河曲马源大肠杆菌耐药相关基因检测及外排泵抑制剂对其生物被膜的影响

doi:10.16431/j.cnki.1671-7236.2024.08.037

摘要/Abstract

摘要： 【目的】了解河曲马源大肠杆菌的耐药情况,为兽医临床合理使用抗菌药物提供基础理论指导。【方法】采用细菌形态学、革兰染色镜检、16S rDNA序列分析等方法进行细菌分离鉴定。采用微量肉汤稀释法和PCR扩增方法对分离鉴定的大肠杆菌进行抗菌药物敏感性试验及耐药基因、整合酶基因、外排泵基因携带情况检测。同时,采用改良结晶紫半定量方法进行生物被膜形成能力测定。利用棋盘法测定13种外排泵抑制剂与多西环素联用对生物被膜的清除作用。【结果】分离株在伊红-美蓝琼脂培养基上为具有绿色金属光泽的紫黑色菌落,在大肠杆菌/大肠菌群显色培养基上为蓝色菌落,革兰染色镜检可见粉红色短杆状细菌。经16S rDNA测序比对与大肠杆菌高度相似的分离株为64株。64株大肠杆菌分离株对酰氨醇类、磺胺类及利福霉素类药物耐药率相对较高并出现多重耐药菌株,其中20.31%(13/64)表现出对5类抗菌药物耐药;检测到21个耐药基因、1个整合酶基因及6个外排泵基因为阳性;整合酶基因intl1检出率为89.06%。生物被膜形成能力为强、中、弱及无成膜能力的大肠杆菌分别为8(12.50%)、34(53.13%)、20(31.25%)和2(3.12%)株。外排泵抑制剂与多西环素联合能增强对生物被膜的清除能力。【结论】河曲马源大肠杆菌耐药较为严重,应进一步加强青海省河南蒙古族自治县赛马的用药监管力度,减少或避免多重耐药菌株的出现及耐药性广泛传播。

关键词: 河曲马; 大肠杆菌; 多重耐药性; 生物被膜; 外排泵

Abstract: 【Objective】 The experiment was conducted to understand the resistance situation of Escherichia coli (E. coli) isolated from Hequ horses and provide theoretical guidance for rational use of antibacterial agents in veterinary medicine.【Method】 Bacterial isolation and identification were carried out using bacterial morphology,Gram staining microscopy and 16S rDNA sequence analysis.Broth micro-broth dilution method and PCR method were conducted for antibacterial sensitivity tests against antibiotics and detection of antibacterial resistance genes,integrase genes,and efflux pump genes in the isolated E. coli.Meanwhile,a modified semi-quantitative crystal violet staining method was employed to assess the biofilm-forming ability.The biofilm removal ability of the combination of 13 efflux pump inhibitors with doxycycline was determined using a checkerboard method.【Result】 The isolates were purple-black colonies with green metallic luster on eosin-mercian blue agar medium,and blue colonies on E.coli/coliform chromogenic medium,and pink short rod-shaped bacteria were seen on Gram staining microscopy.64 strains were highly similar to E.coli by 16S rDNA sequencing.Among 64 strains of E. coli isolates,relatively high resistance rates were observed against quinolones,sulfonamides,and tetracyclines,with multiple drug-resistant strains emerging.Among them,20.31% (13/64) showed resistance to five classes of antibiotics.Twenty-one resistance genes,one integrase gene and six efflux pump genes were positively detected.The detection rate of integrase gene intl1 was 89.06%.The number of E. coli strains with strong, medium, weak and no biofilm forming ability were 8 (12.50%), 34 (53.13%), 20 (31.25%), and 2 (3.12%), respectively.The combination of efflux pump inhibitors with doxycycline enhanced the biofilm removal ability.【Conclusion】 The resistance of E. coli from Hequ horses was severe and it was necessary to further strengthen the supervision of the rational use of antibacterial agents in the racing horses in Henan Mongolian Autonomous county of Qinghai province to reduce and avoid the emergence and widespread transmission of multidrug-resistant strains.

Key words: Hequ horses; Escherichia coli; multi-drug resistance; biofilm; efflux pump

中图分类号:

S852.61

赵星, 梁军, 李阳, 杨丹娇, 陈朝喜. 河曲马源大肠杆菌耐药相关基因检测及外排泵抑制剂对其生物被膜的影响[J]. 中国畜牧兽医, 2024, 51(8): 3603-3614.

ZHAO Xing, LIANG Jun, LI Yang, YANG Danjiao, CHEN Chaoxi. Detection of Drug Resistance Genes and Effect of Efflux Pump Inhibitors on Biofilm of Escherichia coli Isolated from Hequ Horses[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3603-3614.

参考文献

[1] 闫尊强,朱才业,马忠涛,等.河曲马种质资源研究进展[J].甘肃畜牧兽医,2022,52(1):1-5. YAN Z Q,ZHU C Y,MA Z T,et al.Research progress on germplasm resources of Hequ horse[J].Gansu Animal Husbandry and Veterinary Medicine,2022,52(1):1-5.(in Chinese)
[2] TOMPKINS B.E.coli infections[J].Nursing Standard,2011,25(29):51-52.
[3] FRATAMICO P M,DEBROY C,LIU Y,et al.Advances in molecular serotyping and subtyping of Escherichia coli[J].Frontiers in Microbiology,2016,7:644.
[4] BOMBAYWALA S,MANDPE A,PALIYA S,et al.Antibiotic resistance in the environment:A critical insight on its occurrence,fate,and eco-toxicity[J].Environmental Science and Pollution Research,2021,28(20):24889-24916.
[5] LI S,LI X,CHANG H,et al.Comprehensive insights into antibiotic resistance gene migration in microalgal-bacterial consortia:Mechanisms,factors,and perspectives[J].Science of the Total Environment,2023,901:166029.
[6] MARTINSON J,WALK S T. Escherichia coli residency in the gut of healthy human adults[J].EcoSal Plus,2020,9(1):10.
[7] CLINICAL AND LABORATORY STANDARDS INSTITUTE.Performance standards for antimicrobial susceptibility testing[S].28th ed.CLSI supplement M100.Wayne,PA,Clinical and Laboratory Standards Institute,2018.
[8] MESSAI Y,IABADENE H,BENHASSINE T,et al.Prevalence and characterization of extended-spectrum β-lactamases in Klebsiella pneumoniae in Algiers hospitals (Algeria)[J].Pathologie Biologie,2008,56(5):319-325.
[9] SOYER Y,RICHARDS J,HOELZER K,et al.Antimicrobial drug resistance patterns among cattle- and human-associated Salmonella strains[J].Journal of Food Protection,2013,76(10):1676-1688.
[10] YOKE-KQUEEN C,LEARN-HAN L,NOORZALEHA A S,et al.Characterization of multiple-antimicrobial-resistant Salmonella enterica subsp.enterica isolated from indigenous vegetables and poultry in Malaysia[J].Letters in Applied Microbiology,2008,46(3):318-324.
[11] PARK H,KIM J,RYU S,et al.The rate of frequent co-existence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) genes in Escherichia coli isolates from retail raw chicken in South Korea[J].Food Science and Biotechnology,2022,31(6):739-743.
[12] EL-BADAWY M F,TAWAKOL W M,EL-FAR S W,et al.Molecular identification of aminoglycoside-modifying enzymes and plasmid-mediated quinolone resistance genes among Klebsiella pneumoniae clinical isolates recovered from Egyptian patients[J]. International Journal of Microbiology,2017,2017:8050432.
[13] LIAO H,LU X,RENSING C,et al.Hyperthermophilic composting accelerates the removal of antibiotic resistance genes and mobile genetic elements in sewage sludge[J].Environmental Science & Technology,2018,52(1):266-276.
[14] 余娴,袁斌,雷军.产"超-超广谱"β-内酰胺酶革兰阴性杆菌的耐药表型及基因分析[J].中国抗生素杂志,2011,36(1):56-59. YU X,YUAN B,LEI J.Resistant phenotype and genotype analyses of super-spectrum β-lactamase-producing Gram-negative bacilli[J].Chinese Journal of Antibiotics,2011,36(1):56-59.(in Chinese)
[15] CHEN S,ZHAO S,WHITE D G,et al.Characterization of multiple-antimicrobial-resistant Salmonella serovars isolated from retail meats[J].Applied and Environmental Microbiology,2004,70(1):1-7.
[16] CHEN H,ZHANG M.Effects of advanced treatment systems on the removal of antibiotic resistance genes in wastewater treatment plants from Hangzhou,China[J].Environmental Science & Technology,2013,47(15):8157-8163.
[17] 曾杨梅.AcrAB-TolC外排系统在大肠埃希菌对苯扎溴铵和土霉素交叉耐药中的作用机制研究[D].重庆:西南大学,2015. ZENG Y M.Study on the resistance mechanism of AcrAB-TolC efflux system in Escherichia coli that was tolerdined on oxytetracycline and benzalkonium bromide[D].Chongqing:Southwest University,2015.(in Chinese)
[18] 武珊珊,敬文宪,陈启伟,等.鼠伤寒沙门氏菌双组分系统arcB基因缺失株的构建及药物敏感性分析[J].中国预防兽医学报,2020,42(9):879-886. WU S S,JING W X,CHEN Q W,et al.Construction of a two-component system arcB gene deletion strain of Salmonella Typhimurium and sensitivity analysis of antimicrobial agents[J].Chinese Journal of Preventive Veterinary Medicine,2020,42(9):879-886.(in Chinese)
[19] 代冰洁,祁萌,贾宇驰,等.大肠埃希菌外排泵AcrAB-TolC调控基因的表达与季铵盐化合物抗性关系的研究[J].中国病原生物学杂志,2020,15(5):541-545. DAI B J,QI M,JIA Y C,et al.Study on the relationship between overexpression of AcrAB-TolC efflux pump regulatory genes and resistance to quaternary ammonium compounds in Escherichia coli[J].Journal of Pathogen Biology,2020,15(5):541-545.(in Chinese)
[20] 辛娇娇,索南顿珠,娄永志,等.西藏牦牛粪便大肠杆菌毒力特性、耐药性与Ⅰ类整合子分子特征相关性分析[J].中国畜牧兽医,2023,50(10):4210-4222. XIN J J,SUO N D Z,LOU Y Z,et al.Correlation analysis on virulence characteristics,drug resistance and molecular features of class Ⅰ integrator of Escherichia coli isolated from feces in Tibetan yak[J].China Animal Husbandry & Veterinary Medicine,2023,50(10):4210-4222.(in Chinese)
[21] MANALO R V M,JOSOL V J D,GLORIANI N G.The differential effects of atorvastatin co-administered with ampicillin on the bacterial growth and biofilm formation of Staphylococcus aureus[J].Current Medicine Research and Practice,2017,7(5):178-183.
[22] SCHNEPF A,BIENERT-ZEIT A,ERTUGRUL H,et al.Antimicrobial usage in horses:The use of electronic data,data curation,and first results[J].Frontiers in Veterinary Science,2020,7:216.
[23] 石彬,孙艳,泽仁拥忠,等.凉山州不同动物源大肠杆菌耐药性调查[J].黑龙江畜牧兽医,2018,4:115-119. SHI B,SUN Y,ZE R Y Z,et al.Antimicrobial resistance of E.coli from different animals in Liangshan prefecture[J].Heilongjiang Animal Science and Veterinary Medicine,2018,4:115-119.(in Chinese)
[24] 刘鏊,马丁允,陀海欣,等.广州市某马术俱乐部马源大肠杆菌的分离鉴定、毒力基因检测及耐药性分析[J].畜牧与饲料科学,2023,44(3):109-115. LIU A,MA D Y,TUO H X,et al.Isolation,identification,virulence genes detection and antimicrobial resistance profile of horse origin Escherichia coli isolates from an equestrian club in Guangzhou city[J]. Animal Husbandry and Feed Science,2023,44(3):109-115.(in Chinese)
[25] CHUNG Y S,SONG J W,KIM D H,et al.Isolation and characterization of antimicrobial-resistant Escherichia coli from national horse racetracks and private horse-riding courses in Korea[J].Journal of Veterinary Science,2016,17(2):199-206.
[26] YIGIN A.Antimicrobial resistance and extended-spectrum beta-lactamase (ESBL) genes in E.coli isolated from equine fecal samples in Turkey[J].Journal of Equine Veterinary Science,2021,101:103461.
[27] WONGTAWAN T,NARINTHORN R,SONTIGUN N,et al.Characterizing the antimicrobial resistance profile of Escherichia coli found in sport animals (fighting cocks,fighting bulls,and sport horses) and soils from their environment[J].Veterinary World,2022,15(11):2673-2680.
[28] AHMED M O,CLEGG P D,WILLIAMS N J,et al.Antimicrobial resistance in equine faecal Escherichia coli isolates from North West England[J].Annals of Clinical Microbiology and Antimicrobials,2010,9:12.
[29] GHARAIBEH M H,ABUTARBUSH S M,MUSTAFA F G,et al.Identification of risk factors associated with antimicrobial resistance in equine fecal Escherichia coli isolates[J].Infection,Genetics and Evolution,2020,83:104317.
[30] 王继学,吴自豪,王雪,等.马乳源大肠杆菌的毒力基因检测和耐药性分析[J].畜牧与饲料科学,2023,44(1):24-31. WANG J X,WU Z H,WANG X,et al.Virulence genes detection and antimicrobial resistance analysis of Escherichia coli isolated from horse milk[J].Animal Husbandry and Feed Science,2023,44(1):24-31.(in Chinese)
[31] 崔艳丽,马策,崔云昊,等.青岛地区鸭源四环素类耐药大肠杆菌流行情况调查及耐药基因分析[J].中国畜牧兽医,2023,50(11):4681-4692. CUI Y L,MA C,CUI Y H,et al.Epidemiological investigation and resistance gene analysis of tetracycline-resistant from ducks in Qingdao[J].China Animal Husbandry & Veterinary Medicine,2023,50(11):4681-4692.(in Chinese)
[32] 朱琪琪,张继瑜,王承业,等.河北地区动物源大肠杆菌多重耐药性与流行性分析[J].中国畜牧兽医,2023,50(3):1081-1092. ZHU Q Q,ZHANG J Y,WANG C Y,et al.Analysis of multidrug resistance and prevalence of animal-derived Escherichia coli in Hebei[J].China Animal Husbandry & Veterinary Medicine,2023,50(3):1081-1092.(in Chinese)
[33] HALL R M,COLLIS C M.Mobile gene cassettes and integrons:Capture and spread of genes by site-specific recombination[J].Molecular Microbiology,1995,15(4):593-600.
[34] 苏志国,张衍,代天娇,等.环境中抗生素抗性基因与Ⅰ型整合子的研究进展[J].微生物学通报,2018,45(10):2217-2233. SU Z G,ZHANG Y,DAI T J,et al.Antibiotic resistance genes and class 1 integron in the environment,research progress[J].Microbiology China,2018,45(10):2217-2233.(in Chinese)
[35] NOCERA F P,MAURIZI L,MASULLO A,et al.Genotypic and phenotypic characterization of Escherichia coli isolates recovered from the uterus of mares with fertility problems[J].Animals,2023,13(10):1639.
[36] REZA A,SUTTON J M,RAHMAN K M.Effectiveness of efflux pump inhibitors as biofilm disruptors and resistance breakers in Gram-negative (ESKAPEE) bacteria[J].Antibiotics,2019,8(4):229.
[37] LOMOVSKAYA O,ZGURSKAYA H I,TOTROV M,et al.Waltzing transporters and 'the dance macabre’ between humans and bacteria[J].Nature Reviews Drug Discovery,2007,6(1):56-65.
[38] ALAV I,KOBYLKA J,KUTH M S,et al.Structure,assembly,and function of tripartite efflux and type 1 secretion systems in Gram-negative bacteria[J].Chemical Reviews,2021,121(9):5479-5596.
[39] HARMON D E,RUIZ C.The multidrug efflux regulator AcrR of Escherichia coli responds to exogenous and endogenous ligands to regulate efflux and detoxification[J].mSphere,2022,7(6):e47422.
[40] CHOWDHURY N,SUHANI S,PURKAYSTHA A,et al.Identification of AcrAB-TolC efflux pump genes and detection of mutation in efflux repressor AcrR from omeprazole responsive multidrug-resistant Escherichia coli isolates causing urinary tract infections[J].Microbiology Insights,2019,12:1690380579.
[41] 李佳瑞,杨雨齐,李思鸿,等.AcrAB-TolC主动外排系统与猪源耐药大肠杆菌多重耐药的相关性[J].中国兽医学报,2020,40(3):595-601. LI J R,YANG Y Q,LI S H,et al.Correlation between AcrAB-TolC active efflux system and multidrug resistance in porcine drug-resistant Escherichia coli[J].Chinese Journal of Veterinary Science,2020,40(3):595-601.(in Chinese)
[42] SHAHEEN A,AFRIDI W A,MAHBOOB S,et al.Reserpine is the new addition into the repertoire of AcrB efflux pump inhibitors[J].Molecular Biology,2019,53(4):674-684.
[43] ZHANG X,ZHAO Y,FENG L,et al.Combined with mefloquine,resurrect colistin active in colistin-resistant Pseudomonas aeruginosa in vitro and in vivo[J].Frontiers in Microbiology,2021,12:790220.
[44] OKAMOTO-SHIBAYAMA K,YOSHIDA A,ISHIHARA K.Inhibitory effect of resveratrol on Candida albicans biofilm formation[J].The Bulletin of Tokyo Dental College,2021,62(1):1-6.
[45] RUAN X,DENG X,TAN M,et al.In vitro antibiofilm activity of resveratrol against avian pathogenic Escherichia coli[J].BMC Veterinary Research,2021,17(1):249.