86株痤疮丙酸杆菌的耐药性及生物膜成膜能力分析

Clinical characteristics and drug resistance analysis of 86 clinical isolates of Propionibacterium acnes

摘要:

目的 研究临床分离痤疮丙酸杆菌的耐药性及其生物膜成膜能力。方法 选取2020年12月—2022年1月长沙市第四医院皮肤科门诊诊治的114例痤疮患者为研究对象。分离出痤疮丙酸杆菌，通过琼脂稀释法检测其对常用抗生素的耐药性；采用结晶紫染色法检测其生物膜形成能力；通过激光共聚焦显微镜观察生物膜结构；选取8株生物膜成膜能力较强的菌株Pa01、Pa02、Pa03、Pa04、Pa05、Pa06、Pa07和Pa08检测体外抗生素的敏感性，随机选取成膜能力阴性菌株（浮游菌）Pa12和成膜能力阳性菌株Pa03进行大鼠实验，复制痤疮丙酸杆菌菌株感染模型。将感染模型大鼠随机分为2组，每组6只，分别皮下注射30 mg/kg的米诺环素（米诺环素组）或1% DMSO（溶媒组），感染后第5天处死大鼠，测量脓肿的面积，并分离脓肿组织进行均浆计数和HE染色。结果 本研究共分离出86株痤疮丙酸杆菌，分离率为75.4%。痤疮丙酸杆菌对甲硝唑和夫西地酸的耐药性最高，分别为93.0%和90.7%；对大环内酯类抗生素的耐药率为75.6%～88.4%。对米诺环素和多西环素的敏感性最高，耐药率仅分别为7.0%和8.1%。86株临床分离痤疮丙酸杆菌中，34株（39.53%）生物膜成膜能力阳性，其中，成膜能力强、中和弱的占比分别为9.30%（8/86）、12.79%（11/86）和17.44%（15/86）。不同成膜能力组的生物膜吸光度比较，差异有统计学意义（P <0.05），且成膜能力强、中和弱组的吸光度比成膜能力阴性组均显著升高（P <0.05）；对照组与米诺环素实验组（8 μg/mL）菌株Pa01、Pa02、Pa03、Pa04、Pa05、Pa06、Pa07和Pa08的生物膜总量比较，差异有统计意义（P <0.05）。成膜能力阴性菌株Pa12中，米诺环素组的脓肿大小较溶媒组降低（P <0.05），米诺环素可显著抑制浮游菌Pa12脓肿的形成；米诺环素组的菌株载量较溶媒组减少（P <0.05），米诺环素能显著降低其脓肿痤疮丙酸杆菌菌株载量。成膜能力阳性菌株Pa03中，溶媒组和米诺环素组的脓肿大小和菌株载量比较，差异无统计学意义（P >0.05）。结论 痤疮丙酸杆菌对米诺环素和多西环素的敏感性较高，且该菌易形成生物膜导致耐药性增加。

Abstract:

Objective To explore the resistance characteristics and biofilm forming ability of Propionibacterium acnes.Methods One hundred and fourteen acne patients from the outpatient clinic of the Fourth Hospital of Changsha from December 2020 to January 2022 were included in this study. The isolated Propionibacterium acnes were tested for resistance to commonly used antibiotics by agar dilution method. The ability of biofilm formation was detected by crystal violet staining. Observation of biofilm structure using laser confocal microscopy. Strains of Pa01, Pa02, Pa03, Pa04, Pa05, Pa06, Pa07, and Pa08 with positive biofilm-forming ability were selected for in vitro antibiotic susceptibility test. The biofilm-forming negative strain (planktonic bacteria) Pa12 and biofilm-forming positive strain Pa03 were randomly selected to construct Propionibacterium acnes rat infection model in vivo. The rats were divided into two groups (6 rats per group), one was treated with 30 mg/kg minocycline, the other was treated with 1% DMSO as a control. Five days post infection, the rats were euthanized and the abscess area and bacterial loads were measured. In addition, HE staining of the abscess was also performed.Results A total of 86 strains of Propionibacterium acnes were isolated in this study, with an isolation rate of 75.4%. Propionibacterium acnes showed the highest resistance rate to metronidazole and fusidic acid with 93.0% and 90.7%, respectively. The resistance rate of the bacterium to macrolide antibiotics ranged from 75.6% to 88.4%. The bacterium showed the highest susceptibility to minocycline and doxycycline with the resistance rate of 7.0% and 8.1%. Among the 86 clinical isolates, positive rate of biofilm-forming ability of Propionibacterium acnes accounted for 39.53% (34 strains), and the strong, moderate and weak biofilm forming strains were accounted for 9.30% (8/86), 12.79% (11/86), and 17.44% (15/86), respectively. The significance of absorbance was found among different biofilm forming ability groups (P < 0.05), and the absorbance at the groups with strong, moderate, and weak biofilm forming strains was significantly higher compared with the biofilm forming negative strains (P < 0.05). Significant differences (P < 0.05) of the biofilm biomass were found between the control group and minocycline treated groups (including Pa01, Pa02, Pa03, Pa04, Pa05, Pa06, Pa07, and Pa08 ). The abscess size of the minocycline treated group was reduced compared with the control group (P < 0.05) by Pa12 infection. And the bacterial loads in the abscess were also significantly decreased (P < 0.05). As in the infection model by the biofilm forming negative strain Pa03, no statistical difference was found in the abscess area and bacterial loads (P > 0.05).Conclusion Propionibacterium acnes has a high sensitivity to minocycline and doxycycline, and the bacterium is prone to forming biofilms resulting in increased drug resistance.