Analysis of topoisomerase mutations in fluoroquinolone-resistant and -susceptible Campylobacter jejuni strains isolated in Senegal

International Journal of Antimicrobial Agents 29 (2007) 397–401

Analysis of topoisomerase mutations in fluoroquinolone-resistant and -susceptible Campylobacter jejuni strains isolated in Senegal Alfred Dieudonn´e Kinana a,∗ , Eric Cardinale b , Ibrahim Bahsoun a , Fatou Tall b , Jean-Marie Sire a , Benoit Garin a , Cheikh Saad-Bouh Boye c , Jacques-Albert Dromigny a , Jean-David Perrier-Gros-Claude a a

c

Laboratoire de Biologie M´edicale, Institut Pasteur de Dakar, Senegal b CIRAD, BP 2057, Dakar, Senegal Laboratoire de Bact´eriologie–Virologie, Hˆopital Aristide Le Dantec, Dakar, Senegal Received 17 April 2006; accepted 7 November 2006

Abstract In this study, topoisomerase mutations in ciprofloxacin-resistant and -susceptible Campylobacter jejuni were analysed by DNA sequencing. In certain ciprofloxacin-resistant C. jejuni, the mechanism of resistance was complex. The Thr86-Ala substitution in the GyrA protein appears to play a role in increasing the minimum inhibitory concentration of nalidixic acid only. In addition, isolates with this amino acid change and those resistant to quinolones but lacking a mutation in the GyrA quinolone resistance-determining region could be derived from two different clones. Based on gyrA and gyrB polymorphisms, C. jejuni isolates from the Dakar region of Senegal appeared to be less diverse than those from other countries. Moreover, C. jejuni isolates in Senegal appeared to differ from European isolates by lack of a silent mutation at codon 120 of the gyrA gene. © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Campylobacter jejuni; Fluoroquinolones; Resistance; Topoisomerases; Mutation; Senegal

1. Introduction In developing countries, the estimated incidence of Campylobacter in diarrhoea reaches between 40 000 and 60 000 per 100 000 for children under 5 years old and 90/100 000 in the general population [1]. Consumption of poultry products appears to be one of the main sources of human infection [1]. Fluoroquinolone resistance in Campylobacter is increasing all over the world and has been linked to the introduction of these agents in veterinary medicine [2]. In Senegal, fluoroquinolones (enrofloxacin, norfloxacin) were introduced in poultry production in 1996 and the incidence of quinolone-resistant Campylobacter jejuni isolated from commercial chicken reached 40% [3]. In C. jejuni, quinolone resistance is mediated by mutations within the ∗

Corresponding author. Tel.: +221 839 92 00; fax: +221 822 70 52. E-mail address: [email protected] (A.D. Kinana).

quinolone resistance-determining region (QRDR) of the gyrA gene [4,5]. The main mutation associated with high-level resistance to fluoroquinolones in clinical isolates is a C to T transition at codon 86 within the QRDR of the gyrA gene, leading to a Thr86-Ile substitution [5,6]. Several other mutations within the gyrA QRDR have been described at positions 86 (Thr86-Ala), 90, 70 and 104 in clinical isolates or only in laboratory mutants [5–7], but multiple associated GyrA substitutions have been rarely described. However, the Thr86-Ile substitution as well as the Thr86-Ala substitution appear to confer different quinolone minimum inhibitory concentration (MICs) in different strains [6,7]. To explain the wide range of quinolones MICs for the same mutation as well as the existence of quinolone-resistant Campylobacter strains lacking mutations in the GyrA QRDR [6], it has been suggested that mutations outside the gyrA QRDR or in the other topoisomerase genes or efflux pumps might modulate the ultimate MIC. Indeed, in Escherichia coli the presence of

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A.D. Kinana et al. / International Journal of Antimicrobial Agents 29 (2007) 397–401

a single mutation in the gyrA QRDR results only in resistance to nalidixic acid. To achieve high-level resistance to fluoroquinolones, additional mutations in the gyrA QRDR and/or gyrB gene or in topoisomerases IV (parC, parE) are required [8,9]. To date, no mutation in gyrB has been associated with fluoroquinolone resistance in C. jejuni [4,6] and the role of mutations in topoisomerase IV, previously reported [10], has not been confirmed by others studies. However, there has been one report describing mutations outside the GyrA QRDR (Asn203-Ser and/or Ala206-Thr) associated with fluoroquinolone resistance [11]. The role of efflux pumps in intrinsic and acquired resistance of Campylobacter to antibiotics (including quinolones) has recently been shown [12–14]. The purpose of this study was to assess the possible role of gyrB, parC and the downstream part of the gyrA QRDR in quinolone resistance among C. jejuni isolates from poultry in Senegal. Furthermore, polymorphism of gyrA and gyrB genes was explored in order to trace the epidemiology of Senegalese isolates.

ditions (5% O2 , 10% CO2 and 85% N2 ) at 42 ◦ C for 48 h. 2.2. Antibiotic susceptibility The antibiotic susceptibility of each isolate was determined by the disk diffusion method. MICs of ciprofloxacin and nalidixic acid were determined by the Etest method (AB Biodisk, Solna, Sweden). Isolates were incubated on Mueller–Hinton blood agar medium and Etest strips were applied to each plate. Campylobacter jejuni subsp. jejuni ATCC 33560 (NCTC 11351) was included as a quality control strain. Plates were incubated at 42 ◦ C in microaerophilic conditions for 24 h. The following breakpoints were used: ciprofloxacin MIC ≥ 4 ␮g/mL [14] and nalidixic acid MIC > 16 ␮g/mL [15]. 2.3. Detection of mutations within the QRDR of gyrA (codons 54–126) Chromosomal DNA from each isolate was prepared by boiling as described by Bachoual et al. [4]. Amplification of the 220 bp fragment of the QRDR of gyrA (codons 54–126) from C. jejuni were performed using primers and polymerase chain reaction (PCR) conditions already described [5], except that the annealing temperature was 50 ◦ C.

2. Materials and methods 2.1. Bacterial isolates Campylobacter jejuni isolates originating from chicken carcasses were collected during 2000–2002 in the Dakar region (Senegal) and suburbs [3]. This is a limited study using isolates from a restricted geographical location. A total of 54 isolates, randomly selected from amongst 99 C. jejuni isolates, were included in the study and covered 14 dispersed collection sites over a 3-year period. Isolates were cultured on Columbia agar plates (Becton Dickinson, Heidelberg, Germany) containing 5% sheep blood and incubated under microaerophilic con-

2.4. Detection of mutations outside the QRDR of gyrA (codons 107–239) and in gyrB and parC The downstream fragment of gyrA (codons 107–239) was amplified using the primers IPDgyrA1 (5 -ACAGGACAAGGCAACTTTGG-3 ) and IPDgyrA2 (5 -CCCTGTGCGATAAGCTTCTAT-3 ). PCR cycling conditions were as follows: initial denaturation at 95 ◦ C for 5 min, followed by 35 cycles of 95 ◦ C for 50 s, 56 ◦ C for 1 min

Table 1 Minimum inhibitory concentration (MIC) ranges and mutations within the gyrA quinolone resistance-determining region (QRDR) of Campylobacter jejuni isolates Phenotype

Cip-S and Nal-S Cip-S and Nal-I Cip-R and Nal-R

Cip-S and Nal-S

No. of isolates

MIC range (␮g/mL)

Nucleic acid codons and corresponding amino acids of C. jejuni QRDR of gyrA

Cip

Nal

Codon

Amino acid

CAC

His-81

ACA

Thr-86

GGC

Gly-110

AGT

Ser-119

24

0.062–0.5

1–2

– –T

–

–––

–

–––

–

– –C

–

1 0.125

0.125 16

– –T – –T

– –

––– G– –

– Ala-86

– –T – –T

– –

– –C – –C

– –

15

32 to >32

64 to >256

– –T

–

–T–

Ile-86

–––

–

– –C

–

3 4 5 45

16 to >32 8 to >32 8 to >32 0.032–0.5

>256 32–128 64 to >256 1–8

– –T – –T – –T N.D.

– – – N.D.

–T– G– – ––– N.D.

Ile-86 Ala-86 – N.D.

– –T – –T ––– N.D.

– – – N.D.

– –C – –C – –C N.D.

– – – N.D.

1 2

Codon

Amino acid

Cip, ciprofloxacin; Nal, nalidixic acid; S, susceptible; R, resistant; I, intermediate; N.D., not determined.

Codon

Amino acid

Codon

Amino acid

(C–T) (C–T) – (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) MIC, minimum inhibitory concentration; Cip, ciprofloxacin; Nal, nalidixic acid. a Transitions in parentheses are silent mutations.

469 454 436

(C–T) (C–T) – – – (C–T) (C–T) – (C–T) – – – – – – (A–G) (A–G) – – (A–G) – (A–G) (A–G) (A–G)

403 389

(C–T) (C–T) (C–T) – – (C–T) (C–T) (C–T) (C–T) – (C–T) – (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) – (C–T)

208 206

– – – Thr Thr – – – – – – Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser

203

– – – – – – – (T–C) – – (T–C) –

1 1 2 3 3 4 4 5 6 7 8 9

399

(T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C)

161 157

(C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C) (T–C)

119 110

– – – – – (C–T) (C–T) – (C–T) – – (C–T) – – – – – Ala Ala Ile Ile – – Ala (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T) (C–T)

86 Nal

2 >256 2 8 128 16 32–128 >128 >256 >256 >256 128

Muted codons of gyrB (codons 389–469)a

81

Within the QRDR of gyrA (codons 54–126), three silent mutations were identified (Table 1). With a larger fragment of gyrA (codons 54–239) and gyrB (codons 371–540), isolates showed at most 10 non-coding mutations (Table 2). Nine genotypes were identified.

0.125 >32 0.125 0.5 8 0.125 >32 >32 16 >32 >32 8

3.3. Polymorphism of gyrA and gyrB genes

Cip

To assess the possible role of mutations in gyrB, parC and the downstream part of gyrA QRDR (codons 107–239) in quinolone resistance, five quinolone-resistant isolates lacking a mutation in the GyrA QRDR, the six isolates carrying the Thr86-Ala substitution, three isolates randomly picked amongst quinolone-resistant isolates with the usual Thr86-Ile substitution and three other isolates chosen amongst susceptible isolates were analysed (a total of 17 isolates). For the downstream part of GyrA QRDR, all 17 isolates carried the Asn203-Ser substitution, and only three had an additional substitution at codon 206 (Ala206-Thr) (Table 2). The QRDR of gyrB from these isolates was sequenced but showed silent mutations only. Despite repeated and prolonged efforts, it was not possible to amplify the QRDR of parC using primers and conditions described by Gibreel et al. [10].

1 1 1 1 1 2 3 2 1 2 1 1

3.2. Mutations outside the QRDR of gyrA (codons 107–239) and in gyrB and parC

Muted codons of gyrA (codons 81–208)a

Among the highly ciprofloxacin-resistant isolates (MICs 8 ␮g/mL to >32 ␮g/mL), 18 exhibited the Thr86-Ile substitution, 4 had the Thr86-Ala substitution and 5 showed no mutation in the GyrA QRDR (Table 1). However, two isolates susceptible to ciprofloxacin (MIC = 0.125 ␮g/mL) but intermediate to nalidixic acid (MIC = 16 ␮g/mL) also had the Thr86-Ala substitution in the GyrA protein. The 25 susceptible isolates showed silent mutations only.

MIC (␮g/mL)

3.1. Mutations within the QRDR of gyrA (codons 54–126)

No. of isolates

3. Results

Table 2 Mutations within and outside the quinolone resistance-determining region (QRDR) of gyrA, and in the gyrB gene from 17 ciprofloxacin-resistant and -susceptible Campylobacter jejuni isolates

and 72 ◦ C for 30 s, with a final step 72 ◦ C for 10 min. A 358 bp fragment of the gyrB gene was amplified using PCR conditions and primers as previously described [4], except that the annealing temperature was 53 ◦ C for 50 s. The parC gene was amplified using primers and PCR conditions defined by Gibreel et al. [10]. After purification of the PCR products using QIAquick® Gel extraction Kit (Qiagen, Hilden, Germany), DNA sequences of both strands were determined in an ABI PRISM® 310 (Applied Biosystems, Courtaboeuf, France) and compared with the published DNA sequences of C. jejuni (GenBank accession numbers L04566 and AL139074 for gyrA and gyrB, respectively) using the CLUSTALW program (http://bioweb. pasteur.fr/seqanal/interfaces/clustalw.html).

Genotype

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4. Discussion In this study, topoisomerase mutations in quinoloneresistant and -susceptible C. jejuni isolates were analysed. As previously reported [4,5], the Thr86-Ile substitution was the main substitution associated with high-level resistance to quinolones. In addition, another substitution was found in quinolone-resistant isolates, Thr86-Ala. This substitution was first reported in a resistant clinical strain with an elevated nalidixic acid MIC (64 mg/L) and a small increase in the ciprofloxacin MIC (2 mg/L) [4], and recently in chicken isolates expressing high resistance to nalidixic acid (MICs > 32 mg/L) whilst remaining susceptible to ciprofloxacin [7,16]. The particular finding in this study was that Thr86-Ala was expressed among two isolates with a small increase in the nalidixic acid MIC (16 ␮g/mL), not sufficient enough to reach the resistance breakpoint (MIC > 16 ␮g/mL). Nevertheless, these two isolates have an eight-fold increase in the MIC of nalidixic acid compared with the modal MIC observed for susceptible isolates from Senegal (2 ␮g/mL). Thus, the Thr86-Ala substitution appears to play a role in increasing the MIC of nalidixic acid in C. jejuni isolates and this substitution appears to be mainly observed among isolates of chicken origin. It has been suggested that, similar to Enterobacteriaceae, mutations in gyrB or parC genes or outside the QRDR of gyrA may modulate the ultimate MIC conferred by the critical amino acid change within the GyrA QRDR [5,8]. Indeed, C. jejuni isolates with the same mutation had variable MICs. Moreover, certain resistant isolates lacked a mutation in the GyrA QRDR. The QRDR of the gyrB gene from 17 isolates (described above) was sequenced but showed silent mutations only. Furthermore, using primers and conditions described by Gibreel et al. [10], we were unable to amplify the QRDR of parC as has been reported elsewhere [4,6]. Changing PCR conditions such as annealing temperature (range of temperatures 53–63 ◦ C) and MgCl2 concentration (1–2 mM) did not allow any product recovery. It should be noted that the work of Gibreel et al. [10] is unique work describing the parC gene from Campylobacter. Consecutive studies failed to amplify the QRDR of parC from Campylobacter using the same primers described by Gibreel et al. Furthermore, Bachoual et al. [4], using various degenerate oligonucleotide primers, could not amplify the QRDR of parC from Campylobacter and concluded that Campylobacter lacks genes for topoisomerase IV. Our results, in accordance with previous studies [4,7], confirm that gyrB is not involved in quinolone resistance and that parC may not be present in Campylobacter. Indeed, a search in the published genome of C. jejuni NCTC 11168 failed to identify any genes with homology to parC. In addition, genes for topoisomerases IV are absent from the published genomes of an increasing number of bacteria (Treponema pallidum, Mycobacterium tuberculosis and Helicobacter pylori), and DNA gyrase presumably assumes the function of topoisomerase IV in these organisms. It is likely that C. jejuni also lacks topoisomerase IV. On the

other hand, we sequenced a downstream part of the gyrA gene (codons 107–239), identifying Asn203-Ser and Ala206Thr substitutions regardless of quinolone susceptibility. This result, in accordance with a previous study [16], suggests that these amino acid changes are not linked with fluoroquinolone resistance. Other factors, such as membrane permeability or efflux pumps, may contribute to the resistance phenotype in these isolates. Campylobacter jejuni is known to be intrinsically less susceptible to quinolones probably owing to the difference in GyrA sequence compared with E. coli [5]. It has been argued that gyrA polymorphism could be of use in epidemiological studies [6,17,18]. Several silent mutations were found in this study and no isolate was identical to the wild-type [19]; and except for gyrB polymorphism, which has been rarely studied, these silent mutations have already been reported by others [6,7,17,18]. In terms of the number of gyrA alleles, C. jejuni isolates from the Dakar region appeared to be less diverse than isolates described in other countries. Indeed, even studying a larger part of gyrA, the number of gyrA alleles among Senegalese isolates appears to be lower than that previously reported: 8 alleles for Hakanen et al. [17], 15 for Griggs et al. [7], 7 for Piddock et al. [6] and 14 for Dionisi et al. [16]. Moreover, analysis of gyrB polymorphism confirmed the relatively low diversity of our C. jejuni isolates and suggested that isolates with the Thr86-Ala substitution and those resistant to quinolones but lacking a mutation in GyrA QRDR could be derived from two different clones. As in isolates from Europe and the USA [6,7,18], the mutation at Thr-86 was associated with silent mutations at His-81 and Ser-119 (but not at codon 120 for isolates from Senegal). Although the number of isolates was small and the study was limited to the Dakar region and suburbs, it appears that C. jejuni isolates from Senegal differ from European isolates by lack of a silent mutation at codon 120 of gyrA. Further studies with a larger number of isolates, including isolates from other regions of Senegal, should reveal whether or not that this reflects the real tendency in C. jejuni isolates from Senegal.

Acknowledgments We thank Rokhaya Mbaye, Modou Diagne, Armel Bissila and Fatou Kin´e Loum for their technical help. We also thank Elisa Deriu for useful discussions. This work was supported by the Pasteur Institute in Dakar, Senegal.

References [1] Coker AO, Isophi RD, Thomas BN, Amisu KO, Obi L. Human campylobacteriosis in developing countries. Emerg Infect Dis 2002;8: 237–43. [2] Engberg J, Aarestrup FM, Taylor DE, Gerner-Smidt P, Nachamkin I. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerg Infect Dis 2001;7:24–34.

A.D. Kinana et al. / International Journal of Antimicrobial Agents 29 (2007) 397–401 [3] Cardinale E, Dromigny JA, Tall F, Ndiaye M, Konte M, Perrier-GrosClaude JD. Fluoroquinolone susceptibility of Campylobacter strains, Senegal. Emerg Infect Dis 2003;9:1479–81. [4] Bachoual R, Ouabdesselam S, Mory F, Lascols C, Soussy CJ, Tankovic J. Single or double mutational alterations of gyrA associated with fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli. Microb Drug Resist 2001;7:257–61. [5] Wang Y, Huang WM, Taylor DE. Cloning and nucleotide sequence of the Campylobacter jejuni gyrA gene and characterization of quinolone resistance mutations. Antimicrob Agents Chemother 1993;37:457–63. [6] Piddock LJV, Ricci V, Pumbwe L, Everett MJ, Griggs DJ. Fluoroquinolones resistance in Campylobacter species from man and animals: detection of mutations in topoisomerase genes. J Antimicrob Chemother 2003;51:19–26. [7] Griggs DJ, Johnson MM, Frost JA, Humphrey T, Jorgensen F, Piddock LJV. Incidence and mechanism of ciprofloxacin resistance in Campylobacter spp. isolated from commercial poultry flocks in the United Kingdom before, during, and after fluoroquinolone treatment. Antimicrob Agents Chemother 2005;49:699–707. [8] Friedman SM, Lu T, Drlica K. Mutations in DNA gyrase A gene of Escherichia coli that expands the quinolone resistance-determining region. Antimicrob Agents Chemother 2001;45:2378–80. [9] Ruiz J. Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection. J Antimicrob Chemother 2003;51:1109–17. [10] Gibreel A, Sj¨ogren E, Kaijser B, Wretlind B, Sk¨old O. Rapid emergence of high-level resistance to quinolones in Campylobacter jejuni associated with mutational changes in gyrA and parC. Antimicrob Agents Chemother 1998;42:3276–8. [11] Ge B, White DG, McDermott PF, et al. Antimicrobial-resistant Campylobacter species from retail raw meats. Appl Environ Microbiol 2003;69:3005–7.

401

[12] Ge B, McDermott PF, White DG, Meng J. Role of efflux pumps and topoisomerase mutations in fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli. Antimicrob Agents Chemother 2005;49:3347–54. [13] Lin J, Michel OL, Zhang Q. CmeABC functions as a multidrug efflux system in Campylobacter jejuni. Antimicrob Agent Chemother 2002;46:2124–31. [14] Luo N, Sahin O, Lin J, Michel LO, Zhang Q. In vivo selection of Campylobacter isolates with high levels of fluoroquinolone resistance associated with gyrA mutations and the function of the CmeABC efflux pump. Antimicrob Agents Chemother 2003;47: 390–4. [15] Corcoran D, Quinn T, Cotter L, Fanning S. Relative contribution of target gene mutation and efflux to varying quinolone resistance in Irish Campylobacter isolates. FEMS Microbiol Lett 2005;253: 39–46. [16] Dionisi AM, Luzzi I, Carattoli A. Identification of ciprofloxacinresistant Campylobacter jejuni and analysis of the gyrA gene by the LightCycler mutation assay. Mol Cell Probes 2004;18: 255–61. [17] Hakanen A, Jalava J, Kotilainen P, Jousimies-Somer H, Siitonen A, Huovinen P. gyrA polymorphism in Campylobacter jejuni: detection of gyrA mutations in 162 C. jejuni isolates by single-strand conformation polymorphism and DNA sequencing. Antimicrob Agents Chemother 2002;46:2644–7. [18] Zirnstein G, Li Y, Swaminathan B, Angulo F. Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis. J Clin Microbiol 1999;37:3276–80. [19] Parkhill J, Wren BW, Mungall K, et al. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 2000;403:665–8.