鸽源奇异变形杆菌中磷霉素耐药基因fosA3的流行与传播特征

Prevalence and dissemination of fosA3 gene in Proteus mirabilis isolates from a pigeon field

为研究鸽源奇异变形杆菌中磷霉素耐药基因fosA3的流行与传播特征,从广东省佛山市某鸽场采集鸽子及环境样品72份,采用选择性培养基及MALDI-TOF-MS分离鉴定奇异变形杆菌。采用PCR检测质粒介导磷霉素耐药基因fosA3、fosC2以及fosA,采用微量肉汤稀释法测定阳性菌株耐药表型。采用脉冲场凝胶电泳(PFGE)、全基因组测序以及构建核心基因组进化树分析菌株的亲缘关系。采用接合转移、Southern杂交及PCR-mapping方法探究fosA3的分子传播特征。结果显示,72份样品中共获得22株奇异变形杆菌,16株菌中检出fosA3,没有检出fosC2和fosA。fosA3阳性菌株均对磷霉素表现高水平耐药(MIC>256 mg/L),对受试9种抗菌药物耐药,呈现多重耐药表型。16株fosA3阳性菌株主要分为2种不同PFGE谱型,随机挑选2株代表性菌株进行全基因组测序并进行遗传进化树分析,发现其中1株fosA3阳性菌与数据库中1株人源奇异变形杆菌之间亲缘关系最近。16株阳性菌株中fosA3均位于染色体上,不能发生接合转移。有阳性菌株中均检测到IS26-orf3-Δorf2-orf1-fosA3-IS26的遗传结构。本研究鸽场奇异变形杆菌中染色体编码的fosA3存在较高的流行性,该基因主要通过菌株的克隆传播以及插入元件IS26介导的水平转移进行传播,应引起高度重视。

To investigate the prevalence and dissemination of fosfomycin resistant gene fosA3 in Proteus mirabilis from a pigeon farm,72 samples were collected from pigeons and environment of a pigeon farm from Foshan,Guangdong Province.The Proteus mirabilis was isolated and identified by selective medium and MALDI-TOF-MS.The plasmid-mediated fosfomycin resistance gene fosA3,fosC2 and fosA were detected by PCR,and the antibiotic-resistant phenotype of the fosA3 positive strains were determined using the broth microdilution method.The phylogenetic relationship of the strains harboring fos genes was identified by pulsed-field gel electrophoresis(PFGE),whole genome sequencing,and the construction of core-genome phylogenetic tree.The characteristics of molecular transmission of fosA3 were explored by conjugative transfer,Southern hybridization and PCR-mapping.The results showed that a total of 22 Proteus mirabilis strains were obtained from 72 samples,and 16 ones were identified to carry fosA3,while no one carried fosC2 and fosA.All the fosA3-positive strains showed high level resistance to fosfomycin(MIC >256 mg/L)and multidrug resistant phenotypes with resistance to 9 kinds of antimicrobial agents.The 16 fosA3-positive strains were divided into two different PFGE patterns.Two representative fosA3-positive P.mirabilis strains were randomly selected for whole genome sequencing and genetic phylogenetic tree analysis,revealing one was closely genetically related to a human Proteus mirabilis strain in GenBank.Among the 16 fosA3-positive strains,fosA3 gene was located on the chromosome and could not be transferred by conjugation,but the genetic structure of IS26-orf3-Δorf2-orf1-fosA3-IS26 was found in all positive strains.These results demonstrate a high prevalence of the chromosomally encoded fosA3 gene in Proteus mirabilis isolates from a pigeon farm,and the fosA3 gene is transmitted mainly through clonal spread of the strain and horizontal transfer mediated by the insertion element IS26,indicating this should be received great attention.