Study On The Resistance Mechanisms And Relateness Of Carbapenem-resistant Acinetobacter Baumannii

In the last dacade,an increasing incidence ofnonfermentative gram negative bacillus emerged,especially Acinetobacter spp.(mostly Acinetobacter baumannii) and Pseudomonas aeruginosa.Acinetobacter spp.is an opportunistic pathogen,it is found in many health care environments,soil,and water,and is a very effective human colonizer in the hospital.A.baumannii currently became one of the most important clinical pathogens,such as Escherichia coli,P aeruginosa,and Staphylococcus aureus.A.baumannii is emerging as a cause of numerous global outbreaks,displaying ever-increasing rates of resistance.There are reports of MDR A.baumannii from hospitals in Europe,North America,Argentina,Brazil,China,Japan,and Korea and from areas as remote as Tahiti in the South Pacific.These MDR strains often spread to cause outbreaks throughout entire cities,countries,and continents.Carbapenems are often the only active agents against many multi-drug resistant strains.The emergence of carbapenem resistance in A.baumannii had become of global concern.INSPEAR(the International Network for the Study and Prevention of Emerging Antimicrobial Resistance)called the occurrence of carbapenem resistance in A.baumanii a sentry envent of global resistance and urged essential intervention both in epidemiology and microbiology.Carbapenemases are a class ofβ-lactamases that is able to hydrolyse one or more carbapenems,include class A,B,and D enzymes in Ambler molecular classification. In Acinetobacter spp.class D carbapenemases are most popular.Class D carbapenemases were divided into 8 clusters.Among A.baumannii,oxa-23-like, oxa-24-like,oxa-51-like,oxa-58-like genes are predominant.Class D carbapenmases have weak hydrolyze activity to carbapenem antibiotics.Usually,calss D carbapemases together with absence of OMPs and activation of efflux pumps could induce high level carbapenems resistance in clinical isolates.The presence of insertion sequence ISAbal in the upstream of oxa-23-like and oxa-51-like genes might result in overexpression of OXA beta-lactamases and decreased levels of susceptibility to ceftazidime and carbapenems.In 2003,a new mechanism of aminoglycoside resistance was identified in P. aeruginosa,16S rRNA methylase rmtA.16S rRNA methylases protect 16S rRNA within the 30S ribosome subunit which is the target site of aminoglycosides.This mechanism results in high-level resistance to multiple aminoglycosides.These enzymes were found to confer extraordinarily high levels of resistance to clinically useful aminoglycosides,such as amikacin,tobramycin,and gentamicin.From 2003, total of 6 kinds of 16S rRNA methylases were identified,armA,rmtA,rmtB,rmtC, rmtD,and npmA in clinical pathogens such as P.aeruginosa,Citrobacter freundii,K. pneumoniae,Serratia marcescens,Proteus mirabilis,E.coli,A.baumannii.Most 16S rRNA methylase genes were found to locate in conjugative plasmid and associated with transpons.In the study,700 isolates of A.baumannii were recovered from 25 hospitals of 6 provinces.Among them,342 isolates of carbapenem-resistant A.baumannii were screened.The carbapenemase genotypes,16S rRNA methylase genotypes,and relateness of these carbapenemase-resistant A.baumannii were investigated.1.The screen of carbapenem-resistant A.baumannii and Minimal Inhibitory Concentrations determinationTotal of 342 carbapenem-resistant A.baumannii were screened from 700 isolates by using K-B method.The MICs of 14 antimicrobials to 342 carbapenem-resistant A. baumannii were detected by agar dilution or Etest strips.All imipenem-resistant isolates were also resistant to meropenem.The rates of resistance to ampicillin-sulbactam,cefoperazone-sulbactam,and minocycline were 68.0%,54.2%, and 75.9%,respectively.The rate of resistance to polymyxin E was 10.8%,the lowest among the tested agents.The rates of resistance to all other tested antimicrobial agents were more than 90%.2.The relatenes of carbapenem-resistant A.baumanniiPulse field gel electrophoresis(PFGE)was performed to analyze the relateness of 342 isolates of carbapenem-resistant A.baumannii.According to the PFGE DNA patterns,29 distinct clones of A.baumannii were identified.Total of 303 strains belonged to the popular 6 clones.Clone A,62 strains,distributed in 3 provinces (Beijing,Chongqing,and Zhejiang)and spread to 6 district hospitals in Zhejiang Province.Clone B,32 strains,spread to 3 hospitals of 3 Provinces(Zhejiang, Shanghai,and Liaoning).Clone C,160 strains,spread in Shanghai Ruijin Hospital and 10 hospitals of 8 cities in Zhejiang Province.Clone D,31 strains,spread to 3 hospitals of 2 cities in Zhejiang Province.Clone E and F,both only one sub-clone, spread in 1^stAffiliated Hospital of Sun-Yat-Sen University and Jinghua People's Hospital,respectively.We found that clones A,B,C,and D were the dominant isolates and had spread widely among hospitals,cities,and even remote areas.3.Investigation of carbapenemase genotypes and ISAbal element in carbapenem-resistant A.baumanniiCrudeβ-lactamase preparations were extracted,pI values and three-dimensional tests were performed to detected carbapenemases.Metallo-β-lactamase-producing isolates were screened by the imipenem-EDTA double-disk synergy test.Blaimp-type, blavim-type,blasim-1,blaoxa-23-like,blaoxa-24-like,blaoxa-58-like,and blaoxa-51-like were analyzed by PCR.PCR mapping was performed using the ISAbal-oxa-23-like primers or the ISAbal-oxa-51-like primers.Plasmid extraction, conjugation test,electrotransformation,and sourthern blot were performed to locate the oxa gene.All the isolates contained blaoxa-51-like but were negative for the oxa-24-like,oxa-58-like,sim-1,imp-type,or vim-type gene.322 isolates contained the blaoxa-23-like gene.A majority(314)of the clones contain the oxa-23 gene with ISAbal upstream of it.ISAbal-oxa-66 was detected in 13 strains.There was a 34-bp nucleotide sequence between ISAbal and blaoxa-23 and a 7-bp nucleotide sequence between ISAbal and blaoxa-66.Attempts to transfer blaoxa-23 by conjugation using the rifampin-resistant E.coli 600 as the recipient failed.Repeated attempts to detect the presence ofplasmids capable of hybridizing with the blaoxa-23 and blaoxa-66 probes also failed.The fragments of the ApaI-digested PFGE DNA of pulsotypes A and B showed hybridization bands at about 220 kb in clone A,about 300 kb in clone B,respectively.4.Study on 16S rRNA methylase genes in carbapenem-resistant A.baumanniiMICs of the 5 aminoglycosides to 342 isolates of carbapenem-resistant A. baumannii were determinated by agar dilution,armA,rmtA,rmtB,rmtC,rmtD,and npmA were analyzed by PCR and sequencing.Plasmid extraction,conjugation test, electrotransformation,and sourthem blot were performed to locate the armA gene. Resistance rates to amikacin,gentamicin,tobramycin,isepamicin and netilmicin were 92.6%,98.6%,87.4%,90.9%and 92.4%,respectively.287 out of the 342 CRABs were found to be resistant to all of the 5 tested aminoglycoside agents.Among 342 CRABs,armA gene was detected in 221 isolates.All isolates were negative for rmtA, rmtB,rmtC,rmtD,and npmA genes.PFGE patterns showed that 207 of 221 armA-positive CRABs could be classified into three major pulsotypes,which were designated as pulsotypes A,B and C.The fragments of the ApaI-digested PFGE DNA of pulsotypes A,B,and C showed hybridization bands at about 220 kb in clone A, about 300 kb in clone B,and about 220 kb in clone C,respectively.The above results showed:1,Carbapenem-resistant A.baumannii were resistant to most antimicrobial agents. Sulbactam combinations or colistin could be selected to treat infections caused by A. baumannii according to susceptibility test.2,Clones A,B,C,and D were the dominant isolates and had spread widely among hospitals,cities,and even remote areas.Some isolates with the same genetic basis spread in many hospitals in China,suggesting that the spread of isolates plays an important role in the increase of CRABs.So,it is important to monitor and control the spread of CRAB.3,In China,blaoxa-23 and blaoxa-51-like were the most popular carbapenemase genotypes.There were only 15 isolates(4.39%)that lacked ISAbal upstream of OXA-type carbapenemase coding genes,suggesting that an insertion of ISAbal may be a major factor responsible for the carbapenem resistance.4,ArmA-positive CRABs were detected in 19 of the 21 hospitals.PFGE analysis suggested that 93.7%of armA-positive A.baumannii belonged to three pulsotypes that were spread widely in China.The armA-coding gene may be located on the chromosomes of the isolates.Pulsotype dissemination occurred not only within or among hospitals but also among cities and possibly played the most important role in the prevalence of the armA gene.