Fluoroquinolone and multidrug resistance phenotypes associated with the overexpression of AcrAB and an orthologue of MarA in Yersinia enterocolitica

Abstract

Quinolone resistance among clinical isolates is of increasing importance. This phenotype particularly affects the nalidixic acid resistance levels and is also increasing among Yersinia enterocolitica strains. This study investigated the quinolone resistance mechanisms acquired in vitro by a Y. enterocolitica clinical isolate exposed to increasing concentrations of ciprofloxacin in a multi-step selection process. The fluoroquinolone-susceptible clinical isolate, Y.83-wt, the fluoroquinolone-resistant mutant, Y.83-64, and intermediate mutants were analysed by QRDR sequencing and MIC determination. Four different QRDRs (quinolone resistance-determining regions) mutations were characterised in Y.83-64: one in gyrA, one in gyrB, and two in parC. A significant increase in the MICs of ciprofloxacin, norfloxacin, nalidixic acid, and other unrelated antibiotics was detected in Y.83-64. Furthermore, the bacterial growth rate was assessed for strains Y.83-wt and Y.83-64. The analysis reported no significant differences between both strains. Cell envelope protein approach revealed an overexpression of both AcrA and AcrB proteins in Y.83-64. RT-PCR analyses were also carried out as was sequencing of known AcrAB regulators in Yersinia. The RT-PCR analysis showed an increased transcriptional level of a marA orthologue, marA_Ye, in Y.83-64. The sequencing results reported no change in the acrR gene or in the promoter sequence of the acrAB operon when comparing Y.83-wt with Y.83-64. One differential mutation was detected within the marA_Ye promoter in Y.83-64. Thus, the fluoroquinolone resistance phenotype acquired by Y.83-64 relies on the acquisition of 4 QRDR mutations in addition to the overexpression of AcrAB, which is likely triggered by increased expression levels of marA_Ye.

Introduction

Yersinia enterocolitica is a facultative anaerobic pathogen that has been divided into several bioserotypes, only a few of which are associated with human disease (Fredriksson-Ahomaa and Korkeala, 2003). Antibiotic treatment can be required in particular situations in which ceftriaxone and ciprofloxacin have shown to be efficient therapies (Jimenez-Valera et al., 1998). However, despite the lack of nalidixic acid resistance among Y. enterocolitica clinical isolates reported in Spain before 1995 (Prats et al., 2000, Capilla et al., 2004), several studies have described an increasing number of isolates showing nalidixic acid resistance over recent years, representing approximately 5% during the period 1995–2000 (Fernandez-Roblas et al., 2000, Prats et al., 2000), and reaching approximately 23% in 2002 (Capilla et al., 2004). Similar results have been detected among other enteric pathogens, such as Salmonella spp., in which the percentage of nalidixic acid-resistant strains rose from 14% in 2000 to 20% in 2004. However, rates of ciprofloxacin resistance are significantly low and represent around 0.8% in Salmonella (Threlfall et al., 2003, Meakins et al., 2008), whereas none have been reported for Y. enterocolitica (Rastawicki et al., 1999, Fernandez-Roblas et al., 2000, Abdel-Haq et al., 2006). Nonetheless, decreased susceptibility to ciprofloxacin (MIC 0.125–1 mg/L) is significantly prevalent, not only because all nalidixic acid-resistant strains usually show this phenotype, but also because this can be detected among nalidixic acid-susceptible isolates (Sanchez-Cespedes et al., 2003, Capilla et al., 2004, Abdel-Haq et al., 2006). Thus, in-depth characterisation of quinolone resistance mechanisms is becoming significantly important.

The genetic determinants responsible for quinolone resistance acquisition in most Enterobacteriaceae include chromosomal mutations and transferable genetic elements encoded in plasmids. Chromosomal mutations, the most important mechanism, were initially localized within the quinolone resistance-determining regions (QRDRs) of the target genes (those encoding the A and B subunits for the DNA gyrase, gyrA and gyrB, and the topoisomerase IV, parC and parE, respectively). Alternatively, other kinds of chromosomal mutations have been reported to impair the expression of the proteins determining quinolone uptake by overexpressing efflux pumps or repressing the expression of outer membrane porins (Fabrega et al., 2009b). AcrAB/TolC is the main efflux pump characterised to confer the multidrug resistance (MDR) phenotype among Enterobacteriaceae (Okusu et al., 1996). Three regulators belonging to the AraC/Xyls family (Gallegos et al., 1997), i.e. MarA (George and Levy, 1983), SoxS (Amabile-Cuevas and Demple, 1991), and Rob (Skarstad et al., 1993), have been found to activate acrAB transcription in Escherichia coli by binding to the marbox sequence characterised in its promoter (Martin et al., 1999).

These resistance mechanisms have not been extensively studied among the human pathogenic yersiniae species. However, few studies analysing the mutations acquired within the gyrA gene among a set of ciprofloxacin-resistant Y. pestis mutants (derivatives of the avirulent strain KIM5) obtained in vitro have been performed (Lindler et al., 2001, Hurtle et al., 2003). On the other hand, more recent studies performed with Y. enterocolitica clinical isolates have also been carried out (Sanchez-Cespedes et al., 2003, Capilla et al., 2004). They showed that target gene mutations were only acquired in the QRDR of gyrA, whereas none was detected in parC. Furthermore, the use of 20 mg/L of the efflux pump inhibitor PAβN (Phe-Arg-β-naphthylamide) resulted in a decrease in the MICs of nalidixic acid, but no change was detected in the MICs of ciprofloxacin. In addition, the locus YPO2243 of Y. pestis CO92 (NC 003143) has recently been reported to encode an orthologue of MarA with the same ability to induce multidrug resistance through AcrAB overexpression as the E. coli MarA (Udani and Levy, 2006). However, the role played by AcrAB in Y. enterocolitica has yet to be studied, despite full-genome sequencing of the Y. enterocolitica strain 8081 revealing the presence of an orthologous locus (Thomson et al., 2006).

The main objective of this study was to characterise the fluoroquinolone resistance mechanisms acquired in an “in vitro” selected high-level ciprofloxacin-resistant mutant of Y. enterocolitica.