| Background:In recent years, due to the widely use of antibiotic, the resistance pathogens rapidly increased, carbapenem-resistant pathogens is also sharply increasing. The main mechanisms of carbapenem-resistance are:bacterias can produce beta lactamase hydrolysis enzyme, cell membrane permeability of a variety of bacterial get reduced and the ability of protein binding bacterial decline. Carbapenem belongs to the beta lactamase. According to its molecular structure of Ambler,we can divide carbapenem into A, B, D types and class B enzyme belongs to metal enzyme MBLs. Metal-beta lactamase also can be called metal enzyme, which can hydrolyse most antibiotics, can be restrained by metal chelating agents. The first two cases of New Delhi beta lactamase-1 called Klebsiella pneumoniae producing NDM-1 (KP05-506) and Escherichia coli (NF-NDM-1) were reported in 2008, which quickly spreaded all over the world.Europe, Asia, North and South America, Latin America and Australia as followed reported that bacteria carrying NDM-1 gene. At present, China has reported bacteria producing NDM-1 includes Enterococcus, Acinetobacter baumannii, Klebsiella pneumoniae.However, Enterobacter cloacae producing NDM-1 was only found in India. There was no relevant event report in our country.NDM-1 usually exist in Klebsiella pneumoniae and E. coli and other gram-negative bacteria. Other bacterias such as acidification klebsiella bacillus, vuitton, bacteria, and so on also have been reported to produce NDM-1.Since 1990s, we have found Metal-beta lactamase like NDM-1, SPM, SIM-1, GIM1, DIM, TMB-1, SPM, IMP, KHM-1 and so on, which are mainly distributed in the gram-negative bacterias. These drug resistant genes can be horizontal diffused through the plasmid or integron between different bacterias. The experiment isolated a strain Enterobacter cloacae producing NDM-1 from a 67-year-old woman. She had coughed with sputum for 18 days.She also suffered Type Ⅱ diabetes, chronic renal insufficiency and coronary heart disease (CHD). Blood routine showed WBC 10.3 × 109/L, N 85.9%, PCT 0.45 ng/ml.Chest CT:left lung and the middle,lower leave of the right lung have inflammation.Sputum culture showed E. Clocoe existed. After admission, according to the clinical experience, we used cefazolin sodium anti-infection treatment, after two days there was no obvious effect;Routine blood showed N81.9%, then we used moxifloxacin 400 mg qd to continue the treatment.Patient got better after receiving this treatment dramatically and blood routine got normal;Chest CT:lung inflammation significantly reduced. Then we switched to cefoperazone sodium for reduction treatment, after eight days blood routine reviewed N70.7%.The patient recovered.This patient readmission on May 4,2012. The etiology was uremia, cough and sputum, but blood routine and sputum culture was normal. Urine contained E. coli, but did not find gene NDM-1. After investigation,we found the patient had not been to India or Pakistan. Sensitive test showed that E. Cloacae 413 resistants almost all antibiotic except fosfomycin and polymyxin B.We modified Hodge test and Etest confirmed E. Cloacae 413 can produce the c carbapenem. PCR confirmed E. Cloacae 413 DNA can amplify bands blaNDM-1 Joint experiment confirmed blaNDM-1 plasmids in E. Cloacae 413 can transfer between different bacterias, and the size of plasmid approximately was 20 KB. Pulsed field gel electrophoresis showed E. Cloacae 413 was type B, Southern blotting results also showed that blaNDM-1 exists at 250 to 300 KB in the bacterial chromosome.By PCR method and BLAST we found E. Cloacae 413 also carry blaCTX-M-14, 413, rmtA qnrA, qnrB, aac6’-Ib-cr five kinds of resistance genes.NDM-1 can cause bacteria resistant to nearly all kinds of carbapenem antibiotics, so the presence of E. Cloacae 413 may lead to the spread of drug-resistant bacteria, cause great impact to infectious diseases, so finding the source and route of this strain of NDM-1 is essential to us.This article will focus on discussing the pathogenic of E. Cloacae 413 and analysing the gene sequence features.Object:To analyze pathogenicity, bioinformatics and resistance mechanism of E. Cloacae 413, so as to explore possible sources of the resistance gene and control the possible route of transmission. Methods:The first part:Use MTT method to determine rhe growth curve of Enterobacter cloacae 413 producing the NDM-1 and the Enterobacter cloacae standard strain 4533, mouse peritonitis model is used to test the virulence of Enterobacter cloacae 413 and standard strain 4533. Then compare the growth ability and virlence of the two strains.The third Part:Sequence the whole genome of Enterobacter cloacae 413 and analyse the sequences, compare with the other plasmid sequences of the strains production NDM-1,then we conduct bioinformatics analysis to find the source of the resistance genes and genetic characteristics.Results:The first part:Completely lethal dose of Enterobacter cloacae 413 is 109 and totally not lethal dose is 104. Completely lethal dose and totally not lethal of Enterobacter cloacae 4533 strains is 1010 and 105 respectively. Data showed that complete lethal dose of 413 strains is much smaller than 4533 strains.The growth curve showed the growth ability of 413 strains is stronger than strain 4533,so 413 strains has stronger virulence and growth ability. The second part:Genome sequencing produced clear genetic profile, phylogenetic trees and building groups.The plasmid containing NDM-1 of Enterobacter cloacae 413 has high homology with Acinetobacter PM131 plasmids containing NDM-1, Klebsiella pneumoniae PKP-STM29 plasmid containing NDM-1, Escherichia coli PEC STM17 plasmid containing NDM-1 and Enterobacter cloacae strains TP15 plasmid containing NDM-1, but these plamids contain different resistence genes.So we conclude the NDM-1 transfered through different plamids by gene insertion, gene lost or genetic variation; Finally we resolved the whole metabolic pathways, and the majority of the function of genes, and antibiotic production of secondary metabolites such as the control network and the distribution of resistance genes, to discover new drug targets and interpreted from the perspective of genome bacterial drug resistance, bacteria and drug interaction, bacteria and host adaptability mechanism provides new train of thought.Conclusion:Enterobacter cloacae 413 has stronger virulence and growth ability than Enterobacter cloacae standard strain 4533. Genome sequencing produced clear genetic profile, phylogenetic trees and building groups.The plasmid containing NDM-1 of Enterobacter cloacae 413 has high homology with acinetobacter PM131 plasmids containing NDM-1, klebsiella pneumoniae PKPSTM29 plasmid containing NDM-1, escherichia coli PEC STM17 plasmid containing NDM-1, Enterobacter cloacae strains TP15 plasmid containing NDM-1, but these plamids contain different resistence genes.So we conclude NDM-1 passed through different plamids by gene insertion,gene lost or genetic variation; Finally we resolved the whole metabolic pathways, and the majority of the function of genes, and antibiotic production of secondary metabolites such as the control network and the distribution of resistance genes, to discover new drug targets and interpreted from the perspective of genome bacterial drug resistance, bacteria and drug interaction, bacteria and host adaptability mechanism provides new train of thought. Help us to strengthen the monitoring of drug-resistant bacteria and rational use of antibiotics. |
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