1 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany; 2 Department of Obstetrics, Reproduction and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium; 3 Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium
Received 28 April 2005; returned 26 May 2005; revised 27 May 2005; accepted 27 May 2005
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Abstract |
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Methods: Mannheimia and Pasteurella isolates were investigated for their MICs of tetracycline and their plasmid content. Identification of tet genes was achieved by PCR. Plasmids mediating tetracycline resistance were identified by transformation and hybridization experiments. Plasmid pCCK3259 from Mannheimia haemolytica was sequenced completely and analysed for its structure and organization.
Results: All tetracycline-resistant isolates carried the gene tet(L) either on plasmids or on the chromosome. Two M. haemolytica isolates and one Mannheimia glucosida isolate harboured a common 5.3 kb tet(L) plasmid, designated pCCK3259. This plasmid was similar to the tet(B)-carrying tetracycline resistance plasmid pHS-Tet from Haemophilus parasuis and the streptomycin/spectinomycin resistance plasmid pCCK647 from Pasteurella multocida in the parts coding for mobilization functions. The tet(L) gene was closely related to that of the Geobacillus stearothermophilus plasmid pTB19. However, the translational attenuator responsible for the tetracycline-inducible expression of tet(L) was missing in plasmid pCCK3259. A recombination site was identified downstream of tet(L), which might explain the integration of the tet(L) gene region into a basic pCCK3259 replicon.
Conclusion: A tet(L) gene was shown for the first time to be responsible for tetracycline resistance in Mannheimia and Pasteurella isolates. This report demonstrates a lateral transfer of a tetracycline efflux gene in Gram-negative bovine respiratory tract pathogens, probably originating from Gram-positive bacteria.
Keywords: respiratory tract pathogens , antimicrobial resistance , gene transfer , recombination , cattle
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Introduction |
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To date, very little is known about the presence and functional activity of tetracycline efflux genes of Gram-positive bacteria in Gram-negative pathogens. In this study, we investigated six naturally occurring, tet(L)-carrying isolates of the bovine respiratory tract pathogens Mannheimia haemolytica, Mannheimia glucosida, and Pasteurella multocida with particular reference to the plasmid location of the tet(L) gene and its genetic environment.
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Materials and methods |
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Results and discussion |
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The six isolates included in this study originated from different calves on the same farm and included three M. haemolytica, one M. glucosida and two P. multocida isolates. Their MICs of tetracycline ranged between 16 and 64 mg/L (Table 1). PCR screening for the tet genes of classes B, G, H and M previously detected among bovine Pasteurella and Mannheimia isolates7 revealed no amplicons. Further PCR analysis for tet genes of classes A, C, D, E and O also failed to yield the expected amplicons. However, a PCR assay for the simultaneous detection of tet(K) and tet(L)10 revealed the presence of an amplicon of 1.05 kb in all six isolates. Restriction analysis of the amplicon with either ClaI or BclIrestriction sites for both enzymes are located in tet(L), but not in tet(K)resulted in the tet(L)-specific fragments of
0.29 and 0.76 kb for ClaI and 0.08 and 0.97 kb for BclI for all isolates tested. One such amplicon from a M. haemolytica isolate was sequenced and proved to be 1048 bp in size. It showed a 1 bp difference to the corresponding part of the tet(L) sequence of Geobacillus stearothermophilus (accession no. M63891). Plasmid location of the tet(L) gene was confirmed in two M. haemolytica and the single M. glucosida isolate by electrotransformation into a P. multocida recipient strain and by hybridization of plasmid profiles with the specific tet(L) gene probe. Susceptibility testing of the transformants confirmed that these plasmids conferred only tetracycline resistance (Table 1). The tet(L) genes in the remaining M. haemolytica and P. multocida isolates were assumed to be located on the chromosome. Macrorestriction analysis revealed that the SmaI patterns of the two P. multocida isolates differed by three bands (data not shown). The two M. haemolytica isolates that harboured a tet(L)-bearing plasmid exhibited the same SmaI pattern whereas the third M. haemolytica isolate and the M. glucosida isolate showed unique fragment patterns (Table 1).
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The tet(L)-carrying plasmids of the two M. haemolytica and the M. glucosida isolates were subjected to restriction analysis with 19 different endonucleases. Since the three plasmids proved to be indistinguishable by their restriction patterns, a common designation, pCCK3259, was chosen. The plasmid of one of the M. haemolytica isolates was sequenced completely and proved to be 5317 bp in size. A search for open reading frames led to the detection of three reading frames for mobilization proteins (Figure 1a). The mobC reading frame coded for a protein of 102 amino acids which showed 95% and 91% identity to the MobC proteins from the P. multocida plasmid pCCK647 (accession no. AJ884726)11 and the Haemophilus parasuis plasmid pHS-Tet (accession no. AY862435),12 respectively. The largest reading frame coded for a 468 amino acid MobA protein which revealed 89% identity to MobA from pHS-Tet and 79% identity to MobA from pCCK647. Within the mobA gene, there was a reading frame for a 160 amino acid MobB protein which showed 88% and 86% identity to the MobB proteins from plasmids pHS-Tet and pCCK647, respectively.
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The results of this study showed that tet(L) genes are also present in Mannheimia and Pasteurella isolates. Even if they do not confer high-level tetracycline resistance as in the Gram-positive hosts, these genes are expressed in Mannheimia and Pasteurelladespite the lack of the translational attenuatorand allow the bacteria to survive in the presence of the tetracycline levels achievable by application of tetracyclines to the calves.13 The results of macrorestriction analysis strongly suggested that tet(L)-carrying Pasteurella and Mannheimia strains are spread between the calves within this specific farm. The detection of plasmid pCCK3259 in strains of M. haemolytica and M. glucosida also confirmed horizontal transfer of this plasmid between members of different Mannheimia species.
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Acknowledgements |
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References |
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