猪源肠外致病性大肠杆菌药物敏感性及遗传相关性研究
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摘要
大肠埃希氏菌(Escherichia coli,E.coli)通常称为大肠杆菌,是一类常见的人畜共患病原菌。在兽医临床诊断方面,该菌引起的猪水肿病,仔猪黄、白痢是长期困扰猪场的老病;随着养猪业的迅速发展和规模化养殖的深入,近年发现猪大肠杆菌病出现了更为严重的临床表现:如引起猪的肺炎,肾炎,脑膜炎,败血症等;这些菌株同样可以导致人的肠外感染。研究者将引发该类病症的大肠杆菌分类为肠外致病性大肠杆菌(Extraintestinal Pathogenic Escherichia coli,ExPEC),其发病率与死亡率在人畜临床都较高,且没有特定的血清型。目前,针对ExPEC的研究较少,还没有疫苗问世,因而多采用药物治疗。抗生素的大量不合理使用;链霉素类等药物被广泛用作动物饲料添加剂,使得动物源性耐药菌株越来越多,耐药谱也越来越广;产超广谱β-内酰胺酶(Extend-Spectrumβ-Lactamase,ESBL)大肠杆菌的出现;以及耐药元件的转移性在食品链上的潜在危害;都严重威胁着该病的治疗,成为公共卫生事业上的隐患。
在本实验室已经对分离的猪源肠外致病性大肠杆菌进行了血清分型、毒力基因鉴定、动物感染实验的基础上,为进一步了解该类菌株耐药的真实情况和传播的可能性,本研究对临床猪源肠外致病性大肠杆菌分离株进行了药物敏感性及遗传相关性检测,以提供可靠的临床用药和防治依据,具体工作如下:
1、完成了315株猪源肠外致病性大肠杆菌(ExPEC)的MIC测定。结果表明我实验室分离ExPEC菌株耐药谱广,耐药性强,多数菌株仅对头孢菌素敏感;分离菌株耐药谱有46种,95.2%的菌株可以耐受5种以上药物,60.6%的菌株耐受10种以上药物;AMP+STR+KAN+NEO+GEN+CIP+OFX+TE+TMP+SDM是最为流行的耐药谱,占到26%。
2、完成315株猪源肠外致病性大肠杆菌(ExPEC)对6大类27种耐药基因的检测。单一抗性携带多基因现象普遍存在,这表明大肠杆菌的耐药呈现多种机制混合的复杂情况。β-内酰胺类抗性的耐药基因以bla_(TEM)为主,氨基糖苷类耐药基因以aac(3)-Ⅳ,strAstrB,aphA1为主,喹诺酮类耐药以gyrA突变为主,四环素耐药基因以tet(B)和tet(G)为主,甲氧苄啶类dfrA7,dfrA12,dfrA13分布广泛,磺胺类sul1,sul2分布最广。
3、利用intI基因检测315株猪源肠外致病性大肠杆菌(ExPEC)的I型整合子携带情况,并对扩增阳性菌株进行可变区基因盒定性。86(27.3%)株ExPEC含有intI基因即I型整合子阳性。对VR的检测发现其主要含有0.5kb,08kb,1.0kb1.5kb和1.8kb五种基因盒,分别携带dfrA14;aadA2;aadA22,aadA5,dfrA17;dfrA17-aadA2,dfrA1-aadA1;dfrA12-aadA2,dfrA17-aadA5。
4、检测产ESBL菌株并完成其基因分型。检出2株产ESBL菌株,耐药谱均在10以上,PCR分型测序结果表明其含有国内广泛流传的普通β-内酰胺酶bla_(TEM-1)和超广谱β-内酰胺酶bla_(CTX-M-14)。
5、完成了对分离自3个省份12个猪场(区)的38株高度耐药菌株的遗传特性研究,其质粒图谱检测结果显示同一场(区)流行1-2种质粒,不同场(区)质粒图谱不同;BOX-PCR指纹显示同场(区)菌株同源性高于不同场(区),且同场(区)菌株的MIC值和耐药基因差异不显著(P＞0.05)。证明菌株、质粒、整合子和耐药性具有场(区)内区域性且可在一定区域内传播。
Escherichia coli is an important zoonotic pathogen.Piglets edema disease,yellow or white scour of newborn piglets are typical diseases caused by E.coli.They trouble the veterinary clinic for long.New symptoms such as Pneumonia,nephritis, meningitis and septicemia caused by E.coli are increasing with the development of swine industry.And these extraintestinal infections are also similar to human.The specialized strains of E.coli that cause most extraintestinal diseases were classified as extraintestinal pathogenic E.coIi(ExPEC),which shows higher morbidity,mortality and no typical serotypes.Now antimicrobial therapy is the only available tool for treating clinical infections of ExPEC because of no vaccine.The resistance of ExPEC emerged due to the wrong use of the antibiotics and the feedstuff additive at the clinic. Extended-spectrumβ-lactamase(ESBL)-producing strains and the dissemination of resistant vectors are of increasing concern of the failure of antibiotic therapy and public health.
It's helpful for clinical treatment to detect the resistance of ExPEC to common antibiotics and the possibility of resistance transmission,based on the detecting of serotype,virulence factors and animal experiments of ExPEC.so we carried out the following work to analyse the drug senstivity and molecular characteristics for swine ExPEC:
1.We researched the drug minimum inhibitory concentration of 315 swine ExPEC isolates from the clinic.These isolates of Expec showed multiple drug resisitance, and only sensitived to cephalosporins.95.2%were resistant to at least five antimicrobials and 60.6%resistant to more than 10.the most popular multiresistant patterns was AMP+STR+KAN+NEO+GEN+CIP+OFX+TE+TMP+SDM.
2.We established the PCR methods for detection of 27 resistance genes includingβ-lactams,aminoglycosides,tetracyclines,quinolones,trimethoprim and sulfonamides,then appliesed them to the ExPEC.For each antimicrobial agent, ampicillin resistance was primarily mediated by bla_(TEM),aminoglycosides by aac(3)-Ⅳ, strAstrB and aphA1,quinolones resistance by mutations in gyrA,tetracycline resistance by tet(B) and tet(G),trimethoprim resistance by dfrA7,dfrA12,dfrA13,and sulfadimidine resistance by sul1 and sul2.Strains which harbored several genes that conferred resistance to the same antimicrobial agent were often significantly more multiresistant than others.
3.We detected the intⅠgene to identify integron,and amplified variable region to determine gene cassettes.86(27.3%) ExPEC isolates were integron positive,which harbored dfrA14,aadA2,aadA22,aadA5,dfrA17,dfrA17-aadA2,dfrA1-aadA1, dfrA12-aadA2,dfrA17-aadA5 gene cassettes in five major different variable regions.
4.We identified ESBL isolates and classified their genotypes.Two isolates with bla_(TEM-I)+bla_(CTX-M-I4)-genotype ESBL were selected.They were both resistant to more than 10 drugs.
5.We reseached genetic correlation for 38 swine ExPEC isolated from twelve pig farms of three provinces.Analysis of plasmid map proved that 1 to 2 different plasmids were popular in a farm,but distinguishing to the other farms.BOX-PCR demonstrated that the genetic correlation was higher in the same farm than in the others.Resistant phenotype and genotype in the same farm were not significantly (P＞0.05).These above paved a way to certify that strains,plasmids and antibiotic resistance are regional and transmissible in the pig farm.
在本实验室已经对分离的猪源肠外致病性大肠杆菌进行了血清分型、毒力基因鉴定、动物感染实验的基础上,为进一步了解该类菌株耐药的真实情况和传播的可能性,本研究对临床猪源肠外致病性大肠杆菌分离株进行了药物敏感性及遗传相关性检测,以提供可靠的临床用药和防治依据,具体工作如下:
1、完成了315株猪源肠外致病性大肠杆菌(ExPEC)的MIC测定。结果表明我实验室分离ExPEC菌株耐药谱广,耐药性强,多数菌株仅对头孢菌素敏感;分离菌株耐药谱有46种,95.2%的菌株可以耐受5种以上药物,60.6%的菌株耐受10种以上药物;AMP+STR+KAN+NEO+GEN+CIP+OFX+TE+TMP+SDM是最为流行的耐药谱,占到26%。
2、完成315株猪源肠外致病性大肠杆菌(ExPEC)对6大类27种耐药基因的检测。单一抗性携带多基因现象普遍存在,这表明大肠杆菌的耐药呈现多种机制混合的复杂情况。β-内酰胺类抗性的耐药基因以bla_(TEM)为主,氨基糖苷类耐药基因以aac(3)-Ⅳ,strAstrB,aphA1为主,喹诺酮类耐药以gyrA突变为主,四环素耐药基因以tet(B)和tet(G)为主,甲氧苄啶类dfrA7,dfrA12,dfrA13分布广泛,磺胺类sul1,sul2分布最广。
3、利用intI基因检测315株猪源肠外致病性大肠杆菌(ExPEC)的I型整合子携带情况,并对扩增阳性菌株进行可变区基因盒定性。86(27.3%)株ExPEC含有intI基因即I型整合子阳性。对VR的检测发现其主要含有0.5kb,08kb,1.0kb1.5kb和1.8kb五种基因盒,分别携带dfrA14;aadA2;aadA22,aadA5,dfrA17;dfrA17-aadA2,dfrA1-aadA1;dfrA12-aadA2,dfrA17-aadA5。
4、检测产ESBL菌株并完成其基因分型。检出2株产ESBL菌株,耐药谱均在10以上,PCR分型测序结果表明其含有国内广泛流传的普通β-内酰胺酶bla_(TEM-1)和超广谱β-内酰胺酶bla_(CTX-M-14)。
5、完成了对分离自3个省份12个猪场(区)的38株高度耐药菌株的遗传特性研究,其质粒图谱检测结果显示同一场(区)流行1-2种质粒,不同场(区)质粒图谱不同;BOX-PCR指纹显示同场(区)菌株同源性高于不同场(区),且同场(区)菌株的MIC值和耐药基因差异不显著(P＞0.05)。证明菌株、质粒、整合子和耐药性具有场(区)内区域性且可在一定区域内传播。
Escherichia coli is an important zoonotic pathogen.Piglets edema disease,yellow or white scour of newborn piglets are typical diseases caused by E.coli.They trouble the veterinary clinic for long.New symptoms such as Pneumonia,nephritis, meningitis and septicemia caused by E.coli are increasing with the development of swine industry.And these extraintestinal infections are also similar to human.The specialized strains of E.coli that cause most extraintestinal diseases were classified as extraintestinal pathogenic E.coIi(ExPEC),which shows higher morbidity,mortality and no typical serotypes.Now antimicrobial therapy is the only available tool for treating clinical infections of ExPEC because of no vaccine.The resistance of ExPEC emerged due to the wrong use of the antibiotics and the feedstuff additive at the clinic. Extended-spectrumβ-lactamase(ESBL)-producing strains and the dissemination of resistant vectors are of increasing concern of the failure of antibiotic therapy and public health.
It's helpful for clinical treatment to detect the resistance of ExPEC to common antibiotics and the possibility of resistance transmission,based on the detecting of serotype,virulence factors and animal experiments of ExPEC.so we carried out the following work to analyse the drug senstivity and molecular characteristics for swine ExPEC:
1.We researched the drug minimum inhibitory concentration of 315 swine ExPEC isolates from the clinic.These isolates of Expec showed multiple drug resisitance, and only sensitived to cephalosporins.95.2%were resistant to at least five antimicrobials and 60.6%resistant to more than 10.the most popular multiresistant patterns was AMP+STR+KAN+NEO+GEN+CIP+OFX+TE+TMP+SDM.
2.We established the PCR methods for detection of 27 resistance genes includingβ-lactams,aminoglycosides,tetracyclines,quinolones,trimethoprim and sulfonamides,then appliesed them to the ExPEC.For each antimicrobial agent, ampicillin resistance was primarily mediated by bla_(TEM),aminoglycosides by aac(3)-Ⅳ, strAstrB and aphA1,quinolones resistance by mutations in gyrA,tetracycline resistance by tet(B) and tet(G),trimethoprim resistance by dfrA7,dfrA12,dfrA13,and sulfadimidine resistance by sul1 and sul2.Strains which harbored several genes that conferred resistance to the same antimicrobial agent were often significantly more multiresistant than others.
3.We detected the intⅠgene to identify integron,and amplified variable region to determine gene cassettes.86(27.3%) ExPEC isolates were integron positive,which harbored dfrA14,aadA2,aadA22,aadA5,dfrA17,dfrA17-aadA2,dfrA1-aadA1, dfrA12-aadA2,dfrA17-aadA5 gene cassettes in five major different variable regions.
4.We identified ESBL isolates and classified their genotypes.Two isolates with bla_(TEM-I)+bla_(CTX-M-I4)-genotype ESBL were selected.They were both resistant to more than 10 drugs.
5.We reseached genetic correlation for 38 swine ExPEC isolated from twelve pig farms of three provinces.Analysis of plasmid map proved that 1 to 2 different plasmids were popular in a farm,but distinguishing to the other farms.BOX-PCR demonstrated that the genetic correlation was higher in the same farm than in the others.Resistant phenotype and genotype in the same farm were not significantly (P＞0.05).These above paved a way to certify that strains,plasmids and antibiotic resistance are regional and transmissible in the pig farm.
引文
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