- 學位論文
豬隻分離胸膜肺炎放線桿菌，大腸桿菌，豬霍亂沙氏桿菌之抗菌劑感受性與抗藥性基因檢測
Antimicrobial Susceptibility and Resistance Determinants of Porcine Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella enterica serovar Choleraesuis
摘要
台灣養豬業以抗菌劑預防或治療細菌性疾病已使用數十年，使用抗菌劑所造成的篩選壓力促使抗藥性細菌性病原的出現。本實驗探討三種豬隻常見病原，胸膜肺炎放線桿菌（Actinobacillus pleuropneumoniae），大腸桿菌（Escherichia coli），豬霍亂沙氏桿菌（Salmonella enterica serovar Choleraesuis）的抗菌劑感受性與抗藥性產生機制。對具nalidixic acid抵抗性豬霍亂沙氏桿菌的gyrA基因之QRDRs（quinolone resistance-determining regions）序列分析發現共有四種不同的點突變，位在第83與87個胺基酸，Ser-83-to-Phe（TCC→TTC），Ser-83-to-Tyr（TCC→TAC），Asp-87-to-Gly （GAC→GGC），Asp-87-to-Asn（GAC→AAC）。建立PCR-RFLP與MAS-touch down PCR方法以檢測gyrA基因之QRDRs中的點突變，共有50株自1997年至2002年豬隻分離之豬霍亂沙氏桿菌受檢，其中7株於第83個胺基酸具點突變，13株於第87個胺基酸具點突變，30株於第83與第87個胺基酸具點突變。於第83或第87個胺基酸具點突變的分離株，其enrofloxacin的最小抑菌濃度（minimum inhibition concentrations，MICs）均小於2 mg/ml，而具有雙重點突變之分離株的MIC值則由2到大於128 mg/ml。在豬與人的豬霍亂沙氏桿菌分離株均發現有第一型integron存在，此第一型integron攜帶dhfr，orfF，與aad2基因。另外，自1997年至2001年之豬隻分離胸膜肺炎放線桿菌，大腸桿菌，豬霍亂沙氏桿菌各60株，針對16種常用抗菌劑的感受性分析。由MIC值顯示，ceftiofur與amikacin對胸膜肺炎放線桿菌，大腸桿菌，豬霍亂沙氏桿菌分離株具高度活性；cephalothin，doxycycline，florfenicol與gentamicin則對胸膜肺炎放線桿菌有高度活性，對大腸桿菌與豬霍亂沙氏桿菌分離株具中等活性；trimethoprim只對胸膜肺炎放線桿菌分離株具有高度活性；flumequine只對豬霍亂沙氏桿菌分離株具有中等活性； ampicillin，chloramphenicol，kanamycin與tetracycline只對胸膜肺炎放線桿菌分離株具有中等活性；其餘抗菌劑對胸膜肺炎放線桿菌，大腸桿菌，豬霍亂沙氏桿菌分離株不具活性。建立以multiplex PCR檢測大腸桿菌與豬霍亂沙氏桿菌分離株的抗藥基因、integrase基因與毒力基因，大腸桿菌與豬霍亂沙氏桿菌分離株的integrase基因盛行率分別為97﹪與95﹪，大腸桿菌分離株ESBL基因（extended-spectrum β-lactamase）－tem與florfenicol抗藥性基因－floR的盛行率分別為38﹪與55﹪，豬霍亂沙氏桿菌分離株的tem，floR基因與質體毒力基因（spvC）分別為60﹪，25﹪與98﹪。由本實驗的結果可發現，台灣豬隻分離之胸膜肺炎放線桿菌，大腸桿菌，豬霍亂沙氏桿菌分離株出現多重抗藥性，因此必須對豬隻細菌性病原建立一套監測系統以監控多重抗藥性與fluoroquinolone抗藥性的出現。
並列摘要
Antimicrobial usage in pig industry in Taiwan for prevention and treatment of bacterial diseases has been applied for decades. The pressure of antimicrobial selection contributes to the emergence of antimicrobial-resistant porcine bacterial pathogens. Three common bacterial pathogens, Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella enterica serovar Choleraesuis, were investigated for antimicrobial susceptibility and mechanisms of antimicrobial resistance in this study. The quinolone resistance-determining regions (QRDRs) of the gyrA gene of nalidixic acid-resistant S. enterica serovar Choleraesuis isolated from swine were sequenced. Four types of point mutation were found in this regions: Ser-83-to-Phe (TCC→TTC), Ser-83-to-Tyr (TCC→TAC), Asp-87-to-Gly (GAC→GGC), and Asp-87-to-Asn (GAC→AAC). PCR-RFLP and MAS-touch down PCR were developed for detected of point mutations in QRDRs of the gyrA gene. Fifty swine isolates of S. enterica serovar Choleraesuis collected from 1997 to 2002 were examined by PCR-RFLP and MAS-touch down PCR. The result indicated 7 isolates with point mutations in codon 83, 13 with point mutations in codon 87, and 30 with double mutations in both codons 83 and 87. The minimum inhibition concentrations (MICs) of enrofloxacin of the isolates with a single mutation in codon 83 or 87 were < 2 mg/ml, while the MICs of the isolates with double mutations in both codon 83 and 87 ranged from 2 to > 128 mg/ml. A class I integron comprised of dhfr, orfF and aad2 was also identified in both human and swine S. enterica serovar Choleraesuis isolates. In addition, the susceptibility of 16 common antimicrobials among 60 isolates of each of A. pleuropneumoniae, E. coli, and S. enterica serovar Choleraesuis isolated from diseased pigs from 1997 to 2001 were tested. The MIC values indicated that ceftiofur and amikacin were highly active against isolates of A. pleuropneumoniae, E. coli, and S. enterica serovar Choleraesuis; cephalothin, doxycycline, florfenicol, and gentamicin were highly to A. pleuropneumoniae but moderately active to E. coli, and S. enterica serovar Choleraesuis; trimethoprim was only highly active against A. pleuropneumoniae; flumequine was only moderately active against S. enterica serovar Choleraesuis; ampicillin, chloramphenicol, kanamycin, and tetracycline were moderately active against A. pleuropneumoniae only. The other antimicrobials tested were inactive. E. coli and S. enterica serovar Choleraesuis isolates were analyzed for antimicrobial resistance genes, integrase gene and virulence factor by multiplex PCR. The prevalence of integrase gene in E. coli and S. enterica serovar Choleraesuis were 97% and 95%, respectively. The prevalence of extended- spectrum β-lactamase (tem) and florfenicol resistance (floR) in E. coli were 38% and 55%, respectively. The prevalence of tem, floR and spvC (Salmonella plasmid virulence C gene) in S. enterica serovar Choleraesuis were 60%, 25%, and 98%, respectively. This study indicated the emergence of multidrug resistance in A. pleuropneumoniae, E. coli and S. enterica serovar Choleraesuis is a serious problem in swine industry. A surveillance system is necessary on the swine industry to monitor the emergence of fluoroquinolone and/or multidrug resistant bacterial pathogens in Taiwan.
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