抗生素压力下不同血清型沙门氏菌耐药性研究

Study on Resistance in Salmonella with Different Serovar Types under Ciprofloxacin Pressures

本研究旨在探讨不同血清型沙门氏菌在环丙沙星抗生素压力下突变频率及在耐药发展过程中靶位基因突变、外排泵及调控基因表达的差异。选取临床分离的印第安纳型、肠炎型和鼠伤寒型沙门氏菌的敏感菌株,在环丙沙星压力下诱导耐药突变,分别获得一系列不同程度的耐药突变株。分别检测不同血清型沙门氏菌突变株的突变频率、靶位基因喹诺酮耐药决定区(QRDRs)和外排泵调控基因ramR-ramA突变及外排泵相关基因的表达水平;同时检测了母株在羰基氰化物间氯苯腙(CCCP)存在情况下环丙沙星药物的蓄积浓度,以确定母株是否存在外排泵的作用。结果表明,在环丙沙星压力下,印第安纳型沙门氏菌较肠炎型和鼠伤寒型有更高的突变频率,易获得耐药株;印第安纳血清型菌株耐药性的获得主要是由于靶位基因gyrA发生单突变,协同外排泵外排作用增强而获得高水平耐药;肠炎型沙门氏菌耐药性获得主要是由于靶位基因gyrA发生83和87位双位点突变,并随着gyrB和parC基因的多位点同时突变而获得高水平耐药,耐药性的发展过程中没有外排泵作用参与;而鼠伤寒沙门氏菌在抗生素压力下不易发展成耐药菌,耐药性发生主要是由于靶位基因gyrB发生突变,而伴随parC基因突变及微弱的外排泵作用导致耐药水平增加。

The objective of this study was to determine the mutation frequencies of three Salmonellastrains with different serotypes（Indiana,Enteritidis and Typhimurium）when selected under increasing ciprofloxacin pressures,as well the mutations in the quinolone resistance determining regions（QRDRs）in four target genes,mutations in ramA,ramR and ramA-ramR intergenic region,and the AcrAB-TolC efflux activity of the respective mutants of different serotypes during resistance development.PCR and DNA sequencing were applied to determine mutations within the above mentioned genes,and RT-PCR amplification of16 SrRNA,tolC,acrB,acrA and ramA were used to detect the activities of AcrAB-TolC of each mutants.Accumulation of ciprofloxacin in each Salmonella parent strains in the presence and absence of carbonyl cyanide 3-chlorophenylhydrazone（CCCP）was detected using HPLC to determine the presence of active efflux pump.The results showed that the Indiana serotype Salmonellastrain presented a much higher mutation frequency and was easily ready to develop resistance to ciprofloxacin comparing with Enteritidis and Typhimurium serotype strains.The development of resistance in Indiana strains were mainly due to the single mutation in gyrA,and further high level resistance were obtained with additional activated AcrAB-TolC pump.While the resistance to ciprofloxacin in Enteritidis strains was mainly resulted from the double mutations in amino 83 and 87in gyrA,and additional multiple mutations in gyrB and parC were contributed to the high level resistance in Enteritidis strains.However,Typhimurium strains were hard to development resistance under ciprofloxacin pressures.The development of resistance in Typhimurium strains in vitro was firstly derived from a single mutation in gyrB,and subsequently increasing resistance was obtained with additional mutation in parC and a weak AcrAB-TolC role.