The Mechanism of Action of Ginkgolic Acid (15:1) against Gram-Positive Bacteria Involves Cross Talk with Iron Homeostasis.

With the increasing reports of community-acquired and nosocomial infection caused by multidrug-resistant Gram-positive pathogens, there is an urgent need to develop new antimicrobial agents with novel antibacterial mechanisms. Here, we investigated the antibacterial activity of the natural product ginkgolic acid (GA) (15:1), derived from Ginkgo biloba, and its potential mode of action against the Gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus. The MIC values of GA (15:1) against clinical E. faecalis and S. aureus isolates from China were ≤4 and ≤8 µg/mL, respectively, from our test results. Moreover, GA (15:1) displayed high efficiency in biofilm formation inhibition and bactericidal activity against E. faecalis and S. aureus. During its inhibition of the planktonic bacteria, the antibacterial activity of GA (15:1) was significantly improved under the condition of abolishing iron homeostasis. When iron homeostasis was abolished, inhibition of planktonic bacteria by GA (15:1) was significantly improved. This phenomenon can be interpreted as showing that iron homeostasis disruption facilitated the disruption of the functions of ribosome and protein synthesis by GA (15:1), resulting in inhibition of bacterial growth and cell death. Genetic mutation of ferric uptake regulator (Fur) led to GA (15:1) tolerance in in vitro-induced resistant derivatives, while overexpression of Fur led to increased GA (15:1) susceptibility. Additionally, GA (15:1) significantly decreased the bacterial loads of S. aureus strain USA300 in the lung tissues of mice in a pneumonic murine model. Conclusively, this study revealed an antimicrobial mechanism of GA (15:1) involving cross talk with iron homeostasis against Gram-positive pathogens. In the future, the natural product GA (15:1) might be applied to combat infections caused by Gram-positive pathogens. IMPORTANCE The increasing emergence of infectious diseases associated with multidrug-resistant Gram-positive pathogens has raised the urgent need to develop novel antibiotics. GA (15:1) is a natural product derived from Ginkgo biloba and possesses a wide range of bioactivities, including antimicrobial activity. However, its antibacterial mechanisms remain unclear. Our current study found that the function of ferric uptake regulator (Fur) was highly correlated with the antimicrobial activity of GA (15:1) against E. faecalis and that the antibacterial activity of GA (15:1) could be strengthened by the disruption of iron homeostasis. This study provided important insight into the mode of action of GA (15:1) against Gram-positive bacteria and suggested that GA (15:1) holds the potential to be an antimicrobial treatment option for infection caused by multidrug-resistant Gram-positive pathogens.

Changes in cecum microbial community in response to total sulfur amino acid (TSAA: DL-methionine) in antibiotic-free and supplemented poultry birds.

The effect of essential total sulfur amino acids (TSAA) like methionine and cysteine on the cecal microbiome of broilers was investigated at 2 different time points (days 21 and 42) of broiler rearing. A total of 360-day-old Cobb male broiler chicks were randomly distributed to 6 dietary treatments in a 2 × 3 factorial arrangement, with 2 levels of antibiotic growth promoters (AGP: 0 and 0.05%) and 3 levels of TSAA (DL-methionine) either for starter (0.7, 0.8, and 0.9%) or finisher chicks (0.52, 0.62, and 0.72%), labeled as diets 1 to 6. Cecal digesta from each replicate (n = 10) were sampled on days 21 and 42. DNA was extracted for the amplification of the V4 region of bacterial 16S rRNA genes and subjected to Illumina sequencing. Bioinformatic analyses were performed using QIIME, Mothur, and ad hoc tools and functional profiles of the inferred metagenome were analyzed using PICRUST. Statistical difference was determined by 2-way ANOVA and PERMANOVA. Clustering of cecal communities using PCoA showed clear separation of microbial communities based on age (P < 0.05) of birds and between low and medium/ high levels of TSAA (DL-methionine). At day 21, bacterial richness and diversity were higher than at day 42 where Clostridium cluster XI and Lactobacillus were found most abundant. No variability in taxonomic richness at the genus level was observed with AGP and DL-methionine supplementation. Interbird variation for richness was greater at day 42 compared to day 21. The mean fold difference of richness was greater (1.5 mean fold) with diets 1 and 6, suggesting interactive effects of AGP and TSAA (DL-methionine) in the diet. KEGG function profiles calculated by PICRUST suggest that the cecal microbiome increased glycolysis and energy generation correlated with increased dietary TSAA (DL-methionine) supplementation levels during the late broiler growth period (day 42). This study increases our knowledge of microbial dynamics and functions that are relevant to host nutrition and performance that may help us tailoring alternative strategies for raising poultry birds under antibiotic-free conditions.

Comparison of performance and feed digestibility of the non-antibiotic feed supplement (Novacid) and an antibiotic growth promoter in broiler chickens.

Antibiotic growth promoters have been widely used in poultry to improve overall performance. The emergence of antibiotic resistance has resulted in sanctions imposed on the use of antibiotics in poultry diets, and alternatives such as herbal extracts are being considered to improve growth performance. The aim of this study was to compare the performance and feed digestibility of the feed supplement Novacid, which contains organic acids, glucomannan, and phytochemicals, with that of the antibiotic growth promoter bacitracin methylene disalicylate (BMD) in commercial broiler chickens. Six hundred 1-d-old Ross × Ross 308 male broiler chicks were randomly and equally assigned to six treatment groups with five replicates each (20 chicks per replicate). The chicks were fed a corn-soybean meal basal diet, and divided into two groups: unchallenged and challenged with E. coli (400 mg/kg Escherichia coli inoculation). Each of these groups was divided into three study groups: untreated, treated with 0.05% Novacid, and treated with 400 mg/kg BMD. At day 42, inclusion of Novacid or BMD significantly (P < 0.05) improved the performance in the unchallenged groups relative to the control group. However, in E. coli-challenged groups, Novacid and BMD did not improve performance. Ileal digestibility of crude fat, crude protein, and gross energy were reduced in the Novacid group (P < 0.05). BMD and Novacid were equally effective in controlling ileal nutrient digestibility and feed coliform count (P < 0.05). Novacid reduced cecal E. coli and Salmonella count compared to BMD and control. Thus, a phytochemical feed supplement with organic acids and glucomannan could be an effective substitute for antibiotic growth promoters in broiler diets, but cannot replace antibiotics to counter potent infectious agents such as E. coli.

Topical Treatment of Degenerative Knee Osteoarthritis.

This article reviews topical management strategies for degenerative osteoarthritis (OA) of the knee. A search of Pubmed, Embase and the Cochrane library using MeSH terms including "topical," "treatment," "knee" and "osteoarthritis" was carried out. Original research and review articles on the effectiveness and safety, recommendations from international published guidelines and acceptability studies of topical preparations were included. Current topical treatments included for the management of knee OA include topical nonsteroidal anti-inflammatory drugs, capsaicin, salicylates and physical treatments such as hot or cold therapy. Current treatment guidelines recommend topical nonsteroidal anti-inflammatory drugs as an alternative and even first-line therapy for OA management, especially among elderly patients. Guidelines on other topical treatments vary, from recommendations against their use, to in favor as alternative or simultaneous therapy, especially for patients with contraindications to other analgesics. Although often well-tolerated and preferred by many patients, clinical care still lags in the adoption of topical treatments. Aspects of efficacy, safety and patient quality of life data require further research.

PEG modification of Amorfrutin B from Amorpha fructicosa increases gastric absorption, circulation half-life and glucose uptake by T3T-L1 adipocytes.

Through a simple PEG-conjugation of the natural product Amorfrutin B, we enhanced its pharmacokinetic profile. The PEGylated molecule displayed significantly improved gastrointestinal absorption (p<0.05) and had a longer systemic circulation life (p<0.05). Oral glucose tolerance study showed PEGylated Amorfrutin B displayed longer protection against oral glucose load compared to Amorfrutin B (p<0.05). It also showed significant improvement in glucose uptake in-vitro by T3T-L1 adipocytes (p<0.05). The PEGylated molecule also showed reduced propensity of crossing the blood brain barrier and accumulating in the brain (p<0.05). It also showed reduced accumulation in the adipose tissue. Preliminary liver and kidney toxicity screening showed no significant alteration in liver or kidney function of Amorfrutin B or its PEGylated form. In conclusion, PEG modification can be an attractive strategy to reduce lipophilicity and enhance pharmacokinetic properties of natural products, derived from traditional medicine.

Effects of bacitracin methylene disalicylate and diet change on gastrointestinal integrity and endotoxin permeability in the duodenum of broiler chicken.

OBJECTIVE: To determine the effect of bacitracin methylene disalicylate (BMD) and feed changes on gastrointestinal integrity, endotoxin permeability, and morphometric parameters in the duodenum of broilers. RESULTS: Birds were raised on a starter diet without growth promoting antibiotics for 31 days then switched to a grower diet. Four of the pens including 50 g/ton of BMD while 4 pens remained antibiotic free. Eight birds per treatment were sampled prior to the feed change and at 3 and 7 days following the feed change. Gastrointestinal integrity and endotoxin permeability in the duodenum were determined using a modified Ussing Chamber and an adjacent section fixed in 10% formalin for morphometric analysis. Data were analyzed using Proc Glimmix of SAS with the model fitting BMD treatment, time, and the interaction of BMD treatment and time as fixed effects. Intestinal integrity increased at d 3 and 7 compared to prior to the feed change and addition of BMD (P > 0.001) and villus height was decreased with BMD supplementation (P = 0.049). All other tested effects similar (P > 0.1). In conclusion, the practice of changing feed had a greater effect on intestinal health than addition of BMD. However, the factors driving these differences 42 are unclear.

Use of Bacillus Subtilis PB6 as a potential antibiotic growth promoter replacement in improving performance of broiler birds.

The intestinal gut health is one of the primary determinants of broiler growth and performance. Among the various enteric diseases, necrotic enteritis (NE) is an enterotoxemic disease caused by Clostridium perfringens, which can result in severe economic losses in poultry farming. Antibiotics like bacitracin methylene disalicylate (BMD) and avilamycin (AVL) are commonly used antibiotic growth promoters (AGP) in poultry feed to control necrotic enteritis in birds. Bacillus subtilis PB6 was reported to prevent necrotic enteritis and improve performance in birds. This paper investigated the influence of Bacillus subtilis PB6 in improving the performance of broiler birds in comparison with BMD and avilamycin. A 35 day trial was conducted with 240 day-old commercial broiler chicks (VenCobb 400), which were divided into four treatment groups, where each treatment group was composed of 6 replicates each containing 10 birds, for a total of 60 birds per treatment. The treatment groups included a negative control (no AGP), Bacillus subtilis PB6, BMD, and avilamycin. The parameters analyzed included body weight, feed conversion ratio (FCR), mortality, villus histomorphometry, and European efficiency factor (EEF). Bacillus subtilis PB6 significantly (P < 0.05) improved body weight and FCR (8 points) compared to the control. The group supplemented with B. subtilis PB6 or BMD had higher (P < 0.05) body weight compared to all other treatment groups. The supplementation of B. subtilis PB6 significantly improved the villus height (P < 0.05) compared to control and other AGP groups. The EEF was found to be the highest in the B. subtilis PB6 supplemented group at 35th day as compared to other treatment groups. The combined data from this study indicate that supplementation of B. subtilis PB6 improves overall performance of broilers compared to BMD and avilamycin, and can be used as potential AGP replacement in poultry farming.

Avi-Lution® supplemented at 1.0 or 2.0 g/kg in feed improves the growth performance of broiler chickens during challenge with bacitracin-resistant Clostridium perfringens.

Avi-Lution® is a defined, patented, synbiotic product containing Saccharomyces cerevisiae, Enterococcus faecium, and Bacillus spp. Broiler chickens (n = 1,250) were experimentally treated as uninoculated controls (uCon), inoculated controls (iCon) with Clostridium perfringens, or inoculated and treated with bacitracin methylene disalicylate (BMD) at 55 mg/kg as an infected/treated control or Avi-Lution® at 1.0 (AvL1) or 2.0 (AvL2) g/kg in feed for 42 d. Each treatment was applied to 10 replicate pens of 25 straight-run, newly hatched chicks. Pens treated with AvL1, AvL2, or BMD showed improved growth, feed efficiency, or mortality from necrotic enteritis compared with iCon pens at d 14, 28, and 42. No differences in these measurements, however, were observed between pens treated with AvL1 and AvL2, which suggests that Avi-Lution® was effective at 1.0 g/kg in feed. Despite improved performance, BMD, AvL1, and AvL2 treatments did not decrease the severity of intestinal lesion scores through 42 d of age compared with the infected control. These results demonstrate that Avi-Lution® improved growth performance and feed conversion rates in broilers challenged with Clostridium perfringens despite no difference in severity of intestinal lesion scores.

Specific immediate early gene expression induced by high doses of salicylate in the cochlear nucleus and inferior colliculus of the rat / Expressão específica de genes precoces imediatos induzida por doses elevadas de salicilato no núcleo coclear e colículo inferior de rato

Abstract Introduction: Salicylate at high doses induces tinnitus in humans and experimental animals. However, the mechanisms and loci of action of salicylate in inducing tinnitus are still not well known. The expression of Immediate Early Genes (IEG) is traditionally associated with long-term neuronal modifications but it is still not clear how and where IEGs are activated in animal models of tinnitus. Objectives: Here we investigated the expression of c-fos and Egr-1, two IEGs, in the Dorsal Cochlear Nucleus (DCN), the Inferior Colliculus (IC), and the Posterior Ventral Cochlear Nucleus (pVCN) of rats. Methods: Rats were treated with doses known to induce tinnitus in rats (300 mg/kg i.p. daily, for 3 days), and c-fos and Egr-1 protein expressions were analyzed using western blot and immunocytochemistry. Results: After administration of salicylate, c-fos protein expression increased significantly in the DCN, pVCN and IC when assayed by western blot. Immunohistochemistry staining showed a more intense labeling of c-fos in the DCN, pVCN and IC and a significant increase in c-fos positive nuclei in the pVCN and IC. We did not detect increased Egr-1 expression in any of these areas. Conclusion: Our data show that a high dose of salicylate activates neurons in the DCN, pVCN and IC. The expression of these genes by high doses of salicylate strongly suggests that plastic changes in these areas are involved in the genesis of tinnitus.

Resumo Introdução: Salicilato em doses elevadas induz zumbido nos seres humanos e em animais experimentais. No entanto, os mecanismos e loci de ação do salicilato na indução de zumbido ainda não são bem conhecidos. A expressão dos genes precoces imediatos (GPIs) está tradicionalmente associada a alterações neuronais em longo prazo, mas ainda não está claro como e onde os GPIs são ativados em modelos animais de zumbido. Objetivos: No presente estudo investigamos a expressão de c-fos e Egr-1, dois GPIs, no núcleo coclear dorsal (NCD), colículo inferior (CI) e núcleo coclear ventral posterior (NCVp) de ratos. Métodos: Os ratos foram tratados com doses que, conhecidamente, induzem zumbido em ratos (300 mg/kg IP/dia, por três dias) e as expressões das proteínas c-fos e Egr-1 foram analisadas por meio de Western blot e imunoistoquímica. Resultados: Após a administração de salicilato, a expressão da proteína c-fos aumentou significativamente no NCD, NCVp e CI, quando analisados por Western blot. A coloração imunoistoquímica mostrou uma marcação mais intensa de c-fos no NCD, NCVp e CI e um aumento significativo de núcleos positivos de c-fos no NCVp e CI. Não detectamos aumento da expressão de Egr-1 em qualquer dessas áreas. Conclusão: Nossos dados mostram que uma dose alta de salicilato ativa neurônios no NCD, NCVp e CI. A expressão desses genes por doses altas de salicilato sugere que as alterações plásticas nessas áreas estão envolvidas na gênese do zumbido.

Specific immediate early gene expression induced by high doses of salicylate in the cochlear nucleus and inferior colliculus of the rat.

INTRODUCTION: Salicylate at high doses induces tinnitus in humans and experimental animals. However, the mechanisms and loci of action of salicylate in inducing tinnitus are still not well known. The expression of Immediate Early Genes (IEG) is traditionally associated with long-term neuronal modifications but it is still not clear how and where IEGs are activated in animal models of tinnitus. OBJECTIVES: Here we investigated the expression of c-fos and Egr-1, two IEGs, in the Dorsal Cochlear Nucleus (DCN), the Inferior Colliculus (IC), and the Posterior Ventral Cochlear Nucleus (pVCN) of rats. METHODS: Rats were treated with doses known to induce tinnitus in rats (300mg/kg i.p. daily, for 3 days), and c-fos and Egr-1 protein expressions were analyzed using western blot and immunocytochemistry. RESULTS: After administration of salicylate, c-fos protein expression increased significantly in the DCN, pVCN and IC when assayed by western blot. Immunohistochemistry staining showed a more intense labeling of c-fos in the DCN, pVCN and IC and a significant increase in c-fos positive nuclei in the pVCN and IC. We did not detect increased Egr-1 expression in any of these areas. CONCLUSION: Our data show that a high dose of salicylate activates neurons in the DCN, pVCN and IC. The expression of these genes by high doses of salicylate strongly suggests that plastic changes in these areas are involved in the genesis of tinnitus.