The extent of contribution from Kristina Kadlec to the article is evaluated according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: B
2. Performing of the experimental part of the study: C
3. Analysis of the experiments: C
4. Presentation and discussion of the study in article form: C
Tetracycline resistance chapter 4
Sir,
Bordetella bronchiseptica is often involved in respiratory tract infections of farm animals (i.e.
pigs and rabbits) and pets (i.e. cats and dogs). Infections in humans are seen mainly in older and immunocompromised patients. Although antimicrobial agents are commonly applied to control B. bronchiseptica infections,1 little is known about the molecular basis of antimicrobial resistance in these bacteria. Although tetracycline resistance in B.
bronchiseptica was already described in 1981 to be associated with a non-conjugative plasmid,2 it took until 1997 when the first and so far only tetracycline resistance gene, tet(C), was identified in B. bronchiseptica isolates from cats.3 In the present study, we investigated two isolates from pigs for the molecular basis of tetracycline resistance with particular reference to the type of tet gene present, its location on a mobile genetic element and the possibility of horizontal transfer.
A recent survey revealed that tetracycline resistance was seen in < 1% of 349 porcine B.
bronchiseptica isolates.1 A tetracycline MIC value of 64 mg/L had been reported for the two isolates, nos. 958 and V4037/8.1 PCR screening for tet genes, plasmid profiling, conjugation, cloning and sequencing followed previously described protocols.4,5 PCR confirmed that both B. bronchiseptica isolates carried a tet gene of hybridization class A. Both tet(A)-carrying isolates were subjected to macrorestriction analysis.4 Their XbaI fragment patterns differed by ten bands and thus confirmed that the two B. bronchiseptica isolates were unrelated.
In isolate no. 958, the tet(A) gene was located on the conjugative 38 kb plasmid pKBB958, previously described to harbour also a class 1 integron with genes for resistance to trimethoprim, chloramphenicol and sulphonamides.4 Using tetracycline (20 mg/L) as selective agent, transfer of plasmid pKBB958 into Escherichia coli recipient strains HK225, JM109 and JM101 was achieved by conjugation and transformation. Restriction analysis showed that the tet(A) gene was located on a 16 kb XbaI fragment of which we sequenced a 4338 bp segment. Comparisons revealed that 3445 bp were homologous to the tet(A)-carrying prototype transposon Tn1721.6 The transposon Tn1721 consists of 11139 bp and is divided by two terminal and one central 38 bp repeat into two parts (Figure 1).6 Homology (>99%) started immediately downstream of the internal 38 bp repeat of Tn1721 and ended upstream of the truncated transposase gene ∆tnpA in the right-hand portion of the transposon (Figure 1).
While the part including tetR and tet(A) was indistinguishable from that of Tn1721 (accession
no. X61367), minor variations were detected in the non-coding regions. The sequences flanking this Tn1721-homologous part exhibited neither similarities to other sequences deposited in the databases, nor sequence features that might give a hint to the processes that led to the truncation of Tn1721.
In isolate no. V4037/8, the tet(A) gene was located on a non-conjugative plasmid of ~24 kb, designated pKBB4037, which conferred only tetracycline resistance. Cloning of restriction fragments of pKBB4037 produced an EcoRI self-ligand which replicated and conferred tetracycline resistance in E. coli JM101. Sequencing of this self-ligand revealed the presence of open reading frames for a resolvase Res, two partition proteins ParA1 and ParC and a plasmid replication protein Rep within the initial 4936 bp (Figure 1). The 295-amino-acid resolvase protein showed 82% identity to a putative plasmid-borne 283-amino-295-amino-acid resolvase from Pseudomonas aeruginosa (accession no. AAP22618). The 210-amino-acid ParA1 protein is 95% identical to ParA1 located on plasmid pXcB from Xanthomonas citri (AAO72130) while the 106-amino-acid ParC protein showed 55% identity to the 128-amino-acid plasmid-borne ParC proteins from P. aeruginosa (CAI46991) and Pseudomonas alcaligenes (AAD40336). The N-terminal 395 amino acids of the 488-amino-acid plasmid replication protein Rep exhibited 79% identity to a 452-amino-acid hypothetical protein from Nitrosomonas eutropha C71 (ZP_00671175). Lesser degrees of identity of 72 % and 59%
were seen with plasmid replication protein of P. aeruginosa (CAI46990) and Aeromonas hydrophila (ABD64829). The adjacent 5490 bp segment was virtually identical to Tn1721 (>99% homology) and included almost the entire right-hand portion of the transposon with the tetracycline resistance gene region, the truncated ∆tnpA gene, and the right terminal 38 bp repeat. A stretch of 739 bp which included most of the right terminal repeat of Tn1721 and the sequences downstream of the Tn1721-homologous segment closely resembled an internal segment of the transposase gene of Tn5393 (∆tnpA*). The nucleotide sequences of the tetracycline resistance gene region and their flanking areas of plasmids pKBB958 and pKBB4037 have been deposited in the EMBL database under accession nos. AM183165 and AJ877266.
Trimethoprim, chloramphenicol and sulphonamide resistancechap
Figure 1. Comparison of Tn1721 (accession no. X61367) and the sequenced parts of the resistance plasmids pKBB958 (AM183165) and pKBB4037 (AJ877266) from B.
bronchiseptica. A distance scale in kb is given below each map. The genes tetR, tet(A), mcp, tnpR, tnpA, ∆tnpA, res, parA1, parC and ∆tnpA* are presented as arrows with the arrowhead indicating the direction of transcription. The ∆ symbol indicates a truncated, functionally inactive gene. The black boxes represent the terminal or internal 38 bp repeats of Tn1721. The grey shaded areas indicate the homologous parts between the B. bronchiseptica plasmids and Tn1721.
Truncated forms of the transposon Tn1721 have been described in various Gram-negative bacteria.5,7 In all those cases, the tetracycline resistance gene region was intact whereas the genes coding for transposition functions were deleted in part or completely.
Although the Tn1721 relics found in the two plasmids from B. bronchiseptica differed from all previously described ones, we also noticed the presence of an intact resistance gene region and the lack of the transposase part.
In contrast to feline B. bronchiseptica isolates, where a plasmid-borne tet(C) gene was identified,3 we found a different type of tet gene, tet(A), in two unrelated porcine B.
bronchiseptica isolates. This is to the best of our knowledge the first report of a tet(A) gene in B. bronchiseptica and extends the knowledge of the distribution of this transposon-associated tet gene (http://faculty.washington.edu/marilynr/tetweb2.pdf). Although tet genes of various classes have been detected in animal isolates of the family Pasteurellaceae,8 which share the same habitat with B. bronchiseptica, the tet(A) gene has not yet been detected in these bacteria. Its presence on a conjugative plasmid, however, may further the dissemination of the tet(A) gene not only to other Bordetella isolates, but also to other Gram-negative respiratory tract pathogens.
Acknowledgements
We thank Jürgen Wallman, Frederique Pasquali and Petra Lüthje for helpful discussions. K.
K. is supported by a scholarship of the H. Wilhelm Schaumann foundation.
Tetracycline resistance chapter 4
References
1. Kadlec K, Kehrenberg C, Wallmann J et al. Antimicrobial susceptibility of Bordetella bronchiseptica isolates from porcine respiratory tract infections. Antimicrob Agents Chemother 2004; 48: 4903-6.
2. Terakado N, Araki S, Mori Y et al. Non-conjugative R plasmid with five drug resistance from Bordetella bronchiseptica of pig origin. Jpn J Vet Sci 1981; 43: 971-4.
3. Speakman AJ, Binns SH, Osborn AM et al. Characterization of antibiotic resistance plasmids from Bordetella bronchiseptica. J Antimicrob Chemother 1997; 40: 811-6.
4. Kadlec K, Kehrenberg C, Schwarz S. Molecular basis of resistance to trimethoprim, chloramphenicol and sulphonamides in Bordetella bronchiseptica. J Antimicrob Chemother 2005; 56: 485-90.
5. Waturangi DE, Schwarz S, Suwanto A et al. Identification of a truncated Tn1721-like transposon located on a small plasmid of Escherichia coli isolated from Varanus indicus. J Vet Med B 2003; 50: 86-9.
6. Allmeier H, Cresnar B, Greck M et al. Complete nucleotide sequence of Tn1721: gene organization and a novel gene product with features of a chemotaxis protein. Gene 1992; 111: 11-20.
7. Ojo KK, Kehrenberg C, Odelola HA et al. Structural analysis of the tetracycline resistance gene region of a small multiresistance plasmid from uropathogenic Escherichia coli isolated in Nigeria. J Antimicrob Chemother 2003; 52: 1043-4.
8. Kehrenberg C, Walker RD, Wu CC et al. Antimicrobial resistance in members of the family Pasteurellaceae. In: Aarestrup FM, ed. Antimicrobial resistance in bacteria of animal origin. Washington DC: ASM Press, 2005; 167-86.
Florfenicol and chloramphenicol resistance chapter 5