中华临床感染病杂志
中華臨床感染病雜誌
중화림상감염병잡지
CHINESE JOURNAL OF CLINICAL INFECTIOUS DISEASES
2014年
5期
387-392
,共6页
沈晓强%陈琼%周华%蒋琰%俞云松
瀋曉彊%陳瓊%週華%蔣琰%俞雲鬆
침효강%진경%주화%장염%유운송
鲍氏不动杆菌%抗药性,多药%耐药结节细胞分化超家族%替加环素
鮑氏不動桿菌%抗藥性,多藥%耐藥結節細胞分化超傢族%替加環素
포씨불동간균%항약성,다약%내약결절세포분화초가족%체가배소
Acinetobacter baumannii%Drug resistance,multiple%Resistance nodulation division%Tigecycline
目的 研究碳青霉烯类耐药鲍曼不动杆菌中耐药结节细胞分化超家族(RND)外排泵介导菌株对替加环素敏感性降低的机制.方法 连续收集2010年7个省市16家医院鲍曼不动杆菌631株.采用PCR检测oxa-51和oxa-23基因,并对菌株进行多位点序列分型(MLST).用纸片药敏法检测β-内酰胺类、氨基糖苷类、大环内酯类、四环素类、碳青霉烯类抗生素、替加环素和多黏菌素的抑菌圈直径.对替加环素抑菌圈直径≤12 mm的菌株采用E-test法检测替加环素最小抑菌浓度(MIC).使用外排泵抑制剂1-(1-萘甲基)哌嗪(NMP)检测操纵子基因中adeB、adeG和adeJ的表达,确定外排泵抑制剂抑制表型的分布.选取对替加环素和碳青霉烯类均耐药,且具有外排泵抑制剂抑制表型的菌株作为实验组;对替加环素敏感而对碳青霉烯类耐药的菌株作为对照组,并以对替加环素敏感的ATCC1 9606菌株作为标准菌株.采用实时荧光定量PCR (qPCR)检测实验组和对照组外排泵adeABC、adeFGH和adelJK的转录水平并进行比较.采用PCR法对adeR、adeS和adeL进行全基因长度测序,查找突变位点或插入序列.结果 631株鲍曼不动杆菌中,共筛选出32株对替加环素敏感性降低的碳青霉烯类耐药鲍曼不动杆菌,其中8株具有外排泵抑制剂抑制表型.同时选取对替加环素敏感而对碳青霉烯类耐药的4株作为对照组.实验组A518、Z1219和A527菌株adeABC表达明显增高,分别是标准株ATCC19606的13,5和7倍;adeFGH和adeIJK表达量在部分菌株中稍有上调.对照组A207和A1731菌株在转录水平未检测到adeABC,其他菌株adeABC、adeFGH和adeIJK表达没有上调.在adeR和adeS分别发现多态性位点E220K和A130D.在adeABC高表达菌株中发现了adeS的ISAbal插入突变.adeL中未发现突变位点.结论 adeABC高表达在碳青霉烯类耐药鲍曼不动杆菌替加环素耐药机制中发挥重要作用,主要是调控基因adeS中存在ISAbal插入突变导致adeABC高表达所致,但不排除其他替加环素耐药机制的存在.
目的 研究碳青黴烯類耐藥鮑曼不動桿菌中耐藥結節細胞分化超傢族(RND)外排泵介導菌株對替加環素敏感性降低的機製.方法 連續收集2010年7箇省市16傢醫院鮑曼不動桿菌631株.採用PCR檢測oxa-51和oxa-23基因,併對菌株進行多位點序列分型(MLST).用紙片藥敏法檢測β-內酰胺類、氨基糖苷類、大環內酯類、四環素類、碳青黴烯類抗生素、替加環素和多黏菌素的抑菌圈直徑.對替加環素抑菌圈直徑≤12 mm的菌株採用E-test法檢測替加環素最小抑菌濃度(MIC).使用外排泵抑製劑1-(1-萘甲基)哌嗪(NMP)檢測操縱子基因中adeB、adeG和adeJ的錶達,確定外排泵抑製劑抑製錶型的分佈.選取對替加環素和碳青黴烯類均耐藥,且具有外排泵抑製劑抑製錶型的菌株作為實驗組;對替加環素敏感而對碳青黴烯類耐藥的菌株作為對照組,併以對替加環素敏感的ATCC1 9606菌株作為標準菌株.採用實時熒光定量PCR (qPCR)檢測實驗組和對照組外排泵adeABC、adeFGH和adelJK的轉錄水平併進行比較.採用PCR法對adeR、adeS和adeL進行全基因長度測序,查找突變位點或插入序列.結果 631株鮑曼不動桿菌中,共篩選齣32株對替加環素敏感性降低的碳青黴烯類耐藥鮑曼不動桿菌,其中8株具有外排泵抑製劑抑製錶型.同時選取對替加環素敏感而對碳青黴烯類耐藥的4株作為對照組.實驗組A518、Z1219和A527菌株adeABC錶達明顯增高,分彆是標準株ATCC19606的13,5和7倍;adeFGH和adeIJK錶達量在部分菌株中稍有上調.對照組A207和A1731菌株在轉錄水平未檢測到adeABC,其他菌株adeABC、adeFGH和adeIJK錶達沒有上調.在adeR和adeS分彆髮現多態性位點E220K和A130D.在adeABC高錶達菌株中髮現瞭adeS的ISAbal插入突變.adeL中未髮現突變位點.結論 adeABC高錶達在碳青黴烯類耐藥鮑曼不動桿菌替加環素耐藥機製中髮揮重要作用,主要是調控基因adeS中存在ISAbal插入突變導緻adeABC高錶達所緻,但不排除其他替加環素耐藥機製的存在.
목적 연구탄청매희류내약포만불동간균중내약결절세포분화초가족(RND)외배빙개도균주대체가배소민감성강저적궤제.방법 련속수집2010년7개성시16가의원포만불동간균631주.채용PCR검측oxa-51화oxa-23기인,병대균주진행다위점서렬분형(MLST).용지편약민법검측β-내선알류、안기당감류、대배내지류、사배소류、탄청매희류항생소、체가배소화다점균소적억균권직경.대체가배소억균권직경≤12 mm적균주채용E-test법검측체가배소최소억균농도(MIC).사용외배빙억제제1-(1-내갑기)고진(NMP)검측조종자기인중adeB、adeG화adeJ적표체,학정외배빙억제제억제표형적분포.선취대체가배소화탄청매희류균내약,차구유외배빙억제제억제표형적균주작위실험조;대체가배소민감이대탄청매희류내약적균주작위대조조,병이대체가배소민감적ATCC1 9606균주작위표준균주.채용실시형광정량PCR (qPCR)검측실험조화대조조외배빙adeABC、adeFGH화adelJK적전록수평병진행비교.채용PCR법대adeR、adeS화adeL진행전기인장도측서,사조돌변위점혹삽입서렬.결과 631주포만불동간균중,공사선출32주대체가배소민감성강저적탄청매희류내약포만불동간균,기중8주구유외배빙억제제억제표형.동시선취대체가배소민감이대탄청매희류내약적4주작위대조조.실험조A518、Z1219화A527균주adeABC표체명현증고,분별시표준주ATCC19606적13,5화7배;adeFGH화adeIJK표체량재부분균주중초유상조.대조조A207화A1731균주재전록수평미검측도adeABC,기타균주adeABC、adeFGH화adeIJK표체몰유상조.재adeR화adeS분별발현다태성위점E220K화A130D.재adeABC고표체균주중발현료adeS적ISAbal삽입돌변.adeL중미발현돌변위점.결론 adeABC고표체재탄청매희류내약포만불동간균체가배소내약궤제중발휘중요작용,주요시조공기인adeS중존재ISAbal삽입돌변도치adeABC고표체소치,단불배제기타체가배소내약궤제적존재.
Objective To investigate the effect of resistance nodulation division (RND) efflux pumps on reduced susceptibility to tigecycline in carbapenem-resistant Acinetobacter baumanii.Methods Totally 631 isolates of Acinetobacter baumanii were collected from 16 hospitals in 7 provinces in 2010.Genes oxa-51 and oxa-23 were detected by PCR method,and the ST profiles were determined by multilocus sequence typing (MLST).The disk susceptibility assay was used to determine the inhibition zone diameters of β-lactams,aminoglycosides,macrolides,tetracyclines,carbapenems,tigecycline and polymyxin.The minimum inhibitory concentration (MIC) of tigecycline was determined by E-test in strains with inhibition zone diameters ≤ 12 mm on tigecycline.The expression of operon genes adeB,adeG and adeJ was determined with efflux pump inhibitor NMP (N-methyl-2-pyrrolidone) for detection of efflux pump inhibitor phenotype.The isolates of Acinetobacter baumanii which were resistant both to tigecycline and carbapenems and with the inhibited phenotype of efflux pump inhibitor were collected as the experiment group,the isolates which were susceptible to tigecycline but resistant to carbapenems were collected as the control group,and ATCC 19606 was used as the reference strain.The expressions of adeABC,adeFGH and adeIJK were quantified by q-PCR at the transcriptional level.Genes adeR,adeS and adeL were amplified and sequenced using PCR method to find polymorphic locus and insertion sequences.Results There were 32 isolates of Acinetobacter baumanii with reduced susceptibility to tigecycline and carbapenem-resistant.Eight isolates were with the inhibited phenotype by efflux pump inhibitor.And 4 strains which were susceptible to tigecycline but resistant to carbapenems were selected as the control.The expressions of adeABC in A518,Z1219 and A527 of experiment group were 13-fold,5-fold and 7-fold higher than reference strain ATCC19606,respectively.The expressions of adeFGH and adeIJK were up-regulated slightly in some isolates.Transcript of adeABC was not found in control group strains A207 and A1731,and the expressions of adeABC,adeFGH and adeIJK were not up-regulated in other isolates.The single nucleotide polymorphism (SNP) was detected in adeR (E220K) and adeS (A130D),respectively.ISAab1 insertion sequence was identified in adeS of adeABC-over expressed isolates.No mutation was found in adeL.Conclusion High expression of adeABC pump may play an important role in tigecycline resistance in carbapenem-resistant Acinetobacter baumannii,mainly due to the insertion of ISAba1 sequence in its regulator gene adeS,but other mechanism of tigecycline resistance may not be excluded.
