遗传
遺傳
유전
HEREDITAS(BEIJING)
2014年
6期
604-611
,共8页
张跃进%常清利%汪倩%卢俊婉%王欢%李佩珍%应俊%包其郁%胡云良
張躍進%常清利%汪倩%盧俊婉%王歡%李珮珍%應俊%包其鬱%鬍雲良
장약진%상청리%왕천%로준완%왕환%리패진%응준%포기욱%호운량
I类整合子%基因盒%克隆%耐药性
I類整閤子%基因盒%剋隆%耐藥性
I류정합자%기인합%극륭%내약성
class 1 integron%gene cassette%cloning%antibiotic resistance
为了探索细菌多重耐药性的产生和播散的分子机制,文章对2002~2007年间179株临床分离的肺炎克雷伯菌进行耐药性、I 类整合子可变区基因盒结构以及基因盒携带的耐药性基因进行分段克隆和耐药性功能测定。结果显示:65.9%(118/179)的肺炎克雷伯菌表现出对至少两种以上的抗生素(主要为β-内酰胺类、氨基糖苷类和喹诺酮类抗菌药物)的耐药性;36.3%(65/179)的菌株检出单条或者双条I类整合子基因盒条带;对整合子阳性组与阴性组的耐药率进行比较发现,除氨基糖苷类、喹诺酮类和复方新诺明等药物的耐药性存在显著性差异(P<0.01)外,其余药物的差异不显著;共发现15种耐药基因构成形式的整合子基因盒,其中以dfrA17-aadA5最为多见,实验证明整合子可由接合转移耐药性质粒携带;对整合子基因盒(dhfr17-orfF-aadA2)分段克隆的耐药性功能研究发现,3个克隆重组子(pET28a-dhfr17、pET28a-dhfr17-orfF和 pET28a-dhfr17-orfF-aadA2)对复方新诺明的抗性(MIC值)均为256μg/mL,重组子pET28a-dhfr17-orfF与重组子pET28a-dhfr17对链霉素的抗性无明显区别,和受体菌一样MIC值均为8μg/mL,而pET28a-dhfr17-orfF-aadA2对链霉素的抗性则明显提高, MIC值为256μg/mL。结果表明, I类整合子在肺炎克雷伯菌中较常见,携带氨基糖苷类和甲氧苄啶类的耐药基因盒在数量上占优势,且整合子携带的耐药基因具有耐药性功能,位于可水平转移耐药性质粒的耐药性基因相关的整合子对病原菌耐药性播散具有重要意义。
為瞭探索細菌多重耐藥性的產生和播散的分子機製,文章對2002~2007年間179株臨床分離的肺炎剋雷伯菌進行耐藥性、I 類整閤子可變區基因盒結構以及基因盒攜帶的耐藥性基因進行分段剋隆和耐藥性功能測定。結果顯示:65.9%(118/179)的肺炎剋雷伯菌錶現齣對至少兩種以上的抗生素(主要為β-內酰胺類、氨基糖苷類和喹諾酮類抗菌藥物)的耐藥性;36.3%(65/179)的菌株檢齣單條或者雙條I類整閤子基因盒條帶;對整閤子暘性組與陰性組的耐藥率進行比較髮現,除氨基糖苷類、喹諾酮類和複方新諾明等藥物的耐藥性存在顯著性差異(P<0.01)外,其餘藥物的差異不顯著;共髮現15種耐藥基因構成形式的整閤子基因盒,其中以dfrA17-aadA5最為多見,實驗證明整閤子可由接閤轉移耐藥性質粒攜帶;對整閤子基因盒(dhfr17-orfF-aadA2)分段剋隆的耐藥性功能研究髮現,3箇剋隆重組子(pET28a-dhfr17、pET28a-dhfr17-orfF和 pET28a-dhfr17-orfF-aadA2)對複方新諾明的抗性(MIC值)均為256μg/mL,重組子pET28a-dhfr17-orfF與重組子pET28a-dhfr17對鏈黴素的抗性無明顯區彆,和受體菌一樣MIC值均為8μg/mL,而pET28a-dhfr17-orfF-aadA2對鏈黴素的抗性則明顯提高, MIC值為256μg/mL。結果錶明, I類整閤子在肺炎剋雷伯菌中較常見,攜帶氨基糖苷類和甲氧芐啶類的耐藥基因盒在數量上佔優勢,且整閤子攜帶的耐藥基因具有耐藥性功能,位于可水平轉移耐藥性質粒的耐藥性基因相關的整閤子對病原菌耐藥性播散具有重要意義。
위료탐색세균다중내약성적산생화파산적분자궤제,문장대2002~2007년간179주림상분리적폐염극뢰백균진행내약성、I 류정합자가변구기인합결구이급기인합휴대적내약성기인진행분단극륭화내약성공능측정。결과현시:65.9%(118/179)적폐염극뢰백균표현출대지소량충이상적항생소(주요위β-내선알류、안기당감류화규낙동류항균약물)적내약성;36.3%(65/179)적균주검출단조혹자쌍조I류정합자기인합조대;대정합자양성조여음성조적내약솔진행비교발현,제안기당감류、규낙동류화복방신낙명등약물적내약성존재현저성차이(P<0.01)외,기여약물적차이불현저;공발현15충내약기인구성형식적정합자기인합,기중이dfrA17-aadA5최위다견,실험증명정합자가유접합전이내약성질립휴대;대정합자기인합(dhfr17-orfF-aadA2)분단극륭적내약성공능연구발현,3개극륭중조자(pET28a-dhfr17、pET28a-dhfr17-orfF화 pET28a-dhfr17-orfF-aadA2)대복방신낙명적항성(MIC치)균위256μg/mL,중조자pET28a-dhfr17-orfF여중조자pET28a-dhfr17대련매소적항성무명현구별,화수체균일양MIC치균위8μg/mL,이pET28a-dhfr17-orfF-aadA2대련매소적항성칙명현제고, MIC치위256μg/mL。결과표명, I류정합자재폐염극뢰백균중교상견,휴대안기당감류화갑양변정류적내약기인합재수량상점우세,차정합자휴대적내약기인구유내약성공능,위우가수평전이내약성질립적내약성기인상관적정합자대병원균내약성파산구유중요의의。
To investigate molecular mechanism of multi-resistance of Klebsiella pneumoniae and its spreading, 179 strains isolated from different clinical samples in the period of 2002-2007 with serious resistance to 14 anti-bacterial agents were examined. Among them, 118 (65.9%) were resistant to at least two anti-bacterial agents;36.3%(65/179) were found to contain class 1 integrons. There was a significant difference for resistance rate between the integron positive and the neg-ative groups, especially for antimicrobial agents of aminoglycosides, quinolones and sulfonamides (P<0.01). Gene cassette structures of the class 1 integrons in these bacteria were analyzed and their resistance genes were further cloned and tested for antibiotic resistance activities. Fifteen gene cassettes were identified with dfrA17-aadA5 being the most popular form. Three recombinant plasmids pET28a-dhfr17, pET28a-dhfr17-orfF and pET28a-dhfr17-orfF-aadA2 were cloned from a gene cassette of dhfr17-orfF-aadA2. When introduced into a recipient E. coli strain BL21, all of them rendered resistance to co-trimoxazole, with minimum inhibitory concentration (MIC) value up to 256 μg/μL. The E. coli BL21 carrying pET28a-dhfr17 or pET28a-dhfr17-orfF had the same MIC value of 8 μg/μL to streptomycin as the recipient strain without plasmid. However, the E. coli carrying pET28a-dhfr17-orfF-aadA2 was resistant to streptomycin with MIC level up to 256 μg/μL. In conclusion, class 1 integrons were regularly identified in Klebsiella pneumoniae. They mainly carry resistance genes against antimicrobial agents of aminoglycosides and sulfonamide. Transferable plasmid carrying integrons with resistance genes may play an important role in resistance spreading among bacterial species.