沙门菌传代中(氟)喹诺酮类抗生素筛选株药敏性及相关基因突变和表达
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(1.西北农林科技大学食品科学与工程学院,陕西 杨凌 712100;2.中国食品药品检定研究院, 北京 102629;3.国家食品安全风险评估中心,北京 100021)

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牛沁雅 女 硕士生 研究方向为食品安全和食源性致病菌防控 E-mail:nqy736461094@163.com 杨保伟 男 教授 研究方向为食品安全和致病菌防控 E-mail:ybw090925@163.com

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国家自然科学基金(31671956)


Antimicrobial susceptibility, variation and relative expression of relative genes of Salmonella screened from different quinolone and fluoroquinolones
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(1.College of Food Science and Engineering, Northwest A&F University, Shaanxi Yangling 712100, China;2.National Institute for Food and Drug Control, Beijing 102629, China;3.China National Center for Food Safety Risk Assessment, Beijing 100021, China)

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    摘要:

    目的 研究在不同浓度(氟)喹诺酮类抗生素存在条件下,沙门菌在传代时得到的筛选株中与耐药性相关部分基因的变异和表达水平变化规律及其与耐药性间的关联性。方法 使用含有一定浓度(氟)喹诺酮类抗生素的肉汤培养基对沙门菌进行培养,在含有相同浓度同类抗生素的平板上划线筛选突变株,微量肉汤稀释法测定筛选株的药敏性,聚合酶链式反应(PCR)结合DNA测序确定喹诺酮耐药决定区(quinolone resistance-determining region,QRDR)基因突变,实时荧光定量PCR(real-time qPCR)法检测外排泵AcrAB-TolC编码基因表达水平。结果 沙门菌(ATCC 14028s)在含有不同代、不同浓度(氟)喹诺酮类抗生素的LB培养基中培养、筛选后,筛选株对抗生素耐受性均有不同程度增加。萘啶酮酸第5~7代筛选株gyrA突变,发生Asp87Tyr变异;环丙沙星第4~7代筛选株gyrA突变,发生Asp87Asn变异;加替沙星、左氧氟沙星和德拉沙星第1~7代筛选株gyrA中均未检出核苷酸突变。随着抗生素浓度增加,各抗生素相应筛选株中外排泵AcrAB-TolC编码基因表达水平较原始菌株增加,差异有统计学意义(P<0.05),第7代菌株acrAB-tolC表达量间差异无统计学意义(P>0.05)。(氟)喹诺酮类抗生素对不同代筛选株的最小抑菌浓度(MIC)与其对沙门菌(ATCC 14028s)的MIC值比值、gyrA突变、acrAB-tolC表达水平与抗生素作用浓度和筛选代数间呈正相关。结论 在(氟)喹诺酮类抗生素作用下,沙门菌可通过QRDR基因突变及增加acrAB-tolC表达适应抗生素胁迫环境;新一代(氟)喹诺酮类抗生素作用于沙门菌时,菌株QRDR靶位点突变概率降低;多次重复作用后,菌株acrAB-tolC表达量虽然增加,但表达量间差异无统计学意义(P>0.05),避免了因突变导致耐药性的进一步增强。

    Abstract:

    Objective To study the mutation and expression of genes of Salmonella when screened by different concentrations of quinolone and fluoroquinolones during propagation and their relation with antibiotic resistance. MethodsSalmonella strain was cultured in broth medium and screened on nutrition plate with different concentration of quinolone and fluoroquinolones. Antimicrobial susceptibility of the screened subculture was tested by broth microdilution method, mutation of genes in quinolone resistance-determining region (QRDR) was detected using polymerase chain reaction(PCR)and DNA sequencing method, and expression level of the encoding genes of multi-drug associated efflux pump AcrAB-TolC was detected by real-time qPCR. Results Antibiotic resistance level of the subcultures screened from LB plate with quinolone and fluoroquinolones inducement increased in different extents. Mutation of Asp87Tyr in gyrA in QRDR was detected from the fifth to the seventh generation of nalidixic acid screened strains. Mutation of Asp87Asn in gyrA in QRDR was detected from the fourth to the seventh generation of ciprofloxacin screened strains. No amino acid mutation was detected from gyrA in the first to the seventh generation of gatifloxacin, levofloxacin and delafloxacin screened strains. Compared to the expression level of the multi-drug efflux pump AcrAB-TolC encoding genes of the original strain, those of the screened strains had significantly (P<0.05) increased resistance. No significant difference was detected among the expression level of AcrAB-TolC encoding genes in the seventh generation of screened strains. The ratio of the minimal inhibitory concentrations (MICs) of the screened strains and that of Salmonella Typhimurium(ATCC 14028s), gene variation and relative expression level of acrAB-tolC of Salmonella significantly positive correlated with subculture generation and antibiotic concentration. Conclusion Under the selective pressure of antibiotics, Salmonella strain could adapt the stress environment through QRDR mutation and increase the expression level of multi-drug efflux pump AcrAB-TolC. When the next generation fluoroquinolone was used, the mutation frequency of QRDR decreased. After subcultured several times, the expression level of acrAB-tolC of the screened strains increased, however, no significant difference was detected among the expression level, which avoided the antibiotic resistance of Salmonella to be further increased.

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牛沁雅,李珂婷,曹晨阳,盛焕精,李伟,崔生辉,李凤琴,杨保伟.沙门菌传代中(氟)喹诺酮类抗生素筛选株药敏性及相关基因突变和表达[J].中国食品卫生杂志,2020,32(5):484-492.

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  • 收稿日期:2020-09-18
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  • 在线发布日期: 2020-11-18
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