aDivision of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT; bHospital Infection Laboratory, City Hospital, Dudley Road, Birmingham B18 7QH, UK
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Abstract |
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Introduction |
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High-level gentamicin resistance (HLGR; MIC > 1000 mg/L) in enterococci was first reported in E. faecalis by Horodniceanu et al. 3 In-vitro transfer of the gentamicin resistance marker from E. faecalis to E. faecium was successfully carried out by Chen & Williams. 4 This was subsequently followed by the first report of a clinical E. faecium strain with HLGR. 5
HLGR in enterococci results from the production of a bifunctional aminoglycoside modifying enzyme (AME), AAC6'-APH2''. This enzyme confers resistance to all clinically useful aminoglycosides, with the exception of streptomycin. High-level resistance to streptomycin can be mediated by either a chromosomal mutation resulting in a single amino acid change on the 30S ribosomal subunit or by the synthesis of the AAD6 AME. 6 Another AME commonly found in enterococci is APH3' which confers resistance to paromomycin. 7 The AAC6'-APH2'' AME does not modify paromomycin, presumably because of the bulkiness of the paromomycin side chain. 8
The aac6'-aph2''gene has, in most cases, been associated with plasmids. Recently, Hodel-Christian & Murray showed that aac6'-aph2'' was associated with a transposon, Tn5281, in E. faecalis. 9 To our knowledge the only other enterococcal species shown to possess Tn5281 is Enterococcus avium. 10
Woodford and colleagues reported that gentamicin resistance-encoding plasmids isolated from different continents were homologous with respect to size, resistance markers and restriction digestion patterns. 11 In the present study we report on ten E. faecium, isolated in the UK between 1993 and 1995. Clonal relatedness of HLGR-conferring plasmids was investigated by restriction endonuclease analysis. We also investigated the possibility of transposon-mediated HLGR in E. faecium by hybridization studies of restriction endonuclease-digested plasmid DNA.
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Materials and methods |
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Ten HLGR E. faecium isolates from six different hospitals throughout the UK, collected between 1993 and 1995, were studied. The identification of these isolates was confirmed using the API 20STREP kit (Bio-Merieux, Basingstoke, UK). E. faecalis strain HH22, 9 containing the HLGR- conferring transposon Tn5281 on plasmid pBEM10, was used to compare hybridization patterns and subsequently to establish the possibility of the presence of a Tn5281-like element in E. faecium.
Susceptibility testing
Antibiotic susceptibilities were determined by an agar dilution method using Isosensitest agar supplemented with 5% lysed horse blood (Oxoid, Basingstoke, UK). The following ranges of antibiotic concentrations were tested: amikacin, 1-1024 mg/L; kanamycin, 1-4096 mg/L; paromomycin, 1-4096 mg/L and streptomycin, 0.25- 4096 mg/L. Gentamicin was tested in the range 0.5-128 mg/L and also at a breakpoint concentration of 1000 mg/L. Media containing the appropriate antibiotic (Sigma, Basingstoke, UK) were inoculated with a multipoint inoculator at 10 4-10 5 cfu/spot. The MIC was defined as the lowest concentration of antibiotic to give complete inhibition of bacterial growth after incubation at 37°C for 18 h. High-level gentamicin resistance was defined as an MIC > 1000 mg/L.
Identification of AMEs by the PCR
PCR was used to amplify genes encoding AMEs commonly found in enterococci; these included aph3', aad6 and aac6'-aph2''.The amplification protocols were those previously described. 12,13
Conjugation experiments
A filter mating method was used to investigate the transfer of gentamicin resistance by conjugation. 14 The recipient was a laboratory-derived, plasmid-free strain of E. faecium, strain GE-1, resistant to rifampicin and fusidic acid. 5 Transconjugant selection was carried out on blood agar number 2 (Oxoid) supplemented with 5% lysed horse blood, containing gentamicin (250 mg/L), rifampicin (100 mg/L) and fusidic acid (25 mg/L). Twelve transconjugants per mating were restreaked on to fresh selective media before plasmid profiles and antibiotic susceptibilities were determined.
Plasmid analysis
Plasmid DNA was extracted as described by Woodford et al. 11 Two strains of Escherichia coli, strain V517 and strain 39R861, 15,16 harbouring plasmids of known sizes were used as plasmid size markers. Electrophoresis of plasmid DNA was carried out on 0.7% agarose gels at 90 V for 2 h using 1 x TBE (89 mM Tris, 89 mM orthoboric acid, 2 mM EDTA (pH 8.0)) as electrophoresis buffer and visualized under UV light following ethidium bromide staining. Plasmid DNA (2 µg) was digested using either restriction endonucleases commonly used to identify Tn5281-like structures (HaeIII, HindIII, HincIIor ScaI) 9 or restriction endonucleases known not to cut within Tn5281 (EcoRI, PvuII, SalI, PstI or BamHI) according to the specifications of the manufacturer (Boehringer-Mannheim, Lewes, UK). Digested plasmid DNA was separated by electrophoresis on 1% agarose gels at 90 V for 2 h. A 1 kb fluorescein-labelled DNA ladder (Amersham plc, Amersham, UK) was used as a molecular size marker.
IS256 detection
To establish whether IS256 elements were present in E. faecium, PCR amplification for a 468 bp fragment specific to IS256 was performed as previously described. 17
Hybridization studies
The probes used for Southern blot analysis were generated by PCR using plasmid pBEM10 as template. A 220 bp region of the aac6'-aph2''gene was amplified by PCR as described by Jos et al. 13 and a 440 bp region of the IS256 gene was amplified as described above. The amplified products were purified using a QIAquick PCR Purification Kit (Qiagen, Crawley, UK) and labelled with fluorescein using the ECL Random Prime Labelling Kit (Amersham).
Digested or intact plasmid DNA was transferred from agarose gels to Hybond-N+ nylon membrane (Amersham) using a VacuGene blotter (Phamacia-LKB, St Albans, UK). The transferred DNA was fixed to the membrane using an ultraviolet cross linker (UV Stratalinker 1800, Pharmacia) set at 1200 µJ for 45 s. Hybridization was carried out at 60°C overnight before washing the blot under high stringency (in 0.1 x SSC for 15 min at 60°C). Hybridized probe was detected using the ECL Random Prime Detection System (Amersham).
Pulsed-field gel electrophoresis
Preparation and digestion of genomic DNA, using SmaI restriction endonuclease (Boehringer-Mannheim), were as described by Murray et al. 18 Electrophoresis was carried out in two steps using the CHEF DRII apparatus (Bio-Rad, Hemel Hempsted, UK). In the first step the pulse time was increased from 1 s to 10 s over 15 h, whilst in the second step the pulse time was increased from 10 s to 30 s over 7 h using 6 V/cm.
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Results |
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The antibiotic susceptibility patterns are shown in Table I. All the strains exhibited high-level resistance to gentamicin (MIC>1000 mg/L). The resistance to streptomycin ranged from mid-level resistance (MIC 64 mg/L) to high-level resistance (MIC>1000 mg/L).
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The aac6'-aph2''and aad6 genes were detected by PCR in all ten E. faecium isolates. However, only six of the ten isolates were positive when screened for the aph3'gene; this gene was not detected in isolates SS1, SS2, SS23 or SS24.
Plasmid analysis and hybridization studies with the aac6'-aph2'' probe
Plasmid analysis showed that each isolate contained one to three plasmids, ranging in size from 3.5 to 70 kb. Hybridization studies, using a aac6'-aph2probe (Gm r), indicate that the aac6'-aph2''gene was located on a 70 kb plasmid in all ten clinical isolates.
Conjugation experiments
The gentamicin resistance marker was transferable from all ten clinical isolates, but with varying transfer frequencies (10 -6 to 10 -9/recipient cell). Nine of the ten clinical isolates were able to transfer the HLGR genes in association with the 70 kb plasmid. In strains SS1 and SS2 two other plasmids of 3.5 kb and 9 kb were also transferred. No plasmid DNA was detected in the transconjugants derived from strain SS25, but the Gm r probe hybridized to the chromosomal DNA.
With the exception of streptomycin, the aminoglycoside susceptibilities of the transconjugants were identical to those of the donor strains. All transconjugants maintained the recipient strain streptomycin MIC (32 mg/L). Additionally, the transconjugants were resistant to rifampicin (MIC >25 mg/L) and fusidic acid (MIC >100 mg/L). This indicated that the aminoglycoside resistance in the transconjugants resulted from the conjugal transfer of the AME genes into the recipient GE-1 strain.
Detection of IS256 and identification of Tn5281-like structures
PCR detected IS256 in all isolates studied. This led us to examine the isolates for the possible presence of transposons.
In E. faecalis strain HH22 the HLGR marker resides on a transposon designated Tn5281 carried on plasmid pBEM10. In order to determine whether the aac6'-aph2''gene in the HLGR E. faecium isolates resided on a similar transposon, the aac6'-aph2''carrying plasmids from all ten clinical isolates were studied further following restriction endonuclease digestion. The choice of restriction endonucleases were those commonly used to identify Tn5281-like elements. Digested plasmid DNA was probed using the Gm r and IS256 fluorescein-labelled probes (Figures1 and 2). The hybridization patterns represented by the principal bands were then compared with those obtained with plasmid pBEM10. Faint bands were consistent with partial digestion of target DNA.
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The hybridization patterns obtained for the 70 kb plasmid in the remaining six isolates showed no similarities to those obtained for pBEM10 in strain HH22. Thus aac6'- aph2''was not part of an Tn5281-associated transposon in these six isolates. The hybridization results obtained for these six isolates are summarized in Table II.
There are several points of interest to note in the hybridization patterns obtained with these six isolates: (i) Loss of one of the IS256 elements caused loss of one of the HindIII sites and hence the fragment hybridizing with the Gm r probe changed from 2.5 kb to 3.4 kb. (ii) If IS256 were present at both ends of aac6'-aph2''then following ScaI digestion the Gm r probe would hybridize with a 1.5 kb and a 1.8 kb fragment indicating the presence of two IS256 elements. (iii) In six strains (SS1, SS2, SS16, SS23, SS24 and SS41) the Gm r probe hybridized to 1.8 kb and 1.2 kb fragments. Loss of the 3' IS256 element and the presence of a ScaI site outside the aac6'-aph2''would account for this observation. (iv) That the 3' IS256 element was missing can further be substantiated by the fact that if the 5' IS256 were missing from the transposon the HincII fragment hybridizing to the Gm r probe would be unaltered from that in Tn5281. Loss of the 3' IS256, however, would alter the HincII fragment with which the Gm r probe hybridized, as was observed. (v) HincII digests probed with IS256 resulted in the Tn5281 2.2 kb fragment changing to 3.8 kb. The 1.2 kb fragment, from the 5' end of Tn5281, hybridizing with IS256 remained that size in all six E. faecium isolates. These findings are all consistent with those expected in a Tn5281-truncated structure.
The hybridization patterns were used to construct a restriction map of the aac6'-aph2''gene and its flanking regions (Figure 3). From the restriction map it appears that aac6'-aph2''may be part of a Tn5281-truncated structure which comprises the aac6'-aph2''gene flanked by an IS256 element at the 5' end.
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Having identified the possibility that the aac6'-aph2'' may be associated with two different plasmid types in E. faecium, the 70 kb plasmid from strains SS2 and SS25 was further analysed by restriction endonuclease digestion using EcoRI, PvuII, SalI, PstI or BamHI. These strains harboured only the 70 kb plasmid. There were several band differences with each of the five restriction endonucleases used, suggesting that the two plasmids are not related. Figure 4a is a schematic representation of the restriction endonuclease digestion patterns obtained with EcoRI. Each restriction enzyme used gave two different patterns and allowed the plasmids to be allocated to groups cI and cII. All plasmids from each of these groups had identical restriction patterns. Group cI comprised the 70 kb plasmid harbouring the Tn5281-like structure. The cII plasmids contained the Tn5281-truncated structure. In addition, hybridization studies on the 70 kb plasmid cut with enzymes that do not cut within Tn5281 and probed for IS256, were also used as a means of typing the 70 kb plasmid. This IS256 typing method showed four different profiles which were allocated to groups dI to dIV (Figure 4b). Group dI contained the 70 kb plasmid found in isolates SS1, SS2, SS23 and SS24. Group dII had plasmids from isolates SS16 and SS41, group dIII SS17 and SS29, and group dIV SS25 and SS39. The results of the restriction endonuclease digestion and IS256 typing on the 70 kb plasmid are summarized in Table III.
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PFGE results identified three different restriction patterns in the ten E. faecium isolates studied. A three-band difference rule was employed to distinguish the isolates and the patterns obtained were allocated arbitrary designations of patterns 1-3 (Table III). Based on PFGE results, strains SS1, SS2, SS23 and SS24, all isolated from the same hospital, were indistinguishable from each other and were allocated to group 1.
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Discussion |
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Plasmid analysis of the transconjugants confirmed that the 70 kb plasmid had transferred during conjugation from nine of the ten clinical isolates. Interestingly, one isolate, SS25, grew on selective media but plasmid analysis failed to identify the presence of plasmid DNA in the resultant transconjugant. Hybridization studies on this isolate showed that aac6'-aph2'' was located on the chromosome. This would suggest that aac6'-aph2''may be part of a conjugative transposable element in SS25.
The MICs for the transconjugants showed that although HLGR was acquired, high-level streptomycin resistance (HLSR) was not co-transferred. This indicates that the HLSR marker is not located on the aac6'-aph2''-harbouring plasmid. The failure to transfer the HLSR marker in E. faecium is in agreement with previous reports. 11 The HLSR marker may be present on the chromosome in the clinical strains as either point mutations in ribosomal protein genes or aad6. 6
Hybridization analysis of the gentamicin resistance-encoding plasmids, isolated from the clinical isolates, showed that there are two means by which the aac6'-aph2'' gene can be associated with plasmids. The first method, seen in four of the isolates (SS17, SS25, SS29 and SS39), showed a hybridization pattern similar to that seen with plasmid pBEM10, harbouring transposon Tn5281. This suggested that in these isolates of E. faecium aac6'-aph2'' is part of a transposon similar to Tn5281. However, the hybridization patterns obtained, when compared with those obtained for pBEM10, showed that the Tn5281-like transposon identified in the UK E. faecium isolates lacked the symmetrically located HaeIII sites found in Tn5281 (Figure 3a). This would suggest that there are some sequence discrepancies at the terminal ends of the IS256 elements that flank aac6'-aph2'' in the UK Tn5281-like transposon. One isolate, SS39, had a ca. 13.0 kb fragment which hybridized to both the Gm r and IS256 probes following HaeIII digestion. This would suggest that this plasmid may not share the same HaeIII restriction sites as the three other 70 kb plasmids which carry the Tn5281-like transposon.
A second difference was observed in hybridization patterns when comparing the Tn5281-like transposons in E. faecium with Tn5281 found in pBEM10 following HindIII restriction endonuclease digestion and probing with the IS256 probe. Since there is a single HindIII site in IS256 (Figure 3a), the second HindIII restriction fragment would be located outside of the Tn5281-like structure on the plasmid. Variation in HindIII fragments hybridizing to an IS256 probe indicates plasmid sequence variation. These differences are of interest as they further substantiate the restriction endonuclease analysis of the 70 kb plasmid in confirming that the aac6'-aph2''gene-harbouring plasmids in E. faecium are not homogeneous. A 1.0 kb fragment was noted when a pBEM10 HindIII digest was hybridized with the IS256 probe. This can be accounted for by the fact that in pBEM10 the Tn5281 transposon has another IS256 element inserted at one end of the transposon. As a result this forms a 1.0 kb HindIII fragment which is seen to hybridize with the IS256 probe.
A different genetic structure was seen in the remaining six isolates. These showed common hybridization patterns with each other, but these patterns were different from those obtained for pBEM10. In Tn5281, aac6'-aph2'' is flanked by IS256 elements in inverse orientation (Figure 3a). It appears that in the remaining six E. faecium strains aac6'-aph2'' may be part of a truncated Tn5281-like structure. Hybridization patterns suggest that this Tn5281-truncated structure comprises aac6'-aph2'' flanked by an IS256 sequence at the 5' end. A similar structure has previously been reported by Straut et al. in an isolate of E. hirae. 10 The Tn5281-truncated structure may be the remnants of an incomplete transposon formation process. The failure of a second IS256 element to insert so as to flank aac6'-aph2'' may be due to the lack of sequence homology that may be required for IS256 insertion. As a result only certain plasmid groups may be able to carry complete Tn5281 transposons while other plasmids may not be able to facilitate complete transposon formation.
To establish the clonal relatedness of the aac6'-aph2'' gene-harbouring plasmids two studies were carried out to type the 70 kb aac6'-aph2''-harbouring plasmid. The first method relied on restriction endonuclease digestion patterns obtained when the 70 kb plasmid was treated with nine different restriction endonucleases (five that did not cut within Tn5281). The second method used an IS256 typing protocol where restriction endonuclease-treated 70 kb plasmid DNA was probed with a fluorescein-labelled IS256 probe. IS256 elements are mobile DNA elements which have the potential to cause genetic rearrangement and therefore would appear not to be the typing method of choice. However, IS256 typing studies by Arpin et al. 20 have shown that there was no change in a profile of a strain after 18 months and of another strain after 20 in-vitro subcultures. Similarly Dyke et al. 17 found no evidence of mobility of IS256 after a total of 126 generations. These two studies would suggest that IS256 is relatively stable during a considerable number of in-vitro and in-vivo generations. Previous reports on the aac69'-aph2''-carrying plasmids in E. faecium have concluded that aac6'-aph2'' is harboured on a homogeneous group of plasmids. 11 The two typing methods used in this study showed that the 70 kb plasmids from the ten E. faecium were heterogeneous.
Using nine restriction endonucleases (five of which do not cut within Tn5281) to type the 70 kb plasmid, two distinct plasmid types (cI and cII) were apparent. The group cI plasmids identified in E. faecium were those that harboured the Tn5281-like transposon. The group cII plasmids harboured a Tn5281-truncated structure. Differences in the flanking regions of aac6'-aph2'', i.e. the aac6'-aph2'' gene being flanked by an IS256 element on either side in the Tn5281-like transposon and a single IS256 element in the Tn5281-truncated structure would perhaps have altered the restriction fragment patterns by one or two bands only. The fact that there were several distinct band differences in the restriction fragment patterns would repudiate the idea that the differences could be attributed to the different number of IS256 elements flanking the aac6'-aph2'' gene.
When the E. faecium plasmids were typed by the IS256 typing method the plasmids could be categorized into four groups. Groups dI and dII contained plasmids from isolates that carried the Tn5281-truncated structure. The isolates in group dI were all collected from the same hospital and therefore the typing results would suggest dissemination of a plasmid clone. This finding is supported by the fact that PFGE analysis showed these four isolates to be indistinguishable from one another. PFGE analysis found that the strains harbouring the group dII plasmids were also indistinguishable from each other. The plasmids allocated to groups dIII and dIV were those plasmids in which the Tn5281-like transposon has been identified. The strains from which these plasmids originated could not be distinguished from one another by PFGE. This would suggest that there are two plasmid types harbouring the Tn5281-like transposon in E. faecium which are being disseminated in a clone of E. faecium.
The four E. faecium isolates harbouring the Tn5281-like transposon were indistinguishable from each other by PFGE. However, they could be distinguished by PFGE from the remaining six E. faecium isolates which carry the Tn5281-truncated structure. This further substantiates the original hypothesis that the aac6'-aph2'' gene has become part of a heterogeneous group of plasmids which is being disseminated in E. faecium which are not clonally related.
Our high-stringency hybridization and restriction endonuclease analysis demonstrate that aac6'-aph2'' in our E. faecium strains is indistinguishable from those found in other enterococcal species. 10 Although the HLGR determinant in E. faecium appears to be consistent with those found in other enterococcal species, the observed hybridization patterns and endonuclease restriction analysis of the plasmids in this study suggest heterogeneity in the pattern of resistance transfer and may consequently suggest different sources of origin. Although this is the first report of plasmid heterogeneity in E. faecium, a heterogeneous set of plasmids conferring HLGR have previously been described in E. faecalis 21,22 and recently a heterogeneous HLGR-conferring plasmid group has been described in E. hirae. 23 The plasmid heterogeneity described in this study may be due to the acquisition of HLGR-conferring plasmids from one of these other species.
As with E. faecalis, it is inevitable that we will see several different types of plasmids carrying the aac6'-aph2'' gene appearing in isolates of E. faecium possibly by the acquisition of these plasmids from E. faecalis or E. hirae. This is because composite transposons, like Tn5281, are promiscuous and appear to show no preference as to the plasmid type into which they integrate, as has been recently demonstrated by the appearance of Tn5281 in E. avium as part of a 90 kb plasmid. 10
The origin of the HLGR genes in enterococci remains uncertain. The appearance of a transposon similar to Tn5281, and Tn4001 9 found in S. aureus, suggest either direct gene exchange between E. faecalis or S. aureus and E. faecium or a common ancestral origin for gentamicin resistance.
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Acknowledgments |
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Notes |
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References |
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Received 9 July 1998; returned 21 August 1998; revised 16 November 1998; accepted 28 January 1999