1 Departamento de Microbiología, Facultad de Medicina, Universidad de Zaragoza, 50009 Zaragoza; 2 Secció de Medicina Tropical, C.S.I., U.A.S.P., IDIBAPS, Hospital Clínic, Villarroel 170, 08036 Barcelona; 3 Servei de Microbiologia, Institut Clínic Infeccions i Inmunología, IDIBAPS, Facultad de Medicina, Universitat de Barcelona, Villarroel 170, 08036 Barcelona, Spain
Received 31 January 2004; returned 24 February 2004; revised 1 March 2004; accepted 5 March 2004
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Abstract |
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Results: All the nalidixic acid-resistant strains showed mutations in the gyrA gene and none in the parC gene. The presence of the inhibitor produced decreases in the MIC values of nalidixic acid by two to six serial dilution steps in 37 of the 41 nalidixic acid-resistant strains. Meanwhile, the MIC value of ciprofloxacin was affected in two strains whose values diminished three serial dilution steps. The nalidixic acid-resistant mutant obtained in vitro was also affected by the inhibitor decreasing the MIC value of nalidixic acid three serial dilutions steps whereas the MICs for the nalidixic acid-susceptible strains were not affected.
Conclusions: Our results show that the high level of resistance to nalidixic acid is likely due to an overexpression of an efflux pump plus a mutation in the gyrA gene, whereas decreased susceptibility to ciprofloxacin is only associated with the presence of a mutation in the gyrA gene.
Keywords: Y. enterocolitica, Phe-Arg-ß-naphthylamide, quinolones
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Introduction |
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Y. enterocolitica O:3 is a common enteric pathogen in children and adults mainly causing gastrointestinal infection.1 The usual route of acquisition of this microorganism is through ingestion of contaminated food or water.1 Antibiotics are usually not required for gastrointestinal disease; however, they are necessary for treating systemic infections in immunocompromised patients.1 Among these antibacterial agents, fluoroquinolones play a major role owing to their potent activity against Y. enterocolitica.2 Quinolones act by inhibiting type II topoisomerase (DNA gyrase and topoisomerase IV). Both are tetrameric enzymes composed of two A subunits and two B subunits (GyrA and GyrB for DNA gyrase and ParC and ParE for topoisomerase IV).
The mechanisms for quinolone resistance described in Y. enterocolitica O:3 have been limited to mutations located in a region of the gyrA gene known as the quinolone resistance-determining region (QRDR).3 Other mechanisms of resistance to quinolones described in Gram-negative bacteria are the presence of mutations in the other genes encoding the type II topoisomerases (the gyrB, parC and parE genes) and the decreased accumulation inside the bacteria due to impermeability of the membrane and/or an overexpression of efflux systems.46 Furthermore, plasmids carrying the qnr gene, which confers resistance to quinolones, have also been described and are the first clearly established transferable mechanism of resistance to quinolones.7 Moreover, resistance associated with the level of target expression has recently been described.8
All clinical strains of Y. enterocolitica O:3 isolated in the geographical area of Zaragoza, Spain from 1979 to 1993 were susceptible to nalidixic acid, ciprofloxacin and norfloxacin.2 However, in recent years clinical isolates resistant to nalidixic acid have been recovered from this region of Spain.
The aim of this study was to determine the mechanisms of resistance to quinolones in a series of Y. enterocolitica O:3 clinical isolates recovered in the area of Zaragoza, Spain.
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Materials and methods |
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From 1996 to 2002, 271 strains of Y. enterocolitica O:3 were isolated from stools of patients with gastroenteritis. Sixty-two (23%) were nalidixic acid-resistant, 41 of which were included in this study. Three nalidixic acid-susceptible strains were used as controls.
In vitro development of a nalidixic acid-resistant mutant
A nalidixic acid-susceptible strain (2S) was grown in subinhibitory concentrations of nalidixic acid to obtain a nalidixic acid-resistant mutant (2R).
Characterization of mutations in the gyrA and parC genes
Mutations in the gyrA and parC genes were detected by PCR amplification of their QRDR. The QRDR of the gyrA gene was amplified using the primers and conditions previously described3 while to amplify the parC gene, the primers 5'-CGC GAC GGC CTG AAG CCG GTG CA-3'and 5'-GTG CGG CGG GAT GTT GGT GGC CAT-3' and the same conditions were used. The software used to design the primers was Primer Express 1.5 (Applied Biosystems, Foster City, CA, USA).
PCR products were recovered using Wizard SV gel and PCR Clean-Up minicolumns (Promega, Madison, WI, USA) and directly sequenced using Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems).
Antimicrobial susceptibility
Susceptibility to nalidixic acid and ciprofloxacin in the presence and absence of 20 mg/L of the efflux pump inhibitor Phe-Arg-ß-naphthylamide was also determined using an agar dilution method according to the NCCLS guidelines9 as described by Ribera et al.10 We included the E. coli strain NorE5, an in vitro selected quinolone-resistant mutant of the wild-type strain PS15 in which previous studies have showed the great effect of this inhibitor.5 Differences of one serial dilution were considered to be within the margins of error for the method and were therefore not taken into account. The MIC of Phe-Arg-ß-naphthylamide was established by the same method.
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Results and discussion |
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Forty out of 41 nalidixic acid-resistant isolates (showing a MIC of 128 mg/L) carried a single mutation in the gyrA gene, affecting the amino acid codons Ser-83 or Asp-87.
Thirty-seven isolates had a mutation at codon Ser-83 (AGC). In 30 out of these 37 (81%) strains, the mutation generated a change to Arg (16 to AGG, and 14 to AGA) whereas six strains had a mutation producing a change to Ile (ATC). Finally, in one strain a change from Ser-83 to Cys (TGC) was observed. To our knowledge, this is the first description of substitution of Ser-83 by Cys in the GyrA protein. Three strains had a mutation in the amino acid codon Asp-87 changing to glycine (GGC) in two strains and to asparagine (AAC) in the remaining strain. Another strain (strain 42, Table 1) presented a double mutation leading to amino acid changes Ser-83 to Ile and Val-85 to Ile. This double mutation was associated with nalidixic acid and ciprofloxacin MICs of 128 and 0.25 mg/L, respectively, without differences on comparing the MIC levels of the strains carrying a single amino acid substitution. These results indicate that substitution in amino acid codon 85 to Ile does not play a role in the development of quinolone resistance.
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The nalidixic acid-resistant mutant obtained in vitro (strain 2R) presented a mutation at position Gly-81 changing to Cys (TGT). This substitution has been found in other in vitro quinolone-resistant microorganisms.4,6 This fact might reflect the differences in the in vivo and in vitro environments.
In quinolone-resistant strains, the MIC of nalidixic acid ranged from 128 to 2048 mg/L. These differences were not associated with the presence of different amino acid substitutions suggesting the presence of other mechanisms of resistance that were able to modulate the final MIC.
When the MICs of nalidixic acid and ciprofloxacin were determined in the presence of 20 mg/L of Phe-Arg-ß-naphthylamide, the MIC of nalidixic acid was more uniform, although slight differences could also be observed among isolates carrying mutations, probably associated with efflux pumps not affected by Phe-Arg-ß-naphthylamide or to differences in the permeability of the outer membrane. However, no effect was observed on the MIC of ciprofloxacin.
Decreases by two to six serial dilution steps in 37 out of 41 nalidixic acid-resistant strains (90%), were observed in the MIC of nalidixic acid. Meanwhile, the MIC of ciprofloxacin was only affected in two of the 41 strains whose values diminished by three serial dilution steps (Table 1). Thus, this efflux pump was able to pump nalidixic acid out, with only a poor or null effect in the pumping of ciprofloxacin. Moreover, the nalidixic acid-resistant mutant obtained in vitro was also affected by the inhibitor decreasing the MIC of nalidixic acid three serial dilutions steps, whereas the MICs of the nalidixic-susceptible parental strain and the other nalidixic acid-susceptible clinical isolates were not affected by the inhibitor.
To avoid a possible intrinsic activity of this inhibitor against Y. enterocolitica its MIC level was established. Our results (Table 1) showed the inability of Phe-Arg-ß-naphthylamide to affect the growth of Y. enterocolitica.
Thus, these results provide evidence that an efflux-mediated resistance mechanism contributes to the development of high level resistance to nalidixic acid. Nevertheless, Phe-Arg-ß-naphthylamide was only able to produce the change from resistant to susceptible in one of the analysed isolates that carried the substitution Asp-87 to Gly. Interestingly, this high effect was also observed in another strain carrying this substitution and in addition these two strains present the lowest MICs of ciprofloxacin. Although a greater number of isolates is needed for confirmation, these results indicate that this specific substitution produces a minor change in the interaction between quinolones and DNA gyrase.4,6
This decrease in the MIC of nalidixic acid is in agreement with the results obtained in other microorganisms such as E. coli5 or S. maltophilia.10 However, in isolates of E. coli carrying one mutation in GyrA, the Phe-Arg-ß-naphthylamide was able to revert nalidixic acid-resistant isolates to nalidixic acid susceptibility.5 Moreover, the small effect on ciprofloxacin MIC observed in our study is also similar to that found in other studies.5
In conclusion, our results show that although a mutation in the gyrA gene is the primary mutation generating resistance to nalidixic acid, the overexpression of an efflux pump can modulate the final MIC of this antimicrobial agent in most Y. enterocolitica clinical isolates.
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Acknowledgements |
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Footnotes |
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References |
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Ribera, A., Ruiz, J., Jimenez de Anta, M. T. et al. (2002). Effect of an efflux pump inhibitor on the MIC of nalidixic acid for Acinetobacter baumanii and Stenotrophomonas maltophilia clinical isolates. Journal of Antimicrobial Chemotherapy 49, 6978.