tet(L)-mediated tetracycline resistance in bovine Mannheimia and Pasteurella isolates

Abstract

Objectives: Tetracycline-resistant Mannheimia and Pasteurella isolates, which were negative for the tetracycline resistance genes (tet) commonly detected among these bacteria, were investigated for other tet genes present and their location.

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.

Introduction

The tetracycline resistance gene tet(L) codes for a membrane-associated efflux protein composed of 14 transmembrane segments. It has been detected on plasmids and on the chromosome of members of various Gram-positive genera.¹ Analysis of tet(L)-carrying plasmids from Bacillus, Staphylococcus, Streptococcus and Enterococcus revealed that these plasmids varied distinctly in their sizes and occasionally also carried additional resistance genes. This observation supported the assumption that interplasmid recombination events may play an important role in the spread of the tet(L) gene. A model for such a recombination event has already been described and supported by sequence data.² Expression of tet(L) is usually inducible via translational attenuation.³ The corresponding regulatory region upstream of the tet(L) gene consists of a reading frame for a small peptide of 20 amino acids and three pairs of inverted repeated sequences which can form different mRNA secondary structures in the absence or presence of tetracyclines, thereby allowing or preventing the translation of the tet(L) transcripts.³ In contrast to numerous reports on the occurrence of this gene in Gram-positive bacteria, it has rarely been detected in Gram-negative bacteria. So far, its occurrence has been reported in Fusobacterium and Veillonella,¹ and more recently also in Morganella morganii⁴ and Actinobacillus pleuropneumoniae.⁵ Studies on the expression of a cloned tet(L) gene from Staphylococcus hyicus revealed that this gene was active in Escherichia coli, although the MICs were distinctly lower than in the staphylococcal host and induction was not detectable.³

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.

Materials and methods

The six Pasteurella and Mannheimia isolates were obtained from nasal swabs of 6-week-old male Holstein Friesian calves from a veal calf farm in Belgium in 2003. The calves were housed within one stable. They had received a prophylactic medication consisting of colistin (polymyxin E), oxytetracycline and flumequin for 13 days. Immediately thereafter, the calves received an oral treatment with sulphonamides and trimethoprim for another 4 days. The application of antimicrobial agents was stopped ∼3 weeks before sampling. Species identification was performed as previously described.⁶ Resistance phenotypes were determined by disc diffusion and MICs of tetracycline by broth macrodilution according to the NCCLS document M31-A2 with Staphylococcus aureus ATCC 29213 as quality control strain.⁷ PCR analyses for the tet genes of hybridization classes A–E, G, H, K, L, M and O followed previously described protocols.^4,8–10 PCR products were confirmed by restriction analysis. Plasmid preparation by alkaline lysis and electrotransformation experiments into P. multocida P4000 were conducted as previously described.⁴ Transformants were selected on blood agar plates (Oxoid, Wesel, Germany; 5% v/v sheep blood) supplemented with 5–10 mg/L tetracycline. Plasmid DNA obtained from the transformants was subjected to restriction mapping. Sequence analyses were started with four oligonucleotide primers derived from the tet(L) gene sequence from S. hyicus (accession no. X60828). Another six primers designed from the sequences obtained with the aforementioned primers were used to complete the sequencing of the tet(L) plasmid pCCK3259 (MWG, Ebersberg, Germany). Sequence comparisons were performed with the BLAST programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/; last accessed 23 April 2005) and with the ORF finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; last accessed 23 January 2005). The nucleotide sequence of plasmid pCCK3259 has been deposited in the European Molecular Biology Laboratory (EMBL) database under accession number AJ966516. Macrorestriction analysis with SmaI and hybridization studies with a digoxigenin-labelled internal 1046 bp BclI fragment of the tet(L) gene from S. hyicus⁴ followed previously described protocols.⁸

Results and discussion

Identification and location of tet genes

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 BclI—restriction 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).

Structure and organization of the tet(L)-carrying plasmid pCCK3259

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.

Further downstream of mobA, the reading frame for a 458 amino acid tetracycline efflux protein of hybridization class L was detected (Figure 1a). The TetL protein was indistinguishable from most TetL proteins deposited in the databases, including those from G. stearothermophilus plasmid pTB19 (accession no. M63891), Bacillus cereus plasmid pBC16 (accession no. NP_043524), Streptococcus agalactiae plasmid pLS1 (accession no. NP_040422), or Enterococcus faecalis plasmid pAMα1 (accession no. NP_863350). TetL from pCCK3259 differed by one amino acid from the TetL proteins of Enterococcus faecium (accession no. AY081910) and G. stearothermophilus plasmid pTHT15 (accession no. M11036) and by eight amino acid substitutions from that of the S. hyicus plasmid pSTE1.⁴ The entire tet(L) upstream part in pCCK3259 (positions 2973–3483) differed distinctly from that of the corresponding tet(L) upstream sequences known from Gram-positive bacteria and showed no homology to sequences deposited in the databases. The translational attenuator usually present in the tet(L) upstream region was lost completely in the M. haemolytica plasmid pCCK3259. However, a putative promoter with a −35 region (TAGACA, positions 3368–3373), a −10 region (TTTAAT, positions 3395–3400) and G at position 3408 as a potential start for transcription of tet(L) was detected in this region. Moreover, a suitable ribosome binding site, AGAAGG (positions 3475–3480), was located 6 bp upstream of the tet(L) translational start codon GTG. Homology to known tet(L) genes started 3 bp upstream of this start codon. Downstream of the tet(L) gene, a Rho-independent transcriptional terminator consisting of a pair of imperfect inverted repeats of 12 bp and followed by a stretch of seven thymine residues⁴ was detected. Homology to known tet(L) downstream sequences ended 193 bp downstream of the tet(L) stop codon at the sequence TTTTATTC (positions 5049–5056). This sequence was identified by sequence comparisons as a potential recombination site (Figure 1b) which might have played a role in the integration of the tet(L) gene area into a basic pCCK3259 replicon.

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 Pasteurella—despite the lack of the translational attenuator—and 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.

We thank Vera Nöding and Roswitha Becker for excellent technical assistance.

References

Author notes

1Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany; 2Department of Obstetrics, Reproduction and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium; 3Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium