Origin of oqxAB and its contribution in fluoroquinolone resistance in salmonella spp.

Abstract

Bacterial infections have become a serious public health issue worldwide. Antimicrobial agents may not be necessary for treatment of mild cases, they can be life-saving in systemic infections or diseases affecting immunocompromised patients. Fluoroquinolones have been regarded as one of the frontline antimicrobial drugs. Resistance to this category of antibiotic is mediated by a variety of mechanisms, amongst them, acquisition of Plasmid Mediated Quinolone Resistance (PMQR) determinants is a major route by which bacteria evolve to become fluoroquinolone resistant. A PMQR, oqxAB, which encodes a mobile Resistance-nodulation-division family efflux pump, has become increasingly prevalent amongst fluoroquinolone resistant Gram negative bacteria including Escherichia coli and Klebsiella pneumoniae. The works described in this thesis aimed to assess the prevalence of oqxAB in environmental and clinical Salmonella isolates, determine the relative functional role of this element in fluoroquinolone resistance development, identify its evolutionary origin, and elucidate the mechanisms by which expression of PMQR genes are regulated in Salmonella spp.. Prevalence and antimicrobial resistance profile of Salmonella spp. in meat products being sold in Hong Kong was investigated. The oqxAB element, which was encoded in transposon Tn6010, was detectable in two Salmonella isolates for the first time. The study was then expanded to include clinical S. Typhimurium isolates recovered in Hong Kong and China during the period of 2005-2011, with results showing that clinical S. Typhimurium isolates were increasingly resistant to ciprofloxacin and antibiotics of the ACSSuT group. Importantly, we found that this trend of increasing resistance rate correlated with an increasing prevalence of oqxAB and another PMQR gene aac(6')-Ib-cr amongst clinical Salmonella isolates; in particular, oqxAB became detectable only from 2006 onwards. In addition, these two PMQR determinants exhibited strong linkage with the ACSSuT resistance phenotype and were mainly confined to the S. Typhimurium ST34 strain. It was also found that oqxAB and aac(6')-Ib-cr contributed significantly to the formation of ciprofloxacin resistance in S. Typhimurium by facilitating acquisition of an additional single mutation in the gyrA gene. Both oqxAB and aac(6')-Ib-cr were encoded on plasmids of various sizes, which upon transformation into a oqxAB-negative Salmonella host led to a 4-fold increase in CIP MIC. Furthermore, the presence of oqxAB and aac(6')-Ib-cr in Salmonella caused an dramatic increase in the mutation prevention concentration (MPC) of ciprofloxacin. Attempt was then made to probe the origin of the mobile RND-type efflux pump oqxAB. It was found that oqxAB had been harboured by K. pneumoniae isolates recovered before the year 1984. Sequence and phylogenetic analysis confirmed that the oqxAB operon in K. pneumoniae was genetically closest to their Tn6010 counterparts recoverable from other Enterobacteriaceae since 2003. K. pneumoniae strains generally did not exhibit a typical oqxAB-mediated phenotype despite harboring oqxAB chromosomally. The data therefore suggests that oqxAB originated from K. pneumoniae and does not necessarily function as a PMQR determinant in this bacterial species under normal circumstances. Contrary to the chromosomally-encoded oqxAB element, the activator gene rarA was not captured by Tn6010. However, we demonstrated in S. Typhimurium by gene knockout study that the global regulator ramA has a role in activating oqxAB expression, unveiling the role of global regulatory mechanisms in controling the expression of foreign resistance genes.