| Hospital-acquired infections caused by multidrug-resistant (MDR) Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae, have gradually increased over the past decade. In contrast, no novel antimicrobial agent has been specifically developed for these bacteria in recent years. It is exactly this mismatch between increasing multidrug resistance and the dry antibacterial-drug development pipeline, which led the IDS A to place P. aeruginosa, A. baumannii and K. pneumoniae on a "hit list" of the six top-priority dangerous MDR microorganisms. Now, polymyxins are increasingly being used as a last-line therapy for infections caused by Gram-negative bacteria that are resistant to almost all available antibiotics. Although the toxicity of polymyxins may not be as high as that observed in early clinical applications, it may still complicate patient therapy or even lead to treatment failure. Thus, there is an urgent need for the development of novel analogs of polymyxins with less acute toxicity or other new antibiotics against MDR Gram-negative bacteria. During a screening program of microbial resources, two strains (F6-B70and B27) that showed potent antimicrobial activity against all tested pathogens, including P. aeruginosa and E. coli, were obtained in our lab. In the present study, the work was mainly carried out on the characterization of strain F6-B70and lipopeptide antibiotics produced by this strain. The results were as following:1、Polyphasic taxonomic analysis of strains F6-B70and B27Based on the morphology, physiological and biochemical characteristics, cell wall types, whole cellular fatty acid profiles, and DNA G+C mol%contents of these two strains, combined with the analysis of16S rDNA sequence, rpoB gene, and DNA-DNA homo logy studies, strains F6-B70and B27were identified as a novel Paenibacillus species, and named Paenibacillus tianmuensis sp. nov..2、The preparation and structure elucidation of lipopeptide antibiotics produced by F6-B70To induce strain F6-B70to produce active compounds active against Gram-negative bacteria, KL synthetic medium, in which NH4SO2was the only source of nitrogen, was selected. Five lipopeptide antibiotics were obtained from the fermentation supernatant of F6-B70through several columns, including Amberlite XAD-16resin, C18silica, and HPLC column. LC-MS, MS-MS, GC-MS, and amino acid analysis indicated that two of them were known antibiotics pelgipeptins B and D, the other (octapeptin B5, B6, and B7) were novel analogs of polymyxin B. In the current condition, the high test yield of octapeptin B5(battacin) was obtained. Further study indicated that the activity of battacin against Gram-negetive bacteria was more potent than any others.3、In vitro activities and antibacterial mechanism of battacin Battacin kills bacteria in vitro and has potent activity against Gram-negative bacteria, including multidrug-resistant and extremely drug-resistant clinical isolates. Hospital strains of E. coli and P. aeruginosa are the most sensitive pathogens to battacin, with minimal inhibitory concentrations of (2to4) μg/mL. The ability of battacin to disrupt the outer membrane of Gram-negative bacteria is comparable with that of polymyxin B. However, the capacity of battacin to permeate bacterial plasma membranes is less extensive than that of polymyxin B. The bactericidal kinetics of battacin correlate with the depolarization of the cell membrane, suggesting that battacin kills bacteria by disrupting the cytoplasmic membrane.4、The acute toxicity and in vivo protective activity of battacin To further evaluate the potential clinical application of battacin, large-scale preparation of battacin was carried out, and sufficient amount of sample was obtained. The acute toxicity of battacin was evaluated in mice by intravenous administration, and its LD50for mice was15.46mg/kg (95%confidence interval:14.47mg/kg to16.52mg/kg), whereas that of polymyxin B was6.52mg/kg (95%confidence interval:6.07mg/kg to7.01mg/kg). Mouse protection assays indicated that battacin has potent in vivo biological activity against E. coli. Based on the findings of the current study, battacin may be considered as a potential therapeutic agent for the treatment of infections caused by antibiotic-resistant Gram-negative bacteria. |
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