| Summary: | 博士 === 國立臺灣大學 === 獸醫學研究所 === 100 === Riemerella anatipestifer is an etiological agent that can cause serious disease especially in waterfowl. Due to the existence of complex serotypes and limited cross reaction between serotypes, the vaccine cannot protect completely from the attack by wild R. anatipestifer. Therefore, chemotherapy and assessment of antimicrobial susceptibility are very important in the treatment of R. anatipestifer infection. In addition, the antimicrobial-resistance mechanisms in R. anatipestifer have not yet been reported, indicating the need for investigation. This study evaluated the susceptibility of 222 Taiwanese R. anatipestifer isolated from domestic waterfowl between 1999 and 2009 to 22 antimicrobials by agar disk diffusion method. Results showed that isolates were most resistant to colistin, nalidixic acid and kanamycin and most susceptible to nitrofurantoin which has been banned for use in domestic animals since 2004, doxycycline and amoxicillin/clavulanic acid (clavulanic acid is not approved for animals). Furthermore, the resistance rates to several antimicrobials and multi-resistance were increased obviously in 2009. In addition, from the minimum inhibitory concentration (MIC) test, 86.4% of isolates collected between 2005 and 2009 showed intermediate or resistance to chloramphenicol which has been prohibited for use in food-producing animals in Taiwan since 2003. The resistance gene was identified as catB gene with the prevalence of 78.4%. The position of the catB gene was then determined within a novel plasmid, designated pRA0511. pRA0511 also encoded other putative drug-resistance-associated proteins which were a TetX2 and a multi-drug ABC transporter permease/ATPase. CATs, however, are unable to inactivate florfenicol. Thus, 66 R. anatipestifer isolates collected between 1999 and 2009 were investigated for their susceptibility to chloramphenicol and florfenicol and the presence of floR gene. Results showed nine florfenicol intermediate or resistant isolates were all floR positive. The floR was located either in plasmid or chromosomal DNA and was as an efflux pump conferring resistance to both chloramphenicol and florfenicol. Furthermore, two novel floR-carrying plasmids designated pRA0726 and pRA0846 were sequenced completely. pRA0726 was 11,704 bp in size with 10 putative ORFs including a floR, a catB and a novel blaOXA-209 resistance genes. To our knowledge, this is the first report of presence of the floR and blaOXA-209 resistance genes in R. anatipestifer. The most differences between sequences of pRA0846 and pRA0726 were the absence of a blaOXA-209 gene in pRA0846. Plasmid curing tests demonstrated that pRA0726 carried functional coding proteins for resistance to phenicol and ß-lactam antimicrobials. On the other hand, this study also investigated the mutations of topoisomerases associated with quinolone resistance in clinical or laboratory-mutated R. anatipestifer. Most clinical isolates with reduced susceptibility to quinolones possessed amino acid alterations at Ser10 to Pro and/or Ser83 to Arg or Ile in GyrA and at Thr140 to Ile, Ile503 to Val and/or Gln155 to Val in GyrB. In laboratory mutants, all of the mutations were found in GyrA and GyrB but none in ParC and ParE. The most frequent mutation in GyrA was alterations at Ser83 to Ile or Asn which was concurred with aforementioned clinical strains, followed by alterations at Asp87 to Ala, Gly or Tyr and alterations at Ser84 to Pro. In addition, the mutations at positions 183 in GyrA and 452 and 471 in GyrB were newly described. Most mutants induced by higher concentration of quinolones and with more mutation points showed higher MIC values, although some mutants with identical mutation type showed different MIC profiles, suggesting that overexpression of the efflux system or other potential mechanisms might have also been induced. Conclusively, this study has described the trends in antimicrobial susceptibility of R. anatipestifer in Taiwan and the mechanisms of resistance to phenicols and quinolones for the first time in R. anatipestifer. In addition, three novel multidrug-resistance plasmids were characterized, more specifically associated with phenicol resistance.
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