Detection Of Drug-resistant Bacteria Based On Target Capture And Highthroughput Squencing

Objectives To establish the method combining target capture with high-throughput sequencing for clinical detection of resistant bacteria, for the purposes of obtaining strain identification results, drug-resistant genes and housekeeping gene sequences for MLST, in order to provide a scientific basis for clinical rational medication and epidemiological surveillance.Methods Refer to the relevant literature and collect sequence data of E.coli frequent resistant genes, 16 S rRNA gene sequence and eight housekeeping genes, send the sequences to the Agilent company for designing Sureselect target acquisition kit adapting to high-throughtput sequencing. Establish the target acquisition- high-throughput sequencing method, detecte four clinical ESBLs-producing E.coli and verify the accuracy and repeatability of the method. Apply the method to detecting 22 clinical ESBLs-producing E.coli, obtain resistant genotypes, housekeeping gene sequence and MLST results.Results(1) Use the target acquisition-high-throughput sequencing method to detecte four clinical ESBLs-producing E.coli, 16 S rRNA gene sequences show that they are all E.coli isolate, which is consistent with the results from VITEK-2 detection system. Part of the resistant gene sequences are consistent with PCR-Sanger sequencing results. Repeat this method testing the 4 strains, two results are consistent.(2) Use the target acquisition- high-throughput sequencing method to detecte 22 clinical ESBLs-producing E.coli, 16 S rRNA gene sequences show that they are all E.coli isolate, which is consistent with the results from VITEK-2 detection system.(3) BLAST the resistent gene sequencing, and receive 24 kinds of resistant genotypes. There are 20 isolates carry 8 kinds of ESBLs genes, in which the positive rate of CTX-M-14, CTX-M-55, CTX-M-15, CTX-M-27, CTX-M-65, SHV-12, OXA-1, OXA-10 is 68.18%, 45.45%, 13.64%, 4.55%, 9.09%, 18.18%, 4.55% and 4.55%. There are 3 isolates carry AmpC gene and all of them are DHA-1 gene. There are 20 isolates carry 6 kinds of aminoglycoside resistance genes, the positive rate of aac(3)-II、aac(6’)-Ib、ant(3’’)-I、aph(3’)-I、aadA、rmtB is 63.63%、18.18%、13.64%、9.09%、31.82% and 4.55%. There are 17 isolates carry 6 kinds of quinolone resistance genes, and the positive rate of gyrA、gyrB、qnrB、qnrS、aac(6’)-Ib-cr、qepA is 45.45%、4.55%、4.55%、9.09%、36.36% and 22.73%. There are 18 isolates carry 2 kinds of tetracycline resistance genes, and the positive rate of tet(A) and tet(B) is 68.18%、45.45%. The rate of gene TEM-1 is 90.91%. Other resistant genes are not detected. All of the strains detected with three or more different kinds of drug resistant genes. The drug resistance of the three generation of cephalosporins is grim, and multiple drug resistant strains of more than 50%.(4) Upload housekeeping gene sequences to Pasture software and receive 16 different ST types. The highest detection rate is ST38, which consist of 3 isolates, following by ST131, ST405. The 2 strains of ST38 are from ICU department.Conclusions(1) Successfully construct the target capture- high-throughput sequencing method which can strain identification, genetic testing and epidemiological credits typing at the same time. Prove that the method has good accuracy and repeatability, and preliminary confirm that the method can be applied to the monitoring of clinical drug resistant bacteria.(2) The resistance situation of ESBLs-producing E.coli is grim and resistance gene detection rate is high. TEM-1 and CTX-M-14 are closely related with β-lactam resistance, aac(3)-II and aadA are closely related with aminoglycoside resistance, gyrA and aac(6’)-Ib-cr are closely related with quinolone resistance, while tet(A) and tet(B) are closely related with tetracycline resistance.(3) ESBLs-producing E.coli has genetic diversity in this study, ST38 type is popular among these E.coli. There are certain cross infection in ICU, we should pay sufficient attention to it and strengthen monitoring.