多重耐药弗劳地枸橼酸杆菌的基因组及耐药机制分析

Genome and resistance mechanism analysis of multidrug-resistant Citrobacter freundii

目的对多重耐药弗劳地枸橼酸杆菌进行全基因组分析,为研究其耐药机制提供依据。方法采用含美罗培南的MH培养基对粪便标本进行初筛,经BD Phoenix100全自动微生物鉴定仪进行菌种鉴定及药敏试验,提取耐药菌总DNA进行二代测序和细菌多位点序列分型(MLST),经质粒全基因组序列分析检测质粒的组分和功能,并与参考质粒进行比较,通过质粒接合转移实验分析耐药质粒传递情况。结果413份粪便标本中分离出1株多重耐药弗劳地枸橼酸杆菌,该菌株对头孢菌素类、碳青霉烯类抗菌药物、复方磺胺甲噁唑的耐药率均为100%,对氨曲南、环丙沙星、左氧氟沙星、庆大霉素等抗菌药物呈现不同程度耐药;MLST分型的耐药弗劳地枸橼酸杆菌为ST22型,该菌株共含有5412个基因,含耐药基因367个,包括碳青霉烯酶类耐药基因、AmpC酶基因、大环内酯类耐药基因、氨基糖苷类耐药基、喹诺酮类耐药基因等。质粒的全基因组分析结果显示,该质粒含碳青霉烯类耐药基因blaNDM-1、blaSHV12,该质粒与泄殖腔肠杆菌菌株ECN49质粒和肺炎克雷伯菌菌株质粒pA575-NDM有极高的同源性;质粒接合实验显示,blaNDM-1和blaSHV-12可以通过质粒转移。结论弗劳地枸橼酸杆菌耐药的主要原因之一是含有blaNDM-1、blaSHV-12等耐药基因,并且该耐药性能通过质粒进行水平转移。

Objective To analyze the whole genome of multidrug resistant Citrobacter freundii and to provide a basis for the study of its drug resistance mechanism.Methods The fecal samples were screened with MH medium containing meropenem,strains were identified and drug susceptibility tested by BD Phoenix100 automatic microbial identification apparatus.The total DNA of drug-resistant bacteria was extracted for second-generation sequencing and bacterial multi-site sequence typing(MLST).The components and function of the plasmids were detected by whole genome sequence analysis.Compared with the reference plasmid,plasmid conjugation transfer assay was used to analyze the transmission of drug-resistant plasmid.Results One strain of multidrug-resistant Citrobacter freundii was isolated from 413 fecal samples.The resistance rate of the strain to cephalosporins,carbapenems and compound sulfa methoxazole was 100%,and and it showed different degrees of resistance to amtronam,ciprofloxacin,levofloxacin and gentamicin.The drug-resistant Citrobacter freundii of MLST type was ST22.The strain contained 5412 genes,including 367 drug-resistant genes,including carbapenem enzyme resistance genes,AmpC enzyme genes,macrolide resistance genes,aminoglycoside resistance groups,quinolone resistance genes,etc.The whole genome analysis of the plasmid showed that the plasmid contained carbapenem resistance genes blaNDM-1 and blaSHV12.The plasmid had high homology with Entero bacter cloaca strain ECN49 and Klebsiella pneumoniae pA575-NDM.Plasmid conjugation experiments showed that blaNDM-1 and blaSHV-12 could be transferred by plasmid.Conclusion One of the main causes of drug resistance of Citrobacter freundii is that it contains blaNDM-1,blaSHV-12 and other resistance genes,and the resistance can be transferred horizontally through the plasmid.