Patterns of quinolone susceptibility in Campylobacter jejuni associated with different gyrA mutations

Pathology (April 2004) 36(2), pp. 166–169

MICROBIOLOGY

Patterns of quinolone susceptibility in Campylobacter jejuni associated with different gyrA mutations CHRISTOPHER J. MCIVER*{{, TIFFANY R. HOGAN*, PETER A. WHITE{ AND JOHN W. TAPSALL*{

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*Department of Microbiology (SEALS), Prince of Wales Hospital, Randwick, NSW; {School of Medical Sciences, Faculty of Medicine, University of New South Wales, NSW and {School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, NSW, Australia

Summary Aims: To investigate the diversity of genetic mutation in the quinolone resistance-determining region (QRDR) of gyrA in clinical isolates and laboratory-derived mutants of Campylobacter jejuni resistant to ciprofloxacin (CipR) and to determine the influence of this mutation on the susceptibility of the organisms to different quinolone antibiotics. Methods: Laboratory-derived CipR mutants were obtained from C. jejuni NCTC 11 168 and six quinolone-sensitive faecal isolates (parent prototypes) grown in sub-inhibitory concentrations of ciprofloxacin. Initial mutants found to be CipR were designated ‘primary mutants’ and subjected to a repeat of this process to select ‘secondary mutants’ with increased resistance. The susceptibility of the mutants and an additional six clinical isolates of CipR C. jejuni to seven quinolone antibiotics was determined by measuring their MICs. The QRDR of gyrA in all strains was amplified by PCR, sequenced and compared with that of the L04566 C. jejuni gyrA gene. Results: All six CipR clinical isolates contained a Thr-86-Ile mutation. This was also the commonest mutation found amongst the laboratory derived CipR strains. Other derived mutations in the in vitro derived CipR group included Asp90-Asn, Thr-86-Ala, and a previously unreported double mutation, Asp-85-Tyr and Thr-86-Ile. Strains with the Thr86-Ile mutation had the highest MICs to seven different quinolones. CipR strains with other single mutations had a lower range of MICs. There were no additional QRDR mutational changes detected in secondary mutants even where MICs to the fluoroquinolones were higher than in primary mutants. Conclusions: Thr-86-Ile mutations were common in both clinical and laboratory derived CipR strains. Other mutations found amongst the latter strains were more sensitive to the fluoroquinolones. Different QRDR changes in gyrA differentially affected the susceptibility of CipR C. jejuni to the various fluoroquinolones. Key words: Campylobacter jejuni, gyrA mutants, quinolone antimicrobial agents. Received 20 June, revised 16 October, accepted December 2003

INTRODUCTION Gastrointestinal infections caused by Campylobacter jejuni are common. For the year 2000, there were 107.1 cases per 100 000 in Australia, three times that reported for salmonella infections.1 Infections are characterised by diarrhoea, vomiting and abdominal pain, and stools are often liquid and show gross blood and mucus. The infection is usually self-limiting, but when chemotherapy is indicated, quinolone antibiotics including norfloxacin and ciprofloxacin are commonly used. In the past decade there has been an emergence of resistance to ciprofloxacin in C. jejuni in both developed and developing countries.2 The prevalence of ciprofloxacin-resistant (CipR) C. jejuni is low in Australia and is attributable to the strict regulation of antibiotic usage in both the treatment of human infections and in the animal food industry. Fluorinated quinolones have increased activity against a broad spectrum of organisms of clinical importance, including anaerobes. These antibiotics inhibit the activity of topoisomerases, vital to the control of the conformation and replication of bacterial DNA. In campylobacter, quinolone resistance is strongly associated with point mutations in the quinolone resistance-determining region (QRDR) of gyrA. To investigate the diversity of mutations that can occur within the QRDR, we examined recent CipR isolates of C. jejuni. Because CipR C. jejuni are rarely encountered in Australia, resistant mutants were also derived from sensitive prototypes to determine the range of gyrA substitutions that may occur in this organism. In Neisseria gonorrhoeae and also in Streptococcus pneumoniae, it has been shown that the nature of the amino acid substitution in the QRDR has differential effects on the susceptibility of the organism to different quinolone antibiotics.3,4 We therefore determined the susceptibility of these CipR strains to a range of quinolones that are representative of four generations of this antibiotic family and correlated these determinations with QRDR changes.

MATERIALS AND METHODS Strains Mutation experiments were performed using six randomly selected quinolone-sensitive faecal isolates of C. jejuni and a control strain

ISSN 0031-3025 printed/ISSN 1465–3931 # 2004 Royal College of Pathologists of Australasia DOI: 10.1080/00313020410001672019

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NCTC 11 168. The molecular analysis of these strains was undertaken with an additional six (CipR) isolates (five from faeces and one blood) two of which have been previously described.5 Isolates were identified using conventional methods and established criteria.6

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Derivation of ciprofloxacin-resistant mutants CipR mutants were derived from the NCTC 11 168 control strain and six sensitive isolates (parent prototypes) by selecting colonies growing on blood-enriched sensitivity agar in the presence of sub-inhibitory concentrations of this antibiotic. Plates of this medium were inoculated as described for the Calibrated Dichotomous Sensitivity (CDS) method7 and an E test strip (AB Biodisk, Sweden) applied to provide a concentration gradient of 0.016–32 mg/mL of ciprofloxacin. Cultures were examined after 7 days incubation at 42‡C under micro-aerophilic conditions. CipR colonies occurring within the zone of inhibition were designated ‘primary mutants’. These colonies were subcultured and subjected to a second round of incubation under the same conditions. CipR strains exhibiting any further resistance were designated ‘secondary mutants’.

Antimicrobial susceptibility testing Ciprofloxacin susceptibility was determined by the CDS method7 and for seven quinolones by means of MIC determinations using two-fold serial dilutions of antibiotic in Sensitest agar (Oxoid, UK) supplemented with 4% horse blood. Isolates were inoculated onto the media using a Steers replicating device8 and incubated at 42‡C for 48 h. The preparation of the antibiotic stocks and determination of the MIC was as has been previously described.9,10 Control strains used included C. jejuni NCTC 11 168,7 Staphylococcus aureus ATCC 29 213 and Escherichia coli ATCC 25 922.11

Molecular detection of quinolone resistance determining region Chromosomal DNA was extracted from a pinhead-sized suspension of a bacterial colony in 30 ml RNAse-free water, and boiled for 5 min. The QRDR of the gyrA gene of all strains was amplified by PCR using the following primers: GYR-A (forward) 5’ GCT ATG CAA AAT GAT GAG GCA 3’ and GYR-A (reverse) 5’ CAG TAT AAC GCA TCG CAG CG 3’. The preparation of the PCR reaction mix and the thermocycling conditions were as previously described.12 Products were purified by polyethylene glycol precipitation and sequenced.12 The sequence of the L04566 C. jejuni gyrA gene in GenBank13 was used to derive the primers and for sequence comparisons of quinolone-susceptible and resistant strains.

RESULTS All six clinical CipR isolates contained only a single Thr86-Ile non-synonymous substitution. Amongst the laboratoryderived CipR strains, three further non-synonymous mutations were identified: Asp-90-Asn, Asp-85-Tyr and Thr-86-Ala. All sequence changes (compared with the prototype) detected in the primary and secondary mutants derived from the control and six sensitive isolates are shown in Table 1. A primary mutant of the control strain (NCTC 11 168) contained two mutations involving amino acid changes, and a single synonymous change at amino acid position 84 (GGApGGT). Synonymous sequence changes at amino acid positions 81 (CACpCAT) and 119 (AGTpAGC) were common. Both these sequence differences were found in two of the six clinical isolates resistant to ciprofloxacin. The results of the susceptibility testing of all strains examined are shown in Table 2. All seven parent prototypes without a detectable amino acid substitution within the QRDR of gyrA were susceptible to the quinolones tested. Table 2 also shows that the nature of the mutation present influenced the MIC for each quinolone. All CipR clinical isolates and laboratory mutants containing the Thr-86-Ile mutation, either alone or else in combination with the Asp-85-Tyr mutation showed raised MICs. Primary laboratory mutants with Thr-86-Ala and Asp-90-Asn substitutions had a MIC to nalidixic acid w128 mg/mL but lower MIC ranges to the fluoroquinolones of generations 2–4. Though the MICs were raised in the presence of these latter mutations, they were at least one-doubling dilution less than the mode MIC for the more common Thr-86-Ile mutants. There was no additional sequence change in the amplified QRDR in gyrA secondary mutants, even though MICs were higher by at least one doubling dilution.

DISCUSSION The ease with which laboratory-derived CipR C. jejuni mutants were obtained here demonstrates the vulnerability of the QRDR of gyrA to mutation and thus quinolone resistance. The Thr-86-Ile substitution is the most

TABLE 1 Comparison of sequence changes detected in induced ciprofloxacin-resistant mutants from sensitive strains of Campylobacter jejuni (control strain NCTC 11 168 and six sensitive faecal isolates) No. of mutations examineda

Sequence changes Prototype Control strain NCTC 11 168

Synonymous

Non-synonymous

Amino acid change

Prim.

Sec.

Nil Nil 84 (GGApGGT)

86 (ACApATA) 90 (GATpAAT) 85 (GATpTAT), 86 (ACApATA)

Thr-86-Ile Asp-90-Asn Asp-85-Tyr; Thr-86-Ile

3 3 1

5 1 0

Asp-90-Asn Thr-86-Ile Thr-86-Ile Thr-86-Ile Thr-86-Ala Asp-90-Asn Asp-90-Asn

5 2 1 1 1 1 2

0 0 1 0 0 0 1

Sensitive faecal isolates POWCJ023 81 (CACpCAT), 119 (AGTpAGC) POWCJ027 119 (AGTpAGC) POWCJ030 Nil POWCJ039 Nil Nil POWCJ041 81 (CACpCAT), 119 (AGTpAGC) POWCJ046 81 (CACpCAT), 119 (AGTpAGC)

90 86 86 86 86 90 90

a Mutants selected on sub-inhibitory concentrations of ciprofloxacin. Abbreviations: Prim., primary mutants; Sec., secondary mutants.

(GATpAAT) (ACApATA) (ACApATA) (ACApATA) (ACApGCA) (GATpAAT) (GATpAAT)

Pathology (2004), 36(2), April

a

Number of strains examined in each category; bfold increase of doubling dilutions in MIC compared with the mode MIC of sensitive parent prototypes; cprimary mutants; dsecondary mutants.

5 2 8 8 5 2 11 7 w128 w7

w9

16

64

5 6 4 6 2 2 –4 2 –8 1 2 –4 0.25 8 10 5 6 4 8 –16 16 –32 0.5–1 2 0.5 6 7 4 7 2 2 –8 4 –8 1 –2 4 –8 0.25 11 12 6 9 5 8 9 6 8 3 w128 w128 32 –64 128 –w128 4 w7 w7 w7 w7 w7

w9 w9 6 w9 3

16 –32 32 –64 8 16 –32 1

32 –128 32 –128 2 –8 16 1

6 2 –4 9 4 –16 6 2 –8 11 32 –64 7 16 –32 w9 128 –w128 w7

– 0.06 – 0.015–0.06 – 0.06 –0.125 – 0.03–0.125 – 0.125 – 0.5–1 –

Parent prototypes Sensitive strains 7 4–8 Ciprofloxacin-resistant clinical isolates Thr-86-Ile 6 w128 Ciprofloxacin-resistant laboratory-derived mutants c Thr-86-Ile (prim. ) 7 w128 Thr-86-Ile (sec.d) 6 w128 Asp-90-Asn (prim.) 11 w128 Asp-90-Asn (sec.) 2 w128 Thr-86-Ala (prim.) 1 w128 Asp-85-Tyr and Thr-86-Ile (prim.) 1 w128

Increase Range Increase Range Increase Range Increase Range Increase Range Increase Range Increaseb Range na Mutation

Generation

1

Nalidixic acid

2

Norfloxacin

2

Ciprofloxacin

3

Grepafloxacin

3

Gatifloxacin

4

Trovafloxacin

4

Moxifloxacin

MCIVER et al.

TABLE 2 The MICs to a panel of quinolones for ciprofloxacin-resistant clinical isolates and laboratory derived mutants of Campylobacter jejuni and their parent prototypes. Also showing the fold-increase of doubling dilutions in MIC compared with the mode MIC of sensitive parent prototypes for each quinolone

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commonly reported mutation amongst clinically isolated CipR strains.13–16 The other mutations detected, including Asp-90-Asn and Thr-86-Ala, are less frequently encountered,14,17 and to our knowledge the Asp-85-Tyr and Thr86-Ile double mutation has not been previously reported in clinical isolates. It is possible that the sequence changes found in these in vitro-derived mutants do not occur in clinical isolates and that differences in environments induce the different QRDR-derived resistance. This phenomenon was demonstrated by Bjo¨rkman et al.18 in studies of resistant salmonella where different fitnesscompensatory mutations were selected depending on whether the bacteria evolved through serial passage in mice (in vivo) or in broth (in vitro). Also, of interest here is the influence of the nature of the amino acid substitution on the differential expression of MICs to the various quinolones. This effect has been noted in gonococci and pneumococci3,4 where the relative activity of different quinolones alters with different amino acid substitutions. As is shown in Table 2, this phenomenon was also observed in this study. When compared with ciprofloxacin, MIC ranges for Thr-86-Ile mutants for gatifloxacin and moxifloxacin (a third- and fourth-generation quinolone, respectively) showed similar MIC reductions. However, for grepafloxacin (a third-generation quinolone) the MIC ranges were actually higher for this mutation. The MIC range for trovafloxacin (a fourthgeneration quinolone) was between those of these two groups of quinolones. This trend was also seen with the Asp-90-Asn and Thr-86-Ala mutants, even though these changes were associated with MICs of at least two doubling dilutions less than those seen with Thr-86-Ile mutants. The rarity of the Thr-86-Ala mutation, which is more quinolone sensitive, may be a result of it being overlooked in clinical isolates. There was no additional sequence change detected in the QRDR in gyrA of the secondary mutants derived from CipR Thr-86-Ile and Asp-90-Asn primary mutants. However, this was despite the MICs for the secondary mutants being raised by at least one dilution for the newer fluoroquinolones (Table 2). Thus, it is likely that other influences, such as decreased outer membrane permeability or export through an active efflux mechanism, may be involved in the resistance.19,20 Resistance to quinolones associated with mutations within the QRDR of the parC gene have been described in C. jejuni by Gibreel et al.15 These workers used oligonucleotide primers targeted to sequences in Escherichia coli parC. In this study, we used the same primers and a re-designed set from archived sequences in GenBank (Seq: CJE18300). However, parC was only amplified in E. coli control strains NCTC 10 418 and ATCC 11 560 but not in C. jejuni NCTC 11 168 and five other strains of campylobacter from which gyrA was successfully amplified. This difficulty has also been previously reported15,21 and to date the complete C. jejuni genome does not include parC (National Center for Biotechnology Information). The association with high-level resistance to different quinolones helps to account for the predominance of Thr86-Ile mutations amongst clinical isolates of Campylobacter.16 However, the ease with which other mutations can also be derived in sub-inhibitory concentrations of ciprofloxacin demonstrated here, highlights the

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vulnerability of Campylobacter to acquire quinolone resistance. Further, it is evident by the diversity of QRDR mutations amongst the laboratory-derived strains that environment may exert a selective pressure for mutation type. Other studies have also shown that fluoroquinolones such as enrofloxacin—a metabolic precursor to ciprofloxacin that is used in the poultry industry in some countries—will rapidly select for resistant mutants, thereby rendering fluoroquinolones ineffective for the treatment of campylobacteriosis.22 Recent surveys in the Australian states of New South Wales, Western Australia and the Australian Capital Territory did not find CipR amongst locally acquired strains of Campylobacter.23 The prevalence of CipR strains in these studies was low (3.2%) and found to be predominantly acquired overseas. Furthermore, in a clinical study in immuno-compromised patients with Campylobacter diarrhoea, resistance emerged in vivo following quinolone therapy.24 These reports coupled with our observations reinforce the continuing need for judicious use of these therapeutic agents in Campylobacterassociated infection. Further, the diversity of the different effects of QRDR changes means that results of testing one fluoroquinolone agent cannot necessarily be extrapolated to another. Thus, individual quinolone agents used for therapeutic purposes must be tested separately. Address for correspondence: Dr Christopher J. McIver, Department of Microbiology (SEALS), Prince of Wales Hospital, Barker St, Randwick, NSW 2031, Australia. E-mail: [email protected]

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