摘要目的 观察烧伤创面常用外用药对鲍氏不动杆菌(AB)游离菌及生物膜内菌的抗菌活性,以及协同应用氨溴索对AB生物膜内菌的影响. 方法 11株AB均分离自2005年8月-2007年4月笔者单位烧伤住院患者创面、呼吸道和血液标本.(1)稀释法测定醋酸磺胺米隆溶液和醋酸氯己定溶液对AB游离菌(敏感株、标准株、耐药株)的MIC和最低杀菌浓度(MBC).(2)以LB或TSB培养液培养AB 12、24、48 h形成生物膜,采用MBC的上述2种外用药分别处理30 min(磺胺米隆组和氯己定组),以不加外用药处理的生物膜作为对照组,激光扫描共聚焦显微镜检测生物膜厚度,计算生物膜内活菌比例.将每个外用药组生物膜混匀并接种于LB培养皿,观察有无细菌生长.(3)另以LB培养液培养AB 48 h形成生物膜,采用MBC的上述2种外用药单独(磺胺米隆组和氯己定组)及分别联合终浓度3.75 mg/mL氨溴索溶液(氨溴索+磺胺米隆组和氨溴索+氯己定组)处理30 min,以不加任何药物处理的生物膜作为对照组,噻唑蓝法检测生物膜内菌增殖情况(数据以吸光度值表示).对数据行单因素方差分析和LSD-t检验. 结果 (1)醋酸磺胺米隆溶液和醋酸氯己定溶液对AB游离菌的MIC分别为25.00、0.03 mg/mL,且每种药物的MIC和MBC相同.(2)各组耐药株生物膜厚度于多数时相点大于敏感株.与对照组比较,2个外用药组各时相点下3种菌株生物膜厚度及膜内活菌比例均有所降低.各外用药组生物膜培养均可见大量细菌生长.(3)对照组耐药株吸光度值为0.776±0.071,磺胺米隆组和氯己定组耐药株吸光度值分别为0.625±0.063、0.420±0.068；氨溴索+磺胺米隆组、氨溴索+氯己定组耐药株吸光度值分别为0.174±0.089、0.178±0.044,显著低于对照组(t值分别为11.823、16.009,P值均为0.000)和对应的单独用药组(t值分别为9.248、6.681,P值均小于0.01). 结论 耐药AB易形成生物膜,并可阻碍创面外用药物对细菌的杀灭作用,联合应用氨溴索与创面外用药能起到协同灭菌作用.

abstractsObjective To observe the antimicrobial activity of topical agents commonly used for burns on Acinetobacter baumannii (AB) in both free and biofilm states,and their synergistic effect with ambroxol on AB within biofilm. Methods Eleven AB strains were isolated from wound excretion,respiratory tract,and blood of patients hospitalized in our hospital from August 2005 to April 2007. ( 1 ) The minimal inhibitory concentration ( MIC ) and minimal bactericidal concentration (MBC) of mafenide acetate and chlorhexidine acetate to free AB (including drug-resistant,drug-sensitive,and standard strains) were determined by dilution method.(2) AB was cultured with LB or TSB medium for 12,24,and 48 h to form biofilm,and it was treated with above-mentioned two topical agents in MBC ( mafenide group and chlorhexidine group) for 30 min.Biofilm not treated by topical agent was used as control group.The biofilm thickness was determined with confocal laser scanning microscope.The proportion of living bacteria in biofilm was calculated.AB biofilm in each topical agent group was mixed and inoculated into LB culture dish to observe the growth of bacteria.(3) AB was cultured with LB medium for 48 h to form biofilm,which was respectively treated by above-mentioned two topical agents in MBC (mafenide group and chlorhexidine group) and combination of each topical agent with 3.75 mg/mL ambroxol solution ( ambroxol + mafenide group and ambroxol +chlorhexidine group) for 30 min.Biofilm not treated by topical agents was used as control group.Growth of bacteria in biofilm was detected with MTT method ( denoted as absorbance value).Data were processed with one-way analysis of variance and LSD- t test. Results ( 1 ) MIC of mafenide acetate and chlorhexidine acetate for free AB was respectively 25.00 mg/mL and 0.03 mg/mL.MBC of both agents for free AB was the same as their MIC. (2) Among three groups,the thickness of biofilm of sensitive AB was thicker than that of drug-resistant bacteria at most of the time points.Compared with those in control group,biofilm thickness and proportion of living bacteria in biofilm were slightly decreased in mafenide and chlorhexidine groups.The growth of bacteria was abundant in each group. (3) Absorbance value of drug-resistant bacteria in control,mafenide,and chlorhexidine groups was respectively 0.776 ± 0.071,0.625 ± 0.063,and 0.420 ± 0.068.Absorbance value of drug-resistant bacteria in ambroxol + mafenide group (0.174 ± 0.089) was significantly lower than that of control group ( t =11.823,P =0.000) and mafenide group ( t =9.248,P ＜0.01).Absorbance value of ambroxol + chlorhexidine group (0.178 ± 0.044) was significantly lower than that of control group ( t =16.009,P =0.000) and chlorhexidine group ( t =6.681,P ＜0.01). Conclusions Drug-resistant AB forms biofilm readily,which prevents topical agents from killing the bacteria inside.Combined use of ambroxol with topical agents gives synergistic effect on killing AB in biofilm in the wound.