Structural and biosynthetic diversity of plantaricins from Lactiplantibacillus.

Lactiplantibacillus plantarum (L. plantarum) produces an antimicrobial peptide known as plantaricin. Plantaricin-producing L. plantarum is of interest for its gut-friendly nature, wide range of sugar utilization, palatability, and probiotic attributes, making it a better candidate for the food industry. Numerous strains of plantaricin-producing L. plantarum have been isolated from different ecological niches and found to follow different mechanisms for plantaricin production. The mechanism of plantaricin production is sensitive to environmental factors; therefore, any alteration in the optimum conditions can inhibit/halt bacteriocin production. To regain the lost or hidden plantaricin-producing character of the L. plantarum strains under ideal laboratory conditions, it is essential to understand the mechanism of plantaricin production. Previously, discrete information on various mechanisms of plantaricin production has been elaborated. However, based on the literature analysis, we observed that a systematic classification of plantaricins produced by L. plantarum is not explored. Hence, we aim to collect information about rapidly emerging plantaricins and distribute them among the different classes of bacteriocin, followed by classifying them based on different mechanisms of plantaricin production. This may help scaleup the bacteriocin production at industrial levels, which is otherwise challenging to achieve. This will also help the reader understand plantaricins and their mechanism of plantaricin production to a deeper extent and to characterize/reproduce the peptide where plantaricin production is a hidden character. KEY POINTS: • L. plantarum produces the antimicrobial compound plantaricin. • L. plantarum has different regulatory operons which control plantaricin production. • Based on the regulatory operon, the mechanism of plantaricin production is different.

Probiotic Bacillus licheniformis MCC2514 and Bifidobacterium breve NCIM 5671 Regulates GATA3 and Foxp3 Expression in the Elevated Disease Condition.

TNBS-induced ulcerative colitis was evaluated using Bacillus licheniformis MCC 2514 (B. licheniformis) and Bifidobacterium breve NCIM 5671 (Bf. breve) as immune modulators. The study aims to analyze probiotic efficiency of ulcerative colitis induced by TNBS in Wistar rats. The tumor-like structure was found in the colon of TNBS inflammation-induced rats. Nitric oxide production was inhibited by about 65.2% fed with combination of bacteria and C-reactive protein, and decreased by 12% and 10.8% upon supplementing B. licheniformis and Bf. breve against the TNBS-treated rats, respectively. Liver damage was observed in the TNBS-treated rats; addition of probiotic bacteria reduced SGPT (75.4%) and SGOT (42.5%). On TNBS treatment, the transcriptional factor responsible for Th2 cell immune response (GATA3) was analyzed, and the elevation in gene expression (5.31-fold) was found. The FOXP-3 responsible for T-regulatory cells was expressed about 0.91-fold upon the treatment with a combination of bacteria. The expression of antioxidant genes such as iNOS (1.11-fold), GPx (1.29-fold), and PON1 (1.48-fold) has been increased when compared with that of the TNBS-treated group. The cytokines specific to Th2-driven immune response, such as IL-4, IL-5, and TNF-α, were reduced upon feeding the bacteria. It is observed that the B. licheniformis and Bf. breve used in the study have reduced Th2-driven immune response.

The Combined Effect of Potential Probiotic Bacillus licheniformis MCC 2514 and Bifidobacterium breve NCIM 5671 Towards Anti-inflammatory Activity on HT-29 Cell Lines.

Probiotics are considered a natural source for treating many intestinal disorders, which deliver health benefits in different ways. The study aims to evaluate the immunomodulatory gene expression on HT-29 cell line using Bacillus licheniformis MCC 2514 and Bifidobacterium breve NCIM 5671 as a single culture and in combination. Upon inflammation induced by LPS, the combination of bacteria downregulated the pro-inflammatory cytokines IL-1α (13.4), IL-12 (14.6), IL-8 (2.6), and IL-6 (1.9), and in contrast, TNF-α (21.2) folds has upregulated. However, anti-inflammatory genes such as IL-4 (0.6), IL-10 (2.9), TGF-2 (92.2), and TGF-3 (85.8) folds were upregulated. The combination of bacteria against oxidative stress downregulated the pro-inflammatory cytokines such as IL-1α & ß, IL-6, IL-8, IL-12, and IL-18, and upregulated the anti-inflammatory cytokines IL-10, IL-4, TGF-2, and TGF-3. On the introduction of Kocuria rhizophila, the pro-inflammatory cytokines were upregulated. On supplementation of B. licheniformis and B. breve, the upregulated pro-inflammatory cytokines were decreased, and anti-inflammatory cytokines such as IL-4 (6.2), IL-10 (23.5), TGF-2 (166), and TGF-3(28.4) folds were increased. However, gene expression of toll-like receptor-2 was found high (26 folds) upon introducing probiotic bacteria. ELISA results of Interferon-γ found that the expression was higher (7.19 ng/mL) on the introduction of both the bacteria in combination. The higher anti-inflammatory activity was observed when potential probiotic bacteria were used in combination compared to a single culture. Overall study indicates that the combination of aerobic B. licheniformis and anaerobic B. breve has an anti-inflammatory activity that can sustain an excellent gastrointestinal environment during pathogen invasion and inflammation.

Effect of Lactobacillus fermentum MCC2760-Based Probiotic Curd on Hypercholesterolemic C57BL6 Mice.

Lactobacillus fermentum MCC2760 is a probiotic strain proven earlier for cholesterol-reducing and anti-inflammatory properties in vitro and in vivo. This study investigates L. fermentum MCC2760-based probiotic curd in high-cholesterol diet (HCD)-fed C57BL6 mice. The mice were grouped into normal diet control, high-cholesterol diet control, normal diet with probiotic supplementation, and high-cholesterol diet with probiotic supplementation. Control groups and treatment groups were supplemented with market curd and probiotic curd, respectively, via oral gavage for eight weeks. The probiotic count was maintained at 10.95 log CFU/mL in the developed probiotic curd. The HCD group showed an increase in feed intake and body weight. Reduction in the levels of serum cholesterol, triglycerides, low-density lipoprotein cholesterol, glucose, aspartate aminotransferase, and alanine transaminase was observed in probiotic-supplemented groups. The probiotic-supplemented group resulted in an increase in Lactobacillus spp. count along with reduced pathogen count in the feces. Probiotic supplementation also showed a reduction in the bacterial translocation count in mesenteric adipose tissue. Expression of inflammatory markers by qPCR showed the decline in the fold change of TNF-α, IL-6, and IL-12 and elevation in the fold change of IL-10 in the adipose tissue of the probiotic-treated group. Probiotic supplementation also improved the expression of GLP-1, ZO-1, and CB2 in the intestine. They were thus possibly playing a role in the enhancement of barrier function. Histopathological sections showed improvement in the cellular infiltration and pathological indications due to the high-cholesterol diet intake. Our study also confirmed that probiotics could increase serum antioxidant enzymes in treated groups, showing their beneficial antioxidant activity. It suggests the anti-inflammatory, antioxidant effect, and gut barrier function of the given probiotic formulation, which ameliorate hypercholesterolemia.

In vitro validation studies for adhesion factor and adhesion efficiency of probiotic Bacillus licheniformis MCC 2514 and Bifidobacterium breve NCIM 5671 on HT-29 cell lines.

Probiotic bacterial adhesion to the epithelial cell is a composite process and in vivo adhesion studies can be strengthened with the improved in vitro models for preliminary screening of potentially adherent strains. With this rationale, the study aimed is the first report to demonstrate the colonizing efficiency of probiotic Bacillus licheniformis MCC 2514 in comparison to Bifidobacterium breve NCIM 5671on HT-29 cell line. B. licheniformis (54.28 ± 0.99%) and Bif. breve (70.23 ± 0.85%) adhered in a higher percentage on fibronectin and mucin, respectively. However, the adhesion was higher for B. licheniformis when compared to Bif. breve. In adhesion score, B. licheniformis obtained about 138.85 ± 12.32, whereas Bif. breve got the score of 43.05 ± 9.12. The same trend continued in the adhesion percentage study, where B. licheniformis adhered 75.5 ± 5.2%, higher than Bif. breve which adhered 32.66 ± 3.2%. In invasion assay, both the bacteria significantly decreased the colonization of the pathogen Kocuria rhizophila ATCC 9341 about 97.32 ± 0.81% in the competitive assay, 97.87 ± 0.73% in exclusion assay and 82.19 ± 2.51% in displacement assay. The cytotoxicity effects of the test bacterial strains against HT-29 cell line through MTT assay determined no viability loss in the treated cells. Therefore, the data obtained from the in vitro studies showed that both B. licheniformis and Bif. breve had shown significantly good invasion on pathogen and adhesion capacity on HT-29 cell line.

Lactobacillus fermentum MCC2759 and MCC2760 Alleviate Inflammation and Intestinal Function in High-Fat Diet-Fed and Streptozotocin-Induced Diabetic Rats.

The growing incidence of type 2 diabetes and obesity has become a worldwide crisis with increased socio-economic burden. Changes in lifestyle and food habits resulting in dysbiosis of the gut microbiota and low-grade inflammation are linked to the rising incidence. The aim of this study was to investigate the effects of potential probiotic Lactobacillus fermentum MCC2759 and MCC2760 on intestinal markers of inflammation using a high-fat diet (HFD)-fed model and a streptozotocin (STZ)-induced diabetic model. Lact. fermentum administration showed improved oral glucose tolerance compared with the model controls of HFD (AUC 1518) and STZ (628.8). Plasma insulin levels improved in the Lact. fermentum treated groups of HFD + MCC2759 (129 ± 4.24 pmol/L) and HFD + MCC2760 (151.5 ± 9.19 pmol/L) in HFD study, while in STZ diabetic study, the insulin levels were normalized with Lact. fermentum administration, for D + MCC2759 (120.5 ± 7.77) and D + MCC2760 (138 ± 5.65 pmol/L) groups. The results showed reduction in inflammatory tone in liver, muscle, and adipose tissues of rats in both models with stimulation of anti-inflammatory IL-10 by real-time quantitative polymerase chain reaction. Additionally, the potential probiotic cultures also displayed normalization of markers related to intestinal barrier integrity (ZO-1), TLR-4 receptor, and insulin sensitivity (GLUT-4, GLP-1, adiponectin). Thus, the results suggest that Lact. fermentum could act as potential probiotic for lifestyle-related disorders such as obesity, diabetes, and metabolic syndrome as both prophylactic and adjunct therapies.

Presence of extracellular DNA & protein in biofilm formation by gentamicin-resistant Lactobacillus plantarum.

Background & objectives: Bacterial biofilms a multi-layered defence, comprise extracellular DNA (eDNA) and proteins, protect bacteria from harmful environment and nutrient limitation and utilize the mutual benefits within a community. Bacterial biofilms also defend bacteria from harsh environments such as antibiotic treatment. This leads to poor antibiotic penetration, slow growth, adaptive stress responses, and formation of persister cells. This study was done to determine the relation of antibiotic resistance deciphered by the biofilms in Lactobacillus plantarum, a lactic acid bacteria (LAB) with probiotic significance. Methods: The gentamicin-resistant L. plantarum isolates were allowed to form biofilms and subjected to DNase I and proteinase K treatment. The optical density (OD) values were recorded for the biofilm assay and the cell count for the number of viable cells was taken for the control and the test samples. Percentage reduction was calculated based on the difference between the initial and final OD for both the parameters. Results: The biofilm assay revealed that the native L. plantarum isolates which were phenotypically susceptible, possessed the ability to form biofilms. The OD values were significantly decreased in comparison to the biofilm-forming control culture when these were treated with DNase I and proteinase K. Interpretation & conclusions: The study revealed that the biofilms formed by L. plantarum comprised of eDNA and proteins which was evidenced by the reduction in OD values and percentage in comparison to the control upon DNase I and proteinase K treatment. This indicates that the eDNA and biofilm matrix proteins are vital constituents of biofilms and may carry significant risk when coupled with antibiotic resistance.

Prophylactic effects of probiotic Bifidobacterium spp. in the resolution of inflammation in arthritic rats.

In the present study, the modulatory effects of bifidobacterial spp. (Bifidobacterium breve NCIM 5671, Bifidobacterium longum NCIM 5672 and Bifidobacterium bifidum NCIM 5697) on adjuvant induced arthritis in rats were evaluated. Arthritis was induced in male Wistar rats by injecting 250 µg of Freund's adjuvant directly into the paw. Fifteen days before and 15 days after the induction of arthritis, suspended cultures of bifidobacteria (109 cfu/ml) were administered by oral gavage. Paw volume, bone mineral content, oxidative stress markers, antioxidant enzymes, cytokines, eicosanoids and expression of COX2, as well as bone hydrolytic enzymes, were assessed by RT PCR. Although piroxicam-treated groups (drug control) had better effects than bifidobacteria-treated groups, bifidobacteria probiotics administration exhibited significant (P < 0.05) prophylactic effects in terms of downregulating arthritis markers. Parameters including paw volume, bone mineral content, cytokines, and eicosanoids level were significantly (p < 0.05) modulated in bifidobacteria administered groups compared to arthritic control group. Among the three strains tested, B. breve NCIM 5671 exhibited superior prophylactic effects as assessed in the experimental rat model of arthritis. In conclusion, bifidobacteria probiotics administration can downregulate the markers of arthritis and hence can be a potential therapeutic regimen in the treatment of arthritis.

Identification and characterization of a calcium dependent bacillopeptidase from Bacillus subtilis CFR5 with novel kunitz trypsin inhibitor degradation activity.

The cereals and pulses are considered to be an important component in the food chain due to their proteinaceous nature, but the presence of anti-nutritional factors (KTI) decreases their nutrient absorption rate. Kunitz trypsin inhibitors (KTI) reduce the bioavailability of trypsin and are the primary cause for the existence of various metabolic disorders. To overcome the inhibitory effect of KTI, a KTI degrading protein (BPC) was identified and characterized from Bacillus subtilis CFR5. BPC possesses 60% identity with bacillopeptidase of B. subtilis 168. BPC cleaves at DFVLD and DFFNNY sites of KTI which results in the formation of three inactive KTI fragments. Subsequently, BPC was cloned in pHY300PLK and recombinant protein was used for the biochemical characterization, sequence alignment and mutational studies. The optimal temperature and pH of the BPC was 40°C and 8.0, respectively. BPC is a calcium dependent metalloprotease and its activity was significantly increased by 41.2-fold in the presence of 2.5mM Ca2+. BPC also showed moderate thermostability with the half-life of 4h at 55°C. Site directed mutagenesis studies in recombinant BPC revealed that mutation of Tyr49 with Phe, Tyr64 with Phe, and Pro141 to Arg affects the catalytic activity without affecting the conformation of BPC. Hence, Tyr49, Tyr64 and Pro141 were identified as the unique residues responsible for KTI cleavage. Thus, this study leads to the identification of a novel KTI degrading protease from B. subtilis CFR5 which cleaves and deactivates the kunitz trypsin inhibitor.

Purification and characterization of fibrinolytic protease from Bacillus amyloliquefaciens MCC2606 and analysis of fibrin degradation product by MS/MS.

A serine protease with preference for fibrin protein was purified and characterized from Bacillus amyloliquefaciens MCC2606, isolated from dosa batter. The protease was purified using ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The degradation activity of the protease toward the fibrin was significantly higher compared with other protein substrates in the study. The molecular weight of the CFR15-protease was estimated to be 32 kDa based on SDS-PAGE. The purified enzyme exhibited both fibrinolytic and fibrinogenolytic activity. The optimum pH and temperature for the activity of the enzyme was found to be 10.5 and 45°C. A significant inhibition was seen with the protease inhibitors phenyl methyl sulphonyl fluoride and ethylene diamine tetra acetic acid and the activity of the enzyme was enhanced in presence of Mn2+. There was an observed increase in vitro activated partial thromboplastin time and prothrombin time of both time and dose dependent study. Among the four chains of fibrin, the ß-chain of fibrin appears to be the primary component and site susceptible for CFR15-protease in early action as indicated by MS/MS analysis of initial degradation products. These results indicated that the CFR15-protease have the potential to be an effective fibrinolytic agent.

Sub-inhibitory concentrations of gentamicin triggers the expression of aac(6')Ie-aph(2″)Ia, chaperons and biofilm related genes in Lactobacillus plantarum MCC 3011.

The study aimed to analyze the effects of sub-inhibitory concentrations of gentamicin on the expressions of high level aminoglycoside resistant (HLAR) bifunctional aac(6')Ie-aph(2″)Ia, biofilm and chaperone genes in Lactobacillus plantarum. The analysis of the biofilm formation in five isolates obtained from chicken sausages indicated their role in exhibiting phenotypic resistance based on the varied MIC values despite carrying the bifunctional gene. The biofilm formation significantly increased when L. plantarum MCC 3011 was grown in sub-inhibitory concentrations of gentamicin (4 µg/ml), kanamycin (8 µg/ml) and streptomycin (2 µg/ml). Thirty day gentamicin selection increased minimum inhibitory concentration (MIC) values from 4 to 64 and 2 to 256 fold for gentamicin and kanamycin, respectively when compared to the parental cultures. Expression studies revealed that constant exposure to gentamicin had induced chaperon [groEL] and the bifunctional gene, aac(6')Ie-aph(2″)Ia upto nine fold. Induction of groEL, groES and lamC genes in gentamicin (4 µg/ml) preincubated MCC 3011 indicated their significant role in aminoglycoside mediated response. Our study indicates that constant exposure to sub inhibitory concentrations of gentamicin allows L. plantarum to adapt against higher doses of aminoglycosides. This highlights the risks and food safety issues associated with the use of aminoglycosides in livestock and consumption of farm oriented fermented food products.

Taming C-terminal peptides of Staphylococcus aureus leukotoxin M for B-cell response: Implication in improved subclinical bovine mastitis diagnosis and protective efficacy in vitro.

Leukotoxin M/F'-PV (LukM/F'-PV) produced by bovine mastitis causing Staphylococcus aureus structurally comprises three domains, the ß-sandwich, rim and stem domain. The rim and stem domains interacting with target cell membrane lipid rafts contributes to the virulent trait of the toxin. In the present study, two facts were hypothesized that neutralization of these domains will ebb LukM/F'-PV leukotoxicity. Secondly, the neutralizing antibodies can improve the leukotoxin detection sensitivity in bovine mastitis milk samples. The in silico mapping of S. aureus LukM C-termini comprising these domains predicted seven linear B-cell antigenic epitopes. The immune response of C-terminal truncated recombinant peptides rCtM19 (19 kDa; near carboxy-terminal) having four epitopes and rCtM15 (15 kDa; C-terminal) with three epitopes were evaluated for their diagnostic and neutralization potential. Anti-rCtM19 and anti-rCtM15 antibodies with enhanced immunogenicity had the most striking outcome in IgG-ELISA for detecting native determinants of leukotoxin. For the obtained ELISA values, ROC curve inferred a cut-off score of >0.102 OD405. The assay sensitivity in the range of 90-96% along with 100% specificity and AUC of 0.93-0.98 categorized subclinical and clinical from healthy bovine milk samples. As observed through in vitro neutralization and LDH assays, C-terminus specific antibodies (1:42 titer) deactivating leukotoxicity abolished LukM from interacting with lipid bilayer and LukF for forming pores on bovine neutrophil membrane. As a proof of concept, it was proved that peptide antibodies can be a more specific serodiagnostic and passive therapeutic molecules.