JAC Advance Access originally published online on March 16, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):886-892; doi:10.1093/jac/dkm072
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Molecular mechanisms of decreased susceptibility to fluoroquinolones in avian Salmonella serovars and their mutants selected during the determination of mutant prevention concentrations
1 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany 2 Bayer HealthCare AG, Animal Health Division, Clinical Research & Development Antiinfectives, 40789 Monheim, Germany 3 Unité Infectiologie Animale et Santé Publique, Institut National de la Recherche Agronomique, 37380 Nouzilly, France
* Corresponding author. Tel: +49-5034-871-242; Fax: +49-5034-871-246; E-mail: corinna.kehrenberg{at}fal.de
Received 7 December 2006; returned 26 January 2007; revised 12 February 2007; accepted 14 February 2007
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Abstract |
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Objectives: Salmonella enterica isolates of six serovars and mutants obtained during determination of mutant prevention concentrations (MPCs) were investigated for mechanisms of decreased susceptibility to fluoroquinolones.
Methods: The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE genes were sequenced. MIC values were determined in the presence/absence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or Phe-Arg-ß-naphthylamide (PAßN). PCR assays for the quinolone resistance genes qnrA, qnrB, qnrS or aac(6')-Ib-cr were applied. The MPC values of ciprofloxacin (MPCCIP) were determined for all isolates and selected mutants were investigated for their quinolone resistance mechanisms.
Results: In contrast to 11 nalidixic acid-susceptible isolates, 24 nalidixic acid-resistant isolates exhibited single mutations in gyrA (Asp-87 
Tyr, Gly, Asn or Ser-83 Phe, Tyr) or parC (Thr-57 Ser). While CCCP had no influence on the MICs, PAßN decreased the MICCIP values by 1–3 dilution steps and MICNAL values by up to 6 dilution steps. Of the resistance genes investigated, only qnrS was present, in a single Salmonella Infantis isolate. The MPCCIP values were 4–64-fold higher than the MICs and ranged between 1–16 and 0.12–1 mg/L, respectively, for isolates resistant or susceptible to nalidixic acid. Only mutants obtained from formerly nalidixic acid-susceptible isolates developed single mutations in gyrA or gyrB.
Conclusions: In field isolates and mutants, target site mutations and efflux seem to be important mechanisms for decreased fluoroquinolone susceptibility. Mutants derived during MPC determination from field isolates already harbouring single-step mutations in gyrA did not exhibit further mutations in any target genes.
Keywords: ciprofloxacin , nalidixic acid , QRDR , efflux pump systems , qnr genes , fluoroquinolone modifying enzyme aac(6')-Ib-cr
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Fluoroquinolones are important antimicrobial drugs in human and veterinary medicine. As compared to other Enterobacteria- ceae, high-level resistance to fluoroquinolones is relatively uncommon among Salmonella isolates—probably due to a prohibitive fitness cost.1 However, several studies have demonstrated the existence of Salmonella isolates with reduced susceptibility or, even in rare instances, resistance to fluoroquinolones.1–5
Resistance to fluoroquinolones is believed to arise from an interplay of different resistance mechanisms. Mutational modifications of the target enzymes for fluoroquinolones, the DNA gyrase and topoisomerase IV, were initially associated with quinolone resistance in Salmonella.1 These mutations occur in a specific region, the quinolone resistance determining region (QRDR) of the target genes gyrA, gyrB, parC and parE, and mutations at discrete positions have been described.3,6–8 While resistance to nalidixic acid and decreased susceptibility to fluoroquinolones are usually reported to be the consequence of a single point mutation in gyrA, additional mutations are necessary to obtain high-level fluoroquinolone resistance.2 Active efflux of fluoroquinolones was also identified as major mechanism of resistance and an overproduction of the AcrAB-TolC efflux pump was considered to be mainly responsible for multiple antibiotic resistance in Salmonella.9,10 Overproduction of this pump has also been associated with tolerance to organic solvents like cyclohexane.11,12 The AcrAB-TolC system belongs to the resistance nodulation-division (RND) family of efflux pumps and consists of a cytoplasmic membrane component, AcrB, a membrane fusion protein, AcrA, and the outer-membrane channel, TolC.2 Regulation of this efflux pump in Salmonella is believed to occur independently from the mar and sox regulatory systems, and mutations in local regulators of acrAB, for example in the repressor gene acrR, were also reported.13 Previous studies provided evidence that the AcrAB-TolC system may be inhibited by specific RND efflux pump inhibitors, such as Phe-Arg-ß-naphthylamide (PAßN).1,14
In addition, a protection of target topoisomerases can also be mediated by the products of plasmid-borne resistance genes, named qnrA, qnrB or qnrS. These qnr genes have been reported initially in Enterobacteriaceae species, but have recently been identified in non-Typhi serovars of Salmonella.15–18 Even though qnr genes protect DNA gyrase and topoisomerase IV from the inhibitory effect of quinolones, they produce only low-level resistance. However, additional mutations in the target genes may further raise the level of resistance.19 Recently, a novel plasmid-borne resistance mechanism has been identified in Klebsiella pneumoniae: the modification of certain fluoroquinolones by a specific type of aminoglycoside acetyltransferase.20 In the meantime, the gene aac(6')-Ib-cr coding for this aminoglycoside- and fluoroquinolone-modifying enzyme has also been detected among other Enterobacteriaceae producing extended spectrum ß-lactamases,21,22 but no data are available about the prevalence of this gene in Salmonella isolates.
Since several different mechanisms seem to play a role in decreased fluoroquinolone susceptibility, it was the aim of this study to investigate the resistance mechanisms present in isolates of six Salmonella serovars of avian origin. In addition, the mutant prevention concentrations (MPCs) were determined for all isolates and selected mutants with remarkably higher MICs were investigated for the underlying mechanisms.
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Bacterial isolates and susceptibility testing
A total of 35 Salmonella enterica subsp. enterica isolates from chickens comprising the serovars Enteritidis (n = 5 isolates), Hadar (n = 6), Infantis (n = 6), Livingstone (n = 5), Paratyphi B (n = 7) and Virchow (n = 6) were included in this study. The isolates were obtained from a surveillance study on non-typhoidal Salmonella isolates from Germany during 2001–05. Nalidixic acid-susceptible and -resistant isolates, which in part also exhibited differences in their MIC values of ciprofloxacin, were chosen for this study. One Salmonella Infantis isolate, recently described to carry the plasmid-borne resistance gene qnrS,17 was also included as part of the strain collection. The isolates were cultivated overnight on Luria Bertani (LB) agar plates. MICs of nalidixic acid, ciprofloxacin, ampicillin, chloramphenicol, cefotaxime, gentamicin, kanamycin, tetracycline and sulfadiazine/trimethoprim were determined by broth macrodilution and evaluated according to the guidelines given in the documents M07-A7 and M100-S1723,24 of the CLSI. The reference strain Escherichia coli ATCC 25922 served for quality control purposes.
Isolation of DNA, PCR amplification, hybridization experiments and sequencing
Whole-cell DNA was isolated using a previously described protocol.25 Plasmid DNA was extracted and purified following a modification of the alkaline lysis procedure.17
For the detection of resistance-mediating mutations in the target genes, the QRDR of gyrA, gyrB, parC and parE were amplified and sequenced. For this, previously described primer sets were slightly modified.6,9 Furthermore, the recently described primers specific for the detection of the genes qnrA, qnrB and qnrS were used.17 Since more than one primer set was required to amplify the aac(6')-Ib-cr gene, all the recommended primers as described by Robicsek et al.20 were chosen for the detection of this gene. In addition, the aac(6')-Ib-cr-fw: 5'-GATCTCATATCGTCGAGTGGTGG-3' and aac(6')-Ib-cr-rv: 5'-GAACCATGTACACGGCTGGAC-3' primer combination was used to amplify an internal 435 bp segment of the gene. The PCR conditions included an initial step of 94°C for 1 min, followed by 34 cycles of 1 min at 94°C, 2 min at 58°C annealing temperature and 3 min at 72°C and a final extension step of 7 min at 72°C. Sequencing of PCR products was performed on both strands by primer walking (MWG-Biotech, Martinsried, Germany).
Southern blot hybridization experiments were performed using internal fragments of the genes qnrA, qnrS and qnrB as gene probes. To detect variants of these qnr genes to which the PCR primers might not bind, hybridization experiments were conducted at moderate stringency. The DIG-High-Prime labelling and detection kit (Roche Diagnostics GmbH, Mannheim, Germany) was used as recommended by the manufacturer.
Efflux pump inhibition and cyclohexane resistance
Two different efflux pump inhibitors, PAßN and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) were tested for their ability to influence MIC values of nalidixic acid or ciprofloxacin. While CCCP increases the concentration of accumulated fluoroquinolones by inhibition of proton-motive force driven efflux pumps,3 PAßN inhibits specific RND efflux pumps and is known to be active against the AcrAB-TolC transporters.14 As described by Preisler et al.,26 both efflux pump inhibitors were used in a concentration corresponding to the 0.25-fold MIC. Strain-specific MICs of CCCP and PAßN were determined by following the CLSI methodology for antimicrobial susceptibility testing. The investigation of the 35 Salmonella isolates for tolerance against the organic solvent cyclohexane followed the method of Nakajima et al.27
MPCs and detection of resistance mechanisms in mutants
The MPC was defined as the lowest concentration of an antibiotic that inhibits the emergence of mutants from 
1010 cells.28–30 For the determination of the MPCs, aliquots of 100 µL overnight cultures containing 1010 cfu were plated on 10 agar plates supplemented with 1–128 x the MIC of ciprofloxacin. Plates were incubated and the lowest concentration that inhibited the growth of any mutants after 24 and 48 h was recorded as MPC24 and MPC48 values, respectively. In parallel, the number of colony-forming units per millilitre of inoculum was determined by stepwise dilution of an aliquot of the inoculated suspension. The MPC/MIC ratio was calculated and the frequency at which mutants were selected in the concentration range between the MIC and the MPC was calculated. For this calculation of the mutant frequency, the number of mutants growing in the presence of a specific ciprofloxacin concentration per millilitre inoculum was divided by the colony-forming units per millilitre inoculum.29 Selected mutants were analysed for their molecular basis of decreased ciprofloxacin susceptibility as well.
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Bacterial susceptibility and mutations in the quinolone resistance determining regions
The 35 Salmonella isolates tested were divided on the basis of the approved CLSI breakpoints into 24 nalidixic acid-resistant isolates (MICNAL of 32 mg/L) and 11 nalidixic acid-susceptible isolates (MICNAL of 
16 mg/L) (Table 1). Among the resistant isolates, the MICs of ciprofloxacin (MICCIP) differed between 0.12 and 1 mg/L, whereas the MICCIP values for the susceptible isolates were in the range of 0.015–0.03 mg/L (Table 1). While all Salmonella Livingstone isolates were classified as nalidixic acid-susceptible, one isolate of the serovars Enteritidis, Hadar, Infantis and Virchow as well as two isolates of Salmonella Paratyphi B were classified as nalidixic acid-susceptible. These susceptible isolates served as internal controls for the determination of QRDR mutations in the corresponding resistant isolates.
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Among the 24 nalidixic acid-resistant isolates, single mutations were detected either in the QRDR of gyrA (23 isolates) or parC (1 isolate). The isolates exhibited mutations that resulted in changes of the amino acid sequences Ser-83
Phe, Tyr and Asp-87 Tyr, Gly, Asn in GyrA or Thr-57 Ser in ParC. While the amino acid changes in GyrA have previously been described3,7,8 and assumed to be responsible for a decrease in the susceptibility to fluoroquinolones, the amino acid exchange Thr-57 Ser in ParC was also identified in nalidixic acid-susceptible isolates7,31 and hence is possibly not involved in quinolone resistance. However, no other mutation could be detected in the Salmonella Virchow isolate carrying this parC mutation. No correlation between the type of amino acid exchange and the serovars was seen among the isolates investigated, and isolates showing the same mutation also exhibited different levels of ciprofloxacin susceptibility. No target gene mutations were detected in any of the nalidixic acid-susceptible isolates, including all Salmonella Livingstone isolates. Influence of efflux pump inhibitors
Incubation of the Salmonella isolates in the presence of 0.25-fold the MIC of PAßN decreased the MICCIP values by 1–3 dilution steps. The influence of PAßN on the MICNAL depended on the resistance levels of the isolates. A decrease of 4–6 dilution steps was detected among the nalidixic acid-resistant isolates, whereas the susceptible isolates usually showed only a reduction in their MICNAL values by 1–3 dilution steps. Since PAßN inhibits RND efflux pumps, the decrease in the MICs for the Salmonella isolates may result from inhibition of the RND efflux pump AcrAB-TolC which is able to export quinolones/fluoroquinolones.9 Since expression of the AcrAB-TolC pump is known to vary between Salmonella isolates,1 the differences in the MICCIP values in isolates carrying the same gyrA mutation might be explained by the differential expression of this pump.
The overexpression of the AcrAB-TolC system was suggested to be linked to an intrinsic tolerance to organic solvents in E. coli.10,32 In some studies, statistical analysis showed that cyclohexane-tolerant Salmonella isolates were also significantly less susceptible to many antibiotics, including fluoroquinolones, than susceptible strains.29 The analysis of all Salmonella isolates for their tolerance to cyclohexane did not identify any of the 35 isolates as cyclohexane-tolerant (data not shown). Possible explanations are that overexpression of AcrAB-TolC plays only a minor role in isolates with decreased fluoroquinolone susceptibility or that cyclohexane tolerance is independent of AcrAB-TolC overexpression in Salmonella Typhimurium9 and obviously also other serovars. This second hypothesis is in good agreement with the observation that cyclohexane tolerance can be mediated by an acrB-independent mechanism.11
In contrast to the effect of PAßN, the second efflux pump inhibitor tested, CCCP, did not influence the MICs of nalidixic acid or ciprofloxacin. This was an interesting observation, since CCCP was reported to reduce the fluoroquinolone accumulation in some resistant bacteria to a similar level as seen with wild-type cells.3,10 Whether the concentration of 0.25-fold the MIC of CCCP was not sufficient to see an effect or whether both inhibitors act preferentially on different types of efflux pumps remains to be clarified.
Detection of plasmid-borne resistance genes
PCR assays and hybridization experiments were performed to detect the plasmid-borne resistance genes qnrA, qnrS or qnrB as well as the gene aac(6')-Ib-cr encoding the fluoroquinolone modifying enzyme. Only one isolate exhibited positive signals for the qnrS gene. This Salmonella Infantis isolate was included as part of the strain collection although the qnrS-carrying plasmid pINF5 has recently been analysed in detail.17 All other attempts to detect the genes in the six Salmonella serovars failed. However, single isolates of non-Typhi Salmonella serovars of human origin carrying qnr genes have been described during recent years.15,18 Until now, no information has been available about the presence of the fluoroquinolone and aminoglycoside modifying enzyme Aac(6')-Ib-cr in Salmonella. For the detection of the corresponding gene, the MIC values of kanamycin were determined since the enzyme is known to mediate fluoroquinolone as well as kanamycin resistance. However, only four Salmonella Paratyphi B exhibited MICs of kanamycin of 64 mg/L and were classified as resistant. In addition, all isolates were analysed with four different primer sets to detect the aac(6')-Ib gene and its variant aac(6')-Ib-cr, but no amplification product was obtained from any isolate. Even if the gene was detected among other Enterobacteriaceae,20–22 it has to the best of our knowledge not been identified yet in Salmonella isolates.
MPCs and resistance mechanisms of selected mutants
The MPCs of ciprofloxacin for the nalidixic acid-resistant isolates were in the range of 1–16 mg/L after 24 or 48 h of incubation and ranged between 0.12–1 mg/L for the nalidixic acid-susceptible isolates (Table 2). The calculation of the MPC/MIC ratios resulted in values between 4 and 64. The frequencies at which mutants were selected at the last concentration below the MPC ranged from 1 x 10–10 to 8.3 x 10–16 after 24 h (Table 2). Slightly higher mutant selection frequencies were detected after 48 h of incubation. For each serovar except Salmonella Livingstone, two mutants—one generated from a formerly nalidixic acid-resistant isolate and one from a formerly nalidixic acid-susceptible isolate—were selected for further analysis. For Salmonella Livingstone, only one mutant was investigated since all field isolates were nalidixic acid-susceptible. The mutants were picked after 24 h of incubation from agar plates containing the highest ciprofloxacin concentration below the MPC. The QRDR regions of gyrA, gyrB, parC and parE of the selected mutants were amplified by PCR, sequenced and compared with the nucleotide sequence of the respective original isolate. In addition, MICNAL and MICCIP values were determined in the presence and absence of 0.25-fold the MIC of the efflux pump inhibitors PAßN and CCCP.
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As shown in Table 2, there was a difference between the MPC and the MIC values of ciprofloxacin for some of the mutants. This might be the result of divergences in the testing systems used for determination of MPC (agar dilution) and MIC (broth macrodilution) values. These slight discrepancies of usually not more than one dilution step were, however, confirmed in repeated experiments.
Surprisingly, all mutants derived from formerly nalidixic acid-resistant strains exhibited only the mutation already present in gyrA of the original isolate, whereas mutants derived from formerly nalidixic acid-susceptible isolates exhibited single novel mutations in gyrA and, in the case of a single mutant, in gyrB. All these mutations in the QRDRs resulted in amino acid changes and were located at positions known to mediate quinolone resistance. Amino acid changes were detected in gyrA at positions Asp-87 Tyr in Salmonella Paratyphi B and Salmonella Enteritidis, at Asp-87 Asn in Salmonella Hadar, at Ser-83 Tyr in Salmonella Infantis and Salmonella Living- stone, or in gyrB at position Ser-464 Phe in Salmonella Virchow (Table 2). The mutant that exhibited only the gyrB mutation revealed a distinctly lower MIC of nalidixic acid, compared with the mutants that exhibited alterations in gyrA. As seen earlier with the original isolates (Table 1), mutants carrying the same mutations in gyrA again differed in their MICCIP values. Since repeated MIC determination confirmed these values, it is rather unlikely that the test conditions account for this observation. More probably, additional factors, such as differential expression of efflux pumps that remove ciprofloxacin from the bacterial cell, might play a role.
When looking at the influence of efflux pump inhibitors on the MICs for the mutants, a similar effect of the inhibitors was seen as for the original isolates: CCCP had no influence on the MICNAL and MICCIP values for any mutant (data not shown), suggesting that this substance is unable to inhibit efflux pumps responsible for elevated quinolone MICs in Salmonella. In contrast, incubation of mutants in the presence of PAßN decreased the MICNAL values by 3–6 dilution steps and had variable effects on the MICCIP values. In the mutants of Salmonella Paratyphi B and Salmonella Livingstone, no changes of the MICCIP values were detected, whereas decreases in the MICCIP values by 1–3 dilution steps were seen in the remaining mutants.
Other non-related antimicrobial agents, such as tetracycline, florfenicol and chloramphenicol, have proved to be substrates for the AcrAB-TolC efflux system which in Salmonella is mainly involved in the export of quinolones/fluoroquinolones.9 Analysis of selected mutants obtained during MPC determination revealed 2-fold to 16-fold increases in the MICs of tetracycline, florfenicol or chloramphenicol (data not shown). This observation may underline the role of efflux systems in the mechanism of decreased susceptibility to fluoroquinolones and other antimicrobial agents.
In conclusion, different resistance mechanisms seem to play a role in decreased fluoroquinolone susceptibility of the Salmonella isolates from avian origin included in this study. Mutations in the target genes for DNA gyrase and topoisomerase IV as well as enhanced efflux were identified as important resistance mechanisms, whereas a plasmid-borne qnrS gene was detected in only one Salmonella Infantis isolate. The qnrA, qnrB or aac(6')-Ib-cr genes could not be detected in these isolates. Analysis of the mutants selected during MPCCIP determination suggested that a first step in the development of decreased fluoroquinolone susceptibility is a mutation in the target genes. This was observed in formerly nalidixic acid-susceptible isolates. In nalidixic acid-resistant isolates already harbouring a first mutation in gyrA, the second step is most probably a change in the expression of efflux pumps, since no further mutations could be detected in the target genes of the respective mutants.
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A. de J. and S. F. are Bayer employees. In 2006, C. K. received financial support from Bayer for participation at ICAAC, and S. S. received expenses for speaking at a symposium organized by Bayer.
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Acknowledgements |
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Many thanks to Peter Heisig for helpful discussions and to Vera Nöding for excellent technical assistance. This study was financially supported by the Bayer HealthCare AG.
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References |
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