Molecular Characteristics,Resistance Mechanism Of Carbapenamase-resistant Enterobacteriaceae And Drug Discovery

With improper use and overuse of antibiotics in recent years, antibiotic resistance has become a severe problem in the field of anti-infection worldwide. World Health Organization assembled the accessible information on national antimicrobial resistance (ABR) surveillance and on ABR data for a set of common pathogenic bacteria for the first time, in order to present analysis of the global situation in 2014, of which drug-resistant problem of Gram-negative bacteria was especially acute. As a big antibiotic-consuming country, China has a negative picture on drug-resistant bacteria condition. As one of the most serious countries in abuse of antibiotics, China has been reported that the proportion of hospitalized patients treated with broad-spectrum antibiotics or more than two kinds of combined drugs is up to 58%. The proportion far exceeds the national standards.The data showed that among 55,000 Klebsiella pneumoniae isolates collected in China in 2012, resistance rate of K. pneumoniae against Carbapenemase was 7.1%, and resistance rate against the third-generation cephalosporins was up to 52.5%. According to the CHINET surveillance report, Enterobacteriaceae remain highly sensitive to carbapenem antibiotics, with a rising resistance rate year by year from<1% in 2006 to≤7% in 2013.Antibiotic-resistant Gram-negative bacteria consist of carbapenem-resistant Enterobacteriaceae (CRE), including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and non-fermenters, like Acinetobacter baumannii and Pseudomonas aeruginosa. Among them, the drug-resistant phenomenon in Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa is the worst. Accompanying the increasing emergence of clinical multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates, there is a huge dilemma for the laboratory diagnosis and clinical anti-infective therapy.Carbapenemase, causing Enterobacteriaceae resistant to carbapenem, is a kind of P-lactam that can obviously hydrolyze imipenem and meropenem, which can be divided into three classes (Amber):class A, class B and class D. Class B carbapenemase are metallo-beta-lactamase (MBL), with NDM-1, VIM2 and IMP as key members. MBL can hydrolyze cephalosporins and carbapenemases, being sensitive to monobactams (aztreonam) and EDTA. MBL have been detected in strains of Pseudomonas aeruginosa, Acinetobacter spp. and Enterobacteriaceae.Metallo β-lactamase NDM-1 producing K. pneumonia was first found in New Nehli, India in 2009[4], which have attracted more attention for hydrolyzing nearly all kinds of clinical antibiotics and easy tendency to transfer with other drug-resistant genes. Among ESBL genes accompanying NDM-1, CTX-M-15 is one of the most common genes, which is also the type with the highest detection rate among more than ninety CTX-M types. Following CTX-M-15, TEM-1, OXA-1 and OXA-10 were also detected in NDM-1-harbouring bacteria[5].CTX-M-15 highly hydrolyzes cefotaxime, moderately hydrolyzes aztreonam, with clavulanic acid, sulbactam, tazobactam and other inhibitors inhibiting its activity. Although resistant to carbapenem, relatively sensitive to polymyxin and tigecycline, NDM-1 can’t hydrolyze aztreonam. CTX-M-15 complements NDM-1 with activity on hydrolyzing aztreonam, which enlarges substrate range of NDM-1 and increases the difficulty of treating NDM-1 infected patients. Moreover, some mobile genetic elements associated with NDM-1, such as transposon, integron, virulence factor and transferrable plasmid and other kinds of resistant genes, make NDM-1-harboring bacteria resistant to the third generation cephalosporins, most of antibiotics such as quinolones, aminoglycoside, and present more widely spread. Nowadays, transferrable plasmids carrying blaNDM-1&blaCTX-M-15 have been found in K. pneumoniae[6], E. coli [7] and Citro Bacter[8]. NDM-1 has also been found in Acinetobacter [9-11],E. faecium[12],K. pneumoniae[13] and E. coli[14] in China in recent years. Thus, infection caused by Gram-negative drug-resistant bacteria holding NDM-1 or NDM-1&CTX-M-15 seems to be one of the most tough problems with a wide spread threat.The control of improper use and overuse of antibiotics can rely on the standardization and management of antibiotics. While the resolution of drug-resistant problem should start with multiple aspects:Firstly, strengthen the resistance surveillance of bacteria, conduct the rational administration of clinical antibiotics; secondly, conduct research on mechanisms of transmission of drug-resistant bacteria and develop inhibitors such as hydrolase inhibitors or efflux pump inhibitors; thirdly, seek novel antimicrobial drugs, such as non-antibiotic natural antimicrobial peptides (AMPs), artificially hybrid AMPs and antimicrobial antibodies.Based on the above background information and the advantage of our lab having rich ATCC standards strains and clinical Gram-negative bacteria resources, we conducted the research from three aspects:In the first part, the CRE resistance rate of 625 clinical Enterobacteria collected from 2002-2004 and 2010-2014 in Beijing district were compared. The data showed that the resistance rate increased from 0.8% in 2002-2004 to 4.9% in 2010-2014. We also detected the molecular characterization of 7 typical isolates carrying blaNDM-1&blaCTX-M-15 in terms of integron type, incompatibility group, epidemic plasmid and sequence type (ST) and mechanisms of resistance. Research on this part provide prospective study on the tendency of drug-resistant bacteria and theoretical basis for further studies on mechanism of drug-resistant bacteria.In the second part, we recombinantly expressed carbapenemase in prokaryotic E. coli cells from ATCC standards strains and clinical isolates. The recombinant carbapenemases can be used in setting screening system for carbapenemase stable compounds or inhibitors. The carbapenemase we expressed were CTX-M-15, SHV-18, KPC1/2, KPC3 from K. pneumoniae ATCC(?)BAA2146, K. pneumoniae ATCC(?)700603, K. pneumoniaeATCC(?)BAA1898, K. pneumoniae ATCC(?)BAA1900, and VIM2, OXA23 from clinical P. aeruginosa 08-14, A. baumannii 09-1, respectively. The hydrolysis activity and the protective effect of the inhibitors, as well as the enzymatic kinetics were also checked for recombinant CTX-M-15 and VIM2.In the third part, we recombinantly expressed the natural bactericin, colicin la, KWKAQKRFLK from bactericidal permeability increasing protein and hybrid bactericin colicin Ia-KWKAQKRFLK, detected the MIC values of the three bactericins to ATCC standard strains. The results showed that these bactericins were most active to ATCC(?) 25922, with MIC value at 64μg/ml.