全基因组测序对碳青霉烯类耐药肺炎克雷伯菌T361耐药及毒力基因的分析

Analysis of resistance and virulence genes of carbapenem-resistant Klebsiella pneumoniae T361 by whole-genome sequencing

目的 分析碳青霉烯类耐药肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumoniae, CRKP)T361株的耐药及毒力基因携带情况。方法 肺炎克雷伯菌T361株分离自昆明医科大学第一附属医院干部医疗科患者的痰液标本。采用Vitek-2 compact系统对菌株进行鉴定和药物敏感性试验。使用PCR检测菌株的耐药基因。并用Illumina Hiseq 4000高通量测序平台对其进行全基因组测序,分析菌株携带的耐药及毒力基因。结果 肺炎克雷伯菌T361株对头孢菌素类、碳青霉烯类、氨基糖苷类、氟诺喹酮类、磺胺类、β内酰胺/β内酰胺酶抑制剂复合物、呋喃妥英及替加环素等抗生素均耐药。PCR检出bla_(KPC-2)、bla_(TEM)、bla_(SHV)及bla_(CTX)等耐药基因。全基因组测序得到1条完整的基因组序列(GenBank登录号:CP053016)及4条质粒序列(GenBank登录号:CP053017、CP053018、CP053019、CP053020)。在染色体基因组中发现,有44种耐药相关基因,包括bla_(SHV-11)、ompK36、ompC、ompK37、lamB等;有50多种毒力基因,包括fimD、mrkC、mrkD、ybtE、ybtT等。在质粒1(pKPN361-1)中发现了4种耐药基因,包括bla_(CTX-M-65)、bla_(TEM-1)、rmtB和bla_(KPC-2);质粒2(pKPN361-2)中发现了6种耐药基因,包括tet(A)、sul1、mphA、aph(3′)-Ia、bla_(LAP-2)和aac(3)-IId;质粒3(pKPN361-3)和质粒4(pKPN361-4)中未检测到耐药基因,并且4个质粒中均未发现毒力基因。结论 碳青霉烯类耐药肺炎克雷伯菌T361株中携带了大量耐药及毒力基因且产酶耐药基因位于质粒上,造成其易传播,临床上须引起高度重视。

Objective To analyze the resistance and virulence genes of carbapenem-resistant Klebsiella pneumoniae(CRKP) T361 strain. Methods Klebsiella pneumoniae T361 was isolated from sputum specimen of a patient in the First Affiliated Hospital of Kunming Medical University. The strains were identified and tested for drug susceptibility using the Vitek-2 compact system. The whole genome of the strain was sequenced by Illumina Hiseq 4000 high-throughput sequencing platform to analyze the drug resistance and virulence genes carried by the strain. Results Klebsiella pneumoniae T361 was resistant to cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, sulfonamides, β-lactam/β-lactamase inhibitor complexes, nitrofurantoin and tigecycline. BlaKPC-2, blaTEM, blaSHV and blaCTX were detected by PCR. Whole genome sequencing yielded one complete genome sequence(GenBank accession number: CP053016) and four plasmid sequences(GenBank accession numbers: CP053017, CP053018, CP053019, CP053020).There were 44 resistance-related genes in the genome, including bla_(SHV-11), ompK36, ompC, ompK37, lamB, etc. There are more than 50 virulence genes, including fimD, mrkC, mrkD, ybtE, ybtT, etc. Four resistance genes including bla_(CTX-M-65), bla_(TEM-1), rmtB and bla_(KPC-2)were found in plasmid 1(pKPN361-1). Six genes including tet(A), sul1, mphA, aph(3′)-Ia, bla_(LAP-2) and aac(3)-IId were located in plasmid 2(pKPN361-2). No resistance gene was detected in plasmid 3(pKPN361-3) and plasmid 4(pKPN361-4) and no virulence genes were found in all plasmids. Conclusion Carbapenem-resistant Klebsiella pneumoniae strain T361 carries a large number of resistance and virulence genes and the enzyme-producing resistance gene is located on the plasmid, which makes it easy to spread and should be given high priority in clinical practice.