1 Division of Botany and Zoology, School of Life Sciences, Daley Rd, Australian National University, Canberra, ACT 0200, Australia
2 Department of Microbiology and Infectious Diseases, The Canberra Hospital, Garran, Canberra, ACT 2605, Australia
Correspondence
David Gordon
David.Gordon{at}anu.edu.au
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ABSTRACT |
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INTRODUCTION |
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Levin (1995) made two predictions regarding the evolution of multi-resistance plasmids. Where two incompatible plasmids are simultaneously selected for, he argues that new plasmids will arise by transposition and predicts that the position of resistance genes in otherwise identical plasmids will be highly variable. Where selection for the co-transfer of compatible resistance plasmids is involved, he argues that new multi-resistance plasmids will arise through co-integration and predicts the occurrence of resistance genes in plasmids with multiple replicons that can be identified as the co-integrates of other plasmids.
Both co-integration and transposition have been implicated in empirical studies of plasmid evolution (Berg et al., 1998; Bradley et al., 1986
; Guessouss et al., 1996
; Mitsuhashi et al., 1977
; Schwarz et al., 1996
; Sohail & Dyke, 1995
; Venkatesan et al., 2001
; Woodward et al., 1990
). However, a range of other mechanisms, including recombination and the acquisition of integron cassettes, have also been observed (Boerlin, 1999
; Boyd et al., 1996
; Brown et al., 2000
; Lindler et al., 1998
; Prentice et al., 2001
; Radstrom et al., 1991
; Venkatesan et al., 2001
). While several individual plasmids have been the subject of intensive study, there is limited information available regarding plasmid populations (Blazquez et al., 1996
; Boyd et al., 1996
; Brown et al., 2000
; Carattoli, 2003
; Carattoli et al., 2001
, 2002
; Groves, 1979
; Ling et al., 1993
; Petit et al., 1990
; Preston et al., 2003
; Radstrom et al., 1991
; Saksena & Truffaut, 1992
; Tosini et al., 1998
). To date we know of no study that has attempted to infer the processes underlying plasmid evolution through an investigation of the genetic relationships within a large collection of single- and multiple-resistance plasmids.
Escherichia coli is a prime candidate as a species in which new multi-resistance plasmids may evolve. It is a common enteric commensal of mammals and a common cause of human infection. As such, E. coli strains are routinely exposed to a wide range of antimicrobial agents. E. coli also has a very wide natural distribution (Selander et al., 1987) and a propensity for plasmid carriage (Sherley et al., 2003
). Resistance to tetracycline, chloramphenicol or trimethoprim is relatively common in clinical pathogens in Australia, including E. coli (Bell & Turnidge, 1995
), and is frequently plasmid-mediated (Neu, 1992
). We studied a collection of single- and multi-resistance plasmids isolated from tetracycline-, chloramphenicol- and/or trimethoprim-resistant clinical isolates of E. coli. The plasmids were screened to determine their antibiotic resistance profiles, the genetic relatedness of both the plasmids and their host bacterial strains was determined, and an attempt was made to infer the process by which multiple resistance had evolved within this population.
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METHODS |
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Resistance plasmids were transferred from the resistant isolates to a plasmid-free, rifampicin (200 µg ml1)-resistant, laboratory strain of E. coli K-12 (J53) by mating (conjugation) using rifampicin+tetracycline, rifampicin+chloramphenicol or rifampicin+trimethoprim as selective agents as appropriate. All matings were carried out both in broth culture (Miller's LB broth; Bacto) without shaking and on solid agar plates (tetrazolium-lactose agar; Levin et al., 1979). Putative transconjugants were restreaked onto fresh selective plates and their identity was confirmed on the basis of biochemical profiles. Pure cultures were stored at 70 °C in 6·25 % (v/v) glycerol. The plasmid content of the transconjugants was electrophoretically compared with that of the donors using a modified in-well lysis technique (de Souza et al., 1998
). All transconjugants were also subjected to alkaline-lysis plasmid isolation (Sambrook et al., 1989
) and transconjugants containing multiple plasmids were excluded from the study.
Plasmid characterization.
To determine the resistance profiles conferred by the plasmids, the transconjugant cells were screened for sensitivity to 10 antimicrobials [chloramphenicol, 30 µg ml1; tetracycline, 30 µg ml1; trimethoprim, 5 µg ml1; neomycin, 30 µg ml1; kanamycin, 30 µg ml1; streptomycin, 10 µg ml1; spectinomycin, 100 µg ml1; gentamicin, 10 µg ml1; sulfisoxazole, 0·25 µg ml1; ampicillin, 10 µg ml1 (Bacto)] using a soft-agar disc diffusion method. Briefly, 100 µl of a fresh overnight culture in Miller's LB broth was inoculated into 3 ml soft agar (0·6 %, w/v, in Miller's LB broth), vortexed and decanted onto the surface of a Miller's LB agar plate. Antimicrobial-impregnated filter discs (Bacto) were then deposited onto the surface of the plate, which was incubated overnight. The diameter of the zones of inhibition surrounding the antimicrobial discs was measured. Isolates were deemed resistant only when the zone of inhibition was less than or equal to the resistance breakpoint recommended by the manufacturer (intermediate and sensitive strains were scored as sensitive). The plasmid-free host strain was included as a sensitive control.
A PCR-based method was used to assign incompatibility groupings to the plasmids by determining the presence or absence of incA/C, incFII, incN, incP and incW replicons, as described previously (Sherley et al., 2003). Plasmid sizes were determined by electrophoretic comparison of native plasmid DNA isolated using alkaline lysis (Sambrook et al., 1989
) with plasmids of known size (R388, 31·8 kbp; R136, 62·1 kbp; pIP40a, 145·5 kbp; MIP233, 227·3 kbp).
To determine the degree of genetic similarity between resistance plasmids, they were isolated from the transconjugant cells by alkaline lysis, then subjected to restriction fragment length polymorphism (RFLP) analysis using BamH1, EcoRI, HindIII, PstI and SacI (Roche). Relatedness was determined by pair-wise comparison of banding patterns following restriction digestion and electrophoresis. Given the large degree of variability observed, we chose to score plasmid pairs as identical, highly similar, similar or dissimilar. For each restriction enzyme, identical plasmid pairs were scored as 0, plasmid pairs that were non-identical but shared >50 % of digest bands were scored as 1, plasmid pairs that shared <50 % of digest bands were scored as 2, and plasmid pairs with no shared bands were scored as 3. The scores for each enzyme were then pooled to give an overall relatedness score, ranging from 0 to 15 for every plasmid pair. These data were graphically represented using Unweighted Pair Group Matching Analysis (Rohlf, 1993). A pair-wise comparison of the observed antimicrobial resistance profiles was also determined for each plasmid pair (Jacard scores).
Strain characterization.
The genetic relationships between the donor strains of E. coli were determined by multi-locus enzyme electrophoresis (MLEE) as described previously (Gordon & Lee, 1999). Only strains from which plasmids were successfully isolated were included in this analysis. Thirteen donor strains from The Canberra Hospital perished during storage and were also excluded.
Statistical analyses.
The correlations between the RFLP data, resistance profile data and MLEE data were determined from their respective Euclidean/Jacard distance matrices using a Mantel test (Mantel, 1967). The relationship between individual antimicrobial resistances and the plasmid RFLP data was also determined from their respective distance matrices using a Mantel test. The correlation between pairs of antimicrobial resistances was determined using a Spearman correlation (Sokal & Rohlf, 1969
).
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RESULTS |
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Plasmid incompatibility
PCR-based screening for conserved regions of the incA/C, FII, N, P and W replicons successfully identified 61 % of the plasmids as belonging to one or more of these incompatibility groups. The vast majority (51 % of all plasmids isolated) belonged to incFII, with only a handful of isolates belonging to incN or A/C, and no plasmids belonging to incP or W (Fig. 1). Two clinical resistance plasmids were identified as having multiple replicons: pME001 from Melbourne was identified as incFII, incN and incA/C, while pSE019 from Sydney was identified as incFII and incN.
It is possible that some of the plasmid incompatibility screening results were false negatives, as described by Gotz et al. (1996). We optimized the conditions for each primer pair so that there were no false-negative and no false-positive reactions detected for either of the following two control sets: (i) the set of 20 incompatibility replicon typing probes described by Couturier et al. (1988)
; and (ii) a set of 18 wild-type plasmids, including R805a (IncBI2), pIP40a (IncC), R1 (IncFII), R1drd19 (IncFII), R136 (IncFII), R16 (IncFII), R6 (IncFII), R27 (HI1), R478 (HI2), MIP233 (IncHI3), TP114 (IncI2), R1215 (IncM), R390 (IncN), R1010 (IncN), R751 (IncP), R934 (IncP), pHH1307 (IncW) and R388 (IncW).
The distribution of antimicrobial resistance genes
Plasmid-mediated resistance was observed for all ten of the antimicrobials tested (ampicillin, chloramphenicol, gentamicin, kanamycin, neomycin, streptomycin, spectinomycin, sulfisoxazole, tetracycline and trimethoprim). Resistance was more common to some antimicrobials than others, while the number of antimicrobials that a given plasmid mediated resistance to ranged from one to nine, with the majority of plasmids determining resistance to either three or six antimicrobials (Fig. 2).
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Some discrepancies between the RFLP and resistance data reflect differences in resistance profile that were not observed at the DNA level. In some cases plasmids with identical RFLP profiles differed by the loss or gain of resistance to a single antibiotic. For example, while pCE001 and pCE003 appeared identical from their restriction profiles, pCE003 was not sulfisoxazole-resistant and pCE001 was. Similarly, pSE013 and pSE014 differed only by the presence or absence of tetracycline resistance (Fig. 1).
The relationship between individual antimicrobial resistances and the RFLP data
The relationships between individual antimicrobial resistance markers and the RFLP data were determined using Mantel analysis. Ampicillin, chloramphenicol, neomycin, kanamycin, spectinomycin and tetracycline resistance all correlated significantly with the RFLP data (P<0·05; Fig. 3). In contrast, gentamicin, streptomycin, sulfisoxazole and trimethoprim resistance did not correlate significantly with the RFLP data, suggesting more frequent horizontal movement of these resistance genes. This division between antimicrobial resistances agrees well with the above finding that tetracycline, chloramphenicol, kanamycin and neomycin resistance correlated significantly with each other, as did sulfisoxazole, trimethoprim and streptomycin resistance.
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DISCUSSION |
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These results are in strong agreement with a previous study of conjugative plasmids in environmental isolates of E. coli. A sequence-based analysis of incompatibility group FIA, FIB and FII plasmids carried by strains from the ECOR reference collection (Boyd et al., 1996) found that recombination played a major role in the evolution of these plasmids. Furthermore, the phylogenetic relationships between the plasmids (based on multiple gene sequences) differed from those of the host cells, implying a high rate of plasmid transfer even between the major ECOR groups. Other authors have also highlighted the importance of recombination in the evolution of plasmids from various members of the Enterobacteriaceae (Blazquez et al., 1996
; Boerlin, 1999
; Petit et al., 1990
; Preston et al., 2003
). However, some authors have found that transposons and integron cassette systems are major mediators of resistance plasmid evolution in the Enterobacteriaceae, rather than recombination (Brown et al., 2000
; Carattoli et al., 2002
; Guessouss et al., 1996
; Radstrom et al., 1991
; Tosini et al., 1998
).
In our plasmid population, tetracycline, chloramphenicol, kanamycin and neomycin resistance correlated significantly with one another and with the plasmid RFLP data. This suggests that the plasmids carrying these markers have a shared genetic history. That is to say that tetracycline, chloramphenicol, kanamycin and neomycin resistance appear primarily to be transferred vertically or via large-scale recombination events. Resistance to trimethoprim, sulfisoxazole and streptomycin also tended to co-occur. However, resistance to these antimicrobials did not correlate significantly with the plasmid genetic backbone, suggesting more frequent horizontal movement of the genes conferring resistance to these agents.
The finding that trimethoprim, sulfisoxazole and streptomycin resistance are horizontally mobile and also co-occur would suggest association with transposable genetic elements. Furthermore, the association between the sulI sulphonamide resistance gene and class I integrons in the Enterobacteriaceae (Radstrom et al., 1991), and the frequent association of streptomycin and trimethoprim resistance gene cassettes with these integrons (Radstrom et al., 1991
; White et al., 2001
) strongly suggest that the correlation between these resistances may reflect resistance-cassette arrays within integrons. It is worth noting that it has previously been argued that integrons and their associated cassettes are more likely to move as a group than as independent units (Martinez-Freijo et al., 1999
).
Despite the fact that our results indicate a role for both recombination and transposition events, neither of Levin's predictions for the mechanisms of resistance plasmid evolution (Levin, 1995) fit these data well. We found a large bias in terms of plasmid incompatibility grouping in this plasmid set, but did not find a correspondingly high degree of genetic similarity between the plasmids. Nor did we find evidence that resistance genes had primarily been moving horizontally within the plasmid population. On the contrary, a significant correlation between some antimicrobial resistances and the plasmid backbone (as represented by RFLP data) argues strongly for vertical transmission of the corresponding resistance genes. We did observe some plasmids with multiple replicons, as would be expected where plasmid co-integration had occurred. However, if multi-resistance plasmids in E. coli had been evolving through the co-integrative capture of smaller resistance plasmids we would expect a strong correlation between the size of a plasmid and the number of antimicrobials that it mediated resistance to. No such relationship existed, nor was it evident from the plasmid restriction profiles that any plasmids had co-integrated other members of this plasmid population.
In the case of the plasmid pSE019 (incFII and incN) a highly related plasmid, pSE020 (incFII), that carried two fewer resistance markers (for sulfisoxazole and trimethoprim resistance) was isolated from the same host cell and is almost certainly a breakdown product of pSE019. The question then is whether pSE020 arose through the loss of a co-integrated incN plasmid bearing sulfisoxazole and trimethoprim resistance genes, or through some other means. The most serious argument against pSE019 being a co-integrate is that both pSE019 and pSE020 were very similar in size (90 kbp). They most certainly did not differ by the 2030 kbp that might be expected as a minimum for the size of a second conjugative plasmid. Some mobilizable plasmids transfer by co-integration into conjugative plasmids (Riemmann & Haas, 1993
), so there is the possibility that co-integration of a very low weight mobilizable plasmid is responsible for these results, a solution that would also explain the absence of any similar conjugative incN plasmids.
The stable co-integration of a small mobilizable plasmid and a conjugative plasmid has previously been described in a member of the Enterobacteriaceae (Mitsuhashi et al., 1977), as has the co-integration of relatively small conjugative plasmids resulting in co-integrates no larger than 140 kbp (Chu et al., 2001
). However, co-integration is rarely reported in the Enterobacteriaceae and in at least two cases large plasmid co-integrates have been observed to be unstable (Bradley et al., 1986
; Woodward et al., 1990
). Co-integrate plasmids may be unstable for various reasons. Some incompatibility groups have temperature-dependent transfer systems, so a co-integrate containing one replicon that is temperature-sensitive and a second that is not may be unstable outside of a fixed temperature range (Bradley et al., 1986
). Bacterial species differ significantly in the number and size-range of the plasmids they carry (Sherley et al., 2003
), suggesting that cellular factors in some way limit plasmid size. The presence of multiple replication regions on a single plasmid may also be directly destabilizing in a host species with a tendency to carry multiple plasmids as incompatibility between a plasmid with multiple replicons and a second plasmid can result in the loss of genetic material, including the incompatible replicon, from the hybrid plasmid (Coetzee et al., 1975
).
Whatever the means by which plasmids are evolving at a genetic level, the unequal distribution of plasmid incompatibility groups has important implications. Evolution via the co-selection of incompatible plasmids (Condit & Levin, 1990) requires only two selective pressures, and these need not both be antimicrobial resistance genes. Several factors such as virulence determinants, adhesins, heavy metal resistance determinants and genes involved in the metabolism of unusual substrates are also found on plasmids. Furthermore, according to the local optimization model (Eberhard, 1990
) all plasmids are actively selected for within their own environmental niche. As a result, treatment with a single antimicrobial may be sufficient to select for the movement of genes mediating resistance to that antimicrobial onto other actively selected plasmids. Moreover, selection for horizontally mobile genes such as sulfonamide, streptomycin and trimethoprim resistance genes, will increase the opportunity for associated mobile genetic elements to become involved in the evolution of multi-resistance plasmids, hence increasing the overall rate of horizontal gene movement and particularly the movement of clusters of genes.
In conclusion, these clinically derived plasmids do not belong to distinct plasmid lineages. They exhibit evidence of broad-scale inter-plasmid gene transfer, probably involving a range of mechanisms, including recombination, transposition and integration. The lack of correlation between plasmids and their hosts would suggest that horizontal plasmid transfer is common in clinical E. coli strains, while the substantial bias towards incFII plasmids increases the opportunity for plasmid evolution to occur via exposure to a minimum number of selective forces.
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ACKNOWLEDGEMENTS |
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Received 18 September 2003;
revised 19 December 2003;
accepted 26 January 2004.
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