As the causative bacteria of tuberculosis, Mycobacteriumtuberculosis (M. tb) is aggravated by the emergence of its multidrug-resistant isolates in China. Mutations of six of the most frequently reported resistant genes (rpoB, katG, inhA, embB, gyrA, and rpsL) were detected for rifampicin (RIF), isoniazid (INH), ethambutol (EMB), ofloxacin (OFX), and streptomycin (STR) in this study. The amino acid missense mutations (MMs) and their corresponding single nucleotide polymorphism mutations for all drug-resistant (DR) isolates are described in detail. All isolates were divided into non-extensively drug-resistant (Non-XDR) and preXDR/XDR groups. No statistical differences were detected among MMs and linked MMs (LMs) between the two groups, except for rpsL 88 (p = 0.037). In the preXDR/XDR group, the occurrence of MMs in rpoB, katG, and inhA developed phenotypic resistance and MMs of rpoB 531, katG 315, rpsL 43, and rpsL 88 could develop high levels of DR. It is necessary to carry out epidemiological investigations of DR gene mutations in the local region, and thus provide necessary data to support the design of new technologies for rapid detection of resistant M. tb and the optimization of detection targets.

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.

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