Data_Sheet_1_A Conjugative MDR pMG1-Like Plasmid Carrying the lsa(E) Gene of Enterococcus faecium With Potential Transmission to Staphylococcus aureus.docx

lsa(E) is a pleuromutilin, lincosamide, and streptogramin A (PLSA phenotype) resistance gene that was first described in S. aureus and was thought to have been transferred from Enterococcus sp. This study aimed to elucidate the prevalence of the lsa(E) gene among E. faecium isolates at a tertiary teaching hospital and to evaluate the transferability of the lsa(E) gene from E. faecium to S. aureus in vitro. A total of 96 E. faecium strains isolated from one hospital in Beijing in 2013 were analysed for quinupristin-dalfopristin (QDA) resistance genes, and multilocus sequence typing (MLST) was performed. The transferability of QDA resistance between ten E. faecium strains and four S. aureus strains was determined by filter mating. Genome sequencing of the transconjugant was performed. A total of 46 E. faecium isolates (46/96, 47.92%) tested positive for lsa(E), while two isolates (2/96, 2.08%) tested positive for lsa(A). Thirty-six lsa(E)-positive strains (36/46, 78.3%) belonged to ST78. Among 40 mating tests, lsa(E) was successfully transferred through one conjugation at a frequency of 1.125 × 10–7 transconjugants per donor. The QDA resistance of the transconjugant N7435-R3645 was expressed at a higher level (MIC = 16 mg/L) than that of the parent S. aureus strain (MIC = 0.38 mg/L). Next-generation sequencing (NGS) analysis of the transconjugant N7435-R3645 showed that the complete sequence of the lsa(E)-carrying plasmid pN7435-R3645 had a size of 92,396 bp and a G + C content of 33% (accession no. MT022086). The genetic map of pN7435-R3645 had high nucleotide similarity and shared the main open reading frame (ORF) features with two plasmids: E. faecium pMG1 (AB206333.1) and E. faecium LS170308 (CP025078.1). The rep gene of pN7435-R3645 showed 100% identity with that of pMG1, although it did not belong to the rep1-19 family but instead a unique rep family. Multiple antibiotic resistance genes, including lsa(E), aadE and lnu(B), erm(B), ant6-Ia, and lnu(B), were present on the plasmid. In conclusion, an lsa(E)-carrying plasmid that can be transferred by conjugation from E. faecium to S. aureus in vitro was identified. This multidrug resistance (MDR) pMG1-like plasmid may act as a vector in the dissemination of antimicrobial resistance among species.

lsa(E)基因是一种属于截短侧耳素类（pleuromutilin）、林可酰胺类（lincosamide）及链阳菌素A类（streptogramin A，PLSA表型）的耐药基因，最初于金黄色葡萄球菌（S. aureus）中被报道，此前被认为是从肠球菌属（Enterococcus sp.）转移而来。本研究旨在明确某三级教学医院分离的屎肠球菌（E. faecium）菌株中lsa(E)基因的携带率，并体外评估lsa(E)基因从屎肠球菌向金黄色葡萄球菌的转移能力。本研究对2013年于北京某医院分离的96株屎肠球菌进行了奎奴普丁-达福普汀（quinupristin-dalfopristin，QDA）耐药基因检测，并开展多位点序列分型（MLST）。通过滤膜接合试验，检测10株屎肠球菌与4株金黄色葡萄球菌之间QDA耐药性的可转移性，并对接合子进行全基因组测序。共计46株屎肠球菌（46/96，47.92%）lsa(E)检测呈阳性，另有2株（2/96，2.08%）lsa(A)检测呈阳性。其中36株lsa(E)阳性菌株（36/46，78.3%）属于ST78型。在40次接合试验中，1次成功实现lsa(E)的接合转移，转移频率为每供体菌1.125×10^–7个接合子。接合子N7435-R3645的QDA耐药水平（最低抑菌浓度MIC=16 mg/L）高于亲本金黄色葡萄球菌菌株（MIC=0.38 mg/L）。对该接合子N7435-R3645进行下一代测序（NGS）分析显示，携带lsa(E)的质粒pN7435-R3645全长为92396 bp，G+C含量为33%（登录号MT022086）。pN7435-R3645的遗传图谱与两株屎肠球菌质粒——pMG1（AB206333.1）和LS170308（CP025078.1）——具有较高的核苷酸同源性，且共享主要开放阅读框（ORF）特征。尽管pN7435-R3645的rep基因与pMG1的rep基因同源性达100%，但该基因不属于rep1-19家族，而是隶属于一个独特的复制家族。该质粒上携带多种抗菌耐药基因，包括lsa(E)、aadE、lnu(B)、erm(B)、ant6-Ia及lnu(B)。综上，本研究成功鉴定出一株可通过接合作用在体外从屎肠球菌转移至金黄色葡萄球菌的携带lsa(E)质粒。这种多重耐药（multidrug resistance, MDR）类pMG1质粒可能作为载体，推动跨物种间的抗菌耐药性传播。