JAC Advance Access originally published online on October 2, 2007
Journal of Antimicrobial Chemotherapy 2007 60(6):1251-1257; doi:10.1093/jac/dkm345
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Spontaneous mutation frequency and emergence of ciprofloxacin resistance in Campylobacter jejuni and Campylobacter coli
Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
* Corresponding author. Tel: +358-9-191-57113; Fax: +358-9-191-57101; E-mail: marja-liisa.hanninen{at}helsinki.fi
Received 29 March 2007; returned 11 May 2007; revised 16 July 2007; accepted 13 August 2007
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Abstract |
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Objectives: We analysed the contribution of spontaneous mutation frequency to the evolution of ciprofloxacin resistance and the diversity of mutations in the quinolone resistance-determining region (QRDR) of gyrA and in the intergenic region, cmeR–cmeA, of the CmeABC efflux pump in Campylobacter jejuni and Campylobacter coli.
Methods: The mutation frequency was measured in 11 quinolone-susceptible C. jejuni and 5 C. coli strains, with and without ox bile. MICs and target-specific and efflux-associated mutations were studied for a number of colonies of each strain selected from plates containing 1 mg/L ciprofloxacin.
Results: The spontaneous mutation frequency level among susceptible C. jejuni and C. coli strains ranged from hypomutable (
4 x 10–9) to strongly mutable (7 x 10–3). Ox bile had no effect on mutation frequency. Pre-existing ampicillin and tetracycline resistance increased the MICs of ciprofloxacin for two strains from 0.032–0.125 to 0.5 mg/L. MICs of ciprofloxacin for the colonies selected from plates containing 1 mg/L ciprofloxacin varied from 1 to 16 mg/L, with the Thr-86
Ile or Asp-90Asn mutation detected in the QRDR of gyrA. In 21.5% (14/65) of the selected colonies, no specific mutations existed. Two types of mutations in CmeR promoter-binding inverted sequences were identified both in the parent strains and in the colonies selected from ciprofloxacin plates.
Conclusions: The variation in mutation frequencies between most C. jejuni and C. coli strains was up to 700-fold. Mutation in the QRDR of gyrA or in the intergenic region was not associated with differences in spontaneous mutation frequencies. Previously acquired resistance to tetracycline and ampicillin predisposed strains to high-level ciprofloxacin resistance and to multiple antibiotic resistance.
Keywords: antimicrobials , gyrA , CmeABC
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Introduction |
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Campylobacter jejuni and Campylobacter coli are two of the most important enteric bacterial pathogens in industrialized countries worldwide.1,2 These bacteria have a large variety of animal hosts and are mostly transmitted to humans through contaminated food or water. Poultry and wild birds are major reservoirs of the organisms and play a key role in the epidemiology of infection.1
Fluoroquinolone resistance of human and animal C. jejuni and C. coli isolates has been a growing problem, especially in countries where fluoroquinolones are extensively used to treat infectious animal diseases, such as Escherichia coli infections in poultry.3,4 In earlier studies, in vivo evolution of fluoroquinolone resistance was developed by treating chickens with enrofloxacin, a fluoroquinolone commonly used in veterinary medicine.5–7 As a consequence, fluoroquinolone-resistant C. jejuni populations in both individual chickens and whole flocks became dominant, and the resistance persisted even in the absence of selection pressure.5–8
CmeABC, a resistance-nodulation division-type efflux pump,7,9–11 and alteration of the quinolone-binding site in the gyrA gene by mutation11–14 are involved in fluoroquinolone resistance. CmeABC is a multidrug-resistance efflux pump system encoded by a three-gene operon (cmeABC).9 The expression of CmeABC is repressed by a transcriptional repressor CmeR, encoded by cmeR, that directly binds an inverted repeat sequence localized in the cmeR–cmeA intergenic region.15 Mutations in cmeR or in the CmeR-binding site impede repression and result in overexpression of cmeABC and enhanced resistance to several classes of structurally differing antimicrobials.15 CmeABC is also involved in the resistance of C. jejuni to bile, and bile salts were shown to elevate the expression of this efflux operon, possibly by triggering conformational changes in CmeR, resulting in reduced binding affinity to the promoter region.16 The gyrA gene encodes a subunit of DNA gyrase, an enzyme involved in DNA replication and transcription.4,12 A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA has been shown to be involved in clinically relevant levels of resistance.3,4,7 Mutations include specific point mutations leading to substitutions at Thr-86, Ala-70 and/or Asp-90.4,6,11–14 However, some fluoroquinolone-resistant isolates do not have any resistance-associated mutations in gyrA.14,17,18
Multiresistance among C. jejuni isolates from both animal and human sources is on the rise. In addition to quinolones, isolates are commonly resistant to macrolides, tetracycline and ampicillin.14,17,18 The percentage of multiresistant isolates has ranged from 0.5% to 15% in different studies.17,18
Bacteria are able to increase their rate of mutation in stressful conditions, and, subsequently, the probability of changes arising that have an impact on antimicrobial susceptibility increases.19–21 In vitro studies indicate that under exposure to fluoroquinolones, selective pressure favours the emergence of mutator strains.21,22 A general definition of the term ‘hypermutable’ does not exist, but it has been defined for several bacterial species. For example, studies with E. coli defined normomutable strains as those with a mutation frequency of 8 x 10–9–4 x 10–8, weakly hypermutable as those with a mutation frequency of 4 x 10–8–4 x 10–7, strongly hypermutable strains as those with a frequency of >4 x 10–7 and hypomutable strains had frequencies of <8 x 10–9.23 According to the results of studies performed with E. coli, only a minority of the strains (0.7%) collected from blood cultures, positive urinary cultures or faecal cultures of healthy persons originating from several countries were of the hypermutable type.23 In hypermutation studies involving Helicobacter pylori, the mutation frequencies of the strains varied from 4 x 10–8 to 3 x 10–5, analysed as frequencies of point mutations in the rboB gene after rifampicin challenge. Most H. pylori strains showed higher frequencies than wild-type Enterobacteriaceae,24 suggesting that H. pylori represents a bacterial population with a high spontaneous mutation frequency.
Studies on frequency and formation of resistant Campylobacter isolates in the intestines of animals during antimicrobial treatment are important for the understanding of the evolvement, spreading and persistence of resistant bacteria. The aim of this study was to examine in vitro spontaneous mutation frequencies in a collection of quinolone-susceptible C. jejuni and C. coli isolates, to elucidate the impact of ciprofloxacin on selection of multiresistant strains and to analyse genetic mechanisms behind resistance.
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Bacterial strains
Eleven C. jejuni and five C. coli strains isolated from chickens, human patients or cattle were included in this study. In addition, two variants selected in vitro to ampicillin/tetracycline resistance after serial passage in increasing concentrations of each drug from two parent strains (C. coli 6590AT and C. jejuni 49/7RAT) were included in the study (Table 1).
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Induction of mutations and estimation of mutation frequencies
To investigate mutation frequencies in Campylobacter, the methods of Björkholm et al.24 and Wang et al.25 with minor modifications were applied. Briefly, bacterial strains were grown in 5 mL of Brucella broth for 48 h, after which 30 µL aliquots of each strain were distributed into 20 tubes containing 3 mL of Brucella broth. Cultures were grown for 48 h at 37°C. After incubation, colony counts (cfu) of evolved mutants in each tube were determined by spreading either 500 µL or, in cases where the plates were overgrown by resistant mutants, 100 µL of 10-fold dilutions, on Brucella blood agar plates containing 1 mg/L ciprofloxacin. The number of viable bacteria was determined from three tubes by plating 10–6, 10–7 and 10–8 dilutions on non-selective Brucella blood agar plates. After 3–4 days of incubation in a microaerobic atmosphere at 37°C, colonies were counted. The frequency of resistant mutants was expressed as the mean number of resistant colonies divided by the mean of the total number of viable cells.24,25 Three to 10 colonies of each strain from Brucella blood agar containing 1 mg/L ciprofloxacin were selected and stored at –70°C to await further studies.
For comparison of mutation frequencies caused by some other antimicrobials, C. jejuni ATCC 33650 and C. coli S140R were tested for mutation frequencies for erythromycin (4 mg/L) and ampicillin (8 mg/L) simultaneously with the testing for ciprofloxacin.
Impact of ox bile on mutation frequency
Because bile salts are known to induce activity of CmeABC efflux in C. jejuni,16 we tested the impact of ox bile extracts (Difco) on spontaneous mutation frequencies of five C. jejuni strains (ATCC 33560, B42, N191, 6507 and B67) and C. coli S140R. A test strain was incubated in 20 tubes of 3 mL of Brucella broth containing 20 g/L ox bile (Difco) and in 20 tubes of Brucella broth without ox bile. Mutation frequencies were determined on Brucella blood agar media containing 1 mg/L ciprofloxacin, either with or without ox bile. Testing of the impact of ox bile on mutation frequency was performed simultaneously, and the mutation frequencies were estimated according to the tests described in the previous section.
Antimicrobial susceptibility of the wild-type strains to ciprofloxacin, erythromycin, tetracycline and ampicillin was tested using an agar dilution method on Muller–Hinton agar (Oxoid).26,27 Similarly, the MICs of ciprofloxacin for the colonies isolated from agar plates containing 1 mg/L ciprofloxacin were also determined by the agar dilution method. C. jejuni strain ATCC 33650 was used as a control.26 The breakpoint values used were 4, 16, 8 and 16 mg/L for ciprofloxacin, tetracycline, ampicillin and erythromycin, respectively.26
Identification of mutations in gyrA
For each strain, a 270 bp gyrA sequence containing the QRDR was sequenced both from the original wild-type susceptible strain and from one to five mutated colonies subcultivated on Brucella blood agar. The PCR primers and amplification conditions were as described previously.13,14
Identification of mutations associated with cmeABC regulation
Potential mutations in the promoter-binding region of the transcriptional regulator CmeR of cmeABC was studied by PCR amplification, sequencing of the 97 bp intergenic region between cmeR and cmeA and analysing the inverted repeat region (5'-TGTAATAAATATTACA-3').15
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Bacterial strains and their antimicrobial susceptibility profiles
Bacterial strains and their susceptibilities to selected antimicrobials are shown in Table 1. All parent strains were highly susceptible to ciprofloxacin (MIC 0.032–0.064 mg/L, 9 strains), 0.125 mg/L (5 strains) and 0.250 mg/L (2 strains). In the induced variants 49/7RAT and 6590AT, the MICs of ciprofloxacin had increased to 0.5 mg/L. Two of the C. jejuni strains (B1 and B67) had increased resistance to ampicillin (MIC 16 mg/L), but were susceptible to the other antimicrobials studied. C. coli 6590 was resistant to tetracycline (MIC 32 mg/L; Table 1).
Spontaneous mutation frequencies
All strains were tested for growth on media containing 1 mg/L ciprofloxacin to verify the absence of growth at this concentration before the mutation frequency experiments. Among the C. jejuni strains, the spontaneous mutation frequencies varied from hypomutable (strain 14/8R) to strongly hypermutable (strains ATCC 33560, FB6371 and B5). Five strains were normomutable (5/11) and two strains were weakly hypermutable (Table 2). C. coli strain 6590 was strongly hypermutable and two of the C. coli strains were normomutable and two of them were weakly hypermutable (Table 2). With C. jejuni ATCC 33650 and C. coli S140R strains, challenge with erythromycin or ampicillin produced lower mutation frequencies than that with ciprofloxacin, because no colonies were seen on any of the 20 plates containing either 4 mg/L erythromycin or 8 mg/L ampicillin. The two variants with previously induced resistance to tetracycline and ampicillin (49/7RAT and 6590AT) had similar cfu counts on control and ciprofloxacin-containing media (109/mL). The parent C. jejuni strain 49/7R had a normal spontaneous mutation frequency (1.6 x 10–8), and only 5 of 20 tubes contained cells capable of growing on a medium containing 1 mg/L ciprofloxacin (Table 2). The parent C. coli strain 6590, which had an MIC value of 0.125 mg/L of ciprofloxacin, had a high spontaneous capacity to adapt to growth on 1.0 mg/L ciprofloxacin.
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Effect of ox bile on mutation frequency
The presence of ox bile in Brucella blood agar plates (containing 1 mg/L ciprofloxacin) or in Brucella blood agar plates and Brucella broth had negligible effect on the spontaneous mutation frequencies of any of the five tested C. jejuni strains or the one C. coli strain (data not shown).
MICs and mutations in QRDR and promoter-binding region
MICs for the parent strains and the corresponding colonies isolated from media containing 1 mg/L ciprofloxacin are shown in Table 2. The MICs of ciprofloxacin for the colonies grown in media containing 1 mg/L ciprofloxacin ranged from 1 to 16 mg/L. In 49/7RAT, the MICs did not increase above 1–2 mg/L, even though the MICs of ciprofloxacin for the colonies from the parent strain 49/7R varied from 1 to 8 mg/L. The MICs for the colonies of 6590AT varied from 1 to 16 mg/L after incubation in 1 mg/L ciprofloxacin.
Analysis of QRDR sequences revealed no mutations in this region in the parent strains (Table 2). Among most of the colonies picked from 1 mg/L plates, characteristic mutations had occurred either as a single substitution at codon 86 (ThrIle) or as a single substitution at codon 90 (AspAsn). Challenged colonies of a strain isolated from different plates with 1 mg/L ciprofloxacin often possessed several types of mutations. Interestingly, the studied colonies of 6590AT, ATCC 33650, B25, B67 and 49/7RAT included colonies both with and without mutations within the QRDR of gyrA.
Variations in the 7 bp inverted sequence (5'-TGTAATAAATATTACA-3') in the CmeR-binding region were detected. Several colonies had a transversion mutation TA (5'-AATTACA-3') in the first nucleotide of the second half of the sequence (Table 2). This mutation was, however, present in some of the parent strains (FB6371, B67 and 49/7R). Some strains (ATCC 33650, N191, 14/8R, B5 and 6590) had no mutations in this region in any of the colonies studied. C. jejuni 49/7RAT had a deletion in the first nucleotide of the second half of the inverted sequence (Table 2). The wild-type strain 49/7R possessed the TA mutation, and the two variants cultivated on 1 mg/L ciprofloxacin had unmutated sequences. All 6590AT colonies had unmutated sequences in this region.
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Discussion |
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The frequency of mutants selected under antimicrobial challenge has an important effect on the emergence of resistance. Our in vitro results showed that the spontaneous mutation frequency level among ciprofloxacin-susceptible parent C. jejuni and C. coli strains ranged from hypomutable (
4 x 10–9) to strongly mutable (7 x 10–3). Ciprofloxacin challenge at 1 mg/L represented a challenge with 4–30-fold the MICs for the studied strains, but the level of increase had little effect on the mutation frequencies. Likewise, Yan et al.28 measured mutation frequencies of wild-type C. jejuni NCTC 11168 and concluded that ciprofloxacin used at 5x or at 10x MIC produced mutants at similar frequency. Chopra et al.22 recommended a concentration of at least 4x MIC in the mutant selection medium used for the estimation of mutation frequency. In our study, the concepts normomutable, weakly hypermutable and strongly hypermutable were used in a similar way as previously used for E. coli,22,23 as C. jejuni and C. coli, similar to E. coli, colonize the intestinal tract, which does not select for hypermutable bacterial populations in healthy host animals. This is contrary to, for example, H. pylori, which persists lifelong in a specific niche, the human gastric mucosa, where a niche-associated adaptation may have selected populations with high mutation frequencies.24 The two variants C. jejuni 49/7RAT and C. coli 6590AT resistant to tetracycline and ampicillin grew on 1 mg/L ciprofloxacin with the same frequency as control cultures, indicating that the induction of tetracycline and ampicillin resistance subsequently improved their tolerance to 1 mg/L ciprofloxacin. The MICs of ciprofloxacin for these strains changed from 0.032–0.125 mg/L (parent strains) to 0.5 mg/L (6590AT and 49/7RAT) and finally to 1–16 mg/L after exposure to 1 mg/L ciprofloxacin. Strain 49/7R had a normal mutation frequency (1.6 x 10–8). The parent C. coli 6590 strain was strongly hypermutable (7.0 x 10–3), which may be associated with its pre-existing tetracycline resistance (MIC 32 mg/L). These results indicate that resistance to tetracycline or ampicillin and tetracycline simultaneously increases the MIC of ciprofloxacin, allowing the bacterium to acquire multiresistance. The acquisition of resistance to tetracycline and ampicillin, which are commonly used in veterinary medicine, may be important in the acquisition of low-level fluoroquinolone resistance in vivo. The results of monitoring studies have indicated that multidrug resistance is significantly associated with ciprofloxacin resistance in C. jejuni in both human17,18 and chicken isolates.6 For example, 22% of C. jejuni strains collected from Finnish patients during 1995–2000 were multiresistant, and this phenotype was significantly associated with ciprofloxacin resistance.17
Martinez and Baquero29 have reported that mutation frequencies in vivo during infection are higher than those in vitro under laboratory conditions, because bacteria in vivo are subjected to frequent and numerous stresses. A known stress-associated factor in the intestines is bile.30 Campylobacters as intestinal organisms are known to tolerate high concentrations of bile in the growth environment in vitro.16 Our in vitro studies revealed no marked impact of ox bile extracts on spontaneous mutation frequencies in any of the studied strains. The strains used in the studies had 30-, 15-, 8- or 4-fold lower MICs (0.032–0.250 mg/L) of ciprofloxacin than the concentration (1 mg/L) used in the mutant selection medium. In C. jejuni, bile salts are known to induce activation of the CmeABC efflux pump in vitro and simultaneously increase resistance to fluoroquinolones.16
Determination of MICs for various colonies from plates containing 1 mg/L ciprofloxacin revealed that this level of ciprofloxacin exposure was selected for a range of MIC values from 1 to 16 mg/L. In most cases, the level of resistance was 4–16 mg/L. The range of MICs for the colonies was not associated with the initial susceptibility of the strain. These results are concordant with in vivo studies, in which treatment of chickens with either 25 or 50 ppm of enrofloxacin caused similar levels of resistance among C. jejuni colonizing in chickens.5 The acquisition of ciprofloxacin resistance in vitro is associated with a resistance selection window in a similar way as in vivo.8 Similarly, the recent study of Yan et al.28 also showed that a variety of MICs and gyrA mutations were selected at a constant concentration of 1.25 mg/L ciprofloxacin.
Sequencing and analysis of the inverted repeat of the promoter-binding region of CmeABC15 revealed that certain parent strains had a point mutation TA in the seven-nucleotide inverted sequence (e.g. B67 and 49/7R). Interestingly, 49/7RAT and all its studied colonies from 1 mg/L ciprofloxacin plates possessed a deletion of T instead of the transversion TA at the same location. Among some strains, colonies with both unmutated and mutated sequences were found, suggesting that bacterial populations could be mixtures of both types of sequences. Lin et al.15 induced a high-level resistance to ciprofloxacin (MIC 100 mg/L) in the C. jejuni strain 81-176 and found a deletion in the 2 bp spacer sequence located between the inverted seven-nucleotide sequences. Cagliero et al.31 found another type of point mutation in this region in a C. jejuni strain. Both studies further showed that CmeR was unable to bind to the mutated region, and subsequently, CmeABC efflux was enhanced. The mutations identified in our study may also inhibit binding of CmeR, thus preventing its repressor activity on cmeABC and leading to increased activity of the efflux pump. These mutations could explain the small shift in MIC from highly susceptible (49/7R; 0.032 mg/L) to less susceptible (49/7RAT; 0.5 mg/L), although no clear consistent association was detected between a mutation in the inverted sequence and a level of resistance. Further studies are underway.
In conclusion, our studies revealed that the spontaneous mutation frequencies of quinolone-susceptible C. jejuni and C. coli strains varied from hypomutable to strongly hypermutable and that no particular pattern of mutation within gyrA or the CmeR-binding site was associated with any of these phenotypes.
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M. Hannula, MSc, received funding from the Graduate School of Applied Biosciences.
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None to declare.
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The support from the Academy of Finland and the Walter Ehrström Foundation is gratefully acknowledged.
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  M. Hannula and M.-L. Hanninen Effect of putative efflux pump inhibitors and inducers on the antimicrobial susceptibility of Campylobacter jejuni and Campylobacter coli J. Med. Microbiol., July 1, 2008; 57(7): 851 - 855. [Abstract] [Full Text] [PDF]
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