Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006, Logroño, Spain
Received 8 April 2002; returned 24 July 2002; accepted 20 January 2003
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Abstract |
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Keywords: Escherichia coli, quinolone resistance
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Introduction |
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The most frequent mechanism of resistance to quinolones in E. coli includes alterations in genes that encode subunits of the quinolone targets DNA gyrase (in gyrA and gyrB genes) and topoisomerase IV (in parC and parE).24 These alterations involve mainly mutations located in the quinolone resistance-determining region (QRDR) of the gyrA gene and its homologous region of the parC gene.3,5,6 In contrast, mutations in gyrB and parE genes are of minor importance and are rare contributors to quinolone resistance.4,7 Other mechanisms of resistance, such as efflux pump systems or modifications of porins, can decrease susceptibility to quinolones.2,5,7,8
Most of the studies on mechanisms of quinolone resistance in E. coli have been performed in human clinical strains and very few studies have focused on strains from animals or foods.2,7 In this study, mutations in gyrA, parC and gyrB have been analysed in nalidixic acid-resistant (NALR) E. coli strains from food products, humans and animals.
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Materials and methods |
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The MICs of nalidixic acid and ciprofloxacin were determined by an agar dilution method in MuellerHinton agar, according to NCCLS guidelines.
Mechanisms of resistance to quinolones
Detection of mutations in the QRDR of the gyrA gene, as well as in the analogous region of the parC gene, was performed in the 80 NALR and 13 NALS strains by PCR.4,6 Amplified fragments were purified (Qiagen), and both strands were automatically sequenced (ABI 310, Applied Biosystems, Madrid, Spain) using the same set of primers as for the PCR. Mutations in the gyrB gene were also analysed by sequencing specific amplicons in 18 NALR strains.9 Sequences obtained were compared with those previously reported for gyrA (GenBank accession no. X06373), gyrB (X04341) and parC genes (M58408 with the modification included in L22025).
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Results and discussion |
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Analysis inside the QRDR of the gyrA sequences of all 93 strains (NALR and NALS), in comparison with the gyrA sequence previously reported (GenBank accession no. X06373), revealed five different patterns (AE), according to silent mutations identified at positions 255, 261, 267, 273, 282 and 300 (Table 2). All strains exhibited a TC transition at nucleotide 267. Mutations at positions 261 and 282 have not been reported previously. Table 2 shows the relationship between silent mutation patterns and amino acid substitutions in the GyrA protein.
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Two substitutions outside the QRDR of the ParC protein (Ala-56Thr and Ser-57
Thr) were identified in two strains. The substitution Ala-56
Thr was detected in one NALR E. coli from a pig (ciprofloxacin MIC 0.5 mg/L) and the Ser-57
Thr change in one strain from food (E. coli Co7). This strain also presented a double amino acid substitution in the GyrA protein (Ser-83
Leu + Asp-87
Tyr) and a single amino acid change in the ParC protein (Glu-84
Lys) (Table 1). In addition, E. coli Co7 also showed a total of 14 different silent mutations inside the QRDR of the parC gene.
Analysis inside the QRDR of the parC sequences of all 93 strains, in comparison with the parC sequence previously reported (GenBank accession nos M58408 and L22025) revealed six different patterns (from I to VI) according to the silent mutations identified at positions 240, 243, 273 and 321 (Table 2). No silent mutations were found in the 22 strains included in pattern I, whereas the remaining 71 strains presented a transition (a guanine instead of an adenine) at nucleotide 273. Table 2 shows the relationship between silent mutation patterns and amino acid substitutions in ParC.
No changes were observed in the GyrB protein of the 18 NALR strains.
A correlation between the number of changes in GyrA and ParC proteins and the level of quinolone resistance in E. coli strains was observed. This is in accordance with previous observations.4,5,7 None of the NALS E. coli showed amino acid changes either in GyrA or in ParC. A single substitution in the GyrA protein was associated with a decrease in susceptibility to ciprofloxacin (MIC 0.031 mg/L) and two amino acid changes (one in GyrA and the other in ParC) with a low level of resistance to ciprofloxacin (24 mg/L). Three amino acid substitutions (two in GyrA and one in ParC) were associated with a moderate or high level of ciprofloxacin resistance (832 mg/L), and four substitutions (two in GyrA and two in ParC) with the highest ciprofloxacin MIC observed (64 mg/L). Several strains did not follow the above schedule (Table 1): six strains with ciprofloxacin MICs ranging from 0.5 to 1 mg/L showed two amino acid changes (one in GyrA and other in ParC), and one strain with a ciprofloxacin MIC of 4 mg/L had three amino acid changes (two in GyrA and one in ParC proteins). MICs of all seven strains could be explained by increasing drug permeability.4 However, in contrast to these observations, three amino acid changes were detected in one E. coli strain with a ciprofloxacin MIC of 64 mg/L. The presence of active efflux pumps or mutations that affect regulatory loci (e.g. mar and sox) might be associated with this high ciprofloxacin MIC.2,5,8 These other mechanisms might also be responsible for the wide range of ciprofloxacin MICs (464 mg/L) detected in strains that show the same amino acid substitutions in GyrA (Ser-83Leu + Asp-87
Asn) and in ParC proteins (Ser-80
Ile).
In the present study, a large variety of amino acid changes in GyrA and ParC proteins were detected in the NALR E. coli strains of different origins. A correlation was found between the number of changes in the QRDR of GyrA and ParC proteins and the level of quinolone resistance of the E. coli strains.
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Acknowledgements |
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Footnotes |
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References |
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2 . Everett, M. J., Fang Jin, Y., Ricci, V. & Piddock, L. J. V. (1996). Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrobial Agents and Chemotherapy 40, 23806.[Abstract]
3 . Piddock, L. J. V. (1999). Mechanisms of fluoroquinolone resistance: an update 19941998. Drugs 58, 118.[ISI][Medline]
4 . Vila, J., Ruiz, J., Goñi, P. & Jiménez de Anta, T. (1996). Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrobial Agents and Chemotherapy 40, 4913.[Abstract]
5 . 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, 87985.[Abstract]
6 . Oram, M. & Fisher, L. M. (1991). 4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction. Antimicrobial Agents and Chemotherapy 35, 3879.[ISI][Medline]
7
.
Giraud, E., Leroy-Sétrin, S., Flaujac, G., Cloeckaert, A., Dho-Moulin, M. & Chaslus-Dancla, E. (2001). Characterization of high-level fluoroquinolone resistance in Escherichia coli O78:K80 isolated from turkeys. Journal of Antimicrobial Chemotherapy 47, 3413.
8
.
Kern, W. V., Oethinger, M., Jellen-Ritter, A. S. & Levy, S. B. (2000). Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy 44, 81420.
9 . Vila, J., Ruiz, J., Marco, F., Barcelo, A., Goñi, P., Giralt, E. et al. (1994). Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs. Antimicrobial Agents and Chemotherapy 38, 24779.[Abstract]
10
.
Tavio, M. D., Vila, J., Ruiz, J., Martín-Sánchez, A. M. & de Anta, M. T. J. (1999). Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates. Journal of Antimicrobial Chemotherapy 44, 73542.