Enhancement of host fitness by the sul2-coding plasmid p9123 in the absence of selective pressure

Virve I. Enne1,2,*, Peter M. Bennett1, David M. Livermore3 and Lucinda M. C. Hall2

1 Bristol Centre for Antimicrobial Research, Department of Pathology and Microbiology, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD; 2 Department of Medical Microbiology, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, Turner Street, London E1 2AD; 3 Antibiotic Resistance Monitoring & Reference Laboratory, Specialist and Reference Microbiology Division, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK

Received 17 December 2003; returned 14 January 2004; revised 24 February 2004; accepted 2 March 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Despite a 97% reduction in clinical sulphonamide usage, the prevalence of sulphonamide resistance among Escherichia coli has remained constant in the UK. Genetic linkage of sulphonamide resistance to other resistances is thought important for this maintenance, but the finding also implies that sulphonamide resistance exerts little fitness cost. To test this hypothesis, we examined the fitness impact of four naturally occurring sul2-coding plasmids upon their hosts.

Methods: The fitness impact of the plasmids upon E. coli was determined by pairwise growth competition in a minimal medium. The DNA sequence of plasmid p9123 was obtained by primer walking and PCR.

Results: Three of the four sul2-coding plasmids studied imposed fitness costs on their hosts. The fourth plasmid, a 6.2 kb resistance element carrying sul2, strA and strB designated p9123, conferred a 4% fitness advantage upon its original clinical host and also on E. coli K12 JM109. The complete sequence of p9123 revealed eight open reading frames, including five of unknown function. There was no obvious gene to which the fitness advantage might be attributed.

Conclusions: The novel finding that p9123 can improve host fitness may explain why this plasmid and its close relatives are so widespread among enteric bacteria. In addition to other factors such as co-selection of sulphonamide resistance by other agents, the fitness advantage conferred by plasmids such as p9123 may have contributed to the maintenance of sulphonamide resistance in the UK in the absence of clinical selection pressure. These data indicate that once antibiotic resistance has been established on mobile genetic elements, it may be difficult to eliminate.

Keywords: fitness gain, Escherichia coli, sulphonamides


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Widespread antibiotic use since the middle of the last century has created a powerful selection force driving the evolution of drug-resistant bacterial pathogens.1 This development now seriously threatens the efficacy of antibacterial agents. It has been suggested that it may be possible to reduce the prevalence of established antibiotic resistance by reducing use of the agent(s) concerned.2,3 Such a reduction would however depend upon antibiotic resistance exerting a fitness cost on bacteria in the absence of drug selection, either by reducing the efficiency of a reaction of vital importance to the cell or by imposing an additional metabolic load. Most studies on the fitness cost of antibiotic resistance have concentrated on resistance acquired by mutation of chromosomal genes.3 In general, a fitness cost has been observed,4,5 although some no-cost mutations have been documented.6 Furthermore, after a period of host adaptation, second-site compensatory mutations often arise that reduce or eliminate the cost of the first mutation.46 Fewer studies have examined the fitness costs associated with acquired resistance genes. Several resistance plasmids have been shown to impose a fitness cost upon their hosts.710 However, work with pBR322 and pACYC184 indicates that bacteria can develop chromosomal compensatory mutations during a period of co-evolution, after which the plasmid-carrying strain becomes fitter than the plasmid-free parent strain.7,10

Most studies examining the fitness costs associated with plasmid-borne resistance determinants have used cloning vectors and/or laboratory strains and studies investigating natural plasmid–host interactions are lacking3 as are data relating fitness cost of resistance to prescribing and resistance gene prevalence data.

As a consequence of a national prescribing restriction11 that prompted a switch from co-trimoxazole use to trimethoprim only use, the UK experienced a 97% reduction in clinical sulphonamide use during the 1990s.12 Nevertheless, the prevalence of sulphonamide resistance among clinical Escherichia coli isolates remained constant, at ~40–45%.12 Linkage of sulphonamide resistance genes to determinants conferring resistance to antimicrobials that are still commonly used is thought important for this maintenance, but other factors may also be important. In this context, it is notable that the prevalence of one of the genes responsible for resistance, sul2, increased during the period of falling sulphonamide use, indicating continued selection.12 One factor that could influence the prevalence of sul2 is the fitness cost of sul2-coding plasmids on their hosts. Hence, we examined the fitness impact of four sul2-coding plasmids identified in our previous study.12


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and plasmids

Four plasmids encoding sul2 obtained from clinical E. coli isolates from East London, and forming part of a study on the maintenance of sulphonamide resistance,12 were selected for investigation (Table 1). Two were small and non-conjugative and two were large and conjugative. E. coli K12 JM109 was used as a naive host for some of the competition experiments.


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Table 1. Characteristics of the clinical E. coli isolates, study plasmids and the fitness impact of the plasmids upon their original clinical host strains and E. coli K12 JM109
 
Construction of bacterial strains for fitness assays

Curing of plasmids from their clinical hosts was attempted by several methods described elsewhere.13 Plasmid-containing wild-type strains were grown at 42°C in nutrient broth containing 80 mg/L Acridine Orange at pH 7.6 for 24 h, in nutrient broth containing 10 mg/L rifampicin at pH 7.6 for 24 h or in brain heart infusion broth containing 10% (w/v) sodium dodecyl sulphate for 48 h. These cultures were plated onto nutrient agar and incubated at 37°C overnight. Colonies were replicated onto nutrient agar containing 1 mg/L sulfamethoxazole, to detect loss of the sul2 plasmid.

Plasmids were transformed into electrocompetent E. coli JM109, 91-23 or 99-38 cells by standard procedures.14 Strain construction was verified by plasmid extraction14 and visualization by agarose gel electrophoresis and by PCR amplification of the sul2 gene.15

Rifampicin-resistant derivatives of the study bacteria were selected by plating 107 cfu from overnight nutrient broth cultures onto nutrient agar containing 30 mg/L rifampicin. For E. coli 99-38, which was already rifampicin-resistant, cultures were grown overnight in nutrient broth containing 10 mg/L nalidixic acid and then plated onto nutrient agar containing 200 mg/L nalidixic acid, to isolate nalidixic acid-resistant derivatives.

Screening of experimental plasmids for toxin production

Because plasmid-encoded production of a toxin (e.g. a colicin or a microcin) might interfere with the competition assays, transformants were screened for the production of a bacteriocin. The plasmid-carrying strain was streaked as a strip across a nutrient agar plate, which was incubated overnight at 37°C. A filter paper disc soaked in chloroform was then placed in the lid of the Petri dish for 15 min to kill the bacteria. The plasmid-free strain was streaked at right angles to the original line of growth. The plate was incubated at 37°C overnight. An uninterrupted line of growth of the plasmid-free strain across the plate was taken to indicate that no toxin was produced, whereas a break in the line at the point of intersection implied plasmid-encoded bacteriocin production.

Experiments to assay fitness

Pairwise growth competition in Davis minimal medium (DM) containing glucose (25 mg/mL), to assay the fitness impact of sul2-coding plasmids, was carried out by a modification of the method of Lenski et al.7 Isogenic bacteria with and without a sul2-coding plasmid were mono-cultured overnight in nutrient broth, inoculated at a dilution of 1:104 into DM and grown separately for 24 h. The two cultures were then mixed at a volumetric ratio of 1:1 and the mixture diluted 1:100 into DM. After 24 h of growth, the mixed culture was re-diluted 1:100 into fresh DM. This dilution step was repeated daily for a total of six transfers. After each 24 h period, including the initial day when the strains were grown separately, the cultures were diluted appropriately and 100 µL volumes were spread in triplicate onto Iso-Sensitest agar (Oxoid) with and without 1000 mg/L sulfamethoxazole. Following overnight incubation of the plates at 37°C, the colonies were counted. The mean number of colonies on the agar containing sulfamethoxazole was subtracted from the mean number of colonies on the agar lacking sulfamethoxazole to determine the number of plasmid-free cells in the mixed population. Six replicates of each competition experiment were carried out. The percentage per generation fitness impact of each plasmid was estimated as described by Lenski et al.7 and Reynolds.6

To estimate the extent of plasmid transfer during a competition experiment, a parallel experiment, using a rifampicin- or nalidixic acid-resistant derivative of the plasmid-free competitor strain was carried out. In addition to plating appropriate dilutions onto Iso-Sensitest agar with and without 1000 mg/L sulfamethoxazole, undiluted culture was also plated onto Iso-Sensitest agar containing 1000 mg/L sulfamethoxazole and 30 mg/L rifampicin or 200 mg/L nalidixic acid. Any transfer of plasmid to the plasmid-free rifampicin- or nalidixic acid-resistant competitor would yield colonies on these plates. The experimental design does not rule out the possibility that colonies recovered may represent spontaneous mutation to rifampicin or nalidixic acid resistance by the plasmid-carrying competitor, but these arise only at very low frequencies (10–7 for rifampicin and 10–9 for nalidixic acid) that would not compromise the main findings.

The single culture growth characteristics of E. coli 91-23, E. coli 91-23 cured of p9123, E. coli JM109 and E. coli JM109 transformed with p9123 were examined by monitoring their OD600 in nutrient broth over 24 h. Six replicate cultures per strain were monitored. Generation times were compared by carrying out paired t-tests.

DNA sequencing of p9123

Plasmid p9123 was isolated with a Qiaprep Spin Miniprep kit (Qiagen) according to the manufacturer’s instructions. Sequencing primers were initially designed to target both extremities of the sul2 gene. Purified plasmid DNA (1 µg/reaction) was sent to the Advanced Biotechnology Centre (ABC), Imperial College, London, where sequencing was carried out on an ABI 3100 automated DNA instrument using a BigDye terminator kit (Applied Biosystems). Further sequencing primers were then designed to target the newly obtained sequences and the sequence was extended. This reiterative process was repeated until the entire sequence of the plasmid had been obtained. Segments of the plasmid were then amplified by PCR, using primers based on the preliminary sequence. The PCR mixture consisted of 25 µL of ReadyMix Taq PCR reaction mixture (Sigma), 23 µL of H2O, 0.5 µL of each primer (0.2 µM) and 1 µL of template DNA. Amplification was carried out for 5 min at 95°C for 1 cycle, then for 1 min at 95°C, 1 min at 53–58°C and 1 min at 72°C for 35 cycles, followed by 1 cycle at 72°C for 10 min. Amplification products were purified with a QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions and then sent for sequencing at ABC. Each plasmid segment was sequenced at least twice on both strands. Sequence analysis was carried out using the Lasergene DNASTAR software package. BLAST searches were carried out to identify similarities with known DNA sequences and sequence alignments were carried out using ClustalW. A drawing of the plasmid was produced using pDRAW32. The complete sequence of p9123 has been deposited in the GenBank database under the accession number AY360321.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fitness impact of sul2-coding plasmids upon their hosts

Plasmid-containing and non-containing isogenic pairs were generated by curing wild-type strains of their plasmids and by plasmid transfer into E. coli JM109. The fitness impacts of p9123 and p9938 upon their original clinical E. coli host strains were measured by competing the primary isolates against isogenic derivatives cured of the respective plasmid by Acridine Orange treatment. The clinical host strains were assumed to have shared an evolutionary history with the plasmids carried by them, i.e. they were assumed to be adapted to carriage of the specific plasmid. The curing rates were 55% for p9123 and 92% for p9938. Attempts to cure the clinical isolates of the large sul2-coding plasmids p9118 and p9919 were not successful.

Each of the four study plasmids was successfully transferred into E. coli K12 JM109 and the transformants were competed against plasmid-free JM109 (Table 1). JM109 was assumed to be naive with respect to the plasmids, i.e. it had not shared an evolutionary history with them and was therefore not adapted to their carriage.

Plasmids p9118 and p9919 both imposed a fitness cost on JM109. The fitness cost of p9919 could not be measured accurately because, for three independent transformants tested, the acquisition of plasmid inhibited growth of E. coli JM109 in DM, reflecting a significant cost imposed by p9919. Plasmid p9118 imposed a 5% fitness cost upon E. coli JM109. Likewise, p9938 imposed fitness costs on both its original clinical host and E. coli JM109. In four of six replicate competition experiments with the cured clinical host strain, the proportion of plasmid-carrying clinical isolate in the mixed culture declined steadily throughout the experiment. However, in two of the replicates, the plasmid-carrying strain, after initially being out-competed, gained a fitness advantage over its plasmid-free competitor after 20 generations of mixed growth. The competition dynamics for these two replicates, as well as for one demonstrating a stable fitness cost of p9938 carriage are shown in Figure 1. At present, we have no explanation for the reversal of the cost-benefit of p9938 carriage after 20 generations, or for why this was seen in only two of six cases.



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Figure 1. Dynamics of replicate competition experiments for E. coli 99-38 with and without p9938. The graphs show the ratio of sulphonamide-resistant (sulR) over sulphonamide-susceptible (sulS) cfus over time. Replicate F is representative of the four experiments where the cured derivative outgrew the parent, whereas replicates A and D represent cases in which the plasmid-containing strain gained an advantage over its cured derivative during the course of the experiment.

 
In marked contrast to the results described above, the second small plasmid, p9123, improved the fitness of both its original host and E. coli JM109, by approximately 4% per generation in each case. Thus, whether the host strain was plasmid-adapted or not appeared not to influence the competitive edge conferred by p9123. The growth of E. coli 91-23 and JM109, with and without p9123, was monitored in single culture to determine at which stage of the bacterial growth cycle p9123 might be conferring the observed fitness advantage. No differences were seen in lag or stationary phase, however, the two p9123 carrying strains had generation times approximately 3% shorter than those of their plasmid-free counterparts. Although this result was not statistically significant (P = 0.2640 for E. coli 91-23, P = 0.0764 for E. coli JM109), shorter generation times for the plasmid-carrying strains were found consistently.

Control experiments demonstrated that transfer of the p9118 plasmid may have occasionally occurred in the competition experiments, but at a maximum frequency of 1 transfer event per 106 p9118-carrying cells. Plasmid transfer is therefore too infrequent to contribute significantly to the change in competitor ratio observed during competition assays. None of the four plasmids studied determined production of a colicin or microcin, indicating that the findings reflect real fitness effects of each of the plasmids on its host, rather than the elimination of the competing strain due to the production of a bacteriocin. Plasmid curing by Acridine Orange treatment might potentially mutate the host chromosome, which could influence the outcome of competition experiments. To control this aspect, p9123 and p9938 were transformed back into the clinical hosts from which they were cured, and one competition experiment was carried out, per pairing. In both cases, the fitness impacts observed were within the range of those observed for competition between the original plasmid-carrying isolate and its cured derivative (+4.3% for 91-23 and –7.8% for 99-38).

Analysis of the p9123 sequence

The complete sequence of p9123 was determined. The plasmid was 6222 base pairs in size and its genetic structure is depicted in Figure 2. In silico restriction analysis indicated a close relationship to pBP1,16 a small plasmid conferring streptomycin and sulphonamide resistance that was prevalent among E. coli in the 1970s and 1980s,16 and pSSOJO1, a plasmid isolated from E. coli in different locations in south-western Nigeria.17 Comparative analysis showed that p9123 is 71% homologous to pKKTET7 from Shigella sonnei. The main difference between the two plasmids is that pKKTET7 also possesses the tetracycline resistance genes tetA-1 and tetR. Discounting these two genes, the plasmids are 99% identical. Plasmid pSSTA-1, identical in size to pKKTET7, also seems closely related as the known sequence is identical to pKKTET7 and the remainder accommodates the strA, strB and sul2 genes.18



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Figure 2. Genetic map of p9123 showing the position and direction of genes, putative open reading frames and the position of the putative origin of vegetative replication.

 
The p9123 plasmid had eight putative open reading frames (ORFs) >400 bp in size. The plasmid encoded the sulphonamide resistance gene sul2, which was identical to that of the IncQ plasmid RSF1010.19 The p9123 plasmid also encoded the strA–strB gene pair, which specify the APH-3'' and APH-6 streptomycin phosphotransferase enzymes conferring streptomycin resistance. The strA gene sequence differed by two nucleotides from that of RSF1010 but was identical to the sequence of the strA gene specified by Tn5393.20 The strB nucleotide sequence was identical to that on RSF1010.

Among five unidentified ORFs, ORF1 encoded a hypothetical protein of 179 amino acids. The first 135 amino acids were 98% identical to the N-terminus of orfA, encoding a putative transposase and carried on a plasmid specifying florfenicol resistance in E. coli.21 ORF2 encoded a hypothetical protein of 129 amino acids which showed no similarity to any protein of known function. ORF3 encoded a 162-amino-acid protein exhibiting 52% identity to Exc1, encoded by the plasmid ColE1.22 The exc1 gene was originally proposed as one of two genes responsible for the entry exclusion function of ColE1,23 but this hypothesis was later discounted when the function was attributed to the mbeD gene.24 ORF4 encoded a 148-amino-acid hypothetical protein, which was not related to any protein of known function. ORF5 encoded a 147-amino-acid protein, the carboxy terminal of which showed 78% identity to an open reading frame on ColE1 coding a 13.8 kDa protein of unknown function.22

The putative replication region of p9123 overlapped ORF5 and was 84% identical to the 830 bp replication region of the ColE1-like plasmid p15A.25 The presumed replication origin of p9123 was identical to that of p15A25 and was located at nucleotide 5393.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Three of four sul2-encoding plasmids examined reduced the competitive fitness of their hosts. Interestingly, each of these plasmids encoded resistance to at least one antibacterial agent still in common clinical use, suggesting that they have been maintained in the E. coli population through use of these agents. Moreover, two of the plasmids, p9118 and p9919, could not be cured from their host strains, a factor that may contribute to their stability in the E. coli population.

In contrast, p9123 did not encode resistance to any commonly used antibacterial agents. Accordingly, its survival cannot be attributed to drug selection. Instead it may be maintained because it can improve host fitness. This advantage is not due to the production of a toxic compound encoded by the plasmid but rather an improvement of the inherent fitness of the host strain. Surprisingly, and in contrast to previous findings on the fitness costs of antibiotic resistance plasmids,7,10 p9123 conferred an immediate fitness advantage upon its hosts, in the absence of plasmid–host adaptation as well as in its presence. The results described here suggest that the enhanced fitness observed is due to an increase in growth rate conferred by plasmid carriage.

The fact that plasmids closely related to p9123 are widespread further testifies to its success. The plasmid is very closely related to pBP1, which was prevalent in the E. coli population in the 1970s and 1980s,16 and to pSSOJO1, which was recently found to be prevalent in south-western Nigeria.17 The plasmid pSSTA-1, to which p9123 is also related is widely distributed among enteropathogenic bacteria worldwide.18 The present results with p9123 therefore may have a broad relevance.

Analysis of the p9123 sequence revealed no gene product that will obviously enhance the fitness of its host, although five putative genes of unknown function have been identified. The possibility that the gene(s) mediating the effect is commonly carried on sul2-coding plasmids and may serve to reduce the potential fitness cost even of large plasmids merits further investigation.

The finding of a truncated transposase-like ORF (ORF1) adjacent to the sul2, strA and strB genes may offer a clue as to how these genes have been disseminated. The strA–strB gene pair is widely distributed throughout the bacterial kingdom and is present on Tn5393 in plant pathogenic bacteria.20 In bacteria isolated from humans and animals, the strA–strB gene pair is usually linked to sul226 and this gene assembly occurs in many genetic locations including not only p9123 and related plasmids, but also IncQ plasmids exemplified by RSF1010,19 large conjugative plasmids such as pHCM127 and the Vibrio cholerae SXTMO10 integrative and conjugative element.28 ORF1 is related to orfA from an E. coli florfenicol resistance plasmid,20 which in turn is related to the REP-like transposases of the IS91 family of insertion sequences. ORF1 itself nevertheless lacks obvious transposase-like motifs and hence may be non-functional.

The present results, together with earlier data12 demonstrate the persistence of sulphonamide resistance in the absence of clinical selection pressure, and suggest that this may partly be explained by the fact that not all sulphonamide resistance exerts a fitness cost as well as by co-selection of resistance genes affecting agents that are commonly used.12 Another factor that may contribute to the maintenance of sul2 plasmids is the use of sulphonamides in veterinary medicine. We suggest that, in general, co-selection may be the more important factor for maintenance, as most sul2 genes are on large multi-resistance plasmids.12 On a more general note, these results indicate that attempts to eliminate widely distributed resistance genes that occur on diverse mobile-genetic elements, by reducing antibiotic use, may be unrealistic, especially in the short to medium term.


    Acknowledgements
 
We would like to thank the British Society for Antimicrobial Chemotherapy for financial support.


    Footnotes
 
* Corresponding author. Tel: +44-117-9287522; Fax: +44-117-9287896; E-mail: v.i.enne{at}bristol.ac.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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