四川地区牦牛源肺炎克雷伯氏菌的分离鉴定及其耐药性分析
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摘要
为了分析四川地区牦牛源肺炎克雷伯氏菌耐药性并确定其耐药基因的携带情况,本试验采集了四川地区不同养殖场患呼吸道疾病牦牛肺脏、咽拭子、鼻拭子共127份病料组织,分离得到了43株肺炎克雷伯氏菌,并采用微量肉汤稀释法结合PCR扩增法检测43株肺炎克雷伯氏菌的耐药性和耐药基因携带情况。结果表明:43株肺炎克雷伯氏菌对氨苄西林、阿莫西林、多西环素、磺胺间甲氧嘧啶4种药物耐药率较高,为65.12%～90.70%;对阿米卡星、氟苯尼考、多黏菌素B、大观霉素4种药物的耐药率在20.93%～48.84%之间;对头孢噻呋、恩诺沙星、环丙沙星3药物的耐药率在18.60%～25.58%之间;多数筛选分离得到的菌株均呈现出多重耐药性,耐9、10种药物的菌株最多,分别占分离菌株的23.3%和20.9%。通过使用PCR扩增法检测分离菌携带的耐药基因情况,结果表明:bla_(TEM)、bla_(SHV)、sul2、sul3、floR 5种耐药基因检出率较高,检出率在62.8%～69.8%之间;ant(3″)-Ⅰa、aph(3′)-Ⅱa、aac(6′)-Ⅰb、aacC2 4种耐药基因检出率较低,检出率在7.0%～20.9%;bla_(CTX-M)、Mcr-1、qnrA、qnrS、rmtB 5种耐药基因未检出。综上所述,本试验中分离得到肺炎克雷伯氏菌具有较强的耐药性并携带有较多的耐药基因,为了更好的促进四川省牦牛养殖产业的发展,应注意避免滥用抗生素并通过药敏试验对症下药。
In order to analyze the drug resistance of Klebsiella pneumoniae isolated from yak in Sichuan area,43 Klebsiella pneumoniae were isolated from 127 samples of lung,pharynx and nose swabs of yaks suffering from respiratory diseases in different feeding farms in Sichuan province.Microdilution broth method and PCR method were used to detect drug resistance and resistance genes of 43 strains of Klebsiella pneumoniae.The results showed that 43 strains of Klebsiella pneumoniae were resistant to ampicillin,amoxicillin,doxycycline and sulfamonomethoxine 4 drugs with a high resistance rate of 65.12% to 90.70%;The resistance rate to amikacin,florfenicol,polymyxin B and spectinomycin were 20.93% to 48.84%:And that to cefotafur,enrofloxacin,ciprofloxacin were 18.60% and 25.58%.Most isolates were multidrug resistant and 9 and 10 drug resistant strains accounted for 23.3% and 20.9%,respectively.The detection rates of bla_(TEM),bla_(SHV),sul2,sul3 and floR genes were high,ranging from 62.8% to 69.8%;The detection rates of ant(3″)-Ⅰa、aph(3′)-Ⅱa,aac(6′)-Ⅰb,aacC2 gene were low,ranging from 7.0% to 20.9%,and bla_(CTX-M),Mcr-1,qnrA,qnrS,rmtB genes were not detected.In conclusion,Klebsiella pneumoniae isolates in this experiment had strong drug resistance and carried multiple drug resistance genes.And in order to better promote the development of yak breeding industry in Sichuan province,more attention should be paid to avoid the abuse of antibiotics through drug sensitivity test.
In order to analyze the drug resistance of Klebsiella pneumoniae isolated from yak in Sichuan area,43 Klebsiella pneumoniae were isolated from 127 samples of lung,pharynx and nose swabs of yaks suffering from respiratory diseases in different feeding farms in Sichuan province.Microdilution broth method and PCR method were used to detect drug resistance and resistance genes of 43 strains of Klebsiella pneumoniae.The results showed that 43 strains of Klebsiella pneumoniae were resistant to ampicillin,amoxicillin,doxycycline and sulfamonomethoxine 4 drugs with a high resistance rate of 65.12% to 90.70%;The resistance rate to amikacin,florfenicol,polymyxin B and spectinomycin were 20.93% to 48.84%:And that to cefotafur,enrofloxacin,ciprofloxacin were 18.60% and 25.58%.Most isolates were multidrug resistant and 9 and 10 drug resistant strains accounted for 23.3% and 20.9%,respectively.The detection rates of bla_(TEM),bla_(SHV),sul2,sul3 and floR genes were high,ranging from 62.8% to 69.8%;The detection rates of ant(3″)-Ⅰa、aph(3′)-Ⅱa,aac(6′)-Ⅰb,aacC2 gene were low,ranging from 7.0% to 20.9%,and bla_(CTX-M),Mcr-1,qnrA,qnrS,rmtB genes were not detected.In conclusion,Klebsiella pneumoniae isolates in this experiment had strong drug resistance and carried multiple drug resistance genes.And in order to better promote the development of yak breeding industry in Sichuan province,more attention should be paid to avoid the abuse of antibiotics through drug sensitivity test.
引文
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[6] 曹颖颖,杜泓明,白安斌,等.家养观赏地图鱼肺炎克雷伯氏菌、维氏气单胞菌与黏质沙雷氏菌的分离鉴定及耐药性分析[J].中国畜牧兽医,2015,42(1):215-223.CAO Y Y,DU H M,BAI A B,et al.Isolation,identification and drug resistance of Klebsiella pneumoniae,Aeromonas veronii and Serratia marcescens isolated from domesticated ornamental Astronotus ocellatus[J].China Animal Husbandry & Veterinary Medicine,2015,42(1):215-223.(in Chinese)
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[8] 张智,李再新,曾光菊,等.一株兔源耐药性肺炎克雷伯氏菌的分离与鉴定[J].中国畜牧兽医,2013,40(9):199-203.ZHANG Z,LI Z X,ZENG G J,et al.Isolation and identification of a resistant K.peneumoniae strain from rabbits[J].China Animal Husbandry & Veterinary Medicine,2013,40(9):199-203.(in Chinese)
[9] 马磊,颜其贵,万莉,等.竹鼠肺炎克雷伯氏菌的分离鉴定[J].中国人兽共患病学报,2011,27(9):825-827.MA L,YAN Q G,WAN L,et al.Isolation and identification on K.peneumoniae in Rhizomys[J].Chinese Journal of Zoonoses,2011,27(9):825-827.(in Chinese)
[10] HE T,WANG Y,SUN L,et al.Occurrence and characterization of blaNDM-5-positive Klebsiella pneumoniae isolates from dairy cows in Jiangsu,China[J].Journal of Antimicrobial Chemotherapy,2016,72(1):90-94.
[11] WYRES K L,HOLT K E.Klebsiella pneumoniae as a key trafficker of drug resistance genes from environmental to clinically important bacteria[J].Current Opinion in Microbiology,2018,45:131-139.
[12] YANG Y Q,LI Y X,LEI C W,et al.Novel plasmid-mediated colistin resistance gene mcr-7.1 in Klebsiella pneumoniae[J].Journal of Antimicrobial Chemotherapy,2018,73(7):235-239.
[13] 李富祥,李华春,赵德宏,等.肺炎克雷伯氏菌强毒株的分离鉴定及16-23S rRNA ITS序列分析[J].中国畜牧兽医,2015,42(2):352-357.LI F X,LI H C,ZHAO D H,et al.Isolation,identification and 16-23S rRNA ITS sequence analysis of a strain of highly virulent Klebsiella pneumoniae[J].China Animal Husbandry & Veterinary Medicine,2015,42(2):352-357.(in Chinese)
[14] DONG F,LU J,WANG Y,et al.A five-year surveillance of carbapenemase-producing Klebsiella pneumoniae in a pediatric hospital in China reveals increased predominance of NDM-1[J].Biomedical & Environmental Sciences,2017,30(8):562-569.
[15] SANCHEZ G V,MASTER R N,CLARK R B,et al.Klebsiella pneumoniae antimicrobial drug resistance,United States,1998-2010[J].Emerging Infectious Diseases,2013,19(1):133-136.
[16] QIN O,WEN F L,BEI L,et al.Prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and the distribution of class 1 integron in their strains isolated from a hospital in Central China[J].Chinese Medical Sciences Journal,2017,32(2):107-112.
[17] SHI W,LIU Y,HAN Y,et al.Molecular epidemiological analysis of carbapenem-resistant Klebsiella pneumoniae[J].Laboratory Medicine,2018,33(1):37-43.
[18] BRAHMI S,TOUATI A,DUNYACH-REMY C,et al.High prevalence of extended-spectrum β-lactamase-producing enterobacteriaceae in wild fish from the Mediterranean sea in Algeria[J].Microbial Drug Resistance,2018,24(3):290-298.
[19] ZHAO W,TIAN Q,LUO Y,et al.Isolation,identification,and genome analysis of lung pathogenic Klebsiella pneumoniae (lpkp) in forest musk deer[J].Journal of Zoo and Wildlife Medicine,2017,48(4):1039-1048.
[20] OSMAN K M,HASSAN H M,ORABI A,et al.Phenotypic,antimicrobial susceptibility profile and virulence factors of Klebsiella pneumoniae isolated from buffalo and cow mastitic milk[J].Pathogens & Global Health,2014,108(4):191-199.
[21] LEE C R,LEE J H,PARK K S,et al.Global dissemination of carbapenemase-producing Klebsiella pneumoniae:Epidemiology,genetic context,treatment options,and detection methods[J].Frontiers in Microbiology,2016,7(1):895.
[22] 韩坤,白雪,闫喜军,等.貂源肺炎克雷伯氏菌的分离及毒力和耐药性的分析[J].中国兽医科学,2018,48(8):1019-1023.HAN K,BAI X,YAN X J,et al.Isolation,virulence and drug resistance of Klebsiella pneumoniae from mink[J].Chinese Veterinary Science,2018,48(8):1019-1023.(in Chinese)
[23] 冯娜,高翔,肖敏,等.牛源肺炎克雷伯氏菌的分离鉴定及遗传进化分析[J].中国兽医科学,2016,46(11):1358-1364.FENG N,GAO X,XIAO M,et al.Identification and genetic evolution analysis of Klebsiella pneumoniae strain from calves[J].Chinese Veterianry Science,2016,46(11):1358-1364.(in Chinese)
[24] 刘海林.猪源肺炎克雷伯氏菌分离菌株耐药性调查及防控技术研究[D].南京:南京农业大学,2015.LIU H L.Investigation and prevention and control of drug resistance of Klebsiella pneumoniae isolated from swine[D].Nanjing:Nanjing Agricultural University,2015.(in Chinese)
[25] LU L,FENG Y,LONG H,et al.ST273 carbapenem-resistant Klebsiella pneumoniae carrying blaNDM-1 and blaIMP-4[J].Antimicrobial Agents & Chemotherapy,2018,62(6):e00160.
[26] 李珍珍.兰州市规模化猪场腹泻仔猪粪便中的细菌分离鉴定[D].兰州:甘肃农业大学,2018.LI Z Z.Isolation and identification of bacteria in diarrhea piglet feces from large-scale pig farm in Lanzhou city[D].Lanzhou:Gansu Agricultural University,2018.(in Chinese)
[2] MCDANELD T G,KUEHN L A,KEELE J W.Evaluating the microbiome of two sampling locations in the nasal cavity of cattle with bovine respiratory disease complex (BRDC)[J].Journal of Animal Science,2018,96(4):1281-1287.
[3] 计慧姝,罗光荣,肖敏,等.川西北红原县和理塘县牦牛4类体内寄生虫感染的研究[J].中国畜牧兽医,2017,44(2):561-567.JI H S,LUO G R,XIAO M,et al.Study on infection of 4 kinds of internal parasites in yaks in Hongyuan and Litang county of Northwest Sichuan province[J].China Animal Husbandry & Veterinary Medicine,2017,44(2):561-567.(in Chinese)
[4] PAPADIMITRIOU-OLIVGERIS M,FLIGOU F,BARTZAVALI C,et al.Carbapenemase-producing Klebsiella pneumoniae bloodstream infection in critically ill patients:Risk factors and predictors of mortality[J].European Journal of Clinical Microbiology & Infectious Diseases,2017,36(7):1125-1131.
[5] QUAN J,ZHAO D,LIU L,et al.High prevalence of ESBL-producing Escherichia coli and Klebsiella pneumoniae in community-onset bloodstream infections in China[J].Journal of Antimicrobial Chemotherapy,2017,72(1):273-280.
[6] 曹颖颖,杜泓明,白安斌,等.家养观赏地图鱼肺炎克雷伯氏菌、维氏气单胞菌与黏质沙雷氏菌的分离鉴定及耐药性分析[J].中国畜牧兽医,2015,42(1):215-223.CAO Y Y,DU H M,BAI A B,et al.Isolation,identification and drug resistance of Klebsiella pneumoniae,Aeromonas veronii and Serratia marcescens isolated from domesticated ornamental Astronotus ocellatus[J].China Animal Husbandry & Veterinary Medicine,2015,42(1):215-223.(in Chinese)
[7] 蒙正群,冷依伊,任梅渗,等.一株牛源肺炎克雷伯氏菌的分离鉴定与耐药基因型检测[J].浙江农业学报,2017,29(4):534-541.MENG Z Q,LENG Y Y,REN M S,et al.Isolation,identification and drug resistance detection of a calf strain[J].Acta Agriculturae Zhejiangensis,2017,29(4):534-541.(in Chinese)
[8] 张智,李再新,曾光菊,等.一株兔源耐药性肺炎克雷伯氏菌的分离与鉴定[J].中国畜牧兽医,2013,40(9):199-203.ZHANG Z,LI Z X,ZENG G J,et al.Isolation and identification of a resistant K.peneumoniae strain from rabbits[J].China Animal Husbandry & Veterinary Medicine,2013,40(9):199-203.(in Chinese)
[9] 马磊,颜其贵,万莉,等.竹鼠肺炎克雷伯氏菌的分离鉴定[J].中国人兽共患病学报,2011,27(9):825-827.MA L,YAN Q G,WAN L,et al.Isolation and identification on K.peneumoniae in Rhizomys[J].Chinese Journal of Zoonoses,2011,27(9):825-827.(in Chinese)
[10] HE T,WANG Y,SUN L,et al.Occurrence and characterization of blaNDM-5-positive Klebsiella pneumoniae isolates from dairy cows in Jiangsu,China[J].Journal of Antimicrobial Chemotherapy,2016,72(1):90-94.
[11] WYRES K L,HOLT K E.Klebsiella pneumoniae as a key trafficker of drug resistance genes from environmental to clinically important bacteria[J].Current Opinion in Microbiology,2018,45:131-139.
[12] YANG Y Q,LI Y X,LEI C W,et al.Novel plasmid-mediated colistin resistance gene mcr-7.1 in Klebsiella pneumoniae[J].Journal of Antimicrobial Chemotherapy,2018,73(7):235-239.
[13] 李富祥,李华春,赵德宏,等.肺炎克雷伯氏菌强毒株的分离鉴定及16-23S rRNA ITS序列分析[J].中国畜牧兽医,2015,42(2):352-357.LI F X,LI H C,ZHAO D H,et al.Isolation,identification and 16-23S rRNA ITS sequence analysis of a strain of highly virulent Klebsiella pneumoniae[J].China Animal Husbandry & Veterinary Medicine,2015,42(2):352-357.(in Chinese)
[14] DONG F,LU J,WANG Y,et al.A five-year surveillance of carbapenemase-producing Klebsiella pneumoniae in a pediatric hospital in China reveals increased predominance of NDM-1[J].Biomedical & Environmental Sciences,2017,30(8):562-569.
[15] SANCHEZ G V,MASTER R N,CLARK R B,et al.Klebsiella pneumoniae antimicrobial drug resistance,United States,1998-2010[J].Emerging Infectious Diseases,2013,19(1):133-136.
[16] QIN O,WEN F L,BEI L,et al.Prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and the distribution of class 1 integron in their strains isolated from a hospital in Central China[J].Chinese Medical Sciences Journal,2017,32(2):107-112.
[17] SHI W,LIU Y,HAN Y,et al.Molecular epidemiological analysis of carbapenem-resistant Klebsiella pneumoniae[J].Laboratory Medicine,2018,33(1):37-43.
[18] BRAHMI S,TOUATI A,DUNYACH-REMY C,et al.High prevalence of extended-spectrum β-lactamase-producing enterobacteriaceae in wild fish from the Mediterranean sea in Algeria[J].Microbial Drug Resistance,2018,24(3):290-298.
[19] ZHAO W,TIAN Q,LUO Y,et al.Isolation,identification,and genome analysis of lung pathogenic Klebsiella pneumoniae (lpkp) in forest musk deer[J].Journal of Zoo and Wildlife Medicine,2017,48(4):1039-1048.
[20] OSMAN K M,HASSAN H M,ORABI A,et al.Phenotypic,antimicrobial susceptibility profile and virulence factors of Klebsiella pneumoniae isolated from buffalo and cow mastitic milk[J].Pathogens & Global Health,2014,108(4):191-199.
[21] LEE C R,LEE J H,PARK K S,et al.Global dissemination of carbapenemase-producing Klebsiella pneumoniae:Epidemiology,genetic context,treatment options,and detection methods[J].Frontiers in Microbiology,2016,7(1):895.
[22] 韩坤,白雪,闫喜军,等.貂源肺炎克雷伯氏菌的分离及毒力和耐药性的分析[J].中国兽医科学,2018,48(8):1019-1023.HAN K,BAI X,YAN X J,et al.Isolation,virulence and drug resistance of Klebsiella pneumoniae from mink[J].Chinese Veterinary Science,2018,48(8):1019-1023.(in Chinese)
[23] 冯娜,高翔,肖敏,等.牛源肺炎克雷伯氏菌的分离鉴定及遗传进化分析[J].中国兽医科学,2016,46(11):1358-1364.FENG N,GAO X,XIAO M,et al.Identification and genetic evolution analysis of Klebsiella pneumoniae strain from calves[J].Chinese Veterianry Science,2016,46(11):1358-1364.(in Chinese)
[24] 刘海林.猪源肺炎克雷伯氏菌分离菌株耐药性调查及防控技术研究[D].南京:南京农业大学,2015.LIU H L.Investigation and prevention and control of drug resistance of Klebsiella pneumoniae isolated from swine[D].Nanjing:Nanjing Agricultural University,2015.(in Chinese)
[25] LU L,FENG Y,LONG H,et al.ST273 carbapenem-resistant Klebsiella pneumoniae carrying blaNDM-1 and blaIMP-4[J].Antimicrobial Agents & Chemotherapy,2018,62(6):e00160.
[26] 李珍珍.兰州市规模化猪场腹泻仔猪粪便中的细菌分离鉴定[D].兰州:甘肃农业大学,2018.LI Z Z.Isolation and identification of bacteria in diarrhea piglet feces from large-scale pig farm in Lanzhou city[D].Lanzhou:Gansu Agricultural University,2018.(in Chinese)