作者：肖毅，李 晨，梅其炳，张 峰，周四元，马吉献
作者单位：1(710032)中国陕西省西安市，第四军医大学药理学教研室；2(710032)中国陕西省西安市，第四军医大学西京医院眼科
【摘要】 目的：研究妥布霉素滴眼液的抗菌作用。
方法：体外采用M-H肉汤稀释法，妥布霉素滴眼液对大肠埃希菌、金黄色葡萄球菌、绿脓杆菌、化脓性链球菌、肺炎克雷伯菌、肺炎链球菌进行体外抑菌、杀菌试验。体内用角膜环钻损伤兔眼角膜，感染大肠埃希菌、金黄色葡萄球菌、绿脓杆菌、化脓性链球菌造成角膜炎动物模型。然后用妥布霉素滴眼液滴眼4次/d，每眼0.1mL，连续给药7d。d8处死家兔。以观察角膜病理切片，临床症状评分统计学t检验处理及角膜分泌物细菌培养判断疗效。
结果：体外试验表明妥布霉素滴眼液对上述6种细菌最低杀菌浓度(MBC)分别为0.19，0.39，0.78，46.88，93.75， 187.50μg/mL。体内抗菌试验显示，家兔眼感染大肠埃希菌、金黄色葡萄球菌、绿脓杆菌、化脓性链球菌感染后，妥布霉素滴眼液在治疗3d评分结果与未治疗对照组比较均有显著差异，5d细菌培养转阴率大肠埃希菌、金黄色葡萄球菌、绿脓杆菌均为100%，化脓性链球菌83%。角膜病理结果显示除化脓性链球菌伤口愈合较慢，余3种菌伤口斑痕愈合良好。
结论：妥布霉素滴眼液对大肠埃希菌、金黄色葡萄球菌、绿脓杆菌敏感性较强；所致家兔角膜炎治疗效果较好，化脓性链球菌、肺炎克雷伯菌、肺炎链球菌对其敏感性较弱。
【关键词】 妥布霉素滴眼液；最低抑菌浓度；最低杀菌浓度； 细菌性角膜炎；疗效
Anti-bacterial effect of Tobramycin Eye Drops against rabbit keratitis in vitro and in vivo
Yi Xiao1, Chen Li1, Qi-Bing Mei1，Feng Zhang1, Si-Yuan Zhou1, Ji-Xian Ma2
Department of Pharmacology, the Fourth Military Medical University, Xi'an 710032, Shaanxi Province , China; 2Department of Ophthalmology, Xijing Hospital, the Fourth Military Medical University, Xi'an 710032, Shaanxi Province , China
Abstract AIM: To investigate the anti-bacterial efficacy of Tobramycin Eye Drops against experimental keratitis on rabbits.METHODS: Tobramycin Eye Drops was stocked in 1g/L by PBS, diluted in half concentration by M-H meat soup with addition of six different bacteria (1010CFU/L), which are Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Klebsiella pneumoniae, and Streptococcus pneumoniae, and subject to experimentation. Rabbit model of keratitis was made by scratching parenchymal layer of cornea by corneal trephine with addition of 0.1 mL bacterial dilution（2×1012CFU /L）per eyes, then the eyes were treated with Tobramycin Eye Drops q.i.d. in seven successive days, 0.1mL. At eighth day, the rabbits were executed, and the cornea was sliced for pathological examination. Corneal secretions were subject to bacterial culture one day before treatment and 1, 3, and 5 post-treatment. Evaluation scoring based on rabbit clinical manifestations was processed by t -test. RESULTS: In vitro test, the minimal bactericidal concentration of Tobramycin Eye Drops were 0.19, 0.39, 0.78, 46.88, 93.75, 187.50μg/mL for E. coli, S. aureus, P. aeruginosa, S. pyogenes, K. pneumoniae, and S. pneumoniae, respectively. In vivo test, evaluation scoring of Tobramycin Eye Drops at third day was significantly different compared with the control group, while bacterial culture from corneal secretion were 100% negative for E. coli, S. aureus, P. aeruginosa, and 83% negative for S. pyogenes at fifth day. Pathological examination results of the corneal sections demonstrated that the healing of keratitis was well enough for the first three bacteria and slower for S. pyogenes. CONCLUSION: Tobramycin Eye Drops treats effectively for E. coli, S. aureus, and P. aeruginosa, but poorly for S. pyogenes, K. pneumoniae, and S. pneumoniae.
· KEYWORDS: Tobramycin Eye Drops; minimal inhibitory concentration; minimal bactericidal concentration; bacterial keratitis; efficacy
0引言
近年来，随着抗生素及糖皮质激素在眼科的普遍应用，眼部微生物菌群发生了变迁，由金黄色葡萄球菌、绿脓杆菌、大肠杆菌感染眼部引起角膜炎是目前眼科的常见疾患[1，2]。由于滥用抗生素和耐药菌株出现，临床抗感染治疗失败已屡见不鲜，合理选用抗生素成为眼科医生不得不考虑的问题。妥布霉素滴眼液是近几年用于临床的抗生素，临床常见菌对其敏感性到底如何？我们选用南京恒生制药厂生产妥布霉素滴眼液，分别对金黄色葡萄球菌、绿脓杆菌、大肠杆菌、化脓性链球菌、肺炎链球菌、肺炎克雷伯菌6种菌进行体内外抗菌试验，以便对临床合理使用抗生素提供依据。
1材料和方法
1.1材料 妥布霉素滴眼液 (商品名佳诺泰，含3g/L妥布霉素)，南京恒生制药厂生产。速眠新注射液 5mL/支，长春农牧大学兽医研究所研制，常温避光保存。Mueller-Hinton(M-H)肉汤和营养琼脂培养基由北京奥博星生物技术有限公司生产。大耳白家兔由第四军医大学动物中心提供，雌雄均有，体质量2～2.5kg。实验前单笼常规饲养1wk，室温20℃左右，自由饮水，检查双眼无异常。大肠埃希菌ATCC25922， 金黄色葡萄球菌ATCC25923，绿脓杆菌ATCC27853，化脓性链球菌（临床株），肺炎克雷伯菌（临床株），肺炎链球菌（临床株），由第四军医大学西京医院检验科提供。
1.2方法
1.2.1体外试验 新鲜菌种接种M-H肉汤培养18h作原菌液，取0.1mL测活菌数调整菌液1010CFU/L。用0.1mol/L磷酸盐缓冲液（PBS）将妥布霉素滴眼液稀释成含妥布霉素浓度为1g/L，过滤除菌4℃保存备用，试验时先用M-H肉汤倍比稀释，每管加1.0mL不同浓度的妥布霉素滴眼液，分别加入1010CFU/L大肠埃希菌、金黄色葡萄球菌、绿脓杆菌、化脓性链球菌、肺炎克雷伯菌、肺炎链球菌0.1mL；肺炎链球菌、化脓性链球菌每管各加兔血1滴，最终接种细菌量每管109CFU /L。同时设空白对照和阳性对照管（不加药）。37℃培养18～24h，测定最低抑菌浓度（MIC），继之选出未见细菌生长管中液体0.1mL接种到琼脂培养基平板上（肺炎链球菌和化脓性链球菌接种血琼脂培养板），进一步做最低杀菌浓度（MBC）测定[3]。
1.2.2体内试验 将接种于普通琼脂培养基平板上的大肠埃希菌、金黄色葡萄球菌、绿脓杆菌和血琼脂培养板的化脓性链球菌用接种环刮下，生理盐水分别配成2×1012 CFU/L（光电比浊仪测定浓度）。按方法[4]用角膜环钻致家兔角膜损伤（双眼），家兔每眼感染细菌0.1mL，2d用无菌盐水棉签取眼睛分泌物，放入4mL无菌生理盐水瓶中，用琼脂平板培养法对其液体做细菌培养。同时进行炎症评分，0级：眼明亮无分泌物；0.5级：无分泌物覆盖，眼稍有红肿；1级：分泌物覆盖小于6mm；2级:分泌物覆盖充满6mm；3级：分泌物覆盖大于6mm。根据评分标准随机分组。治疗组（感染细菌+妥布霉素滴眼液），未治疗对照组（感染细菌+滴生理盐水），各组6只家兔。正常对照组（不造模、不感染细菌、滴生理盐水）2只。治疗组滴妥布霉素滴眼液0.8mL（2.4mg）/d（双眼），未治疗对照组和正常对照组滴生理盐水0.8mL/d（双眼），每眼0.1mL，4次/d，连续点药7d。每隔24h双眼进行一次评分，连续观察7d，评分结果作统计学t检验处理。并于给药前1d，给药后1，3，5，7d用无菌盐水棉签取眼分泌物作细菌培养，判断阴阳性结果。d8处死家兔，取角膜放入40g/L甲醛中固定作病理切片，HE染色检查。
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