Study of the resistant mechanisms of fluoroquinolones and chloramphenicols in two indicator bacteria

• Main Authors: Hung-Chih Kuo, 郭鴻志
• Other Authors: Shao-Kuang Chang
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/57607975882429237850

博士 === 臺灣大學 === 獸醫學研究所 === 98 === In recent years, international and domestic surveys to assess antimicrobial susceptibility among indicator bacteria (Escherichia coli and Enterococcus spp.) and zoonotic bacteria (Salmonella spp. and Campylobacter spp.) isolated from sick food-producing animals had been addressed. However, the background information about the bacterial resistance in healthy animals is totally neglected. The major focus of this dissertation is to investigate the levels and mechanisms of resistance of fluoroquinolones (FQ) and chloramphenicols in two indicator bacteria (Escherichia coli and Enterococcus faecalis) from two major food-producing animals, pig and chicken, in Taiwan. Our results indicated that the qnrS genes, but not qnrA, qnrB, and qepA were detected in 12/360 pig E. coli isolates (3.33%) and in 6/300 chicken E. coli isolates (2%). Southern blot hybridization analysis indicated that qnrS was located on plasmids ranging in size from 50-165 kb. Eleven of the 18 qnrS positive isolates which showed high ciprofloxacin resistance phenotype (MIC ≥ 8 mg/L) also had amino acid sequence variations in chromosomal quinolone resistance-determining regions of gyrA and parC. Besides, only two qnrS-positive isolates carried the aac(6’)-Ib-cr variant that mediating FQ acetylation. For the high percentage resistance of cephalosporins, the blaCTX-M gene was also examined from qnrS-positive isolates. The blaCTX-M gene was detected in fifteen isolates (15/18, 83.3%) in which 12 isolates were blaCTX-M-1 and 3 isolates were blaCTX-M-15. These results concluded that a close linkage between qnrS gene and blaCTX-M-1. The CTX-M and Qnr-based mechanisms might be co-emerging in E. coli strains isolated from healthy chickens and pigs under a selective pressure of quinolones and cephalosporines administration. The percentage of E. faecalis isolates resistant to ciprofloxacin was 54.0% (162/300) in pigs and 53.5% (107/200) in chickens. Two hundred sixty-nine ciprofloxacin-resistant isolates showed different levels of resistance to ciprofloxacin (MIC 4-512 mg/L), enrofloxacin (MIC 8-512 mg/L) and moxifloxacin (MIC 0.5-512 mg/L). Two mutations associated with resistance were detected in GyrA at Ser83 (to Arg/Ile) and Glu87 (to Lys/Gly) and one mutation was found in ParC at position 80 (Ser to Ile). In addition, triple-point mutation in DNA gyrase (GyrA) and topoisomerase IV (ParC) of E. faecalis was firstly reported in this paper. In the presence of reserpine, the level of resistance to ciprofloxacin for these 36 strains was decreased. The effect of reserpine on ciprofloxacin resistance was correlated with the level of expression of the emeA gene. In E. faecalis, our results demonstrate that not only point mutations in topoisomerase IV and DNA gyrase but also the efflux pump is the main mechanism for quinolones resistance. A similar approach but different sets of procine samples were used to elucidate the possible genetic determinants of resistance to florfenicol and chloramphenicol in porcine Escherichia coli. Six hundread fecal samples of healthy pigs from 50 different farms were collected from 2003 to 2007. The florfenicol resistance in the isolated E. coli strains doubled from 39.2% in 2003 to 78.3% in 2007. A total of 351 florfenicol-resistant E. coli isolates were isolated from nursery pigs (61.5%), grower-finisher pigs (62.5%), and sows (51.5%). The prevalence of resistance genes, floR, cmlA, cat-1, cat-2 and cat-3, was 82.9, 61.3, 10.8, 3.7, and 0%, respectively. Of the 351 florfenicol-resistant isolates, 184 (52.4%) were positive for both floR and cmlA. Furthermore, the results of efflux inhibitor studies with Phe-Arg-β-naphthylamide showed a 4- to 64-fold decrease in the florfenicol MIC levels. The FloR efflux pump may play a role in phenicol resistance among porcine E. coli isolates in Taiwan. In conclusions, our data clearly indicated that bacteria can continuously use various mechanisms to produce and/or maintain their antimicrobial resistance in healthy food-producing animals. The added risk to animal’s helath from resistance among E. coli and Enterococcus faecalis selected by florfenicol and enrofloxacin use is large. This reservoir phenomenon may directly enhance the expression and/or the levels of resistance/co-resistance as well as indirectly affect the public health, preventive medicine and clinical therapy. Furthermore, other microorganisms that might be expected to have become more susceptible as a consequence of withdrawing of chloramphenicol and water medication of enrofloxacin. Thus, the further adequate studies need to be done.