Objectives: In this study a large random collection (n = 378) of Irish thermophilic Campylobacter isolates were investigated for the presence of integrons, genetic elements associated with the dissemination of antimicrobial resistance.
Methods: Purified genomic DNA from each isolate was analysed by PCR for the presence of class 1 integrons. Four gene cassette-associated amplicons were completely characterized.
Results: Sixty-two of the isolates possessed a complete class 1 integron with a recombined gene cassette located within a 1.0 kb amplicon containing an aadA2 gene. This cassette was present in both Campylobacter jejuni and Campylobacter coli isolates and following sequence analysis was shown to be similar to sequences recently reported in Salmonella enterica Hadar and on an 85 kb plasmid conferring quinolone resistance in Escherichia coli.
Conclusions: Aminoglycoside aadA2-encoding class 1 integrons were identified among unrelated Campylobacter spp. Amino acid sequence comparisons revealed identical structures in both Salmonella and E. coli. The presence of class 1 integrons in Campylobacter spp. may be significant should these organisms enter the food chain and especially when antimicrobial treatment for severe infections is being considered.
Received 17 December 2003; returned 12 January 2004; revised 18 February 2004; accepted 23 February 2004
Campylobacter spp. are now regarded as the leading causative agents of acute diarrhoeal disease in humans worldwide, posing an increasingly significant clinical challenge.1,2 Patients infected with Campylobacter spp. have a higher mortality compared with controls.3 Although the associated gastroenteritis is normally self-limiting, antimicrobial treatment is usually reserved for patients with severe and advanced infection and the drugs of choice often include erythromycin, the fluoroquinolones or tetracycline.4 Intravenous aminoglycoside therapy may also be considered in more serious cases of Campylobacter infection, such as bacteraemia and other systemic infection(s).5 Several studies have recently signalled an increasing incidence of antimicrobial resistance among Campylobacter spp. isolates.6,7 Resistance to trimethoprim is intrinsic and increasing resistance trends for other agents including sulphonamides have been reported.8 Significantly, over the past decade there has been an increase in the number of quinolone-resistant and to a lesser extent macrolide-resistant strains reported, being identified from human infections.1,4,9
In general, bacterial populations respond to the threat of an antimicrobial agent by eventually developing some type of resistance mechanism.10 The imposed selective pressure results in the development of a corresponding resistance determinant that facilitates evasion of the inhibitory substance.8 Horizontal transfer of such resistance determinants together with any genetic modification of pre-existing genes through point mutation or some other genetic event, are thought to be the main mechanisms contributing to bacterial resistance. Self-transmissible elements including plasmids, transposons and bacteriophage all facilitate the acquisition and subsequent dissemination of resistance determinants. In addition, integrons are now considered efficient vehicles for the transfer of resistance markers among unrelated bacterial populations.11 Integron structures are naturally occurring gene expression systems that can potentially capture and integrate one or more gene cassettes and convert them into functionally expressed genes.12 It is these gene cassettes that encode the resistance determinants to several antimicrobial agents.
Nine classes of integrons have been described to date and class 1 integrons are clinically significant. Briefly, the typical structure of a class 1 integron includes two conserved segments (CSs), denoted as 5′- and 3′-CSs, flanking a gene cassette. An int1 gene encoding an integrase enzyme is located within the 5′-CS and this is responsible for the recombination of a gene cassette at a specific att1 attachment site.11 Also within this region is a promoter which facilitates the efficient expression of any integrated gene cassette.11,12 The 3′-CS contains two open reading frames (ORFs) encoding resistance to quaternary ammonium compounds (qac) and sulphonamide (sul1), respectively. Integrons can incorporate and express more than one gene cassette, provided that its location is flanked by the 5′- and 3′-CS domains. Thus integrons may contain a number of recombined gene cassettes, oriented in a classical ‘head-to-tail’ arrangement conferring a multidrug-resistant (MDR) phenotype on any isolate in which these genetic elements are located.13,14
Integron-like structures were reported in Campylobacter isolates, suggesting that gene cassettes encoding antimicrobial resistance may act as a possible vehicle for the dissemination of resistance among Campylobacter spp.15,16 Gibreel & Sköld,17 reported the existence of chromosomally located integrons carrying a dfr1-containing gene cassette in Campylobacter jejuni. This study reports the investigation of a large collection of unrelated Campylobacter spp. isolates (n = 378) of both human and animal origin for the presence of class 1 integrons. Transmission of antimicrobial resistance determinants mediated by integron-containing Campylobacter spp. via the food chain and the associated implications for public health are discussed.
Materials and methods
Three hundred and seventy-eight randomly collected Campylobacter spp. isolates were isolated from human and poultry sources during the year 2000. Isolates were subcultured onto Preston agar which consisted of Campylobacter agar base (Oxoid, Basingstoke, UK) containing Campylobacter modified selective supplement (Oxoid) and 5% (v/v) lysed horse blood (Oxoid). Subcultures were incubated at 42°C for 48 h in a microaerophilic environment. All cultures were examined for purity by carbol fuschin staining, and species identification was performed using the hippurate hydrolysis test and species-specific PCR assays.4,18
Cultures were initially suspended in 1 mL 0.85% (w/v) NaCl and washed twice. DNase activity was inhibited using treatment with formaldehyde according to the method of Gibson et al.19 DNA extraction was performed according to Lind et al.20 and DNA concentrations were determined spectrophotometrically as described previously.18 The integrity of the purified template DNA was assessed by conventional agarose gel (1.5%, w/v) electrophoresis and DNA preparations were stored at 4°C.
Amplification of gene cassettes by PCR
Each isolate was analysed for the presence of gene cassettes associated with class 1 integron structures using a modified version of the PCR assay described by Lévesque et al.21 Briefly, for each isolate, 100 ng of purified template DNA was added to a reaction mixture which contained the following: 5 µL 10× PCR buffer [100 mM Tris–HCl pH 9.0, 500 mM KCl, 1% (v/v) Triton X-100], 2.5 mM MgCl2, 0.2 mM each dNTP, 25 pmol each of the int1 forward primer (5′-GGCATCCAAGCAGCA- AGC-3′) and int1 reverse primer (5′-AAGCAGACTTGACCTGAT-3′), 2.5 U Taq DNA polymerase (Promega, Madison, WI) and sterile distilled water, which was added to a final volume of 50 µL. Thermal cycling reaction parameters included an initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 1 min. A final extension at 72°C was carried out for 7 min and following this step all completed reactions were maintained at 4°C. Amplified DNA products were analysed by conventional agarose gel (1.5%, w/v) electrophoresis and each DNA sample was analysed in duplicate.
DNA sequence analysis
Any amplified PCR product of interest was initially gel extracted using a Qiagen gel extraction kit (Qiagen, West Sussex, UK). Each gel-purified product was re-amplified and subsequently cloned using the TOPO TA Cloning kit (Invitrogen BV, The Netherlands). After verification of the cloned insert the cloned products were sequenced (MWG Biotech, Ebersberg, Germany) and the subsequent data were analysed initially using the DNA Sequencher (version 4.1) sequencing software (Gene Codes Corp., Ann Arbor, MI, USA). Sequences were initially compared with the current GenBank sequence databases using the BLAST suite of programs.22 ClustalW amino acid sequence alignments were produced for comparison.23 These alignments were carried out online using the latter program over the internet at http://www.ebi.ac.uk/clustalw.
Nucleotide accession numbers
Two DNA sequences were submitted to GenBank and assigned the following accession numbers: AF530636 and AF530637.
In this collection 317/378 isolates were identified as C. jejuni, 55 as Campylobacter coli and six cultures were identified as mixed cultures of both species.
All isolates in the collection were analysed in duplicate by PCR for the presence of integrated gene cassettes.21 Several DNA amplicon profiles were identified after gel electrophoresis. These groups were broadly designated as integron pattern (IP) groups, IP-1–IP-4. The amplicon sizes within these groups ranged from 300 bp to larger DNA fragments of 1.4 kb (Figure 1a). Assignment to each group was defined based on the largest amplicon within the profile. IP-1 consisted of amplicons of ≤500 bp (data not shown), IP-2 consisted of amplicons of ≤700 bp. The remaining two groups, IP-3 and IP-4, contained amplicons of ≤1.1 and ≤1.4 kb, respectively (some of the IP-group composite profiles are shown in Figure 1a). Since the average size of a bacterial coding sequence is ∼800 bp, amplified DNA fragments of ≥1.0 kb were investigated further, on the basis that these were more likely to contain a complete ORF corresponding to a potential gene. In total, the IP-3 and IP-4 groups were associated with 16.4% (62/378) of the Campylobacter spp. isolates in this collection. Of these, 54 of the 62 isolates were C. jejuni and the remaining isolates were identified as C. coli. In addition, 55 of these isolates were isolated from poultry and the remaining seven were isolated from humans. A 1.0 kb amplicon common to the IP-3 and IP-4 groups (Figure 1a) was identified and further characterized from four of the study isolates, three C. jejuni (CIT-325C , CIT-195C, CIT-134C) and one C. coli (CIT-181C). The characteristic conserved features associated with class 1 integrons were identified by PCR, in these four isolates. These included the 5′-CS-located integrase, and 3′-CS-located qacΔE1 and sul1 genes (data not shown).
Analysis of the DNA sequence of the 1.0 kb amplicon, from all four isolates, identified two ORFs of 39 and 789 bp. The 59-base element (be) core site necessary for recombination between gene cassettes and integrons was also noted. (A schematic illustration of two of these is shown in Figure 1(b) with the 59-be shown in the box below and compared with the consensus sequence.) When the former ORF was compared with the current databases, BLAST22 searches identified perfectly matching sequences (Figure 1c) of unknown function in two C. jejuni16 isolates along with a recent Salmonella enterica serovar Hadar isolate (accession number AY258269). This sequence was designated ORF-11. In the former, ORF-11 was located proximal to an aacA4-encoding gene in a class 1 integron and in S. enterica Hadar this sequence was similarly located on the proximal side of an aadA2-encoding gene.
BLAST searches with the larger ORF identified it as an aadA2-encoding aminoglycoside adenyltransferase, of 263 residues, which closely matched similar sequences in S. enterica Hadar and in an 11.6 kb In36 integron, on an 85 kb plasmid in Escherichia coli (Figure 1c).24 In each case resistance conferred by a gene cassette was consistent with phenotypic resistance as determined by susceptibility testing.4 Deduced amino acid sequences from all four aadA2-encoding genes from C. jejuni and C. coli were compared with each other using ClustalW.23 The alignment (Figure 2) showed that all of the Campylobacter isolates contained a similar AAD2 protein with a high level of amino acid identity (ranging from 98% to 100%) between the sequences. A small number of amino acid substitutions were identified as indicated in Figure 2 and these were particularly associated with two of the isolates, C. jejuni CIT-134C (five substitutions) and C. jejuni CIT-325C (three substitutions) (indicated in bold in Figure 2). When the Campylobacter spp. sequences were compared against those of S. enterica Hadar and the plasmid containing the aadA2-encoding gene in E. coli a similar amino acid identity was also identified.
Treatment with antimicrobials is a risk factor for infection with organisms that are simultaneously resistant to several drugs and this may contribute to mortality.3 Horizontal gene transfer is a significant mechanism for disseminating antimicrobial resistance among bacterial populations. Integron structures can play a pivotal role and have been identified in several Gram-negative bacterial species including food-borne pathogens such as Salmonella spp., E. coli and Shigella spp.13,14,24 These genetic structures may contain several resistance markers with more than one gene cassette integrated between the conserved domains in a classical ‘head-to-tail’ arrangement. This feature has the potential to confer resistance to several antimicrobial agents simultaneously, including aminoglycosides, cephalosporins, the penicillins and trimethoprim. Until recently, integron structures were not identified in Campylobacter spp. and therefore potential for disseminating resistance by this mechanism was unknown. However, studies are now reporting the existence of these structures in Campylobacter spp. and therefore their role and contribution to antimicrobial resistance must be assessed.15–17
Several aad genes, encoding resistance to streptomycin/spectinomycin, have been located within integrons as gene cassettes in several human and animal bacterial isolates.21,25 In fact these gene cassettes are common among class 1 integrons.11,12 Pinto-Alphandary et al.26 previously mapped aminoglycoside resistance-encoding genes to the chromosome of a number of Campylobacter spp. isolates. These antimicrobial agents are now seldom used therapeutically due to the high level of resistance reported among unrelated bacterial species. Surprisingly however, aad gene cassettes remain prevalent within integrons despite the fact that the selective pressure associated with drug use is no longer a significant factor. White et al.25 suggested that this feature may indicate that even in the absence of selection by antimicrobial agents normally used for therapeutic purposes, genes encoding resistance are not necessarily lost but can persist within bacterial populations. Therefore, integron screening and characterization of gene cassettes may be a useful approach to predict antibiotic resistance at the phenotypic level.
In this study we reported the investigation of a large random collection of Campylobacter spp. isolates for the presence of class 1 integrons. Four unique amplification profiles were identified and amplicons of 1.0 kb were investigated in an attempt to identify any potential coding sequences. Sixty-two isolates were associated with this particular amplicon and this group consisted predominantly of C. jejuni species isolated from poultry sources. Characterization of the 1.0 kb amplicon in four isolates independently identified highly similar aadA2-encoding gene cassettes from C. jejuni and C. coli. This finding demonstrates that identical class 1 integron structures are present in different members of the same genus, suggesting that genetic exchange may have occurred in the gastrointestinal environment. Furthermore, S. enterica Hadar and E. coli were also found to contain identical gene cassettes, with aadA2-encoding resistance determinants, suggesting that this gene cassette was transmitted between these microorganisms.7,10,11,13 Streptomycin resistance is prevalent in class 1 integrons found among poultry E. coli.27 When increased resistance to human antimicrobials occurs in food animals, transmission via food or other routes is more likely.28
As the use of aminoglycoside therapy may be considered as a treatment option for some Campylobacter-related infections our data suggest that the possibility now exists for treatment failure to occur due to these mobile elements. Furthermore, the presence of class 1 integrons in several Campylobacter isolates may in part offer an explanation for the high levels of resistance to sulphonamides frequently reported among these organisms.18 Increasing prevalence of macrolide and quinolone resistance is more usually attributed to specific mutations in chromosomally located genes although the future involvement of plasmid-encoded integrons cannot be ruled out.24 In conclusion, our findings highlight the possibility that integrons may be partly responsible for horizontal gene transfer as a potential vehicle for dissemination of MDR phenotypes among Campylobacter spp. These findings may have further implications for future therapeutic strategies, leading to reduced drug efficacy and/or treatment failures in the case of MDR organisms, whose transmission through the food chain poses a real threat to public health.
We would like to thank Dr Tom Barragry for useful discussions and the staff of the Irish Equine Centre for providing isolates. This work was funded in part by the Food Safety Authority of Ireland (FSAI) (grant number FSAI-86/FS/2001).
Present address. Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland
Correspondence address. Centre for Food Safety, University College, Belfield, Dublin 4, Ireland. Tel: +353-1-716-6082; Fax: +353-1-716-6091; E-mail: email@example.com
1Molecular Diagnostics Unit, Cork Institute of Technology, Bishopstown, Cork; 2Department of Medical Microbiology, Cork University Hospital, Wilton, Cork; 3Irish Equine Centre, Naas, Co. Kildare; 4Centre for Food Safety, Faculties of Agriculture, Medicine and Veterinary Medicine, University College, Belfield, Dublin 4, Ireland