The rumen microbiome: an underexplored resource for novel antimicrobial discovery.

Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic application.

Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis.

Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

A relevant exposure to a food matrix contaminated environmentally by polychlorinated biphenyls induces liver and brain disruption in rats.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants present in dietary fats. Most studies evaluating PCB effects have been conducted with a single compound or a mixture of PCBs given as a single acute dose. The purpose of this study was to evaluate in vivo PCB toxicity in a realistic model of exposure: a low daily dose of PCBs (twice the tolerable daily intake (TDI)), chronically administered (8 weeks) to rats in contaminated goat milk. Liver and brain PCB toxicities were investigated by evaluating oxidative stress status and mitochondrial function. PCB toxicity in the liver was also estimated by transaminase enzymatic activity. This study shows that even at low doses, chronic PCB exposure resulted in a statistically significant reduction of mitochondrial function in liver and brain. In the liver, oxygen consumption in the condition of adenosine triphosphate (ATP) production (state 3) decreased by 22-29% (p < 0.01), according to the respiratory substrates. In the brain, respiratory chain complexes II and III were reduced by 24% and 39%, respectively (p < 0.005). The exposed rats presented higher lipid peroxidation status (+20%, p < 0.05) and transaminase activity (+30%, p < 0.05) in the blood. Thus, our study showed that exposure of rats to a daily realistic dose of PCBs (twice the TDI in a food complex mixture of environmental origin) resulted in multiple disruptions in the liver and brain.

Whole Rye Consumption Improves Blood and Liver n-3 Fatty Acid Profile and Gut Microbiota Composition in Rats.

BACKGROUND: Whole rye (WR) consumption seems to be associated with beneficial health effects. Although rye fiber and polyphenols are thought to be bioactive, the mechanisms behind the health effects of WR have yet to be fully identified. This study in rats was designed to investigate whether WR can influence the metabolism of n-3 and n-6 long-chain fatty acids (LCFA) and gut microbiota composition. METHODS: For 12 weeks, rats were fed a diet containing either 50% WR or 50% refined rye (RR). The WR diet provided more fiber (+21%) and polyphenols (+29%) than the RR diet. Fat intake was the same in both diets and particularly involved similar amounts of essential (18-carbon) n-3 and n-6 LCFAs. RESULTS: The WR diet significantly increased the 24-hour urinary excretion of polyphenol metabolites-including enterolactone-compared with the RR diet. The WR rats had significantly more n-3 LCFA-in particular, eicosapentanoic (EPA) and docosahexanoic (DHA) acids-in their plasma and liver. Compared with the RR diet, the WR diet brought significant changes in gut microbiota composition, with increased diversity in the feces (Shannon and Simpson indices), decreased Firmicutes/Bacteroidetes ratio and decreased proportions of uncultured Clostridiales cluster IA and Clostridium cluster IV in the feces. In contrast, no difference was found between groups with regards to cecum microbiota. The WR rats had lower concentrations of total short-chain fatty acids (SCFA) in cecum and feces (p<0.05). Finally, acetate was lower (p<0.001) in the cecum of WR rats while butyrate was lower (p<0.05) in the feces of WR rats. INTERPRETATION: This study shows for the first time that WR consumption results in major biological modifications-increased plasma and liver n-3 EPA and DHA levels and improved gut microbiota profile, notably with increased diversity-known to provide health benefits. Unexpectedly, WR decreased SCFA levels in both cecum and feces. More studies are needed to understand the interactions between whole rye (fiber and polyphenols) and gut microbiota and also the mechanisms of action responsible for stimulating n-3 fatty acid metabolism.

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

Wheat aleurone polyphenols increase plasma eicosapentaenoic acid in rats.

METHODS: These studies were designed to assess whether wheat polyphenols (mainly ferulic acid [FA]) increased the very-long-chain omega-3 fatty acids (VLC n-3) [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in rats. Wheat aleurone (WA) was used as a dietary source of wheat polyphenols. Two experiments were performed; in the first one, the rats were fed WA or control pellets (CP) in presence of linseed oil (LO) to provide alpha-linolenic acid (ALA), the precursor of VLC n-3. In the second one, the rats were fed WA or CP in presence of control oil (CO) without ALA. The concentrations of phenolic acid metabolites in urine were also investigated. RESULTS: The urinary concentration of conjugated FA increased with WA ingestion (p<0.05). Plasma EPA increased by 25% (p<0.05) with WA in the CO group but not in the LO group. In contrast, there was no effect of WA on plasma DHA and omega-6 fatty acids (n-6). Finally, both n-3 and n-6 in the liver remained unchanged by the WA. CONCLUSION: These results suggest that WA consumption has a significant effect on EPA in plasma without affecting n-6. Subsequent studies are required to examine whether these effects may explain partly the health benefits associated with whole wheat consumption.

Identification and characterization of three novel lipases belonging to families II and V from Anaerovibrio lipolyticus 5ST.

Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14).