Double-Edged Nanobiotic Platform with Protean Functionality: Leveraging the Synergistic Antibacterial Activity of a Food-Grade Peptide to Mitigate Multidrug-Resistant Bacterial Pathogens.

While persistent efforts are being made to develop a novel arsenal against bacterial pathogens, the development of such materials remains a formidable challenge. One such strategy is to develop a multimodel antibacterial agent which will synergistically combat bacterial pathogens, including multidrug-resistant bacteria. Herein, we used pediocin, a class IIa bacteriocin, to decorate Ag° and developed a double-edged nanoplatform (Pd-SNPs) that inherits intrinsic properties of both antibacterial moieties, which engenders strikingly high antibacterial potency against a broad spectrum of bacterial pathogens including the ESKAPE category without displaying adverse cytotoxicity. The enhanced antimicrobial activity of Pd-SNPs is due to their higher affinity with the bacterial cell wall, which allows Pd-SNPs to penetrate the outer membrane, inducing membrane depolarization and the disruption of membrane integrity. Bioreporter assays revealed the upregulation of cpxP, degP, and sosX genes, triggering the burst of reactive oxygen species which eventually cause bacterial cell death. Pd-SNPs prevented biofilm formation, eradicated established biofilms, and inhibited persister cells. Pd-SNPs display unprecedented advantages because they are heat-resistant, retain antibacterial activity in human serum, and alleviate vancomycin intermediate Staphylococcus aureus (VISA) infection in the mouse model. In addition, Pd-SNPs wrapped in biodegradable nanofibers mitigated Listeria monocytogenes in cheese samples. Collectively, Pd-SNPs exhibited excellent biocompatibility and in vivo therapeutic potency without allowing foreseeable resistance acquisition by pathogens. These findings underscore new avenues for using a potent biocompatible nanobiotic platform to combat a wide range of bacterial pathogens.

Bovicins: The Bacteriocins of Streptococci and Their Potential in Methane Mitigation.

Bovicin is a type AII lantibiotic, possessing two ß-methyllanthionine and a disulfide bridge encoded by bovA gene hitherto unknown a couple of decades ago. Bacteriocins can be useful in directly inhibiting methanogens and/or redirecting H2 to other reductive microorganisms, in particular, propionate producers or reductive acetogens. So far, the role of nisin and bovicin to suppress greenhouse gas (GHG) production under in vitro conditions has been documented. GHG emissions from ruminants are a threat to the environment, because of their role in global warming as well as in climate change. Methane (CH4) produced from livestock farming practices is a potent GHG, comprising 18% of total GHG emissions in the world. Therefore, minimizing enteric CH4 production is quite essential from both the economical livestock production as well as environment perspectives. Strategies for the abatement of CH4 have provided two-way opportunities, viz., improved livestock productivity and reduced GHG emissions. In the past, different strategies have been proposed and tested to mitigate CH4, such as the dietary composition of feeds, ionophores, antibiotics, vaccines, analogues, probiotics, and secondary metabolites of plants and fungi. However, quite a few of these strategies have been adopted at farm level due to their varied effect on animal health and/or residues on animal products. The use of bacteriocins might have potential in inhibiting methanogens in the rumen. A bacteriocin produced by Streptococcus bovis (an isolate from rumen) named bovicin HC5 has been exhibited to decrease CH4 production to an extent of 50%. In this review, authors intend to discuss the sources, structure, biochemical properties, and antimicrobial spectra of bovicins, besides the potential applications with special reference to CH4 mitigation.

Cell surface proteins play an important role in probiotic activities of Lactobacillus reuteri

BACKGROUND: Eight Lactobacillus reuteri strains, previously isolated from breast-fed human infant feces, were selected to assess the potential contribution of their surface proteins in probiotic activity. These strains were treated with 5 M LiCl to remove their surface proteins, and their tolerance to simulated stomach-duodenum passage, cell surface characteristics, auto aggregation, adhesion, and inhibition of pathogen adhesion to Caco-2 cells were compared with untreated strains. RESULTS: The survival rates, auto aggregation, and adhesion abilities of the LiCl-treated L. Reuteri strains decreased significantly (p< 0.05) compared to that of the untreated cells. The inhibition ability of selected L. reuteri strains, untreated or LiCl treated, against adherence of Escherichia coli 25922 and Salmonella typh iNCDC113 to Caco-2 was evaluated in vitro with L. reuteri ATCC55730 strain as a positive control. Among the selected eight strains of L.reuteri, LR6 showed maximum inhibition against the E. Coli ATCC25922 and S. typhiNCDC113. After treatment with 5 M LiCl to remove surface protein, the inhibition activities of the lactobacilli against pathogens decreased significantly (p< 0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated thatLR6 strains had several bands with molecular weight ranging from 10 to 100 KDa, and their characterization and functions need to be confirmed. CONCLUSIONS: The results revealed that the cell surface proteins of L. reuteri play an important role in their survivability, adhesion, and competitive exclusion of pathogen to epithelial cells.

Hypocholesterolemic effects of Lactobacillus reuteri LR6 in rats fed on high-cholesterol diet.

The bacterium Lactobacillus reuteri LR6, an isolate from breast-fed human infant feces, was tested positive for bile tolerance and bile salt hydrolase activity. It was also evaluated as a potential probiotic with cholesterol-lowering effect in vivo. In this study, 32 male Albino rats were divided into four groups consisting of eight mice per group. For 60 d, group I was fed with normal synthetic diet, group II was fed with cholesterol-enriched diet only, group III was fed with cholesterol-enriched diet supplemented with skimmed milk, and group IV was fed with cholesterol-enriched diet supplemented with L. reuteri LR6-fermented skimmed milk (10(8) cfu/mL). Blood samples were taken to study lipid profile on 0th, 15th, 30th and 60th day. Compared with the control group, the values for total cholesterol (TC), triglyceride (TG), and LDL were reduced significantly in group fed with L. reuteri LR6 but for HDL this difference was not significant. The results indicated that L. reuteri LR6 might be effective as a probiotic with cholesterol-lowering activities.

Antibacterial efficacy of nisin, pediocin 34 and enterocin FH99 against L. monocytogenes, E. faecium and E. faecalis and bacteriocin cross resistance and antibiotic susceptibility of their bacteriocin resistant variants.

The bacteriocin susceptibility of Listeria monocytogenes MTCC 657, Enterococcus faecium DSMZ 20477, E. faecium VRE, and E. faecalis ATCC 29212 and their corresponding bacteriocin resistant variants was assessed. The single and combined effect of nisin and pediocin 34 and enterocin FH99 bacteriocins produced by Pediococcus pentosaceus 34, and E. faecium FH99, respectively, was determined. Pediocin34 proved to be more effective in inhibiting L. monocytogenes MTCC 657. A greater antibacterial effect was observed against E. faecium DSMZ 20477 and E. faecium (VRE) when the a combination of nisin, pediocin 34 and enterocin FH99 were used whereas in case of L. monocytogenes MTCC 657 a combination of pediocin 34 and enterocin FH99 was more effective in reducing the survival of pathogen. Bacteriocin cross-resistance and the antibiotic susceptibility of wild type and their corresponding resistant variants were assessed and results showed that resistance to a bacteriocin may extend to other bacteriocins within the same class and also the acquired resistance to bacteriocins can modify the antibiotic susceptibility/resistance profile of the bacterial species used in the study. According to the hydrophobicity nisin resistant variant of L. monocytogenes was more hydrophobic (p < 0.001), whereas the pediocin 34 and enterocin FH99 resistant variants were less hydrophobic than the wild type strain. Nisin, pediocin 34 and enterocin FH99 resistant variants of E. faecium DSMZ 20477 and E. faecium VRE were less hydrophobic than their wild type counterparts. Nisin resistant E. faecalis ATCC 29212 was less hydrophobic than its wild type counterpart.

Spore germination based assay for monitoring antibiotic residues in milk at dairy farm.

Spore germination based assay involves the transformation of dormant spores of Bacillus stearothermophilus 953 into active vegetative cells. The inhibition of germination process specifically in presence of antibiotic residues was used as a novel approach for monitoring target contaminants in milk. The indicator organism i.e., B. stearothermophilus 953 was initially allowed to sporulate by seeding in sporulation medium and incubating at 55 °C for 18 ± 2 h. The spores exhibited a typical chain behavior as revealed through phase contrast microscopy. The minimal medium inoculated with activated spores was incubated at 64 °C for 2-3 h for germination and outgrowth in presence of specific germinant mixture containing dextrose, whey powder and skimmed milk powder added in specific ratio along with reconstituted milk as negative control and test milk samples. The change in color of the medium from purple to yellow was used as criteria for detection of antibiotic residues in milk. The efficiency of the developed assay was evaluated through a surveillance study on 228 samples of raw, pasteurized and dried milks and results were compared with AOAC approved microbial receptor assay. The presence of antibiotic level was 10.08 % at Codex maximum residual limit having false positive result only in 0.43 % of the samples. The results of the present investigation suggest that developed spore based assay can be a practical solution to dairy industry for its application at farm level, milk processing units, independent testing and R & D centres in order to comply with the legal requirements set by Codex.

Mechanism of Nisin, Pediocin 34, and Enterocin FH99 Resistance in Listeria monocytogenes.

Nisin-, pediocin 34-, and enterocin FH99-resistant variants of Listeria monocytogenes ATCC 53135 were developed. In an attempt to clarify the possible mechanisms underlying bacteriocin resistance in L. monocytogenes ATCC 53135, sensitivity of the resistant strains of L. monocytogenes ATCC 53135 to nisin, pediocin 34, and enterocin FH99 in the absence and presence of different divalent cations was assessed, and the results showed that the addition of divalent cations significantly reduced the inhibitory activity of nisin, pediocin 34, and enterocin FH99 against resistant variants of L. monocytogenes ATCC 53135. The addition of EDTA, however, restored this activity suggesting that the divalent cations seem to affect the initial electrostatic interaction between the positively charged bacteriocin and the negatively charged phospholipids of the membrane. Nisin-, pediocin 34-, and enterocin-resistant variants of L. monocytogenes ATCC 53135 were more resistant to lysozyme as compared to the wild-type strain both in the presence as well as absence of nisin, pediocin 34, and enterocin FH99. Ultra structural profiles of bacteriocin-sensitive L. monocytogenes and its bacteriocin-resistant counterparts revealed that the cells of wild-type strain of L. monocytogenes were maximally in pairs or short chains, whereas, its nisin-, pediocin 34-, and enterocin FH99-resistant variants tend to form aggregates. Results indicated that without a cell wall, the acquired nisin, pediocin 34, and enterocin FH99 resistance of the variants was lost. Although the bacteriocin-resistant variants appeared to lose their acquired resistance toward nisin, pediocin 34, and enterocin FH99, the protoplasts of the resistant variants appeared to be more resistant to bacteriocins than the protoplasts of their wild-type counterparts.

Characterization of Intestinal Lactobacillus reuteri Strains as Potential Probiotics.

This study was conducted to evaluate the probiotic properties of Lactobacillus reuteri isolated from human infant feces (less than 3 months). Out of thirty-two representative L. reuteri strains isolated from the infant human feces, nine isolates (i.e. LR5, LR6, LR9, LR11, LR19, LR20, LR25, LR26 and LR34) showed survival in acid, bile and simulated stomach-duodenum passage conditions, indicating their high tolerance to gastric juice, duodenal juice and bile environments. The nine isolates did not show strong hydrophobic properties because the percentages of adhesion to the apolar solvent, n-hexadecane, did not exceed 40%, showing that their surfaces were rather hydrophilic. Functionality of these nine probiotic isolates was supported by their antagonistic activity and their ability to deconjugate bile salts. The safety of the nine indigenous L. reuteri isolates was supported by the absence of transferable antibiotic resistance determinants, DNase activity, gelatinase activity and hemolysis. The results obtained so far suggest that the nine strains are resistant to low pH, bile salts and duodenum juice, so they could survive when passing through the upper part of the gastrointestinal tract and fulfill their potential probiotic action in the host organism. According to these results, the L. reuteri strains isolated from human infant feces possess interesting probiotic properties that make them potentially good candidates for probiotics.

Nisin and class IIa bacteriocin resistance among Listeria and other foodborne pathogens and spoilage bacteria.

Food safety has been an important issue globally due to increasing foodborne diseases and change in food habits. To inactivate foodborne pathogens, various novel technologies such as biopreservation systems have been studied. Bacteriocins are ribosomally synthesized peptides or proteins with antimicrobial activity produced by different groups of bacteria, but the bacteriocins produced by many lactic acid bacteria offer potential applications in food preservation. The use of bacteriocins in the food industry can help reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods that are more naturally preserved. However, the development of highly tolerant and/or resistant strains may decrease the efficiency of bacteriocins as biopreservatives. Several mechanisms of bacteriocin resistance development have been proposed among various foodborne pathogens. The acquiring of resistance to bacteriocins can significantly affect physiological activity profile of bacteria, alter cell-envelope lipid composition, and also modify the antibiotic susceptibility/resistance profile of bacteria. This article presents a brief review on the scientific research about the various possible mechanisms involved in the development of resistance to nisin and Class IIa bacteriocins among the foodborne pathogens.

Safety assessment and evaluation of probiotic potential of bacteriocinogenic Enterococcus faecium KH 24 strain under in vitro and in vivo conditions.

In the present investigation, a previously isolated Enterococcus faecium KH 24 strain was evaluated for the presence of virulence determinants (agg, esp, efaAfm, gelE, cylA, cylB, clyM, cpd, cob, ccf, ace and hyl), sensitivity to various antibiotics and production of biogenic amines. No virulence determinants were detected, except efaAfm. KH 24 was found to be sensitive to most of the tested antibiotics and none of the biogenic amines were produced by it. Moreover, KH 24 showed good in vitro tolerance to biological barriers and furthermore, its survival in gut of mice was also evaluated. Mice group fed with E. faecium KH 24 strain showed better weight gain and nearly 1 log cfu/g decrease in Salmonella enteritidis counts in the intestines as compared to control (p<0.05). Enhanced growth of lactobacilli (p<0.05) and decrease in coliform counts (p<0.05) were also observed in test group. E. faecium KH 24 is, therefore, found to be a safe strain and it may be used as protective culture or as a probiotic in food preparations.

Comparison of susceptibility to opsonic killing by in vitro human immune response of Enterococcus strains isolated from dairy products, clinical samples and probiotic preparation.

The genus Enterococcus like other LAB has also been featured in food and probiotic industry for decades due to its specific biochemical traits and beneficial health claims. At the same time, some enterococcal strains present an emerging pool of opportunistic pathogens for humans and are frequently armed with potential virulence factors. Thus, there is a need to assure the safety of enterococci before their use in food and probiotic preparations. Opsonophagocytic assay is an important test for the safety assessment of enterococci. In the present study comparative safety assessment of the different enterococcal strains isolated from dairy products, faeces, clinical samples and a commercial probiotic preparation was carried out by in vitro opsonophagocytic assay. Eleven strains of Enterococcus spp. were tested for their susceptibility to killing by opsonophagocytic assay. Among them, six isolates (Enterococcus faecium strain) were from our previous study (isolated from dairy products and faecal sample), four were from clinical samples and one from a probiotic preparation. Five out of six previous isolates and the isolate from probiotic preparation showed higher susceptibility to killing in contrast to the clinical isolates. The difference in the susceptibility to opsonic killing among the clinical and non-clinical Enterococcus isolates may be attributed to the presence of a capsule in the former, which protect them against the opsonophagocytic killing. Thus, these susceptible E. faecium strains may be designated as safe. However, certain other virulence traits must be evaluated prior to their exploitation in food and probiotic preparations.