Antibiotic resistance in thermotolerant campylobacter isolated in 2000/01 and 2010 from patients with diarrhoea in Dunedin, New Zealand.
Thermotolerant Campylobacter species have an optimum growth temperature of 42o C. They are a major cause of foodand water-borne gastrointestinal infections world-wide, C. jejuni being the most prevalent species (1). Foods of animal origin, especially poultry, are significant sources of these organisms (2). Campylobacteriosis is the most frequently reported notifiable disease in New Zealand and accounts for over 70% of the estimated economic cost of foodborne diseases (3). The Campylobacter problem in New Zealand could be further exacerbated by the rapid emergence of antibiotic resistance, as has been reported elswhere in the world (1,4).
Campylobacter enteritis cases are usually self-limiting, but treatment with antibiotics may be required e.g. for pregnant women, young children, immunocompromised patients, prolonged cases of enteritis, and septicaemia (5,6). Fluoroquinolones are often used empirically but bacterial resistance can lead to therapeutic failure (7). Antibiotic resistance arises through a number of genetic mechanisms and spread of resistance genes is believed to occur through natural transformation and conjugation (4,8,9). As resistance limits treatment options for serious infections, it is important to monitor trends and identify the antibiotics to which resistance is most common.
The first aim of this pilot study was to determine whether a trend in increasing antibiotic resistance could be detected among clinical, thermotolerant Campylobacter isolated in Dunedin, New Zealand which might warrant a larger investigation. To this end, the antibiotic susceptibility of 60 isolates collected in 2000/01 were compared with the same number obtained in 2010 by the disc diffusion method, an acceptable alternative to the agar dilution test (10). Minimum inhibitory concentrations (MIC) of resistant isolates were determined by Epsilometer (E)-test methods. A second aim of the project was to investigate the mechanism of quinolone resistance by amplifying and sequencing the quinolone resistance determining region (QRDR) of the DNA gyrase A gene (gyrA). The third aim was DNA fingerprinting of Campylobacter isolated in 2010. Flagellin A gene (flaA) restriction fragment length polymorphism (RFLP) was chosen as a simple and reliable method (11).
Materials and methods
Collection and identification of isolates
Campylobacter were isolated from diarrhoeal stool specimens at Southern Community Laboratories, Dunedin Public Hospital using standard microbiology methods for thermotolerant species. Sixty isolates were randomly selected from an existing collection which was part of a previous study (12). These isolates were collected over the summer months 2000/01. The 60 year-2010 isolates were collected sequentially in spring. Repeat isolates from the same patient were excluded.
Isolates were presumptively identified as thermotolerant Campylobacter using standard microbiology techniques. Species identification was achieved using specific primers for C. jejuni, C. coli, C. lari and C. upsaliensis in a multiplex PCR reaction and by amplification and sequencing of the 16s rRNA gene as previously described (13). Amplification was not achieved for six isolates and these were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) by Canterbury Health Laboratories, Christchurch, New Zealand. All isolates were stored in brain heart infusion broth (BactoTM, Becton Dickinson & Co., Sparks MD, USA) with 20% glycerol (Sigma-Aldrich, Castle Hill, NSW, Australia) at -80[degrees]C without further subculture.
Antibiotic susceptibility testing
Campylobacter isolates were recovered from frozen stocks by plating on Wilkins Chalgren agar (Fort Richard Laboratories, Auckland, New Zealand). The disc diffusion test was performed on Mueller Hinton agar with 5% lysed horse blood plates (Fort Richard Laboratories) as previously described (14). Inoculum densities were prepared using a spectrophotometer ([OD.sub.600nm]) as recommended (14). Discs used were: ciprofloxacin 5 [micro]g; nalidixic acid 30 [micro]g; tetracycline 30 [micro]g; and erythromycin 15 [micro]g (Becton Dickinson & Co.). Ciprofloxacin, erythromycin and tetracycline resistance detected by the disc diffusion test was confirmed using M.I.C.E. E-test strips (Oxoid, Basingstoke, Hampshire, UK) according to the manufacturer's instructions with innocula prepared as for the disc test. Nalidixic acid E-tests were not performed as M.I.C.E. strips were not available. C. jejuni ss. jejuni type strain NZCC 2398 (New Zealand Culture Collection, ESR, Porirua) was used as a susceptible control. Resistance/susceptibility breakpoints were according to Andrews (14).
Polymerase chain reactions
DNA was extracted by boiling bacterial suspensions at 100[degrees]C for 10 min and cooling on ice for 10 min. The PCR reaction used to amplify the QRDR of the gyrA gene, was adapted from Griggs et al (15). The use of primers 293cj (5'GCCTGACGCAAGAGATGGTT-3') and 343cj (5'CATCGCAGCGGCACTATCAC- 3') allowed for the amplification of codons 39 to 123 of the gyrA gene. Thermocycling consisted of an initial denaturation step of 94[degrees]C for 5 min, followed by 25 cycles of 94[degrees]C for 15 s, 55[degrees]C for 30 s and 72[degrees]C for 45 s, with a final extension period at 72[degrees]C for 10 min. The presence of a 259 bp amplicon covering codons 39 to 123 of gyrA was confirmed by 1% agarose gel electrophoresis, ethidium bromide staining and UV transillumination. Sequencing was performed by the Genetic Analysis Services (Department of Pathology, University of Otago) using the ABI 3730xl DNA analyser with the BigDye[R] Terminator Version 3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems[R], Life Technologies Ltd., Mulgrave, Victoria, Australia). Sequences were analyzed using the Lasergene[R] suite of DNA analysis software programmes (DNASTAR Inc., Madison, Wisconsin, USA). To detect mutations, QRDR sequences were aligned with sequence from quinolone susceptible C. jejuni deposited in NCBI GenBank and examined for differences in nucleotides as previously described (15).
Amplification of flaA was performed on all year-2010 isolates. The method was adapted from the method of Pope et al using the primers flaA-F (5'- GGATTTCGTATTAACACAAATGGT- 3') and flaA-R (5'- CTGTAGTAATCTTAAACAATTTTG- 3') (16). Thermocycling consisted of an initial step of 94[degrees]C for 1 min, followed by 35 cycles of 94[degrees]C for 15 s, 53[degrees]C for 45 s and 72[degrees]C for 1 min 45 s with a final extension period at 72[degrees]C for 3 min. The presence of a 1.7 kb amplicon was confirmed by 1% agarose gel electrophoresis followed by ethidium bromide staining and UV transillumination.
For the 29 isolates where flaA PCR was successful, amplicons were digested with the restriction enzyme DdeI (Roche Diagnostics New Zealand Ltd., Auckland, New Zealand), which cuts the DNA into fragments of different lengths depending on the number and location of restriction sites (restriction fragment length polymorphism; RFLP). The resulting restriction fragments were separated by 2% agarose electrophoresis and visualised by ethidium bromide staining and UV transillumination. Comparison of RFLP profiles (DNA fingerprints) obtained from the digests was achieved using Gelcompar II software (Applied Maths BVBA, Sint-Martens-Latem, Belgium). Dice similarity coefficients were calculated and used to generate dendrograms showing per cent similarity of isolates to one another.
Data was analysed using Fisher's exact probability test and Wilcoxon rank sum test (Graphpad Software Inc. 2013, La Jolla, CA, USA).
Identification of isolates
All thermotolerant Campylobacter were identified as C. jejuni apart from one year-2010 isolate which did not amplify with the 16S rDNA primers and yielded uncertain results by MALDITOF. However, this isolate was retained in the study as it was phenotypically a Campylobacter.
The results of the antibiotic disc diffusion and E-tests are shown in Table I. For the year-2010 Campylobacter, resistance to ciprofloxacin occurred in four isolates (6.7%) and nalidixic acid resistance in three (5%). Resistance to tetracycline and erythromycin was not detected. In contrast, there were significantly more resistant isolates (15; 25%) among the year-2000/01 group (P = 0.01), erythromycin resistance being predominant. One year-2000/01 isolate was resistant to all four antibiotics tested. E-tests indicated that MIC were generally high among the antibiotic resistant year-2000/01 Campylobacter, exceeding the resistant breakpoint by at least 8 -fold in all but one case. MIC of the four ciprofloxin resistant Campylobacter isolated in 2010 were significantly less than the four year-2000/01 isolates (P = 0.02) (Table 1).
Amplification and sequence comparison of the QRDR of the gyrA gene showed that the four ciprofloxacin resistant, year-2010 isolates contained a mutation in this region of the bacterial chromosome. A cytosine to thymine nucleotide substitution at codon 86 of the gyrA subunit resulting in an amino acid change of threonine to isoleucine was present. Three of the year-2000/01 ciprofloxacin resistant isolates had the same mutation but mutations were not detected in the fourth resistant isolate, although a complete sequence for this isolate could not be obtained. This isolate was additionally resistant to tetracycline, erythromycin and nalidixic acid.
Twenty-nine of the 60 year-2010 antibiotic susceptible isolates were successfully flaA genotyped using RFLP. Comparison of RFLP profiles indicated the presence of six groups of identical isolates each consisting of two to five isolates (Figure 1). A variety of RFLP profiles were seen but there were two main clusters of Campylobacter profiles (>70% similarity) consisting of 9 and 11 isolates. RFLP profiles were obtained for three of the four ciprofloxacin resistant year-2010 isolates and two of these gave identical profiles (Figure 1).
From the results of this pilot study, there is nothing to suggest antibiotic resistance in thermotolerant Campylobacter increased from 2000/01 to 2010 in Dunedin. In fact, a reverse trend seemed to be evident, with fewer antibiotic resistant isolates occurring in the year-2010 compared to year-2000/01 isolates. In particular, resistance to erythromycin and tetracycline, which occurred in 21.7% of the year-2000/01 isolates, was not detected at all in thermotolerant Campylobacter collected in year-2010. These findings contrast sharply with reports from some other locations in the Pacific Rim, where antibiotic resistance rates of greater than 90% have been reported for thermotolerant Campylobacter (17,18).
Of interest are the changes in policy which occurred just prior to the study period regarding the prophylactic use of antibiotics in chicken feed. A recommendation of the Antibiotic Resistance Steering Group to the New Zealand Animal Remedies Board (1999) was that macrolides should only be used under veterinary prescription and fluoroquinolones should only be used for the treatment of serious infections in individual animals, if at all (19). These restrictions were put in place as a protective measure to prevent the occurrence of antibiotic resistant C. jejuni in poultry because the consumption of poultry meat has been proven to be one of the main sources of Campylobacter infection in New Zealand as elsewhere (11,20) A recent publication and Ministry of Agriculture and Forestry report (2011) indicates prudent use of therapeutic antibiotics and a reduction in feed/water administration of antibiotics has been achieved in New Zealand and that levels of resistence in Gram negative bacteria, including Campylobacter isolated from chicken carcasses, are among the lowest reported in the world (19,21). In our study, a similar trend towards decreasing antibiotic resistance was seen when recent and historical human Campylobacter isolates were compared.
Amplification and sequencing of the QRDR revealed the same Thr-86-Ile (threonine to isoleucine) mutation was present in both year-2000/01 and -2010 ciprofloxacin resistant isolates. This is a common mutation in quinolone resistant campylobacters which usually confers cross-resistance to nalidixic acid (4). The higher ciprofloxacin MIC in year-2000/01 resistant isolates may have been due to concurrent tetracycline resistance (22). The single year-2000/01 isolate not showing the The-86-Ile mutation was multiply resistant and it is likely that an efflux pump such as the cmeABC pump was responsible, although a gyrA mutation could not be ruled out due to incomplete sequencing (23).
Epidemiological subtyping by determination of flaA RFLP profiles had a lower success rate than anticipated, possibly due to polymorphisms in the primer binding sites. flaA RFLP is a low cost, simple method which can be used to predict clonal groupings inferred from the more expensive multi-locus sequence typing method (24). The presence of small groups of identical isolates suggests multiple common sources of infection among these patients. Previous subtyping of a larger group of year-2000/01 isolates by a pulsed field gel electrophoresis method showed the presence of both dominant and unique profiles. This was reasonably similar to the smaller year-2010 group, where two dominant clusters of related isolates along with the presence of subtypes with low similarity were observed (12). These findings reflect both commonality of source (e.g. poultry) and diverse origin of human thermotolerant Campylobacter found in New Zealand (12,25). A limitation of the study was that the 2000/01 isolates were randomly selected from a larger group while the 2010 isolates were collected sequentially.
There was a low, and apparently decreasing, incidence of antibiotic resistance in thermotolerant Campylobacter detected in this investigation. Molecular subtyping of some year-2010 isolates confirmed the pattern of dominant clusters of related Campylobacter along with more unique genotypes as seen previously.
Michael Harrington and Rebecca Lopes were supported by Otago School of Medical Sciences summer scholarships.
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Michael G Harrington, BMLSc, Undergraduate Student in Medicine 
Rebecca M Lopes, Undergraduate Student (BMLSc) 
Kimberley A Hughes, BMLSc, Postgraduate Student (MSc) 
Julie E Weaver, PhD, Teaching Fellow 
Gayleen Parslow, Charge Medical Laboratory Scientist 
Rebekah F Roos, BSc (Hons), Health Intelligence Team ESR 
Heather JL Brooks, PhD, Senior Lecturer 
 Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin
 Department of Microbiology, Southern Community Laboratories, Dunedin Hospital
 Freshwater Ecology Group, Department of Zoology, University of Otago, Dunedin
 Environmental Science and Research, Kenepuru Science Centre, Porirua
Correspondence: HJL Brooks, Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, PO Box 56, Dunedin 9012, New Zealand. E-mail: email@example.com
Table 1. Antibiotic resistance of thermotolerant Campylobacter isolated from patients with diarrhoea in 2000/01 and 2010 determined by the disc diffusion and E-tests. Year of isolation Antibiotic (disc 2000/01 2010 concentration) (n=60; S=45/R=15 ) (n=60; S=56/R=4) Disc test E-test Disc test E-test (n) MIC mg/L (n) MIC mg/L Ciprofloxacin 4 (6.7%) >32 4 (6.7%) 4 (5 [micro]g) Nalidixic acid 4 (6.7%) nd 3 (5.0%) nd (30 [micro]g) Erythromycin 12 (20%) >256 0 -- (30 [micro]g) Tetracycline 4 (6.7%) >256 (n=3) 0 -- (15 [micro]g) 32 (n=1) S = susceptible; R = resistant; nd = not done; MIC = minimum inhibitory concentration
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|Author:||Harrington, Michael G.; Lopes, Rebecca M.; Hughes, Kimberley A.; Weaver, Julie E.; Parslow, Gayleen;|
|Publication:||New Zealand Journal of Medical Laboratory Science|
|Date:||Apr 1, 2014|
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