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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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