Flavomycin restores colistin susceptibility in multidrug-resistant Gram-negative bacteria.

Polymyxin is used as a last resort antibiotics for infections caused by multi-drug resistant (MDR) Gram-negative bacteria and is often combined with other antibiotics to improve clinical effectiveness. However, the synergism of colistin and other antibiotics remains obscure. Here, we revealed a notable synergy between colistin and flavomycin, which was traditionally used as an animal growth promoter and has limited activity against Gram-negative bacteria, using checkerboard assay and time-kill curve analyses. The importance of membrane penetration induced by colistin was assessed by examining the intracellular accumulation of flavomycin and its antimicrobial impact on Escherichia coli (E. coli) strains with truncated lipopolysaccharides. Besides, a mutation in the flavomycin binding site was created to confirm its role in the observed synergy. This synergy is manifested as an augmented penetration of the E. coli outer membrane by colistin, leading to increased intracellular accumulation of flavomycin and enhanced cell killing thereafter. The observed synergy was dependent on the antimicrobial activity of flavomycin, as mutation of its binding site abolished the synergy. In vivo studies confirmed the efficacy of colistin combined with flavomycin against MDR E. coli infections. This study is the first to demonstrate the synergistic effect between colistin and flavomycin, shedding light on their respective roles in this synergism. Therefore, we propose flavomycin as an adjuvant to enhance the potency of colistin against MDR Gram-negative bacteria. IMPORTANCE: Colistin is a critical antibiotic in combating multi-drug resistant Gram-negative bacteria, but the emergence of mobilized colistin resistance (mcr) undermines its effectiveness. Previous studies have found that colistin can synergy with various drugs; however, its exact mechanisms with hydrophobic drugs are still unrevealed. Generally, the membrane destruction of colistin is thought to be the essential trigger for its interactions with its partner drugs. Here, we use clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) for specifically mutating the binding site of one hydrophobic drug (flavomycin) and show that antimicrobial activity of flavomycin is critical for the synergy. Our results first give the evidence that the synergy is set off by colistin's membrane destruction and operated the final antimicrobial function by its partner drugs.

Effects of litter from antimicrobial-fed broiler chickens on soil bacterial community structure and diversity.

This study examined changes in soil bacterial community composition and diversity in response to fertilization with litter from chickens fed a diet without antibiotics and with bambermycin, penicillin, bacitracin, salinomycin, or mix of salinomycin and bacitracin. Litter (27.5 T/ha) was applied to 24 agricultural plots in the Fraser Valley of British Columbia. Nonfertilized plots were used as a negative control. Soil samples collected from the studied plots were used to quantify Escherichia coli by plate counts, and Clostridium perfringens by qPCR. The 16S rRNA gene sequencing was performed for microbiota analysis. Following litter application in December, the population size of E. coli was 5.4 log CFU/g; however, regardless of treatments, the results revealed 5.2 and 1.4 log CFU/g of E. coli in soil sampled in January and March, respectively. Fertilization with litter from antibiotic-treated birds increased (P < 0.05) the relative abundance of Proteobacteria, Actinobacteria, and Firmicutes in soil, but decreased Acidobacteria and Verrucomicrobia groups. The alpha diversity parameters were higher (P < 0.05) in nonfertilized soil compared to the fertilized ones, suggesting that litter application was a major factor in shaping the soil bacterial communities. These results may help develop efficient litter management strategies like composting, autoclaving, or anaerobic digestion of poultry litter before application to land for preservation of soil health and crop productivity.

Harnessing Fluorescent Moenomycin A Antibiotics for Bacterial Cell Wall Imaging Studies.

The imaging of peptidoglycan (PGN) dynamics in living bacteria facilitates the understanding of PGN biosynthesis and wall-targeting antibiotics. The main tools for imaging bacterial PGN are fluorescent probes, such as the well-known PGN metabolic labeling probes. However, fluorescent small-molecule probes for labeling key PGN-synthesizing enzymes, especially for transglycosylases (TGases), remain to be explored. In this work, the first imaging probe for labeling TGase in bacterial cell wall studies is reported. We synthesized various fluorescent MoeA-based molecules by derivatizing the natural antibiotic moenomycin A (MoeA), and used them to label TGases in living bacteria, monitor bacterial growth and division cycles by time-lapse imaging, and study cell wall growth in the mecA-carrying methicillin-resistant Staphylococcus aureus (MRSA) strains when the ß-lactam-based probes were unsuitable.

Salmonella shedding and seropositivity and its association with in-feed flavophospholipol in nursery pigs.

The objective of this study was to assess the impact of in-feed flavophospholipol on Salmonella shedding and antibody response in nursery pigs. Weaned pigs were fed either a diet containing 4 ppm flavophospholipol (n = 16) or a non-medicated feed (n = 16) for 36 d. All pigs were orally challenged with a 2-mL dose of 108 colony-forming units (CFUs)/mL of Salmonella Typhimurium on Days 7 and 8 of the trial. On Day 36, all pigs were euthanized and samples were collected from the liver, spleen, and ileocecal lymph nodes. Fecal and tissue samples were quantitatively cultured for Salmonella and serum samples were tested for the presence of the Salmonella antibody by enzyme-linked immunosorbent assay (ELISA). There was no difference between the 2 groups in antibody response and the presence of Salmonella in feces and tissue (P > 0.05). Medicating nursery diets with flavophospholipol at 4 ppm did not appear to reduce Salmonella infection in nursery pigs.

L'objectif de la présente étude était d'évaluer l'impact de l'ajout de flavophospholipol dans l'aliment sur l'excrétion de Salmonella et la réponse en anticorps chez des porcs en pouponnière. Des porcs sevrés ont été nourris avec soit une diète contenant 4 ppm de flavophospholipol (n = 16) ou une diète non-médicamentée (n = 16) pendant 36 j. Tous les porcs ont reçu oralement une dose de 2 mL de 108 unités formatrices de colonies (UFC)/mL de Salmonella Typhimurium aux Jours 7 et 8 de l'essai. Au Jour 36, tous les porcs ont été euthanasiés et on préleva des échantillons de foie, rate, et noeuds lymphatiques iléo-caecaux. Des échantillons de fèces et de tissus ont été cultivés pour quantifier le nombre de Salmonella et des échantillons de sérum furent testés pour la présence d'anticorps contre Salmonella par épreuve immunoenzymatique (ELISA). Il n'y avait pas de différence entre les deux groupes quant à la réponse en anticorps et la présence de Salmonella dans les fèces et les tissus (P > 0,05). L'ajout de 4 ppm de flavophospholipol à la diète en pouponnière ne semble pas réduire l'infection par Salmonella chez les porcs en pouponnière.(Traduit par Docteur Serge Messier).

Effects of selenium-enriched Bacillus sp. compounds on growth performance, antioxidant status, and lipid parameters breast meat quality of Chinese Huainan partridge chicks in winter cold stress.

BACKGROUND: Both selenium (Se) and probiotic Bacillus regulate the metabolism to help defense clod stress and improve the meat quality in breeding chicks. The purpose of this study was to evaluate the effect of supplemental Se and Bacillus in the form of Se-enriched Bacillus (SECB) on the growth performance, lipid parameters, breast Se and antibiotic levels, and breast meat quality of chicken in winter cold stress. METHODS: Five hundred 1-d-old chickens were divided into five groups randomly: Control, inorganic Se, compound Bacillus, SECB, and antibiotic. The feed duration was 56 d. RESULTS: After 28 d of treatment, chicks feed SECB or compound Bacillus had higher body weights than the control, and after 56 d, chicks given either SECB or compound Bacillus had higher body weights than the control chicks or those given inorganic Se. Adding SECB to feed significantly increased the lightness, redness, and yellowness of breast meat, improved the water-holding capacity, and reduced the shear force and cooking loss. The concentration of Se in the breast muscle very significantly increased after SECB and inorganic Se supplementation, which was opposite to the concentration of flavomycin in antibiotic supplemented chicks. The antioxidative status of plasma and breast meat was significantly improved with added compound Bacillus and SECB: the total antioxidant capacity, total superoxide dismutase, and glutathione peroxidase ability in the breast muscle significantly improved, and the malondialdehyde concentration in plasma decreased. The levels of total cholesterol plasma triglyceride and very-low-density lipoprotein cholesterol in the plasma and breast muscle was decreased compared to that of the control, while the plasma high-density lipoprotein cholesterol concentration increased. CONCLUSIONS: In conclusion, SECB supplementation promoted the body growth, antioxidative status, and Se concentrations in the plasma and breast meat, and also improved the breast meat quality.

Inhibition effect of flavophospholipol on conjugative transfer of the extended-spectrum ß-lactamase and vanA genes.

Flavophospholipol (FPL) is an antimicrobial feed additive that has been approved for use in livestock animals and has the potential to decrease horizontal dissemination of antimicrobial resistance genes. Since previous studies showed that FPL has an inhibitory effect on plasmid transfer, in vitro experiments have proven the efficacy of FPL in reducing the conjugative transfer of plasmids encoding the extended-spectrum ß-lactamase (ESBL) and vanA genes. These are among the most important antimicrobial resistance loci known. ESBL-producing Escherichia coli and vancomycin-resistant Enterococcus faecalis (VRE) were exposed to several concentrations of FPL, and transfer frequency and plasmid curing activity were determined. FPL inhibited the conjugative transfer of plasmids harboring ESBL and vanA genes in a concentration-dependent manner in all strains. Further transfer experiments revealed that FPL could decrease or increase transfer frequency depending on plasmid type when transfer frequency was at low levels. The plasmid curing activity of FPL was also observed in ESBL-producing E. coli in a concentration-dependent manner, suggesting that they partially contribute to the inhibition of conjugative transfer. These results suggest that the use of FPL as a feed additive might decrease the dissemination of ESBL and vanA genes among livestock animals.

A comparison of the effects of antibiotics, probiotics, synbiotics and prebiotics on the performance and carcass characteristics of broilers.

Routine use of the antibiotic flavomycin in broiler production may lead to resistance, and alternative growth promoters are used to enhance performance. Two hundred day-old male Ross 308 broiler chicks were allocated to five dietary supplements included from d 1-42: flavomycin, three possible alternatives, a probiotic, prebiotic and a synbiotic, as well as a control treatment. There were four replicate cages of 10 birds each in each treatment. Compared with the control and antibiotics treatments, the probiotic, prebiotic and synbiotic treatments increased (p = 0.001) weight gain (64, 66, 73, 70 and 74 g/d, respectively). The synbiotic treatment reduced (p = 0.004) the feed conversion ratio, compared with the control and antibiotic treatments (1.70, 1.84, 1.83, respectively). Compared with the control and antibiotic treatments, the birds fed the synbiotic treatment had greater relative gizzard (+47%) and spleen weights (+115%), and lighter kidneys (-47%). The birds fed the symbiotic treatment also had thinner walls of the caudal gut segments. The prebiotic had the most beneficial effect on cecal microbiota, stimulating aerobic and lactic acid producing bacteria and reducing Escherichia coli bacteria. Enterococci were increased in the antibiotic treatment. We conclude that there were significant performance and health benefits of using prebiotics, probiotics and synbiotics for broilers, rather than antibiotics.

In-feed bambermycin medication induces anti-inflammatory effects and prevents parietal cell loss without influencing Helicobacter suis colonization in the stomach of mice.

The minimum inhibitory concentration of bambermycin on three porcine Helicobacter suis strains was shown to be 8 µg/mL. The effect of in-feed medication with this antibiotic on the course of a gastric infection with one of these strains, the host response and the gastric microbiota was determined in mice, as all of these parameters may be involved in gastric pathology. In H. suis infected mice which were not treated with bambermycin, an increased number of infiltrating B-cells, T-cells and macrophages in combination with a Th2 response was demonstrated, as well as a decreased parietal cell mass. Compared to this non-treated, infected group, in H. suis infected mice medicated with bambermycin, gastric H. suis colonization was not altered, but a decreased number of infiltrating T-cells, B-cells and macrophages as well as downregulated expressions of IL-1ß, IL-8M, IL-10 and IFN-γ were demonstrated and the parietal cell mass was not affected. In bambermycin treated mice that were not infected with H. suis, the number of infiltrating T-cells and expression of IL-1ß were lower than in non-infected mice that did not receive bambermycin. Gastric microbiota analysis indicated that the relative abundance of bacteria that might exert unfavorable effects on the host was decreased during bambermycin supplementation. In conclusion, bambermycin did not affect H. suis colonization, but decreased gastric inflammation and inhibited the effects of a H. suis infection on parietal cell loss. Not only direct interaction of H. suis with parietal cells, but also inflammation may play a role in death of these gastric acid producing cells.

Affinity-Based Screen for Inhibitors of Bacterial Transglycosylase.

The rise of antibiotic resistance has created a mounting crisis across the globe and an unmet medical need for new antibiotics. As part of our efforts to develop new antibiotics to target the uncharted surface bacterial transglycosylase, we report an affinity-based ligand screen method using penicillin-binding proteins immobilized on beads to selectively isolate the binders from complex natural products. In combination with mass spectrometry and assays with moenomycin A and salicylanilide analogues (1-10) as reference inhibitors, we isolated four potent antibacterials confirmed to be benastatin derivatives (11-13) and albofungin (14). Compounds 11 and 14 were effective antibiotics against a broad-spectrum of Gram-positive and Gram-negative bacteria, including Acinetobacter baumannii, Clostridium difficile, Staphylococcus aureus, and drug-resistant strains with minimum inhibitory concentrations in the submicromolar to nanomolar range.

A clinical trial investigating the impact of in-feed flavophospholipol on Salmonella shedding and antimicrobial resistance in pigs.

A clinical trial was conducted to assess the effectiveness of in-feed flavophospholipol in reducing Salmonella shedding and antimicrobial resistance (AMR) associated with Salmonella and generic Escherichia coli in naturally infected grower-finisher pigs. Pigs were obtained from a farm with a history of salmonellosis and were housed at a research facility. Over the span of 10 weeks the pigs received either a feed containing 4 ppm of flavophospholipol (treatment, n = 25) or a non-medicated feed (control, n = 20). Weekly fecal samples were collected and cultured for Salmonella and generic E. coli. A subset of Salmonella and E. coli isolates were tested for antimicrobial susceptibility. A multilevel mixed-effects logistic regression model was used to compare the prevalence of Salmonella shedding and AMR in Salmonella and E. coli isolates in treatment and control groups. Overall, the prevalence of Salmonella shedding (P > 0.05) and AMR in Salmonella (P > 0.01) and E. coli (P > 0.005) isolates was not different between the treatment and control groups.

Essai clinique étudiant l'impact du flavophospholipol dans les aliments sur l'excrétion de Salmonellaet l'antibiorésistance chez les porcs. Un essai clinique a été réalisé pour évaluer l'efficacité du flavophospholipol dans les aliments pour réduire l'excrétion de Salmonella et l'antibiorésistance associée à Salmonella et à Escherichia coli générique chez les porcs d'engraissement naturellement infectés. Les porcs obtenus provenaient d'une ferme ayant des antécédents de salmonellose et ils ont été logés à un établissement de recherche. Pendant 10 semaines, les porcs ont reçu soit des aliments contenant 4 ppm de flavophospholipol (traitement, n = 25) ou des aliments non médicamentés (témoin, n = 20). Des échantillons fécaux hebdomadaires ont été prélevés et soumis à des cultures pour Salmonella et E. coli générique. Un sous-groupe d'isolats de Salmonella et d'E. coli ont été testés pour la susceptibilité antimicrobienne. Un modèle de régression logistique à effets contrastés à plusieurs niveaux a été utilisé pour comparer la prévalence d'excrétion de Salmonella et de l'antibiorésistance dans les isolats de Salmonella et d'E. coli dans le groupe de traitement et le groupe témoin. Dans l'ensemble, la prévalence d'excrétion de Salmonella (P > 0,05) et de l'antibiorésistance des isolats de Salmonella (P > 0,01) et d'E. coli (P > 0,005) n'était pas différente entre le groupe de traitement et le groupe témoin.(Traduit par Isabelle Vallières).

Monensin, virginiamycin, and flavomycin in a no-roughage finishing diet fed to zebu cattle.

Two experiments were designed to evaluate the effects of monensin, virginiamycin, and flavomycin on growth performance, carcass characteristics, apparent total tract nutrient digestibility, and rumen fermentation of zebu cattle fed a no-roughage finishing diet (whole shelled corn [WSC] based). In Exp. 1, 100 crossbred bulls (; 392 kg [SD 46.8] average initial BW) were blocked by initial BW in a 101-d feedlot trial. Five treatments were evaluated using 4 pens per treatment (5 bulls/pen): monensin at 30 mg/kg DM, virginiamycin at 25 mg/kg DM, monensin at 20 mg/kg DM plus virginiamycin at 25 mg/kg DM, flavomycin at 4.4 mg/kg DM, and monensin at 20 mg/kg DM plus flavomycin at 2.2 mg/kg DM. There were no differences in growth performance (final BW, ADG, DMI, and G:F; ≥ 0.527) and carcass characteristics (HCW, dressing percent, and 12th-rib fat; ≥ 0.235) among treatments. In Exp. 2, 7 ruminally fistulated steers were used in a 7 × 7 Latin square design to evaluate the 5 treatments of Exp. 1 and 2 additional treatments: monensin at 30 mg/kg DM plus virginiamycin at 25 mg/kg DM and monensin at 20 mg/kg DM plus flavomycin at 4.4 mg/kg DM. Experimental periods were 14 d in length (9 d of adaptation and 5 d of measurements). Apparent total tract DM, OM, CP, and NDF digestibilities were similar among treatments ( ≥ 0.224). There was no treatment effect ( ≥ 0.253) in rumen fermentation responses (ruminal pH, rumen ammonia nitrogen, VFA, and number of protozoa). In conclusion, no evidence of benefits to cattle fed a no-roughage WSC-based diet was found to support the use of monensin combined with virginiamycin or flavomycin in the doses tested herein.

Structure-based design, synthesis, and biological evaluation of isatin derivatives as potential glycosyltransferase inhibitors.

Peptidoglycan glycosyltransferase (PGT) has been shown to be an important pharmacological target for the inhibition of bacterial cell wall biosynthesis. Structure-based virtual screening of about 3,000,000 commercially available compounds against the crystal structure of the glycosyltransferase (GT) domain of the Staphylococcus aureus penicillin-binding protein 2 (S. aureus PBP2) resulted in identification of an isatin derivative, 2-(3-(2-carbamimidoylhydrazono)-2-oxoindolin-1-yl)-N-(m-tolyl)acetamide (4) as a novel potential GT inhibitor. A series of 4 derivatives were synthesized. Several compounds showed more active antimicrobial activity than the initial hit compound 4, in particular 2-(3-(2-carbamimidoylhydrazono)-2-oxoindolin-1-yl)-N-(3-nitrophenyl)acetamide (4l), against Gram-positive Bacillus subtilis and S. aureus with MIC values of 24 and 48 µg/mL, respectively. Saturation transfer difference (STD) NMR study revealed that there is a binding contact between 4l and the GT domain of S. aureus PBP2. Competitive STD-NMR further proved that 4l and moenomycin A bind to GT domain in a competitive manner. Molecular docking study suggests a potential binding pocket of 4l in the GT domain of S. aureus PBP2. Taken together, compound 4l would provide a new scaffold for further development of potent GT inhibitors.

Development of bacterial transglycosylase inhibitors as new antibiotics: moenomycin A treatment for drug-resistant Helicobacter pylori.

The problem of multidrug-resistant Helicobacter pylori requires new antibiotics development. We have evaluated a potential antibiotics, moenomycin A, which is classified as a phosphoglycolipid antibiotics that targets transglycosylase and is previously thought to be limited in Gram-positive bacteria. Herein, we report the activity of moenomycin A against multidrug-resistant H. pylori and the isolates from patients with different gastrointestinal diseases.

Can probiotics improve the environmental microbiome and resistome of commercial poultry production?

Food animal production systems have become more consolidated and integrated, producing large, concentrated animal populations and significant amounts of fecal waste. Increasing use of manure and litter as a more "natural" and affordable source of fertilizer may be contributing to contamination of fruits and vegetables with foodborne pathogens. In addition, human and animal manure have been identified as a significant source of antibiotic resistance genes thereby serving as a disseminator of resistance to soil and waterways. Therefore, identifying methods to remediate human and animal waste is critical in developing strategies to improve food safety and minimize the dissemination of antibiotic resistant bacteria. In this study, we sought to determine whether withdrawing antibiotic growth promoters or using alternatives to antibiotics would reduce the abundance of antibiotic resistance genes or prevalence of pathogens in poultry litter. Terminal restriction fragment length polymorphism (T-RFLP) paired with high throughput sequencing was used to evaluate the bacterial community composition of litter from broiler chickens that were treated with streptogramin growth-promoting antibiotics, probiotics, or prebiotics. The prevalence of resistance genes and pathogens was determined from sequencing results or PCR screens of litter community DNA. Streptogramin antibiotic usage did not elicit statistically significant differences in Shannon diversity indices or correlation coefficients among the flocks. However, T-RFLP revealed that there were inter-farm differences in the litter composition that was independent of antibiotic usage. The litter from all farms, regardless of antibiotic usage, contained streptogramin resistance genes (vatA, vatB, and vatE), macrolide-lincosamide-streptogramin B resistance genes (ermA and ermB), the tetracycline resistance gene tetM and class 1 integrons. There was inter-farm variability in the distribution of vatA and vatE with no statistically significant differences with regards to usage. Bacterial diversity was higher in litter when probiotics or prebiotics were administered to flocks but as the litter aged, diversity decreased. No statistically significant differences were detected in the abundance of class 1 integrons where 3%-5% of the community was estimated to harbor a copy. Abundance of pathogenic Clostridium species increased in aging litter despite the treatment while the abundance of tetracycline-resistant coliforms was unaffected by treatment. However some treatments decreased the prevalence of Salmonella. These findings suggest that withdrawing antibiotics or administering alternatives to antibiotics can change the litter bacterial community and reduce the prevalence of some pathogenic bacteria, but may not immediately impact the prevalence of antibiotic resistance.

Tuning the moenomycin pharmacophore to enable discovery of bacterial cell wall synthesis inhibitors.

New antibiotic drugs need to be identified to address rapidly developing resistance of bacterial pathogens to common antibiotics. The natural antibiotic moenomycin A is the prototype for compounds that bind to bacterial peptidoglycan glycosyltransferases (PGTs) and inhibit cell wall biosynthesis, but it cannot be used as a drug. Here we report the chemoenzymatic synthesis of a fluorescently labeled, truncated analogue of moenomycin based on the minimal pharmacophore. This probe, which has optimized enzyme binding properties compared to moenomycin, was designed to identify low-micromolar inhibitors that bind to conserved features in PGT active sites. We demonstrate its use in displacement assays using PGTs from S. aureus, E. faecalis, and E. coli. 110,000 compounds were screened against S. aureus SgtB, and we identified a non-carbohydrate based compound that binds to all PGTs tested. We also show that the compound inhibits in vitro formation of peptidoglycan chains by several different PGTs. Thus, this assay enables the identification of small molecules that target PGT active sites, and may provide lead compounds for development of new antibiotics.

Effects of flavomycin and probiotic supplementation to diets containing different sources of fat on growth performance, intestinal morphology, apparent metabolizable energy, and fat digestibility in broiler chickens.

This study was conducted with broilers to evaluate the effects of growth-promoting antibiotic (flavomycin) and probiotic (7 bacterial species) supplementation in diets containing soybean oil or free fatty acids (FFA) on performance, morphological parameters of the small intestine, apparent digestibility of gross energy (GE) in the ileum, and apparent digestibility of fat in the ileum and total intestinal tract. Eight-hundred and sixty 4-d-old Ross 308 broiler chicks were used in a 3 × 3 factorial arrangement of dietary treatments that comprised 3 additives (without additive, flavomycin, and probiotic) and 3 fat sources (without fat, 30 g/kg of FFA, and 30 g/kg of soybean oil) with 4 pen replicates per treatment. All diets contained chromic oxide (3 g/kg) as an indigestible marker. Body weight and feed intake were recorded weekly over 40 d. Flavomycin interacted positively with soybean oil and FFA causing improvements (P < 0.05) in BW gain. Among the different fat sources, soybean oil significantly increased (P < 0.05) BW gain and jejunal villi height, whereas flavomycin improved (P < 0.05) BW gain and feed conversion when compared with the remaining dietary additives. However, the probiotic negatively affected (P < 0.05) BW gain and feed conversion despite increased (P < 0.05) villi heights of the duodenum, jejunum, and ileum. At 21 and 38 d of age, fat and GE digestibility were higher (P < 0.05) in the ileum and total intestinal tract of birds fed diets containing soybean oil than those of birds fed FFA. Fat and GE digestibility were highest (P < 0.05) among birds fed flavomycin but lowest (P < 0.05) among probiotic-fed birds. Flavomycin addition to soybean oil or FFA diets significantly increased (P < 0.05) fat and GE digestibility when compared with the same diets containing the probiotic. Therefore, soybean oil is a better energy source than FFA, as indicated by increased growth, nutrient digestibility, and jejunal villi height. However, probiotic supplementation to fat-rich diets caused detrimental effects on nutrient digestibility and growth.

Effects of the antibiotic growth promoters flavomycin and florfenicol on the autochthonous intestinal microbiota of hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂).

The 16S rDNA PCR-DGGE and rpoB quantitative PCR (RQ-PCR) techniques were used to evaluate the effects of dietary flavomycin and florfenicol on the autochthonous intestinal microbiota of hybrid tilapia. The fish were fed four diets: control, dietary flavomycin, florfenicol and their combination. After 8 weeks of feeding, 6 fish from each cage were randomly chosen for the analysis. The total number of intestinal bacteria was determined by RQ-PCR. The results showed that dietary antibiotics significantly influenced the intestinal microbiota and dramatically reduced the intensity of total intestinal bacterial counts. The intensity of some phylotypes (EU563257, EU563262 and EU563255) were reduced to non-detectable levels by both dietary antibiotics, while supplementation of florfenicol to the diet also reduced the intensity of the phylotypes EU563242 and EU563262, uncultured Mycobacterium sp.-like, uncultured Cyanobacterium-like and uncultured Cyanobacterium (EU563246). Dietary flavomycin only reduced the OTU intensity of one phylotype, identified as a member of the phylum Fusobacteria. The antibiotic combination only reduced the phylotypes EU563242 and EU563262. Based on our results, we conclude that the reduced effect of florfenicol on intestinal microbiota was stronger than that of flavomycin, and when flavomycin and florfenicol were added in combination, the effect of florfenicol overshadowed that of flavomycin.

Functional and structural analysis of a key region of the cell wall inhibitor moenomycin.

Moenomycin A (MmA) belongs to a family of natural products that inhibit peptidoglycan biosynthesis by binding to the peptidoglycan glycosyltransferases, the enzymes that make the glycan chains of peptidoglycan. MmA is remarkably potent, but its clinical utility has been hampered by poor physicochemical properties. Moenomycin contains three structurally distinct regions: a pentasaccharide, a phosphoglycerate, and a C25 isoprenyl (moenocinyl) lipid tail that gives the molecule its name. The phosphoglycerate moiety links the pentasaccharide to the moenocinyl chain. This moiety contains two negatively charged groups, a phosphoryl group and a carboxylate. Both the phosphoryl group and the carboxylate have previously been implicated in target binding but the role of the carboxylate has not been explored in detail. Here we report the synthesis of six MmA analogues designed to probe the importance of the phosphoglycerate. These analogues were evaluated for antibacterial and enzyme inhibitory activity; the specific contacts between the phosphoglycerate and the protein target were assessed by X-ray crystallography in conjunction with molecular modeling. Both the phosphoryl group and the carboxylate of the phosphoglycerate chain play roles in target binding. The negative charge of the carboxylate, and not its specific structure, appears to be the critical feature in binding since replacing it with a negatively charged acylsulfonamide group produces a more active compound than replacing it with the isosteric amide. Analysis of the ligand-protein contacts suggests that the carboxylate makes a critical contact with an invariant lysine in the active site. The reported work provides information and validated computational methods critical for the design of analogues based on moenomycin scaffolds.

The molecular biology of moenomycins: towards novel antibiotics based on inhibition of bacterial peptidoglycan glycosyltransferases.

Moenomycins are phosphoglycolipid antibiotics and the only known natural product inhibitors of peptidoglycan glycosytransferases (PGTs). Techniques that would allow facile diversification of the moenomycin structure would facilitate the development of novel antibiotics, which are urgently needed in the wake of multidrug resistant bacterial infections. The cloning and initial characterization of the moenomycin biosynthetic genes has already redefined the minimal moenomycin pharmacophore and now opens the door for the biocombinatorial generation of bioactive moenomycin fragments. Here, we highlight the importance of research on the genetic mechanisms that regulate moenomycin biosynthesis and that confer moenomycin resistance to bacteria in the development of novel anti-infectives based on PGT inhibition.

Synthesis and evaluation of a new fluorescent transglycosylase substrate: lipid II-based molecule possessing a dansyl-C20 polyprenyl moiety.

The preparation of a novel fluorescent lipid II-based substrate for transglycosylases (TGases) is described. This substrate has characteristic structural features including a shorter lipid chain, a fluorophore tag at the end of the lipid chain rather than on the peptide chain, and no labeling with a radioactive atom. This fluorescent substrate is readily utilized in TGase activity assays to characterize TGases and also to evaluate the activities of TGase inhibitors.