水貂沙门氏菌的分离鉴定及药物敏感性分析
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摘要
为指导临床合理用药并为治疗患病水貂提供依据,本试验对山东省某水貂养殖场送检的7只疑似患有沙门氏菌病的病死水貂的肝脏和脾脏样品进行细菌分离鉴定及药物敏感性分析,通过分离纯化方法从样品中分离菌株,并采用革兰氏染色、生化鉴定和PCR方法对分离菌株进行鉴定。运用K-B药敏法检测菌株对临床常用药物的敏感性,并通过PCR方法检测菌株耐药基因及Ⅰ类整合子的携带情况。结果显示,本试验分离得到的7株菌均为革兰氏阴性、短小的杆菌;生化反应检测显示,分离菌株葡萄糖、麦芽糖、甘露醇、MR试验、枸橼酸盐、硫化氢试验均表现为阳性,初步鉴定分离菌株为沙门氏菌;PCR产物测序结果进一步表明7株分离菌均为沙门氏菌;药敏试验结果显示,7株沙门氏菌对头孢曲松、左氧氟沙星、氟苯尼考和多黏菌素较敏感,对氨基糖苷类药物、四环素和氨苄西林表现为耐药;耐药基因检测结果显示,7株沙门氏菌共检测出8种耐药基因blaTEM-1、blaCTX-M-1G、aadA1、aac(3′)-Ⅳ、aac(3′)-Ⅱc、aph(4′)-Ⅰa、aph(3′)-Ⅶ和oqxAB,以及基因盒为aadA1、arr-3-aacA4和blaPSE-1的Ⅰ类整合子。综上可知,本试验在送检的7只患病水貂的肝脏和脾脏样品中分离到7株沙门氏菌,分离菌株均为耐药菌株,且主要表现为多重耐药现象;耐药基因呈多样性,且存在位于质粒上的耐药基因扩大了耐药基因的传播范围,增加了细菌的耐药性,为临床用药治疗带来困难。
In order to guide rational drug use and provide the basis for treatment of diseased mink,the bacteria of seven sick minks suffering from salmonellosis provided by mink farms in Shandong province were isolated and identified,and the drug sensitivity was tested.The strains were isolated from the samples by separation and purification methods,and the isolated strains were identified by Gram staining,biochemical identification and PCR.K-B susceptibility test was used to detectthe sensitivity of strains to commonly used drugs,and the drug resistance gene and classⅠintegrants were detected by PCR.The results showed that 7 strains were isolated,all of which were Gram-negative and short bacilli.Biochemical reaction tests showed that the isolates were positive for glucose,maltose,mannitol,MR test,citrate and hydrogen sulfide test,and the isolates were preliminarily identified as Salmonella.The sequencing results of the PCR products further indicated that 7 isolated strains were Salmonella.The drug sensitivity test showed that 7 strains of Salmonella were sensitive to ceftriaxone,levofloxacin,florfenicol and polymyxin,and resistant to all of the aminoglycosides,tetracycline and ampicillin.The results of drug resistance gene test showed that 8 kinds of resistance genes were detected in 7 strains of Salmonella,blaTEM-1,blaCTX-M-1 G,aadA1,aac(3′)-Ⅳ,aac(3′)-Ⅱc,aph(4′)-Ⅰa,aph(3′)-Ⅶand oqxAB,and the classⅠintegrin of the gene cassettes aaA1,arr-3-aacA4 and blaPSE-1 were detected.According to the above results,7 strains of Salmonella were isolated from the liver and spleen samples of 7 diseased minks,and the isolates were all resistant strains,and mainly showed multi-drug resistance;The drug resistance genes were diverse,and the presence of drug resistance genes on the plasmid expanded the spread of drug resistance genes and increased the drug resistance of bacteria,which brought difficulties for clinical drug treatment.
In order to guide rational drug use and provide the basis for treatment of diseased mink,the bacteria of seven sick minks suffering from salmonellosis provided by mink farms in Shandong province were isolated and identified,and the drug sensitivity was tested.The strains were isolated from the samples by separation and purification methods,and the isolated strains were identified by Gram staining,biochemical identification and PCR.K-B susceptibility test was used to detectthe sensitivity of strains to commonly used drugs,and the drug resistance gene and classⅠintegrants were detected by PCR.The results showed that 7 strains were isolated,all of which were Gram-negative and short bacilli.Biochemical reaction tests showed that the isolates were positive for glucose,maltose,mannitol,MR test,citrate and hydrogen sulfide test,and the isolates were preliminarily identified as Salmonella.The sequencing results of the PCR products further indicated that 7 isolated strains were Salmonella.The drug sensitivity test showed that 7 strains of Salmonella were sensitive to ceftriaxone,levofloxacin,florfenicol and polymyxin,and resistant to all of the aminoglycosides,tetracycline and ampicillin.The results of drug resistance gene test showed that 8 kinds of resistance genes were detected in 7 strains of Salmonella,blaTEM-1,blaCTX-M-1 G,aadA1,aac(3′)-Ⅳ,aac(3′)-Ⅱc,aph(4′)-Ⅰa,aph(3′)-Ⅶand oqxAB,and the classⅠintegrin of the gene cassettes aaA1,arr-3-aacA4 and blaPSE-1 were detected.According to the above results,7 strains of Salmonella were isolated from the liver and spleen samples of 7 diseased minks,and the isolates were all resistant strains,and mainly showed multi-drug resistance;The drug resistance genes were diverse,and the presence of drug resistance genes on the plasmid expanded the spread of drug resistance genes and increased the drug resistance of bacteria,which brought difficulties for clinical drug treatment.
引文
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[12]WANG W,PENG Z,BALOCH Z,et al.Genomic characterization of an extensively-drug resistance Salmonella enterica serotype Indiana strain harboring blaNDM-1gene isolated from a chicken carcass in China[J].Microbiological Research,2017,204(11):48-54.
[13]廖成水,程相朝,张春杰,等.鸡源致病性沙门氏菌新近分离株的耐药性与耐药基因[J].中国兽医科学,2011,41(7):751-755.LIAO C S,CHENG X C,ZHANG C J,et al.Antimicrobial resistance and resistance genes of pathogenic Salmonellarecently isolated from chicken[J].Chinese Veterinary Science,2011,41(7):751-755.(in Chinese)
[14]张洪学,宫彦超,曲宁宁,等.水貂阿留申与沙门氏菌混合感染的诊治[J].山东畜牧兽医,2016,4:33.ZHANG H X,GONG Y C,QU N N,et al.Diagnosis and treatment of mixed infection with Salmonellaand Aleutian in mink[J].Shandong Journal of Animal Science and Veterinary Medicine,2016,4:33.(in Chinese)
[15]PATEL S R,BHARTI S,PRATAP C B,et al.Drug resistance pattern in the recent isolates of Salmonella Typhi with special reference to cephalosporins and azithromycin in the Gangetic plain[J].Journal of Clinical and Diagnostic Research,2017,11(6):DM01-DM03.
[16]张璐,张雪伟,袁宗辉,等.畜禽沙门氏菌病防制研究进展[J].中国畜牧兽医,2017,44(7):2103-2111.ZHANG L,ZHANG X W,YUAN Z H,et al.Research progress on Salmonellosis prevention and control in livestock and poultry[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):2103-2111.(in Chinese)
[17]沈学怀,张丹俊,潘孝程,等.安徽地区禽源沙门氏菌分离鉴定与耐药性研究[J].中国畜牧兽医,2017,44(12):3663-3669.SHEN X H,ZHANG D J,PAN X C,et al.Isolation,identification and drug resistance analysis of avian Salmonellain Anhui[J].China Animal Husbandry&Veterinary Medicine,2017,44(12):3663-3669.(in Chinese)
[18]王海龙,张敏敏.水貂沙门氏菌的分离鉴定与药敏试验[J].中国畜禽种业,2015,7:28-29.WANG H L,ZHANG M M.Isolation,identification and susceptibility test of Salmonellafrom mink[J].The Chinese Livestock and Poultry Breeding,2015,7:28-29.(in Chinese)
[19]董文娟,蔡淑霞.一例水貂感染沙门氏菌的诊治[J].特种经济动植物,2015,10:11-12.DONG W J,CAI S X.Diagnosis and treatment of Salmonellainfection in a mink[J].Special Economic Animal and Plant,2015,10:11-12.(in Chinese)
[20]钟舒红,冯世文,李军,等.广西畜禽产品中沙门氏菌血清型、耐药性及耐药基因调查[J].中国畜牧兽医,2018,45(3):770-780.ZHONG S H,FENG S W,LI J,et al.Investigation on serotype,drug resistance and drug resistance gene of Salmonellain livestock and poultry products of Guangxi[J].China Animal Husbandry&Veterinary Medicine,2018,45(3):770-780.(in Chinese)
[21]HARBOTTLE H,THAKUR S,ZHAO S,et al.Genetics of antimicrobial resistance[J].Animal Biotechnology,2006,17(2):111-124.
[22]刘耀川,于本良,高锋,等.整合子-基因盒系统耐药性传递机制研究新进展[J].中国预防兽医学报,2017,39(3):244-247.LIU Y C,YU B L,GAO F,et al.New progress in research on drug resistance transfer mechanism of integrin-gene box system[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(3):244-247.(in Chinese)
[23]葛琨,武运,杨保伟,等.乌鲁木齐牛羊肉源沙门氏菌对喹诺酮类药物的耐药现状及相关基因分析[J].食品科学,2017,38(4):107-112.GE K,WU Y,YANG B W,et al.Quinolone resistance characteristics and related gene analysis of Salmonellain beef and mutton retailed in Urumqi[J].Food Science,2017,38(4):107-112.(in Chinese)
[24]张纯萍,宋立,崔明全,等.我国鸡源沙门氏菌的血清型分布和对粘菌素耐药性的研究[J].中国兽药杂志,2018,52(1):13-18.ZHANG C P,SONG L,CUI M Q,et al.Serotype distribution and antimicrobial susceptibility to colistin of Salmonella spp.from chicken in China[J].Chinese Journal of Veterinary Drug,2018,52(1):13-18.(in Chinese)
[2]陆承平.兽医微生物学[M].北京:中国农业出版社,2001.LU C P.Veterinary Microbiology[M].Beijing:China Agriculture Press,2001.(in Chinese)
[3]HARBOTTLE H,WHITE D G,MCDERMOTT P F,et al.Comparison of multilocus sequence typing,pulsed-field gel electrophoresis,and antimicrobial susceptibility typing for characterization of Salmonella enterica serotype Newport isolates[J].Journal of Clinical Microbiology,2006,44(7):2449-2457.
[4]HUR J,KIM J H,PARK J H,et al.Molecular and virulence characteristics of multi-drug resistant Salmonella Enteritidis strains isolated from poultry[J].The Veterinary Journal,2011,189(3):306-311.
[5]YU F,CHEN Q,YU X,et al.High prevalence of extended-spectrum beta lactamases among Salmonella enterica Typhimurium isolates from pediatric patients with diarrhea in China[J].PLoS One,2011,6(3):e16801.
[6]PREETHI B,SHANTHI V,RAMANATHAN K,et al.Identification of potential therapeutics to conquer drug resistance in Salmonella Typhimurium:Drug repurposing strategy[J].Biodrugs Clinical Immunotherapeutics Biopharmaceuticals&Gene Therapy,2016,30(6):593-605.
[7]GARCIA V,GARCIA P,RODRIGUEZ I,et al.The role of IS26in evolution of a derivative of the virulence plasmid of Salmonella enterica serovar Enteritidis which confers multiple drug resistance[J].Infection Genetics and Evolution,2016,45(11):246-249.
[8]马婧嘉,施春雷,李可,等.沙门氏菌耐药谱及质粒耐药基因的筛查[J].中国食品学报,2014,14(4):184-190.MA J J,SHI C L,LI K,et al.Screening of antibiotic resistance and plasmid resistance genes in Salmonella isolates[J].Journal of Chinese Institute of Food Science and Technology,2014,14(4):184-190.(in Chinese)
[9]马孟根,王红宁,余勇,等.猪源致病性沙门氏菌耐药基因的分析[J].畜牧兽医学报,2006,37(1):65-70.MA M G,WANG H N,YU Y,et al.Detection of antimicrobial resistance genes of pathogenic Salmonella from swine[J].Acta Veterinaria et Zootechnica Sinica,2006,37(1):65-70.(in Chinese)
[10]SUN N,YANG F,LIN D C,et al.Molecular characterization of the antimicrobial resistance of Riemerella anatipestiferisolated from ducks[J].Veterinary Microbiology,2012,158(8):376-383.
[11]董剑辉,熊惠军,宋立,等.鸡源沙门菌的耐药性及脉冲场凝胶电泳分型研究[J].动物医学进展,2011,32(5):9-13.DONG J H,XIONG H J,SONG L,et al.Drug resistance and molecular typing study of Salmonella Gallinarum by pulsed-field gel electrophoresis[J].Progress in Veterinary Medicine,2011,32(5):9-13.(in Chinese)
[12]WANG W,PENG Z,BALOCH Z,et al.Genomic characterization of an extensively-drug resistance Salmonella enterica serotype Indiana strain harboring blaNDM-1gene isolated from a chicken carcass in China[J].Microbiological Research,2017,204(11):48-54.
[13]廖成水,程相朝,张春杰,等.鸡源致病性沙门氏菌新近分离株的耐药性与耐药基因[J].中国兽医科学,2011,41(7):751-755.LIAO C S,CHENG X C,ZHANG C J,et al.Antimicrobial resistance and resistance genes of pathogenic Salmonellarecently isolated from chicken[J].Chinese Veterinary Science,2011,41(7):751-755.(in Chinese)
[14]张洪学,宫彦超,曲宁宁,等.水貂阿留申与沙门氏菌混合感染的诊治[J].山东畜牧兽医,2016,4:33.ZHANG H X,GONG Y C,QU N N,et al.Diagnosis and treatment of mixed infection with Salmonellaand Aleutian in mink[J].Shandong Journal of Animal Science and Veterinary Medicine,2016,4:33.(in Chinese)
[15]PATEL S R,BHARTI S,PRATAP C B,et al.Drug resistance pattern in the recent isolates of Salmonella Typhi with special reference to cephalosporins and azithromycin in the Gangetic plain[J].Journal of Clinical and Diagnostic Research,2017,11(6):DM01-DM03.
[16]张璐,张雪伟,袁宗辉,等.畜禽沙门氏菌病防制研究进展[J].中国畜牧兽医,2017,44(7):2103-2111.ZHANG L,ZHANG X W,YUAN Z H,et al.Research progress on Salmonellosis prevention and control in livestock and poultry[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):2103-2111.(in Chinese)
[17]沈学怀,张丹俊,潘孝程,等.安徽地区禽源沙门氏菌分离鉴定与耐药性研究[J].中国畜牧兽医,2017,44(12):3663-3669.SHEN X H,ZHANG D J,PAN X C,et al.Isolation,identification and drug resistance analysis of avian Salmonellain Anhui[J].China Animal Husbandry&Veterinary Medicine,2017,44(12):3663-3669.(in Chinese)
[18]王海龙,张敏敏.水貂沙门氏菌的分离鉴定与药敏试验[J].中国畜禽种业,2015,7:28-29.WANG H L,ZHANG M M.Isolation,identification and susceptibility test of Salmonellafrom mink[J].The Chinese Livestock and Poultry Breeding,2015,7:28-29.(in Chinese)
[19]董文娟,蔡淑霞.一例水貂感染沙门氏菌的诊治[J].特种经济动植物,2015,10:11-12.DONG W J,CAI S X.Diagnosis and treatment of Salmonellainfection in a mink[J].Special Economic Animal and Plant,2015,10:11-12.(in Chinese)
[20]钟舒红,冯世文,李军,等.广西畜禽产品中沙门氏菌血清型、耐药性及耐药基因调查[J].中国畜牧兽医,2018,45(3):770-780.ZHONG S H,FENG S W,LI J,et al.Investigation on serotype,drug resistance and drug resistance gene of Salmonellain livestock and poultry products of Guangxi[J].China Animal Husbandry&Veterinary Medicine,2018,45(3):770-780.(in Chinese)
[21]HARBOTTLE H,THAKUR S,ZHAO S,et al.Genetics of antimicrobial resistance[J].Animal Biotechnology,2006,17(2):111-124.
[22]刘耀川,于本良,高锋,等.整合子-基因盒系统耐药性传递机制研究新进展[J].中国预防兽医学报,2017,39(3):244-247.LIU Y C,YU B L,GAO F,et al.New progress in research on drug resistance transfer mechanism of integrin-gene box system[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(3):244-247.(in Chinese)
[23]葛琨,武运,杨保伟,等.乌鲁木齐牛羊肉源沙门氏菌对喹诺酮类药物的耐药现状及相关基因分析[J].食品科学,2017,38(4):107-112.GE K,WU Y,YANG B W,et al.Quinolone resistance characteristics and related gene analysis of Salmonellain beef and mutton retailed in Urumqi[J].Food Science,2017,38(4):107-112.(in Chinese)
[24]张纯萍,宋立,崔明全,等.我国鸡源沙门氏菌的血清型分布和对粘菌素耐药性的研究[J].中国兽药杂志,2018,52(1):13-18.ZHANG C P,SONG L,CUI M Q,et al.Serotype distribution and antimicrobial susceptibility to colistin of Salmonella spp.from chicken in China[J].Chinese Journal of Veterinary Drug,2018,52(1):13-18.(in Chinese)