Pharmacological, Toxicological, and Dose Range Assessment of OG716, a Novel Lantibiotic for the Treatment of Clostridium difficile-Associated Infection.

Lantibiotics present an attractive scaffold for the development of novel antibiotics. We report here a novel lantibiotic for the treatment of Clostridium difficile infection. The lead compounds were selected from a library of over 700 single- and multiple-substitution variants of the lantibiotic mutacin 1140 (MU1140). The best performers in vitro and in vivo were further used to challenge Golden Syrian hamsters orally in a Golden Syrian hamster model of Clostridium difficile-associated disease (CDAD) in a dose-response format, resulting in the selection of OG716 as the lead compound. This lantibiotic was characterized by a 50% effective dose of 23.85 mg/kg of body weight/day (10.97 µmol/kg/day) in this model. Upon oral administration of the maximum feasible dose (≥1,918 mg/kg/day), no observable toxicities or side effects were noted, and no effect on intestinal motility was observed. Compartmentalization to the gastrointestinal tract was confirmed. MU1140-derived variants offer a large pipeline for the development of novel antibiotics for the treatment of several indications and are particularly attractive considering their novel mechanism of action. Based on the currently available data, OG716 has an acceptable profile for further development for the treatment of CDAD.

Current developments in lantibiotic discovery for treating Clostridium difficile infection.

INTRODUCTION: Clostridium difficile is a major cause of healthcare-associated diarrhea linked to the misuse of antimicrobials and the corresponding deleterious impact they have on the protective microbiota of the gut. Resistance to agents used to treat C. difficile including metronizadole and vancomycin has been reported highlighting the need for novel agents. Lantibiotics represent a novel class of agents that many studies have highlighted as effective against C. difficile. Areas covered: In this review lantibiotics including nisin, actagardine, mersacidin, NAI-107 and MU-1140 that exhibit good activity against C.difficile, all of which are currently in the preclinical phase of investigation are discussed. The lantibiotic NVB302, which has completed phase I clinical trials for the treatment of C. difficile, is also described. Expert opinion: Lantibiotics represent promising candidates for the treatment of C. difficile infections due to their novel mode of action, which is thought to decrease the potential of resistance developing and the fact they often possess a less deleterious effect on the protective gut microbiota when compared to traditional agents. They are also extremely amenable to bioengineering approaches and the incorporation of synthetic biology to produce more potent variants.

Seleção e aplicação de bactérias láticas produtoras de folato para obtenção de um leite fermentado bioenriquecido e avaliação de biodisponibilidade do folato produzido / Screening and application of folate-producing lactic acid bacteria for production of bioenriched fermented milk and evaluation of the bioavaliability of the produced folate

A deficiência de folato é um problema de saúde pública que vem sendo combatido através da fortificação obrigatória de ácido fólico. Entretanto, a ingestão excessiva pode causar efeitos colaterais indesejados. A forma natural da vitamina produzida por algumas cepas de bactérias láticas pode ser uma alternativa segura para aumentar a ingestão de folato pela população. Este trabalho visou selecionar as melhores cepas de BAL produtoras de folato, identificar as melhores condições para a produção da vitamina e avaliar a biodisponibilidade do folato produzido pelas cepas no leite fermentado, empregando-se testes em modelos animais. O estudo foi desenvolvido com cinco cepas de Streptococcus thermophilus (34v, 170v, 268v, 361v e 341 pc) e um cepa de Lactobacillus plantarum (16cv), todas boas produtoras de folato. As condições de produção e a combinação de cepas que resultaram na melhor produção folato foram utilizadas para produzir um leite biofermentado (BFM), avaliando-se a biodisponibilidade da vitamina produzida empregando-se um modelo animal de depleção/repleção, com camundongos Balb/c submetidos a 14 dias de depleção, seguido de 21 dias de repleção da vitamina. Após os 35 dias, os animais foram sacrificados, retirando-se sangue, rins, fígado, baço e intestino para quantificação de folato e avaliação histológica da mucosa intestinal. Todas as quantificações de folato foram feitas pelo método microbiológico. A melhor combinação de cepas (St. Thermophilus 34v + Lb. Plantarum 16cv) resultou na produção de 300 ng/mL de folato, sendo a temperatura de 42ºC melhor que 37ºC para a produção da vitamina. A produção de folato no leite na fermentação em pH controlado 6,0 foi 35% superior à observada no leite fermentado em pH livre. As concentrações de folato no baço e glóbulos vermelhos foram mais altas nos camundongos que ingeriram leite fermentado bioenriquecido (BFM) em relação aos demais grupos de animais. A relação vilosidade/cripta nos camundongos dos grupos BFM foi igual à observada nos animais que receberam leite suplementado com ácido fólico. Houve aumento da hemoglobina, hematócrito e hemácias nos camundongos que ingeriram BFM, evidenciando a bioeficácia do folato produzido. Esses resultados indicam que a produção de folato por bactérias láticas selecionadas em produtos fermentados pode ser uma boa alternativa para aumentar a ingestão de vitamina B9 pela população

A deficiência de folato é um problema de saúde pública que vem sendo combatido através da fortificação obrigatória de ácido fólico. Entretanto, a ingestão excessiva pode causar efeitos colaterais indesejados. A forma natural da vitamina produzida por algumas cepas de bactérias láticas pode ser uma alternativa segura para aumentar a ingestão de folato pela população. Este trabalho visou selecionar as melhores cepas de BAL produtoras de folato, identificar as melhores condições para a produção da vitamina e avaliar a biodisponibilidade do folato produzido pelas cepas no leite fermentado, empregando-se testes em modelos animais. O estudo foi desenvolvido com cinco cepas de Streptococcus thermophilus (34v, 170v, 268v, 361v e 341 pc) e um cepa de Lactobacillus plantarum (16cv), todas boas produtoras de folato. As condições de produção e a combinação de cepas que resultaram na melhor produção folato foram utilizadas para produzir um leite biofermentado (BFM), avaliando-se a biodisponibilidade da vitamina produzida empregando-se um modelo animal de depleção/repleção, com camundongos Balb/c submetidos a 14 dias de depleção, seguido de 21 dias de repleção da vitamina. Após os 35 dias, os animais foram sacrificados, retirando-se sangue, rins, fígado, baço e intestino para quantificação de folato e avaliação histológica da mucosa intestinal. Todas as quantificações de folato foram feitas pelo método microbiológico. A melhor combinação de cepas (St. Thermophilus 34v + Lb. Plantarum 16cv) resultou na produção de 300 ng/mL de folato, sendo a temperatura de 42ºC melhor que 37ºC para a produção da vitamina. A produção de folato no leite na fermentação em pH controlado 6,0 foi 35% superior à observada no leite fermentado em pH livre. As concentrações de folato no baço e glóbulos vermelhos foram mais altas nos camundongos que ingeriram leite fermentado bioenriquecido (BFM) em relação aos demais grupos de animais. A relação vilosidade/cripta nos camundongos dos grupos BFM foi igual à observada nos animais que receberam leite suplementado com ácido fólico. Houve aumento da hemoglobina, hematócrito e hemácias nos camundongos que ingeriram BFM, evidenciando a bioeficácia do folato produzido. Esses resultados indicam que a produção de folato por bactérias láticas selecionadas em produtos fermentados pode ser uma boa alternativa para aumentar a ingestão de vitamina B9 pela população

Risks Related to High-Dosage Recombinant Antimicrobial Peptide Microcin J25 in Mice Model: Intestinal Microbiota, Intestinal Barrier Function, and Immune Regulation.

Antimicrobial peptide (AMP) can be a promising alternative in various domains. However, further risk information is required. In this study, mice were orally administrated different dosages of recombinant AMP microcin J25 (4.55, 9.1, and 18.2 mg/kg; MccJ25) for 1 week, and the toxicity risk impacts were examined. We evidenced that middle-dosage administration mice had a lower inflammation, better body weight, and ameliorated mucosal morphology, accompanied by reduced intestinal permeability and tighter intestinal barrier. Fecal microbiota composition analysis in middle- or low-dosage mice revealed the Bifidobacterium count was increased and the coliform bacteria count was decreased, and increased in short-chain fatty acid levels. Unexpectedly, there was a risk that high-dosage mice increased intestinal permeability and imbalance of intestinal bacteria. Taken together, these data indicated a safe threshold for usage of MccJ25 in clinical practice. Such studies can effectively enhance the safety of various aspects such as food preservative and drug.

Biocontrol strategies for Mediterranean-style fermented sausages.

Naturally fermented meat sausages have a long tradition in Mediterranean countries and are one of the most important groups of traditional foods consumed throughout Europe. Despite all the advances in food science and technology and increased regulatory requirements and concerns for safety and quality during the last decades, the challenge to control important foodborne pathogens in this type of meat products still persists. Simultaneously, growing consumer interest in safe, high quality and minimal processed products, with less additives/preservatives have driven the food industry and scientists in a crusade for innovative technologies to maintain the safety of these products by natural means. Biological control (biocontrol) fits well within this tendency. This review summarizes the latest achievements on biocontrol strategies applied to Mediterranean-style fermented sausages, namely: (i) bioprotective cultures; (ii) bacteriocins; and, (iii) essential oils (EOs).

Clinical research on ultrasonically guided intrahepatic injections of HAS in interventional treatment of liver carcinomas.

PURPOSE: To study the clinical value of intrahepatic injections of Highly Agglutinative Staphylococcin (HAS) in the interventional treatment of liver carcinomas. METHODS: Under ultrasonic guidance, intrahepatic injections of HAS were administered in 22 cases of pathologically diagnosed liver carcinomas, 3 days, 7 days, 30 days, 3 months, 6 months, 9 months, and 12 months after microwave coagulation therapy. The dose of each injection was 2000U. RESULTS: Immunohistochemical staining of the sample from the tumor site after HAS administrations demonstrated a significant increase in the number of antitumor immune cells compared with that before the injections (p<0.01) and an improvement in local immune status. One-year survival rate and recurrence rate, which were determined by Kaplan- Meier method, were 93.8% and 81.9% respectively. CONCLUSIONS: As a new route of administration, intrahepatic injections of HAS are a safe and effective procedure and deserves further clinical research and discussion.

Safety evaluation of the antimicrobial peptide bovicin HC5 orally administered to a murine model.

BACKGROUND: Bovicin HC5 is an antimicrobial peptide that shows a broad spectrum of activity and potential for biotechnological and therapeutic applications. To gain insight about the safety of bovicin HC5 application, the histological and immunostimulatory effects of orally administrated bovicin HC5 to BALB/c mice were evaluated. BALB/c mice were divided into three groups: negative control (NC group); mice given purified bovicin HC5 (Bov group); mice given ovalbumin (positive control, PC group; a murine model of enteropathy). The mice were initially pre-sensitized, and PBS, bovicin HC5 or ovalbumin were administered for 30 days by daily gavages. Histological and morphometric analysis were performed and the relative expression of cytokines was analyzed by real-time RT-PCR. RESULTS: The oral administration of bovicin HC5 to BALB/c mice reduced weight gain and caused alterations in the small intestine, although absorptive changes have not been detected. The number of total goblet cells and the mucopolysaccharides production were not affected by bovicin HC5 administration. A hypertrophy of Paneth cells and an increase in the number of mitotic cells were observed in Bov group, while the number of mast cells remained unaltered. Increased expression of TNF-α, INF-γ and IL-12 was observed in the small intestine upon bovicin HC5 administration. CONCLUSION: Bovicin HC5 has only minor effects on intestinal permeability and did not elicit an allergenic response upon oral administration to animal models. Considering the low in vivo toxicity of bovicin HC5, it might be a good candidate for enteral applications.

New developments and applications of bacteriocins and peptides in foods.

There is an increased desire for sophisticated foods, whereby consumers harbor higher expectations of health-promoting benefits above basic nutrition. Moreover, there is a move from the adulteration of foods with chemical preservatives toward biopreservation. Such expectations have led scientists to identify novel approaches to satisfy both demands, which utilize bacteriocin and peptide-based solutions. The best known examples of biopreservation involve bacteriocins. However, with the exception of nisin, bacteriocins have received limited use in the food industry. Peptides can be added to foods to improve consumer health. Some of the best known examples are angiotensin I-converting enzyme (ACE)-inhibitory peptides, which inhibit ACE, a key enzyme involved in blood pressure (BP) regulation. To be effective, these peptides must be bioavailable, but by their nature, peptides are degraded by digestion with proteolytic enzymes. This review critically discusses the use and potential of peptides and bacteriocins in food systems in terms of safety, quality, and improvement of human health.

Effects of albusin B (a bacteriocin) of Ruminococcus albus 7 expressed by yeast on growth performance and intestinal absorption of broiler chickens--its potential role as an alternative to feed antibiotics.

BACKGROUND: Bacteriocins with antimicrobial activity are considered as potential alternatives to antibiotics. The aim of this study was to investigate the effect of albusin B (bacteriocin) of Ruminococcus albus 7 expressed by yeast on the growth performance of broiler chickens. Ninety 1-day-old healthy broiler chickens were randomly divided into three groups: control, albusin B (2.5 g kg(-1)) and nosiheptide (2.5 mg kg(-1), antibiotic control). Growth performance and intestinal functions were measured at 5 weeks of age. RESULTS: Albusin B-supplemented broilers showed increased body weight gain compared with control broilers (54.7 ± 5.3 vs 48.5 ± 6.1 g day(-1) per bird, P < 0.05). Broilers supplemented with nosiheptide had a less developed mucosal layer than broilers in the other two groups. Compared with the control group, broilers supplemented with albusin B or nosiheptide showed increased mRNA expression of sGLT1, GLUT2 and PEPT1 in the jejunum (P < 0.05). The faecal Lactobacillus count was higher in the albusin B group than in the other two groups (P < 0.05). CONCLUSION: Albusin B supplementation increased intestinal absorption and elevated the faecal Lactobacillus count, thereby promoting the growth performance of broiler chickens. These improvements resulting from albusin B supplementation provide evidence of potential alternatives to antibiotics in broiler chicken feed.

Pulmonary pharmacokinetics and safety of nebulized duramycin in healthy male volunteers.

Duramycin (Moli1901) is being developed for the treatment of reduced mucociliary clearance in cystic fibrosis. This study was conducted to estimate lung residence time and systemic exposure and to assess whether duramycin causes an inflammatory response. Six volunteers were administered a single dose (7.5 mg) of nebulized duramycin and underwent bronchoscopies to obtain a composite data set for pharmacokinetic analysis; duramycin was measured in the cellular fraction of bronchoalveolar lavage fluid (BALF) (mainly alveolar macrophages) and brush biopsies (bronchial epithelial cells). The estimated t(1/2) of duramycin was approximately 5 days in brush biopsies and 25 to 91 days in BALF cells. Levels of duramycin in BALF (C (max) 800 ng/mg) exceeded those in brush biopsies by approximately 20-fold. Duramycin was absent from plasma and did not cause any detectable inflammatory response in pulmonary tissue as judged from the BALF profile of 14 relevant cytokines. Our data suggest that duramycin qualifies for intrapulmonary administration in cystic fibrosis (CF) patients.

Effects of duramycin on cardiac voltage-gated ion channels.

The amphipathic peptide duramycin is in clinical development for the treatment of cystic fibrosis. It is deposited in cellular membranes where it binds to phosphatidylethanolamine. Duramycin may thereby change the biophysical membrane properties and perturb the function of ion channels. If so, in heart tissue, its application carries the risk to elicit cardiac arrhythmias. In fact, premature ventricular complexes were observed in the electrocardiogram during toxicological testing in dogs. To study the arrhythmogenic potential of duramycin, we investigated its effects on currents through voltage-gated hERG potassium, sodium, and calcium channels in native cells, and using a heterologous expression system, by means of the whole-cell patch clamp technique; duramycin bath concentrations between 1 nM and 0.1 microM did not generate any effects on these currents. Concentrations >or=0.3 microM, however, reduced the amplitudes of all investigated currents. Moreover, sodium current fast inactivation kinetics was slowed in the presence of duramycin. A further rise in duramycin bath concentration (>or=3.3 microM) induced a leak current consistent with pore formation. The reported effects of duramycin on ion channel function are likely to arise from a change in the biophysical properties of the membrane rather than from a specific interaction of the peptide with ion channel proteins. Under therapeutic conditions (i.e., administration via inhalation), duramycin plasma concentrations are below 0.5 nM. Thus, upon inhalation, duramycin has a large safety margin and is highly unlikely to elicit arrhythmias.

Safety assessment of dairy microorganisms: Streptococcus thermophilus.

Streptococcus thermophilus is a major dairy starter used in yogurt and cheese production. In Streptococcus genus, S. thermophilus is the only one food species among commensal and opportunistic pathogen species. Comparative genomics suggest that this species recently emerged and evolved by combination of loss-of-function and horizontal gene transfer events. These gene transfer events detected in S. thermophilus have originated from other dairy species and might contribute to its adaptation to the milk environment.

Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages.

Bacteriocins, antimicrobial peptides, and bacteriophage have attracted attention as potential substitutes for, or as additions to, currently used antimicrobial compounds. This publication will review research on the potential application of these alternative antimicrobial agents to poultry production and processing. Bacteriocins are proteinaceous compounds of bacterial origin that are lethal to bacteria other than the producing strain. It is assumed that some of the bacteria in the intestinal tract produce bacteriocins as a means to achieve a competitive advantage, and bacteriocin-producing bacteria might be a desirable part of competitive exclusion preparations. Purified or partially purified bacteriocins could be used as preservatives or for the reduction or elimination of certain pathogens. Currently only nisin, produced by certain strains of Lactococcus lactis subsp. lactis, has regulatory approval for use in certain foods, and its use for poultry products has been studied extensively. Exploration of the application of antimicrobial peptides from sources other than bacteria to poultry has not yet commenced to a significant extent. Evidence for the ability of chickens to produce such antimicrobial peptides has been provided, and it is likely that these peptides play an important role in the defense against various pathogens. Bacteriophages have received renewed attention as possible agents against infecting bacteria. Evidence from several trials indicates that phage therapy can be effective under certain circumstances. Numerous obstacles for the use of phage as antimicrobials for poultry or poultry products remain. Chiefly among them are the narrow host range of many phages, the issue of phage resistance, and the possibility of phage-mediated transfer of genetic material to bacterial hosts. Regulatory issues and the high cost of producing such alternative antimicrobial agents are also factors that might prevent application of these agents in the near future.