【作者】 曾宪玉；

【导师】 杨志波；

【作者基本信息】 湖南中医药大学 ， 中医外科学， 2014， 博士

【摘要】 目的：研究从痤疮患者皮损中分离的丙酸杆菌对红霉素、克林霉素和四环素的药敏情况及耐药机制,研究盐酸小檗碱对丙酸杆菌的抑制作用并探讨其可能机制。方法：分离丙酸杆菌,厌氧培养经革兰染色、16SrRNA和23SrRNA测序等方法鉴定,采用E-test方法检测分离株对红霉素、克林霉素和四环素的MIC值,PCR扩增ermX、ermX (cj)、IS1249a、IS1249b并测序。采用微量肉汤稀释法检测盐酸小檗碱对丙酸杆菌的MIC值,采用real time PCR方法研究小檗碱是否调节erm基因的表达和逆转23S rRNA点突变。结果：共分离84株丙酸杆菌,72株、12株分别鉴定为痤疮丙酸杆菌(Propionibacterium acnes, P.acnes)、贪婪丙酸杆菌(Propionibacterium avidum,P.avidum)。所有菌株对四环素敏感。29株(34.52%)对红霉素耐药,均为高度耐药(MIC>25μg/ml)；其余55株对红霉素敏感。19株P.acnes耐药株中,16株对克林霉素高度耐药(MIC>256μg/ml)；10株P. avidum对克林霉素高度耐药(MIC>256μg/ml)。19株P.acnes耐药株中,7株在相当于Escherichia coli23S rRNA2058位点发现由A→G点突变,均对红霉素和克林霉素高度耐药；4株在相当于E.coli23S rRNA2059位点发现由A→G点突变,其中1株对克林霉素耐药,3株敏感；另外8株P.acnes扩增ermX阳性,其序列与Genbank中P.acnes ermX基因100%同源。10株P. avidum中,2株ermX扩增阳性,其序列与P.acnes ermX基因100%同源；另外8株扩增ermX (cj)得到预期片段PCR产物,与Genbank中Corynebacterium jeikeium ermX (cj)99%同源,而与P.acnes ermX仅有94%的同源性。10株扩增ermX基因阳性的菌株扩增IS1249a和IS1249b均阳性,而其余菌株均阴性。盐酸小檗碱对丙酸杆菌的MIC值在1-8μg/ml,未发现其有调控erm基因、逆转23SrRNA点突变的作用。经盐酸小檗碱溶液共孵育后的耐药P. acnes透射电镜下可见DNA扭曲碎裂、细胞核固缩、空泡化。结论：本研究中痤疮患者携带的丙酸杆菌包括P. acnes对红霉素耐和克林霉素耐药率高,对四环素敏感。对红霉素和克林霉素的耐药与在相当于E.coli23S rRNA2058、2059位点由A→G点突变、携带ermX(cj)基因及位于转座子Tn5432中的ermX基因相关。盐酸小檗碱对耐药和敏感丙酸杆菌均有较好的抑制作用,其MIC值在1-8μg/ml,其不是通过抑制erm基因、逆转23SrRNA点突变介导抑制。透射电镜下观察经盐酸小檗碱孵育后,丙酸杆菌DNA结构破坏、胞核固缩空泡化,提示其可能通过整体抑制DNA发挥作用,是否通过抑制FtsZ蛋白或与DNA结合后诱发SOS反应抑制丙酸杆菌,有待在今后的研究中进一步探讨更多还原

【Abstract】 Abjective To evaluate the susceptibility test of propionibacteria and determine whether erythromycin resistant propionibacteria is due to the mutation in23S rRNA and Tn5432with ermX located plus two copies of IS1249flanked was transferred to propionibacteria. To determine the susceptibility test of propionibacteria to berberine.Methods Samples were collected from acne lesions of84acne patients. All samples were cultured in anaerobic conditions and identified by o sequencing of16s rRNA and23s rRNA respectively. The susceptibility test of propionibacteria isolated from acne patients to erythromycin and clindamycin was tested with E-test23S rRNA, ermX, ermX (cj), IS1249a and IS1249b was amplified and sequenced respectively. To determine the susceptibility test of propionibacteria to berberine with broth microdilution method. Real time PCR was evaluated to detect if erm was regulated by berberine.Results72Propionibacterium acnes and12Propionibacterium avidum were isolated respectively. Isolates resistant to erythromycin (29/84,34.52%) were observed and all isolates were susceptible to tetracycline. Isolates resistant to erythromycin were observed in P.acnes (26.39%,19/72) and P.avidum (83.33%,10/12) respectively.19P.acnes isolates and10Ravidum isolates were all high resistant to erythromycin.16P.acnes isolates were high resistant (MIC>256μg/ml) and3susceptible to clindaymcin respectively.10P.avidum isolates were all high resistant to erythromycin and clindaymcin (MIC>25μg/ml). In7P.acnes isolates, cross high resistant to erythromycin and clindamycin was associated with an A→G transition at a position cognate with Escherichia coli23S rRNA2058. These isolates were resistant to>256μg/ml of erythromycin and clindamycin respectively. In4P.anees isolates, high resistant to clindamycin and partly susceptible to clindamycin was associated with an A→G transition at a position cognate with E.coli23S rRNA2059. ermX gene located in Tn5432with two copies of IS1249was detected in the other8P.acnes isolates and2Ravidum isolates. These isolates were all high resistant to clindamycin and erythromycin. ermX(cj) gene was detected in the other8P.avidum isolates which shows99%homology with Corynebacterium jeikeium ermX (cj), while94%with P. acnes ermX. Berberine inhibited propionibacteria(MIC1-8μg/ml). The mutation of23S rRNA and erm gene weren’t regulated by berberine.Conclusion Antimicrobial resistance in propionibacteria in this population has a higher prevalence than those reported in Japan and Korea and follows a similar pattern to findings in Europe. No isolates were resistant to tetracycline. Clinical resistant propionibacteria to erythromycin is associated with an A→G transition at a position cognate with E.coli23S rRNA2058and2059respectively, erm located in Tn5432and ermX(cj) also account for the resistance. Berberine inhibited P.acnes, which provides a good choice for treatment of acne.更多还原