Lack of efflux mediated quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi A

Salmonella enterica serovars Typhi and Paratyphi A isolates from human patients in France displaying different levels of resistance to quinolones or fluoroquinolones were studied for resistance mechanisms to these antimicrobial agents. All resistant isolates carried either single or multiple target gene mutations (i.e., in gyrA, gyrB, or parC) correlating with the resistance levels observed. Active efflux, through upregulation of multipartite efflux systems, has also been previously reported as contributing mechanism for other serovars. Therefore, we investigated also the occurrence of non-target gene mutations in regulatory regions affecting efflux pump expression. However, no mutation was detected in these regions in both Typhi and Paratyphi isolates of this study. Besides, no overexpression of the major efflux systems was observed for these isolates. Nevertheless, a large deletion of 2334 bp was identified in the acrS-acrE region of all S. Typhi strains but which did not affect the resistance phenotype. As being specific to S. Typhi, this deletion could be used for specific molecular detection purposes. In conclusion, the different levels of quinolone or FQ resistance in both S. Typhi and S. Paratyphi A seem to rely only on target modifications.


INTRODUCTION
Enteric fever caused by the human-adapted pathogens Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A), B, and C, remains a major health problem (Crump and Mintz, 2010). A global epidemiologic study estimated that during the year 2000 typhoid fever caused 21.7 million illnesses and 21,7000 deaths and paratyphoid fever caused 5.4 million illnesses (Crump et al., 2004). During the past decade S. Paratyphi A was responsible for a growing proportion of enteric fever in Asia (Ochiai et al., 2005;Crump and Mintz, 2010). Enteric fever being associated with poor sanitation and unsafe food and water, it particularly affects children and adolescents in developing countries of Asia, Africa and Latin America (Crump et al., 2004;Bhan et al., 2005;Crump and Mintz, 2010). In developed countries, patients are most often ill-returned travellers or migrant workers (Bhan et al., 2005;Connor and Schwartz, 2005;Hassing et al., 2013).
To treat these infections, fluoroquinolones (FQ) and thirdgeneration cephalosporins have been considered as first-line drugs, owing to the resistance to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole that appeared during the 1980s (Hassing et al., 2011(Hassing et al., , 2013. Multidrug resistance (MDR) in S. Typhi is encoded mainly by resistance genes carried by large conjugative plasmids and has been reported worldwide (Le et al., 2007). As a consequence of a widespread FQ usage, S. Typhi and S. Paratyphi A isolates resistant to nalidixic acid (NAL R , minimum inhibitory concentration [MIC] > 16 mg/L) and with decreased susceptibility to ciprofloxacin (CIP DS , MIC 0.125-1.0 mg/L) have also emerged. Such NAL R -CIP DS S. Typhi and S. Paratyphi A have been isolated in endemic areas and also in developed countries (Roumagnac et al., 2006;Le et al., 2007;Gaborieau et al., 2010;Accou-Demartin et al., 2011;Hassing et al., 2011Hassing et al., , 2013. Resistance to quinolones in Salmonella spp. is mostly attributed to point mutations in the quinolone resistancedetermining regions (QRDRs) of the target genes gyrA, gyrB, parC, and parE (Cloeckaert and Chaslus-Dancla, 2001;Piddock, 2002;Velge et al., 2005;Giraud et al., 2006). For the gyrA gene, coding the A subunit of DNA gyrase, a single mutation resulting in an amino acid substitution at the position 83 (Serine to Phenylalanine or to Tyrosine) or at the position 87 (Aspartic acid to Asparagine or Glycine) has been the most frequently described in NAL R -CIP DS S. Typhi and S. Paratyphi A isolates (Bhan et al., 2005;Renuka et al., 2005;Le et al., 2007;Gaborieau et al., 2010;Hassing et al., 2011). A second mutation leading to the amino acid change at the position 80 (Serine to Isoleucine or to Arginine) of the ParC subunit of topoisomerase IV was described to increase the CIP MIC (≥ 0.5 mg/L) in S. Typhi and S. Paratyphi A human isolates from India (Gaind et al., 2006). Whereas three mutations, i.e., a double mutation in gyrA at both codons 83 and 87 and one mutation in parC, were shown to confer CIP resistance (MIC >1 mg/L) in S. Typhi and S. Paratyphi A human isolates from India or from Taiwan (Gaind et al., 2006;Lee et al., 2013).
Moreover, the varying levels of CIP resistance observed in S. Typhi and S. Paratyphi A isolates with only a single gyrA mutation suggest that other mechanisms could be involved in quinolone resistance in this serovar (Renuka et al., 2005).
Resistance to FQ in S. Typhimurium has also been attributed to active efflux mechanism, due to overproduction of the AcrAB-TolC efflux system (Giraud et al., 2000(Giraud et al., , 2006Cloeckaert and Chaslus-Dancla, 2001;Piddock, 2006). We have previously reported the contribution of the AcrAB-TolC efflux system in resistance to FQ in several MDR epidemic clones of S. Typhimurium, such as S. Typhimurium of phage types DT204 or DT104 (Baucheron et al., 2002(Baucheron et al., , 2004a. Among the chromosomal loci affecting AcrAB-TolC expression, the ramRA locus appears to be the most important 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;Bailey et al., 2010). The latter is involved in upregulating expression of the AcrAB-TolC system (Nikaido et al., 2008;Bailey et al., 2010). 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 or Kentucky 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;Baucheron et al., 2013).
In the present study, we have characterized mechanisms involved in resistance to quinolones or fluroquinolones in 21 S. Typhi and S. Paratyphi A strains displaying different levels of resistance to these drugs and isolated from patients in France during the period 1997-2008. For a subset of strains, with suspected increased efflux activity, we investigated the occurrence of mutations in the global ram, sox and mar regulatory loci of AcrAB-TolC, and in the local acrR and acrS repressor genes of the AcrAB and AcrEF pumps, respectively (Abouzeed et al., 2008;Kehrenberg et al., 2009).

BACTERIAL STRAINS
The twenty one strains including 16 S. Typhi and 5 S. Paratyphi A selected for this study were collected by the French National Reference Center for Salmonella, Institut Pasteur, Paris, France. They were isolated in France from travellers or migrants between 1997 and 2008 ( Table 1). The selection was made to obtain diversity in terms of geographic origin, year of isolation, genetic lineages (haplotype for S. Typhi; Roumagnac et al., 2006), and phenotype of resistance to quinolones ( Table 1).

ANTIMICROBIAL SUSCEPTIBILITY TESTING
Antimicrobial susceptibility was investigated by the standard disk diffusion method according to the recommendations of the Antibiogram Committee of the French Society for Microbiology (CA-SFM) (www.sfm-microbiologie.org/). The MICs of NAL and CIP were determined by the standard agar doubling dilution method as described previously (Baucheron et al., 2002). The NAL R isolates were defined as having a MIC > 16 mg/L. The CIP R isolates were defined as having a MIC > 1 mg/L and CIP DS isolates as having a MIC comprised between 0.125 and 1.0 mg/L (Accou- Demartin et al., 2011;Hassing et al., 2013). MICs of these antibiotics were also determined in the presence of the efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN, Sigma) at the following concentrations: 10, 20, 30, 40, 50, and 60 mg/L.

RESISTANCE PHENOTYPES AND TARGET-AFFECTING MECHANISMS
The twenty one S. Typhi and S. Paratyphi A strains of this study were isolated in France but acquired abroad, mainly in Asia and Africa (Table 1). Among the S. Typhi strains, all but two were NAL R (MIC > 16 mg/L). One of the two strains was pansusceptible and the second was CIP DS but only categorized as intermediate for NAL (NAL I , MIC 16 mg/L). Of the 14 NAL R strains, one was CIP R , 12 were CIP DS and one was CIP S (MIC 0.03 mg/L). Eight NAL R S. Typhi strains were also multidrug resistant. The majority of the NAL R S. Typhi strains belonged to haplotype H58 which had emerged in Southern Asia during the mid 1990s (Roumagnac et al., 2006;Le et al., 2007). Among the S. Paratyphi A strains, all but two were NAL R . One of the two strains was pansusceptible and the second was NAL I -CIP DS . The three others were NAL R -CIP DS . As shown in Table 1, both NAL I -CIP DS had a mutation resulting in an amino acid substitution at position 464 of GyrB: serine to tyrosine for the S. Typhi isolate and serine to phenylalanine for the S. Paratyphi A isolate. The most frequent mechanism of resistance of NAL R -CIP DS (n = 17) and NAL R -CIP S (n = 1) strains, whatever the serovar, was a substitution at position 83 (serine to phenylalanine, n = 12, 66.6%) of GyrA. Other GyrA modifications were observed at position 83 (serine to tyrosine) in two isolates or at position 87 (aspartic acid to glycine, aspartic acid to asparagine) in one isolate for each. As described previously, a single substitution in GyrA was associated with resistance to nalidixic acid and decreased susceptibility to CIP (Bhan et al., 2005;Le et al., 2007;Gaborieau et al., 2010;Hassing et al., 2011). One exception was the S. Typhi strain 4 (02) MB, which was NAL R -CIP S (and not CIP DS ) despite a mutation in gyrA resulting in substitution serine to tyrosine at position 83.
Additional substitutions were found in ParE of 2 NAL R -CIP DS S. Typhi strains that led to amino acid substitution aspartic acid to asparagine at position 420. In both cases, a 2-fold increase of NAL MICs was observed.
In the CIP R S. Typhi isolate, three mutations leading to a double substitution in GyrA at positions 83 (serine to phenylalanine) and 87 (aspartic acid to asparagine) and one substitution at the position 80 of ParC (serine to isoleucine), as observed in previous studies (Renuka et al., 2005;Gaind et al., 2006;Lee et al., 2013).
The NAL R and CIP DS S. Typhi and S. Paratyphi A strains harboring a single substitution in GyrA showed various values for NAL (64-1024 mg/L) and CIP (0.03-0.5 mg/L) MICs which suggested the presence of other mechanisms of resistance. Since the plasmid-mediated quinolone resistance-conferring genes qnrA, qnrB, qnrD or qnrS, qepA, and aac(6 )-Ib-cr were not detected, we investigated the role of the AcrAB-TolC efflux system.

INVOLVEMENT OF EFFLUX
None of the S. Typhi strains showed significant AcrA overproduction by dot blot, but nevertheless all S. Paratyphi A isolates showed a 2 or 3-fold increased AcrA production relative to the susceptible S. Typhi isolate (Table 1). Thus, overproduction of AcrA seems not to be involved in CIP DS isolates compared with the susceptible isolates of S. Typhi or S. Paratyphi A. In presence of the efflux pump inhibitor PAβN (20 or 40 mg/L), the CIP MICs similarly decreased (4 or 8-fold) in CIP DS and in susceptible strains ( Table 3 and data not shown), which is in accordance with previous studies on S. Typhimurium and corresponds to a decrease of resistance level observed for acrB or tolC deletion mutants (Baucheron et al., 2002(Baucheron et al., , 2004b. Despite a lack of evidence of increased efflux in the resistance phenotype, we measured by qRT-PCR the transcription levels of efflux related genes acrA, acrF, acrB, tolC, ramA, and ramR in CIP DS non-MDR strains and in the CIP R strain. No differences were detected in the transcription levels of these genes, between susceptible, CIP DS and CIP R strains, whatever the serovar ( Table 3). In addition, no mutations were detected in the regulatory regions of the AcrAB-TolC efflux system. However, during the screening of the regulatory regions, we identified a single large deletion of 2334 bp in the acrS-acrE region of all S. Typhi strains, including the susceptible one (Table 3). This deletion encompassed the acrS gene, that encodes a transcriptional repressor, and a large part of the acrE gene that encodes the AcrE periplasmic lipoprotein, which is homologous to AcrA (Olliver et al., 2005). This 2334 bp deletion was also observed in the acrS-acrE region of sequenced genomes of MDR S. Typhi CT18 strain (Parkhill et al., 2001) and pansusceptible TY2 strain (Deng et al., 2003) (Figure 1). Previously, it has been shown that acrS deletion in S. Typhimurium does not affect acrEF expression (Olliver et al., 2005). Similarly the natural acrSE deletion detected in S. Typhi had no impact on the acrF transcription level as observed in this study. To our knowledge, this is the first description of such a natural acrS-acrE chromosomal deletion and seems specific to S. Typhi since it was not detected in all currently sequenced genomes of the other serovars (not shown).

CONCLUSIONS
The main mechanisms involved in quinolone or FQ resistance in both S. Typhi and S. Paratyphi A are target modifications.
In contrast to what is seen in enteric pathogenic serovars, such as Typhimurium or the emerging CIP R Kentucky ST198 clone , increased efflux pump productionmediated mechanisms seem to be totally absent in both S. Typhi and S. Paratyphi A. The deletion identified in the acrSEF region, although not involved in the resistance phenotype, may be helpful for the specific detection of S. Typhi.