外排泵抑制剂对海水养殖源弧菌酰胺醇类药物耐药性的影响

Effects of efflux pump inhibitors on the amphenicols resistance of Vibrio strains isolated from marine aquaculture

通过检测从海水养殖中分离的对氟苯尼考低敏感弧菌菌株中常见酰胺醇耐药基因的携带情况,来分析外排泵抑制剂对弧菌酰胺醇耐药性的影响,旨在研究主动外排机制在我国海水养殖源弧菌耐药中的作用。本研究从前期分离的海水养殖源弧菌中挑选出41株对氟苯尼考低敏感的菌株,运用琼脂稀释法测定了这些菌株对酰胺醇类药物(氯霉素和氟苯尼考)的敏感性;利用PCR方法检测了这些菌株的酰胺醇外排泵耐药基因和整合子的携带情况,包括外排泵基因floR、cmlA、pexA、fexA、fexB和optrA,以及Int1、SXT和ISCR1整合子相关基因;同时研究4种外排泵抑制剂对弧菌氯霉素、氟苯尼考最小抑菌浓度(MIC)的影响,利用琼脂稀释法检测41株弧菌分别加入甲基吡咯烷酮(N-methyl-2-pyrrolidone,NMP)、利血平(reserpine)、羰基氰氯苯腙(carbonyl cyanide m-chlorophenyl hydrazone,CCCP)和苯丙氨酸-精氨酸-β萘酰胺(Phe-Arg-β-naphthylamide,PAβN)4种外排泵抑制剂前后对氯霉素和氟苯尼考的MIC值的变化情况。副溶血弧菌(14株)、溶藻弧菌(17株)、哈维氏弧菌(8株)、创伤弧菌(2株)共41株氟苯尼考低敏感弧菌敏感度检测结果显示,氟苯尼考耐药菌株39株、氯霉素耐药菌株11株。41株氟苯尼考低敏感弧菌菌株中,酰胺醇类外排泵基因floR检出率为100%,而cmlA的检出率仅为17.1%,其他4个外排泵基因均未检出;整合子相关基因Int1、SXT和ISCR1的检出率分别为46.3%(19/41)、46.3%(19/41)和63.4%(26/41)。41株弧菌中,添加NMP、利血平、CCCP和PAβN后,分别有0株、2株、10株和2株弧菌为氯霉素外排阳性菌;分别有1株、1株、15株和3株弧菌为氟苯尼考外排阳性菌。研究表明,CCCP被验证可以作为有效治疗酰胺醇类药物耐药弧菌的外排泵抑制剂。

This study was conducted to determine the role of efflux pump inhibitors in amphenicols resistance of Vibrio species from mariculture.A total of 41 intermediate/high-level florfenicol-resistant Vibrio isolates were tested,including V.parahaemolyticus（14）,V.alginolyticus（17）,V.harveyi（8）,and V.vulnificus（2）.To investigate the effect of efflux pump on amphenicols resistance,minimal inhibitory concentrations of chloramphenicol and florfenicol with/without efflux pump inhibitors were determined using the agar dilution method.NMP,reserpine,CCCP and PAβN were used as efflux pump inhibitors in this study.PCR amplication method was used to detect the efflux pump genes and integron-related genes.The results showed that 39 strains and 11 strains were resistant to florfenicol and chloramphenicol,respectively.As is shown,100%（41/41）of the 41 Vibrio isolates tested were detected positive for amphenicols efflux pump genes flo R,but only 17.1%（7/41）for cml A gene,and pex A,fex A,fex B and optr A could not be detected.Furthermore,Int1,SXT,and ISCR1 were found in 46.3%（19/41）,46.3%（19/41）,and 63.4%（26/41）of the 41 Vibrio isolates tested,respectively.Antibiotic susceptibility testing suggested that CCCP was an effective efflux pump inhibitor in the treatment for the amphenicol resistance of Vibrio species.The prevalence of amphenicol resistance,efflux pump genes and integron-related elements in Vibrio sp.derived from marine aquaculture in China was observed and was likely due to the extensive use of amphencols.The use of efflux pumb inhibitors,e.g.CCCP,might be an effective tool to control the amphenicol resistance in Vibrio species.