Research On The Horizontal Mobile Elements Of Escherichia Coli Producing ESBLs

Objective To understand the clinical distribution characteristics as well as the resistance situation of Escherichia coli producing ESBLs isolated from clinical,and provide reference for clinical reasonable application of antimicrobial agents.Detection of the beta-lactam type of acquired resistance genes and mobile genetic elements of the ESBL-EC strains,to understand the genotype of ESBL-EC strains and the existence of mobile genetic markers,and to analyze the correlation between them.Verification of the possibility of ESBL plasmid transmission by transformation test,the characteristics of transformants were studied,and to provide a reference basis for the prevention and control of the spread of ESBL.Methods1 A total of 409 strains of clinical isolates of Escherichia coli were collected from January 2014 to December 2014.The identification and susceptibility matching card of automatic microbial analysis system of VITEK-Compact 2(bio Merieux,France)were used for identification of bacteria and drug sensitivity test.The detection of ESBLs bacteria using the double disk method recommended by the American Clinical Laboratory Standards Committee(NCCLs).The results were interpreted according to the standards of the American Institute for clinical laboratory standardization(CLSI2014).2 19 kinds of beta lactamase genes,3 kinds of insertion sequence elements(ISEcp1,ISCR1,IS26),6 kinds of transposon genetic markers(mer A,tnp M,tnp R,tnp U,tnp A7,tnp A21),3 kinds of integron genetic markers(intl 1,intl 2,intl 3)were detected by polymerase chain reaction(PCR).The positive gene PCR products were sequenced,and the sequencing results were compared.3 ESBLs gene positive E.coli was used as the plasmid donor and E.coli DH5? as plasmid receptor to carry out the plasmid transformation.Bacterial identification and drug sensitivity test of transformants by VITEK-Compact 2 system.Results1 Clinical Distribution: Totally 409 strains of Escherichia coli were isolated from clinical,among them,ESBLs-producing strains accounted for 23.96%(98/409).ESBL-EC strains were mainly isolated from 50-69 years old age group(36.73%),followed by more than 70 years old age group(28.57%).Mainly from the department of urology(22.45%),followed by gynecology(10.20%),department of orthopedics(9.18%)and general surgery(9.18%).Specimens were isolated from urine(47.96%),followed by vaginal discharge(11.22%)and blood(10.20%).2 Drug sensitivity results: To Ampicillin,Piperacillin,Cefotaxime,Ceftriaxone,Cefazolin and Ceftazidime,the resistance rates were higher than 90%.Resistant to Aztreonam,Cotrimoxazole,Gentamicin,Tobramycin,Ciprofloxacin and Levofloxacin were higher than 60%.To Cefepime,Amoxicillin/Clavulanic acid and Nitrofurantion were 51.02%,46.93% and 34.69% respectively.To Cefoxitin and Piperacillin/Tazobactam resistance rate were relatively low,16.33% and 11.22% respectively,and most of them were significantly higher than that of non-ESBL-producing strains(P<0.05).The strains were very sensitive to Amikacin,Tigecycline and Imipenem.3 Genotype detection results:Of the 98 ESBL-EC strains,the positive rates of genes TEM,CTX-M-9,CTX-M-1,CTX-M-2 and SHV were 61.22%,53.06%,32.65%,4.08% and 3.06% respectively.7 strains were not detected(7.14%).The predominant distribution of genotype in ESBL-EC strains was TEM-1 + CTX-M-9(31.63%).4 Sequencing results of PCR products: The 61 bla TEM positive strains were all bla TEM-1.Among the 52 bla CTX-M-9 positive strains,the main genotype was bla CTX-M-14,accounting for 94.2%(49/52),the other 3 genotypes were bla CTX-M-19,bla CTX-M-21 and bla CTX-M-38,respectively.Among the 32 bla CTX-M-1 positive strains,the main genotype was bla CTX-M-55,accounting for59.4%(19/32),followed by bla CTX-M-15 and bla CTX-M-64,accounting for 31.3%(10/32)and 9.4%(3/32),respectively.5 Detection of mobile genetic elements: In the 98 strains,47 strains(47.96%)were detected containing class?integron,2 strains(2.04%)containing class?integron,one of them both containing classes?and class ? integron.Class ? integron was not detected.The positive rates of Tetracycline and Trimethoprim were higher in integron positive strains than that of integron negative strains(P<0.05).Of the insertion sequence elements and transposon genetic markers,ISEcp1,IS26,ISCR1,Tnp A21 and Tnp M were detected positive.Among them,the detection rate of ISEcp1 gene was the highest,reaching 97.96%.6 The results of transformation test: of the 42 strains of ESBL-EC,39 strains were able to grow on the screen after transformation.Among them,36 strains successfully transferred ESBL gene into transformants.The ESBL gene carried by some wild plants could not be transferred to the transformants.The bla CTX-M genes were successfully transferred,while the bla TEM gene was not successfully transferred.7 To Ampicillin,Cefotaxime and other 7 kinds of antibiotic resistance,the 36ESBL-EC wild strains and corresponding transformants were completely resistant to Ampicillin,Ceftriaxone and Cefotaxime.Obviously,the resistance was spread between wild strains and the corresponding transformants.Conclusions1 The drug resistance of ESBL-EC strains are very serious,usually exhibit multiple resistance to antibiotics.To penicillins,cephalosporins,monocyclic ?-lactam antibiotics-Aztreonam,quinolones and aminoglycosides(except Amikacin)antimicrobial agents,the drug resistance rates are very high.They are extremely sensitive to Tigecycline,Piperacillin/Tazobactam and carbapenem antibiotics.2 The most common of genotype in ESBL-EC strains is bla CTX-M(81.63%),followed by bla TEM(61.22%).Most clinical isolates strains detect two or more than two genotypes(60.20%).The predominant distribution of genotype is TEM+CTX-M-9positive group(31.63%).Multiple drug resistance is common in clinical isolates,It can be seen that simultaneously carrying TEM and CTX-M-9 genes may have been one reason that cause ESBL-EC strains clinical drug resistant.The bla CTX-M gene in our hospital is mainly bla CTX-M-14,followed by bla CTX-M-55 and bla CTX-M-15.The popular bla TEM gene in our hospital is bla TEM-1.3 The distribution of Class I integron in ESBL-EC is extremely widely.The spread of ESBLs in our hospital may be closely related to the presence of ISEcp1 gene.4 The ESBL gene located on the plasmid can be spread by the way of transformation,especially the bla CTX-M.Drug resistance to Ampicillin,Ceftriaxone and Cefotaxime of the ESBL-EC strains closely associated with the presence of bla CTX-M.