Screening Of Microbial - Derived Strains Against Klebsiella Pneumoniae And Study Of Secondary Metabolites

Drug resistance of gram-negative bacteria is a big concern in the clinical infection treatment. In recent years, due to the abuse use of all kinds of antibiotics, drug-resistant strains continue to emerge. However, since the1980s, the number of newly approved antibacterial drugs is reducing. Meanwhile, the new drugs on sale recently have no obvious improvement focus on infection caused by gram-negative drug-resistant bacteria. Klebsiella pneumonia (KPN), an important drug-resistant pathogen of gram-negative bacteria, ranks the second in clinical separation and detection of the gram-negative bacteria. Beyond that, the bacteria has become one of enterobacteriaceae pathogen that grows fast in resistance rates. Therefore, to develop new antibiotics is of great importance to solve the increasing drug-resistant problem of KPN.Microbial secondary metabolite is the important source for new drugs’discovery. Its features of species diversity and abundant structure types provide the possibilities for discovering new compounds against drug-resistant bacteria. In this work, we screened33400samples of16700strains including actinomycetes, bacteria and fungi, to identify positive samples against KPN (ATCC700603) that generate extended spectrum beta-lactamase SHV-18, using microdilution susceptibility testing. A total of101active samples of93active strains were identified.Among them,36active samples of32active strains were validated using a K-B method after repeated fermentation and4strains I09AA-02146, I09AA-02711, I11AB-02500and I11AB-02762show great activity against gram negative bacteria including standard strains and drug-resistant stains of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, etc. The fermentation of I11A-02500(CPCC203718) has better repeatabilityand its broth exhibits the good inhibitory effect on Klebsiella pneumonia ATCC BAA-2146containgNDM-1.Next, we conducted scale-up fermentation of111A-02500and isolated its secondary metabolites. Guided by anti-KPN (ATCC700603) activity,13compounds were isolated by means of normal-phase and reversion-phase silicagel column chromatography, Sephadex LH-20column chromatography, semi-preparative HPLC, etc., followed by the structural identification by modern spectroscopy methods. Among them,2500-1and2500-10show anti-KPN activity with MIC512μg/mL and16μg/mL respectively.