Development and validation of a sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method for quantitative analysis of bambermycin in livestock and aquatic products: Implications for food safety control and regulatory enforcement.

A sensitive and accurate analytical method was developed and validated to detect bambermycin, a commonly used antibiotic in animal feed and livestock. The presence of bambermycin residues in food products can pose health risks to consumers, emphasizing the need for a sensitive and accurate analytical method. A reversed-phase analytical column was utilized with a mobile phase comprising 0.005 mol/L ammonium acetate in 5% acetonitrile (A) and 0.005 mol/L ammonium acetate in 95% acetonitrile (B) to achieve effective chromatographic separation. Quantitative determination of bambermycin in various samples, including beef, pork, chicken, milk, eggs, flatfish, eel, and shrimp, was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry. Sample extraction involved a mixture of methanol and a 25% ammonium hydroxide solution, followed by low-temperature purification and phospholipid removal utilizing a Phree cartridge. The method exhibited a satisfactory recovery rate ranging from 69% to 100%. Validation results demonstrated the reliability, robustness, and accuracy of the method, exhibiting good linearity, precision, and recovery. This validated method can be applied for routine analysis of bambermycin residues, assisting in the development of effective monitoring and control measures to ensure the safety of livestock and aquatic products.

Coordinated peptidoglycan synthases and hydrolases stabilize the bacterial cell wall.

Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that insert new PG strands and hydrolases that generate openings to allow the insertion. However, the mechanisms of their coordination remain elusive. Moenomycin that inhibits a family of PG synthases known as Class-A penicillin-binding proteins (aPBPs), collapses rod shape despite aPBPs being non-essential for rod-like morphology in the bacterium Myxococcus xanthus. Here, we demonstrate that inhibited PBP1a2, an aPBP, accelerates the degradation of cell poles by DacB, a hydrolytic PG peptidase. Moenomycin promotes the binding between DacB and PG and thus reduces the mobility of DacB through PBP1a2. Conversely, DacB also regulates the distribution and dynamics of aPBPs. Our findings clarify the action of moenomycin and suggest that disrupting the coordination between PG synthases and hydrolases could be more lethal than eliminating individual enzymes.

[An LC-MS/MS Analytical Method for Moenomycin A in Livestock Products].

A simple and sensitive method for the determination of moenomycin A residues in livestock products using LC-MS/MS was developed. Moenomycin A, a residual definition of flavophospholipol, was extracted from samples with a mixture of ammonium hydroxide and methanol (1 : 9, v/v) preheated at 50℃. The crude extracted solutions were evaporated and purified by liquid-liquid partitioning between a mixture of ammonium hydroxide, methanol and water (1 : 60 : 40, v/v/v) and ethyl acetate. The alkaline layer was taken, and cleaned up using a strong anion exchange (InertSep SAX) solid phase extraction cartridge. The LC separation was performed on an Inertsil C8 column with liner gradient elution using 0.3 vol% formic acid and acetonitrile containing 0.3 vol% formic acid. Moenomycin A was detected using tandem mass spectrometry with negative ion electrospray ionization. Recovery tests were conducted using three porcine samples (muscle, fat and liver) and chicken eggs. Samples were spiked with moenomycin A at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The trueness ranged from 79 to 93% and precision ranged from 0.5 to 2.8%. The limit of quantification (S/N≥10) of the developed method is 0.01 mg/kg. The developed method would thus be very useful for regulatory monitoring of flavophospholipol in livestock products.

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.

Moenomycin is broadly active against multidrug-resistant Neisseria gonorrhoeae and clears an infection from a murine vaginal tract infection model.

OBJECTIVES: Ceftriaxone therapy for gonorrhoea has become under increasing pressure due to waning susceptibility levels and emergence of high-level resistant strains such as the FC428 clone. Moenomycin was recently identified to display potent anti-gonococcal activity against some reference strains. Therefore, the aim of this study was to investigate moenomycin in vitro and in vivo antimicrobial activity. METHODS: Moenomycin in vitro antimicrobial activity was investigated against 575 clinical isolates, including strains associated with the FC428 clone, using the agar dilution method. Moenomycin in vivo activity was investigated in a mouse vaginal tract gonococcal infection model. RESULTS: The moenomycin MIC range for the strain collection was 0.004-0.06 mg/L, with a MIC50 of 0.016 mg/L and a MIC90 of 0.03 mg/L. The correlation between moenomycin and ceftriaxone susceptibility levels was poor (R = 0.13), while the fractional inhibitory concentration index (FICI) resulted in indifference for all tested strains. Therefore, development of cross-resistance between moenomycin and ceftriaxone is unlikely for N. gonorrhoeae. Determination of the moenomycin mode of activity against N. gonorrhoeae by time-kill assays showed that moenomycin is bactericidal, with over 104-fold inactivation observed after 4 h exposure. Finally, an intramuscular moenomycin dose of 10 mg/kg given on 2 consecutive days was able to clear a gonococcal infection in a mouse vaginal tract infection model within 1-3 days after the second dose, which was significantly faster than for mice treated with the vehicle control (P < 0.0001). CONCLUSIONS: Moenomycin displays potent in vitro and in vivo antimicrobial activity against N. gonorrhoeae, warranting further exploration as alternative therapy.

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.

Adaptive Optimization Boosted the Production of Moenomycin A in the Microbial Chassis Streptomyces albus J1074.

Great efforts have been made to improve Streptomyces chassis for efficient production of targeted natural products. Moenomycin family antibiotics, represented by moenomycin (Moe) and nosokomycin, are phosphoglycolipid antibiotics that display extraordinary inhibition against Gram-positive bacteria. Herein, we assembled a completed 34 kb hybrid biosynthetic gene cluster (BGC) of moenomycin A (moe-BGC) based on a 24 kb nosokomycin analogue biosynthetic gene cluster (noso-BGC). The heterologous expression of the hybrid moe-BGC in Streptomyces albus J1074 achieved the production of moenomycin A in the recombinant strain LX01 with a yield of 12.1 ± 2 mg/L. Further strong promoter refactoring to improve the transcriptional levels of all of the functional genes in strain LX02 enhanced the production of moenomycin A by 58%. However, the yield improvement of moenomycin A resulted in a dramatic 38% decrease in the chassis biomass compared with the control strain. To improve the weak physiological tolerance to moenomycin A of the chassis, another copy of the gene salb-PBP2 (P238N&F200D), encoding peptidoglycan biosynthetic protein PBP2, was introduced into the chassis strain, producing strain LX03. Cell growth was restored, and the fermentation titer of moenomycin A was 130% higher than that of LX01. Additionally, the production of moenomycin A in strain LX03 was further elevated by 45% to 40.0 ± 3 mg/L after media optimization. These results suggested that the adaptive optimization strategy of strong promoter refactoring in the BGC plus physiological tolerance in the chassis was an efficient approach for obtaining the desired natural products with high titers.

A Screen of Natural Product Extracts Identifies Moenomycin as a Potent Antigonococcal Agent.

Increasing multidrug resistance in Neisseria gonorrheae is a growing public health crisis. Resistance to the last line therapies, cephalosporins and azithromycin, are of particular concern, fueling the need to discover new treatments. Here, we identified the phosphoglycolipid moenomycin from a screen of microbial natural products against drug-resistant N. gonorrheae as a potent antigonococcal agent. Moenomycin demonstrates excellent activity (MIC = 0.004-0.03 µg/mL) against a variety of multidrug-resistant N. gonorrheae. Importantly, moenomycin, thought to be a Gram-positive specific antibiotic, penetrates the Gram-negative gonococcal outer membrane. Moenomycin causes intracellular accumulation of peptidoglycan precursors, cell blebbing, and rupture of the cell envelope, all consistent with cell wall biosynthesis inhibition. Serial bacterial exposure to moenomycin for 14 days revealed slow development of resistance (MICDay14 = 0.03-0.06 µg/mL), unlike the clinically used drug azithromycin. Our results offer the potential utility of moenomycin as a lead for antigonococcal therapeutic candidates and warrant further investigation.

Effects of supplementation with narasin, salinomycin, or flavomycin on performance and ruminal fermentation characteristics of Bos indicus Nellore cattle fed with forage-based diets.

The aim of the present study was to evaluate the inclusion of narasin, salinomycin, or flavomycin for 140 d on ruminal fermentation parameters, apparent nutrient digestibility, and performance of Nellore cattle offered a forage-based diet. In experiment 1, 32 rumen-cannulated Bos indicus Nellore steers [initial body weight (BW) = 220 ± 12.6 kg] were assigned to individual pens in a randomized complete block design according to their initial shrunk BW. Within block, animals were randomly assigned to 1 of 4 treatments: (1) forage-based diet without feed additives (CON; n = 8), (2) CON diet plus 13 ppm of narasin (NAR; n = 8), (3) CON diet plus 20 ppm of salinomycin (SAL; n = 8), or (4) CON diet plus 3 ppm of flavomycin (FLA; n = 8). The experimental period lasted 140 d and was divided into 5 periods of 28 d each. The inclusion of feed additives did not impact (P ≥ 0.17) dry matter intake (DMI), nutrient intake, and apparent total tract digestibility of nutrients. Nonetheless, steers fed NAR had lower (P < 0.01) molar proportion of acetate compared with CON, SAL, and FLA steers, whereas ruminal acetate tended to be greater (P < 0.09) for SAL vs. CON and FLA, but did not differ (P = 0.68) between CON vs. FLA steers. Ruminal propionate was the highest (P < 0.01) for steers fed NAR and did not differ (P > 0.20) between CON, SAL, and FLA. Consequently, NAR steers had the lowest (P < 0.01) Ac:Pr ratio, whereas Ac:Pr did not differ (P > 0.18) among CON, SAL, and FLA. Total volatile fatty acids were greater (P < 0.04) for NAR and CON vs. SAL and FLA, but did not differ (P > 0.67) among NAR vs. CON and SAL vs. FLA. In experiment 2, 164 Nellore bulls (initial shrunk BW = 299 ± 2.5 kg) were assigned to feedlot pens for 140 d in a randomized complete block design. Within block (n = 10), animals were randomly assigned to the same treatments used in experiment 1. Average daily gain was greater (P < 0.01) in NAR vs. CON, SAL, and FLA bulls, and did not differ (P > 0.12) between CON, SAL, and FLA bulls. Bulls fed NAR had greater (P < 0.02) DMI (as kg/d or % BW) and final shrunk BW compared with CON, SAL, and FLA bulls, whereas DMI and final shrunk BW did not differ (P > 0.26) between CON, SAL, and FLA bulls. Feed efficiency, however, was not impacted (P = 0.51) by any feed additives used herein. Collectively, narasin was the only feed additive that benefited performance and ruminal fermentation of Nellore animals fed a forage-based diet.

Effect of Bacillus velezensis to substitute in-feed antibiotics on the production, blood biochemistry and egg quality indices of laying hens.

BACKGROUND: The excessive use of antibiotics in the livestock feed industry caused inevitable side effects of microbial resistance. Besides this residual antibiotics in animal-derived foodstuff imposed serious health problems for humans. So this study aimed to investigate the potential use of Bacillus velezensis to substitute antibiotics for poultry production. A total of 468, 49-week-old Hy-Line Brown chickens, were randomly divided into four groups the control group (regular diet), experiment group I (0.1% B. veleznesis), experiment group II (0.2% B. veleznesis), and antibiotic group (50 mg/kg flavomycin), with three replicates per group and trial period consisted on 42 days. RESULTS: The results showed that, compared with the control group, the average egg production rate and daily feed intake of experimental groups I and II increased significantly (P < 0.05), while the average egg weight was increased in experimental group II as compared to (I) (P < 0.01). The feed conversion ratio was decreased (P > 0.05) in group (II) Egg quality parameters such as yolk weight of the experimental group II was increased, but that of the antibiotic group and experiment group I was decreased, neither significant (P > 0.05). Moreover, the eggshell strength, yolk color, albumen height, and Haugh unit were significantly increased (P < 0.05). Compared with the control group, probiotic groups can increase the progesterone and motilin (P > 0.05) but decrease the secretin and cholecystokinin in the blood plasma (P > 0.05). CONCLUSIONS: This study suggested that B. velezensis can substitute in-feed-antibiotics and improved most of the study parameters significantly. Which suggested that B. velezensis has potential future application value to replace the feed antibiotics.

Cyclic di-GMP cyclase SSFG_02181 from Streptomyces ghanaensis ATCC14672 regulates antibiotic biosynthesis and morphological differentiation in streptomycetes.

Streptomycetes are filamentous bacteria famous for their ability to produce a vast majority of clinically important secondary metabolites. Both complex morphogenesis and onset of antibiotic biosynthesis are tightly linked in streptomycetes and require series of specific signals for initiation. Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, one of the well-known bacterial second messengers, has been recently shown to govern morphogenesis and natural product synthesis in Streptomyces by altering the activity of the pleiotropic regulator BldD. Here we report a role of the heme-binding diguanylate cyclase SSFG_02181 from Streptomyces ghanaensis in the regulation of the peptidoglycan glycosyltransferase inhibitor moenomycin A biosynthesis. Deletion of ssfg_02181 reduced the moenomycin A accumulation and led to a precocious sporulation, while the overexpression of the gene blocked sporogenesis and remarkably improved antibiotic titer. We also demonstrate that BldD negatively controls the expression of ssfg_02181, which stems from direct binding of BldD to the ssfg_02181 promoter. Notably, the heterologous expression of ssfg_02181 in model Streptomyces spp. arrested morphological progression at aerial mycelium level and strongly altered the production of secondary metabolites. Altogether, our work underscores the significance of c-di-GMP-mediated signaling in natural product biosynthesis and pointed to extensively applicable approach to increase antibiotic production levels in streptomycetes.

Secondary nucleotide messenger c-di-GMP exerts a global control on natural product biosynthesis in streptomycetes.

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.

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).

Complex structures of MoeN5 with substrate analogues suggest sequential catalytic mechanism.

The antibiotic moenomycin A is a phosphoglycerate derivative with a C25-moenocinyl chain and a branched oligosaccharide. Formation of the C25-chain is catalyzed by the enzyme MoeN5 with geranyl pyrophosphate (GPP) and the sugar-linked 2-Z,E-farnesyl-3-phosphoglycerate (FPG) as its substrates. Previous complex crystal structures with GPP and long-chain alkyl glycosides suggested that GPP binds to the S1 site in a similar way as in most other α-helical prenyltransferases (PTs), and FPG is likely to assume a bent conformation in the S2 site. However, two FPG derivatives synthesized in the current study were found in the S1 site rather than S2 in their complex crystal structures with MoeN5. Apparently S1 is the preferred site for prenyl-containing ligand, and S2 binding may proceed only after S1 is occupied. Thus, like most trans-type PTs, MoeN5 may employ a sequential ionization-condensation-elimination mechanism that involves a carbocation intermediate.

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.

Effects of dietary coated-oleum cinnamomi supplementation on the immunity and intestinal integrity of broiler chickens.

Oleum cinnamomi (OCM) is increasingly used as a feed additive in animal diets. The aim of this study was to investigate the effects of dietary supplementation with coated-OCM (cOCM) on the immunity and intestinal integrity of broiler chickens. A total of 396 one-day-old chicks were randomly assigned into six groups. The basal diets were supplemented with 50 mg/kg of flavomycin (positive control) as well as 0 (control), 50, 100, 200, and 300 mg/kg of cOCM. Compared with the control, both positive control and cOCM treatments did not improve the growth performance. Serum immunoglobulin (Ig) Y levels were decreased by flavomycin and 50 mg/kg of cOCM treatments (p < 0.05). Dietary cOCM decreased ileal secretory IgA contents at d 21 and commonly down-regulated duodenal and ileal mRNA expression of interleukin (IL)-1ß and IL-8 at d 42 (p < 0.05). The 300 mg/kg of cOCM increased jejunal ratio of villus height to crypt depth and upregulated intestinal claudin-1 expression (p < 0.05). Jejunal (at d 21) and duodenal (at d 42) mucin-2 expression was up and downregulated by both 50 and 300 mg/kg of cOCM, respectively (p < 0.05). In conclusion, dietary cOCM addition helped to maintain noninflammatory states of humoral and mucosal immunity, and improved the intestinal integrity of broiler chickens.

Peptidoglycan glycosyltransferase-ligand binding assay based on tryptophan fluorescence quenching.

Peptidoglycan glycosyltransferases (GTase) of family 51 are essential enzymes for the synthesis of the glycan chains of the bacterial cell wall. They are considered potential antibacterial target, but discovery of inhibitors was hampered so far by the lack of efficient and affordable screening assay. Here we used Staphylococcus aureus MtgA to introduce a single tryptophan reporter residue in selected positions flanking the substrates binding cavity of the protein. We selected a mutant (Y181W) that shows strong fluorescence quenching in the presence of moenomycin A and two lipid II analogs inhibitors. The assay provides a simple method to study GTase-ligand interactions and can be used as primary high throughput screening of GTase inhibitors without the need for lipid II substrate or reporter ligands.

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.