Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystrasse 10, 31535 Neustadt-Mariensee, Germany
Received 25 May 2005; returned 23 June 2005; revised 24 June 2005; accepted 25 June 2005
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Abstract |
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Methods: Six B. bronchiseptica isolates with elevated MICs of trimethoprim were investigated by PCR for the presence of trimethoprim resistance genes and their association with class 1 integrons. The amplicons obtained were cloned and sequenced. Plasmid localization of these integrons was confirmed by transformation and conjugation. Isolates carrying the same integron were compared for their genetic relatedness by XbaI and SpeI pulsed-field gel electrophoresis (PFGE).
Results: Five B. bronchiseptica isolates carried a class 1 integron with two gene cassettes, one carrying the trimethoprim resistance gene dfrA1 and the other the chloramphenicol resistance gene catB3. This integron was present on a common conjugative plasmid in four of the five isolates and on the chromosome in the remaining isolate. All five B. bronchiseptica isolates proved to be related on the basis of their PFGE patterns. Another isolate had a class 1 integron with a dfrB1 and a catB2 cassette on a structurally different conjugative plasmid. The sulphonamide resistance gene sul1 was detected in the 3'-conserved segment of both types of integrons.
Conclusions: This is the first report of trimethoprim, chloramphenicol and sulphonamide resistance genes and class 1 integrons in B. bronchiseptica isolates.
Keywords: dfrA1 , dfrB1 , catB2 , catB3 , sul1 , class 1 integrons , conjugation , respiratory tract infections
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Introduction |
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Materials and methods |
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The six isolates included in this study were obtained during 20002003 from diagnostic laboratories in Germany on the basis of one isolate per herd. All isolates were from pigs suffering from respiratory tract infections.7 The initial susceptibility testing was performed by broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI; formerly known as the NCCLS) document M31-A2.8 Since the highest test concentration of trimethoprim in the microtitre plate panels used in the previous study was 64 mg/L, the isolates that grew at 64 mg/L were additionally tested for growth in the presence of 128 and 256 mg/L trimethoprim by broth macrodilution with Escherichia coli ATCC 25922 as a quality control strain.8 Susceptibility testing of the transformants and transconjugants was performed by either broth dilution or disc diffusion.8
DNA preparation and PCR analysis
Isolation of plasmids and whole-cell DNA followed standard protocols.9 To detect the most common trimethoprim resistance genes by PCR, four recently described primer sets each of which allowed the amplification of two to three closely related dfrA or dfrB genes were used.10 Integrase genes of classes 1 and 2, gene cassettes and sulphonamide resistance genes were detected by previously reported PCR assays.1016 All primers used are listed in Table 1. The amplicons obtained were confirmed and compared by restriction analysis. To confirm the linkage between the sequenced variable part of the class 1 integrons, the integrase and the sulphonamide resistance gene sul1, two combinations of primer pairs were used: (i) the 5'-CS primer for class 1 integrons was combined with the sul1 reverse primer and used at an annealing temperature of 55°C; and (ii) the forward primer for the class 1 integrase gene was used with the reverse primer for the detected dfrA or dfrB gene at an annealing temperature of 50°C.
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Conjugation experiments were performed by filter mating with the rifampicin-resistant E. coli HK225 as recipient strain.17 Transconjugants were selected on LB agar plates containing rifampicin (100 mg/L) and trimethoprim (20 mg/L). A donor:recipient ratio of 1:5 was used in this approach. For transformation, competent E. coli JM109 cells (Stratagene, Amsterdam, The Netherlands) were used and transformants were selected on LB agar supplemented with 20 mg/L trimethoprim.18 Amplicons representing the variable parts of the class 1 integrons were cloned into pCR Blunt II TOPO and transformed into E. coli TOP10 cells (Invitrogen, Groningen, The Netherlands).18 The complete sequence of both amplicons was determined by primer walking. Sequence comparisons were carried out using the BLAST® programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/; last accessed 25 May 2005) and with the ORF finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; last accessed 25 May 2005). The sequences of the amplicons have been deposited in the EMBL database under accession numbers AJ844287 and AJ879564.
Pulsed-field gel electrophoresis
For pulsed-field gel electrophoresis (PFGE) with XbaI and SpeI, a standard protocol was used.18 Whole-cell DNA of Staphylococcus aureus 8325 digested with SmaI and of Salmonella Typhimurium LT2 digested with XbaI served as size markers. PFGE was performed in a CHEF DR III system (Bio-Rad, Munich, Germany) using 0.5x TrisborateEDTA buffer as running buffer and 5.6 V/cm. The pulse times were increased from 7 to 20 s for the first 11 h, and from 30 to 50 s for the following 13 h.
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Results |
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Of the 349 B. bronchiseptica isolates originally tested, six isolates proved to be high-level resistant to trimethoprim with MICs of 128 mg/L (one isolate) or 512 mg/L (five isolates). These isolates also had high MICs of 16/30464/1216 mg/L for the combination trimethoprim/sulfamethoxazole (1:19), suggesting that all six isolates were also resistant to sulphonamides.7 Moreover, the six isolates also exhibited elevated MICs of 1632 mg/L for chloramphenicol, whereas their florfenicol MICs were
2 mg/L.7 The PCR assay with consensus primers for the simultaneous detection of the trimethoprim resistance genes dfrA1-dfrA15-dfrA16 yielded the expected amplicon of 414 bp in the five B. bronchiseptica isolates with trimethoprim MICs of
512 mg/L. ClaI digestion of the amplicon was used to discriminate between these three dfrA genes since there was one ClaI site in dfrA1, two ClaI sites in dfrA15 and no ClaI site in the amplicon specific for dfrA16. ClaI fragments of
0.26 and
0.15 kb, which are indicative of dfrA1, were detected in all five amplicons. The remaining B. bronchiseptica isolate that exhibited a trimethoprim MIC of 128 mg/L yielded an amplicon of 205 bp, which was obtained with consensus primers for the genes dfrB1 and dfrB2. Owing to its small size, this amplicon was not subjected to restriction analysis, but was sequenced.
Characterization of class 1 integrons and associated gene cassettes
Since the genes dfrA1 and dfrB1/dfrB2 have previously been found on gene cassettes located in class 1 or class 2 integrons, the six B. bronchiseptica isolates were investigated for the presence of class 1 and class 2 integrons and associated gene cassettes. All six isolates carried a class 1 integron, but were negative for class 2 integrons. Amplicons of 450 and 840 bp, which were specific for the integrase gene and the sulphonamide resistance gene sul1 of class 1 integrons, respectively, were detected by PCR. In addition, two different sized amplicons were obtained by PCR analysis of the variable part located between the 5'-CS and the 3'-CS region.
Each of the five isolates with trimethoprim MICs of 512 mg/L yielded an amplicon of 1445 bp, which comprised two gene cassettes flanked by short sequences of the 5'-CS and 3'-CS regions. Restriction analysis of all five amplicons with ClaI and BclI revealed the same fragment patterns. Therefore, one of the amplicons, namely that of B. bronchiseptica isolate 668, was chosen for sequence analysis. It showed that this integron harboured a first gene cassette of 577 bp, which contained the trimethoprim resistance gene dfrA1, and a second cassette of 715 bp with the chloramphenicol resistance gene catB3. The dfrA1 gene codes for a trimethoprim-resistant class A dihydrofolate reductase consisting of 157 amino acids. The corresponding 59-base element was 95 bp in size. The gene catB3 codes for a type B chloramphenicol acetyltransferase (CAT) of 210 amino acids. The 59-base element of the catB3 cassette was 60 bp in size (Figure 1a).
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Localization and transferability of the integrons
The integron harbouring the dfrA1-catB3 gene cassettes was located on plasmids of 24 kb in four of the five isolates. Since the fifth isolate was plasmid-free, it was assumed that the integron was located in the chromosomal DNA. Comparative restriction analysis using the endonucleases DraI, PvuI, PvuII, ClaI and HindIII showed indistinguishable fragment patterns consisting of two to four fragments among the four plasmids. Therefore, a common designation, pKBB668, was chosen for this type of plasmid. Plasmid pKBB668 was transferred into E. coli JM109, where it expressed its resistance properties. Conjugation experiments with E. coli HK225 as recipient confirmed that plasmid pKBB668 was conjugative and transferred from B. bronchiseptica to E. coli at a frequency of
105 per recipient. The presence of the class 1 integron and its gene cassettes was confirmed by PCR using plasmid DNA from E. coli JM109::pKBB668 transformants and E. coli HK225::pKBB668 transconjugants. Plasmid pKBB668 mediated no resistance properties other than those associated with the class 1 integron. The integron with the dfrB1-catB2 gene cassettes was also located on a conjugative plasmid, designated pKBB958. This plasmid was distinctly larger and structurally different from pKBB668. In addition to the integron-associated resistance properties, the 38 kb plasmid pKBB958 also mediated tetracycline resistance. Again, all resistance properties were expressed in E. coli JM109 transformants or E. coli HK255 transconjugants. Plasmid pKBB958 showed conjugal transfer into E. coli at a high frequency of 103 per recipient.
Genomic relatedness of dfrA1-catB3-carrying B. bronchiseptica isolates
To assess the genomic relatedness of the five isolates that harboured the integron with the dfrA1-catB3 gene cassettes, PFGE was conducted. The results confirmed that all five B. bronchiseptica isolates were related, with isolates 2, 3 and 5 being indistinguishable by their XbaI patterns and isolate 4 differing by two bands. Isolate 1 differed from the others by four bands (Figure 2). Upon SpeI analysis (data not shown) isolates 2, 3 and 5 exhibited the same pattern, whereas isolates 1 and 4 had an additional band. Comparison of these fragment patterns with those of unrelated B. bronchiseptica isolates from pigs and that of the B. bronchiseptica type strain NCTC452 revealed differences of at least eight fragments.
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Discussion |
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A closer look at the dfrB1 cassette detected in the present study revealed that this cassette was 74 bp shorter than the prototype dfrB1 cassette (accession no. U36276).20 This difference in size was based on the loss of a 72 bp tandem duplication and two single base pairs in the part upstream of the dfrB1 gene in the respective cassette from B. bronchiseptica. The dfrB1 cassette described in the present study was indistinguishable from the dfrB1 cassettes found on plasmids pMVH202 or pSp39. Surprisingly, the DfrB1 proteins of pMVH202 and pSp39 were described to be 97 amino acids in size, while that of DfrB1 from B. bronchiseptica was found to be 78 amino acids. This difference is most likely the result of a search for the largest possible open reading frame within the dfrB1 cassette. In this case, an ATG codon (position 111113 in Figure 1b) was recognized as the putative translational start codon of the dfrB1 gene. However, the intact DfrB1 protein from E. coli plasmid R67 had been purified and shown by protein sequencing to be 78 amino acids in size.21 Hence, the start codon at positions 168170 (Figure 1b) is most likely the true translational start codon of the dfrB1 gene.
Since the same type of plasmid-borne class 1 integron was detected in isolates from different farms in the Northern part of Germany, there are two general possibilities: spread of a resistant clone or horizontal dissemination of the plasmid-borne integron into members of different clonal lineages. PFGE strongly suggested a clonal relationship between the five isolates rather than a horizontal spread of the conjugative plasmid pKBB668 between unrelated B. bronchiseptica isolates. The dissemination of closely related B. bronchiseptica isolates within a particular geographic area might be explained by the purchase of piglets already carrying these resistant B. bronchiseptica isolates and originating from the same pig breeder by different commercial pig growers. Another possibility is the transmission via living and non-living vectors. Since three of the farms from which the isolates in question were obtained were located <100 km apart from each other and known to be under support of the same veterinarian, a farm-to-farm spread of the B. bronchiseptica isolates by the veterinarian cannot be excluded. Exchange of pigs between these herds as well as close contacts between people working on these farms could not be confirmed.
Among the antimicrobial agents licensed for the control of bacteria involved in porcine respiratory diseases and atrophic rhinitis, older and comparatively cheaper antimicrobials, such as tetracyclines and the combination trimethoprim/sulphonamides, are often preferred over newer and more expensive agents such as third-generation cephalosporins, tilmicosin or florfenicol. This might explain why plasmids such as pKBB668 and pKBB958, which mediate resistance to trimethoprim, sulphonamides and chloramphenicol (and in the case of pKBB958 also to tetracyclines), are acquired by and stably maintained in B. bronchiseptica. The observation that a gene cassette for chloramphenicol resistancean antimicrobial agent that was banned from use in food animalsis still present in both types of integrons might be explained by co-selection in the presence of selective pressure imposed by the use of sulphonamides and trimethoprim.
In conclusion, the data presented in this study underline that there is a potential resistance gene flow between porcine respiratory tract pathogens and enteric and environmental bacteria, which also includes class 1 integrons and their associated gene cassettes.
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Acknowledgements |
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