Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen V, Denmark
Correspondence
Henrik Hasman
hha{at}dfvf.dk
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ABSTRACT |
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INTRODUCTION |
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Homeostatic mechanisms like the copYZAB system are rarely able to handle artificially elevated concentrations of copper. In response to toxic levels of copper, plasmid-borne copper resistance mechanisms are often employed. The pco system from Escherichia coli, and the cop system from Pseudomonas syringae pv. tomato (Brown et al., 1995; Lee et al., 2002
), are well-known model systems of transferable copper resistance in Gram-negative bacteria (Bender & Cooksey, 1986
; Mellano & Cooksey, 1988
). However, these systems do not involve CPx-type ATPases. The tcrB (transferable copper-resistance gene B) gene is, to date, the only plasmid-encoded and transferable CPx-type copper ATPase gene described. The tcrB gene has been described in Enterococcus faecium (Hasman & Aarestrup, 2002
) and Enterococcus faecalis (Aarestrup et al., 2002
), where it confers copper resistance. Strains harbouring the tcrB gene have an MIC of 24 mmol l1 for CuSO4, whereas strains lacking the tcrB gene have a MIC of 28 mmol l1 (Hasman & Aarestrup, 2002
). The prevalence of tcrB in Denmark among E. faecium is especially high among isolates from pigs compared with other reservoirs: 4679 % of pig isolates examined between 1997 and 2003 were copper resistant (Hasman & Aarestrup, 2005
). The most likely explanation for this high prevalence is the use of CuSO4 as a growth-promoting agent for pigs: piglets in Denmark and most of the European Union receive 175 p.p.m. CuSO4, and slaughter animals receive 35 p.p.m. CuSO4 in their feedstuff. A relationship between copper resistance, and glycopeptide and macrolide resistance in E. faecium has been established previously (Hasman & Aarestrup, 2002
, 2005
). Therefore, a closer examination of the mechanisms and selective concentrations responsible for development of copper resistance in E. faecium is needed.
This paper identifies the tcrB gene to be part of a previously uncharacterized operon called the tcrYAZB operon, with a genetic organization highly similar to the copYZAB operon from E. hirae. This operon has been further characterized, and its ability to select for copper resistance has been examined.
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METHODS |
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PCR and sequencing.
Sequencing of the tcr operon was completed by inverse PCR, as described by Hui et al. (1998). In short, two primers (P511, 5'-GGA AAG GCA ACT GAA TAT CC-3', and P560, 5'-GCC GTC TTG ATG TCA CTT TC-3') were designed to read downstream and upstream, respectively, of the previously sequenced tcrB gene (Hasman & Aarestrup, 2002
). Plasmid DNA from A17sv1 was purified, and 20 µl (50 ng µl1) of this was digested separately with a series of different restriction enzymes that are known not to cut inside the tcrB gene. The digested DNA was purified using a GFX PCR DNA and gel band purification kit (Amersham Biosciences), and eluted in 50 µl double-distilled water. T4 DNA ligase (Invitrogen) was added to this 50 µl eluate, and the DNA was religated to generate circular DNA molecules. Then, 2 µl of the ligation mix was used for PCR with the two primers. In cases where a single PCR product was generated, this was sequenced, and two new primers were designed based on this new sequence, until the complete sequence of the tcr gene operon was obtained. The complete sequence of tcrYAZB has been submitted to GenBank (accession no. AY048044).
Cycle sequencing of the PCR products was carried out according to the manufacturer's instructions, using an AmpliTaq dye terminator kit and a 373A automatic sequencer (Applied Biosystems/Perkin Elmer). The Vector NTI suite v8.0 (Invitrogen) was used to assemble sequencing fragments.
Construction of promoter fusions to pAK80.
The computer program Winseq32 (a kind gift from Flemming G. Hansen, Biocentrum-DTU, Denmark) was used to identify a potential promoter upstream of tcrY. Based on the sequencing result of the complete tcr operon described above, two sets of primers were designed using the computer program Vector NTI suite v8.0. The first set of primers was designed to amplify a 220 bp DNA fragment predicted to carry the copper-responsive promoter Ptcr, including putative regulatory binding sites. The forward primer (P1: 5'-CCC AAG CTT ACA GAG AAG TGT CCG ACG AAC C-3') was designed to carry a HindIII site, and the reverse primer (P2: 5'-CCC GGA TCC TCA TAT TCT CTC CCC CTT TCG TT-3') was designed to contain a BamHI site, for cloning into the erythromycin-resistant promoter-probe vector pAK80 containing the promoterless -galactosidase genes lacL and lacM behind a multicloning site (Israelsen et al., 1995
), thus generating the plasmid pHHA213. The second set of primers was designed to amplify a 679 bp DNA fragment carrying precisely the same region as above, as well as the downstream putative regulatory gene tcrY. Here, the forward primer (P3: 5'-CGC CTC GAG ACA GAG AAG TGT CCG ACG AAC CA-3') was designed to contain an XhoI site, and the downstream primer (P4: 5'-CTC GGA TCC TCG CTC CTT ATT CTC CAT GAT GAT G-3') to contain a BamHI site for insertion into pAK80, which generated the plasmid pHHA218. Amplification of the DNA fragments was done using the EXPAND High FidelityPLUS PCR system (Roche) under standard PCR conditions: 94 °C for 3 min, then 25 cycles (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min), and finally 72 °C for 10 min. The inserts of both plasmids were sequenced to exclude PCR-generated mutations.
-Galactosidase assay.
pAK80, pHHA213 and pHHA218 were electroporated into the copper- and erythromycin-sensitive E. faecalis strain JH2-2 RF as well as the copper-resistant and erythromycin-sensitive E. faecalis strain 9831021-2, as described by Dunny et al. (1991), and selected on 16 µg erythromycin ml1. A single colony from each electroporated strain was inoculated into BHI broth (Oxoid) containing 16 µg erythromycin ml1, and grown overnight at 30 °C, with gentle shaking (125 r.p.m.). A 100 µl volume of each culture was reinoculated into fresh preheated BHI broth containing 0, 4, 8, 12 and 16 mmol CuSO4 l1 (pH 7), and grown at 30 °C, with gentle shaking until an OD600 of 0·30·4 was reached for each culture. Then, three sets of 2 ml culture samples were collected from each concentration, and
-galactosidase activity was measured for each set, as described by Miller (1992)
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Creation of the vanAtcr deletion mutant.
The A17sv1 strain was grown overnight at 37 °C in BHI broth containing 5 µg novobiocin ml1 (Sigma-Aldrich), in an attempt to cure the plasmid carrying the tcr genes. Screening of a large number of colonies led to an isolate (A17sv1-34) that had lost the copper-resistance and vancomycin-resistance phenotypes. The loss of the tcr and van genes was confirmed by Southern blot analysis using specific probes directed towards tcrB and vanA, respectively. The clonal relationship to A17sv1 was confirmed by PFGE using SmaI digestion of chromosomal DNA, as described by Aarestrup (2000).
Conjugation.
In order to ensure plasmid location of the resistance genes, plasmids from A17sv1 and A17sv1-34 were transferred by filter-mating to the plasmid-free E. faecium recipient BM4105RF (rifampicin- and fusidic-acid-resistant), as described by Clewell et al. (1985). Transconjugants were selected on BHI agar containing 16 µg erythromycin ml1, 25 µg rifampicin ml1 and 25 µg fusidic acid ml1.
Plasmid purification.
Plasmids were isolated using Qiagen Plasmid Midi kit (Qiagen), as described previously (Hasman & Aarestrup, 2002).
Growth curves.
The A17sv1 and the A17sv1-34 (tcr) strains were grown on BHI agar (Oxoid) containing 16 µg erythromycin ml1, at 37 °C overnight. The next day, the two strains were inoculated into 10 ml BHI, and grown overnight at 37 °C, with gentle shaking. The OD600 was measured, and the cultures were diluted approximately 3000-fold to an OD600 of 0·001 in preheated BHI broth containing 0, 1, 2, 3 and 4 mM CuSO4 (pH 7·0). Then, 300 µl of each culture was dispensed into separate wells of a 100-well Bioscreen microwell plate. The Bioscreen microwell plate was inserted into a Bioscreen C apparatus (Growth Curves AB), and analysed using the software Research Express (Transgalactic). Hardware settings were as follows: temperature, 37 °C; continuous shaking (medium, 80 steps); measurement of OD492 every 12 min for 18 h. Each strain was tested in the same media at least four times, and the mean generation time (tgen) was calculated.
Competition assay.
A growth competition assay was done between A17sv1 and A17sv1-34 in different concentrations of CuSO4. Overnight cultures of the two strains were mixed with a surplus of the copper-sensitive strain A17sv1-34 (in a 100 : 1 ratio), and 100 µl of this mixed culture was then transferred to eight different flasks. These flasks contained 25 ml preheated BHI broth with 0, 1, 2, 3, 4, 8, 12 or 16 mmol CuSO4 l1(pH 7), and they were incubated at 37 °C, with gentle shaking. After 8 h, 100 µl of each culture was transferred to a fresh flask containing 25 ml preheated medium supplemented with the same copper concentration as the previous flask. This was repeated three times, leading to a total of 32 generations of growth. The cell suspension from the last flask was diluted by an appropriate factor of between 105- and 107-fold, and then plated onto BHI agar containing 16 µg erythromycin ml1, for incubation overnight at 37 °C. From each concentration, 100 colonies were picked randomly, and streaked onto two sets of BHI agar containing either 16 mmol CuSO4 l1or 16 µg erythromycin ml1, and incubated overnight at 37 °C. From these cultures, the ratios of copper-resistant bacteria were calculated. Furthermore, the presence of the tcrYAZB genes in the copper-resistant population was tested by PCR, as previously described (Hasman & Aarestrup, 2002).
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RESULTS AND DISCUSSION |
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tcrZ was a 204 bp gene encoding a putative chaperone protein called TcrZ (68 aa), with homology to other copper chaperones, including CopZ from E. hirae. It also contained the CXXC motif in the N-terminus, which is normally found in CopZ-like chaperones. The last ORF in the operon, tcrB, has been described previously as encoding a copper efflux pump (TcrB), homologous to CopB from E. hirae (Hasman & Aarestrup, 2002).
The close homology to the copYZAB operon of E. hirae makes the structural relationship evident, and gives an indication of the function of the plasmid-located tcrYAZB genes within the cell, but the origin of the operon remains elusive, as the order of the individual genes is not the same as the cop genes in E. hirae. The chaperone tcrZ is located between the tcrA and tcrB genes in the tcr operon, whereas the chaperone copZ is located between the repressor copY and copA in E. hirae. Similar location of the copZ gene after the copA gene is seen among copper-homeostasis genes from S. mutans and Streptococcus gordonii (Vats & Lee, 2001; Mitrakul et al., 2004
), which could give a hint to the possible origin of the plasmid-located tcr operon, especially as the DNA and protein homology to the streptococcal cop gene clusters from S. mutans and S. gordonii are only slightly less than the homology to E. hirae (data not shown).
A truncated ISS1-type transposase (ORF1' in Fig. 1) was located upstream of the tcr promoter, and an IS1216E element was located downstream of the four tcr genes, indicating the termination of the operon (Fig. 1
). This was further supported by the fact that the intergenic region between tcrB and the IS1216E element contained a strong dyad symmetry region (
G 21·4 kcal mol1; 89·5 kJ mol1) able to form a loop structure, which could function as a factor-independent transcriptional terminator.
The tcrYAZB genes are transcribed from a promoter regulated by CuSO4
A putative promoter structure (Ptcr) was located immediately upstream of the tcrY gene (Fig. 1), with the 35 and 10 boxes (underlined in Fig. 2
) separated by a 16 bp spacer region. Again, this region showed strong homology to the promoter region of the cop promoter from E. hirae (Table 1
), including the 35 box, the 10 box and the two repressor operator half-sites (indicated with arrows in Fig. 2
). Two imperfect cop boxes were located within these repressor operator sites (indicated with grey in Fig. 2
). As the cop box has been defined using CopY from E. hirae, this could indicate that TcrY has a slightly altered recognition site compared with CopY. This makes biological sense, as TcrY could otherwise interfere with the normal regulation of copper homeostasis.
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Construction of a tcr-deletion mutant
A tcr-deletion mutant (A17sv1-34; MIC 6 mmol CuSO4 l1) of the copper-resistant E. faecium isolate A17sv1 was isolated. The A17sv1-34 strain had retained its erythromycin-resistance phenotype (MIC 32 µg ml1), indicative of a deletion in, rather than curing of, the plasmid. Conjugational transfer to the plasmid-free E. faecium recipient BM4105RF using erythromycin as selective marker confirmed this. Plasmid purifications of the A17sv1 and A17sv1-34 strains were compared by RFLP analysis using EcoRI and PvuII, and then subjected to Southern blotting to ensure complete deletion of the tcr and vanA genes. Based on the RFLP analysis, the size of the deletion could be estimated to approximately 75 kb, leading to a total plasmid size of approximately 100 kb (data not shown).
The tcr genes confer a growth advantage in sublethal concentrations of CuSO4
The generation times (tgen) of the A17sv1-34 mutant in different concentrations of CuSO4 were compared with the those of the wild-type (Fig. 4). At CuSO4 concentrations between 0 and 2 mmol l1, the doubling times of the two strains were indistinguishable, at around 30 min, but at CuSO4 concentrations equal to or above 3 mmol l1, the tgen of both cultures was influenced by the metal ions. However, the growth of the tcr mutant was significantly more affected than the wild-type strain, with an approximately 25 % reduction in the growth rate at 4 mmol CuSO4 l1 for the tcr mutant compared with the A17sv1 wild-type strain. At CuSO4 concentrations of 6 mmol l1 and above, only the A17sv1 strain was able to grow. So, even at low levels of CuSO4, where both strains were able to grow, and where the tcr promoter was only induced at a low level in the promoter fusion experiment described above, copper did impose a significant effect on the growth of the copper-sensitive strain relative to the copper-resistant strain.
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Based on the observations described above, the question is then, can the use of CuSO4 in concentrations of up to 175 p.p.m. in production animals select for Cu2+ resistance among E. faecium? Interestingly, 175 p.p.m. CuSO4 is equal to 2·8 mmol l1 Cu per kg feedstuff. This concentration is comparable to the selective concentration of 3 mM found in this study, and could therefore lead to selection of copper-resistant E. faecium in the gut of piglets fed 175 p.p.m. CuSO4. However, factors in the gut, such as the pH, copper speciation, adsorption and complex formation to organic material, have an influence on the actual copper concentration. Furthermore, it is unlikely that the concentration in the intestine remains the same as it is in the feed initially, because feed components are removed from the feed for growth of the animal, and also because an unknown volume of water is added during the digestion process. The selective copper concentration found in this study can therefore only serve as an indication for the effect of adding high doses of copper to the feed used in pig production.
Based on the results presented above, the five times lower concentration given to slaughter animals (35 p.p.m.) is more likely to be below the selective concentration, and therefore less likely to select for copper resistance. This could explain why we find a high level of copper-resistant bacteria, but not full resistance, among E. faecium isolated from slaughter pigs as part of the DANMAP programme (Hasman & Aarestrup, 2005).
Since copper resistance is closely linked to resistance to erythromycin (a macrolide) and vancomycin (a glycopeptide) in E. faecium from pigs in Denmark, the results presented here cannot exclude the possibility that addition of CuSO4 to the feed can co-select for these antibiotic resistances. Data regarding co-selection of macrolide and glycopeptide resistance, as well as the level of copper-resistant E. faecium in piglets fed 175 p.p.m. CuSO4 to evaluate such a hypothesis, do not exist, but this is currently under examination in animal feeding studies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 April 2005;
revised 20 May 2005;
accepted 27 May 2005.
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