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Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics 1 . Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins 2 , potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome 3 . Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed 2 . Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus , exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms. Modular synthesis and structural biology are used to design and characterize group A streptogramin antibiotics, one of which has activity against streptogramin-resistant strains and demonstrates efficacy in a mouse model of bacterial infection.

During biosynthesis by multi-modular trans -AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic β-ketones into β-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans -AT polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for β-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second β-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin β-branching programming. Furthermore, variations in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives. Biosynthesis of complex polyketides by polyketide synthases often relies on trans-acting enzymes to modify the intermediates. Here, the authors elucidate how β-methylation enzymes identify their substrates. The recognition is imperfect, resulting in a doubly β-methylated virginiamycin derivative.

Although dietary antibiotic growth promoters have long been used to increase growth performance in commercial food animal production, the biochemical details associated with these effects remain poorly defined. A metabolomics approach was used to characterize and identify the biochemical compounds present in the intestine of broiler chickens fed a standard, unsupplemented diet or a diet supplemented with the antibiotic growth promoters, virginiamycin or bacitracin methylene disalicylate. Compared with unsupplemented controls, the levels of 218 biochemicals were altered (156 increased, 62 decreased) in chickens given the virginiamycin-supplemented diet, while 119 were altered (96 increased, 23 decreased) with the bacitracin-supplemented diet. When compared between antibiotic-supplemented groups, 79 chemicals were altered (43 increased, 36 decreased) in virginiamycin- vs. bacitracin-supplemented chickens. The changes in the levels of intestinal biochemicals provided a distinctive biochemical signature unique to each antibiotic-supplemented group. These biochemical signatures were characterized by increases in the levels of metabolites of amino acids (e.g. 5-hydroxylysine, 2-aminoadipate, 5-hydroxyindoleaceate, 7-hydroxyindole sulfate), fatty acids (e.g. oleate/vaccenate, eicosapentaenoate, 16-hydroxypalmitate, stearate), nucleosides (e.g. inosine, N -methyladenosine), and vitamins (e.g. nicotinamide). These results provide the framework for future studies to identify natural chemical compounds to improve poultry growth performance without the use of in-feed antibiotics.

Antimicrobials are sometimes given to food animals at low doses in order to promote faster growth. However, the mechanisms by which those drugs improve performance are not fully understood. This study aimed to investigate the impact of zinc bacitracin (55g/ton), enramycin (10g/ton); halquinol® (30g/ton); virginiamycin (16,5g/ton) and avilamycin (10g/ton) on the cecal microbiota of broiler chicken, compared to a control group. Six hundred and twenty four chicks (Cobb 500) arriving to an experimental unit were randomly assigned into each treatment with four repetitions per treatment. The cecal content of 16 animals per treatment (n = 96) was used for DNA extraction and sequencing of the V4 region of the 16S rRNA gene using Illumina technology. The use of antimicrobials induced significant changes in membership but not in structure of the cecal microbiota compared to the control group, suggesting a greater impact on the less abundant species of bacteria present in that environment. Halquinol was the only drug that did not affect microbial membership. Firmicutes comprised the major bacterial phylum present in the cecum of all groups. There was no statistical difference in relative abundances of the main phyla between treated animals and the control group (all P>0.05). Treatment with enramycin was associated with decreased richness and with lower relative abundance of unclassified Firmicutes, Clostridium XI, unclassified Peptostreptococcaceae (all P<0.001) and greater abundance of Clostridium XIVb (P = 0.004) and Anaerosporobacter spp. (P = 0.015), and treatment with bacitracin with greater relative abundance of Bilophila spp. (P = 0.004). Several bacterial genera were identified as representative of usage of each drug. This study used high throughput sequencing to characterize the impact of several antimicrobials in broiler chicken under controlled conditions and add new insights to the current knowledge on how AGPs affect the cecal microbiota of chicken.

With increasing pressures to reduce or eliminate the use of antimicrobials for growth promotion purposes in production animals, there is a growing need to better understand the effects elicited by these agents in order to identify alternative approaches that might be used to maintain animal health. Antibiotic usage at subtherapeutic levels is postulated to confer a number of modulations in the microbes within the gut that ultimately result in growth promotion and reduced occurrence of disease. This study examined the effects of the coccidiostat monensin and the growth promoters virginiamycin and tylosin on the broiler chicken cecal microbiome and metagenome. Using a longitudinal design, cecal contents of commercial chickens were extracted and examined using 16S rRNA and total DNA shotgun metagenomic pyrosequencing. A number of genus-level enrichments and depletions were observed in response to monensin alone, or monensin in combination with virginiamycin or tylosin. Of note, monensin effects included depletions of Roseburia, Lactobacillus and Enterococcus, and enrichments in Coprococcus and Anaerofilum. The most notable effect observed in the monensin/virginiamycin and monensin/tylosin treatments, but not in the monensin-alone treatments, was enrichments in Escherichia coli. Analysis of the metagenomic dataset identified enrichments in transport system genes, type I fimbrial genes, and type IV conjugative secretion system genes. No significant differences were observed with regard to antimicrobial resistance gene counts. Overall, this study provides a more comprehensive glimpse of the chicken cecum microbial community, the modulations of this community in response to growth promoters, and targets for future efforts to mimic these effects using alternative approaches.

This study investigated the cecal microbiota and serum metabolite profile of chickens fed with plant essential oils (PEO) or virginiamycin (VIRG) using high-throughput 16S rRNA gene sequencing and untargeted metabolomics approach. The main aim of this work was to explore the biochemical mechanisms involved in the improved growth performance of antibiotics and their alternatives in animal production. The results showed that both PEO and VIRG treatment significantly increased the relative abundance of phyla Bacteroidetes and decreased the abundance of phyla Firmicutes and genus of Lactobacillus in cecal microbiota of chickens. Compared to the control group (CT group), the relative abundance of genus of Alistipes , unclassified Rikenellaceae, Roseburia , and Anaeroplasma was enriched in the PEO group; that of genus Bacteroides , Lachnospiraceae , and unclassified Enterobacteriaceae was enriched in the cecal microbiota of the VIRG group. Untargeted metabolomics analyses revealed that the PEO treatment modified 102 metabolites and 3 KEGG pathways (primary bile acid biosynthesis and phenylalanine metabolism) in the cecal microbiota, and 81 metabolites and relevant KEGG pathways (fructose and mannose metabolism, biosynthesis of unsaturated fatty acids, and linoleic acid.) in the serum of the chicken. Compared to the CT group, VIRG treatment group differed 217 metabolites and 10 KEGG pathways in cecal contents and 142 metabolites and 7 KEGG pathways in serum of chickens. Pearson’s correlation analysis showed that phyla Bacteroidetes and genus of Bacteroides, Alistipes , and unclassified Rikenellaceae (in the VIRG and PE group) were positively correlated with many lipid metabolites. However, phyla Firmicutes and genera Lactobacillus (higher in the CT group) were negatively correlated with the lipid and thymine metabolism, and positively correlated with hydroxyisocaproic acid, cytosine, and taurine. This study shows that dietary supplementation with PEO and VIRG altered the composition and metabolism profile of the cecal microbiota, modified the serum metabolism profile.

Two experiments were conducted to examine the influence of protein and virginiamycin (VM) supplementation on feedlot growth performance, digestion, and metabolizable AA (MAA) supply of calf-fed Holstein steers. Growth performance and dietary energetics were evaluated in 120 Holstein steers (127 ± 9 kg). During the initial 112-d feeding period, a steam-flaked corn-based diet was balanced to meet either 100% (MAB) or 87% (UREA) of MAA requirements. Diets were supplemented with or without 22.5 mg/kg VM in a 2 × 2 factorial arrangement. Subsequently (d 112 to 308), all steers received the UREA diet with or without VM. During the initial 112-d, MAB increased ADG, G:F, and dietary NE ( < 0.01). Thereafter, when all steers received the UREA diet, ADG, G:F, and dietary NE were not different ( > 0.10) across initial supplementation treatments. Overall (d 1 to 308), MAB did not affect ADG ( > 0.10) but enhanced G:F efficiency ( = 0.03) and dietary NE ( = 0.05). During the initial 112-d period and through the remainder of the experiment, VM increased G:F ( < 0.01) and dietary NE ( < 0.01). Four Holstein steers (146 ± 4 kg) with cannulas in the rumen and proximal duodenum were used in a 4 × 4 Latin square design to evaluate initial 112-d treatment effects on digestive function. There were no treatment effects ( > 0.10) on ruminal digestion of OM, NDF, starch, microbial efficiency, or total tract digestion of OM and NDF. The MAB increased indispensable AA flow to the small intestine ( < 0.01) and total tract digestion of N ( < 0.01) and starch ( = 0.04). Observed AA supply to small intestine was in agreement with expected supply ( = 0.96). Virginiamycin decreased ( = 0.04) nonammonia N flow to the small intestine and did not affect ( > 0.10) total tract N digestion. Extrapolating from AA supplies in the metabolism study, MAB satisfied indispensable AA requirements during the initial 112-d period, whereas the UREA diet met 73.5% and 79.2% of methionine and lysine requirements, respectively. During the subsequent periods (d 112 to 308) indispensable AA supplies exceeded theoretical requirements. We conclude that enhancements in energy utilization when diets are balanced to meet MAA requirements of calf-fed Holstein steers during the initial 112-d feedlot period remain appreciable throughout time on feed. Virginiamycin enhanced efficiency of energy utilization throughout the feedlot growing-finishing period.

The rearing of calves is an essential activity of a dairy system, as it impacts the future production of these animals. This study aims to evaluate the incidence of diarrhea, performance, and blood parameters of suckling calves that received mineral-vitamin supplementation in milk plus virginiamycin that was offered in milk (via the abomasum) or by esophageal tube (via the rumen). Twenty-seven calves were used, from the first week to 60 days of age, submitted to the following treatments: CONTROL, without supplementation; MILK, supplementation of 20 g of a mineral-vitamin complex with 100 mg of virginiamycin, diluted in milk; RUMEN, supplementation of 20 g of a mineral-vitamin complex diluted in milk and 100 mg of virginiamycin in gelatin capsules via an esophageal applicator. MILK and RUMEN calves had lower fecal consistency scoring, fewer days with scores 2 and 3 throughout the experimental period, and lower spending on medication compared to the CONTROL animals. Supplemented calves had higher fat and protein intake and reached feed intake of 600 g earlier than CONTROL animals, but did not differ in performance and hematological parameters. Supplementation with virginiamycin and vitamin-mineral complex for suckling calves reduced the incidence and days of diarrhea, and reduced medication costs, with no difference in performance, but the supplemented animals had higher initial protein and fat intake and reached targeted feed intake earlier to begin the weaning process.

Abstract The judicious use of commercial products in livestock operations can be part of a sustainable and environmentally friendly production scenario. This study was designed to gather published data of virginiamycin (VM) used in feedlot conditions of the United States and analyze its effectiveness and optimum dosage in reducing the liver abscess incidence (LAI). The dataset contained 26 studies that evaluated more than 7,156 animals of diverse breeds fed in several regions in the United States under different management. Statistical analyses included contingency tables to assess the nonparametric independence of the LAI, meta regression analysis to remove study effects and to evaluate LAI and animal performance, broken-line analysis to determine thresholds of VM dosage on LAI, and residual-based shading mosaic plots to illustrate the contingency analysis. There were 1,391 of 5,430 animals with LAI scores 1, 2, or 3 (LAI1–3) and 651 of 4,690 animals with LAI A+ (score 3). Our analyses suggested that there was a significant dependency (χ2P-value < 0.001) and significant asymmetry (McNemar’s test P-value < 0.001) between LAI and VM treatment for both LAI1–3 and LAI A+. For the LAI1–3 group, only 22.5% of the treated animals had liver abscesses compared with 31.7% of the control animals. The metaregression analysis indicated that LAI1–3 was linearly reduced (P < 0.001) by about 0.42% per mg/kg of DM of VM. The lower 95% confidence interval of the intercept for LAI1–3 and LAI A+ obtained with a generalized nonlinear mixed regression was 18.7 and 20.3 mg/kg of DM, respectively. The broken-line regression analysis identified 2 thresholds for LAI (23.9 and 12.3 mg/kg of DM) at which the reduction in total LAI1–3 and LAI A+, respectively, would decrease faster as VM dosage increases (from 2.14% to 6% and from 1.91% to 4.33% per mg of VM per kg of DM, respectively). Additionally, our analyses indicated that after accounting for the study effects, VM significantly increased ADG at 2.08 g BW/d per mg/kg DM compared with 0.92 g BW/d per mg/kg DM for monensin (P < 0.001), suggesting that VM was about 2.3 times more effective in increasing ADG for the same dosage and feeding period length. All analyses yielded consistent results that led us to conclude that VM is effective in reducing LAI when fed between approximately 12 and 24 mg/kg of DM, and the maximum reduction might occur at approximately 24 mg/kg of DM or higher.

Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ₂ mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).