Fluoroquinolone resistance in Campylobacter species from man and animals: detection of mutations in topoisomerase genes

Laura J. V. Piddock*, Vito Ricci, Lilian Pumbwe, Martin J. Everett and Deborah J. Griggs

Antimicrobial Agents Research Group, Department of Infection, The Medical School, University of Birmingham, Birmingham B15 2TT, UK

Received 27 June 2002; returned 5 September 2002; revised 25 September 2002; accepted 3 October 2002


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Consecutive isolates of quinolone-resistant campylobacter isolated over a 5 year period (1990–1995) from the faeces of patients with enteritis in Plymouth, UK, were examined for the epidemiology of mutations in gyrA (n = 127). In addition, clinical isolates and poultry isolates from Germany, The Netherlands and other regions of the UK collected before 1995 were examined for mutations in the quinolone resistance-determining region of gyrA by single-stranded conformational polymorphism analysis and direct sequencing of a 270 bp fragment of PCR-generated DNA. The majority of isolates (173/208) carried a mutation at codon 86 in gyrA resulting in substitution of Ile for Thr; all of these were resistant to ciprofloxacin (MIC >= 2 mg/L). One isolate of Campylobacter jejuni had a mutation at Asp-90, and another had a double mutation at Thr-86 and Pro-104. Only two resistant isolates showed no mutation in gyrA. A novel gyrA sequence was amplified from two Campylobacter lari and one C. jejuni, which exhibited a valine at codon 86. Only 8/192 isolates had changes in gyrB; all were shown to relate to silent mutations in gyrB and presumably reflect natural polymorphisms in the gene.

Keywords: fluoroquinolone, resistance, Campylobacter


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Campylobacter is recognized as a major cause of food poisoning, the most common source of which is poultry.1 Whilst gastroenteritis is normally self-limiting, and antibiotic therapy not usually required, treatment is indicated in immunocompromised patients or if the infection is extraintestinal.2 In such cases, fluoroquinolones are increasingly the treatment of choice as campylobacter is naturally susceptible to these agents (ciprofloxacin MIC90 = 0.5 mg/L) and high concentrations can be attained in the lumen.3 However, since the early 1990s there has been a dramatic increase in the prevalence of fluoroquinolone resistance among campylobacters isolated from both man and poultry4 and this has been shown to correlate with the use of fluoroquinolones in poultry.5

In the UK, there have been sporadic reports of fluoroquinolone-resistant campylobacter, and a longitudinal survey clearly demonstrated a trend towards an increased incidence of resistance, despite a low level of incidence amongst UK-bred poultry flocks.6 The risk factors for acquisition of fluoroquinolone-resistant campylobacter were overseas travel and poultry consumption, and the hypothesis was that imported poultry was the primary source amongst those patients without a recent travel history. In Northern Ireland, fluoroquinolone resistance in human isolates rose from <4% in 1992 to 17% in 2000, but there was no parallel rise in resistance in isolates from locally produced poultry.7 Furthermore, analysis of the phenotypes of human and poultry isolates found little identity between strains from the two sources.8 The higher incidence of resistance in human isolates was attributed to the trend of the consumption of poultry meat in preference to beef, and the increased importation of poultry products. Other recent UK studies have reported rates of quinolone resistance among campylobacter ranging from 6.7% to 18%.911 Recently, Smith et al.12 reported that the duration of gastrointestinal symptoms in patients infected with fluoroquinolone-resistant Campylobacter jejuni was longer than that in patients infected with susceptible isolates.

In campylobacter, mutations in gyrA have been shown to confer decreased sensitivity of DNA gyrase to fluoroquinolone antibiotics, and a corresponding increase in the MIC.13 Quinolone resistance is associated with mutations at Thr-86.14 This codon is analogous to Ser-83 in the gyrA gene of Escherichia coli and other Gram-negative bacteria, and is thought to form part of the quinolone-binding pocket that interacts with the antibiotic.14 The majority of highly fluoroquinolone-resistant clinical isolates of C. jejuni have the GyrA substitution Thr-86 to Ile.1519 Other substitutions reported have been Pro-104 to Ser,17,19 Thr-86 to Lys16 or Ala,18 Ala-70 to Thr, Asp-90 to Asn and transitions at codon 11915,19 but the role in fluoroquinolone resistance has not been established for all of these mutations. No changes in the B subunit gene, gyrB, have yet been documented in campylobacter. There has been one report describing mutations in parC associated with fluoroquinolone resistance in C. jejuni,20 and no reports of homologues of parE.

The aim of this study was to assess the prevalence of gyrA mutations in ciprofloxacin-resistant campylobacter isolated from humans and poultry. The majority of clinical isolates originated from the Plymouth Public Health Laboratory (PHL), which serves a population of 450 000, in urban and rural locations in the south-west of England. These were consecutive ciprofloxacin-resistant strains, isolated between 1990 and 1995, and represented 4% of the total campylobacters isolated by Plymouth PHL during this time period and approximately one-third of these were acquired abroad.6 In the course of our investigations, it became clear that possession of a gyrA mutation, whilst being almost universal, is not the sole mediator of ciprofloxacin resistance in campylobacter, hence the study was broadened to search for mutation(s) in other topoisomerase genes.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Bacterial strains and cultivation

Clinical isolates were obtained from Plymouth PHL (n = 127, isolated 1990–1995),6 Cardiff PHL (n = 3) and Westminster Hospital (n = 4, isolated 1990) in the UK. All isolates from Plymouth were unlinked and non-replicate, acquired by individual patients and were previously shown to be resistant to ciprofloxacin.6 Of these, 107 isolates had been previously identified as C. jejuni, eight as Campylobacter lari and five as Campylobacter coli; species data were not available for seven isolates. Poultry isolates (three C. jejuni, one C. lari, one C. coli, five unknown) have been described previously.6 In addition, 55 campylobacter from Germany (35 human isolates, 20 from animals isolated in 1993; kindly donated by B. Geilhausen, Institute of Medical Microbiology and Hygiene, University of Cologne, Germany), and 10 isolates from The Netherlands (five from humans and five from poultry isolated before 1995; kindly donated by H. P. Endtz, Department of Microbiology and Infectious Diseases, Erasmus University, Rotterdam, The Netherlands) were investigated. All isolates were maintained and cultivated as described previously.6 Type strains of C. jejuni subsp. jejuni (NCTC 11351), C. jejuni subsp. doylei (NCTC 11951), C. coli (NCTC 11366), Campylobacter fetus (NCTC 10842) and C. lari (NCTC 11352) were obtained from the PHLS, Colindale, UK.

Antibiotic susceptibility

The MICs of ciprofloxacin, tetracycline, erythromycin, chloramphenicol and cefotaxime for all isolates were determined by the agar dilution method as described previously 6 as there is no standardized method for this species. However, this method is identical to that currently being evaluated by the NCCLS Veterinary Antimicrobial Susceptibility Testing Campylobacter Working Group, except that the inoculum was 106 cfu/spot. All determinations were repeated on at least two occasions.

DNA isolation

Individual colonies were subcultured from campylobacter selective medium on to Mueller–Hinton agar (Unipath, UK) and incubated in an anaerobic jar with a CampPak (Oxoid Ltd, Basingstoke, UK) at 37°C for 2 days. A cell suspension was made in Mueller–Hinton broth and centrifuged to give an ~50–100 µL cell pellet. Cells were then resuspended in Tris–ethylenediaminetetraacetic acid (TE) buffer and genomic DNA prepared using cetyltrimethylammonium bromide (CTAB)/chloroform extraction.21

Analysis of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB and parC

A 270 bp fragment of the QRDR of gyrA (codons 38–126) was amplified by PCR using primers cjgyrA1 and cjgyrA2 (Figure 1), derived from the C. jejuni gyrA sequence (GenBank accession number L04566).14 An alternative gyrA forward primer (clgyrA1) was used in certain circumstances (see Results) to generate a 235 bp product (codons 50–126; Figure 1). PCR was carried out in a 50 µL volume containing 100 ng genomic DNA, 1 x buffer, 25 pmol each primer, 200 µM each deoxynucleoside triphosphate, 3 mM MgCl2 and 1 U Taq polymerase (Bioline, London) with an initial denaturation at 94°C for 5 min, 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, then a final extension step at 72°C for 10 min. A 382 bp fragment of the QRDR of gyrB (codons 387–499) was amplified using forward primer 5'-ATGGCAGCTAGAGGAAGAGA-3' and reverse primer 5'-GTGATCCATCAACATCCGCA-3', derived from the gyrB sequence obtained from the C. jejuni genome. PCR was carried out in a 50 µL volume containing 100 ng genomic DNA and 50 pmol each primer using 2 x PCR Mastermix (ABgene, Epsom, UK) with an initial denaturation at 95°C for 5 min, 30 cycles of 95°C for 30 s, 52.3°C for 30 s and 72°C for 30 s, then a final extension step at 72°C for 10 min. The QRDR of parC was amplified exactly as described by Gibreel et al.20 using the same primers and conditions for PCR. Mutations present in each gene were detected initially by single-stranded conformational polymorphism (SSCP) analysis and confirmed by DNA sequencing. The SSCP of each amplimer was determined on a Multiphor II with polyacrylamide Cleangel and silver staining (Pharmacia LKB). The DNA sequencing of all amplimers with individual SSCP patterns was carried out by MWG Biotech (Ebersberg, Germany). RT–PCR was carried out to detect transcription of parC in selected isolates. Superscript (Roche, Lewes, UK) was used for first strand synthesis to generate cDNA, which was used as a template for PCR of parC.



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Figure 1. DNA sequence alignment of gyrA from C. jejuni (L04566), C. coli (AF092101), C. fetus (U25640), C. lari (U63412) and Campylobacter spp. (U63414). The positions of primers cjgyrA1 and cjgyrA2 are shown in bold, that of primer clgyrA1 is underlined. Thr-86 is shaded in dark grey, and codons at which silent mutations were observed in SSCP type I isolates are shaded in light grey.

 

    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
SSCP and sequence analysis of gyrA

Chromosomal DNA was prepared from all campylobacter isolates, including the NCTC wild-type strains of C. jejuni, C. jejuni subsp. doyleii, C. fetus, C. lari and C. coli, and a 270 bp fragment of gyrA including the QRDR was amplified from most samples using primers cjgyrA1 and cjgyrA2. However, despite repeated attempts, DNA could not be amplified from 12 isolates, including the C. fetus and C. coli control strains, using these primers. Therefore, a new forward primer (clgyrA1) was designed based on a region of strong homology between the three gyrA sequences of C. lari, C. jejuni and C. fetus. PCR products were obtained using clgyrA1 and cjgyrA2 on 11/12 isolates for which the original PCR had yielded very little or no product. No product could be amplified from one C. jejuni isolate with either set of primers.

PCR amplimers were analysed by SSCP to identify different alleles of gyrA. Several distinct SSCP patterns were identified, which were designated I–VII (Figure 2 and Table 1). In theory, SSCP analysis of a double-stranded PCR product should yield patterns consisting of two bands, each representing the structural conformation of one DNA strand; however, under our experimental conditions, patterns I, III, IV, VI and VII reproducibly consisted of more than two bands, suggesting that certain DNA sequences can adopt more than one stable conformation. The separation of one DNA strand into two SSCP bands due to formation of different stable conformations has also been reported for gyrA in Staphylococcus aureus.22 Analysis of PCR fragments of gyrA, corresponding to each SSCP pattern, by denaturing HPLC23 confirmed that each pattern was distinct (data not shown). Thirty isolates gave the same pattern as the wild-type C. jejuni control strain (type III pattern). By far the greatest number of isolates gave either a type I (n = 122) or type II (n = 46) SSCP pattern. Direct sequencing was carried out on 13 type I and 12 type II PCR products, chosen at random, as well as all those that showed an alternative SSCP pattern. Type I sequences contained four nucleotide changes compared with the published C. jejuni wild-type sequence,14 although only one resulted in a putative amino acid substitution (Thr-86 to Ile; ACA->ATA). Type II sequences contained only a single nucleotide change, again resulting in substitution of Ile for Thr-86. Two isolates gave unique patterns: type IV corresponded to an Asp-90 to Asn change (GAT->AAT), analogous to Asp-87 in E. coli, and type V corresponded to a double mutation of Thr-86 to Ile, and Pro-104 to Ser (CCA->TCA).



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Figure 2. Single-stranded conformational polymorphism analysis of Campylobacter spp. showing patterns I–VII. Pattern types correspond to the following mutations in gyrA: I, C. jejuni Thr-86->Ile plus three silent mutations; II, C. jejuni Thr-86->Ile; III, C. jejuni wild-type; IV, C. jejuni Asp-90->Asn; V, C. jejuni Thr-86->Ile and Pro-104->Ser; VI, C. lari wild-type; VII, C. lari Thr-86->Ile.

 

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Table 1.  SSCP patterns, corresponding DNA sequences and MICs of ciprofloxacin (CIP) for human and poultry isolates and control strains
 
Antibiotic susceptibility

Twenty-eight of 30 isolates with the C. jejuni wild-type (type III) pattern were susceptible to ciprofloxacin (MICs < 0.12–0.5 mg/L; Table 1). All isolates having the Thr-86 to Ile substitution in GyrA (SSCP types I, II and VII) were resistant to ciprofloxacin (MICs 2–>128 mg/L). The range of ciprofloxacin MICs seen in isolates with the Thr-86 to Ile substitution, and the observation that some ciprofloxacin-resistant strains lack a mutation in gyrA, suggests that mutations in other genes may contribute to fluoroquinolone resistance in campylobacter. Ciprofloxacin MICs of 16 and 32 mg/L were determined for two clinical isolates (P129 and P88, respectively), which had no mutation in gyrA. Both isolates were also resistant to tetracycline (>=128 mg/L) and erythromycin (>16 mg/L).

Identification of C. lari gyrA sequence

Sequencing of PCR products giving type VII SSCP patterns (n = 4) revealed a sequence which differed from that of C. lari NCTC 11352 (type VI) by a single nucleotide, but both sequences were significantly different from that of C. jejuni gyrA.14 Superimposing the C. jejuni gyrA reading frame showed the type VII sequence encoded Ile-86 instead of Thr (ACT->ATT), seen in type VI. Two of the four isolates having this SSCP pattern were typed as C. lari, and two as C. coli.

Identification of novel gyrA sequence

PCR using primers clgyrA1 and cjgyrA2 allowed amplification of gyrA from 11/12 isolates for which the original PCR had been unsuccessful. Sequencing of the product from C. coli NCTC 11366 yielded an almost identical sequence to that for C. lari gyrA, except for a single non-substituting nucleotide change (Figure 1). Sequencing the PCR amplimers from three isolates (two C. lari and one C. jejuni) revealed a novel sequence sharing 88% nucleotide identity with the C. lari and C. coli gyrA sequences, and 83% with that of C. jejuni. The putative amino acid translation showed 97% identity with that of C. lari gyrA; two amino acid substitutions were found at Thr-86 and Ile-106, both of which encoded Val. It is not clear whether this represents a wild-type sequence or that of a fluoroquinolone-resistant mutant, as only one allele has been identified; however, it is interesting to note that a Thr-86 to Val change cannot be achieved by a single point mutation (ACA or ACT->GTT; Figure 1). The DNA sequences were lodged with GenBank in July 1996 with the following accession numbers: C. lari U63412, C. coli U63413 and the unknown Campylobacter sp. as U63414. More recently the C. coli gyrA sequence has been published24 but ours remains the only C. lari sequence in GenBank.

Analysis of gyrA from poultry isolates

Analysis of gyrA from poultry isolates showed a similar distribution of alleles as for the clinical isolates with all 10 UK isolates, all five isolates from The Netherlands and 12/20 German isolates possessing the substitution Thr-86 to Ile. The remaining eight German strains had wild-type gyrA and were all susceptible to ciprofloxacin (MIC <= 0.5 mg/L; Table 1).

Comparison of genetic and species data

In general, the gyrA genotype determined for each isolate corresponded to the species data, where known, as determined by traditional microbiological/biochemical tests; however, there were a number of exceptions among isolates of C. coli and C. lari (Table 2). The speciation of the strains with anomalous gyrA sequences was further confirmed with the specific PCR identification method of Fermer and Engvall.25 The species determined by the PCR method confirmed the typing in three isolates and confirmed that suggested by the gyrA sequence in four isolates (Table 2).


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Table 2.  Discrepancies between genetic and species data for isolates of C. coli and C. lari
 
Role of mutation in parC

Despite repeated and prolonged efforts with a variety of experimental strategies, we were unsuccessful in cloning parC from the NCTC type strains of C. jejuni, C. coli, C. lari and C. fetus. We also attempted to amplify parC with primers based on the consensus regions of parC from several different species but no product could be obtained. Gibreel et al.20 published a DNA sequence of a parC gene from C. jejuni; however, this sequence bears far greater homology with E. coli parC and gyrA, than with campylobacter gyrA or than campylobacter gyrA does with E. coli gyrA. A search of the complete genome database of C. jejuni NCTC 11168 with the parC sequence published by Gibreel et al.20 does not reveal any homology other than with gyrA. Oligonucleotide primers designed from the C. jejuni parC DNA sequence (GenBank accession number Y18300) were used in a PCR with genomic DNA from the type strains of all four campylobacter species. A touchdown PCR was also carried out that allows a range of annealing temperatures to be used. PCR amplification of a product using these primers was inconsistent and invariably gave a low yield; however, products were amplified more readily from clinical strains than from C. jejuni NCTC 11168. The nucleotide sequence of the product from a clinical isolate (P9) was highly homologous (99% identity) to that of E. coli parC, with only one nucleotide difference. RT–PCR of C. jejuni NCTC 11168 gave no product and although a product was amplified from cDNA from two clinical isolates and C. jejuni NCTC 11351, DNA sequencing revealed poor homology with parC (28%). No further experiments were carried out.

Role of mutation in gyrB

Silent mutations in gyrB were seen in few isolates (8/192), and presumed to reflect natural polymorphisms in the gyrB gene.


    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
We present here a novel and broad study investigating a large number of fluoroquinolone-resistant campylobacter isolates, of other species in addition to C. jejuni, from humans, animals and food, for mutations in topoisomerase genes. Previous studies have focused upon C. jejuni, as this is the species that causes most gastrointestinal infections in humans.16,17 However, in the present study we examined all species isolated from humans by the Plymouth PHL from 1990 to 1995 plus isolates from retail poultry, and from Germany and The Netherlands. In this study, a region of gyrA was examined encompassing all known loci described previously as containing a mutation conferring nalidixic acid or fluoroquinolone resistance in other bacteria (codons 38–126). We have demonstrated that a Thr-86 to Ile change is present in the vast majority of fluoroquinolone-resistant campylobacter, whether isolated from humans or poultry. One C. jejuni had an alternative mutation at Asp-90 (analogous to Asp-87 in E. coli), and one had a double mutation at Thr-86 and Pro-104. No analogous mutation to that at codon 104 has been reported in the gyrA QRDR of other bacteria; however, Zirnstein et al.17 also reported a C. jejuni with this double mutation. It is unclear whether the Pro-104->Ser substitution contributes to fluoroquinolone resistance in this isolate. Only two resistant isolates (MICs of ciprofloxacin of 16 and 32 mg/L) had no gyrA mutation, suggesting that the development of resistance in the absence of a mutation in gyrA is extremely rare. The mechanism of resistance in these isolates is the subject of further study.

Two alleles of the amplified C. jejuni gyrA fragment were identified; one identical to the published sequence, the other containing three silent mutations at His-81, Ala-119 and Ser-120. Silent mutations at these codons have recently been described in campylobacter isolated in Finland.19 This difference may be of use in differentiating isolates in epidemiological studies. During the course of the study, two new gyrA sequences were identified, one of which was identified as belonging to C. lari. The C. lari gyrA wild-type sequence was most similar to that of C. coli and C. fetus and contained a Thr at codon 86, like both C. jejuni and C. fetus. As in C. jejuni, resistant isolates contained a substitution at this codon of Ile for Thr. Comparison of the predicted amino acid sequence of GyrA for C. lari with that of C. jejuni14 showed that between codons 68 and 124, there were four residues different (codons 75, 79, 107 and 119) and that between codons 56 and 67, the two sequences were very different, which may account for the intrinsic resistance to nalidixic acid of C. lari. The novel sequence, which was amplified from three isolates, was very similar to that identified as the C. lari gyrA sequence, the most noticeable difference being that it encoded Val at codon 86. It is not known whether this sequence represents a wild-type or resistant allele; however, the substitution of Val for Thr cannot be achieved by a single point mutation, and it is possible that this is the sequence of an intrinsically resistant organism.

Despite most isolates having identical amino acid substitutions in the QRDR of gyrA, the MIC of ciprofloxacin varied greatly from 2 to >128 mg/L, suggesting that other factors contribute to the resistance phenotype. In E. coli and other bacteria, topoisomerase IV has been shown to be a secondary target for fluoroquinolone action. High-level resistance is commonly associated with at least one mutation in gyrA and another mutation in the A subunit gene of topoisomerase IV (usually termed parC). In S. aureus, the primary target of many fluoroquinolones is topoisomerase IV, and DNA gyrase is regarded as the secondary target.26 In campylobacter, the overwhelming abundance of gyrA mutations in the isolates we have analysed suggests that mutation in gyrA is probably a first step in resistance and that DNA gyrase is the primary target. We spent considerable effort and a variety of strategies to identify campylobacter parC. To date, we have been largely unsuccessful and are of the opinion that this gene may not be present in all campylobacter, despite topoisomerase IV being an essential enzyme for chromosome partitioning in E. coli. However, genes for topoisomerase IV are absent from the published genomes of Mycobacterium tuberculosis, Treponema pallidum and Helicobacter pylori, and it is thought that DNA gyrase can serve the decatenase functions normally carried out by topoisomerase IV in these species. A thorough and extensive search of the complete genome database of C. jejuni NCTC 11168 failed to identify any genes with homology to parC and it appears most likely that C. jejuni also lacks topoisomerase IV. Only using the exact primers used by Gibreel et al.,20 were we able to amplify any product from campylobacter that was so similar to that of the E. coli parC gene that we suspect it is not native to the campylobacter genome, particularly as this gene is absent from the genome database. Recently, other workers have also described being unable to amplify parC from C. jejuni or C. coli.18

Finally, we add a note of caution with regard to the speciation anomalies with the gyrA data: it is suggested that as some antibiotic-resistant campylobacter may harbour mutations affecting the cell envelope that this may interfere with speciation as defined serologically. As in C. jejuni, fluoroquinolone resistance in C. coli and C. lari is primarily associated with a single Thr-86 to Ile substitution in GyrA in isolates from both humans and animals. Despite an earlier report linking a mutation in parC to fluoroquinolone resistance in C. jejuni,20 we were unable to confirm the role of this gene in campylobacter.


    Acknowledgements
 
This study was supported in part by a grant from Bayer AG, and also by the British Society for the Study of Infection. M.J.E. was a Lilly Industries Research Fellow (1993–1996).


    Footnotes
 
* Corresponding author. Tel: +44-121-414-6969; Fax: +44-121-414-3454; E-mail: l.j.v.piddock{at}bham.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
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