a Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT; b Hospital Infection Research Laboratory, City Hospital, Dudley Road, Birmingham B18 7QH, UK
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Abstract |
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Introduction |
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Resistance to the aminoglycosides usually occurs by enzymatic modification of the aminoglycoside by aminoglycoside-modifying enzymes (AME). The first high-level gentamicin-resistant (HLGR) E. faecalis isolate was reported in France in 1979.3 Subsequent studies confirmed that the high-level resistance to gentamicin was due to the fusion of two AME genes.4 The resultant bifunctional AAC6'-APH2'' AME is highly potent and confers resistance to all the clinically useful aminoglycosides with the exception of streptomycin. High-level resistance to streptomycin (MIC > 1000 mg/L) can be mediated either by a chromosomal mutation resulting in a single amino acid change on the 30S ribosomal subunit or by the synthesis of the AAD6' AME.5
The genes for aac6'-aph2'' have in most cases been identified on plasmids. Studies by Hodel-Christian & Murray6 identified the aac6'-aph2'' gene as being part of a transposon, which they designated Tn5281. Association of aac6'-aph2'' with a transposon further aids the rapid dissemination of the resistance marker. Recently, there have been reports of Tn5281-truncated structures being identified in enterococcal species.710 There has been no consistency in the location of the IS256 that flanks the aac6'-aph2'' gene in these Tn5281-truncated structures and the IS256 sequence has been found on either the 5' or 3' side of the aac6'-aph2'' gene. The significance of these Tn5281-truncated structures is as yet unknown but we have previously suggested that the truncated structures may be remnants of incomplete transposon formation.8
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Material and methods |
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A total of 42 HLGR E. faecalis isolates were collected from 17 different hospitals from diverse geographical regions of the UK between 1993 and 1995. The identification of these isolates was confirmed with the API 20STREP kit (bioMérieux, Basingstoke, UK). E. faecalis strain HH22,6 containing the HLGR-conferring transposon Tn5281 on plasmid pBEM10, was used as a reference to compare restriction endonuclease digestion and Southern hybridization patterns and subsequently to establish the possible presence of a Tn5281-like element in UK isolates of HLGR E. faecalis.
Susceptibility testing
Antibiotic susceptibility was determined by an agar dilution method with Iso-Sensitest agar supplemented with 5% lysed horse blood (Oxoid, Basingstoke, UK). The following ranges of antibiotic concentration were tested: amikacin 12048 mg/L; kanamycin and paromomycin 14096 mg/L; and streptomycin 0.254096 mg/L. Gentamicin was tested in the range 0.5128 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 104105 cfu/spot. The MIC was defined as the lowest concentration of antibiotic to give complete inhibition of visible bacterial growth after incubation at 37°C for 18 h. High-level gentamicin resistance was defined as an MIC > 1000 mg/L.12
Identification of AMEs by PCR
PCR was utilized to amplify the AME sequences commonly found in enterococci; these included aph3' (conferring resistance to paromomycin), aad6 (conferring resistance to streptomycin), aad4' (conferring resistance to amikacin) and aac6'-aph2''. The primers and amplification protocols were those previously described.13
Conjugation experiments
A filter mating method14 was used to investigate the transfer of gentamicin resistance by conjugation. The recipient was a laboratory-derived plasmid-free strain of E. faecalis, strain JH2-2,15 resistant to rifampicin and fusidic acid. Transconjugant selection was carried out on blood agar base (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.16 Two strains of Escherichia coli, strains V51717 and 39R861,18 harbouring plasmids of known sizes were used as plasmid size markers. Electrophoresis of plasmid DNA was carried out through 0.7% agarose gels at 90 V for 2 h with 1 x TBE as electrophoresis buffer (89 mM Tris, 89 mM orthoboric acid and 2 mM EDTA, pH 8.0) and visualized under UV light following ethidium bromide staining. Two micrograms of plasmid DNA was digested with restriction endonucleases commonly used to identify Tn5281-like structures (HaeIII, HindIII, HincII, ScaI and ScaI + HindIII) according to the specifications of the manufacturer (Boehringer-Mannheim, Lewes, UK). In order to type plasmids associated with Tn5281, plasmid DNA was digested with restriction enzymes that do not cut within the transposon (EcoRI, PvuII, SalI, PstI or BamHI). Digested plasmid DNA was separated by electrophoresis in 1% agarose gels at 90 V for 2 h. A 1 kb fluorescein-labelled DNA ladder (Amersham plc, Amersham, UK) was used as molecular size marker.
Screening of E. faecalis for a Tn5281-like transposon by long-PCR
A long-PCR (L-PCR) protocol was established to identify the presence of Tn5281. Chromosomal DNA was used as template to amplify a 3.5 kb fragment of Tn5281 by the Expand Long Template PCR System (Boehringer-Mannheim). The method utilizes the fact that in Tn5281 the aac6'-aph2'' gene is flanked by IS256 sequences in inverse orientation. Therefore any PCR primer used to act as a forward primer will also anneal to the inverted IS256 sequence and subsequently act as a reverse primer. Based on this, the L-PCR method developed utilized only a single IS256 primer for the amplification reaction. The IS256 primer we used was the forward primer previously described by Dyke et al.19
Each PCR reaction contained 350 µM of each dNTP, 300 nM of IS256 forward primer, 5 µL of x10 reaction buffer containing 1.75 mM magnesium chloride, 250 ng of chromosomal DNA and 2.5 units of reaction enzyme (Taq and Pwo DNA polymerases). The final volume was made up to 50 µL using distilled water. Each reaction mixture was overlaid with 40 µL of mineral oil. The amplification protocol consisted of (i) one cycle of 94°C for 2 min, (ii) ten cycles of 92°C for 10 s, 57°C for 30 s and 68°C for 45 s, (iii) 30 cycles of 92°C for 10 s, 57°C for 30 s and 68°C for 8 min (with the elongation time increased by 20 s per cycle) and (iv) a final elongation cycle of 68°C for 7 min.
Hybridization studies
The probes used for Southern hybridization 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 Klundert & Vliegenthart.20 (Gmr probe) and a 440 bp region of IS256 was amplified as described previously by Dyke et al.19 (IS256 probe). The PCR-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).
Restriction endonuclease-treated or intact plasmid DNA was transferred from agarose gels to a Hybond-N+ nylon membrane (Amersham) with a VacuGene blotter (Phamacia-LKB, St Albans, UK). The transferred DNA was fixed to the membrane with an ultraviolet cross linker (UV Stratalinker 1800; Stratagene Europe, Amsterdam, The Netherlands) set at 1200 µJ for 45 s. Hybridization was carried out at 60°C overnight before washing the blot under high stringency (0.1 x SSC (15 mM sodium chloride, 15 mM sodium citrate, pH 7.0), 15 min, 60°C). Detection of the hybridized probe was by the ECL Random Prime Detection System (Amersham).
IS256 detection
To establish whether IS256 elements were present in isolates, PCR amplification of a 468 bp fragment specific to IS256 was performed as previously described.19
Pulsed-field gel electrophoresis (PFGE) analysis
Preparation and digestion of genomic DNA, using SmaI restriction endonuclease (Boehringer-Mannheim), were as described by Murray et al.21 Electrophoresis was carried out in two steps using the CHEF DRII apparatus (Bio-Rad, Hemel Hempstead, UK). The pulse time was increased from 5 to 35 s over 35 h using 6 V/cm.
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Results |
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Strain origins and antibiotic susceptibility patterns are shown in Table I. All 42 isolates used in this study exhibited high-level gentamicin resistance (MIC > 1000 mg/L). The MICs of amikacin varied from mid-level to high-level resistance (MIC 64>1000 mg/L) as did the resistance to streptomycin (MIC 32>1000 mg/L). However, four E. faecalis isolates (SS14, SS28, SS36 and SS49) remained susceptible to streptomycin (MICs
1 mg/L). High-level resistance to kanamycin and paromomycin was observed in all E. faecalis isolates apart from strain SS49, which remained susceptible to paromomycin (MIC < 1 mg/L) but showed high-level resistance to kanamycin (MIC > 4096 mg/L).
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Plasmid analysis and hybridization studies with the aac6'-aph2'' probes
Plasmid analysis showed that the majority of the E. faecalis isolates harboured a single plasmid of 70 kb. However, some isolates possessed a second plasmid of 5 kb. The plasmid profiles of all 42 E. faecalis isolates are summarized in Table I. In some instances isolates collected from the same hospital did not necessarily show identical plasmid profiles. For example, although the eight isolates SS4 to SS11 collected from the same hospital all had a common c. 70 kb plasmid, a second plasmid of 5 kb could be identified only in isolates SS7, SS8 and SS11. Similarly, isolates SS42 and SS43 both had the common c. 70 kb plasmid but only isolate SS42 carried a second plasmid of 5 kb. Hybridization studies, using a aac6'-aph2'' probe (Gmr), indicated that the aac6'-aph2'' gene was exclusive to the 70 kb plasmid in all 42 E. faecalis clinical isolates.
Conjugation experiments
The gentamicin resistance marker was transferable in all 42 clinical isolates studied, but with varying transfer frequencies (104107 per recipient cell). Thirty-nine of the 42 clinical isolates were able to transfer the HLGR marker in association with the 70 kb plasmid. Transfer of the 5 kb plasmid was not observed from any isolate. In three isolates, SS8, SS42 and SS43, no plasmid DNA was detected in the transconjugants, but the Gmr probe hybridized to the chromosomal DNA. The aminoglycoside susceptibilities of the transconjugants were identical to those of the donor strains, except in the case of streptomycin. The majority of the transconjugants maintained the recipient strain streptomycin MIC (32 mg/L). However, the transconjugants of three isolates, strains TSS5, TSS12 and TSS32, acquired the donor strain streptomycin MICs.
Detection of IS256 and screening for a Tn5281-like transposon by L-PCR
IS256 was detected by PCR in all the strains studied. The isolates were examined for the presence of transposons by L-PCR. By this technique a 3.5 kb fragment of Tn5281 was amplified from strain HH22. Similarly, a 3.5 kb fragment was obtained for each of the 42 clinical strains examined. The L-PCR results for strains HH22, SS8, SS42 and SS43 are shown in Figure 1(a). Several faint bands visible following electrophoresis of PCR products may have been due to multiple copies of IS256 elements present in enterococci annealing to the L-PCR primers. Restriction endonuclease analysis of the L-PCR products with ScaI, HincII, HindIII and AluI restriction endonucleases gave digested fragments that were comparable to those obtained with the HH22-digested L-PCR products (Figure 1b
). However, again there were faint bands visible, possibly due to the presence of digested products from the faint bands observed in Figure 1
(a). The L-PCR products from the remaining 39 isolates were also subjected to restriction endonuclease analysis with these same four restriction endonucleases and gave patterns identical to those obtained from strain HH22.
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The IS256 forward primer, used for L-PCR, begins at position 520 of the IS256 element that flanks aac6'-aph2''. As a consequence, any variations that were present outside of the amplification region of Tn5281 would not be identified by L-PCR. Hybridization studies were undertaken, using the Gmr and IS256 probes, to identify the possibility of any variations being present in the regions not amplified by L-PCR.
The digested HLGR-conferring plasmids from SS8, SS42 and SS43 are shown in Figure 2(a) along with the autoradiographs following hybridization with the Gmr probe (Figure 2b
) and the IS256 probe (Figure 2c
). The restriction endonucleases were those commonly used to identify the Tn5281 transposon. The hybridization patterns represented by the principal bands were compared with those obtained with plasmid pBEM10. Faint bands were consistent with partial digestion of target DNA.
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The PFGE patterns were analysed as suggested by Tenover et al.22 Seven different restriction patterns were identified among the 42 E. faecalis isolates (Table I).
Clonal relatedness of aac6'-aph2'' plasmids
The 70 kb plasmids from all 42 E. faecalis strains were further analysed by restriction endonuclease digestion with EcoRI, PvuII, SalI, PstI or BamHI; these enzymes do not digest within Tn5281. There were five distinct restriction endonuclease digestion patterns noted, each with several band differences with each of the five restriction endonucleases used, suggesting that the 70 kb plasmids are not all clonally related. Each restriction enzyme used gave five different patterns and allowed the plasmids to be allocated to groups aI and aV. All plasmids from each of these groups had identical restriction patterns. In addition, hybridization studies on the 70 kb plasmid cut with enzymes that do not cut within Tn5281 and probed for IS256 were used as a means of typing the 70 kb plasmid. This IS256 typing method showed nine different profiles, which were allocated to groups bI to bIX. The results of the restriction endonuclease digestion and IS256 typing on the 70 kb plasmid are summarized in Table I.
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Discussion |
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Plasmid analysis showed that the majority of the isolates harboured a single plasmid of 70 kb. However, several isolates carried a second plasmid of 5 kb. Hybridization studies with the Gmr probe showed that in all cases aac6'-aph2'' was associated exclusively with the 70 kb plasmid. The function of the 5 kb plasmid is not clear. The 5 kb plasmid did not appear to be mobilizable, as it could not be detected in any of the transconjugants. There did not appear to be any significant differences between the MICs for isolates harbouring only the 70 kb plasmid and those harbouring both the 70 and 5 kb plasmids. Additionally, the aminoglycoside susceptibility profiles of the transconjugants, which harboured the 70 kb plasmids alone, were no different from the donor strains, which harboured both the 70 and 5 kb plasmids. It can be concluded that the 5 kb plasmid does not contain genes conferring aminoglycoside resistance
The gentamicin resistance marker was transferable by conjugation in all 42 isolates, with the transfer frequencies varying from 104 to 107 per recipient. These transfer frequencies are comparable to those previously reported for E. faecalis.1011 In 39 of the isolates aac6'-aph2'' was transferred in association with the 70 kb plasmid. However, in the transconjugants derived from three isolates, high-level gentamicin resistance was observed but plasmid isolation procedures failed to yield plasmid DNA. In hybridization studies the Gmr probe was seen to hybridize to chromosomal DNA of these three isolates, suggesting the presence of a conjugative transposon.
Although high-level resistance to gentamicin and most other aminoglycosides was observed in all the transconjugants, strain SS49 maintained the donor strain MIC of paromomycin and the majority of the transconjugants maintained the recipient strain MIC of streptomycin (32 mg/L). The transconjugants obtained from the mating of three of the donor strains, however, acquired the donor strain MICs of streptomycin. This would suggest that in these isolates aad6, which confers high-level resistance to streptomycin, may be part of the 70 kb plasmid.
Although the L-PCR method was successful in amplifying the 3.5 kb fragment of Tn5281, the L-PCR primers probably also annealed with other, non-Tn5281-related IS256 elements owing to the possible presence of multiple copies of IS256 elements in enterococci. This might explain the various-sized PCR products that appeared as faint bands. The L-PCR screening method confirmed that a transposon comparable to Tn5281 was present in all the HLGR E. faecalis isolates used in the present study. Restriction endonuclease analysis of the L-PCR products showed no significant differences between digested products obtained with strain HH22, isolated in the USA, and the 42 clinical isolates from the UK. This is further evidence that the UK E. faecalis isolates have a transposon that is comparable to Tn5281. In Tn5281 aac6'-aph2'' is flanked by IS256 elements in inverse orientation. The IS256 forward primer, used for L-PCR, begins at position 520 of the IS256 element that flanks aac6'-aph2''. As a consequence, any variations that may be present in the first 520 bp region of IS256 could not be identified by L-PCR. The Southern hybridization analysis identified two differences between the USA Tn5281 transposon and the UK Tn5281-like transposon. The first was seen when the isolates were probed with the IS256 probe following HindIII digestion. The differences in the HindIII patterns would also suggest that the Tn5281-like transposon in the UK E. faecalis isolates is not harboured on a plasmid similar to pBEM10. Figure 3(a) shows the restriction map of Tn5281 as it is found on pBEM10. The hybridization results shown in Table II
were used to construct a restriction map of the Tn5281-like structure identified in the UK E. faecalis isolates (Figure 3b
). As seen in Figure 3
(a), there is a single HindIII site in IS256; the second HindIII lies outside the Tn5281-like structure. Several previous studies have shown that aac6'-aph2'' is part of a heterogeneous group of plasmids of various sizes.8,11,24 As a consequence we would not expect the Southern hybridization patterns of the regions flanking the Tn5281-like structure to be comparable between the HLGR E. faecalis isolates studied. The differences cannot be due to missing HindIII sites on the Tn5281-like transposon, since the Gmr probe hybridized both to a 2.5 kb fragment of pBEM10 and to the 70 kb plasmids identified in this study.
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Our findings suggest that it is very unlikely that the aac6'-aph2'' gene in UK E. faecalis isolates is part of a truncated Tn5281-like transposon as described by Hodel-Christian & Murray,25 and Thal et al.10 The UK E. faecalis isolates studied show that both the 1.5 and 1.8 kb ScaI fragments are present. In the two studies in which the Tn4001-IS257 hybrids were identified, the ScaI restriction endonuclease sites were missing. Also, there is a HincII site located between the two HaeIII sites, which are c. 0.3 kb apart, in each of the IS256 elements. Southern hybridization patterns of the UK E. faecalis isolates showed that following HincII digestion the IS256 probe hybridized to a fragment of c. 0.3 kb. This indicates that the HincII site is present in UK Tn5281-like structures and it may be concluded that the UK Tn5281-like structure is not truncated. We have, however, recently reported a study of ten HLGR E. faecium isolates of which four harboured a Tn5281-like transposon, similar to the one described in the present study, and six carried a Tn5281-truncated structure.8
PFGE studies identified seven distinct patterns. The fact that isolates from the same geographical location did not necessarily belong to the same PFGE group suggests either that they were not clonally related or else that the isolates were collected in different time periods.
Using nine different restriction endonucleases, five of which do not cut with the Tn5281 transposon, to type the 70 kb plasmid, the digestion patterns showed that the 70 kb plasmid in E. faecalis could be classified into five groups, aI to aV. However, in typing the 70 kb plasmid using the IS256 typing method the plasmids could be separated into nine different groups, bI to bIX. The restriction endonuclease digestion patterns were difficult to interpret due to the close proximity of many of the bands, and consequently misinterpretation is more likely. The IS256 typing method proved to be more discriminatory. The plasmid typing results are consistent with previous reports that in E. faecalis the aac6'-aph2'' gene is on a heterogeneous group of plasmids.12,24 It was noted that the plasmid type had no correlation with PFGE patterns. This is contrary to what we previously reported in E. faecium8 and further emphasizes the fact that in the UK there is a heterogeneous group of E. faecalis isolates habouring a heterogeneous group of 70 kb plasmids carrying the Tn5281-like transposon.
The association of aac6'-aph2'' with a conjugative transposon is of concern, since this will assist in the rapid dissemination of the AAC6'-APH2'' AME. The data presented in this study suggest that in HLGR strains of E. faecalis isolated in the UK the aac6'-aph2'' gene is predominantly part of a transposon that appears to be closely related to the USA Tn5281 transposon. However, the UK Tn5281-like transposon has the HaeIII sites of Tn5281 missing. This study demonstrates that there may be genetic diversity in the HLGR-conferring Tn5281 transposon.
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Acknowledgments |
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Notes |
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References |
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Received 24 February 1999; returned 7 July 1999; revised 25 August 1999; accepted 27 November 1999