养殖鱼塘中铜绿假单胞菌致病性及耐药相关基因的研究

Pathogenicity and multidrug resistance of Pseudomonas aeruginosa isolated from fishery pond

旨在分析养殖鱼塘水体中铜绿假单胞菌(Pseudomonas aeruginosa,PA)分离菌株的致病性、耐药性及其可能机制,为保障水产食品的安全提供科学依据。使用美国临床与实验室标准研究所的标准纸片扩散法,以及聚合酶链反应技术对PA分离菌株进行了抗菌素耐药性,以及毒性、固有和获得性耐药相关基因的检测与分析。结果显示,受试PA分离菌株的50%为exoS+/exoU-侵染型分子型,无临床分离菌株的exoS-/exoU+的细胞毒型分子型。PA菌株对6大类9种抗菌素的耐药性存在明显差异,其中甲氧胺苄嘧啶和利福平的耐药率最高为100%,其次是氨苄西林、卡那霉素和四环素,分别为90%、90%和80%,庆大霉素的耐药率最低为10%。多药抗性PA菌株均含有MexAB-OprM、MexXY-OprM和MexVW-OprM外排泵系统,其中20%菌株检测为β-内酰胺酶基因(ampC)阳性;而MexEF-OprN、MexJK-OprM、MexCD-OprJ和MexGHI-OpmD外排泵基因全部或部分缺失。此外,在PA菌株中均未检测到Ⅰ～Ⅲ类整合子的整合酶基因(comINT),但是整合接合元件(ICEs)保守模块功能基因(ICEint、soj、pilS2、pilD)均检测为阳性,提示PA菌株携带的ICEs具有潜在的转移活性,为进一步探讨PA多药抗性的散播奠定了基础。

The aim of this study was to investigate virulence and drug resistant mechanism of multidrug resistant Pseudomonas aeruginosa（PA） isolated from water sample of fishery pond,in order to provide scientific support for practical aquaculture disease treatment,effective management of aquaculture environment and aquatic food safety control.Antimicrobial susceptibility of PA isolates under this study were examined by using standard Kirby Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute（USA,2010 Edition）,showing distinct susceptibility to nine antimicrobial agents belonging to six drug classes tested.All PA isolates were resistant to trimethoprim and rifampin,of which,90% were to ampicilin and kanamycin,and 10% were to gentamycin.The genes involved in pathogenicity,intrinsic and transferred antibiotic resistance were detected by polymerase chain reaction（PCR）.The toxA and exoU genes were absent from the PA isolates tested,50% of which were exoS＋/exoU-infectious genetype,and none of which were exoS-/exoU＋ cell toxic genetype that usually existed in clinical PA isolates.Subsequent analysis revealed that the multidrug efflux pumps including MexAB-OprM,MexXY-OprM and MexVW-OprM were present in all PA isolates,20% of which were positive for ampC gene,whereas the other four efflux pumps including MexEF-OprN,MexJK-OprM,MexCD-OprJ and MexGHI-OpmD were fully or partially deficient.In addition,the comINT gene of class Ⅰ-Ⅲ integrons was negative in the PA isolates,however,the functional genes（int,soj,pilS2,pilD）of conserved module structures of integrative conjugative elements（ICEs） were detected positive,suggesting possible transfer activity of the ICEs detected in the PA isolates.This study revealed the major mechanism under multidrug resistance of the PA isolates mediated by efflux pumps encoded by chromosome genes,and constituted the first evidence for mobile genetic element ICEs in PA isolates of environmental origin.