ramR mutations affecting fluoroquinolone susceptibility in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198

A screening for non-target mutations affecting fluoroquinolone susceptibility was conducted in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198. Among a panel of representative isolates (n = 27), covering the epidemic, only three showed distinct mutations in ramR resulting in enhanced expression of genes encoding the AcrAB-TolC efflux system and low increase in ciprofloxacin MIC. No mutations were detected in other regulatory regions of this efflux system. Ciprofloxacin resistance in serovar Kentucky ST198 is thus currently mainly due to multiple target gene mutations.


INTRODUCTION
Fluoroquinolones, together with extended-spectrum cephalosporins, are the treatment of choice for nontyphoid salmonellosis, as stable resistance to the most common members of different families of antimicrobial agents (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) has developed during the 1990s with the epidemic Salmonella enterica serovar Typhimurium phage type DT104 (Cloeckaert and Schwarz, 2001). Emerging resistance to fluoroquinolones in Salmonella spp. has been reported for both human and animal cases and is thus threatening to become a serious public health problem (Cloeckaert and Chaslus-Dancla, 2001;Piddock, 2002;Velge et al., 2005;Giraud et al., 2006). Of particular concern is the international spread of ciprofloxacin-resistant serovar Kentucky ST198 (Le Hello et al., 2011). This clone is not only highly resistant to ciprofloxacin but also multidrug-resistant (MDR) due to the presence of the Salmonella genomic island 1 (SGI1) carrying a multiple antibiotic resistance gene cluster, mostly variant SGI1-K carrying another resistance gene cluster (Doublet et al., 2008;Le Hello et al., 2011). SGI1 was initially identified in MDR serovar Typhimurium DT104 (Boyd et al., 2001), but nor the MDR serovar Typhimurium DT104 clone neither other MDR S. enterica serovars carrying SGI1 or variants of it, have to our knowledge been reported to display this high-level ciprofloxacin resistance.
In Salmonella spp., quinolone/fluoroquinolone resistance is mostly attributed to point mutations in the quinolone resistancedetermining regions (QRDRs) of the target genes gyrA, gyrB, parC, and parE. For the gyrA gene, coding for the A subunit of DNA gyrase, mutations resulting in amino acid changes at Ser83 (to Phe, Tyr, or Ala) or at Asp87 (to Gly, Asn, or Tyr) are the most frequently observed in nalidixic acid-resistant strains (Cloeckaert and Chaslus-Dancla, 2001;Piddock, 2002;Velge et al., 2005;Giraud et al., 2006). High-level fluoroquinolone resistance has been reported in several S. enterica serovars (Choleraesuis, Schwarzengrund, Typhimurium) and is essentially due to the combination of several target gene mutations of which the most frequent are double mutations resulting in modifications of both residues 83 and 87 of GyrA together with one mutation leading to the amino acid change Ser80Ile in the ParC subunit of topoisomerase IV (Baucheron et al., 2002(Baucheron et al., , 2004Chu et al., 2005). In addition two main other mechanisms have been reported consisting of active afflux mediated by the chromosomally-encoded AcrAB-TolC efflux system and target protection by Qnr proteins which are mostly encoded by plasmids acquired by horizontal transfer (Giraud et al., 2006). However, according to the literature over 15 years, these mechanisms appear less frequently and thus from an epidemic point of view seem of lesser importance than multiple target gene mutations to reach high-level ciprofloxacin resistance and compromise treatment.
In the case of ciprofloxacin resistance in serovar Kentucky ST198, three combinations of multiple target modifications, acquired in a possible sequential way, have been reported consisting of a first GyrA Ser83Phe modification, followed by three different situations of a second GyrA modification at position 87, i.e., Asp87Asn, Asp87Gly, or Asp87Tyr, and finally the ParC modification Ser80Ile (Le Hello et al., 2011). Qnr proteins have not been reported yet as additional mechanism for this epidemic clone, and active efflux has been suspected in a previous study due to a moderate increase of production in some isolates of the AcrA protein belonging to the AcrAB-TolC efflux system (Weill et al., 2006). In the present study we assessed the frequency of enhanced efflux by AcrAB-TolC in a subset of serovar Kentucky ST198 strains of the 2000-2005 period of the epidemic. In case of significant increased production of AcrAB-TolC we investigated more deeply the regulatory mechanisms behind this overproduction, in particular the involvement of the ram, sox, and mar regulatory loci (Abouzeed et al., 2008;Kehrenberg et al., 2009). Among these loci, the ramRA locus appears to be the most important in regulating AcrAB-TolC expression in Salmonella spp. (Abouzeed et al., 2008;Kehrenberg et al., 2009). ramR encodes a repressor protein (RamR) belonging to the TetR family of repressor proteins, and has been shown to be the local repressor protein of ramA transcription (Abouzeed et al., 2008;Baucheron et al., 2012); while ramA encodes a transcriptional activator protein (RamA) belonging to the AraC/XylS family of regulatory proteins (Nikaido et al., 2008). The latter is involved in upregulating expression of the AcrAB-TolC system (Nikaido et al., 2008). Several mutations in ramR or its binding site upstream of ramA, affecting expression of this efflux system, have been detected in clinical isolates of serovar Typhimurium and of minor serovars Hadar, Infantis, Livingstone, or Schwarzengrund (Abouzeed et al., 2008;Kehrenberg et al., 2009;Hentschke et al., 2010;Akiyama and Khan, 2012).

MATERIALS AND METHODS
The 27 serovar Kentucky ST198 strains selected for this study are shown in Table 1. Bacterial isolates were selected for this study, based on their evolutionary history following the emergence of target gene mutations initially in gyrA at the commencement of the epidemic in 2000-2002, followed by isolates with additional mutations (in gyrA and parC) toward the end in [2002][2003][2004][2005] and which demonstrated a higher MIC toward ciprofloxacin. An additional criterion for selection consisted of the differences observed in ciprofloxacin MICs suggestive for another resistance mechanism than target gene mutation. MICs were determined as described previously (Baucheron et al., 2002(Baucheron et al., , 2004. SGI1 detection and characterization were performed as described previously (Boyd et al., 2001;Doublet et al., 2008). Efflux pump production was assessed by Dot blot using an anti-AcrA polyclonal antibody as described previously (Abouzeed et al., 2008). Occurrence of mutations affecting acrAB and tolC expression was determined by PCR and sequencing the regulatory regions ramR-ramA, acrR-acrA, marC-marO-marR-marA, soxS-soxR, and acrS-acrE using primers listed in Table 2. Transcription levels of ramA, acrA, and tolC were determined by qRT-PCR as described previously (Giraud et al., 2013).

RESULTS AND DISCUSSION
As shown in the Table 1 most of the strains selected carried SGI1 or variants of it and were thus MDR. They were all from human cases in France who acquired their infection during travel to Africa or India. As assessed by Dot blot, most of the strains (n = 24) did not show significant increased production of AcrA relative to susceptible serovar Kentucky reference strain 98K (AcrA production ratios from 1 to 2; Table 1). Relative to strain 98K, three strains showed a 3-fold increased AcrA production, and more suggestive for increased active efflux three strains a 5-to 6-fold increased production of AcrA (Table 1). Among these regulatory regions, mutations were detected only in the ramR open reading frame and in only three strains of this study ( Table 3). The mutations were distinct frame shift mutations and consisted of a GATC duplication for strain 02-2818, a G insertion for strain 05-8560, and a 91 bp deletion for strain 02-8141 (Figure 1). The role of these mutations in upregulating acrAB and tolC expression, and consecutive enhanced effluxmediated resistance, was further assessed by: (i) complementing with the wild-type ramR gene (using plasmid pRamR Abouzeed et al., 2008); (ii) determining the MICs of ciprofloxacin and unrelated antibiotic florfenicol shown to be substrate of AcrAB-TolC (Baucheron et al., 2002); and (iii) measuring expression of ramA, acrA, and tolC by qRT-PCR (Giraud et al., 2013). The results shown in Table 3 are in agreement with data published previously for other S. enterica serovars (Abouzeed et al., 2008;Kehrenberg et al., 2009), i.e. ramR mutations observed account for a 2-to 4fold increased resistance level by active efflux through enhanced expression of AcrAB-TolC. As also observed in previous studies, the effect of such mutations on ramA transcription level was significantly higher than on acrA or tolC transcription levels. It is somehow expected considering the direct local repressor activity of RamR on ramA transcription and the distant RamA transcriptional activator activity on acrAB and tolC (Abouzeed et al., 2008;Baucheron et al., 2012;Giraud et al., 2013). Non-target mutations as assessed in this study confirm they are infrequent in Salmonella spp. but seem nevertheless www.frontiersin.org July 2013 | Volume 4 | Article 213 | 3  mostly restricted to the ram regulatory region. Most mutations in the ramR-ramA region reported to date, as also shown in this study, are distinct and found in single isolates. To our knowledge only independent isolates of the epidemic ciprofloxacin-resistant serovar Typhimurium DT204 clone from the 1990s have been shown to carry the same mutation in ramR consisting of an insertion by an IS1 element (Abouzeed et al., 2008). We may nevertheless expect that the further global spread of ciprofloxacin-resistant serovar Kentucky ST198 and its resistance evolution will possibly, like in the case of serovar Typhimurium DT204, result in successful ramRmutation-carrying subclones.