Role of TolC and parC mutation in high-level fluoroquinolone resistance in Salmonella enterica serotype Typhimurium DT204

Sylvie Baucheron, Elisabeth Chaslus-Dancla and Axel Cloeckaert*

Unité BioAgresseurs, Santé et Environnement, Institut National de la Recherche Agronomique, 37380 Nouzilly, France

Received 27 November 2003; returned 13 December 2003; revised 18 December 2003; accepted 19 December 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: To study the role of TolC and of parC mutation in high-level fluoroquinolone resistance in clonal clinical strains of Salmonella enterica serotype Typhimurium phage type DT204 (S. Typhimurium DT204).

Methods: Deletion of the tolC gene ({Delta}tolC) was first performed in a susceptible S. Typhimurium DT104 strain lacking target gene mutations involved in fluoroquinolone resistance. P22 transduction was further used to transduce {Delta}tolC from this strain to a high-level fluoroquinolone-resistant S. Typhimurium DT204 strain carrying several target gene mutations, including one in parC (ciprofloxacin MIC of 32 mg/L).

Results: Deletion of tolC in the high-level fluoroquinolone-resistant S. Typhimurium DT204 strain resulted in the same decrease in resistance levels (16- to 32-fold) as shown previously for an acrB mutant of the same strain, suggesting that AcrAB-TolC is the main efflux system involved in high-level fluoroquinolone resistance of S. Typhimurium DT204 strains. In some S. Typhimurium DT204 {Delta}tolC transductants, concomitant loss of the parC (Ser-80->Ile) mutation, located ~9.3 kb upstream of tolC, resulted in a further 16- to 32-fold decrease in resistance levels to fluoroquinolones and thus a hypersusceptible phenotype (ciprofloxacin MIC of 0.063 mg/L).

Conclusion: The AcrAB-TolC efflux system, together with multiple target gene mutations, including the parC mutation, appear essential to confer high-level fluoroquinolone resistance in S. Typhimurium DT204.

Keywords: salmonellosis, multidrug resistance, AcrAB-TolC efflux system, multidrug transporter AcrB, outer membrane component TolC, target gene


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Emergence of resistance to fluoroquinolones in Salmonella spp. is a problem in both veterinary and human medicine since these are often the treatment of choice in cases of multidrug-resistant life-threatening salmonellosis.

High-level fluoroquinolone resistance in Salmonella has been documented rarely. The first reported example was the emergence and probable clonal spread of Salmonella enterica serotype Typhimurium variant Copenhagen of phage type DT204 (hereafter referred to as S. Typhimurium DT204) in limited areas in Europe.15 These strains were mainly isolated between 1991 and 1995 from animals and humans and were highly resistant to ciprofloxacin (MIC of 32 mg/L).

In Salmonella, quinolone resistance has been attributed to point mutations in the gyrA gene coding for the A subunit of DNA gyrase. These mutations cluster in a region of the gyrA gene resulting in amino acid changes between amino acid positions 67 and 106. This region has been termed the quinolone resistance-determining region (QRDR). Amino acid changes at Ser-83 (to Phe, Tyr or Ala) or at Asp-87 (to Gly, Asn or Tyr) are the most frequently observed in nalidixic acid-resistant strains.6 Double mutations at both residues 83 and 87 have been identified in clinical isolates of S. Typhimurium DT204 showing high-level resistance to fluoroquinolones.15 The double mutation was identical in several isolates (Ser-83->Ala and Asp-87->Asn), thereby suggesting the clonality of the strains. Additionally, high-level fluoroquinolone-resistant isolates have shown one mutation in the QRDR of gyrB, encoding the B subunit of DNA gyrase, leading to amino acid change Ser-464->Phe.14 More recently, these high-level fluoroquinolone-resistant isolates have been shown to carry a fourth mutation in the QRDR of parC coding for the ParC subunit of topoisomerase IV, which is the secondary target for quinolones. The mutation identified led to amino acid change Ser-80->Ile.13

We have recently reported the participation of active efflux as an important mechanism of resistance to fluoroquinolones in S. enterica serotype Typhimurium.1,7 Inactivation of the gene coding for the AcrB multidrug transporter in S. Typhimurium DT204 strains resulted in a 16- to 32-fold reduction in resistance to several fluoroquinolones (see Table 2).1


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Table 2. Characteristics of the Salmonella enterica serotype Typhimurium strains studied
 
In the present study, we analysed the role of the outer membrane component, TolC, in this high-level fluoroquinolone resistance of S. Typhimurium DT204 strains. Since TolC might interact with other potential multidrug transporters involved in fluoroquinolone resistance, it could thus be expected that inactivation of the tolC gene would reduce the resistance level to fluoroquinolones below that observed by inactivation of the multidrug transporter acrB gene.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Deletion of the tolC gene was first performed according to the method described by Datsenko & Wanner.8 Briefly, the kanamycin resistance gene kan flanked by FRT (FLP Recognition Target) sites was amplified by PCR under standard conditions (initial denaturation: 5 min at 94°C; 30 cycles of amplification: denaturation, 1 min at 94°C, annealing, 0.5 min at 52°C, extension, 1.5 min at 72°C; and final extension: 7 min at 72°C), using the template plasmid pKD4 and hybrid primers. These primers, P1TolC and P2TolC (Table 1), were synthesized with 20 nucleotides (nt) of priming sites 1 and 2 of pKD4 and with 50 nt of the 5' and 3' ends of the tolC gene (GenBank accession number NC_003197). The 1.6 kb long PCR fragment was purified and electroporated into the S. Typhimurium DT104 S/921495 antibiotic-susceptible strain lacking target gene mutations for fluoroquinolone resistance (Table 2), in which the Red recombinase expression plasmid pKD46 was introduced. Transformants were selected at 37°C on LB agar medium containing kanamycin 50 mg/L. Homologous recombination between the genomic DNA and PCR product resulted in the deletion of the tolC gene from bp 70 to bp 1426 (1356 bp deletion) and its replacement with the kanamycin resistance gene. This was confirmed by three different PCRs. Deletion of tolC in the transformants was first shown by a negative PCR using primers TolC3 and TolC4 (Table 1), corresponding to internal sequences of the deleted tolC gene. A second PCR was carried out to confirm the deletion using primers TolC5 and TolC6 (Table 1), corresponding to sequences flanking the tolC deletion, and which resulted in a 2400 bp fragment for the parental strains and in a 2644 bp fragment when tolC was deleted and replaced by the kan gene flanked by FRT sites. The third control PCR, with the primers k2 and kt (Table 1), was used to detect the 471 bp kan fragment. The tolC gene deletion was further transduced in S. Typhimurium DT204 strain 102SA00 using phage P22 as described previously.1 MICs of nalidixic acid, flumequine, marbofloxacin, enrofloxacin and ciprofloxacin were determined by the standard agar doubling dilution method, as described previously.1 Identification of mutations in the QRDRs of gyrA, gyrB, parC and parE was performed by nucleotide sequencing, as described previously.1


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Table 1. Primers used for PCRs
 

    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
P22 transduction of the tolC deletion ({Delta}tolC) in S. Typhimurium DT204 strain 102SA00 resulted in the selection of two populations of colonies exhibiting two susceptibility patterns to fluoroquinolones. In the first population (102SA00{Delta}tolC1::kan), transduction of {Delta}tolC resulted in a 16- (ciprofloxacin and marbofloxacin) to 32-fold (enrofloxacin) reduction in resistance levels to fluoroquinolones, as was previously observed for acrB-constructed mutants of this strain (Table 2). In the second population (102SA00{Delta}tolC2::kan), transduction of this deletion resulted in a further 16- to 32-fold decrease in resistance levels to fluoroquinolones, and thus in a hypersusceptible phenotype (ciprofloxacin MIC of 0.063 mg/L) (Table 2). Nucleotide sequencing of the QRDRs of target genes showed that mutations identified in the parental S. Typhimurium DT204 strain 102SA00 were not affected in the first population of strains (Table 2). However, nucleotide sequencing revealed that the second population of {Delta}tolC transduced strains had lost the parC mutation leading to amino acid change Ser-80->Ile (Table 2). The other mutations in the QRDRs of gyrA and gyrB were not affected. In fact, parC is located 9.3 kb upstream of tolC in the S. enterica serotype Typhimurium strain LT2 genome (GenBank accession number NC_003197). Since P22 transduction may mobilize up to 40 kb of DNA, transduction of {Delta}tolC in the second population of strains probably resulted, in addition to deletion of tolC, in the replacement of the parC-resistant allele by the susceptible parC allele of strain S/921495{Delta}tolC::kan (Table 2). These results indicate the importance of the parC mutation at codon position 80 in reaching high-level fluoroquinolone resistance in S. Typhimurium DT204 strains, as was previously reported for E. coli.9,10 The absence of differences in the MIC levels observed between 102SA00acrB::kan and 102SA00{Delta}tolC1::kan suggests that AcrAB-TolC is the main efflux system involved in high-level fluoroquinolone resistance in S. Typhimurium DT204 strains. When comparing the resistance levels between strain 102SA00{Delta}tolC2::kan and strain S/921495{Delta}tolC::kan—lacking mutations in the QRDRs of target genes—the remaining target gene mutations, i.e. double mutation in QRDR of gyrA and single mutation in QRDR of gyrB, together could count for an eight- to 32-fold increase in resistance levels to quinolones (Table 2).

In conclusion, both the AcrAB-TolC efflux system and accumulation of multiple target gene mutations, including parC mutations, appear essential to confer high-level fluoroquinolone resistance in S. Typhimurium DT204.


    Acknowledgements
 
We thank C. Schouler and S. Payot for helpful suggestions in the construction of the tolC mutants. We thank H. Imberechts and S. Rankin for supplying strains 102SA00 and S/921495. The pKD4 and pKD46 plasmids were provided by the E. coli Genetic Stock Center, Yale University. We also thank C. Mouline for expert technical assistance. This study was funded by INRA, projet Transversalité.


    Footnotes
 
* Corresponding author. Tel: +33-2-47-42-77-50; Fax: +33-2-47-42-77-74; E-mail: cloeckae{at}tours.inra.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Baucheron, S., Imberechts, H., Chaslus-Dancla, E. et al. (2002). The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar Typhimurium phage type DT204. Microbial Drug Resistance 8, 281–9.[CrossRef][ISI][Medline]

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5 . Heisig, P., Kratz, B., Halle, E. et al. (1995). Identification of DNA gyrase A mutations in ciprofloxacin-resistant isolates of Salmonella typhimurium from men and cattle in Germany. Microbial Drug Resistance 1, 211–8.[ISI][Medline]

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7 . Giraud, E., Cloeckaert, A., Kerboeuf, D. et al. (2000). Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar Typhimurium. Antimicrobial Agents and Chemotherapy 44, 1223–8.[Abstract/Free Full Text]

8 . Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences, USA 97, 6640–5.[Abstract/Free Full Text]

9 . Bagel, S., Hullen, V., Wiedemann, B. et al. (1999). Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli. Antimicrobial Agents and Chemotherapy 43, 868–75.[Abstract/Free Full Text]

10 . Heisig, P. (1996). Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy 40, 879–85.[Abstract]