Prevalence and characteristics of ESBL-producing Escherichia coli in clinically healthy pigs: implications for antibiotic resistance spread in livestock.

AIM: This study aimed to compare and characterize the resistance profile and the presence of extended-spectrum beta-lactamase (ESBL) related genes in Escherichia coli isolated from healthy finishing pigs fed with or without antibiotics in their diets. METHODS AND RESULTS: A total of 27 ceftiofur-resistant E. coli isolates were obtained from 96 healthy pigs. The antibiotic resistance profile was tested, and all 27 isolates were classified as multidrug-resistant (MDR). A high proportion of isolates were resistant to cephalosporins, ampicillin, ciprofloxacin, and tetracyclines. The ESBL production was observed in 85% of isolates by double-disc synergy test. The MDR-E. coli isolates harbored ESBL genes, such as blaTEM, blaCTX-M-1, blaCTX-M-2, and blaCTX-M-8,25. In addition, other antibiotics resistance genes (ARGs) were also detected, such as sul2, ant(3″)-I, tetA, and mcr-1. The mobilization of the blaCTX-M gene was confirmed for nine E. coli isolates by conjugation assays. The presence of blaCTX-M on mobile genetic elements in these isolates was demonstrated by Southern blot hybridization, and the resistance to cephalosporins was confirmed in the transconjugants. Our results indicate the prevalence of CTX-M-producing E. coli strains harboring mobile genetic elements in the normal microbiota of healthy pigs. CONCLUSIONS: These findings highlight the significance of ESBL genes as a global health concern in livestock and the potential spread of antimicrobial resistance to other members of the gastrointestinal tract microbiota.

Antimicrobial Resistance Profiles of Multidrug-Resistant Enterobacteria Isolated from Feces of Weaned Piglets.

Nosocomial infections caused by multidrug-resistant enterobacteria have become a major challenge in global public health. Previous studies have indicated that use of antibiotics in livestock production chains is linked to the rising threat of antibiotic resistance in humans. In this study, we aimed to evaluate the distribution of genes encoding resistance to tetracycline, ß-lactams, and colistin in multidrug-resistant enterobacteria isolated from feces of weaned pigs. Ninety-four enterobacteria isolates were submitted to antibiotic susceptibility test by minimum inhibitory concentration (MIC). In addition, we performed conjugation experiments to verify if plasmid-bearing isolates containing the mcr-1 gene could transfer their resistance determinant to a colistin-sensitive recipient strain. Our results demonstrated a positive association between the detection of antibiotic resistance genes in enterobacteria and the phenotypic resistance profiles of the bacterial isolates. At least one of the extended-spectrum ß-lactamases (ESBL) genes (blaCTX-M, blaTEM, or bla SHV) and tetA was found among most bacterial genera analyzed. In addition, results revealed that the mcr-1 gene can be transferred from E. coli UFV-627 isolate to an F- recipient (Escherichia coli K12) by conjugation. Our findings support the hypothesis that swine represents an important reservoir of antibiotic resistance genes and suggest that horizontal transfer mechanisms (e.g., conjugation) may mediate the spread of these genes in the swine gastrointestinal tract.

Anti-virulence compounds against Staphylococcus aureus associated with bovine mastitis: A new therapeutic option?

Bovine mastitis represents a major economic burden faced by the dairy industry. S. aureus is an important and prevalent bovine mastitis-associated pathogen in dairy farms worldwide. The pathogenicity and persistence of S. aureus in the bovine mammary gland are associated with the expression of a range of virulence factors involved in biofilm formation and the production of several toxins. The traditional therapeutic approach to treating bovine mastitis includes the use of antibiotics, but the emergence of antibiotic-resistant strains has caused therapeutic failure. New therapeutic approaches targeting virulence factors of S. aureus rather than cell viability can have several advantages including lower selective pressure towards the development of resistance and little impact on the host commensal microbiota. This review summarizes the potential of anti-virulence therapies to control S. aureus associated with bovine mastitis focusing on anti-toxin, anti-biofilm, and anti-quorum sensing compounds. It also points to potential sources of new anti-virulence inhibitors and presents screening strategies for identifying these compounds.

Selection of Multidrug-Resistant Enterobacteria in Weaned Pigs and Its Association With In-feed Subtherapeutic Combination of Colistin and Tylosin.

In-feed antibiotics are administered to piglets to improve performance and production efficiency. However, the use of growth promoters in the swine industry can select for multidrug-resistant (MDR) bacteria. Here, we evaluate the resistance profile of enterobacteria isolated from fecal samples of weaned pigs (21-35 days) fed or not with antibiotics (colistin and tylosin) and investigated the piglets gut microbiota in both groups. Six hundred and eighteen bacterial cultures were isolated from the control group (CON; n = 384) and antibiotic-fed pigs (ATB; n = 234). All isolates were tested for resistance to 12 antibiotics belonging to six distinct antibiotic classes. Isolates were highly resistant to ampicillin (90%; n = 553), amoxicillin (85%; n = 525), and tetracycline (81%; n = 498). A significant increase (P < 0.05) in resistance to cephalexin, kanamycin, doxycycline, and colistin was observed for bacteria from the ATB group. Piglets allocated in the ATB and CON groups shared similar intestinal microbiota, as revealed by alpha- and beta-diversity analyses. Our findings demonstrate that colistin and tylosin contribute to select MDR enterobacteria in weaned piglets. The high frequency of antibiotic resistance among isolates from the CON group suggests that environmental sources (e.g., fecal contents, aerosols, soil, water, food) also represent a potential reservoir of multidrug-resistant enterobacteria in pig production systems.

Microbiome-derived antimicrobial peptides offer therapeutic solutions for the treatment of Pseudomonas aeruginosa infections.

Microbiomes are rife for biotechnological exploitation, particularly the rumen microbiome, due to their complexicity and diversity. In this study, antimicrobial peptides (AMPs) from the rumen microbiome (Lynronne 1, 2, 3 and P15s) were assessed for their therapeutic potential against seven clinical strains of Pseudomonas aeruginosa. All AMPs exhibited antimicrobial activity against all strains, with minimum inhibitory concentrations (MICs) ranging from 4-512 µg/mL. Time-kill kinetics of all AMPs at 3× MIC values against strains PAO1 and LES431 showed complete kill within 10 min to 4 h, although P15s was not bactericidal against PAO1. All AMPs significantly inhibited biofilm formation by strains PAO1 and LES431, and induction of resistance assays showed no decrease in activity against these strains. AMP cytotoxicity against human lung cells was also minimal. In terms of mechanism of action, the AMPs showed affinity towards PAO1 and LES431 bacterial membrane lipids, efficiently permeabilising the P. aeruginosa membrane. Transcriptome and metabolome analysis revealed increased catalytic activity at the cell membrane and promotion of ß-oxidation of fatty acids. Finally, tests performed with the Galleria mellonella infection model showed that Lynronne 1 and 2 were efficacious in vivo, with a 100% survival rate following treatment at 32 mg/kg and 128 mg/kg, respectively. This study illustrates the therapeutic potential of microbiome-derived AMPs against P. aeruginosa infections.

In silico identification of two peptides with antibacterial activity against multidrug-resistant Staphylococcus aureus.

Here we report two antimicrobial peptides (AMPs), HG2 and HG4 identified from a rumen microbiome metagenomic dataset, with activity against multidrug-resistant (MDR) bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) strains, a major hospital and community-acquired pathogen. We employed the classifier model design to analyse, visualise, and interpret AMP activities. This approach allowed in silico discrimination of promising lead AMP candidates for experimental evaluation. The lead AMPs, HG2 and HG4, are fast-acting and show anti-biofilm and anti-inflammatory activities in vitro and demonstrated little toxicity to human primary cell lines. The peptides were effective in vivo within a Galleria mellonella model of MRSA USA300 infection. In terms of mechanism of action, HG2 and HG4 appear to interact with the cytoplasmic membrane of target cells and may inhibit other cellular processes, whilst preferentially binding to bacterial lipids over human cell lipids. Therefore, these AMPs may offer additional therapeutic templates for MDR bacterial infections.

Bovicin HC5 and nisin reduce cell viability and the thermal resistance of Alicyclobacillus acidoterrestris endospores in fruit juices.

BACKGROUND: Alicyclobacillus acidoterrestris is an important thermoacidophilic spore-forming bacterium in fruit-juice deterioration, and alternative non-thermal methods have been investigated to control fruit juice spoilage. This work aimed to evaluate the capacity of bovicin HC5 and nisin to inhibit the growth of vegetative cells and reduce the thermal resistance of endospores of A. acidoterrestris inoculated (107 CFU mL-1 ) in different fruit juices. The number of viable cells was determined after 12 h incubation at 43 °C in the presence and absence of nisin or bovicin HC5 (10-100 AU mL-1 ). The exposure time (min) required to kill 90% of the initial population (reduction of one log factor) at 90 ºC (D90ºC ) was used to assess the thermal resistance of A. acidoterrestris endospores exposed (80 AU mL-1 ) or non-exposed to the bacteriocins. Additionally, the effect of bovicin and nisin on the morphology and cell structure of A. acidoterrestris was evaluated by atomic force microscopy (AFM). RESULTS: Bovicin HC5 and nisin were bactericidal against A. acidoterrestris inoculated in fruit juices and reduced the D90°C values up to 30-fold. AFM topographical images revealed substantial structural changes in the cellular framework of vegetative cells upon treatment with bovicin HC5 or nisin. CONCLUSIONS: These results emphasize the potential application of lantibiotics as additional hurdles in food processing to control thermoacidophilic spoilage bacteria in fruit juices. © 2022 Society of Chemical Industry.

In silico Screening Unveil the Great Potential of Ruminal Bacteria Synthesizing Lasso Peptides.

Studies of rumen microbial ecology suggest that the capacity to produce antimicrobial peptides could be a useful trait in species competing for ecological niches in the ruminal ecosystem. However, little is known about the synthesis of lasso peptides by ruminal microorganisms. Here we analyzed the distribution and diversity of lasso peptide gene clusters in 425 bacterial genomes from the rumen ecosystem. Genome mining was performed using antiSMASH 5, BAGEL4, and a database of well-known precursor sequences. The genomic context of the biosynthetic clusters was investigated to identify putative lasA genes and protein sequences from enzymes of the biosynthetic machinery were evaluated to identify conserved motifs. Metatranscriptome analysis evaluated the expression of the biosynthetic genes in the rumen microbiome. Several incomplete (n = 23) and complete (n = 11) putative lasso peptide clusters were detected in the genomes of ruminal bacteria. The complete gene clusters were exclusively found within the phylum Firmicutes, mainly (48%) in strains of the genus Butyrivibrio. The analysis of the genetic organization of complete putative lasso peptide clusters revealed the presence of co-occurring genes, including kinases (85%), transcriptional regulators (49%), and glycosyltransferases (36%). Moreover, a conserved pattern of cluster organization was detected between strains of the same genus/species. The maturation enzymes LasB, LasC, and LasD showed regions highly conserved, including the presence of a transglutaminase core in LasB, an asparagine synthetase domain in LasC, and an ABC-type transporter system in LasD. Phylogenetic trees of the essential biosynthetic proteins revealed that sequences split into monophyletic groups according to their shared single common ancestor. Metatranscriptome analyses indicated the expression of the lasso peptides biosynthetic genes within the active rumen microbiota. Overall, our in silico screening allowed the discovery of novel biosynthetic gene clusters in the genomes of ruminal bacteria and revealed several strains with the genetic potential to synthesize lasso peptides, suggesting that the ruminal microbiota represents a potential source of these promising peptides.

Genomic and gene expression evidence of nonribosomal peptide and polyketide production among ruminal bacteria: a potential role in niche colonization?

Genomic and transcriptomic analyses were performed to investigate nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) in 310 genomes of ruminal/fecal microorganisms. A total of 119 biosynthetic genes potentially encoding distinct nonribosomal peptides (NRPs) and polyketides (PKs) were predicted in the ruminal microbial genomes and functional annotation separated these genes into 19 functional categories. The phylogenetic reconstruction of the 16S rRNA sequences coupled to the distribution of the three 'backbone' genes involved in NRPS and PKS biosyntheses suggested that these genes were not acquired through horizontal gene transfer. Metatranscriptomic analyses revealed that the predominant genes involved in the synthesis of NRPs and PKs were more abundant in sheep rumen datasets. Reads mapping to the NRPS and PKS biosynthetic genes were represented in the active ruminal microbial community, with transcripts being highly expressed in the bacterial community attached to perennial ryegrass, and following the main changes occurring between primary and secondary colonization of the forage incubated with ruminal fluid. This study is the first comprehensive characterization demonstrating the rich genetic capacity for NRPS and PKS biosyntheses within rumen bacterial genomes, which highlights the potential functional roles of secondary metabolites in the rumen ecosystem.

Efficacy of a Ruminal Bacteriocin Against Pure and Mixed Cultures of Bovine Mastitis Pathogens.

Bacteriocins have been suggested as an alternative to conventional antibiotics for the prevention and treatment of mastitis infections. Predominant bacteria associated with bovine mastitis (n = 276 isolates) were evaluated for their susceptibility to bovicin HC5, a ruminal bacteriocin produced by Streptococcus equinus HC5. Bovicin HC5 inhibited most (> 80%) of the streptococcal and staphylococcal strains tested, but showed no effect against Escherichia coli strains. Susceptibility and resistance testing indicated that approximately 95% of the S. aureus strains were inhibited by concentrations of bovicin HC5 varying from 40 to 2560 AU ml-1. Bovicin HC5 (62.50 AU ml-1) also inhibited the growth of aerobic and anaerobic mixed cultures of S. aureus and S. agalactiae, but the combination with 0.25 mmol l-1 of EDTA showed even greater bactericidal activity. These results demonstrate that bovicin HC5 is effective against the most prevalent pathogens found in contagious udder infections and could complement the use antibiotics in mastitis prophylaxis and therapy.

Nisin penetration and efficacy against Staphylococcus aureus biofilms under continuous-flow conditions.

Biofilms may enhance the tolerance of bacterial pathogens to disinfectants, biocides and other stressors by restricting the penetration of antimicrobials into the matrix-enclosed cell aggregates, which contributes to the recalcitrance of biofilm-associated infections. In this work, we performed real-time monitoring of the penetration of nisin into the interior of Staphylococcus aureus biofilms under continuous flow and compared the efficacy of this lantibiotic against planktonic and sessile cells of S. aureus. Biofilms were grown in Center for Disease Control (CDC) reactors and the spatial and temporal effects of nisin action on S. aureus cells were monitored by real-time confocal microscopy. Under continuous flow, nisin caused loss of membrane integrity of sessile cells and reached the bottom of the biofilms within ~20 min of exposure. Viability analysis using propidium iodide staining indicated that nisin was bactericidal against S. aureus biofilm cells. Time-kill assays showed that S. aureus viability reduced 6.71 and 1.64 log c.f.u. ml-1 for homogenized planktonic cells in exponential and stationary phase, respectively. For the homogenized and intact S. aureus CDC biofilms, mean viability decreased 1.25 and 0.50 log c.f.u. ml-1, respectively. Our results demonstrate the kinetics of biofilm killing by nisin under continuous-flow conditions, and shows that alterations in the physiology of S. aureus cells contribute to variations in sensitivity to the lantibiotic. The approach developed here could be useful to evaluate the antibiofilm efficacy of other bacteriocins either independently or in combination with other antimicrobials.

Compositional and structural dynamics of the ruminal microbiota in dairy heifers and its relationship to methane production.

BACKGROUND: Heifers emit more enteric methane (CH4 ) than adult cows and these emissions tend to decrease per unit feed intake as they age. However, common mitigation strategies like expensive high-quality feeds are not economically feasible for these pre-production animals. Given its direct role in CH4 production, altering the rumen microbiota is another potential avenue for reducing CH4 production by ruminants. However, to identify effective microbial targets, a better understanding of the rumen microbiota and its relationship to CH4 production across heifer development is needed. RESULTS: Here, we investigate the relationship between rumen bacterial, archaeal, and fungal communities as well as CH4 emissions and a number of production traits in prepubertal (PP), pubertal (PB), and pregnant heifers (PG). Overall, PG heifers emitted the most CH4 , followed by PB and PP heifers. The bacterial genus Acetobacter and the archaeal genus Methanobrevibacter were positively associated, while Eubacterium and Methanosphaera were negatively associated with raw CH4 production by heifers. When corrected for dietary intake, both Eubacterium and Methanosphaera remained negatively associated with CH4 production. CONCLUSION: We suggest that Eubacterium and Methanosphaera represent likely targets for CH4 mitigation efforts in heifers as they were negatively associated with CH4 production and not significantly associated with production traits. © 2018 Society of Chemical Industry.

Addressing Global Ruminant Agricultural Challenges Through Understanding the Rumen Microbiome: Past, Present, and Future.

The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in "omic" data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent "omics" approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.

Nutritive value and fermentation quality of palisadegrass and stylo mixed silages.

The nutritive value and fermentation quality of palisadegrass (Brachiaria brizantha cv. Xaraes) and stylo (Stylosanthes capitata × S. macrocephala cv. Campo Grande) mixed silages were evaluated. The experiment was analyzed in a factorial scheme (5 × 2) in a completely randomized design using increasing levels of stylo (0, 25, 50, 75 and 100% on a fresh matter basis) on palisadegrass silages, with and without microbial inoculants (MI). With the increased ratio of stylo in mixed silages, dry matter (DM), crude protein (CP), acid detergent fiber (ADF), and lignin content increased in silages. The presence of MI promoted lower DM content, and higher neutral detergent fiber corrected for ash and protein, ADF and lignin content. The acid detergent insoluble nitrogen content and the lactic acid bacteria populations were not affected by treatments. The in vitroDM digestibility was affected by the interaction of levels of the stylo and MI. The pH, NH3 -N/total nitrogen and butyric acid concentrations decreased with increasing levels of stylo. Better nutritive value and quality of fermentation was found in the silage containing higher proportions of this stylo mixed with palisadegrass. The microbial inoculant evaluated did not alter the nutritive value or quality of the fermentation of the silages in this experiment.

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.

Assessing the impact of rumen microbial communities on methane emissions and production traits in Holstein cows in a tropical climate.

The evaluation of how the gut microbiota affects both methane emissions and animal production is necessary in order to achieve methane mitigation without production losses. Toward this goal, the aim of this study was to correlate the rumen microbial communities (bacteria, archaea, and fungi) of high (HP), medium (MP), and low milk producing (LP), as well as dry (DC), Holstein dairy cows in an actual tropical production system with methane emissions and animal production traits. Overall, DC cows emitted more methane, followed by MP, HP and LP cows, although HP and LP cow emissions were similar. Using next-generation sequencing, it was found that bacteria affiliated with Christensenellaceae, Mogibacteriaceae, S24-7, Butyrivibrio, Schwartzia, and Treponema were negatively correlated with methane emissions and showed positive correlations with digestible dry matter intake (dDMI) and digestible organic matter intake (dOMI). Similar findings were observed for archaea in the genus Methanosphaera. The bacterial groups Coriobacteriaceae, RFP12, and Clostridium were negatively correlated with methane, but did not correlate with dDMI and dOMI. For anaerobic fungal communities, no significant correlations with methane or animal production traits were found. Based on these findings, it is suggested that manipulation of the abundances of these microbial taxa may be useful for modulating methane emissions without negatively affecting animal production.

Genome analysis reveals insights of the endophytic Bacillus toyonensis BAC3151 as a potentially novel agent for biocontrol of plant pathogens.

Diseases caused by phytopathogenic microorganisms account for enormous losses for agribusiness. Although Bacillus species are recognized as being antimicrobial producers and some may provide benefits to plants, the association between Bacillus toyonensis and plants has not been studied. In this study, the whole-genome sequenced endophytic B. toyonensis BAC3151, which has demonstrated antimicrobial activity and quorum sensing inhibition of phytopathogenic bacteria, was investigated for its potential for the production of compounds for biocontrol of plant pathogens. Four whole-genome sequenced B. toyonensis strains shared 3811 protein-coding DNA sequences (CDSs), while strain-specific CDSs, such as biosynthetic gene clusters of antimicrobials, were associated with specific chromosomal regions and mobile genetic elements of the strains. B. toyonensis strains had a higher frequency of putative bacteriocins gene clusters than that of Bacillus species traditionally used for the production of antimicrobials. In addition, gene clusters potentially involved in the production of novel bacteriocins were found in BAC3151, as well as biosynthetic genes of several other compounds, including non-ribosomal peptides, N-acyl homoserine lactonase and chitinases, revealing a genetic repertoire for antimicrobial synthesis greater than that of other Bacillus strains that have demonstrated effective activity against phytopathogens. This study showed for the first time that B. toyonensis has potential to produce various antimicrobials, and the analyses performed indicated that the endophytic strain BAC3151 can be useful for the development of new strategies to control microbial diseases in plants that are responsible for large damages in agricultural crops.

Effect of Pre-weaning Diet on the Ruminal Archaeal, Bacterial, and Fungal Communities of Dairy Calves.

At birth, calves display an underdeveloped rumen that eventually matures into a fully functional rumen as a result of solid food intake and microbial activity. However, little is known regarding the gradual impact of pre-weaning diet on the establishment of the rumen microbiota. Here, we employed next-generation sequencing to investigate the effects of the inclusion of starter concentrate (M: milk-fed vs. MC: milk plus starter concentrate fed) on archaeal, bacterial and anaerobic fungal communities in the rumens of 45 crossbred dairy calves across pre-weaning development (7, 28, 49, and 63 days). Our results show that archaeal, bacterial, and fungal taxa commonly found in the mature rumen were already established in the rumens of calves at 7 days old, regardless of diet. This confirms that microbiota colonization occurs in the absence of solid substrate. However, diet did significantly impact some microbial taxa. In the bacterial community, feeding starter concentrate promoted greater diversity of bacterial taxa known to degrade readily fermentable carbohydrates in the rumen (e.g., Megasphaera, Sharpea, and Succinivribrio). Shifts in the ruminal bacterial community also correlated to changes in fermentation patterns that favored the colonization of Methanosphaera sp. A4 in the rumen of MC calves. In contrast, M calves displayed a bacterial community dominated by taxa able to utilize milk nutrients (e.g., Lactobacillus, Bacteroides, and Parabacteroides). In both diet groups, the dominance of these milk-associated taxa decreased with age, suggesting that diet and age simultaneously drive changes in the structure and abundance of bacterial communities in the developing rumen. Changes in the composition and abundance of archaeal communities were attributed exclusively to diet, with more highly abundant Methanosphaera and less abundant Methanobrevibacter in MC calves. Finally, the fungal community was dominated by members of the genus SK3 and Caecomyces. Relative anaerobic fungal abundances did not change significantly in response to diet or age, likely due to high inter-animal variation and the low fiber content of starter concentrate. This study provides new insights into the colonization of archaea, bacteria, and anaerobic fungi communities in pre-ruminant calves that may be useful in designing strategies to promote colonization of target communities to improve functional development.

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.

Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes.

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides.