Epidemiological Distribution Of Carrying Bla_KPC-2 Resistant Bacteria In Our Hospital And The Study Of Bla_KPC-2 Gene Environment

Background and purpose:The discovery and use of antibiotics has pushed human medical care to an unprecedented new height,but the problem of bacterial resistance that accompanies the widespread use or even abuse of antibiotics has become a problem that is urgently needed to be solved in the global medical field today.The carbapenem antibiotics,nown as the last defengding line of negative bacilli,are now also on the verge of being defeated.The main mechanism of the resistance of carbapenem antibiotics is the bacterial production of carbapenemases that hydrolyze this class of antibiotics,since Class A carbapenem KPC enzyme was reported in 2001,D carbapenemase OXA-48 enzyme in 2004,and then to class B carbapenemase NDM-1 enzyme in 2009,as the time goes,various types of carbapenema Enoneases have been reported one after another,and most of the carbapenemase resistance genes are located on plasmids of drug-resistant strains,and plasmid self-replication and horizontal propagation capabilities have enabled this type of drug resistance to spread more efficiently and rapidly.Since the first report of the type A carbapenemase KPC in China in 2007,it has been reported and researched continuously across the country,but the research within the regional system and the research on the genetic environment of these drug resistance genes are still relatively rare.This study mainly collected the resistant strains of KPC-2 enzyme produced in our hospital and studied its current status in a regional system such as a hospital within the First Affiliated Hospital of Nanchang University in Jiangxi Province.Based on this,to deeply study the mechanism of drug-resistance and dissemination,to further study the genetic environment of KPC-2 drug resistance genes by using genome-wide sequencing and bioinformatics analysis,and make analysis by comparing the results of the study with the relevant domestic and foreign research.From that,we can reveal the trend of epidemic,spread,and evolution of KPC-2 drug resistance genes from macroscopic to microscopic perspectives,It provides a scientific and valuable research basis for formulating effective prevention and control measures.Methods:1.by screening bacterial isolates that are insensitive to carbapenem antibiotics from clinically isolated bacterial specimens from January to December 2016 in our hospital(Eliminate the repeated strains),and screening the bla KPC-2 gene positive samples by PCR(PCR)technique,and identifying all bla KPC-2 positive strains and performing the in vitro drug susceptibility test by the Vitek2 compact automatic microbe identification instrument,by performing Carbapenemase phenotype screening experiments with the help of modified carbapenemase inactivation experiment,hrough performing drug sensitivity experiments and carbapenemase phenotypic screening experiments,the data of the above-mentioned drug-resistant strains containing bla KPC-2 gene were obtained and analyzed in combination with clinical data.2.The Klebsiella pneumoniae carrying bla KPC-2 gene screened in our hospital were analyzed for homology of the strains by ERIC-PCR technique.Plasmid analysis,bla KPC-2 gene mapping,and bla KPC-2 were performed using the S1-PFGE-backend blot technique and plasmid conjugation experiments.bla KPC-2 gene transmission studies.3.After the bla KPC-2 gene was identified,then the plasmids containing the bla KPC-2 gene were constructed to construct a genomic sequencing library,and the bla KPC-2 plasmid was subjected to high-throughput sequencing.Sequencing results were spliced and compared.Plasmid gap filling and differential sequence verification were performed,and plasmid series genes were annotated.The obtained plasmid sequences were input into the NCBI online website for Blast comparison,and comparisons were made with the Mauve software to examine different points in detail.Result:1.Among 568 strains of carbapenem antibiotic-insensitive strains,63 strains of bla KPC-2 positive strains were screened,with a total positive rate of 11.09%;of these,98 strains of Klebsiella pneumoniae detected 59 strains of bla KPC-2 positive strains.The positive rate was 60.20%.Among the 186 strains of Acinetobacter baumannii bla KPC-2 positive strains were 2 strains,the positive rate was 1.08%;72 strains of Escherichia coli were bla KPC-2 positive strains,the positive rate was 2.78%;125 cullots Enterobacteriaceae and 87 Pseudomonas aeruginosa strains were not screened to bla KPC-2 positive strains;all bla KPC-2 positive strains mCIM experiments were all positive;by querying the clinical data of all bla KPC-2 positive strain specimens,we found 18 clinical families in our hospital.There are epidemic distributions of bla KPC-2 positive strains in the department,among which the ICU department is the key epidemic department of this type of drug-resistant strains;susceptibility tests show that the resistant strains are carbapenems in imipenem,meropenem,quinolones Among the antibiotics,levofloxacin,ciprofloxacin,amikacin,gentamycin,and tobramycin in aminoglycoside antibiotics showed high levels of resistance,and other commonly used clinical antibiotics were mostly moderately resistant.2.The bla KPC-2 gene Klebsiella pneumoniae found in our hospital can be roughly divided into six ERIC-PCR fingerprints(A ~ F)based on ERIC-PCR results.Type A is divided into three types: A1,A2,and A3.Subtypes,A1 type accounted for 45.76%(27/59),A2 type accounted for 18.64%(11/59),A3 type accounted for 3.39%(2/59),type A accounted for 67.80%(40/59);type B accounted for 18.64%(11/59);C type accounted for 5.08%(3/59),D type accounted for 3.39%(2/59),E type was 1.69%(1/59),F type was 3.39%(2/59).Type A1 was mainly distributed in the neurological ICU of our hospital.,Emergency ICU,Burn ICU,Respiratory ICU,Neurosurgery,Critical Care,NICU,Respiratory,Orthopedics,Burns,among which NICU is the most widely distributed;A2 was mainly distributed in Neurology,Respiratory ICU,Neurosurgery,NICU,Infectious Diseases;A3 was mainly distributed in digestion of ICU,intensive medicine;B type distributed departments for endocrinology,respiratory ICU,NICU,dermatology,orthopedics,rehabilitation;C type was mainly distributed in emergency ICU,NICU;D type was mainly distributed in neurosurgical;E type is distributed in department of neurosurgery;F type is distributed in intensive medicine and neurology departments;among them,A1,A2,B and C type are mainly distributed in the ICU departments of our hospital;S1 nucleases are digested by PFGE according to plasmids.The number of bands and plasmid size were labeled as type I~V.Among them,there were three types of plasmids for type I plasmids,including 12 strains;two types of plasmids for type II plasmids,a total of 37 strains;and a type III plasmid plasmid.3 strains;Type IV plasmids with 3 plasmids,2 strains;V-plasmid spectrum with 2 plasmids;A total of 7 plasmid strains were not run out of the experiment,and the distribution of different plasmid strains in each clinical department of our hospital In the study,the plasmid type II strain was found to be the most widely distributed in our hospital,mainly in the ICU department,and the most distributed is the NICU department;the plasmid spectrum type I strains are distributed in neurological ICU,emergency ICU,respiratory ICU,neurosurgery,and severe Department of Medicine,NICU and other departments;the plasmid type III strains are distributed in two departments of neurosurgery and intensive care medicine.The plasmid strain type IV strains are distributed in the emergency ICU and NICU departments;the plasmid profile V strains are distributed in two departments: intensive medicine and dermatology;The strains that did not run out of the plasmid band were distributed in the endocrinology,digestion ICU,neurology,NICU,respiratory,and rehabilitation departments;some plasmids carrying the bla KPC-2 gene strain were subjected to Southern blot hybridization to find the bla KPC-2 gene.Both were located on plasmids with a size of approximately 160-170 kb in resistant strains;plasmids carrying bla KPC-2 and E.coli J53 were not successfully tested.3.The plasmids of Klebsiella pneumoniae 30388 and Acinetobacter baumannii 28784 bla KPC-2 were subjected to full-plasmid high-throughput sequencing.Two plasmids with sizes of 162,874 bp and 155,460 bp were obtained.Sequencing showed that the environment around the two sequencing bla KPC-2 genes is exactly the same.The bla KPC-2 gene is located in repeated insert-fragment with IS26 genes at both ends.The entire fragment is also consist of ISKpn27,ISKpn6 and an unclearly annotated gene structure,There are differences between other bla KPC-2 gene environments in domestic and foreign studies.The homology of the two sequencing plasmids was very high,but Klebsiella pneumoniae bla KPC-2 plasmid had two gene structure inversion and two insertion sequences compared with the Bauman immovable bla KPC-2 plasmid.Compared with other bla KPC-2 plasmids,the two plasmids were partially similar to other bla KPC-2 plasmids but the homology was less than two strains of our hospital.Conclusion:1.The drug-resistant strains carrying bla KPC-2 gene in our hospital were distributed in Klebsiella pneumoniae,Acinetobacter baumannii,and Escherichia coli,which were mainly distributed in Klebsiella pneumoniae,whose resistance in the first affiliated hospital of Nanchang universivy is prevalent;drug susceptibility shows that it is highly resistant to carbapenems,quinolones,aminoglycosides,and other antibiotics.The drug-resistant strains are widely distributed in our hospital.The ICU department is the focus of its epidemic spread.The region should pay attention to the prevention and control measures.2.The Klebsiella pneumoniae found in our hospital carrying bla KPC-2 gene was cloned and transmitted in our hospital.the A clonal was the most distributed,mainly distributed in the ICU;Plasmid polymorphism exists in plasmids of bla KPC-2 gene strain.Among them,the type II plasmid strains were most widely distributed in our hospital,and were mainly distributed in the ICU department.Among them,NICU was the most widely distributed one.the transmission of bla KPC-2 gene in the same strain may not be mediated by simple plasmids.3.There is a difference between the surrounding environment of bla KPC-2 gene in our hospital and in domestic and foreign studies.suggesting the existence of variability and diversity around the environment of bla KPC-2 gene.The diversity of the bla KPC-2 gene environment may affect the epidemic of the bla KPC-2 gene;and the two plasmids sequenced in our hospital have high homology,suggesting that the bla KPC-2 gene has plasmid transmission in our hospital,the bla KPC-2 plasmid of Klebsiella pneumoniae has the inversion and insertion sequence of the gene structure of the bla KPC-2 plasmid of Acinetobacter Bauman.It is speculated that the change of the gene structure and the insertion sequence may affect the epidemic distribution of the bla KPC-2 gene in different strains;its bla KPC-2 plasmid and our hospital bla KPC-2 plasmid There are some similar structures,but there are still some differences.