC 2514 facilitated by a genetic algorithm optimized medium.

One of the significant challenges during the purification and characterization of antimicrobial peptides (AMPs) from Bacillus sp. is the interference of unutilized peptides from complex medium components during analytical procedures. In this study, a semi-synthetic medium was devised to overcome this challenge. Using a genetic algorithm, the production medium of AMP is optimized. The parent organism, Bacillus licheniformis MCC2514, produces AMP in very small quantities. This AMP is known to inhibit RNA biosynthesis. The findings revealed that lactose, NH4Cl and NaNO3 were crucial medium constituents for enhanced AMP synthesis. The potency of the AMP produced was studied using bacterium, Kocuria rhizophila ATCC 9341. The AMP produced from the optimized medium was eightfold higher than that produced from the unoptimized medium. Furthermore, activity was increased by 1.5-fold when cultivation conditions were standardized using the optimized medium. Later, AMP was produced in a 5 L bioreactor under controlled conditions, which led to similar results as those of shake-flask production. The mode of action of optimally produced AMP was confirmed to be inhibition of RNA biosynthesis. Here, we demonstrate that improved production of AMP is possible with the developed semi-synthetic medium recipe and could help further AMP production in an industrial setup.

Lactic acid bacteria in some Indian fermented foods and their predictive functional profiles.

Lactic acid bacteria (LAB) were isolated from naturally fermented foods of India, viz., sidra, a dried fish product; kinema, a naturally fermented sticky soybean food; and dahi, a naturally fermented milk product. Five strains of LAB, based on 16S rRNA gene sequence, were identified: Lactococcus lactis FS2 (from sidra), Lc. lactis C2D (dahi), Lc. lactis SP2C4 (kinema), Lactiplantibacillus plantarum DHCU70 (=Lactobacillus plantarum) (from dahi), and Lactiplantibacillus plantarum KP1 (kinema). The PICRUSt2 software, a bioinformatic tool, was applied to infer the raw sequences obtained from LAB strains mapped against KEGG database for predictive functionality. Functional features of LAB strains showed genes associated with metabolism (36.47%), environmental information processing (31.42%), genetic information processing (9.83%), and the unclassified (22.28%). KEGG database also showed abundant genes related to predictive membrane transport (29.25%) and carbohydrate metabolism (11.91%). This study may help in understanding the health-promoting benefits of the culturable LAB strains in fermented foods.

Structural characteristics and functional properties of a fucose containing prebiotic exopolysaccharide from Bifidobacterium breve NCIM 5671.

AIM: To evaluate the structure and functions of capsular exopolysaccharide (CPS) from Bifidobacterium breve NCIM 5671. METHODS AND RESULTS: A CPS produced by the probiotic bacteria B. breve NCIM 5671 was isolated and subjected to characterization through GC analysis, which indicated the presence of rhamnose, fucose, galactose, and glucose in a molar ratio of 3:1:5:3. The average molecular weight of the CPS was determined to be ∼8.5 × 105 Da. Further, NMR analysis revealed the probable CPS structure to be composed of major branched tetra- and penta-saccharide units alternately repeating and having both α- and ß-configuration sugar residues. CPS displayed an encouraging prebiotic score for some of the studied probiotic bacteria. Compared to standard inulin, CPS showed better resistance to digestibility against human GI tract in vitro. DPPH, total antioxidant, and ferric reducing assays carried out for CPS displayed decent antioxidant activity too. CONCLUSION: This study indicates that the CPS from B. breve NCIM 5671 has the potential to be utilized as a prebiotic food supplement. It is a high-molecular-weight (∼8.5 × 105 Da) capsular heteropolysaccharide containing rhamnose, fucose, galactose, and glucose.

In vitro fermentation of glycosaminoglycans from mackerel fish waste and its role in modulating the antioxidant status and gut microbiota of high fat diet-fed C57BL/6 mice.

Bioactive polysaccharides such as glycosaminoglycans (GAGs) exhibit potential health benefits for several health complications including obesity. The gut microbiota plays a key role in regulating host metabolism, nutrition and immunity. The present work assessed the potential of extracted GAGs (e-GAGs) in maintaining the gut microbiota and ameliorating the effects of high fat diet in in vitro and in vivo models. The in vitro fermentability of e-GAGs extracted from mackerel fish waste was analyzed with Lactobacillus plantarum (LP) and Bifidobacterium bifidum (BB); e-GAGs at 0.5 and 1% proved their prebiotic nature up to 48 h. The pH value decreased from 6.23 to 3.32, the cell density increased from 1.70 to 2.32, the viable cell count increased from 8 to 12 log CFU mL-1, and short chain fatty acid (SCFA) production was ≈33, 31 and 36% for LP and ≈37, 29 and 34% for BB in terms of acetic acid, propionic acid and butyric acid, respectively. In vivo studies on high fat diet (HFD)-fed C57BL/6 mice with e-GAGs (380 and 760 mg kg-1 diet) showed ameliorated gut microbiome and tissue/plasma antioxidant enzyme activities, and also the e-GAG-fed group showed significantly (P < 0.05) decreased lipid peroxidation. Cecal microbial analysis showed the health-promoting effects of e-GAGs in reducing (P < 0.05) the obesity ratio of Firmicutes to Bacteroidetes (F/B) within the range (5.32 and 5.26) compared with HFD (6.23). Hence, e-GAGs can be a potential molecule for the treatment of obesity by restoring the redox status under oxidative stress and ameliorating the gut microbes that produce SCFAs which are known to have health beneficial effects.

Effect of probiotics as an immune modulator for the management of COVID-19.

COVID-19, an acute respiratory viral infection conveyed by pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected millions of individuals globally, and is a public health emergency of international concern. Till now, there are no highly effective therapies for this infection without vaccination. As they can evolve quickly and cross the strain level easily, these viruses are causing epidemics or pandemics that are allied with more severe clinical diseases. A new approach is needed to improve immunity to confirm the protection against emerging viral infections. Probiotics can modify gut microbial dysbiosis, improve the host immune system, and stimulate immune signaling, increasing systemic immunity. Several probiotic bacterial therapies have been proven to decrease the period of bacterial or viral infections. Superinduction of inflammation, termed cytokine storm, has been directly linked with pneumonia and severe complications of viral respiratory infections. In this case, probiotics as potential immunomodulatory agents can be an appropriate candidate to improve the host's response to respiratory viral infections. During this COVID-19 pandemic, any approach that can induce mucosal and systemic immunity could be helpful. Here, we summarize contexts regarding the effectiveness of various probiotics for preventing virus-induced respiratory infectious diseases, especially those that could be employed for COVID-19 patients. In addition, the effects of probiotics, their mechanisms on different aspects of immune responses against respiratory viral infection, and their antiviral properties in clinical findings have been described in detail.

Prevalence of macrolide-lincosamide-streptogramin resistant lactic acid bacteria isolated from food samples.

Lactic acid bacteria (LAB) being a reservoir of antibiotic resistance genes, tend to disseminate antibiotic resistance that possibly pose a threat to human and animal health. Therefore, the study focuses on the prevalence of macrolide-lincosamide-streptogramin- (MLS) resistance among LAB isolated from various food samples. Diverse phenotypic and genotypic MLS resistance were determined among the LAB species (n = 146) isolated from fermented food products (n = 6) and intestine of food-producing animals (n = 4). Double disc, triple disc diffusion and standard minimum inhibitory concentration (MIC) tests were evaluated for phenotypic MLS resistance. Specific primers for MLS resistance genes were used for the evaluation of genotypic MLS resistance and gene expressions using total RNA of each isolate at different antibiotic concentrations. The isolates identified are Levilactobacillus brevis (n = 1), Enterococcus hirae (n = 1), Limosilactobacillus fermentum (n = 2), Pediococcus acidilactici (n = 3), Enterococcus faecalis (n = 1). The MIC tests along with induction studies displayed cMLSb, L phenotype, M phenotype, KH phenotype, I phenotype resistance among MLS antibiotics. Genotypic evaluation tests revealed the presence of ermB, mefA/E, msrA/B and msrC genes. Also, gene expression studies displayed increased level of gene expression to the twofold increased antibiotic concentrations. In the view of global health concern, this study identified that food samples and food-producing animals represent source of antibiotic resistant LAB that can disseminate resistance through food chain. This suggests the implementation of awareness in the use of antibiotics as growth promoters and judicious use of antibiotics in veterinary sectors in order to prevent the spread of antibiotic resistance.

Bacillus licheniformis MCC 2514 genome sequencing and functional annotation for providing genetic evidence for probiotic gut adhesion properties and its applicability as a bio-preservative agent.

Bacillus licheniformis is a well-known probiotic that can be found in a variety of foods. The strain Bacillus licheniformis MCC 2514 was previously characterized by our group for its bio-physiological capabilities establishing it as a promising probiotic, but information on the genetic evidence for its attributes was lacking. In the current study, whole genome analysis identified the underlying molecular determinants responsible for its probiotic potential. The circular genome of MCC 2514 was 4,230,480 bp with 46.2% GC content, 24 rRNA, and 83 tRNA genes. The pangenome analysis between B. licheniformis MCC 2514 and 12 other B. licheniformis strains revealed a pangenome of 6008 genes and core genome of 3775 genes. Genome mining revealed NRPS and bacteriocins producing gene clusters indicating its biocontrol properties. Several genes encoding carbohydrate degrading enzymes, which aid in proper food degradation in the intestine, were also observed. Stress tolerance, vitamin, and essential amino acids biosynthesis related genes were found, which are important characteristics of a probiotic strain. Additionally, vital genes responsible for gut adhesion and biofilm formation were observed in its genome. The bacterium has been shown to improve the shelf life of idli batter by preventing whey separation, CO2, and odour production while maintaining the pH of 3.96-4.29, especially at cold temperatures. It has significantly reduced coliform contamination at both room and low temperatures, demonstrating its bio-preservative ability, which is also corroborated by the presence of the NRPS and bacteriocin gene clusters in its genome. The present study helped to understand both, the ability of B. licheniformis MCC 2514 to adapt the intestinal gut environment and its probiotic functionality for food preservation.

A machine learning-based approach to determine infection status in recipients of BBV152 (Covaxin) whole-virion inactivated SARS-CoV-2 vaccine for serological surveys.

Data science has been an invaluable part of the COVID-19 pandemic response with multiple applications, ranging from tracking viral evolution to understanding the vaccine effectiveness. Asymptomatic breakthrough infections have been a major problem in assessing vaccine effectiveness in populations globally. Serological discrimination of vaccine response from infection has so far been limited to Spike protein vaccines since whole virion vaccines generate antibodies against all the viral proteins. Here, we show how a statistical and machine learning (ML) based approach can be used to discriminate between SARS-CoV-2 infection and immune response to an inactivated whole virion vaccine (BBV152, Covaxin). For this, we assessed serial data on antibodies against Spike and Nucleocapsid antigens, along with age, sex, number of doses taken, and days since last dose, for 1823 Covaxin recipients. An ensemble ML model, incorporating a consensus clustering approach alongside the support vector machine model, was built on 1063 samples where reliable qualifying data existed, and then applied to the entire dataset. Of 1448 self-reported negative subjects, our ensemble ML model classified 724 to be infected. For method validation, we determined the relative ability of a random subset of samples to neutralize Delta versus wild-type strain using a surrogate neutralization assay. We worked on the premise that antibodies generated by a whole virion vaccine would neutralize wild type more efficiently than delta strain. In 100 of 156 samples, where ML prediction differed from self-reported uninfected status, neutralization against Delta strain was more effective, indicating infection. We found 71.8% subjects predicted to be infected during the surge, which is concordant with the percentage of sequences classified as Delta (75.6%-80.2%) over the same period. Our approach will help in real-world vaccine effectiveness assessments where whole virion vaccines are commonly used.

Preventive and therapeutic aspects of fermented foods.

In recent times, the status of some fermented foods which are considered as functional foods that confer health benefits in certain disease conditions has grown rapidly. The health benefits of fermented foods are due to the presence of probiotic microbes and the bioactive compounds formed during fermentation. Microbes involved and metabolites produced by them are highly species specific and contribute to the authenticity of the fermented foods. Several studies pertaining to the effect of fermented foods on various disease conditions have been conducted in recent years using both animal models and clinical trials on humans. This review focuses on the impact of fermented foods on conditions such as diabetes, cardiovascular disease, obesity, gastrointestinal disorder, cancer and neurodegenerative disorders.

Prevalence of polymyxin resistance through the food chain, the global crisis.

Antimicrobial resistance is one of the vital challenges facing global health today. Multi-drug resistant (MDR) infections are often treated with the narrow-spectrum drugs, colistin (polymyxin E) or polymyxin B, which are last-resort antibiotics for human therapeutics that are effective against Gram-negative bacteria. Unfortunately, resistance to these polymyxins has occurred because of selective pressure caused by the inappropriate use of those antibiotics, especially in farming. The mechanisms of resistance to polymyxins are mediated through intrinsic, mutational, or genetic alteration in chromosomal genes. The mechanism includes the regulatory network controlling chemical modifications of lipid A moiety of lipopolysaccharide, reducing the negative charge of lipid A and its affinity for polymyxins. Additionally, the unique mobile colistin/polymyxin B resistance (mcr) gene reported in Enterobacteriales is responsible for the horizontal dissemination of resistance to polymyxins via the food chain. There is now an urgent need to increase surveillance for detecting resistance to polymyxins. Therefore, this review presents an overview of presently available scientific literature on the mechanism of resistance to polymyxins, with their associated gene variants, evaluation methods, resistance transmission through the food chain via food bacteria, and related risk factors. We further focus on the significant implications of polymyxins usage in India and future views for food safety to preserve polymyxin activity.

Novel pathways in bacteriocin synthesis by lactic acid bacteria with special reference to ethnic fermented foods.

Ethnic fermented foods are known for their unique aroma, flavour, taste, texture and other sensory properties preferred by every ethnic community in this world culturally as parts of their eatables. Some beneficial microorganisms associated with fermented foods have several functional properties and health-promoting benefits. Bacteriocins are the secondary metabolites produced by the microorganisms mostly lactic acid bacteria present in the fermented foods which can act as lantibiotics against the pathogen bacteria. Several studies have been conducted regarding the isolation and characterization of potent strains as well as their association with different types of bacteriocins. Collective information regarding the gene organizations responsible for the potent effect of bacteriocins as lantibiotics, mode of action on pathogen bacterial cells is not yet available. This review focuses on the gene organizations, pathways include for bacteriocin and their mode of action for various classes of bacteriocins produced by lactic acid bacteria in some ethnic fermented foods.

Genome of Bifidobacterium longum NCIM 5672 provides insights into its acid-tolerance mechanism and probiotic properties.

Bifidobacterium longum NCIM 5672 is a probiotic strain isolated from the Indian infant feces. The probiotic efficacy of Bifidobacteria is majorly affected by its acid tolerance. This study determined the probiotic properties and acid-tolerance mechanism of B. longum NCIM 5672 using whole-genome sequencing. The genome annotation is carried out using the RAST web server and NCBI PGAAP. The draft genome sequence of this strain, assembled in 63 contigs, consists of 22,46,978 base pairs, 1900 coding sequences and a GC content of 59.6%. The genome annotation revealed that seven candidate genes might be involved in regulating the acid tolerance of B. longum NCIM 5672. Furthermore, the presence of genes associated with immunomodulation and cell adhesion support the probiotic background of the strain. The analysis of candidate acid- tolerance-associated genes revealed three genes, argC, argH, and dapA, may play an essential role in high acid tolerance in B. longum NCIM 5672. The results of RT-qPCR supported this conclusion. Altogether, the results presented here supply an effective way to select acid-resistant strains for the food industry and provide new strategies to enhance this species' industrial applications and health-promoting properties.

Distribution and Diversity of Nisin Producing LAB in Fermented Food.

An attempt was made, to characterize natural antibiotics or lantibiotics from unconventional sources and its antibacterial spectrum against food borne pathogens and drug resistant bacteria. Six different traditional fermented foods i.e., fermented fish, fermented soybeans, Soibum (fermented bamboo shoots), milk, idly and dosa batter were used for the isolation of bacteriocin producing Lactic acid bacteria (LAB). Among all bacterial cultures isolated from the various sources, 129 cultures have found to produce antimicrobial compounds. Nisin specific reporter bacteria was utilized as biosensor to identify the Nisin like bacteriocin, where 10 cultures found to be positive Nisin producer. Identified Nisin like bacteriocin was partially concentrated by using ammonium sulphate followed by butanol extraction. Minimum inhibitory concentration (MIC) was analyzed against food borne pathogen and drug resistant bacteria. MIC of partially purified Nisin (pp-Nisin) of all the LAB isolates against food-borne pathogens are ranged between 0.5 and 92 µg/ml respected to various Gram-positive bacteria. Similarly, the drug resistant bacteria were also inhibited by pp-Nisin (MIC ranged between 15 and 175 µg/ml). All samples of ppnisin exhibited auto induction ability. Taxonomic identification of the nisin producers was done by whole genome sequencing which reveals that cultures belongs to Lactococcus lactis ssp. lactis. Also it was found that Lactococcus lactis ssp. lactis C2d and Lactococcus lactis ssp. lactis SP2C4 harbor nisA gene and Lactococcus lactis ssp. lactis FS2 (L. lactis FS2) harbor nisQ gene. The finding of this study highlights the first case of L. lactis FS2 isolated from fermented fish harbor nisQ gene. Antibacterial activity of pp-Nisin against drug resistant LAB is also reported.

Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV2.

To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India) conducted a serosurvey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS-CoV2 anti-nucleocapsid (anti-NC) antibodies, 95% of which had surrogate neutralization activity. Three-fourth of these recalled no symptoms. Repeat serology tests at 3 (n = 607) and 6 (n = 175) months showed stable anti-NC antibodies but declining neutralization activity. Local seropositivity was higher in densely populated cities and was inversely correlated with a 30-day change in regional test positivity rates (TPRs). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of seropositivity were high-exposure work (odds ratio, 95% confidence interval, p value: 2.23, 1.92-2.59, <0.0001), use of public transport (1.79, 1.43-2.24, <0.0001), not smoking (1.52, 1.16-1.99, 0.0257), non-vegetarian diet (1.67, 1.41-1.99, <0.0001), and B blood group (1.36, 1.15-1.61, 0.001).

Role of probiotics to combat viral infections with emphasis on COVID-19.

Interspecies transmissions of viruses between animals and humans may result in unpredictable pathogenic potential and new transmissible diseases. This mechanism has recently been exemplified by the discovery of new pathogenic viruses, such as the novel severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) pandemic, Middle-East respiratory syndrome-coronavirus epidemic in Saudi Arabia, and the deadly outbreak of Ebola in West Africa. The. SARS-CoV-2 causes coronavirus disease-19 (COVID-19), which is having a massive global impact in terms of economic disruption, and, above all, human health. The disease is characterized by dry cough, fever, fatigue, myalgia, and dyspnea. Other symptoms include headache, sore throat, rhinorrhea, and gastrointestinal disorders. Pneumonia appears to be the most common and severe manifestation of the infection. Currently, there is no vaccine or specific drug for COVID-19. Further, the development of new antiviral requires a considerable length of time and effort for drug design and validation. Therefore, repurposing the use of natural compounds can provide alternatives and can support therapy against COVID-19. In this review, we comprehensively discuss the prophylactic and supportive therapeutic role of probiotics for the management of COVID-19. In addition, the unique role of probiotics to modulate the gut microbe and assert gut homeostasis and production of interferon as an antiviral mechanism is described. Further, the regulatory role of probiotics on gut-lung axis and mucosal immune system for the potential antiviral mechanisms is reviewed and discussed.Key points• Gut microbiota role in antiviral diseases• Factors influencing the antiviral mechanism• Probiotics and Covid-19.

Genome Analysis of Lactobacillus plantarum Isolated From Some Indian Fermented Foods for Bacteriocin Production and Probiotic Marker Genes.

In this study, Lactobacillus plantarum strain DHCU70 isolated from dahi, a fermented milk product and L. plantarum strain DKP1 isolated from kinema, a fermented soybean food of India, respectively were evaluated for their bacteriocin production and probiotic properties. Both strains of L. plantarum (DHCU70 and DKP1) were found to have potent antimicrobial activity against Kocuria rhizophila ATCC 9341. Bacteriocin produced by L. plantarum strains DHCU70 and DKP1 did not exhibit inhibition of cell wall, DNA and fatty acids biosynthesis mechanisms as evaluated by whole cell reporter assays. We characterized the bacteriocin encoding genes in L. plantarum strains DHCU70 and DKP1 by whole genome sequence which consisted of a single and circular chromosome with genome size of 3.38 Mb (GC content of 44.3%) and 3.39 Mb, respectively and a GC content of 44.3%. L. plantarum DHCU70 has 3252 number of protein encoding genes comprising 89 number of RNA genes (69tRNA, 16rRNA, 4nc RNA) whereas L. plantarum DKP1 has total of 3277 number of protein encoding genes with 89 number. of RNA genes (69tRNA, 16S rRNA, 4nc RNA). Analysis revealed the presence of 20.5 kb long and 23 numbers of plantaricin encoding locus (pln locus) for production of antimicrobial compound. BAGEL analysis has shown that the pln locus of both the strains of L. plantarum showed maximum sequence similarity with plantaricin NC8 of L. plantarum NC8, originally isolated from grass silage. Annotated whole genome sequence of both strains DHCU70 and DKP1 was analyzed for the presence of probiotic marker genes. The probiotic properties of these strains of were also evaluated in vitro. Due to the presence of genes responsible for antimicrobial activity and probiotic properties, both strains of L. plantarum may be considered as a suitable probiotic candidate in food industry.

In vivo safety assessment of Lactobacillus fermentum strains, evaluation of their cholesterol-lowering ability and intestinal microbial modulation.

BACKGROUND: Despite the growing importance of probiotics apparent health benefits, an impediment to the use of new probiotic cultures is their safety. Hence there is a need to strictly examine the biosafety as well as health benefits of probiotics in in vivo model systems. RESULTS: In this study, two lactic acid bacterial (LAB) cultures Lactobacillus fermentum NCMR 2826 and FIX proven for their in vitro probiotic properties were investigated for their in vivo safety in Wistar rats. An acute toxicity study (14 days) with a high dose of biomass (1016 colony-forming units (CFU) mL-1 ) followed by a subchronic test for 13 weeks with oral feeding of the probiotic cultures in three different doses (107 , 108 and 1010 CFU mL-1 ) on a daily basis revealed the safety of the L. fermentum cultures. The probiotic feeding had no toxic effects on survival, body weight and food consumption with any of the dosages used throughout the treatment period. No statistically significant changes in relative organ weights and serum biochemical and hematological indices were found between the control and the probiotic fed animals. In addition to the safety attributes, the L. fermentum culture fed rats showed reduced serum cholesterol levels, macrovesicular steatosis and hepatocyte ballooning compared with control animals. Further, quantification of intestinal microbiota using real-time polymerase chain reaction (PCR) analysis from animal feces indicated a significant increase and stability of Lactobacillus and Bifidobacterium counts but a decrease of Escherichia coli numbers. CONCLUSION: This study of safety and beneficial features highlights the use of the two native L. fermentum isolates as potential probiotic food supplements. © 2019 Society of Chemical Industry.

Heterogeneity of macrolide-lincosamide-streptogramin phenotype & conjugal transfer of erm(B) in Pediococcus pentosaceus.

Background & objectives: Pediococcus pentosaceus has been reported to cause clinical infections while it is being promoted as probiotic in food formulations. Antibiotic resistance (AR) genes in this species are a matter of concern for treating clinical infections. The present study was aimed at understanding the phenotypic resistance of P. pentosaceus to macrolide-lincosamide-streptogramin B (MLSB) antibiotics and the transfer of AR to pathogens. Methods: P. pentosacues isolates (n=15) recovered from fermented foods were screened for phenotypic resistance to MLSBantibiotics using disc diffusion and microbroth dilution methods. Localization and transferability of the identified resistance genes, erm(B) and msr(C) were evaluated through Southern hybridization and in vitro conjugation methods. Results: Four different phenotypes; sensitive (S) (n=5), macrolide (M) (n=7), lincosamide (L) (n=2) and constitutive (cMLSB) (n=1) were observed among the 15 P. pentosaceus isolates. High-level resistance (>256 µg/ml) to MLSBwas observed with one cMLSBphenotypic isolate IB6-2A. Intermediate resistance (8-16 µg/ml) to macrolides and lincosamides was observed among M and L phenotype isolates, respectively. Cultures with S phenotype were susceptible to all other antibiotics but showed unusual minimum inhibitory concentration (MIC) values of 8-16 µg/ml for azithromycin. Southern hybridization studies revealed that resistance genes localized on the plasmids could be conjugally transferred to Enterococcus faecalis JH2-2. Interpretation & conclusions: The study provides insights into the emerging novel resistance patterns in P. pentosaceus and their ability to disseminate AR. Monitoring their resistance phenotypes before use of MLS antibiotics can help in successful treatment of Pediococcal infections in humans.

Conjugal transfer of erm(B) and multiple tet genes from Lactobacillus spp. to bacterial pathogens in animal gut, in vitro and during food fermentation.

Three strains of Lactobacillus comprising Lactobacillus salivarius (CHS-1E and CH7-1E) and Lactobacillus reuteri (CH2-2) previously isolated from chicken meat were analyzed for their transferability of antibiotic resistance (AR) genes to pathogenic strains under in vivo, in vitro, and during food fermentation. For in vivo model, Albino Wistar rats were inoculated with 1010 CFU/g/ml of Enterococcus faecalis JH2-2 (recipient). After 7 days, either of two donors L. salivarius CH7-1E or L. reuteri [harbouring erythromycin and tetracycline resistance genes] were introduced at a concentration of 109 CFU/ml daily for 1 week. Two days after donor introduction, there was a stable increase in the number of transconjugants in the animal faeces from 102 to 103 CFU/g and presented erm(B), tet(M), tet(L) and tet(W) in their genome like donor strains. Similar observations were made with in vitro filter mating between CHS-1E, CH2-2 and CH7-1E and E. faecalis JH2-2 with transfer frequencies of 1 × 10-4, 3.8 × 10-3 and 2 × 10-3 per donor cell respectively. With the results obtained in vivo and in vitro, the AR transferability of donor strains was estimated during food fermentation (chicken sausage, fermented milk or idli batter) with pathogenic recipient strains added as contaminants. At the end of mating period, phenotypic resistance to erythromycin and tetracycline in Listeria monocytogenes and Yersinia enterocolitica strains was observed. This study showed the ability of food borne Lactobacillus in diffusing their AR traits in diverse natural environments increasing their concern of AR dissemination in the food chain when used as food additives and/or probiotics.