餐饮从业人员耐喹诺酮类药物大肠埃希菌基因突变研究
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摘要
目的调查和分析餐饮从业人员体检人群分离的大肠埃希菌gyrA、gyrB、parC、parE基因的突变特征。方法用加入4μg/mL环丙沙星营养琼脂平板筛选耐环丙沙星大肠埃希菌耐药菌株,PCR方法扩增其gyrA、gyrB、parC、parE基因,琼脂稀释法测定环丙沙星的最小抑菌浓度(MIC)值。结果共分离到112株环丙沙星耐药大肠埃希菌,所有菌株的MIC值都介于16～512μg/mL之间。gyrA检测到3种突变类型:112株都出现Ser83→Leu替代,103株发生Asp87→Asn替代,7株菌出现Asp87→Tyr替代。gyrB检测到2种突变类型:6株菌出现Ser343→Phe替代,9株菌出现Ser492→Asn替代。parC共检测到7种突变类型:112株菌都发生Ser80→Ile代替,12株大肠埃希菌发生Glu84→Val代替,5株菌发生了Ala56→Thr替代,各有1株菌发生Glu84→Gly和Glu84→Lys代替,另外各有1株发生Ala108→Thr和Val144→Gly替代。parE基因共检测到2种突变类型:有2株菌发生了Leu445→His代替,49株菌发生Ser458→Ala代替。其中gyrA Ser83→Leu、Asp87→Asn、Asp87→Tyr替代和parC Ser80→Ile、Glu84→Val、Glu84→Gly、Glu84→Lys代替,为gyrA和parC喹诺酮耐药决定区(QRDR)突变引起的氨基酸替代类型。所有菌株都有2个及以上氨基酸位点的突变,都是高耐药菌株。环丙沙星的MIC值32μg/mL以上的耐药菌株都发生了3个以上位点的突变。Spearman相关系数显示,gyrB492、parE458的突变与环丙沙星的MIC值成正相关,差异有统计学意义(r=0.226 9,P=0.016 1;r=0.273 7,P=0.003 5)。结论餐饮从业人员耐喹诺酮药物的大肠杆菌gyrA、gyrB、parC、parE基因具有多种氨基酸替代类型,gyrB492、parE458的突变与环丙沙星的MIC值呈正相关。
Objective Fluoroquinolone resistance is mainly due to the DNA gyrase and topoisomerase IV mutation. This study investigated the mutation characteristics of the gyrA, gyrB, parC and parE genes of Escherichia coli strains isolated from food and beverage industry workers. Methods The nutrient agar with 4 μg/mL ciprofloxacin was used for the screening of ciprofloxacin-resistant E. coli strains from anal swabs. PCR assays were performed with the isolates to detect gyrA, gyrB,parC,and parE genes. The ciprofloxacin MIC values of isolates were determined by agar dilution method.Results A total of 112 ciprofloxacin-resistant E. coli strains were isolated, and the MIC values of all the strains ranged from 16 to 512 μg/mL. In these isolates, 3 amino acid sites of gyrA for ciprofloxacin-resistance were found changing from Ser83 to Leu( 112 in 112), from Asp87 to Asn( 103/112) and from Asp87 to Tyr(7/112), while 2 amino acid sites of gyrB have been found changing from Ser343 to Phe( 6 in 112) and from Ser492 to Asn( 9 in 112). The parC gene exhibits a high diversity of 7 amino acid substitutions that Ser80 was substituted by Ile in all 112 isolates, while Glu84 by Val in 14 isolates, Ala56 by Thr in 5 isolates, and Glu84 by Gly, Glu84 by Lys, Ala108 by Thr and Val144 by Gly only in 1 isolate,respectively. Two amino acid substitution were found in parE that the Leu445 was substituted by His in 2 isolates while Ser458 by Ala in 49 isolates. The substitutions of Ser83 to Leu, Asp87 to Asn, Asp87 to Tyr in gyrA and parC Ser80 to Ile,Glu84 to Val, Glu84 to Gly, Glu84 to Lys in the quinolone resistance determinant region(QRDR). All the ciprofloxacinresistant isolates had two or more amino acid substitutions. And the isolates with the MIC value ≥32 μg/mL had at least 3 amino acid substitutions. Spearman correlation coefficient showed that the mutations of Ser492 in gyrB and Ser458 in parE were highly correlated with the MIC values of ciprofloxacin(r=0.2269, P=0.0161; r=0.2737, P=0.0035). Conclusions A variety of amino acid changes in gyrA, gyrB, parC and parE were detected in the fluoroquinolone-resistant E. coli isolates from catering staffs. The mutation of gyrB492 and parE458 was positively correlated with the MIC value of ciprofloxacin,and the difference was statistically significant.
Objective Fluoroquinolone resistance is mainly due to the DNA gyrase and topoisomerase IV mutation. This study investigated the mutation characteristics of the gyrA, gyrB, parC and parE genes of Escherichia coli strains isolated from food and beverage industry workers. Methods The nutrient agar with 4 μg/mL ciprofloxacin was used for the screening of ciprofloxacin-resistant E. coli strains from anal swabs. PCR assays were performed with the isolates to detect gyrA, gyrB,parC,and parE genes. The ciprofloxacin MIC values of isolates were determined by agar dilution method.Results A total of 112 ciprofloxacin-resistant E. coli strains were isolated, and the MIC values of all the strains ranged from 16 to 512 μg/mL. In these isolates, 3 amino acid sites of gyrA for ciprofloxacin-resistance were found changing from Ser83 to Leu( 112 in 112), from Asp87 to Asn( 103/112) and from Asp87 to Tyr(7/112), while 2 amino acid sites of gyrB have been found changing from Ser343 to Phe( 6 in 112) and from Ser492 to Asn( 9 in 112). The parC gene exhibits a high diversity of 7 amino acid substitutions that Ser80 was substituted by Ile in all 112 isolates, while Glu84 by Val in 14 isolates, Ala56 by Thr in 5 isolates, and Glu84 by Gly, Glu84 by Lys, Ala108 by Thr and Val144 by Gly only in 1 isolate,respectively. Two amino acid substitution were found in parE that the Leu445 was substituted by His in 2 isolates while Ser458 by Ala in 49 isolates. The substitutions of Ser83 to Leu, Asp87 to Asn, Asp87 to Tyr in gyrA and parC Ser80 to Ile,Glu84 to Val, Glu84 to Gly, Glu84 to Lys in the quinolone resistance determinant region(QRDR). All the ciprofloxacinresistant isolates had two or more amino acid substitutions. And the isolates with the MIC value ≥32 μg/mL had at least 3 amino acid substitutions. Spearman correlation coefficient showed that the mutations of Ser492 in gyrB and Ser458 in parE were highly correlated with the MIC values of ciprofloxacin(r=0.2269, P=0.0161; r=0.2737, P=0.0035). Conclusions A variety of amino acid changes in gyrA, gyrB, parC and parE were detected in the fluoroquinolone-resistant E. coli isolates from catering staffs. The mutation of gyrB492 and parE458 was positively correlated with the MIC value of ciprofloxacin,and the difference was statistically significant.
引文
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[10]Korona-Glowniak I,Skrzypek K,Siwiec R,et al.Fluoroquinolone-resistance mechanisms and phylogenetic background of clinical Escherichia coli strains isolated in south-east Poland[J].New Microbiol,2016,39(3):210-215.
[11]Fu Y,Zhang W,Wang H,et al.Specific patterns of gyrAmutations determine the resistance difference to ciprofloxacin and levofloxacin in Klebsiella pneumoniae and Escherichia coli[J].BMC Infect Dis,2013,13:8.
[12]R?derova M,Halova D,Papousek I,et al.Characteristics of quinolone resistance in Escherichia coli Isolates from humans,animals,and the environment in the Czech Republic[J].Front Microbiol,2017,7:2147.
[13]Sorlozano A,Gutierrez J,Jimenez A,et al.Contribution of a new mutation in parE to quinolone resistance in extended-spectrumbeta-lactamase-producing Escherichia coli isolates[J].J Clin Microbiol,2007,45(8):2740-2742.
[2]龙永艳,吴葵,傅松哲,等.质粒介导的喹诺酮耐药细菌的耐药机制[J].热带医学杂志,2018,18(1):122-126,138.
[3]Zhang QQ,Ying GG,Pan CG,et al.Comprehensive Evaluation of Antibiotics Emission and Fate in the River Basins of China:Source Analysis,Multimedia Modeling,and Linkage to Bacterial Resistance[J].Environ Sci Technol,2015,49(11):6772-6782.
[4]Fàbrega A,Madurga S,Giralt E,et al.Mechanism of action of and resistance to quinolones[J].Microb Biotechnol,2009,2(1):40-61.
[5]戚文丰.妇科住院患者分离的大肠埃希菌氟喹诺酮耐药机制与MIC值分布关系的研究[J].中国卫生检验杂志,2015,25(14):2436-2439.
[6]李维正,孙琳,陈蓓,等.上海市一区级医院老年患者尿路致病性大肠埃希菌分子分型和耐药性分析[J].中华传染病杂志,2016,34(6):337-343.
[7]桓新,马颖,冯彩丽.不同人群大肠埃希菌耐药性变迁趋势分析[J].临床合理用药杂志,2018,11(2):95-96.
[8]Hu Y,Yang X,Qin J,et al.Metagenome-wide analysis of antibiotic resistance genes in a large cohort of human gut microbiota[J].Nat Commun,2013,4:2151.
[9]Balakrishnan S,Antony PX,Mukhopadhyay HK,et al.Genetic characterization of fluoroquinolone-resistant Escherichia coli associated with bovine mastitis in India[J].Vet World,2016,9(7):705-709.
[10]Korona-Glowniak I,Skrzypek K,Siwiec R,et al.Fluoroquinolone-resistance mechanisms and phylogenetic background of clinical Escherichia coli strains isolated in south-east Poland[J].New Microbiol,2016,39(3):210-215.
[11]Fu Y,Zhang W,Wang H,et al.Specific patterns of gyrAmutations determine the resistance difference to ciprofloxacin and levofloxacin in Klebsiella pneumoniae and Escherichia coli[J].BMC Infect Dis,2013,13:8.
[12]R?derova M,Halova D,Papousek I,et al.Characteristics of quinolone resistance in Escherichia coli Isolates from humans,animals,and the environment in the Czech Republic[J].Front Microbiol,2017,7:2147.
[13]Sorlozano A,Gutierrez J,Jimenez A,et al.Contribution of a new mutation in parE to quinolone resistance in extended-spectrumbeta-lactamase-producing Escherichia coli isolates[J].J Clin Microbiol,2007,45(8):2740-2742.
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