| Over the past decade, antibiotic resistance in Gram-negative bacilli has been increasingly serious, especially reflects in the carbapenem-resistant Enterobacteriaceae, which includes Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter aerogenes, and the carbapenem-resistant non-fermenters, like Acinetobacter baumannii and Pseudomonas aeruginosa. Among these, resistance in Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa is the most serious, and it is very difficult to treat infections due to these bacteria.Carbapenemases is a very important mechanism causing carbapenem-resistance in Gram-negative bacilli, like Enterobacteriaceae. Since the first report of extended spectrum β-lactamases (ESBLs) found in Enterobacteriaceae in1983, resistance in Enterobacteriaceae has been increasingly serious. In2001, KPC, Klebsiella pneumoniae carbapenemase, was firstly found in United States, and soon disseminated all over the world, including many countries and regions throughout North America, South America, Asia and Europe. In China, KPC-producing Klebsiella pneumoniae, which then caused a pandemic, was firstly found in2007, with the gene blaKPC spreading among Escherichia coli, Klebsiella spp., and other Enterobacteriaceaes. In2009, the first infection case, caused by metallo-β-lactamase NDM-1-producing multidrug-resistant Klebsiella pneumoniae, was reported by the British to be found in New Delhi, India. After this, such infections were found in United States, Canada, Japan, Korea, Australia, Hong Kong and other regions, leading the great concerns from all over the world. The NDM-1-producing bacteria can be resistant to almost all the antibiotics including carbapenems, except colistin and tigecycline, which are relatively effective against the NDM-1producers. However, there’re some multidrug-resistant bacteria that show resistance to colistin and tigecycline. Therefore, it is very urgent to development new drugs against multidrug-resistant bacteria. Under such background, we did mainly two parts of studies as follows:Part one:Establishment of evaluation model for drugs against multidrug-resistant bacteria based on NDM-1The main purpose is to lay a foundation for the development of novel drugs against NDM-1-producing bacteria. Firstly, NDM-1protein expression systems were successfully constructed by molecular cloning techniques and pure NDM-1enzyme was got through overexpression and purification. The kinetic parameters of NDM-1with meropenem as the substrate were determined as Kin=63.3±7.5μM, Kcat=42.6±1.8s-1, and Kcat/Km=0.67s-1/μM. It was also confirmed that EDTA was the inhibitor of NDM-1, while tazobactam and clavulanic acid were not. Based on this, the evaluation model for NDM-1inhibitors at the enzyme level was established and then applied to evaluate the activity of7compounds. The results demonstrated that none of the7compounds could inhibit the activity of NDM-1effectively. Furthermore, transformant with full-length blaNDM-1showed resistances to P-lactams including carbapenems, the characteristic of NDM-1producers, and hence could be used to assist drug evaluation at the cellular level.Part two:Experimental induction, antimicrobial susceptibility analysis and mechanism study of Enterobacteriaceae with decreased susceptibility to antibiotics.The overall purposes are to provide a reference for the rational clinical use of carbapenems, colistin and tigecycline, and hence to reduce or mitigate the problem of carbapenem resistance and also to provide a prospective study for the potential resistance occuring during anti-NDM-1producer infection treatment. We did mainly two aspects of studies as follows:1. Selection of Escherichia coli and Klebsiella pneumoniae with reduced susceptibility to carbapenems by in vitro induction and study of the mechanisms.The purposes are to evaluate that whether Enterobacteriaceae can become less susceptible or even resistant to carbapenems by in vitro induction under laboratory conditions, and to study the mechanisms involved. Firstly,11mutants were isolated after induction of Escherichia coli and Klebsiella pneumoniae by carbapenems in vitro. Antimicrobial susceptibility testing showed that mutants became less susceptible to the carbapenems used in the induction, and also showed decreased susceptibilities to other P-lactams, including other carbapenems. PFGE confirmed the isogeny of the parents and the mutants. PFGE confirmed the isogeny of the parents and the mutants. Further studies, including PCR, DNA sequencing, SDS-PAGE analysis of outer membrane proteins, MALDI-TOF/TOF and Real-time PCR, demonstrated that efflux pump and β-lactamases did not have much effect on the reduced susceptibility of the mutants to carbapenems, while loss or decreased expression of OMPs (including OmpK36, OmpC, NmpC, LamB and OmpF) caused by mechanisms like insertional inactivation associated with the reduced susceptibility.2. Selection of NDM-1-producing Klebsiella pneumoniae with reduced susceptibility to colistin and tigecycline by in vitro induction and study of the mechanisms. The purposes are to evaluate that whether NDM-1-producing Klebsiella pneumoniae can become less susceptible or even resistant to colistin and tigecycline by in vitro induction under laboratory conditions, and to study the mechanisms involved. Firstly, mutants were isolated after NDM-1-producing Klebsiella pneumoniae were induced by colistin or/and tigecycline in vitro. Antimicrobial susceptibility testing showed that the mutants had a reduced susceptibility to colistin and tigecycline, and became (highly) resistant to the two antibiotics. Subculturing of mutants in medium without antibiotics demonstrated that the reduced susceptibility to colistin was irreversible, while that to tigecycline was reversible. PFGE confirmed the isogeny of the parents and the mutants. Mouse system infection assay indicated that the "pan-drug resistant" mutants, induced by both colistin and tigecycline, showed decreased virulence, but still can be lethal to mice. To investigate the mechanisms involved, SDS-P AGE of outer membrane proteins and2-DE of bacteria total proteins were conducted, and the results showed that the reduced susceptibility of the mutants to both drugs had no relationship with outer membrane proteins. The exact mechanisms involved remain to be studied.In conclusion, in this study, the evaluation model for drugs against multidrug-resistant bacteria basing on NDM-1was established, laying a foundation for the development of novel drugs. Additionally, this study also demonstrated that after the in vitro induction by carbapenems, colistin and tigecycline, Enterobacteriaceae showed reduced susceptibility to these drugs, issuing a warning for the clinical treatment. |
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