b 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.T he production of low-molecular-weight antimicrobial peptides is a trait widely distributed among species of bacteria and archaea (1). Although a variety of functions has been assigned to these compounds (e.g., toxins, virulence factors, bacterial hormones), the bacteriocins have mainly been investigated due to their potential as alternatives to antibiotics and food preservatives (2-4). With the rise in antibiotic resistance among commensal and pathogenic strains of bacteria, there is an urgent need to identify and develop novel therapeutic strategies for clinical applications in human and animal health (5).Bacteriocins are often defined as ribosomally synthesized antimicrobial peptides produced by bacteria or archaea (6). Bacteriocins show great diversity in their chemical structures and mechanisms of action, and at least four main groups have been proposed to classify these antimicrobial agents (7). Class I contains posttranslationally modified antimicrobial peptides, such as lanthipeptides, sactipeptides, and lasso peptides, which differ in their molecular structures, their mechanisms of action, and the enzymatic apparatuses involved in modifying the precursor peptides (7,8). Class II bacteriocins consist of antimicrobial peptides without posttranslationally modified residues and ca...

BackgroundDietary protein plays a major role in ruminant nutrition, and protein supplementation is a widespread practice among farmers in the tropics. Ruminal bacteria are the main agents of dietary protein and amino acid degradation, yet few studies have focused on the isolation and characterization of hyper-ammonia-producing bacteria in animals fed tropical diets or supplemented with rumen-degradable proteins. This work investigated the bacterial community diversity of the rumen of Nellore steers fed tropical forages, with or without casein supplementation. We also isolated and characterized ruminal bacteria showing high levels of ammonia production.ResultsPolymerase chain reaction–denaturing gradient gel electrophoresis analysis indicated no differences in the ruminal bacterial community composition between the control and supplemented animals. Amino acid-fermenting bacteria (n = 250) were isolated from crossbred Nellore steers fed Tifton 85 (Cynodon sp.) using trypticase as the sole carbon and organic nitrogen source in the enrichment and isolation media. The deamination rates in isolates obtained from steers supplemented with casein showed a higher incidence of deamination rates >350 nmol NH3 mg protein−1 min−1 (P < 0.05), whereas isolates obtained from steers without supplementation showed deamination rates <200 nmol NH3 mg protein−1 min−1. Although most isolates (84%) could ferment carbohydrates, none could hydrolyze proteins or use urea to sustain growth. All isolates were sensitive to lasalocid and monensin (1 μmol l−1), and similarity analysis of the 16S rRNA sequences indicated a predominance of bacteria from the order Clostridiales, with variable homology (73–99%) to known bacterial species.ConclusionsThese results expand what is known about the biochemical and genetic diversity of hyper-ammonia-producing bacteria, and emphasize the role of carbohydrate-fermenting bacteria in ammonia production in the rumen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0369-9) contains supplementary material, which is available to authorized users.

In tropical regions, protein supplementation is a common practice in dairy and beef farming. However, the effect of highly degradable protein in ruminal fermentation and microbial community composition has not yet been investigated in a systematic manner. In this work, we aimed to investigate the impact of casein supplementation on volatile fatty acids (VFA) production, specific activity of deamination (SAD), ammonia concentration and bacterial and archaeal community composition. The experimental design was a 4 × 4 Latin square balanced for residual effects, with four animals (average initial weight of 280 ± 10 kg) and four experimental periods, each with duration of 29 days. The diet comprised Tifton 85 (Cynodon sp.) hay with an average CP content of 9.8%, on a dry matter basis. Animals received basal forage (control) or infusions of pure casein (230 g) administered direct into the rumen, abomasum or divided (50 : 50 ratio) in the rumen/abomasum. There was no differences ( P > 0.05) in ruminal pH and microbial protein concentration between supplemented v. non-supplemented animals. However, in steers receiving ruminal infusion of casein the SAD and ruminal ammonia concentration increased 33% and 76%, respectively, compared with the control. The total concentration of VFA increased ( P < 0.05) in steers receiving rumen infusion of casein. SAD and the microbial protein concentration did not vary significantly among treatments during the feeding cycle, but mean SAD values were greater in steers supplemented in the rumen and rumen/abomasum. Ruminal ammonia concentration was positively correlated with SAD in animals receiving ruminal infusion of casein. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis revealed low similarity between treatments, animals and time of sample collection. Richness analysis and determination of the Shannon-Wiener index indicated no differences ( P > 0.05) in species richness and diversity of γ-proteobacteria, firmicutes and archaea between non-supplemented Nellore steers and steers receiving casein supplementation in the rumen. However, species richness and the Shannon-Wiener index were lower ( P < 0.05) for the phylum bacteroidetes in steers supplemented with casein in the rumen compared with non-supplemented animals. Venn diagrams indicated that the number of unique bands varied considerably among individual animals and was usually higher in number for non-supplemented steers compared with supplemented animals. These results add new knowledge about the effects of ruminal and postruminal protein supplementation on metabolic activities of rumen microbes and the composition of bacterial and archaeal communities in the rumen of steers.Keywords: beef cattle, casein, rumen degradable protein, deamination, microbial fingerprint ImplicationsFormulation of cattle diets requires balancing the availability of carbohydrates and protein in order to maximize microbial growth in the rumen. Increasing the availability of rumen degradable protein (RDP) is useful to red...

Antibiotic resistance poses an important threat to global public health and has become a challenge to modern medicine. The occurrence of antibiotic-resistant bacteria in a broad range of foods has led to a growing concern about the impact that food may have as a reservoir of antibiotic resistance genes. Considering Minas Frescal Cheese (MFC)—a typical Brazilian white soft cheese—and its economic and cultural values, in this study, medically relevant antimicrobial-resistance genetic markers (AR genes) were screened, and the occurrence of integrons were evaluated in manufactured MFC using culture-independent approaches. Through a fingerprinting analysis, the tested MFCs were brand-clustered, indicating reproducibility along the production chain. A common core of resistance markers in all brands evaluated and related antimicrobials such as β-lactams, tetracyclines, quinolones, and sulfonamide was detected. Several other markers, including efflux pumps and aminoglycosides-resistance were distributed among brands. Class 1 and 2 integrons were observed, respectively, in 77% and 97% of the samples. The presence of AR genes is of special interest due to their clinical relevance. Taken together, the data may suggest that the production chain of MFC might contribute to the spread of putative drug-resistant bacteria, which could greatly impact human health. Furthermore, detection of class 1 and class 2 integrons in MFC has led to discussions about resistance gene spread in this traditional cheese, providing evidence of potential horizontal transfer of AR genes to human gut microbiota.

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Streptococcus equinus (Streptococcus bovis) HC5 is a bacteriocinogenic lactic acid bacterium with simple growth requirements. The draft genome sequence of S. equinus HC5 consists of 1,846,241 bp, with a G+C content of 37.04%. In silico analysis indicated that S. equinus HC5 might be useful to control bacteria that are detrimental to livestock animals.

Antibiotics are used as feed additives for cattle to alter rumen fermentation and increase weight gain. However, this practice can potentially lead to the presence of antibiotic residues in milk and meat and the selection of multiresistant bacteria. Bacteriocins have been suggested as an alternative to antibiotics used in animal production. This work aimed to evaluate the in vitro effects of bovicin HC5 and virginiamycin on ruminal fermentation and on microbial community composition. Ruminal fluid was collected from fistulated cows fed corn silage and incubated with Trypticase (15 g L -1). Cultures treated with bovicin HC5 or virginiamycin decreased (P < 0.05) ammonia accumulation by 47.46% and 66.17%, respectively. Bovicin HC5 and virginiamycin also decreased (P < 0.05) the concentration of organic acids and gas production, but the effects were somewhat distinct. Molecular fingerprinting of the microbial community using PCR-DGGE revealed that community structure varied between treatments and were distinct from the controls. These results demonstrate that bovicin HC5 and virginiamycin have distinct effects on ruminal fermentation and modify differently the microbial community composition. These results also expand the knowledge about the effects of antibiotics and bacteriocins on bacterial and archaeal communities involved in protein metabolism in the rumen.

Aims To investigate the inhibitory activity and the distribution of biosynthetic genes encoding bovicin‐like bacteriocins among ruminal Streptococcus isolated from beef and dairy cattle. Methods and Results Most isolates were classified as Streptococcus equinus and Streptococcus lutetiensis based on 16S rRNA sequencing. The antimicrobial activity of 150 ruminal streptococci isolated from beef and dairy cattle were tested by deferred inhibition assays and their genetic diversity was characterized by BOX‐PCR. The frequency of biosynthetic genes associated with the biosynthesis of bovicin‐like bacteriocins (bovicin HC5 and bovicin 255) was investigated by PCR screening. Approximately 33% of the ruminal streptococci isolated from Nellore heifers showed inhibitory activity in vitro with the majority harbouring genes for bacteriocin biosynthesis. In contrast, streptococci from Holstein cows showed limited inhibitory activity and a lower frequency of bacteriocin biosynthetic genes. Conclusions Streptococcus from the rumen of beef and dairy cattle exhibit remarkable differences in inhibitory activity and distribution of genes associated with the biosynthesis of prototypical bovicins (bovicin HC5 and bovicin 255). Significance and Impact of the Study Our findings demonstrate that bovicin HC5 is distributed among ruminal streptococci from different breeds of cattle. The high degree of conservation of the bovicin HC5 structural gene among strains of ruminal streptococci suggests that random genetic drift is not a dominant force in the evolution of this bacteriocin.