Characterization of high-level fluoroquinolone resistance in Escherichia coli O78:K80 isolated from turkeys

Etienne Giraud, Sabine Leroy-Sétrin, Géraldine Flaujac, Axel Cloeckaert, Maryvonne Dho-Moulin and Elisabeth Chaslus-Dancla,*

Station de Pathologie Aviaire et de Parasitologie, Institut National de la Recherche Agronomique, Centre de Recherche de Tours-Nouzilly, 37380 Monnaie, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Fluoroquinolone resistance was characterized in Escherichia coli O78:K80 isolated from diseased turkeys. The level of resistance to fluoroquinolones of the isolates appeared closely correlated with substitutions in GyrA and ParC, but not with the production of the AcrAB efflux pump. Among isolates highly resistant to ciprofloxacin (MIC 8 mg/L) and harbouring identical substitutions (two in GyrA and one in ParC), two close but distinguishable ribotypes were identified. This indicated that at least two independent selection events may have occurred.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In the mid-1990s, a high incidence of Escherichia coli O78:K80 strains was observed in France in turkeys affected with colibacillosis, an avian systemic disease responsible for important economic losses in poultry breeding.

Enrofloxacin, the first fluoroquinolone authorized as a veterinary drug, has been used in France since 1992. Its use may lead to the selection of resistant strains, which may spread inside and outside poultry flocks. The effect of such antibiotic use on human health has to be considered, as a relationship has been reported between septicaemic human and animal E. coli O78 strains.1 Most of these E. coli O78 were multiply antibiotic resistant.

The acquisition of fluoroquinolone resistance in E. coli is linked to the sequential selection of mutations in the gyrA and parC genes and less frequently in the gyrB and parE genes, which encode subunits of the target topoisomerases, gyrase and topoisomerase IV.25 However, non-target mutations can also have an important role in the development of fluoroquinolone resistance. Mutations that affect marOR, soxRS and presumably other unidentified chromosomal loci cause an increase in the expression of the AcrAB multidrug efflux system, whose role in both the intrinsic and the acquired fluoroquinolone resistance of E. coli is critical.6,7

In this study we analyse the topoisomerase mutations, the production of the AcrAB efflux system and the clonal relationships of E. coli O78:K80 strains isolated in turkeys during episodes of colibacillosis in the mid-1990s.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
One hundred and seventy E. coli O78:K80 strains isolated from diseased turkeys were collected in France through local diagnostic laboratories. Antibiotic susceptibility was evaluated by the disc diffusion method according to the guidelines of the Comité de l'Antibiogramme de la Société Française de Microbiologie.8 Twenty-three isolates representative of the different levels of resistance to quinolones were chosen for further studies (TableGo). The MICs of nalidixic acid, flumequine, enrofloxacin and ciprofloxacin were determined by the standard agar doubling dilution method.8 The strains were classified as susceptible, intermediate or resistant as described previously.9


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Table. Susceptibility to quinolones and amino acid substitutions within GyrA, GyrB, ParC and ParE of 23 E. coli O78:K80 isolates
 
For each of the twenty-three isolates, a 595 bp fragment (from nucleotides –19 to 576) containing the quinolone resistance-determining region (QRDR) of gyrA was amplified using primers 5'-GTAGAGGGATAGCGGTTAG-3' and 5'-CGTTGATGACTTCCGTCAG-3'. gyrB was amplified between nucleotides 1223 and 2438 with primers 5'-CAGACTGCCAGGAACGCCAT-3' and 5'-GCACGC TCGCATGGTTAG-3'. Amplifications of the QRDR of parC and parE were achieved as described previously.2

Immunoblotting experiments were performed as described previously using an anti-AcrA polyclonal antibody.10

For ribotyping, genomic DNA was digested with HindIII or PvuII, electrophoresed and blotted on to nylon membranes. The blots were hybridized with a cDNA probe reverse transcribed from a mixture of 16S + 23S rRNA of E. coli and labelled using the Dig-High Prime system (Boehringer-Mannheim, Mannheim, Germany).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Quinolone susceptibility testing

Among the 170 clinical isolates, 84 (49%), 82 (48%) and 68 (40%) were resistant to nalidixic acid, flumequine and enrofloxacin, respectively. For the twenty-three isolates chosen on the basis of the antibiogram results, the quinolone MICs covered a wide range, from full susceptibility to a high level of resistance to fluoroquinolones (MICs of enrofloxacin and ciprofloxacin: 8 mg/L), with all the intermediates represented (TableGo).

Mutations in the QRDRs of the gyrA, gyrB, parC and parE genes

Mutations leading to amino acid substitutions were found only in the gyrA and parC genes (TableGo). Six isolates that were resistant to nalidixic acid but showed only a decreased susceptibility to enrofloxacin and ciprofloxacin had a Ser83->Leu substitution in GyrA. Two isolates highly resistant to nalidixic acid and to flumequine but not clinically resistant to fluoroquinolones had an additional Ser80->Ile substitution in ParC. For the 13 most resistant isolates, an additional Asp87->Tyr change was found in GyrA. The occurrence of these mutations is in good agreement with the ‘ping-pong’ model proposed by Heisig:3 the gyrase, initially more susceptible to quinolones than topoisomerase IV, is therefore the first target enzyme to be modified (primary target). Then, the wild-type topoisomerase IV, which is more susceptible than the mutated version of the gyrase, is secondarily modified.

Level of production of the AcrAB efflux pump

In order to assess whether different levels of resistance to quinolones could be linked to different levels of production of the AcrAB efflux pump, we performed immunoblotting experiments using an anti-AcrA antibody. The results clearly indicated a poor correlation between the levels of resistance to quinolones and the levels of production of the AcrAB pump. This production appeared similar among isolates exhibiting very different susceptibilities to quinolones (FigureGo, a), and also among highly fluoroquinolone-resistant isolates (FigureGo, b). However, three isolates exhibiting a full (BN177) or a decreased (BN49 and BN6) susceptibility to fluoroquinolones apparently produced less AcrAB. In the absence of an E. coli control strain with low-level AcrAB production such as shown previously for susceptible Salmonella enterica serovar Typhimurium strains,10 it is not clear whether the production of AcrAB in the three isolates above corresponds to a basal production level in this species. Thus, the different levels of resistance to fluoroquinolone apparently do not depend on the AcrAB production level as previously shown for S. enterica serovar Typhimurium.10



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Figure. Immunoblot analysis of the AcrA protein of selected E. coli O78:K80 isolates exhibiting increasing (a) or high (b) levels of resistance to quinolones (see TableGo). BN177 is a quinolone-susceptible strain used as control. As additional internal control, a set of previously described S. enterica serovar Typhimurium strains producing different levels of AcrA were included (not shown).10

 
Clonal relationships between the isolates

We used ribotyping to investigate whether these E. coli isolates could be clonally related. Combining the results obtained with the two restriction enzymes, a total of 11 ribotypes could be distinguished among the 170 E. coli O78:K80 isolates. Two ribotypes, H1P1 and H3P1, were highly predominant, with 82 (48%) and 67 (39%) of the isolates, respectively. These two major ribotypes were closely related, as they only differed by one band for one restriction enzyme (data not shown). Among the 68 enrofloxacin-resistant isolates, 14 were of the H1P1 ribotype, 47 of the H3P1 ribotype and seven were of other ribotypes. Of the 13 highly fluoroquinolone-resistant isolates whose topoisomerase genes were sequenced and appeared identical, eight were of the H3P1 ribotype and five were of the H1P1 ribotype (TableGo). Thus, if a clonal identity between the O78:K80 fluoroquinolone-resistant isolates presenting the same mutations and the same ribotype cannot be excluded, it appears that at least two epidemiologically distinct populations, characterized by their ribotypes H1P1 and H3P1, have acquired exactly the same mutations during independent selection events toward fluoroquinolone resistance.


    Acknowledgments
 
We thank Annie Brée and Christian Mouline for skilful technical assistance and Hiroshi Nikaido (University of California, Berkeley) for providing the anti-AcrA antibody.


    Notes
 
* Corresponding author. Tel: +33-2-47-42-77-65; Fax: +33-2-47-42-77-74; E-mail: chaslus{at}tours.inra.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Cherifi, A., Contrepois, M., Picard, B., Goullet, P., Orskov, I. & Orskov, F. (1994). Clonal relationships among Escherichia coli serogroup O78 isolates from human and animal infections. Journal of Clinical Microbiology 32, 1197–202.[Abstract]

2 . Everett, M. J., Jin, Y. F., Ricci, V. & Piddock, L. J. (1996). Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrobial Agents and Chemotherapy 40, 2380–6.[Abstract]

3 . 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]

4 . Yoshida, H., Bogaki, M., Nakamura, M. & Nakamura, S. (1990). Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrobial Agents and Chemotherapy 34, 1271–2.[ISI][Medline]

5 . Breines, D. M., Ouabdesselam, S., Ng, E. Y., Tankovic, J., Shah, S., Soussy, C. J. et al. (1997). Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV. Antimicrobial Agents and Chemotherapy 41, 175–9.[Abstract]

6 . Oethinger, M., Kern, W. V., Jellen-Ritter, A. S., McMurry, L. M. & Levy, S. B. (2000). Ineffectiveness of topoisomerase mutations in mediating clinically significant fluoroquinolone resistance in Escherichia coli in the absence of the AcrAB efflux pump. Antimicrobial Agents and Chemotherapy 44, 10–3.[Abstract/Free Full Text]

7 . 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, 814–20.[Abstract/Free Full Text]

8 . Comité de l'Antibiogramme de la Société Française de Microbiologie. (1998). Communiqué 1998. Pathologie Biologie 46, I–XVI.

9 . Giraud, E., Brisabois, A., Martel, J. L. & Chaslus-Dancla, E. (1999). Comparative studies of mutations in animal isolates and experimental in vitro- and in vivo-selected mutants of Salmonella spp. suggest a counterselection of highly fluoroquinolone-resistant strains in the field. Antimicrobial Agents and Chemotherapy 43, 2131–7.[Abstract/Free Full Text]

10 . Giraud, E., Cloeckaert, A., Kerboeuf, D. & Chaslus-Dancla, E. (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]

Received 24 July 2000; returned 24 September 2000; revised 10 November 2000; accepted 14 November 2000