一株样品分离菌-洋葱伯克霍尔德菌对抑菌效力评价体系的启示

Enlightenment of Burkholderia Cepacia Isolated from a Sample on the Evaluation System for Bacteriostasis Efficacy

目的:通过对一株样品分离菌株洋葱伯克霍尔德菌耐受力的考察及其对苯扎氯铵防腐体系的挑战,对产品抑菌效力评价体系的一些思考和建议,以供业内借鉴和参考。方法:采用微量稀释法测定质控菌株(洋葱伯克霍尔德菌)、样品中及其分离传代后的洋葱伯克霍尔德菌对苯扎溴铵的MIC值,并通过活菌计数法考察了回接到未受污染样品中的存活状态;在《中国药典》2015年版第四部1121抑菌效力检查法的基础上,增加洋葱伯克霍尔德菌作为挑战菌,对三种含不同浓度苯扎氯铵的玻璃酸钠滴眼液的抑菌效力进行评价。结果:苯扎溴铵对未经传代培养的野生型洋葱伯克霍尔德菌MIC值为128μg·mL^-1,高于分离传代后的洋葱伯克霍尔德菌和质控菌株的MIC值(均为64μg·mL^-1);回接试验表明,在接种后24 h内,菌量下降,24 h后菌量逐渐回升,28天菌量回升至初始水平;洋葱伯克霍尔德菌能耐受0.03 g·L^-1和0.05 g·L^-1苯扎氯铵防腐体系,只有最高浓度的的苯扎氯铵防腐体系(0.10g·L^-1)抑菌效力符合药典规定。结论:环境或样品中分离出来的野生菌株对抑菌剂的耐受性有可能会高于实验室培养菌株或标准菌株,提示我们在进行产品的防腐剂抑菌效力评价时,企业应考虑环境和样品中的常见分离菌株,并针对相应的添加菌株建立相对完善的文件体系,以保障抑菌效力评价体系的有效性和完整性。

Objective:To put forward some thoughts and suggestions on the evaluation system for bacteriostasis efficacy of the product through the investigation of the tolerance of Burkholderia cepacia isolated from a sample and its challenge to the bacteriostatic system of benzalkonium chloride so as to provide references for the industry.Methods:The micro-dilution method was used to determine the MIC value of the control strain(Burkholderia cepacia),sample and its isolated and subcultured strains against benzalkonium bromide.The isolated and subcultured strains were returned to the uncontaminated samples,and the viability of the strains was observed by counting the viable bacteria.According to the 1121 preservative effectiveness test method of the Chinese Pharmacopeia(2015 edition)part IV,the Burkholderia cepacia was used as challenge bacteria to evaluate the antibacterial effect of three different concentrations of benzalkonium chloride.Results:The MIC value of benzalkonium bromide to wild-type Burkholderia cepacia that was not subcultured was 128μg·mL^-1,which was higher than the MIC value of Burkholderia cepacia and isolated and subcultured quality control strain(both 64μg·mL^-1).The back-test showed that the amount of bacteria decreased within 24 hours after inoculation,and the amount of bacteria gradually increased after 24 hours.The number of bacteria returned to the initial level in 28 days.Burkholderia cepacia could tolerate 0.03 g·L^-1 and 0.05 g·L^-1 bacteriostatic system of benzalkonium chloride.The antibacterial effect of benzalkonium chloride with the highest concentration 0.10 g·L^-1 was in line with the requirement of Chinese Pharmacopeia,while the other two concentrations were not.Conclusion:The tolerance of wild strains isolated from environment or samples for antimicrobial agents may be higher than that of laboratory cultured strains or standard strains.This suggests that manufacturers should consider common strains isolated from environment or samples when evaluating the antimicrobial efficacy of products’antiseptic prescriptions and establish a relatively complete documentation system for the corresponding added strains so as to ensure the effectiveness and integrity of the evaluation system for bacteriostasis efficacy.