CAJ | 학술논문

目的分析铜绿假单胞菌在耐药性变化时菌株Ⅲ型分泌系统(T3SS)毒力相关蛋白的表达差异,结合患者的临床资料,探讨耐药性变化与细菌毒力变化的相关性.方法筛选源于同一患者呼吸道、多次分离、药敏谱发生动态改变的铜绿假单胞菌,用肠杆菌基因间重复一致序列PCR(ERIC-PCR)方法确定菌株的同源性,以同一克隆的菌株体系为研究对象,Kirby-Bauer纸片法检测菌株对药物的敏感性,PCR方法检测T3SS系统和菌株的毒力基因型,双向蛋白电泳比较菌株的全菌蛋白表达谱,质谱分析差异蛋白点,并在蛋白质数据库进行检索.收集患者临床信息,了解临床特征、细菌耐药性、毒力相关蛋白三者间的关系.结果筛选出1例慢性阻塞性肺疾病急性加重期(AECOPD)合并肺炎的患者符合入选条件,其1年内3次呼吸道分离出铜绿假单胞菌,药敏谱依次为:敏感→多重耐药(MDR)→泛耐药(PDR).3株菌株毒力基因属exo U+/exo S-型,经ERIC-PCR证实为同一克隆.双向凝胶电泳显示3株菌有21个差异表达蛋白.质谱分析发现其中11个蛋白功能明确(9个与细菌基础代谢有关).仅毒力相关蛋白二硫化物氧化还原酶A(DsbA)在PDR株时表达明显上调,在敏感株表达最低.临床信息显示该患者在分离出PDR时,肺部感染最重,并于1个月后死亡.结论在长期定植、感染过程中,生存环境的变化可能导致铜绿假单胞菌的耐药表型发生变化,同时菌株毒力加强,共同导致感染难于控制.DsbA在铜绿假单胞菌毒力变化中可能发挥了重要的作用.
목적 분석동록가단포균재내약성변화시균주Ⅲ형분비계통(T3SS)독력상관단백적표체차이,결합환자적림상자료,탐토내약성변화여세균독력변화적상관성.방법 사선원우동일환자호흡도、다차분리、약민보발생동태개변적동록가단포균,용장간균기인간중복일치서렬PCR(ERIC-PCR)방법학정균주적동원성,이동일극륭적균주체계위연구대상,Kirby-Bauer지편법검측균주대약물적민감성,PCR방법검측T3SS계통화균주적독력기인형,쌍향단백전영비교균주적전균단백표체보,질보분석차이단백점,병재단백질수거고진행검색.수집환자림상신식,료해림상특정、세균내약성、독력상관단백삼자간적관계.결과 사선출1례만성조새성폐질병급성가중기(AECOPD)합병폐염적환자부합입선조건,기1년내3차호흡도분리출동록가단포균,약민보의차위:민감→다중내약(MDR)→범내약(PDR).3주균주독력기인속exo U+/exo S-형,경ERIC-PCR증실위동일극륭.쌍향응효전영현시3주균유21개차이표체단백.질보분석발현기중11개단백공능명학(9개여세균기출대사유관).부독력상관단백이류화물양화환원매A(DsbA)재PDR주시표체명현상조,재민감주표체최저.림상신식현시해환자재분리출PDR시,폐부감염최중,병우1개월후사망.결론 재장기정식、감염과정중,생존배경적변화가능도치동록가단포균적내약표형발생변화,동시균주독력가강,공동도치감염난우공제.DsbA재동록가단포균독력변화중가능발휘료중요적작용.
Objective To analyze the difference of virulence-related protein concerned with type Ⅲ secretion system (T3SS) in Pseudomonas aeruginosa during the changes of antibiotic sensitivity and interpret the clinical patient data to explore the relationship between the changes in resistance and variance of virulence. Methods The isolates of Pseudomonas aeruginosa was isolated from the respiratory tract of a same patient with an altered sensitivity of antibiotics. It turned out to be one clone. The homolog of isolates was determined by ERIC-PCR. The Kirby-Bauer antibiotic testing was employed to detect the sensitivity of antibiotics of isolates. PCR was used to detect the gene of T3SS and virulence of isolates and two-dimensional gel electrophoresis to compare the whole-cell proteins. The mass spectrometry was employed to analyze a variety of protein spots. The relevant information was retrieved from protein databases. Clinical record was collected to study the relationship of clinical features, bacteria resistance and virulence-asseciated protein. Results One subject was diagnosed with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated with pneumonia. Psendomonas aeruginosa was isolated from his respiratory tract three times in one year. Sensitivity spectrum of isolates were as follows: sensitivity, multi-drug resistant (MDR) and pan-drug resistance (PDR). Virulence gene was exo U +/exo S -. Twenty-one differentially expressed proteins were revealed by two-dimensional gel electrophoresis in three isolates. The functions of 11 proteins were definite. Only 9 proteins were associated with basal bacterial metabolism. Disulfide oxidoreductase A (DsbA) corresponded to the variation of virulence and sensitivity spectrum. Clinical record revealed that the severe lung infection was caused by the PDR strain and the patient died within one month. Conclusions The sensitivity spectrum and virulence of Pseudomonas aerug/nosa may undergo changes when there is an alteration of eco-environment during colonization and infection over a long period of time. DsbA plays a key role in the variety of virulence.