中国首株副瘰疬分枝杆菌临床分离株的生物学特征和毒力研究

Biological characteristics and toxicity of first Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient

目的 对中国肺病患者体内首次分离到的1株副瘰疬分枝杆菌进行了生物学特征与毒力研究,以提高对该分枝杆菌的认识.方法 2013年对源于2007 2008年全国耐药基线调查的140株非结核分枝杆菌（NTM）,通过联合测定16S rRNA、16S～23S rRNA间隔区（ITS）和hs筇5基因序列进行菌种鉴定,分离到1株副瘰疬分枝杆菌.通过开展一系列标准生化试验,包括生长试验、硝酸还原酶测定、芳基硫酸酯酶测定、碲酸钠还原试验、吐温水解试验、尿素酶试验,以及在50 mg/ml对硝基苯甲酸（PNB）、10 mg/ml噻吩-2-羧酸肼（TCH）、5％ NaCl和麦康凯琼脂上进行生长试验,阐明其生物学特征.通过Alamar blue微量稀释法测定其对利福平、乙胺丁醇、异烟肼、阿米卡星、磺胺甲恶唑、氧氟沙星、对氨基水杨酸、利福喷丁、卷曲霉素、头孢西丁、亚胺培南、米诺环素、多西环素、克拉霉素、阿奇霉素、美罗培南、甲氧苄氨嘧啶、利福布汀、利奈唑胺、链霉素、丙硫异烟胺、莫西沙星和左氧氟沙星共23种抗生素的药物敏感性.为了研究副瘰疬分枝杆菌的毒力,通过尾静脉和腹腔注射两种途径感染C57BL/6小鼠,在小鼠感染后的第2、4、8周取肺脏和脾脏检测细菌载量和病理组织学变化,确定其对小鼠的致病性.结果 该株副瘰疬分枝杆菌在37℃生长旺盛,菌落光滑并呈半球形,呈黄至橘黄色.在体外对利福布丁、利福喷丁、克拉霉素、阿奇霉素、头孢西丁和莫西沙星敏感.在C57BL/6小鼠感染后的第2周,病理组织学结果显示小鼠肺组织出现局限性炎症反应,表现为外周支气管炎性渗出.在感染后的第4和第8周,肺组织表现为外周支气管炎性渗出伴有大量炎性细胞聚集,部分组织有肉芽肿生成,但无坏死.在小鼠感染的8周时间内,肺组织和脾组织中细菌载量呈逐渐上升趋势.结论 该副瘰疬分枝杆菌在体外对利福布汀、利福喷丁、克拉霉素、阿奇霉素、头孢西丁和莫西沙星敏感;可在小鼠体内感染扩增,并可引起小鼠肺组织病变.

Objective A Mycobacterium parascrofulaceum strain was isolated from a pneumonia patient--the first such reported case from China. The biological characteristics and toxicity of the strain were determined. Methods A total of 140 nontuberculous mycobacteria （NTM） strains isolated from sputa during the 2007--2009 nationwide surveillance of drug-resistant tuberculosis in China were analyzed. Species identification was performed by homologue gene sequence comparison, then a series of biochemical tests was conducted to elucidate the bacterio logical characteristics. Drug susceptibility and pathogenicity to mice of the strain were tested. Results Vigorous growth was observed when the bacteria were grown at 37℃. Colonies grown on Middlebrook 7H10 agar were smooth, domed, and scotochromogenic with yellow to orange pigmentation. The M. parascrofulaceum strain was sensitive to rifabutin, rifapentine, elarithromycin, azithromycin, cefoxitin, and moxifloxacin in vitro. At week 2 post-infection, the lung tissues of mice demonstrated a local inflammatory response denoted by peribronchiolar inflammatory infiltrates. At weeks 4 and 8, the lung tissues showed peribronchiolar inflammatory infiltrates with large aggregates of Iymphocytes and part of the tissue showed granulomatous lesions; there was no appreciable necrosis. The colony-forming units （CFU） count of infected lung and spleen increased gradually during the 8 weeks of the experiment. Conclusion The M. parascrofulaceum strain isolated in China was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, cefoxitin, and moxifloxacin. The mycobacteria were capable of proliferating in mice and could lead to pathological changes in the lungs of the mice.