JAC Advance Access originally published online on May 7, 2008
Journal of Antimicrobial Chemotherapy 2008 62(2):303-315; doi:10.1093/jac/dkn190
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Original research |
Persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline
1 Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, Birmingham, UK 2 Health Protection Agency Foodborne Zoonoses Unit, School of Clinical Veterinary Science, University of Bristol, Bristol, UK 3 Centre for Infections, Health Protection Agency, London, UK 4 Foodborne Zoonoses Group, School of Clinical Veterinary Science, University of Bristol, Bristol, UK
* Corresponding author. Tel: +44-121-414-6966; Fax: +44-121-414-6819; E-mail: l.j.v.piddock{at}bham.ac.uk
Received 20 December 2007; returned 5 February 2008; revised 20 March 2008; accepted 4 April 2008
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Abstract |
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Objectives: The aim of this study was to investigate the persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline.
Methods: Three commercially reared broiler flocks, naturally colonized with Campylobacter, were treated with chlortetracycline under experimental conditions. The numbers of Campylobacter isolated, and the species, flaA short variable region allele, and antimicrobial resistance of isolates were determined.
Results: For two of three flocks, tetracycline-resistant strains predominated prior to chlortetracycline exposure. Presence of the antibiotic had no discernible effect on the numbers or types of Campylobacter and the tetracycline-resistant strains persisted in numbers similar to those observed before treatment. With all flocks, some faecal samples were obtained that contained no Campylobacter, irrespective of exposure to chlortetracycline; this was more common as the birds grew older. For the third flock, tetracycline-resistant Campylobacter were in the minority of samples before and during exposure to chlortetracycline, but at sampling times after this, no resistant strains were found in the treated (or untreated) birds, irrespective of exposure to the antibiotic. All tetracycline-resistant isolates (MICs 16 to >128 mg/L) contained tet(O) and, for some isolates, this was transferable to Campylobacter jejuni 81116. The efflux pump inhibitor PAβN reduced the MICs of tetracycline for these isolates by 4-fold, suggesting that an intact efflux pump, presumably CmeABC, is required for high-level tetracycline resistance.
Conclusions: Our data indicate that chlortetracycline treatment does not eradicate tetracycline-resistant Campylobacter spp. from poultry. However, if a low number of resistant isolates are present, then the antibiotic pressure appears insufficient to select such strains as the dominant population.
Keywords: chickens , typing , tetracycline-resistant
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Introduction |
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Campylobacter jejuni causes the greatest number of food-borne bacterial infections in humans in England and Wales.1 Infections by this pathogen also had the greatest impact on the healthcare sector in England and Wales between 1996 and 2000, giving rise to just under 16 000 hospitalizations and 80 deaths. Adak et al.1 showed that the most important cause of indigenous food-borne disease was contaminated chicken. Campylobacteriosis is usually self-limiting but antibiotic treatment is required for chronic and serious infections. Resistance to antibiotics has become a serious problem worldwide, and the numbers of Campylobacter resistant to multiple antibiotics continue to increase. Antibiotics are administered to treat infections in animals caused by other bacteria, and antibiotic-resistant Campylobacter can emerge as a consequence of this, contaminate food and enter the human food chain.
Chlortetracycline is a broad-spectrum antibacterial agent, with activity against respiratory and enteric bacteria, and is the most commonly used therapeutic antibiotic in poultry production. In 2004, 243 tonnes of antimicrobials belonging to the tetracycline class were sold in the UK.2 Tetracycline-resistant C. jejuni isolated from chickens have been described by various authors since Taylor et al.3 The rate of resistance in live chickens and meat at retail sale varies from country to country. Various studies have shown that depending on the origin and type of poultry meat, between 3% and 35% of the Campylobacter are resistant to tetracycline.4–7 Of interest, Anderson et al.4 have shown that there has been an increase in the numbers of resistant isolates in Denmark since 2001, which coincides with the withdrawal of antimicrobial growth promoters in Europe. In the USA and Canada, Campylobacter were highly prevalent in both conventional and organic poultry production, and large numbers of isolates were resistant to tetracycline.8,9 Approximately 25% of the Campylobacter isolates from humans have also been reported as tetracycline-resistant. Most recently, 27.3% of the C. jejuni isolated from patients in England and Wales with non-travel-associated cases of enteritis were resistant to tetracycline compared with 55% of the C. jejuni isolated from travel-associated cases (Iain Gillespie, HPA, Colindale, UK, personal communication).
Taylor et al.3 were the first to show that poultry isolates of C. jejuni contained a conjugative plasmid that transferred tetracycline resistance. Numerous studies have since shown that tetracycline resistance is typically transferable, and a variety of plasmids have been implicated. Manavathu et al.10 found that the gene transferred was tet(O). Avrain et al.11 showed that there was horizontal transfer of tet(O)-positive Campylobacter between chickens. However, tet(O) can also be located on the chromosomes.12–14 Decreased susceptibility to tetracycline can also be mediated by the CmeABC efflux pump along with resistance to several other drugs.15,16
It has been suggested that in the absence of an antimicrobial agent, antibiotic-resistant bacteria are less prevalent. Therefore, it has been postulated that in the presence of a drug (e.g. tetracycline), drug-resistant bacteria would be maintained. In our previous work on fluoroquinolone resistance in C. jejuni, we monitored the persistence of resistant strains before, during and after poultry flocks were exposed to a fluoroquinolone and showed a clear association between exposure to fluoroquinolones and the presence of resistant strains which persisted in the birds to slaughter and entry into the human food chain.17,18 Based on our previous work, we hypothesized that exposure to any antibiotic used to treat infections in poultry would allow resistant Campylobacter to dominate the population. In this study, we investigated the persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline.
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Treatment and sampling of chicken flocks
All three flocks, A, B and C, were broiler birds removed from commercial poultry flocks reared for human consumption. The treatment histories of the birds were obtained and none had previous exposure to tetracycline or other antimicrobials. These birds were housed at the School of Clinical Veterinary Science of the University of Bristol, at a stocking density of 12.6 kg/m2 to meet the UK Home Office requirements and experimentally treated with chlortetracycline. All animal experiments were conducted according to the requirements of the Animals (Scientific Procedures) Act 1986 and were approved by the local Ethics Review Committee. Each group of birds was housed independently in separate rooms. The University of Bristol’s Animal Services Units biosecurity protocol was followed. This included the use of protective clothing (overalls, gloves and disposable boot socks), which was worn throughout husbandry and sampling. Separate protective clothing and footwear was worn for each room, and disinfectant boot dip used upon every entry and exit of the rooms. The footwear worn was dedicated to the animal house and had not previously been worn outside. Flock A consisted of 20 birds brought on-site at 21 days of age from a commercial free-range organic broiler flock consisting of 7500 birds in September 2004. All birds were treated with a therapeutic dose of chlortetracycline at 40 mg/kg/day for 6 days administered in the feed as recommended by the manufacturer (Aurogran; Novartis, Hertfordshire, UK). All birds were weighed before therapy commenced. Food consumption was not measured; however, animal care staff confirmed that the animals fed normally. Flock B consisted of 28 birds brought on-site in October 2005, purchased from a commercial free-range broiler flock of 6300 birds at 22 days old. Fourteen of these birds were treated with a therapeutic dose of chlortetracycline and 14 birds, housed independently, remained untreated. Flock C consisted of 30 birds brought on-site at 49 days old in May 2006, purchased from a commercial housed corn-fed ‘freedom foods’ flock consisting of 31 000 birds. Fifteen of these birds were treated with a therapeutic dose of chlortetracycline and 15 birds, housed independently, remained untreated.
All samples were transported to the laboratory within 3 h of collection. At least 14 freshly voided faecal samples were collected before, during (2 days after treatment started) and every 7 days thereafter up to 4 weeks post-treatment, and Campylobacter spp. were isolated on modified charcoal cefoperazone deoxycholate agar (mCCDA; Oxoid, Basingstoke, UK) as described previously.17 At 4 weeks post-treatment sampling, the caeca from flock B were removed post mortem to enable analysis of each chicken’s flora for Campylobacter spp.
Enumeration of tetracycline-resistant Campylobacter
The proportion of tetracycline-resistant Campylobacter was determined in the faecal sample isolates by replica-plating (using sterile furniture grade velvet) colonies from the ‘master plate’ of bacteria growing on mCCDA onto Mueller–Hinton agar (Oxoid), containing CCDA selective supplement (Oxoid), 5% defibrinated horse blood (Oxoid) and 8 mg/L tetracycline (Sigma, Dorset, UK). This cut-off concentration of 8 mg/L tetracycline was used as a review of the literature had indicated that this differentiated tetracycline-resistant strains from those that were tetracycline-susceptible. Replica plates were incubated at 37°C for 48 h in a microaerobic atmosphere (5% to 6% O2, 3% to 7% CO2 and 7% H2, in a balance of nitrogen19). The number of colonies growing on the replica plate was determined, and their morphology compared with growth on the ‘master’ mCCDA plates from which they were produced and the proportions of resistant colonies in the faecal samples were determined by subtraction. Usually, the lowest dilution was replica plated, but for some samples from flock C, this was not possible due to overgrowth of other faecal flora; hence, the different detection limits for the resistant population are indicated by horizontal lines on the bars in Figure 3 for this flock.
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Identification of presumptive colonies as Campylobacter spp.
Presumptive Campylobacter colonies (up to three colonies per sample) were subcultured from mCCDA to Columbia blood agar containing 5% defibrinated horse blood (Oxoid) and incubated at 37°C for 48 h in a microaerobic atmosphere. Isolates were confirmed as Campylobacter using light microscopy for motility and cell morphology and lack of growth in air at 37°C after 48 h.
Speciation of C. jejuni and Campylobacter coli was performed using the method of Best et al.,20 employing a real-time PCR assay based on the ABI PRISM 7700 Sequence Detection System (Taqman) platform. Isolates that were not identified as Campylobacter spp. were subcultured under aerobic conditions, and any isolates that were aerotolerant were speciated as Arcobacter butzleri, Arcobacter cryaerophilus or Arcobacter skirrowii, according to the multiplex PCR assay of Houf et al.21
Flagellin gene (flaA) short variable region (SVR) sequence typing
Genotyping based on flaA SVR type was performed as follows. DNA templates were generated from chromosomal DNA recovered from boiled whole-cell suspensions using PCR primers and protocols described by Nachamkin et al.22 Template preparations were purified using the Whatman® 96 Well PCR Cleanup Kit, and quantification of the products was performed by electrophoresis on a 1% (w/v) agarose gel. Sequencing primers and protocols for the SVR analysis were carried out as described by Meinersmann et al.23 Sequencing was performed using a Beckman CEQ800 Genetic Analysis System, and also commercially (K-Biosciences, Hoddesdon, UK). Forward and reverse sequences were aligned and trimmed to a 321 bp region covering the flaA SVR of genome-sequenced strain C. jejuni NCTC 11168 using Bionumerics V2.0 software (Applied Maths, Kortrijk, Belgium). The 321 bp sequences were then compared with flaA sequences (870 alleles at the time of writing) held in an online database (http://phoenix.medawar.ox.ac.uk/flaA/), and the corresponding allele numbers were recorded. The flaA SVR types were designated based on 100% homology to the Oxford reference sequences.
PFGE was performed according to the protocol of Gibson et al.,24 when it was considered necessary to check any anomalous results. DNA was digested with SmaI. For example, when isolates had the same flaA SVR type but different antibiotic susceptibility patterns (resistance phenotype) or when an isolate was non-typeable by flaA SVR. PFGE gel profiles were arbitrarily assigned numbers by comparison (visually and by using BioNumerics V2.0) with profiles of other isolates tested as part of a larger collaborative study examining additional flocks (data not shown).
Determination of antimicrobial resistance
Initially, all isolates were screened for antibiotic susceptibility (no growth) or resistance (growth) by the breakpoint screening method of Thwaites and Frost,25 with minor modifications such that the conditions were the same as those used to determine the MIC values (see below). The following concentrations of antimicrobials were used: ampicillin, 8 and 32 mg/L; chloramphenicol, 8 mg/L; gentamicin, 4 mg/L; kanamycin, 16 mg/L; neomycin, 8 mg/L; tetracycline, 8 and 128 mg/L; nalidixic acid, 16 mg/L; ciprofloxacin, 1 mg/L; and erythromycin, 4 mg/L. Isolates were selected for further study on the basis of species, flaA SVR type and resistance phenotype determined by breakpoint screening. MICs were determined for every strain of each flaA SVR and PFGE type from each and every Campylobacter-positive sample from each treatment phase of all flocks. Where there was more than one phenotype (by flaA SVR, PFGE or breakpoint screening) present within a sample, each phenotype was examined. Bacteria were grown on Mueller–Hinton agar containing 5% horse blood at 36°C in 7.5% CO2. The agar doubling dilution procedure recommended by the CLSI (formerly the NCCLS) Campylobacter Working Group26 was used throughout the study, as described previously,13 to determine the MICs of selected antibacterial agents and dyes (chlortetracycline, tetracycline, ampicillin, amoxicillin, ciprofloxacin, nalidixic acid, erythromycin, chloramphenicol, kanamycin, lincospectin, triclosan and ethidium bromide). C. jejuni NCTC 11168 and C. coli NCTC 11366 were used as control strains. Designation of strains as antibiotic-susceptible or -resistant was made as described previously,18 with reference to the guidelines of the BSAC and CLSI as available at the start of this study in 2003.
PCR detection of tet(O) and transfer of tetracycline resistance
All isolates inhibited by 
16 mg/L tetracycline were examined for the presence of tet(O) in cell lysates using primers based on the sequence published by Manavathu et al.10 and amplified from nucleotides 513–1146. Furthermore, 13 isolates representative of resistant strains isolated from each flock were examined for their ability to transfer tetracycline resistance to C. jejuni 81116 as described by Taylor et al.3 These were examined in parallel to 11 tetracycline-resistant isolates from our previous study.17,18 The DNA sequence of tet(O) of four isolates from our previous studies and three isolates from flock A was sequenced. These represented isolates with a range of MIC values from 16 to >128 mg/L tetracycline and examples of transferable and non-transferable tet(O).
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Effect of treatment with chlortetracycline upon numbers of Campylobacter isolated
During the course of this study, we were alerted to three commercial poultry flocks being reared for human consumption that were naturally infected with Campylobacter. Birds were purchased from these flocks and experimentally treated with a therapeutic dose of chlortetracycline exactly as if treated in the commercial environment. For flock A, data were only obtained for the birds treated with chlortetracycline (Figure 1). For flocks B (Figure 2) and C (Figure 3), sufficient birds were obtained to allow them to be divided into two groups: one group remained untreated and one was treated with chlortetracycline. The treated and untreated groups were housed independently.
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Pre-treatment, 19/20 faecal samples from flock A contained tetracycline-resistant Campylobacter. Except for one sample where a small proportion of the colonies were susceptible to tetracycline, replica plating showed that in all samples, every colony was resistant to tetracycline (Figure 1 and Table 1). During treatment, all samples contained Campylobacter spp. but the number of Campylobacter isolated from each varied, and some samples contained tetracycline-susceptible isolates (indicated by white bars in Figure 1). Faecal samples were obtained up to 4 weeks post-treatment, during which time the number of samples containing Campylobacter reduced, although tetracycline-resistant strains were isolated up to slaughter. For example, 2 weeks post-treatment in 3/20 samples, no Campylobacter were detected (indicated by grey bars in the figures), and by 4 weeks post-treatment, 8/20 samples contained no detectable Campylobacter.
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Tetracycline-resistant strains were isolated from most samples from flock B pre-treatment and during treatment, irrespective of whether the birds had been exposed to chlortetracycline (Figure 2 and Table 1). Unfortunately, despite repeated attempts with the raw material, due to overgrowth of contaminants on the replica plates 1 week post-chlortetracycline treatment, the proportion of samples from the treated birds containing tetracycline-resistant Campylobacter could not be calculated with accuracy (indicated by hatched bars in Figure 2). However, all samples from the untreated flock taken at the same time period contained tetracycline-resistant Campylobacter. Two weeks after treatment with chlortetracycline and up to slaughter, samples containing Campylobacter were obtained from both the treated and untreated birds, and all these contained tetracycline-resistant isolates. As with flock A, in the latter stages of sampling (2–4 weeks post-therapy), some samples from both untreated and treated birds contained no Campylobacter (5/42 and 8/41 samples, respectively). Birds from the source flock were also sampled at the original farm immediately prior to slaughter; all 14 samples contained Campylobacter, of which 2 contained tetracycline-resistant Campylobacter.
Flock C contained both tetracycline-susceptible and -resistant Campylobacter (Figure 1 and Table 1) but in some samples from this flock no Campylobacter were detected. There was a different detection limit for resistant Campylobacter in some samples as overgrowth of contaminating faecal flora prevented replica plating of the lower dilutions. Prior to treatment, 2/15, and 2/15, faecal samples from untreated and treated birds, respectively, contained tetracycline-resistant Campylobacter. During treatment, 4/10 samples from the untreated birds and 4/8 from treated birds contained tetracycline-resistant Campylobacter. Following treatment, all samples contained Campylobacter, of which only one contained tetracycline-resistant Campylobacter; thereafter, no tetracycline-resistant Campylobacter were isolated. Over the same time period, no tetracycline-resistant Campylobacter were isolated from the untreated birds. As with flocks A and B, there was a decline in the number of samples from the birds (treated or untreated) from which Campylobacter were detected. Despite the decline, greater numbers of Campylobacter were obtained from the chlortetracycline-treated birds than the untreated birds.
Effect of treatment with chlortetracycline upon species, type and antimicrobial susceptibility of Campylobacter isolated
Up to three colonies from each of the 14 faecal samples per sampling time were flaA SVR genotyped. PFGE was also performed on representative isolates of each flaA type from each sampling period, from treated and untreated birds, and for those isolates for which no flaA type could be obtained. The PFGE patterns were consistently associated with the same flaA type, so that for those isolates for which a flaA type could not be obtained, the PFGE pattern indicated the strain type, which was further confirmed by MIC testing.
Samples from birds in flock A contained two flaA SVR types, 16 and 18, that were previously recorded by others in the Oxford database. flaA18 was further divided into P3a and P3b types by PFGE. These PFGE types differed by the size of a single band only, and both PFGE types had the same phenotype upon MIC testing. The majority of the isolates from the faecal samples from flock A were flaA18. Not only were there fewer isolates of C. jejuni flaA16, but this strain was not detected from faecal samples taken at 2, 3 and 4 weeks after treatment. On initial breakpoint testing, three phenotypes were identified (Table 1). However, the determined MIC values of the 12 agents indicated that they were indistinguishable from each other (Table 2). All flaA18 isolates, irrespective of when isolated from flock A, were resistant to chlortetracycline, tetracycline, ciprofloxacin, nalidixic acid and triclosan. Interestingly, the C. jejuni flaA18 isolated became progressively less susceptible to triclosan as sampling progressed (Table 2). The MIC values were typically 16 mg/L triclosan pre-treatment and by 2 weeks post-treatment the MICs for isolates of this strain were consistently 64 mg/L. This decline in susceptibility was not observed with any other agent or with the isolates from flocks B and C.
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Samples from treated and untreated birds in flock B contained two species of Campylobacter, C. jejuni and C. coli, each with previously recorded flaA SVR types flaA21 and flaA66, respectively. Each type was associated with unique PFGE patterns P22 and P23, respectively (Table 2). As with the strains from flocks A and B, these flaA types had been found previously by others. Before treatment, no C. coli flaA66 were isolated from faeces from the untreated birds, but it was recovered from the treated birds. However, this strain was isolated at all other times from both groups of birds. Consistently, at each sampling time, greater numbers of C. jejuni flaA21 were isolated than C. coli flaA66. All C. jejuni flaA21 were resistant to chlortetracycline, tetracycline, amoxicillin and triclosan. All isolates of C. coli flaA66 were resistant to the same agents plus nalidixic acid. Samples taken from the source flock 4 weeks after treatment also contained Campylobacter; all were C. jejuni flaA14 PFGE 8a (Table 2). Five isolates were resistant to triclosan only and one isolate was resistant to chlortetracycline, tetracycline, nalidixic acid, amoxicillin and triclosan.
Samples from treated and untreated birds in flock C contained two species of Campylobacter, C. jejuni and C. coli (flaA255). Three strains of C. jejuni (flaA85, flaA189 and flaA239) were isolated, each associated with unique PFGE patterns; P24a, P25 and P26, respectively (Table 2). Except for one sampling time (untreated birds at the pre-treatment sampling point), C. coli flaA255 was isolated at all times from both groups of birds. Overall, for both groups, higher numbers of C. coli flaA255 were isolated. Of the C. jejuni, flaA85 was the most commonly isolated strain of this species. Except for triclosan, all isolates of C. coli flaA255, C. jejuni flaA189 and flaA239 (also resistant to amoxicillin) were susceptible to all other agents tested. Two isolates of C. jejuni flaA85 and one of C. coli flaA255 from the treated birds’ faecal samples had a markedly different phenotype upon MIC testing compared with the other isolates of the same flaA type. These isolates were less susceptible to chlortetracycline, tetracycline and ciprofloxacin or chloramphenicol.
Mechanisms and transfer of tetracycline resistance
Of the tetracycline-resistant isolates (MIC 16 mg/L) examined by PCR for the presence of tet(O), all were found to contain the gene (Table 2). Those isolates not tested were of the same genotype and phenotype obtained from the same sample and considered likely to be identical.
Tetracycline-susceptible isolates of each type and from the same sampling time did not contain tet(O). Eight of 13 isolates from the present study and 6/11 isolates from our previous study13 were able to transfer tetracycline resistance to C. jejuni 81116 at a frequency transfer of 1.3 x 10–7–6.4 x 10–10. The MIC of tetracycline for three transconjugants was reproducibly higher than that for the original isolate. The DNA sequences of tet(O) were also examined for seven representative isolates to determine whether there was any sequence variation associated with a particular MIC of tetracycline. The most resistant isolates (MIC 128 mg/L) contained an adenine at nucleotides 884, 1036 and 1784 of tet(O) and thymidine at nucleotide 1111. This is identical to the tet(O) sequence deposited in GenBank (accession no. M18896
[GenBank]
). Other sequence differences that gave amino acid substitutions were found (nucleotides 680, 910, 993 and 1063), but none could be correlated with tetracycline MIC values.
In the present study, all tetracycline-resistant strains contained tet(O), but in a previous study,18 there were eight tetracycline-resistant isolates for which tet(O) was not detectable by PCR. Likewise in studies on the effect of amoxicillin and tylosin performed in parallel to the present study, there were nine tetracycline-resistant isolates for which tet(O) was not detectable by PCR. To determine the role of efflux, and as variable MICs of ethidium bromide had been obtained (suggesting active efflux), in this phenotype MICs of tetracycline were determined in the presence and absence of the efflux pump inhibitor PAβN. For comparison, the effect upon susceptibility of PAβN (20 mg/L) was also examined for one tetracycline-resistant isolate of each flaA type from the present study. The MICs of the isolates for which tet(O) was not detectable by PCR ranged from 16 to 128 mg/L, but in the presence of PAβN, all values were reduced by 4- to 16-fold. PAβN had no effect upon the tetracycline susceptibility of C. jejuni 11168 cmeB::aph, but for a cmeB-overexpressing mutant, P1048,16 the MIC was reduced from 8 to 0.12 mg/L. For those isolates containing tet(O), the MICs of tetracycline were reduced 4-fold in the presence of PAβN.
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In this study, we examined the effect of chlortetracycline treatment upon the numbers of Campylobacter isolated, their species, type and antimicrobial resistance from three naturally colonized flocks. The flocks were housed at commercially relevant stocking densities and treated exactly as in the commercial environment. Therefore, this study is the first to replicate the on-farm situation. For each, the source flock was reared commercially for entry into the human food chain. For flocks A and B, tetracycline-resistant strains predominated prior to chlortetracycline exposure and perhaps, not surprisingly, the presence of the antibiotic had no discernible effect on the numbers of Campylobacter and tetracycline-resistant strains persisted. However, for all three flocks, some faecal samples were obtained that contained no Campylobacter, irrespective of exposure to chlortetracycline; this was more common as the birds grew older. For flock C, low-level tetracycline-resistant Campylobacter were detected in a few samples before and during exposure to chlortetracycline, but at sampling times after this, no resistant strains were found in the treated birds, irrespective of exposure to the antibiotic. These data suggest that for these tetracycline-resistant Campylobacter, the resistance was not sufficient to allow the strain to multiply in the presence of the drug. This is opposite to the situation found previously with fluoroquinolones where the antibiotic pressure allowed the resistant strains to proliferate and predominate.17
By performing genotyping, it was possible to determine whether tetracycline-resistant strains emerged during antibiotic exposure or if original resistant strains were selected and became the dominant population during therapy. It was also possible to establish if these persisted once the antibiotic had been withdrawn. Data obtained indicate that the original tetracycline-resistant strains persisted in flocks A and B. Only for flock C were Campylobacter of the same strain type but with different phenotypes (antibiotic susceptibilities) isolated, and these were only obtained from samples from the treated birds. However, only low numbers of isolates of each phenotype were obtained, so it is not possible to state whether these were present but not detected prior to antibiotic exposure or they emerged as a consequence of chlortetracycline treatment.
Most of the tetracycline-resistant strains were cross-resistant to at least one other class of antibiotics, either a quinolone or β-lactam. No multidrug-resistant isolates (i.e. resistant to three or more classes of antimicrobial agent) were obtained. Although there is no recommended breakpoint concentration for triclosan, all isolates from the three test flocks required at least 32 mg/L triclosan for inhibition. Therefore, these isolates were compared with those from our previous study on the effect of fluoroquinolone treatment upon Campylobacter isolated from poultry flocks.18 In that study, the MIC of triclosan for all multidrug-resistant isolates (n = 97) was determined and a range of 2–128 mg/L was identified; with an MIC at which 50% of the isolates were inhibited (MIC50; median), MIC90, and mode MIC of 64 mg/L. Taken together, these data indicate that irrespective of any antibiotic resistance, Campylobacter spp. are relatively insensitive to this biocide when compared with other Gram-negative bacteria. This may have implications for the eradication of food-borne Campylobacter from the domestic and industrial kitchen, where triclosan is commonly used.
As has been found in previous studies, the presence of tet(O) in the current study was always associated with tetracycline resistance and could be transferred to C. jejuni 81116. The fact that not all tetracycline resistance was transferable suggests that for some isolates, tet(O) is chromosomally encoded, similar to the findings of others.13,14,27 The MIC of tetracycline for some transconjugants was reproducibly higher than that of the original isolate, suggesting that the host strain can influence the level of resistance expressed. Unlike Gibreel and Taylor,27 we did not find any variant tet(O) sequences associated with high-level resistance, because no isolates required 512 mg/L tetracycline for inhibition.
The present study gave different conclusions to those from a previous study investigating the effect of tetracycline antibiotic (oxytetracycline) exposure upon Campylobacter in poultry flocks.12 However, in their study, the single flock examined had no tetracycline-resistant strains before, during or after antibiotic exposure. As none of the Campylobacter became tetracycline-resistant during or after antibiotic exposure, it was concluded that no strains acquired a resistance gene such as tet(O) from the commensal enterococcal flora that were present concurrently.12
Although in the current study all tetracycline-resistant strains contained tet(O), previous and parallel studies by ourselves with other antibiotics also provided strains that were tetracycline-resistant that were determined as tet(O)-positive by PCR. The presence of the efflux pump inhibitor PAβN reproducibly reduced the MICs of all strains tested, irrespective of the presence of tet(O). These data suggest that overexpression of an efflux pump can confer tetracycline resistance in the absence of tet(O) and that an intact efflux pump, presumably CmeB, is required for high-level tetracycline resistance even when the tet(O) gene is present. These data are similar to those of previous studies that indicated that CmeB was required for macrolide resistance in Campylobacter28 and for various plasmid and chromosomally mediated antibiotic resistances in clinical isolates of Salmonella.29
In summary, our data indicate that chlortetracycline treatment does not eradicate tetracycline-resistant Campylobacter spp. from poultry. However, if a low number of resistant isolates are present, then the antibiotic pressure appears insufficient to select such strains as the dominant population. Further studies to determine the Campylobacter population dynamics in flocks containing birds colonized with low numbers of antibiotic-resistant Campylobacter are now required.
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This work was supported by Defra project VM2200. No other support for this work was received.
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None to declare.
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Present address. Health Protection Agency Yorkshire and the Humber, Leeds, UK. 
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Acknowledgements |
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We thank Sangita Bhattarai Sapkota, Zaahira Gani and Saba Ghori for providing technical support.
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1 Adak GK, Meakins SM, Yip H, et al. Disease risks from foods, England and Wales, 1996–2000. Emerg Infect Dis (2005) 11:365–72.[Web of Science][Medline]
2 Anonymous. Veterinary Medicines Directorate. http://www.vmd.gov.uk/Publications/Antibiotic/antimicrob120707.pdf(20 December 2007 date last accessed).
3
Taylor DE, de Grandis SA, Karmali MA, et al. Transmissible plasmids from Campylobacter jejuni. Antimicrob Agents Chemother (1981) 19:831–5.
4 Anderson SR, Saadbye P, Shukri NM, et al. Antimicrobial resistance among Campylobacter jejuni isolated from raw poultry meat at retail level in Denmark. Int J Food Microbiol (2006) 107:250–5.[CrossRef][Web of Science][Medline]
5
Bywater R, Deluyker H, Deroover E, et al. A European survey of antimicrobial susceptibility among zoonotic and commensal bacteria isolated from food-producing animals. J Antimicrob Chemother (2004) 54:744–54.
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Desmonts M-H, Dufour-Gesbert F, Avrain L, et al. Antimicrobial resistance in Campylobacter strains isolated from French broilers before and after antimicrobial growth promoter bans. J Antimicrob Chemother (2004) 54:1025–30.
7
Luber P, Wagner J, Hahn H, et al. Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli strains isolated in 1991 and 2001–02 from poultry and humans in Berlin, Germany. Antimicrob Agents Chemother (2003) 47:3825–30.
8 Guevremont E, Dadeau E, Sirois M, et al. Antimicrobial susceptibilities of thermophilic Campylobacter from humans, swine and chicken broilers. Can J Vet Res (2006) 70:81–6.[Web of Science][Medline]
9
Luangtongkum T, Morishita TY, Ison AJ, et al. Effect of conventional and organic production practices on the prevalence and antimicrobial resistance of Campylobacter spp. in poultry. Appl Environ Microbiol (2006) 72:3600–7.
10 Manavathu EK, Hiratsuka K, Taylor DE. Nucleotide sequence analysis and expression of a tetracycline-resistance gene from Campylobacter jejuni. Gene (1988) 62:17–26.[CrossRef][Web of Science][Medline]
11 Avrain L, Vernozy-Rozand C, Kempf I. Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens. J Appl Microbiol (2004) 97:134–40.[CrossRef][Medline]
12
Fairchild AS, Smith JL, Idris U, et al. Effects of orally administered tetracycline on the intestinal community structure of chickens and on tet determinant carriage by commensal bacteria and Campylobacter jejuni. Appl Environ Microbiol (2005) 71:5865–72.
13 Lee Y-C, Tai L-C, Lin C-S, et al. Occurrence of plasmids and tetracycline resistance among Campylobacter jejuni and Campylobacter coli isolated from whole market chickens and clinical samples. Int J Food Microbiol (1994) 24:161–70.[CrossRef][Web of Science][Medline]
14
Pratt A, Korolik V. Tetracycline resistance of Australian Campylobacter jejuni and Campylobacter coli isolates. J Antimicrob Chemother (2005) 55:452–60.
15 Pumbwe L, Piddock LJV. Identification and molecular characterisation of CmeB, a Campylobacter jejuni multidrug efflux pump. FEMS Microbiol Lett (2002) 206:185–9.[CrossRef][Web of Science][Medline]
16
Pumbwe L, Randall LP, Woodward MJ, et al. Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiple-antibiotic-resistant Campylobacter jejuni. J Antimicrob Chemother (2004) 54:341–7.
17
Humphrey TJ, Jorgensen F, Frost JA, et al. Prevalence and subtypes of ciprofloxacin-resistant Campylobacter spp. in commercial poultry flocks before, during, and after treatment with fluoroquinolones. Antimicrob Agents Chemother (2005) 49:690–8.
18
Griggs DJ, Johnson MM, Frost JA, et al. 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.
19 Bolton FJ, Wareing DRA, Skirrow MB, et al. Identification and biotyping of campylobacters. In: Identification Methods in Applied and Environmental Microbiology—Board GR, Jones D, Skinner FA, eds. (1992) Oxford: Blackwell Scientific Publications. 151–61.
20 Best EL, Powell EJ, Swift C, et al. Applicability of a rapid duplex real-time PCR assay for speciation of Campylobacter jejuni and Campylobacter coli directly from culture plates. FEMS Microbiol Lett (2003) 229:237–41.[CrossRef][Web of Science][Medline]
21 Houf K, Tutenel A, De Zutter L, et al. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS Microbiol Lett (2000) 193:89–94.[CrossRef][Web of Science][Medline]
22
Nachamkin I, Bohachick K, Patton CM. Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis. J Clin Microbiol (1993) 31:1531–6.
23 Meinersmann RJ, Helsel LO, Fields PI, et al. Discrimination of Campylobacter jejuni isolates by fla gene sequencing. J Clin Microbiol (1997) 35:2810–4.[Abstract]
24 Gibson JR, Sutherland K, Owen RJ. Inhibition of DNAse activity in PFGE analysis of DNA from Campylobacter jejuni. Lett Appl Microbiol (1994) 19:357–8.[Web of Science][Medline]
25 Thwaites RT, Frost JA. Drug resistance in Campylobacter jejuni, Campylobacter coli, and Campylobacter lari from humans in north west England and Wales, 1997. J Clin Pathol (1999) 52:812–4.[Abstract]
26 McDermott PF, Bodeis SM, Aarestrup FM, et al. Development of a standardized susceptibility test for Campylobacter with quality control ranges for ciprofloxacin, doxifloxacin, erythromycin, gentamicin and meropenem. Microb Drug Res (2004) 10:124–31.[CrossRef]
27
Gibreel A, Taylor DE. Macrolide resistance in Campylobacter jejuni and Campylobacter coli. J Antimicrob Chemother (2006) 58:243–55.
28 Cagliero C, Mouline C, Payot S, et al. Involvement of the CmeABC efflux pump in the macrolide resistance of Campylobacter coli. J Antimicrob Chemother (2005) 6:948–50.
29
Baucheron S, Tyler S, Boyd D, et al. AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar typhimurium DT104. Antimicrob Agents Chemother (2004) 48:3729–35.
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