Analysis On Carbapenem Resistance And Carbapenemase Gene Diversity Of Three Gram-Negative Bacteria

Acinetobacter baumannii,Klebsiella pneumoniae,and Pseudomonas aeruginosa are the three most common gram-negative bacteria causing nosocomial infections.Carbapenem is the irreplaceable drug for treating these three pathogen infections,but the emergence of carbapenem-resistant Acinetobacter baumannii,Klebsiella pneumoniae,and Pseudomonas aeruginosa has greatly limited the use of carbapenem antibiotics.These three bacteria used multiple resistance mechanisms to acquire carbapenem resistance;among them,producing carbapenemases is a key factor in having a high-level carbapenem resistance.More than 10 types of carbapenemase genes have been found,which have different distributions and popularity in different parts of the world.Furthermore,drug-resistance genes are considered novel environmental pollutants,as antibiotics are used more frequently in hospitals,drug-resistant bacteria with resistant genes are denser,and hospitals have become the most important source of drug-resistant genes.However,the carbapenem resistance mechanisms,epidemic and carbapenemase gene diversity of these three bacteria are yet unclear in Yunnan province,China.In this study,carbapenem-resistant Acinetobacter baumannii,Klebsiella pneumoniae,and Pseudomonas aeruginosa strains were isolated from the 3A hospital in Yunnan Province（the First People’s Hospital of Yunnan Province）,and the epidemic of carbapenem resistance and carbapenemase diversity were surveyed.A retrospective analysis（from June 2016 to December 2017）found that these three bacteria have similar sample sources,mainly from sputum,urine,and blood.All three strains showed resistance to carbapenems:Acinetobacter baumannii had the highest imipenem resistance（64.73%）compared to others;the imipenem resistance rate for Klebsiella pneumoniae was 18.08%.However,the total number of Klebsiella pneumoniae strains is large,resulting in a high number of drug-resistant strains;59.95%of Pseudomonas aeruginosa were not sensitive to imipenem,a rate much higher than that of other-lactams.We observed that the trend of drug resistance of these three bacteria in the hospital was stable.However,drug-resistant strains that may have potential risks need urgent investigation to avoid the hidden dangers of medical malpractice.Producing carbapenemase is the most important resistance mechanism of Acinetobacter baumannii in this hospital.blaOXA-23 is the most prevalent carbapenemase gene type,which is mainly located in the Tn2006 complex transposition structure.In addition,blaNDM-1 was also detected.CC92 complex clones were the most prevalent in this hospital,including ST^Oxf191,ST^Oxf195,ST^Oxf208,and ST^Oxf381.An indigenous plasmid of CC92 became the important vector for transferring Tn2006 between plasmid and chromosome,between different clones.And it is also the reason for the prevalence of Tn2006 and CC92 in this hospital.In addition,blaNDM-1（located on chromosome）carrying strains were collected,which ST belongs to ST^Pas150/ST^Oxf718,and a Tn2009 harbored plasmid was co-occurring in these stains,and this clone is at the risk of outbreaking.Carbapenems,carried by plasmid,are also a key resistance mechanism in Klebsiella pneumoniae.In this research,blaKPC-2 was the most prevalent carbapenemase gene,and blaNDM-1 and blaIMP-4 were also detected.ST11 was the most prevalent clone,and carried an IncFⅡ type plasmid,which harbored blaKPC-2,catA2,blaCTX-M-65,fosA3,blaTEM-1B,and rmt B.These resistance genes,together with mobile genetic elements,make up three transferable regions.Bla IMP-4 was located in an unknown plasmid in the ST704 clone and the noval genetic environment of blaIMP-4 was identified.ST2526 belonged to the novel high-risk ST101 clone members and carried 12 types of resistance genes.Its carbapenem resistance was contributed by blaNDM-1,which is located in a～40k-bp plasmid（IncN2）.This plasmid was also detected in the ST11 clone and became the vector that transferred blaNDM-1 among different clones and species.As opposed to what was mentioned above,producing carbapenemase is not the main resistance mechanism of Pseudomonas aeruginosa.However,blaIMP-14 carbapenemase producing clone ST3390 became the most common clone in this hospital.blaIMP-14 was located in a novel complex transposition structure,Tn7468,which was composed of two reverse IS6100and an int1 integron.The integron gene cassette array is blaIMP-14-ant（2’’）-Ia-blaOXA-10-aac（6’）-Ib3,which has the same origin as the Thai isolates.Pseudomonas aeruginosa is the greatest threat to burn patients.The ST292 clone,with defective Opr D,was the most prevalent clone in the burn center of Yunnan province.A novel deletion of 233＿245 del13 in oprD caused an early termination codon.ST244 harbored a megaplasmid that carried blaIMP-45,and other multiple resistance genes and mobile genetic elements.This megaplasmid may lead to ST244 clone multiple drug resistance and even pan-drug resistance.For the first time,we have isolated an ST2446 clone from clinical sample.Its chromosome harbored blaIMP-87,which was similar to ST3390,together with a 1-bp deletion in oprD,led to a high level of carbapenem resistance.The above research results show that these three bacteria adopted different mechanisms to acquire carbapenem resistance in hospitals in Yunnan.And we reported several dangerous and potentially important ST clones.The obtained results supplement information on molecular mechanisms and the epidemic of drug-resistant bacteria in Yunnan,and also provide important materials to the diversity for carbapenase genes.This study emphasized the risk of carbapenemase-producing strains in hospitals and provided a research basis for preventing the outbreak of carbapenemase-resistant strains and an important reference for laboratory isolation and identification of carbapenemase-producing strains.