Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus.

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Unraveling the diversity and dissemination dynamics of antimicrobial resistance genes in Enterobacteriaceae plasmids across diverse ecosystems.

AIM: The objective of this study was to investigate the antimicrobial resistance genes (ARGs) in plasmids of Enterobacteriaceae from soil, sewage, and feces of food-producing animals and humans. METHODS AND RESULTS: The plasmid sequences were obtained from the NCBI database. For the identification of ARG, comprehensive antibiotic resistance database (CARD), and ResFinder were used. Gene conservation and evolution were investigated using DnaSP v.6. The transfer potential of the plasmids was evaluated using oriTfinder and a MOB-based phylogenetic tree was reconstructed using Fastree. We identified a total of 1064 ARGs in all plasmids analyzed, conferring resistance to 15 groups of antibiotics, mostly aminoglycosides, beta-lactams, and sulfonamides. The greatest number of ARGs per plasmid was found in enterobacteria from chicken feces. Plasmids from Escherichia coli carrying multiple ARGs were found in all ecosystems. Some of the most abundant genes were shared among all ecosystems, including aph(6)-Id, aph(3'')-Ib, tet(A), and sul2. A high level of sequence conservation was found among these genes, and tet(A) and sul2 are under positive selective pressure. Approximately 62% of the plasmids carrying at least one ARG were potentially transferable. Phylogenetic analysis indicated a potential co-evolution of Enterobacteriaceae plasmids in nature. CONCLUSION: The high abundance of Enterobacteriaceae plasmids from diverse ecosystems carrying ARGs reveals their widespread distribution and importance.

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.

Exopolysaccharides produced by Bacillus spp. inhibit biofilm formation by Staphylococcus aureus strains associated with bovine mastitis.

Bovine mastitis is a costly disease in the dairy sector worldwide. Here the objective was to identify and characterize anti-biofilm compounds produced by Bacillus spp. against S. aureus associated with bovine mastitis. Results showed that cell-free supernatants of three Bacillus strains (out of 33 analysed) reduced S. aureus biofilm formation by approximately 40 % without affecting bacterial growth. The anti-biofilm activity was associated with exopolysaccharides (EPS) secreted by Bacillus spp. The EPS decreased S. aureus biofilm formation in a dose-dependent manner, inhibiting biofilm formation by 83 % at 1 mg/mL. The EPS also showed some biofilm disruption activity (up to 36.4 %), which may be partially mediated by increased expression of the aur gene. The characterization of EPS produced by Bacillus velezensis 87 and B. velezensis TR47II revealed macromolecules with molecular weights of 31.2 and 33.7 kDa, respectively. These macromolecules were composed mainly of glucose (mean = 218.5 µg/mg) and mannose (mean = 241.5 µg/mg) and had similar functional groups (pyranose ring, beta-type glycosidic linkage, and alkynes) as revealed by FT-IR. In conclusion, this study shows the potential applications of EPS produced by B. velezensis as an anti-biofilm compound that could contribute to the treatment of bovine mastitis caused by S. aureus.

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.

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.

Characterization of antibiotic resistance genes in the species of the rumen microbiota.

Infections caused by multidrug resistant bacteria represent a therapeutic challenge both in clinical settings and in livestock production, but the prevalence of antibiotic resistance genes among the species of bacteria that colonize the gastrointestinal tract of ruminants is not well characterized. Here, we investigate the resistome of 435 ruminal microbial genomes in silico and confirm representative phenotypes in vitro. We find a high abundance of genes encoding tetracycline resistance and evidence that the tet(W) gene is under positive selective pressure. Our findings reveal that tet(W) is located in a novel integrative and conjugative element in several ruminal bacterial genomes. Analyses of rumen microbial metatranscriptomes confirm the expression of the most abundant antibiotic resistance genes. Our data provide insight into antibiotic resistange gene profiles of the main species of ruminal bacteria and reveal the potential role of mobile genetic elements in shaping the resistome of the rumen microbiome, with implications for human and animal health.