Epidemic Situation Of NDM Producing Strains In China And Resistance Mechanisms Of KPC-2 Producing Strains In Our Hospital

Background and ObjectionThe β-lactamases, including ESBLs and carbapenemases, are the main mechanism of resistance to β-lactams. They can hydrolyze or inactivate the β-lactams and cephalosporins. The resistance to β-lactams has become the public health crisis worldwide with the rapid spread of ESBLs and carbapenemases, and the emergence of resistance to carbapenems has almost broken through the last line of anti-infective therapy.Three main classes of carbapenemases have been identified:Ambler class A β-lactamase (KPC), class B (metallo-enzymes), and class D (OXA-48 type). Since the first discovery of KPC-type carbapenemases in North Carolina.17 types of it, only little difference among their amino acid, over the past years have been discovered worldwide. And the KPC-2 carbapenemases is the most popular type. KPC plasmid strains have spread largely, as all enteric bacteria, via colonized patients and air transportation. In China, the First Affiliated Hospital, Zhejiang University School of Medicine reported the first case of KPC-2-producing Klebsiella pneumoniae and Pseudomonas aeruginosa in 2007 and in 2011 respectively. But the KPC-producing strains spreading in our hospital are still unknown. Carbapenemases of the oxacillinase-48 type (OXA-48) have been identified mostly in Mediterranean, and southern European countries with rapid spread. Class B carbapenemases were known as metallo-enzymes. The New Delhi metallo-β-lactamases (NDM), especially NDM-1, is one of the currently best known metallo-β-lactamases. Since the first discovery of NDM-type carbapenemases,10 different types have been discovered. The NDM strains are usually resistant to almost all antibiotics, except tigecycline and colistin. And some NDM-1 strains often carried with many other β-lactamase genes in the transferable genetic elements which leaded them spreaded all over the world quickly. Now,blaNDM-1 has been identified in various species of bacteria:Klebsiella pneumoniae, Escherichia coli, Serratia, Citrobacter, Pseudomonas, Acinetobacter strains, etc. And these strains may develop and raise unsolvable therapeutic issues, when the patients suffering severe invasive, septicemia or neurosurgical infections especially. The may concern with NDM-1 resistance is that it is now carried by E. coli, ubiquitous and non-nosocomial bacterium, community-acquired infections, the major pathogen responsible for urinary tract infections and bacterial diarrhea. Now it has become the public health crisis worldwide, because of their rapid spread and the lack of development of new antimicrobial drugs. As reported by epidemiological survey of India, Pakistan, Britain in 2010, the positive of NDM-1 bacterium, mainly including Escherichia coli, Klebsiella pneumonia, Citrobacter freundii, Morganella morganii, Providencia spp, was accounted for 1.2%-13% in carbapenem-resistant Enterobacteriaceae. In China, according to the survey carried out by Zhejiang University, Fu Ying,13(0.1%) of 12024 carbapenem-resistant Gram-negative bacteria collected from January 2009 to September 2010 were positive for NDM-1, and all were Acinetobacter spp. As reported by Academy of Military Medical Sciences, Xuesong Wang,14.8% of 10273 clinical fecal samples collected from separate individuals in 52 hospitals representing 11 provinces from January to October 2011 was positive for NDM-1.120 (7.9%) positive samples was isolated and identified. However there were no positive isolates among Escherichia coli and Klebsiella pneumonia. The epidemic status of the NDM strains spreading in China in recently years and whether other P-lactamase genes carried by the positive strains or not is unclear.There are two objections in our study. The first one is to investigate the current situation of the NDM-producing bacteria in our country, including the detection rate, the NDM type, species distribution and other common β-lactamase genes of the positive strains in Gram-negative bacteria. The second one is to explore the resistance mechanisms of the KPC-producing isolations which has been screened no blaNDM gene in the first part study in our hospital.Methods and materialsPartⅠ1. Collection of the strainsA total of 1150 strains, containing 541 Acinetobacter spp,178 K pneumonia,241 P.aeruginosa, and 190 other strains of Enterobacteriaceaes, were collected from separate individuals in 61 hospitals representing 38 cities from January 2009 to March 2014. They were isolated from clinical specimens including fecal, sputum, urine, blood, secretion, catheter, drainage and so on. The identification and the minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by VITEK 2 Compact System. And the resistance to Carbapenem was in accordance with CLSI-2013.2. Resistance gene screeningPCR amplification, cloning and sequencing of blaNDM and other β-lactamase genes, including carbapenem gene (blaGES, blaKPC, blaBIC, blaBIC, blaIMP, blaVIM, blaTMB, blaFIM, blaSPM, blaDIM, blaGIM, blaSIM, blaAIM, blaSMB, blaOXA-23-like,blaOXA-24-like, blaOXA-48-like,blaOXA-58-like, blaOXA-143-like, blaOXA-235-like)and ESBLs gene (blaCTX-M-l1group, blaTX-M-2 group,blaCTX-M-8 group,blaCTX-M-9 group, blaCTX-M-25 group、blaTEM, blaSHV, blaGES, blaPER, blavEB, blaOxA-1-like) of the blaNDM-producing strains, the whole sequence alignments and phylogenetic tree of blaNDM gene were applied to discover the resistance genes.3. Identification of the blaNDM-producing strainsTo clarify the species of positive ones correctly, there are four methods utilized in this study:Specific gene amplification,16SrDNA sequencing, partial rpoB sequence analysis and Mastrix assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS).PartⅡ1. Selection of the strains155 multidrug-resistant Gram-negative strains isolated in our hospital from June to September in 2013 and identified without blaNDM gene in the first part was collected for the blaKCc gene. The strains contain Acinetobacter spp, Klebsiella pneumoniae, Pseudomonas spp and others, which were isolated from fecal, sputum, urine, blood, secretion, catheter, drainage and so on.2. Resistance gene screening and transmission mechanismPCR amplification, cloning and sequencing were utilized for the detection of the blaKPC and other β-lactamase genes containing carbapenem gene (blaGES, blaIMI, blaIMP, blaVIM, blaSPM, blaDIm, blaAIM, blaAMB, blaOXA-23a-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like, blaOXA-143-like, blaOXA,-235-like) and ESBLS gene(blaCTX-M-1 group, blaCTX-M-2 group, blaCTX-M-25 group, blaTEM, blaSHV, blaGES, blaPER, blaVEB,blaOXA-1-like) of the blaKPC-producing strains. The transferability of the postive gene harbouring plasmid was confirmed by electrotransformation, CarbaNP test and drug sensitivity test.3. Identification of the blaKPC-producing strainsThree methods beside the phenotypic test using the VITEK 2 system were applied to identify the species of the KPC-producing ones collectly:Specific gene amplification,16SrDNA sequencing and Mastrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). ResultsPartⅠ1. The dectection result of blaNDM geneThe results showed that all the 46 positive isolates carried blaNDM-1 by the whole sequence alignments and phylogenetic tree of blaNDM.These 46 isolates were collected from 14 cities in China and were scattered 15 different wards. And the samples obtained for the positive isolates included fecal (n=16), sputum(n=7), urine(n=6), blood(n=4), secretion(n=2), catheter(n=1), drainage (n=1). The species identification of the positive shows 11 Acinetobacter towneri,2 Acinetobacter lwoffii, 2 Acinetobacter baumannii,3 Acinetobacter pittii,1 Acinetobacter johnsonii,11 Klebsiella pneumoniae,6 Citrobacter freundii,4 Escherichia coli,2 Enterobacter cloacae,2 Leclercia adecarboxylata, I Raoultella ornithinolytica,1 Enterobacter aerogenes.2. The dectection result of other β-lactamase genesThere may be one or more β-lactamase genes coexist in part of the blaNDM-1-producing bacterias:2 Citrobacter freundii carried with blaKPC-2;6 Acinetobacter towneri and 2 Acinetobacter pittii carried with blaOXA-58;1 Citrobacter freundii carried with blaIMP-4;13 blaCTX-M-lgroup positive, including 1 Klebsiella pneumonia and 1 Citrobacter freundii carried with blaCTX-M-15,6 Klebsiella pneumoniae,2 Citrobacter freundii,1 Escherichia coli,1 Leclercia adecarboxylata and 1 Enterobacter cloacae with blaCTX-M-3 positive; 16 blaTEM positive, including 7 Klebsiella pneumoniae,4 Citrobacter freundii,2 Escherichia coli,2 Enterobacter cloacae,1 Leclercia adecarboxylata;6 blaSHV positive, including 4 Klebsiella pneumonia and 1 Enterobacter cloacae with blaSHV-12 positive and 1 Klebsiella pneumonia with blaSHV-5 positive; 13 blaoXA-1 positive, including 4 Klebsiella pneumoniae,4 Citrobacter freundii,2 Acinetobacter spp,1 Escherichia coli,1 Enterobacter cloacae and 1 Leclercia adecarboxylata.PartⅡ The result showed that one Klebsiella pneumonia isolated from sputum carried with blaKPC-2 and blaSHV-11, and one Pseudomonas aeruginosa isolated from urine carried with blaKPC-2 only. All the maximum ident and query cover value of the positive P-lactamases gene exceeded 99%.The electroporation experiment, CarbaNP test and antimicrobial susceptibility test confirmed only the blaKPC-1 which can lead the resistence to Carbapenem could be identified in transformants.ConclusionsIt was confirmed that the type of blaNDM carried by all the 46 positive isolates is blaNDM-1 by the whole sequence alignments and phylogenetic tree of blaNDM.The detection rate of blaNDM-1 in Gram-negative bacteria increased rapidly in recently years in China, from 0.1% in 2009-2010 up to 4% in 2009-2014.And the blaNDM-1 positive strains which no longer confined to Acinetobacter spp developed to Enterobacteriaceae:Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, etc. there may be one or more β-lactamase genes,blaCTX-M-1 group、blaTREM、blaOXA-1、 blasSHV、blaIMP、blaKPC and blaOXA-58, coexist in the positive ones. In summary, the NDM-1-producing isloations have been become the public health crisis in our country.Although we screened no blaNDM-1 in our hospital, but the plasmid encoded gene blaKPC-2 which can transfer one by one is a huge threat in our hospital. The protection and monitoring measures should be undertaken to reduce or avoid the outbreaks of drug-resistant in our hospital. The ESBLs gene blaSHV-11, which did not discovered in the blaKPC-2-producing transformants but could coexist with the blaKPC-2 strains isolated in our hospital, might be located on the chromosome or not in the plasmid carrying with blaKPC-2.