Research On Mechanism Of Carbapenem-resistant Gram Negative Bacteria

Background and objectiveAccording to China bacterial drug resistance monitoring network(CHINET) 2005-2013 continuous monitoring result, about 70% of the clinical isolated bacteria were gram-negative bacilli, which has a slight upward trend(66.9% in 2005,71.6% in 2010,73.0% in 2013) in large teaching hospital in China. Carbapenem resistance Enterobacteriaceae isolation ratio was about 0%~3% in 2007, and rise to 7% in 2013, and the carbapenem resistance fermentation bacteria was from 30% in 2007 to 60% in 2013. The most common type of gram negative bacteria were E. coli, Klebsiella bacteria genera, Acinetobacter baumannii, Pseudomonas aeruginosa. For 30 years, carbon alkene drugs were the last line to treat gram negative bacillus infections, and these drugs have high effective to treat multiple drug-resistant bacteria. However, in recent years, carbapenem drugs resistant bacteria was reported increasingly. Therefore, investigate、detect and suppress drug resistance bacteria has become research hotspot in the field of microorganism.Carbapenem drug resistance gram negative bacillus are always multiple drug resistance/extrem drug resistant strains, The reason are mainly because of carrying and expression resistance genes. Produce hydrolase, channel protein changes, expression of efflux pump, penicillin binding protein change, and so on, is the key mechanism.The reason why bacteria become resistant to the drug is due to carrying a large number of resistant genes, result in various mechanisms of antimicrobial drug resistance. Treatment of gram-negative bacilli infections were mainly beta lactam drugs, which were penicillin, broad-spectrum penicillin, cephalosporin and carbapenem drugs, and have strong antibacterial sterilization effects in turn. A variety of mechanisms lead to different bacterial drug resistance. Given the clinical treatment of the actual needs and test maneuverability, acquire such strains resistant phenotypes, is one of the main content of the current study. According to the result of drug sensitivity test in vitro, analyze the characteristics of all kinds of drug resistance of clinical isolates and the regional difference of resistance, which can effectively reflect the drug resistance of isolates in different regions, meanwhile, has the important value to guide local clinicians rational antimicrobial treatment.For carbapenem drugs, carbapenemase is the main mechanism of bacterial drug resistance. At present, the study of these strains has focused on carbapenemase distribution in different strains and different regions. According to Ambler classification, carbapenemases can be divided into three groups, respectively, A, B and D. class A and D were serine enzymes, the class B was metal beta lactamase. In class B enzyme, NDM-1 is the most common type of enzyme, which was reported from many countries and regions in the world. And blaNDM gene variant is gradually found and reported(including NDM-1 to NDM-14). There has shown that individual amino acid substitution between subtype lead to different drug resistance. In addition, from the point of view of bioinformatics, different types of carbapenemase by antimicrobial effect for a long time, each have a relatively stable evolutionary relationships. So figured out regional popular of carbapenemase, which kinds of the enzyme and the types of different strains with different enzyme, analysis of popular type in the position of volutionary tree, can predict possible mutation evolution direction, as well as help the infection treatment.Based on extensively carbapenem-resistant gram-negative bacilli, such strains hold the important position in the nosocomial infection, also the nosocomial infection lead to spread of such strains and its drug resistance. Therefore, the research on classification of clinical isolates, figure out whether there is any carbapenem drug resistant strains, and the strain nosocomial infection trend, can provide reasonable guidance for clinical experience in treatment, also has important value for the prevention and control of nosocomial infection.To solve the problems, we have designed this research, aims to detect the clinical isolation resistant to carbapenem gram-negative bacilli, analyze the resistant phenotype, and dectect the existence of carbapenem drug-resistant genes and these distribution, inquire whether there is the existence of nosocomial infection. The final objection was help for clinical anti-infection treatment and epidemiological studies.Research method(1) strain selection Gram negative bacteria resistance to imipenem or meropenem were isolated from the first affiliated hospital of ZHENGZHOU university in bacteria department from July to December in 2013, including 10 Escherichia coli, 19 Klebsiella pneumonia, 30 Pseudomonas aeruginosa and 62 Acinetobacter baumannii. All 121 strains were identified by Vitek 2 Compact.(2) Bacterial drug resistance and epidemiological analysis were analyzed using WHOnet5.0 software. Studying all strains popular distribution characteristics and drug resistance phenotype. Drug sensitive test quality control strains was E.coli ATCC 25922.(3) Extracti whole genome DNA in serious accordance with the specification operation with DNA extraction kit.(4) Carbapenem resistance strain preliminary screening Using modified Hodge and EDTA synergistic inhibition experiments for phenotypic screening. Modified Hodge experiments for measuring bacteriostatic circle and the cross junction with detected strain and positive control have enhanced growth phenomenon were defined screening positive. EDTA coordination experiment are positive for screening further bacteriostatic ring near the EDTA pieces.(5) Confirmation of carbapenem resistance strains For preliminary screening positive strains, extracting genome DNA, using PCR to analyze class A(KPC), class B(NDM, VIM, SPM, IMP, GIM) and class D(OXA23 OXA24, OXA51, OXA58) enzyme gene, then sequencing the amplification product and Blast to definite genotype.(6) Molecular evolution prediction Analyze all NDM variants difference in amino acid sequence and structure, using software analyse drugs suppression site and simulation variant enzyme protein crystal structure.(7) Strain classification research RAPD-PCR method was used to do random primers amplification with Pseudomonas aeruginosa and Acinetobacter baumannii, thus to simple classification the strains, and epidemiological studies. Research results 1 Carbapenemase detectionDetect class A KPC positive 11 strains, 10 strains of pneumonia klebsiella bacteria and 1 strain of E.coli. Detect class B NDM positive 7 strains, three pneumonia klebsiella bacteria strains and 4 strains of E. coli, including two strains detected NDM-5(the domestic only two cases reported). Detection of pseudomonas aeruginosa carbapenemase gene amplification were all negative, among amplification of acinetobacter baumannii of class D enzyme gene, 55 strains are OXA23 and OXA51 positive, one was OXA24 positive strains and an OXA58 positive. 2 Metal enzyme NDM variantsThrough the sequencing of the PCR positive specimens, this research found blaNDM-5, when there is no domestic reports, only a few cases reported in the world. 3 strains of typingAccording to RAPD-PCR results, Pseudomonas aeruginosa is divided into 14 type, Avcinetobacter baumannii for 4 type conclusion(1) Enterobacteriaceae hold high separation rate with carbapenemase gene, which was 62%. Producing carbapenemase is primary mechanism of carbapenem resistance with enterobacteriaceae. In which we found two strains of E.coli carrying blaNDM-1variant gene blaNDM-5, and resistance is different. Resistance genes can through one single nucleotide mutation to produce new resistant genotypes.(2) Do not found carbapenemase genes in P.aeruginosa, and detection of Channel protein gene are negative. Hence we can infer the carbapenem resistance of pseudomonas aeruginosa is given priority to lack of channel proteins.(3) According to RAPD-PCR results, Pseudomonas aeruginosa strains are divided into 14 type, acinetobacter baumannii are for 4 type, in which one type occupied 88.7%. Pseudomonas aeruginosa was disperses in the occurrence, but acinetobacter baumannii has obvious advantages in cloning strains, which should strengthen the monitoring, to prevent the the popularity of cloning plants and nosocomial infection.