Microbial Glycobiology, Institute for Glycomics, Griffith University, Gold Coast Campus, PMB 50 Gold Coast Mail Centre, Queensland, 9726, Australia
Received 26 October 2004; accepted 30 December 2004
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Abstract |
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Methods: Tetracycline MICs were determined for each isolate using an agar dilution method. The distribution and localization of tet(O) on plasmid and chromosomal DNA was determined by Southern-blot experiments. The ability to transfer resistance to recipient strains was examined through conjugation studies. Identity of transconjugants was confirmed by PCR and flaA-restriction fragment length polymorphism analysis.
Results: High-level tetracycline resistance was observed, ranging from 32 to >256 mg/L. Plasmids were detected in 74% of isolates with plasmids between 30 and 40 kb in size most frequently isolated. tet(O) was present in all tetracycline-resistant isolates. In the majority of strains under study the tet(O) gene was chromosomally encoded. Tetracycline resistance of six C. jejuni strains in which tet(O) was plasmid mediated was transferred by conjugation to a C. jejuni recipient strain. Transfer did not occur between tetracycline-resistant C. jejuni strains and a C. coli recipient. No difference in MICs, plasmid carriage and tet(O) localization was detected between human and chicken isolates.
Conclusions: These data indicate that the tet(O) gene, previously reported in Campylobacter strains throughout the world, is present in Australian Campylobacter. This study will lead to a greater understanding of antibiotic resistance distribution in Campylobacter spp. in Australia.
Keywords: tet(O) , plasmids , conjugation
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Introduction |
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Campylobacter species are considered normal intestinal biota of animals used for food production, particularly poultry. Transmission to humans occurs by ingestion of contaminated foods of animal origin and water-based environmental sources, with 5070% of cases due to ingestion of undercooked contaminated poultry.68 Infection is self-limiting and therapy with antibiotics such as erythromycin and ciprofloxacin is reserved for immunocompromised patients or those with unresolving diarrhoea.9
Resistance of Campylobacter spp. to a number of antibiotics, such as tetracycline, erythromycin, ciprofloxacin, kanamycin, nalidixic acid and chloramphenicol has been reported.911 Emergence and dissemination of antibiotic resistance among Campylobacter spp. have been linked to the use of antibiotics in veterinary medicine and use as prophylactics and growth promoters in animal husbandry.9,11 The increasing rate of human infections caused by antimicrobial-resistant strains of C. jejuni makes clinical management of cases of campylobacteriosis more difficult, prolonging illness and compromising treatment of patients with bacteraemia.3,12,13
Resistance to tetracycline in Campylobacter spp. has been reported in many countries. Large geographical differences in susceptibility patterns have been observed.14 High-level tetracycline resistance is usually associated with the tet(O) gene carried on transmissible plasmids in both C. jejuni and Campylobacter coli.15 tet(O) has also been found to be chromosomally located.16,17 The tet(O) gene in campylobacter encodes the Tet(O) protein that protects the ribosome from the inhibitory effect of tetracycline.1820 The presence of tet(O) has been detected in Campylobacter strains throughout the world indicating a wide distribution.1719,21 The presence of tet(O) on conjugative plasmids may explain the distribution of this resistance determinant.
Little was known about tetracycline resistance in Australian Campylobacter spp. Therefore we examined tetracycline resistance in Australian C. jejuni and C. coli isolates. Plasmid carriage, distribution of the tet(O) gene and tetracycline MICs were determined.
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Materials and methods |
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Species and source data for each strain under study are displayed in Table 1. Strains under study were previously identified as tetracycline-resistant by a disc diffusion test. The majority of strains were obtained from humans with gastroenteritis; however, isolates from healthy chickens were also included.
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Campylobacter stains were grown on Columbia agar (Oxoid), supplemented with 5% horse blood (bioMérieux Australia), termed horse blood agar (HBA) and incubated at 37°C before tetracycline MIC determination in a microaerophilic atmosphere (5% O2, 10% CO2, 85% N2). MuellerHinton agar (MHA) (Oxoid) supplemented with 5% horse blood with varying tetracycline concentrations was used for determination of MICs. Culturing of strains for plasmid DNA extractions was carried out on tryptone soya agar (Oxoid) with tetracycline (1 mg/L) to maintain resistance at 37°C and on HBA at 37°C for genomic DNA extractions. Donor and recipient strains in conjugation experiments were cultured on HBA at 42°C. Transconjugants were cultured on HBA with tetracycline (16 mg/L) and erythromycin (20 mg/L) at 42°C for plasmid and genomic DNA extractions.
MIC determination
Tetracycline MICs for strains under study were determined using the agar dilution method complying with methods recommended by the National Committee for Clinical Laboratory Standards.22 MHA plates with tetracycline (Sigma) concentrations of 8, 16, 32, 64, 128 and 256 mg/L were inoculated with 1 µL of a spectrophotometrically determined 12 x 107 cfu/mL suspension in Brucella broth (Difco), and incubated at 37°C under microaerophilic conditions for 4048 h. Control strains consisted of two C. jejuni strains known to be tetracycline-resistant and a C. jejuni strain considered tetracycline-susceptible (MIC 8 mg/L). Control 2% MHA plates were also inoculated with bacterial suspensions. MICs were defined as the lowest concentration of antimicrobial agent producing no visible growth in accordance with Tee et al.23 No internationally accepted criteria for breakpoints are available for Campylobacter spp.24 Therefore we used 8 mg/L as a breakpoint concentration, where isolates with a tetracycline MIC greater this were considered resistant.
Preparation of DNA from cell lysates
Crude cell lysates were prepared by the boiling method as follows. A small loopful of cultured bacteria was washed with 500 µL of sterile phosphate buffered saline (PBS) and pelleted by centrifugation at 9000 g for 5 min in a Sigma 1-15 centrifuge (Nr. 12124 rotor) at room temperature. The pellet was resuspended in 100 µL of sterile water and boiled for 5 min to release the DNA. Final centrifugation at 9000 g for 5 min at room temperature pelleted cell debris and supernatant with bacterial DNA was collected.
Plasmid isolation and analysis
Plasmid DNA was extracted using a modified alkaline lysis method of Sambrook & Russell,25 with the following modifications. Cells from one plate were harvested into 1 mL of PBS and centrifuged for 2 min at 18 000 g in a Sigma 1-15 centrifuge (Nr. 12124 rotor) at room temperature. Pellets were resuspended by vortexing in 200 µL of ice-cold Solution I containing 4 mg/mL of lysozyme and stored for 5 min at room temperature. A 400 µL volume of Solution II was added and the mixture was incubated on ice for 5 min. Then 300 µL of ice-cold Solution III was added to the lysate, which was placed on ice for 5 min, then centrifuged for 7 min at 18 000 g at room temperature. Lysates were treated with 810 µL of 10 mg of pancreatic RNase A per mL for 3045 min at 37°C. Following phenol/chloroform extractions and ethanol precipitation, pellets were resuspended in 20 µL of sterile water and stored at 20°C.
Small-scale genomic DNA extraction
Genomic DNA was prepared by a modified large-scale extraction method of Korolik et al.26 A lawn culture was flooded with 3 mL of PBS and 1.5 mL of cells was harvested and centrifuged for 1.5 min at 18 000 g in a Sigma 1-15 centrifuge (Nr. 12124 rotor) at room temperature. The pellet was resuspended by vortexing in 150 µL of glucose buffer and stored for 5 min at room temperature; 300 µL of TE buffer (pH 7.4) containing 0.51% SDS was added and the lysate was incubated for 15 min at 37°C. Following addition of 100 µL of 1 mg of RNase per mL and incubation for 15 min at 37°C, 100 µL of 1 mg of Pronase per mL was added and the mixture was incubated for 1.5 h at 37°C. A 225 µL volume of ice-cold potassium acetate, pH 4.8, was added and the mixture was gently vortexed and then centrifuged for 10 min at 18 000 g at room temperature. DNA was extracted three times with phenol/chloroform/isoamyl alcohol and once with chloroform/isoamyl alcohol. DNA was precipitated with an equal volume of ice-cold isopropanol and pelleted by centrifugation for 10 min at 18 000 g at room temperature. DNA pellets were washed with 1 mL of isopropanol and resuspended in 30 µL of sterile water and stored at 20°C.
PCR analysis
Primers used for the detection of tet(O) were, tet(O) F 5'-GGCGTTTTGTTTATGTGCG-3' and tet(O) R 5'-ATGGACAACCCGACAGAAGC-3',21 which produced a 559 bp product. Also included in the reaction were internal control primers for the 23S rRNA of thermophilic Campylobacter spp. The primer sequence for the forward primer Therm 1.1 was 5'-TATTCCAATACCAACATTAGT-3', and for the reverse primer Therm 2.1 was 5'-GAAGATACGGTGCTATTTTG-3'.27 This primer pair amplifies a 306309 bp fragment. Standard concentrations of PCR reagents were used with the exception of Mg, which was included in reactions at a concentration of 3.3 mM; 1 U of Taq polymerase (Eppendorf) was used in each reaction; 2 µL of crude lysate extracted DNA was used in each PCR. Genomic DNA was used if amplification from crude lysate extracted DNA was unsuccessful. Japanese isolates, JC6 and JC8, were employed as positive controls and tetracycline-susceptible strain 331 was used as a negative control.
flaA PCR was carried out with primer sequences stated in Wassenaar & Newell.28 The sequence of the forward primer was 5'-ATGGGATTTCGTATTAACAC-3' and the sequence of the reverse primer was 5'-CTGTAGTAATCTTAAAACATTTTG-3'. PCR components consisted of 200 µM dNTPs, 2 mM MgCl2, 25 pmol of primer, 0.5 U of Taq polymerase (Invitrogen) and 5 µL of PCR buffer; 2 µL of crude lysate extracted DNA was used as template. PCRs were carried out in volumes of 50 µL. PCR parameters were initial denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 60°C for 1 min and 72°C for 2 min. Final extension was at 72°C for 5 min. An approximately 1700 bp fragment was amplified.
Southern-blot analysis
Plasmid DNA, ClaI-digested plasmid DNA and ClaI-digested genomic DNA were transferred to nitrocellulose membranes (Hybond+, Amersham) by neutral transfer.25,29 Gels were subjected to depurination in 0.1250.2 M HCl for 1015 min before denaturation and neutralization. The tet(O) PCR product amplified from positive control JC8 was purified using a Wizard PCR Preps DNA Purification System kit (Promega). The probe was radiolabelled using a Nick Translation kit (Promega) with [32 P]dCTP (Amersham). Solutions and conditions were used according to Sambrook & Russell,25 with modification. Hybridization solution contained 100 µg/mL of herring sperm DNA. Hybridizations were carried out at 65°C for 18 h. Membranes were washed twice for 10 min in 2x SSC, 0.1% SDS at 65°C and once for 15 min in 1x SSC, 0.1% SDS at 65°C. Blots were exposed to phosphor screens (Bio-Rad) for 1 to 24 h and analysed with a Bio-Rad Personal Imager FX and the Quantity One program (Bio-Rad).
DNA manipulation
Restriction endonuclease ClaI was obtained from Promega and New England BioLabs. ClaI digestions of genomic and plasmid DNA were carried out according to the manufacturer's recommendations with 40 U and 20 U, respectively. Restriction fragments, plasmid DNA and PCR products were resolved using agarose gel electrophoresis as described by Sambrook & Russell.25 For flaA-restriction fragment length polymorphism (RFLP) analysis, 10 µL of PCR product was digested for 1.5 h at 37°C with 2 U of DdeI (Promega). Restriction fragments were resolved using 3% agarose gels in TAE buffer.
Conjugation of tetracycline-resistant plasmids
Three C. jejuni strains and one C. coli strain were chosen as recipients in intraspecies and interspecies transfer studies. Resistance phenotypes and plasmid carriage of recipient strains are contained in Table 1. Resistance determinants and localization to plasmid or chromosome were unknown for recipient strains. Ten donor strains, including control strain JC6, were mated with the four recipient strains. The two remaining donor strains were only mated with recipients 887 and NCTC C. coli 11366 due to resistance patterns.
Conjugations were carried out according to the plate-mating method of Taylor et al.,30 with the following modifications. Strains were suspended in 1 mL of Brucella broth (Difco) to 108109 cells per mL; 200 µL of donor and recipient strains were mixed and treated with 10 U of DNase for 10 min at 37°C. Cells were spread on HBA plates and incubated for 6 h at 42°C. Cells were washed off plates with Brucella broth, diluted, and spread onto transconjugant selecting medium. Suspensions of donor and recipient strains were also spread on selective medium to detect antibiotic resistance by mutation. Transconjugants were selected on 2% HBA plates containing 16 mg/L of tetracycline and 20 mg/L of erythromycin (Sigma), 10 mg/L of streptomycin (Progen, Archerfield, Queensland, Australia) or 5 mg/L of chloramphenicol (Progen) and incubated for 48 h at 42°C. Matings were also spread on 2% HBA plates as controls.
Verification of transconjugants
Transconjugant colonies from successful matings were verified by plasmid analysis, tet(O) amplification, flaA-RFLP typing and restriction endonuclease (RE) profiling. If flaA-RFLP analysis failed to discriminate between transconjugants and donor and recipient strains, RE profiling was used. Genomic DNA from undistinguished isolates was cleaved with ClaI and resolved as described above, and profiles were compared.
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Results |
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Tetracycline MICs were determined for 41 C. jejuni and five C. coli isolates first identified to be tetracycline-resistant by a disc diffusion test (Table 2). Tetracycline MICs ranged from 32 to > 256 mg/L. Twenty-three strains had an MIC of 128 mg/L, eight strains had an MIC of 256 mg/L and eight strains had an MIC of 64 mg/L. MICs of C. jejuni isolates ranged from 32 to > 256 mg/L and MICs of C. coli isolates ranged from 32 to 256 mg/L. MICs of clinical isolates ranged from 32 to > 256 mg/L and MICs of chicken isolates ranged from 32 to 128 mg/L.
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All isolates and controls were screened for the presence of plasmid DNA. Plasmid carriage and size data are summarized in Table 2. Plasmid profiles of six representative strains are shown in Figure 1. Thirty C. jejuni strains and four C. coli strains carried plasmids, of which 25 C. jejuni and four C. coli strains were isolated from humans with gastroenteritis. Twelve strains carried no detectable plasmids. Approximate sizes of plasmids were determined by the addition of fragment sizes from ClaI plasmid digestions. Where ClaI digestions did not produce distinguishable fragments, approximate plasmid sizes were determined by comparing covalently closed circular forms with those of plasmids that were digested with ClaI. Plasmids ranged from small plasmids (for which sizes were not determined) to larger plasmids 2150 kb in size. The most frequently isolated plasmids were between 30 and 40 kb. Thirty isolates carried single plasmids and four strains harboured more than one plasmid.
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All strains in this study were found to carry the tet(O) gene. Forty-five of the 46 strains were positive for tet(O) when analysed by PCR employing primers described in Bacon et al.21 (Table 2 and Figure 2). The remaining strain was shown to carry the tet(O) gene by Southern-blot analysis using the tet(O) gene as a probe. To determine the localization of tet(O) in the genome, plasmid DNA and ClaI-digested plasmid and genomic DNA was analysed by Southern-blot hybridization at medium stringency, using the tet(O)-coding region as a probe. All strains hybridized with the tet(O) probe. Complete data are listed in Table 2 and hybridization results of a selection of strains are presented in Figure 3. Of the 34 strains that harboured plasmids, plasmids from only 11 strains hybridized to tet(O). Labelled tet(O) hybridized with chromosomal DNA from the remaining 23 plasmid harbouring strains and the 12 strains that lacked plasmids. ClaI fragments of plasmid and genomic DNA that hybridized to tet(O) in nine strains were of similar size, indicating that tet(O) is plasmid mediated. Comparisons could not be made for the two remaining strains with plasmid-borne tet(O).
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The 11 tetracycline-resistant C. jejuni strains that harboured plasmids carrying the tet(O) gene were analysed for the ability to transfer plasmid-encoded tetracycline resistance.
Results of conjugation experiments and transfer frequencies are shown in Table 2. Intraspecies conjugative transfer of tetracycline resistance occurred between control strain JC6, GC5252, GC6108, GC18943, GC21098, GC28719 and GC31677 and recipient strain 887. Transfer frequencies ranged from 0.8 x 108 to 2 x 106 transconjugants per recipient in 6 h matings. Resulting transconjugants were both tetracycline-resistant and erythromycin-resistant. Two transconjugants from each successful mating were verified using tet(O) PCR, flaA-RFLP typing or RE profiling and plasmid carriage analysis. The tet(O) gene was amplified from transconjugants from all successful matings. flaA-RFLP typing verified transconjugants from six of the successful matings. Transconjugants from the remaining mating were verified through RE profiling with ClaI. flaA-RFLP profiles of transconjugants were that of recipient strain 887. Patterns of selected donor and recipient strains and transconjugants are displayed in Figure 4.
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All 11 C. jejuni strains, and control strain JC6, which were examined for conjugative ability, failed to transfer resistance to recipient C. jejuni strains RM10 and 9126. Interspecies transfer of tetracycline resistance between nine of the C. jejuni strains under study, control strain JC6 and the C. coli recipient strain, NCTC 11366, also did not occur. Conclusions could not be drawn from matings between donor strains QH120 and GC28719 and recipient strain NCTC 11366 due to resistance of the donor strains to the selecting antibiotic.
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Discussion |
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PCR amplification and Southern-blot analysis demonstrated that tetracycline resistance in Australian Campylobacter isolates was due to the Tet O determinant. Other investigators have also reported the presence of the tet(O) gene in Campylobacter isolates from Canada, the USA, the UK, Germany, Taiwan, France, Vietnam, Thailand, Switzerland and Algeria.1719,21,33
We have shown that PCR amplification was successful in detecting tet(O) in 97.8% of strains under study. We found one isolate that was consistently negative for tet(O) by PCR, but was subsequently shown to carry the tet(O) gene by Southern-blot analysis. Heterogeneity between the tet(O) sequence in this strain and primers used in PCR detection may account for the lack of amplification. The possibility of a hybrid tetracycline resistance gene in C. jejuni could also possibly account for conflicting results between tet(O) PCR and Southern-blot results observed in our study. Stanton & Humphrey34 reported a mosaic tetracycline determinant in the swine anaerobic bacterium, Megasphaera elsdenii. The tetracycline resistance determinant of M. elsdenii was initially identified as tet(O) by PCR; however, subsequent sequence analysis revealed that the gene consisted of specific regions matching both tet(O) of C. jejuni and tet(W) of Butyrivibrio fibrisolvens.
As tetracycline resistance in Campylobacter has been previously reported to be plasmid mediated,30,35 we examined plasmid carriage in Australian strains. Approximately 74% of the Campylobacter strains in our study harboured plasmids. A similar carriage rate of 70% in Israeli tetracycline-resistant C. jejuni was described by Schwartz et al.32 Plasmid carriage rates of between 80% and 100% for tetracycline-resistant C. jejuni and C. coli, with sizes ranging from 1.7 to 133 kb, have been reported in other countries worldwide.17,32,3638 We have shown that plasmid carriage rates for both species and isolation sources were high. The most frequently isolated plasmids in our study were approximately 3040 kb in size.
In contrast to other studies, only 32.3% of Australian plasmid-carrying isolates had tetracycline resistance mediated by a tet(O)-harbouring plasmid (Table 2), whereas Lee et al.17 reported high rates of plasmid-borne tet(O) carriage, where 96% and 88% of plasmid-harbouring tetracycline-resistant chicken and clinical isolates of C. jejuni carried tet(O) on a plasmid.
We have shown that 26% of total strains did not harbour detectable plasmids. Schwartz et al.32 found a similar rate (30%) in Israel, and Gibreel et al.16 reported that 33% of Canadian tetracycline-resistant isolates of C. jejuni lacked plasmids. Other investigators have also reported plasmid-free tetracycline-resistant Campylobacter.17,36,37,39
It is particularly interesting to note that in the Australian isolates examined in this study, tet(O), and therefore tetracycline resistance, was mostly (76%) chromosomally encoded. Gibreel et al.16 and Lee et al.17 reported chromosomally mediated tet(O) in C. jejuni and C. coli strains that lacked plasmids, but not at such a high rate. It has been suggested that recombination events between plasmid and chromosome or integration of a tetracycline resistance plasmid into the chromosome may occur and this could explain plasmid-free tetracycline-resistant isolates.3941 Richardson & Park,42 observed that even in the absence of functional homology, heterologous plasmid DNA introduced into C. coli by natural transformation could integrate into the genome at random sites by illegitimate recombination.
It is possible that mobile genetic elements other that transmissible plasmids may be involved in the acquisition and dissemination of antibiotic resistance genes, including tet(O), in campylobacter. Ng et al.43 speculated that the tetracycline resistance determinant [tet(O)] may be located on a transposable element, but this has yet to be proven. Of particular interest, Gibreel et al.44 reported the presence of an insertion element, IS607*, similar to IS607 found on the chromosome of Helicobacter pylori, on tet(O)-carrying plasmids of tetracycline-resistant C. jejuni. Parkhill et al.45 reported minimal similarity to IS605 tnpB from H. pylori in sequenced strain NCTC 11168, but no phage-associated sequences and very few repeats. In a study of trimethoprim resistance in Campylobacter, Gibreel & Skold46 found foreign dfr genes in the context of remnants of a transposon and integron. Bacon et al.47 reported that an organization of direct repeats on the virulence plasmid pVir from C. jejuni strain 81-176 was reminiscent of a transposable element.
Six of the 11 C. jejuni strains in which tet(O) was plasmid- mediated were able to transfer tetracycline resistance to a recipient C. jejuni strain by conjugation. Transfer of tetracycline resistance by conjugation has previously been reported for Campylobacter species from many countries including Spain, India, Japan and Canada.16,37,48,49 Spontaneous in vivo transfer of tet(O) between C. jejuni strains in the chicken digestive tract was reported by Avrain et al.50
In this study, donor strains transferred resistance to a recipient strain that harboured native plasmids. A study by Wang & Taylor51 on natural transformation of campylobacter with plasmid DNA found that transformation frequencies were increased 100-fold when the recipient strain harboured similar plasmids. It was suggested that plasmids native to a recipient strain might act as a rescue plasmid by recombining with the incoming plasmid.51 The presence of competing endogenous plasmids in H. pylori strains was found to not constitute a substantial barrier to transformation by an E. coliH. pylori shuttle vector.52
We observed that a small plasmid was co-transferred with the larger conjugative plasmid when Japanese strain JC6, a tet(O)-harbouring strain used as a control, was mated with the recipient strain. Ansary & Radu31 reported that Malaysian tetracycline-resistant isolates also co-transferred small plasmids (3.3 and 12.6 kb) with a larger plasmid (78 kb) by conjugation.
Transfer of tetracycline resistance between the C. jejuni strains under study, control strain JC6, and the remaining C. jejuni and C. coli recipient strains could not be demonstrated. The six strains under study and control strain JC6 that transferred tetracycline resistance to recipient strain 887 did not transfer resistance to these recipient strains. Of these recipient strains, one strain was plasmid free and two strains harboured plasmids (Table 1). Failure of tetracycline resistance plasmids to transfer resistance has been reported by Bacon et al.21 and Sagara et al.37 Plasmids harboured by some donor strains may be non-conjugative and non-mobilizable and therefore unable to transfer. Taylor et al.53 reported that tet(O) in a Canadian tetracycline-resistant C. coli isolate was located on a non-transmissible plasmid.
Barriers to conjugal transfer such as the host range of the plasmid, incompatibility between plasmids, the inability of plasmids to replicate in the recipient, strain specificity, and restriction modification systems may be responsible for lack of tetracycline resistance transfer in some strains. Incompatibility between plasmids may prevent transfer as many conjugative plasmids exhibit surface exclusion against plasmids of the same incompatibility group.54 Differences in restriction modification systems between strains may determine whether conjugal transfer takes place, as restriction modification systems enable cells to selectively destroy unmethylated foreign DNAs.55 Differences between strains have previously been observed in both campylobacters and H. pylori.52,5557 Predicted DNA restriction and DNA modification systems in C. jejuni strain NCTC 11168 appeared to be particularly variable, with many of these systems absent or highly divergent in other strains examined by Dorrell et al.57
Although conjugal transfer of tetracycline resistance between different species of Campylobacter has been reported by a number of investigators, with C. jejuni donor strains transferring tetracycline resistance plasmids to Campylobacter fetus subsp. fetus, C. coli and Campylobacter lari recipient strains,31,35,38,58 we did not observe such intraspecies transfer among our isolates.
In conclusion, we found high-level tetracycline resistance in Australian strains under study. Although 74% of strains analysed harboured plasmids, the tet(O) gene was mostly chromosomally encoded. Six C. jejuni strains transferred a tetracycline resistance plasmid to a C. jejuni recipient strain; however transfer to other C. jejuni and C. coli recipient strains did not occur.
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Acknowledgements |
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References |
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2 . Skirrow, M. (1977). Campylobacter enteritis; a new disease. British Medical Journal 2, 911.[ISI][Medline]
3 . Altekruse, S., Stern, N., Fields, P. et al. (1999). Campylobacter jejunian emerging foodborne pathogen. Emerging Infectious Diseases 5, 2835.[ISI][Medline]
4 . Frost, J. (2001). Current epidemiological issues in human campylobacteriosis. Journal of Applied Microbiology 90, Suppl., 8595.[CrossRef]
5 . Communicable Diseases Network Australia. (1999). National Notifiable Diseases Surveillance System Annual Report 1998. Communicable Diseases Intelligence 23, 28390.
6 . Ketley, J. (1995). Virulence of Campylobacter spp.: a molecular genetic approach. Journal of Medical Microbiology 42, 31227.[ISI][Medline]
7 . Blaser, M. (1997). Epidemiologic and clinical features of Campylobacter jejuni infections. Journal of Infectious Diseases 176, Suppl. 2, 1035.[ISI][Medline]
8 . Hudson, J., Nicol, C., Wright, J. et al. (1999). Seasonal variation of Campylobacter types from human cases, veterinary cases, raw chicken, milk and water. Journal of Applied Microbiology 87, 11524.[CrossRef][ISI][Medline]
9
.
Piddock, L., Ricci, V., Stanley, K. et al. (2000). Activity of antibiotics used in human medicine for Campylobacter jejuni isolated from farm animals and their environment in Lancashire, UK. Journal of Antimicrobial Chemotherapy 46, 3036.
10
.
Gaudreau, C. & Gilbert, H. (2003). Antimicrobial resistance of Campylobacter jejuni subsp. jejuni strains isolated from humans in 1998 to 2001 in Montreal, Canada. Antimicrobial Agents and Chemotherapy 47, 20279.
11
.
Saenz, Y., Zarazaga, M., Lantero, M. et al. (2000). Antibiotic resistance in Campylobacter strains isolated from animals, foods and humans in Spain in 19971998. Antimicrobial Agents and Chemotherapy 44, 26771.
12 . Murphy, G., Echeverria, P., Jackson, L. et al. (1996). Ciprofloxacin- and azithromycin-resistant Campylobacter causing traveller's diarrhoea in U.S. troops deployed in Thailand in 1994. Clinical Infectious Diseases 22, 8689.[ISI][Medline]
13 . Piddock, L. (1995). Quinolone resistance and Campylobacter spp. Antimicrobial Agents and Chemotherapy 36, 8918.
14 . Nachamkin, I., Engberg, J. & Aarestrup, F. (2000). Diagnosis and antimicrobial susceptibility of Campylobacter species. In Campylobacter, 2nd edn (Nachamkin, I. & Blaser, M., Eds), pp. 4566. American Society for Microbiology, Washington, DC, USA.
15 . Taylor, D. & Courvalin, P. (1988). Mechanisms of antibiotic resistance in Campylobacter spp. Antimicrobial Agents and Chemotherapy 32, 110712.[ISI][Medline]
16
.
Gibreel, A., Tracz, D., Nonaka, L. et al. (2004). Incidence of antibiotic resistance in Campylobacter jejuni isolated in Alberta, Canada, from 1999 to 2002, with special reference to tet(O)-mediated tetracycline resistance. Antimicrobial Agents and Chemotherapy 48, 344250.
17 . Lee, C.-Y., Tai, C.-L., Lin, S.-C. et al. (1994). Occurrence of plasmids and tetracycline resistance among Campylobacter jejuni and Campylobacter coli isolated from whole market chickens and clinical samples. International Journal of Food Microbiology 24, 16170.[CrossRef][ISI][Medline]
18 . Sougakoff, W., Papadopoulou, B., Nordmann, P. et al. (1987). Nucleotide sequence and distribution of gene tet(O) encoding tetracycline resistance in Campylobacter coli. FEMS Microbiology Letters 44, 1539.[CrossRef][ISI]
19 . Manavathu, E., Hiratsuka, K. & Taylor, D. (1988). Nucleotide sequence analysis and expression of tetracycline-resistance gene from Campylobacter jejuni. Gene 62, 1726.[CrossRef][ISI][Medline]
20
.
Connell, S., Tracz, D., Neirhaus, K. H. et al. (2003). Ribosomal protection proteins and their mechanism of tetracycline resistance. Antimicrobial Agents and Chemotherapy 47, 367581.
21
.
Bacon, D., Alm, R., Burr, D. et al. (2000). Involvement of a plasmid in virulence of Campylobacter jejuni 81-176. Infection and Immunity 68, 438490.
22 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA, USA.
23 . Tee, W., Mijch, A., Wright, E. et al. (1995). Emergence of multidrug resistance in Campylobacter jejuni isolates from three patients infected with human immunodeficiency virus. Clinical Infectious Diseases 21, 6348.[ISI][Medline]
24 . Aarestrup, F., Neilsen, E., Madsen, M. et al. (1997). Antimicrobial susceptibility patterns of thermophillic Campylobacter spp. from humans, pigs, cattle and broilers in Denmark. Antimicrobial Agents and Chemotherapy 41, 224450.[Abstract]
25 . Sambrook, J. & Russell, D. (2001). Molecular Cloning: A Laboratory Handbook, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
26 . Korolik, V., Moorthy, L. & Coloe, P. (1995). Differentiation of Campylobacter jejuni and Campylobacter coli strains by using restriction endonuclease DNA profiles and DNA fragment polymorphisms. Journal of Clinical Microbiology 33, 113640.[Abstract]
27 . Korolik, V., Friendship, D., Peduru Hewa, T. et al. (2001). Specific identification, grouping and differentiation of Campylobacter jejuni among thermophilic campylobacters using multiplex PCR. Epidemiology and Infection 127, 15.[CrossRef][ISI][Medline]
28
.
Wassenaar, T. & Newell, D. (2000). Genotyping of Campylobacter spp. Applied and Environmental Microbiology 66, 19.
29 . Southern, E. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 50317.[CrossRef][ISI][Medline]
30 . Taylor, D., de Grandis, S., Karmali, M. et al. (1981). Transmissible plasmids from Campylobacter jejuni. Antimicrobial Agents and Chemotherapy 19, 8315.[ISI][Medline]
31 . Ansary, A. & Radu, S. (1992). Conjugal transfer of antibiotic resistances and plasmids from Campylobacter jejuni clinical isolates. FEMS Microbiology Letters 91, 1258.[CrossRef][ISI]
32 . Schwartz, D., Goossens, H., Levy, J. et al. (1993). Plasmid profiles and antimicrobial susceptibility of Campylobacter jejuni isolated from Israeli children with diarrhoea. Zentralblatt fur Bakteriologie 279, 36876.[ISI][Medline]
33
.
Pumbwe, L., Randall, L., Woodward, M. et al. (2004). Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiple-antibiotic-resistant Campylobacter jejuni. Journal of Antimicrobial Chemotherapy 54, 3417.
34
.
Stanton, T. & Humphrey, S. (2003). Isolation of tetracycline-resistant Megasphaera elsdenii strains with novel mosaic gene combinations of tet(O) and tet(W) from swine. Applied and Environmental Microbiology 69, 387482.
35 . Taylor, D., Garner, R. & Allan, B. (1983). Characterisation of tetracycline resistance plasmids from Campylobacter jejuni and Campylobacter coli. Antimicrobial Agents and Chemotherapy 24, 9305.[ISI][Medline]
36 . Cabrita, J., Rodrigues, J., Braganca, F. et al. (1992). Prevalence, biotypes, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from Northern Portugal. Journal of Applied Bacteriology 73, 27985.[ISI][Medline]
37 . Sagara, H., Mochizuki, A., Okamura, N. et al. (1987). Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli with special reference to plasmid profiles of Japanese clinical isolates. Antimicrobial Agents and Chemotherapy 31, 7139.[ISI][Medline]
38 . Tenover, F., Williams, S., Gordon, K. et al. (1985). Survey of plasmids and resistance factors in Campylobacter jejuni and Campylobacter coli. Antimicrobial Agents and Chemotherapy 27, 3741.[ISI][Medline]
39 . Bossinger, T., Blevins, W., Heron, J. et al. (1990). Plasmid profiles of six species of Campylobacter from human beings, swine and sheep. American Journal of Veterinary Research 51, 71822.[ISI][Medline]
40 . Taylor, D. (1992). Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli to tetracycline, chloramphenicol and erythromycin. In Campylobacter jejuni: Current Status and Future Trends (Nachamkin, I., Blaser, M. & Tompkins, L., Eds), pp. 7486. American Society for Microbiology, Washington, DC, USA.
41 . Walker, R., Caldwell, M., Lee, E. et al. (1986). Pathophysiology of Campylobacter enteritis. Microbiological Reviews 50, 8194.[ISI]
42
.
Richardson, P. & Park, S. (1997). Integration of heterologous plasmid DNA into multiple sites on the genome of Campylobacter coli following natural transformation. Journal of Bacteriology 179, 180912.
43 . Ng, L.-K., Stiles, M. & Taylor, D. (1987). DNA probes for identification of tetracycline resistance genes in Campylobacter species isolated from swine and cattle. Antimicrobial Agents and Chemotherapy 31, 166974.[ISI][Medline]
44 . Gibreel, A., Skold, O. & Taylor, D. (2004). Characterisation of plasmid-mediated aphA-3 kanamycin resistance in Campylobacter jejuni. Microbial Drug Resistance 10, 98105.[CrossRef][ISI][Medline]
45 . Parkhill, J., Wren, B., Mungall, K. et al. (2000). The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403, 6658.[CrossRef][ISI][Medline]
46
.
Gibreel, A. & Skold, O. (1998). High-level resistance to trimethoprim in clinical isolates of Campylobacter jejuni by acquisition of foreign genes (dfr1 and dfr9) expressing drug-insensitive dihydrofolate reductases. Antimicrobial Agents and Chemotherapy 42, 305964.
47
.
Bacon, D., Alm, R., Hu, L. et al. (2002). DNA sequence and mutational analysis of the pVir plasmid of Campylobacter jejuni 81-176. Infection and Immunity 70, 624250.
48 . Velazquez, J., Jimenez, A., Chomon, B. et al. (1995). Incidence and transmission of antibiotic resistance in Campylobacter jejuni and Campylobacter coli. Antimicrobial Agents and Chemotherapy 35, 1738.
49 . Prasad, K., Mathur, S., Dhole, T. et al. (1994). Antimicrobial susceptibility and plasmid analysis of Campylobacter jejuni isolated from diarrhoeal patients and healthy chickens in northern India. Journal of Diarrhoeal Disease Research 12, 2703.[ISI]
50 . Avrain, L., Vernozy-Roland, C. & Kempf, I. (2004). Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens. Journal of Applied Microbiology 97, 13440.[CrossRef][ISI][Medline]
51 . Wang, Y. & Taylor, D. (1990). Natural transformation in Campylobacter species. Journal of Bacteriology 172, 94955.[ISI][Medline]
52 . Ando, T., Xu, Q., Torres, M. et al. (2000). Restriction-modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer. Molecular Microbiology 37, 105265.[CrossRef][ISI][Medline]
53 . Taylor, D., Yan, W., Ng, L. et al. (1988). Genetic characterisation of kanamycin resistance in Campylobacter coli. Annales de l'Institut Pasteur Microbiology 139, 66576.[CrossRef]
54 . Van der Hoeven, N. (1985). Evolution of bacterial surface exclusion against incompatible plasmids. Journal of Theoretical Biology 117, 43152.[ISI][Medline]
55 . Edmonds, P., Hall, B., Edwards, W. et al. (1992). Presence of methylated adenine in GATC sequences in chromosomal DNA from Campylobacter species. Journal of Bacteriology 174, 81567.[Abstract]
56 . Ahmed, I., Manning, G., Wassenaar, T. et al. (2002). Identification of genetic differences between two Campylobacter jejuni strains with different colonization potentials. Microbiology 148, 120312.[ISI][Medline]
57
.
Dorrell, N., Mangan, J., Laing, K. et al. (2001). Whole genome comparison of Campylobacter jejuni human isolates using a low-cost microarray reveals extensive genetic diversity. Genome Research 11, 170615.
58 . Taylor, D., Chang, N., Garner, R. et al. (1986). Incidence of antibiotic resistance and characterisation of plasmids in Campylobacter jejuni strains from clinical sources in Alberta, Canada. Canadian Journal of Microbiology 32, 2832.[ISI][Medline]