背景：利奈唑胺是一种新型恶唑烷酮类抗生素，常用来治疗多重耐药的革兰阳性菌感染。美国和中国分别在2000年和2007年引入使用，它被认为是治疗VRE、MRSA和青霉素耐药的肺炎链球菌引起感染的有效药剂。近年来，利奈唑胺耐药的葡萄球菌和肠球菌在多个国家出现，给临床治疗带来了挑战。

方法：对北京协和医院2015-2017年间75份病历（包括15例LNSE患者病例和60例LSE的患者病例）及2012-2017年分离出16株LNSE（14株粪肠球菌、1株屎肠球菌和1株铅黄肠球菌）和56株LSE进行分析。对前者运用卡方检验及T检验分析临床资料的差异性。后者在准确鉴定菌株的基础上，检测15种抗菌药物的最小抑菌浓度。对纳入菌株进行分子分型，进而进行聚类及同源性分析。在耐药机制方面对耐药相关基因及毒力基因进行PCR扩增测序。对4株临床分离粪肠球菌进行利奈唑胺诱导耐药实验并检测23S rRNA编码基因位点突变情况。对14株临床分离的LREfs进行接合实验，分析耐药基因在菌株间的传递情况。

结果：在LNSE和LSE两组中年龄、住院天数、侵入性操作、患者基础情况、利奈唑胺的使用等因素无统计学差异。

分离的16株LNSE多来自妇科（25%，4/16）和外科(25%，4/16)。标本类型主要为尿液（4/16）、引流液(4/16)和血液(2/16)。大部分菌株利奈唑胺MIC为 8μg/ml，氯霉素非敏感率为100%，此外耐药率较高的有四环素、氯霉素、左氧氟沙星和红霉素，分别为100%、94.8%、62.5%和62.5%。万古霉素、替考拉宁、氨苄西林、替加环素、呋喃妥因、达托霉素都有较高的敏感性，分别为93.8%、93.8%、87.5%、100%、93.8%和100%。optrA、fexA、ermA基因仅在耐药菌中为阳性，阳性率分别为100%、100%和35.7%。PFGE对菌株同源性分析具有更高的分辨率，以80%相似度为标准时与MLST有较好的一致性。

临床分离的LRE中只有两株检测出G2237A位突变，其他突变未见。cfr阴性，核糖体蛋白L3和L4氨基酸水平也没有改变，optrA基因均为阳性，其蛋白具有5种不同型别，分别为OptrAE₃₄₉、KD、RDK、EDM和D。

诱导耐药试验说明optrA基因阴性的菌株在长时间药物诱导后主要出现G2576T突变，也存在C2610T突变，optrA基因阳性的菌株则未见G2576T突变，出现的突变为G2237A和G2504A。optrA基因定位在细菌的质粒上，接合成功率为50%(7/14)。

结论：本研究中利奈唑胺使用史不是耐药产生的危险因素，利奈唑胺非敏感肠球菌的耐药机制有点突变和optrA，optrA为主要耐药机制且可在不同菌株之间传播。

Background: Linezolid is an oxazolidinone antimicrobial agent, has been used for treatment of complicated Gram-positive infection. In 2000, linezolid was approved by the American Food and Drug Administration (FDA) and in 2007,it was allowed to be used in Chnia. Linezolid is considered to be effective for treating infection of VRE、MRSA and penicillin-resistant Pneumococcus. In recent years, reserchers have found linezolid-nonsusceptible Staphylococcus and Enterococcus in several counties. It brings challenges to clinical therapy .

Methods: In this study, we collected 75 cases, including 15 cases of LNSE infection and 60 cases of LSE infection from Peking union medical college hospital from 2015 to 2017 ,we isolated 16 strains of LNSE (including 14 strains of Enterococcus faecalis , 1 strain of Enterococcus faecium and 1 strain of Enterococcus casselifavus)and 56 strains of LSEfs from 2012 to 2017, using the chi-square test and T-test to analysis the difference of clinical data.On the basis of the accurate identification of these strains, drug sensitivity test, clustering and homology analysis were done. In terms of resistance mechanism, amplification and sequencing analysis were also performanced for genes of drug resistance and virulence factor. Four Enterococcus faecalis strains of clinical isolates were cultured in different concentration of linezolid medium to induced resistance mutation. Conjugation experiments was done in 14 strains of linezolid-resistant Enterococcus faecalis to analysis the transferability of optrA among strains.

Results: There were no difference in age, hospitalization days, invasive operation, patients' basic situation and the use of lineolid between the two groups of LNSE and LSE. In the LNSE, 50% strains were from gynecology（25%, 4/16） and surgery（25%, 4/16）, the specimen types were mainly urine(25%, 4/16) and drainage fluid (25%, 4/16).Tetracycline and chloramphenicol had high resistance rate, 100% and 94.8%, respectively. The sensitivity rates of teicoplanin and daptomycin were both 100%, and vancomycin, teicoplanin and nitrofurantoin were all 93.8%. optrA, fexA and ermA gene were positive in LREfs, and positive rate were 100%, 100% and 35.7%, respectively. The PFGE clustering analysis has higher resolution, and it has good consistency with MLST on the basis of 80% similarity.
We only detected the G2237A mutation in two clinical isolated Enterococcus strains, optrA genes were all positive, with five different types OptrAE349, KD, RDK, EDM, and D.
Induced tests in vitro suggested that strains without optrA gene might have G2576T and C2601T mutation in a long period of time of inducing, but strains with optrA gene had G2237A and G2504A mutations, but had no G2576T mutations. One strain with optrA gene found no mutations after the inducing , it suggests that there may be other resistance mechanisms. optrA gene location on plasmids, the rate of conjugation was 50% (7/14).

Conclusion: This study found no significant differences in the factor of linezolid using. Virulence factors also showed no significant differences between the two groups. The main resistant mechanism is the carring of optrA gene and the gene can be transmitted between different strains. Mutation can also lead to resisitant of linezolid.