J Med Microbiol 56 (2007), 833-837; DOI: 10.1099/jmm.0.47103-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Role of the plasmid-encoded tet(O) gene in tetracycline-resistant clinical isolates of Campylobacter jejuni and Campylobacter coli
Javid Iqbal Dasti,
Uwe Groß,
Sven Pohl,
Raimond Lugert,
Michael Weig and
Ruprecht Schmidt-Ott
Institute of Medical Microbiology, University of Göttingen, D-37075 Göttingen, Germany
Correspondence
Uwe Groß
ugross{at}gwdg.de
Received 30 November 2006
Accepted 2 February 2007
The prevalence of tetracycline resistance, tetracycline MICs and tet(O) gene localization were investigated in 83 Campylobacter isolates from patients suffering from acute gastroenteritis in Germany. Combined biochemical and molecular markers identified 74 isolates (89 %) as Campylobacter jejuni, including seven atypical isolates that failed to hydrolyse hippurate, and nine isolates (11 %) as Campylobacter coli. Tetracycline resistance was detected in six out of nine Campylobacter coli isolates (67 %) and 13 out of 74 C. jejuni isolates (18 %). Low-level tetracycline resistance was observed for C. coli (MIC 16 µg ml–1 for all strains), whereas C. jejuni showed high-level resistance (MIC >256 µg ml–1 for all strains). Both low- and high-level tetracycline resistance was associated with the presence of the tet(O) gene. In C. jejuni, tet(O) was plasmid-encoded in 54 % of tetracycline-resistant isolates, whereas in C. coli, tet(O) appeared to be located on the chromosome.
Present address: GlaxoSmithKline GmbH & Co. KG, Theresienhöhe 11, D-80339 München, Germany. 
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INTRODUCTION
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Over the last few decades, Campylobacter jejuni and Campylobacter coli have emerged as important food-borne pathogens and are of major public health concern. Both species cause gastrointestinal infections as well as post-infection manifestations, such as Guillain–Barré syndrome and reactive arthritis (Schmidt-Ott et al., 2006). Annually, approximately 60 000 cases of Campylobacter enteritis are reported in Germany (RKI, 2006). Campylobacter enteritis is a zoonotic disease, and poultry, cattle and pigs can be the source of infection (Mead et al., 1999). Antibiotic supplementation in animal feed constitutes more than half of the total antimicrobial use worldwide (Wegener et al., 1999). Transmission of antimicrobial resistance via the food chain can occur from food animals to humans (Pezzotti et al., 2003; Putnam et al., 2003). In the past few years, increased antibiotic resistance has been reported in C. jejuni, particularly tetracycline resistance (Gibreel et al., 2004). In Campylobacter species, high-level tetracycline resistance is usually associated with the tet(O) gene carried on transmissible plasmids (Taylor & Courvalin, 1988). The Tet(O) protein belongs to the class of ribosomal protection proteins that confer resistance by dislodging tetracycline from its primary binding site on the ribosome (Connell et al., 2003). Previously, it has been shown that 38.5 % of C. jejuni isolates in Germany are resistant to tetracycline (Wagner et al., 2003). However, the role of the plasmid-encoded tet(O) gene in tetracycline-resistant clinical isolates of Campylobacter in Germany has not been well described. Precise species differentiation is an important prerequisite for such investigations. We therefore combined biochemical and molecular markers for precise differentiation of clinical isolates of Campylobacter to determine their prevalence, with a focus on plasmid-encoded tet(O)-mediated tetracycline resistance in German clinical isolates.
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METHODS
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Campylobacter strain collection.
Eighty-three isolates of Campylobacter were collected from patients suffering from gastroenteritis in the university hospital of Göttingen. Faecal specimens from these 83 patients were examined for the presence of common faecal pathogens. During sample collection, no outbreak of campylobacteriosis was reported in the area.
Media and growth conditions.
Prior to tetracycline MIC determination, Campylobacter isolates were cultured on Columbia agar base (Merck) supplemented with 5 % sheep blood, polymyxin B (2.5 IU ml–1), trimethoprim (5 µg ml–1) and vancomycin (10 µg ml–1), and incubated at 42 °C under microaerophilic conditions (5 % O2, 10 % CO2, 85 % N2) for 48 h. Campylobacter isolates were biochemically differentiated at the species level by Gram staining, oxidase and catalase activities, hippurate hydrolysis, hydrogen sulfide production and susceptibility to nalidixic acid using a commercially available species differentiation kit (API Campy; bioMérieux).
Determination of antibiotic resistance and tetracycline MICs.
Campylobacter isolates were initially tested for resistance to ampicillin, ciprofloxacin, erythromycin, gentamicin and tetracycline using a disc-diffusion method (Gaudreau & Gilbert, 1997). The MIC of tetracycline was subsequently determined by an agar dilution method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) guidelines (NCCLS, 1997; CLSI, 2006). Mueller–Hinton blood agar supplemented with tetracycline concentrations of 4, 8, 16, 32, 64, 128 and 256 µg ml–1 was inoculated with 1 µl brain–heart infusion broth containing 1x107–2x107 bacteria and incubated at 37 °C under microaerophilic conditions for 40–48 h. Two C. jejuni tetracycline-resistant strains and one C. jejuni strain known to be tetracycline susceptible (MIC 4 µg ml–1) were used as controls. Each experiment was conducted in triplicate. The lowest concentration of the antimicrobial agent that produced no visible growth was considered to be the MIC for the relative isolate. The CLSI has previously described testing conditions for Campylobacter spp., but did not define interpretive breakpoints (CLSI, 2006). Therefore, in agreement with a previously published study (Gaudreau & Gilbert, 1997), we considered isolates having a tetracycline MIC of 
4 µg ml–1 to be sensitive to tetracycline.
Genomic DNA preparation.
Genomic DNA was prepared by a previously described CTAB (hexadecyltrimethyl ammonium bromide) genomic DNA isolation method (Colegio et al., 2001). A lawn culture of Campylobacter grown overnight on Columbia agar base was flooded with physiological 0.9 % NaCl and pelleted at room temperature. The pellet was resuspended in 564 µl TE buffer (pH 7.4), with 30 µl 10 % SDS and 6 µl proteinase K (10 mg ml–1; Qiagen) and incubated for 1 h at 37 °C. Subsequently, 100 µl 5 M NaCl and 80 µl CTAB/NaCl solution were added, followed by a 1 h incubation at 65 °C. Genomic DNA was extracted with chloroform : isoamyl alcohol (1 : 24), ethanol precipitated, resuspended in 50 µl sterile water and stored at –20 °C.
PCR and Southern blot analysis of the hipO gene.
A previously established PCR method was used to detect the hipO gene (Linton et al., 1997). The presence of the hipO gene in isolates lacking biochemical hippurate hydrolase activity was confirmed by Southern blot analysis. For Southern blot analysis of the hipO gene, a PCR product labelled with digoxigenin-11-UTP (Roche Diagnostics) was used as a probe and hybridized with the BglII-digested genomic DNA of Campylobacter isolates. The DNA was blotted on a nitrocellulose membrane (Optitran BA-S 85; Schleicher & Schuell). Solutions and conditions were used according to a standard protocol (Sambrook & Russell, 2001). Hybridization was performed at 42 °C for 18 h. Washing of membranes was carried out twice at 37 °C in 2x SSC, 0.5 % SDS for 15 min and twice at 65 °C in 0.1x SSC, 0.5 % SDS for 30 min. Digoxigenin was detected with specific peroxidase-labelled antibodies using an enhanced chemiluminescence analysis system (Amersham Pharmacia Biotech) according to the recommendations of the supplier. Hippurate hydrolase-negative isolates were confirmed as C. coli by PCR. Genomic DNA was isolated by the CTAB method and a previously reported primer pair (CC18F, 5'-GGTATGATTTCTACAAAGCGA-3'; CC519R, 5'-ATAAAAGACTATCGTCGCGTG-3') was used to amplify the expected 583 bp fragment of the aspartokinase (aspA) gene (Linton et al., 1997).
Plasmid preparation from C. coli isolates.
Plasmid DNA from Campylobacter isolates was purified from an overnight culture on blood agar using mini Qiagen columns as recommended by the manufacturer. The plasmids were designated ‘pCj’ or ‘pCc’ for C. jejuni or C. coli, respectively, following species differentiation. Restriction digestion of plasmid DNA was performed using HindIII, Bglll, PstI and AccI (New England Biolabs) and analysed on 1.2 % agarose gels with TAE buffer (0.04 M Tris/acetate, 0.001 M EDTA).
Detection and localization of the tet(O) gene in C. coli isolates.
The presence of the tet(O) gene in tetracycline-resistant C. coli isolates was confirmed by a tet(O)-specific PCR (Schmidt-Ott et al., 2005). Southern blot analysis was performed to determine the localization of the tet(O) gene. DNA probes were generated and labelled with digoxigenin-11-dUTP by PCR using the above-mentioned primer pair. Plasmid pCjA13 carrying the tet(O) gene was used as a template for this PCR (Schmidt-Ott et al., 2005). The generated probes were hybridized as described above with the HindIII-digested C. coli plasmids. DNA was then blotted onto Optitran BA-S 85 nitrocellulose membranes and detection of the tet(O) gene was performed with a specific peroxidase-labelled antibody using an enhanced chemiluminescence analysis system.
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RESULTS AND DISCUSSION
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Species identification of thermophilic Campylobacter strains
Precise species identification of Campylobacter is an important prerequisite for epidemiological and resistance studies. Eighty-three thermophilic Campylobacter isolates were identified and tested for the presence of the hipO gene using phenotypic and molecular methods. On the basis of their hippurate hydrolase activity, 67 isolates (81 %) were identified as C. jejuni. Sixteen Campylobacter isolates (19 %) did not show any hippurate hydrolase activity. However, in seven of these hippurate hydrolase-negative isolates, the hipO gene was detected by probing genomic DNA with a digoxigenin-11-dUTP-labelled hipO probe (Fig. 1a
). These isolates were identified as atypical isolates of C. jejuni and the remaining nine hippurate hydrolase-negative isolates were confirmed as C. coli (Fig. 1b) using a specific PCR (Linton et al., 1997).
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Fig. 1. Genotypic species differentiation of hippurate hydrolase-negative thermophilic Campylobacter strains. (a) BglII-digested genomic DNA probed with a digoxigenin-labelled hipO probe. (b) C. coli-specific PCR amplification of genomic DNA.
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Antibiotic resistance in C. jejuni and C. coli
Tetracyclines have been used as an alternative choice in the treatment of C. jejuni and C. coli enteritis. Large geographical variation in the susceptibility patterns of C. jejuni and C. coli to tetracycline has been observed. The rate of resistance in Denmark ranges from 0 to 11 % (Aarestrup et al., 1997), in Spain it is 25 % (Gomez-Garces et al., 1995) and in the USA it is 48 % (Nachamkin, 1994). After precise identification at the species level, tetracycline MICs were determined for 19 Campylobacter isolates that were identified as tetracycline resistant by the disc-diffusion test. Tetracycline MICs ranged from 16 to >256 µg ml–1. High-level tetracycline resistance was found in C. jejuni, whereas in C. coli isolates, tetracycline resistance was significantly lower. For the 13 C. jejuni strains, the MIC was determined as >256 µg ml–1, whilst the six C. coli isolates had an MIC of 16 µg ml–1. The frequency of tetracycline resistance was significantly higher (P <0.001, 
2 test) in C. coli (67 %) than in C. jejuni (18 %).
C. coli is frequently found in pigs (Moore & Madden, 1998), and it is known that the regular use of antimicrobial agents for therapeutic purposes and growth promotion can play a role in the prevalence of antimicrobial-resistant strains of C. coli in pigs (Harvey et al., 1999; Payot et al., 2001). Therefore, a higher frequency of tetracycline resistance in clinical isolates of C. coli might be linked to the use of related antibiotics in the food chain.
Plasmid prevalence and tetracycline resistance
Bacterial resistance to tetracycline commonly arises through one of four identified mechanisms: efflux of tetracycline, modification of tetracycline, ribosomal protection or mutation of the 16S rRNA (Burdett, 1991; Ross et al., 1998; Schnappinger & Hillen, 1996). However, plasmid-mediated tet(O)-encoded tetracycline resistance is reported quite frequently in Campylobacter spp. (Manavathu et al., 1988; Lee et al., 1994). Plasmids bearing the tet(O) determinant have also been isolated from other bacteria, such as Enterococcus faecalis and Streptococcus spp., and the plasmids were shown to have similar sizes and restriction profiles to those isolated from C. jejuni and C. coli (Zilhao et al., 1988). Previously, the isolation rate of plasmids from Campylobacter species has been reported to be quite variable, ranging from 44 to 91 % for clinical and poultry isolates (Gaudreau & Gilbert, 1998). In this study, approximately 23 % (n=19) of Campylobacter isolates harboured plasmids, ranging in size from 5 to 66 kb. Significant differences among plasmids were detected in both species of Campylobacter: 19 % (n=14) of the C. jejuni isolates and 56 % (n=5) of C. coli isolates harboured plasmids. Instead of the 33–66 kb plasmids found in C. jejuni, C. coli isolates harboured plasmids of 5–9 kb. Fifty per cent (n=7) of plasmid-harbouring C. jejuni and 60 % (n=3) of the plasmid-positive C. coli isolates were resistant to tetracycline (Table 1). To determine the localization of the tet(O) gene, plasmid DNA from C. jejuni and C. coli isolates was probed with digoxigenin-11-dUTP-labelled tet(O) (Fig. 2). Our results revealed that 54 % (n=7) of the tetracycline-resistant C. jejuni isolates carried the tet(O) gene on their plasmids. Surprisingly, in C. coli none of the plasmids carried the tet(O) gene. Amplification of the tet(O) gene from genomic DNA of tetracycline-resistant C. coli isolates indicated a chromosomal localization of the tet(O) gene. However, considering the limitation of the alkaline lysis method for plasmid isolation, the presence of low-copy-number plasmids larger than 70 kb cannot be totally excluded. It has been suggested previously that recombination events between plasmids and the chromosome, or integration of a plasmid, might occur, which could explain chromosomally mediated tetracycline resistance in these isolates (Boosinger et al., 1990). It is also known that illegitimate recombination can cause integration of a heterologous plasmid in C. coli (Richardson & Park, 1997) and this would ultimately lead to a higher frequency of chromosomally mediated tetracycline resistance in C. coli. We previously confirmed conjugation in two isolates having plasmids of 40.5 kb (pCjA9) and 41.9 kb (pCjA13) (Schmidt-Ott et al., 2005). In this study, Southern blot analysis showed that tetracycline resistance in these isolates was tet(O)-encoded and plasmid-mediated, which ultimately confirmed conjugation transfer of tet(O) in these C. jejuni isolates.
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Table 1. Characterization of tetracycline-resistant Campylobacter isolates
The method applied in this study allowed the detection of plasmids of 1–66 kb.
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Fig. 2. Correlation of tetracycline resistance of C. jejuni isolates with the presence of the tet(O) gene. BglII-digested C. jejuni plasmid DNA was probed with a digoxigenin-labelled tet(O) gene probe. For pCjA5 and pCjA6, two bands are visible, which is probably due to incomplete digestion of the plasmid DNA.
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In conclusion, resistance against tetracycline in C. jejuni and C. coli isolates was associated with the tet(O) gene in all cases and there was a strong correlation between tetracycline resistance and plasmid carriage in C. jejuni isolates. Although all plasmid-containing isolates of C. coli were resistant to tetracycline, none of the C. coli isolates carried the tet(O) gene on their plasmid. Instead, the tet(O) gene appeared to be chromosomally encoded in all tetracycline-resistant C. coli isolates.
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ACKNOWLEDGEMENTS
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This study was supported by the Deutsche Forschungsgemeinschaft (GRK335).
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INTRODUCTION
METHODS
