Rifampicin resistance and its fitness cost in Enterococcus faecium

V. I. Enne1,*, A. A. Delsol2, J. M. Roe2 and P. M. Bennett1

1 Department of Pathology and Microbiology, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD; 2 Division of Farm Animal Science, Department of Clinical Veterinary Science, University of Bristol, Langford BS40 5DU, UK

Received 8 August 2003; returned 8 October 2003; revised 24 October 2003; accepted 3 November 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The genetic basis of rifampicin resistance and the associated fitness cost in Enterococcus faecium were investigated.

Methods: Twelve spontaneous rifampicin-resistant E. faecium mutants were selected from four parent strains recently isolated from porcine faecal material. The DNA sequence of the complete rpoB gene from the parent strains and of nucleotides –189 to +1785 from the mutants was determined from PCR amplicons. The fitness of the mutants was assessed by determining growth rate, by direct growth competition and by the ability of some of the mutants to survive in the pig intestine.

Results: The rpoB genes of the parent strains diverged from each other by 1–10% and each encoded proteins that were 1208 amino acids in length. All mutants had a single amino acid substitution in the region implicated in rifampicin resistance in other organisms. Six mutants carried the substitution H489Y/Q, two mutants carried the substitution R492H, one mutant carried the substitution Q480H, two mutants carried the substitutions S494L and V224I, and one mutant carried the substitutions G485D and V224I. Per generation fitness costs of the mutants ranged from a gain of 2.5% to a cost of 10%. Mutants with the substitution H489Y/Q were the most fit, whereas the double mutants were the least fit. The mutant with the substitution H489Q was able to survive in the pig gut for 12 days. There was some correlation between the rifampicin MIC and fitness cost, with higher MICs being associated with higher fitness costs.

Conclusions: Substitutions in RpoB are associated with rifampicin resistance in E. faecium. The fitness cost of resistance is variable and can sometimes be absent.

Keywords: rpoB, biological cost, in vivo fitness


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Enterococcus faecium is a commensal organism of both human and animal guts. However, it is also a common cause of nosocomial infections such as bloodstream infections, urinary tract infections, wound infections and endocarditis. Antibiotic-resistant strains of this organism, especially those resistant to vancomycin, are of particular concern.

Although rifampicin is not routinely used to treat infection caused by E. faecium, the organism is nevertheless susceptible to this drug. However, acquired resistance is common. For example, a study of 71 E. faecium isolates from UK hospitals found that 78.9% were resistant to rifampicin.1 Rifampicin acts by binding to the ß-subunit of the DNA-dependent RNA polymerase. In most bacteria, rifampicin resistance is mediated via mutations in the rpoB gene encoding the ß-subunit of RNA polymerase. Such mutations have been described in a wide variety of organisms including Escherichia coli,2 Mycobacterium tuberculosis,3 Streptococcus pneumoniae4 and Legionella pneumophila.5 Other mechanisms of rifampicin resistance have also been described,6,7 but these are much rarer. Mutations in rpoB mediating rifampicin resistance tend to cluster to specific regions of the gene, designated clusters I, II and III,2 irrespective of the bacterial species reported. As an essential housekeeping gene, mutations in this gene can be expected to compromise transcription efficiency and hence the fitness of the organism. This has been reported in E.coli,8 Salmonella typhimurium,9 M. tuberculosis10 and Staphylococcus aureus.11 However, a fitness cost is not always associated with rpoB mutations8 and the adverse effects of mutation can sometimes be reduced by the acquisition of second-site compensatory mutations.8,9 Detrimental effects on growth have also been observed for mutations mediating resistance to other antibiotics such as streptomycin12 and fusidic acid.13

In this paper, we describe a set of mutations in the rpoB gene of E. faecium that confer resistance to rifampicin and assess the fitness cost of these mutations both in the laboratory and in pigs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Selection of rifampicin-resistant mutants

In order to isolate E. faecium strains from the pig intestine, porcine faecal material was collected in November 2001, resuspended in saline and plated onto Slanetz and Bartley agar (Merck). Putative enterococci were then identified using API-20Strep strips (bioMérieux). Four independent isolates, E. faecium strains 38–15, 40–4, 40–8 and 343–3 were used in the study. Independent rifampicin-resistant mutants were selected from the four parent strains by growing organisms overnight in nutrient broth and plating onto agar containing 20 mg/L rifampicin. The rifampicin MIC of each of the mutants was determined using Etest strips (AB Biodisc). Twelve of the mutants obtained were studied further.

Sequencing of the rpoB gene

The sequence of the entire rpoB gene of the parent strains was obtained from four PCR amplification products covering bases –189 to +3852. For the rifampicin-resistant mutants, two PCR amplification products (with primers RPOB1F&R and RPOB2F&R) were obtained to cover bases –189 to +1785 to obtain sequence for the rifampicin resistance determining region (RRDR).2 PCR primers were designed from sequence obtained from the incomplete enterococcal genome sequencing project by searching for homology to E. coli rpoB. The primers are described in Table 1. Template DNA was prepared by suspending one colony in 100 µL of water and boiling for 5 min. The PCR mixture consisted of 25 µL of ReadyMix Taq PCR reaction mixture (Sigma), 23 µL of water, 0.5 µL of each primer (0.2 µM) and 1 µL of template DNA. Amplification was carried out by heating for 5 min at 95°C for 1 cycle, for 1 min at 95°C, 1 min at 50°C and 1 min at 72°C for 35 cycles, followed by 1 cycle at 72°C for 10 min. PCR amplification products were visualized by gel electrophoresis on 1% agarose gels in Tris boric acid/EDTA buffer (pH 7.0) and stained with 1% ethidium bromide. Amplification products were purified using a Qiaquick purification kit (Qiagen) according to manufacturer’s instructions. They were sent for sequencing at the Advanced Biotechnology Centre, Imperial College, London, where sequencing was carried out on an ABI 3100 automated DNA sequencer using a BigDye terminator kit (Applied Biosystems, Warrington, UK). For each primer pair, at least two independent PCR products were sequenced on both strands. Sequence analysis was carried out using the Lasergene DNASTAR software package. Sequence alignments were done using ClustalW. The rpoB sequences of the parent strains have been deposited in GenBank and have accession numbers AY167138AY167141.


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Table 1. Sequences of primers used to amplify the rpoB gene
 
Determination of fitness in vitro and in vivo

The fitness of each mutant in laboratory culture was assessed by determining its growth rate in nutrient broth in triplicate and by direct growth competition in peptone water using a modified method of that described previously.14 Briefly, mutants and parents were grown separately in peptone water at 37°C with shaking at 170 r.p.m. for 24 h and then mixed at a ratio of 1:1. The mixture, comprising 107 cfu in total, was diluted at a ratio of 1:100 into 50 mL of fresh peptone water. Thereafter, 0.5 mL of culture was transferred to 50 mL of fresh peptone water daily and grown at 37°C shaking at 170 r.p.m. for a total of 10 transfers. Ratios of the competing organisms were determined on days 1, 3, 6 and 10 of the experiment by plating in triplicate onto nutrient agar and nutrient agar containing 20 mg/L rifampicin. Following overnight incubation of the plates at 37°C the colonies were counted. The mean number of colonies on the plates containing rifampicin was subtracted from the mean number of colonies on plates not containing rifampicin in order to obtain an estimate of the number of rifampicin-susceptible colonies. Per generation fitness costs were then calculated using the formula (1–(b/T)) x 100, where b is the slope of the regression ln(RifR/RifS) and T is the number of cell generations per transfer, equal to ln(100)/ln(2). Six independent replicate experiments were carried out for each competing pair.

In order to test the ability of the mutants to survive in vivo, a mixture of the three fittest mutants at a ratio of 1:1:1, comprising 108 cfu in total, was inoculated into four 7-week-old organic piglets. In order to monitor the numbers of rifampicin-resistant faecal streptococci, faecal samples were taken regularly, resuspended in saline and plated onto Slanetz and Bartley agar containing 50 mg/L rifampicin. At the end of the experiment, on day 12, 25 colonies were picked and identified more closely using API-20Strep strips (bioMérieux). All procedures complied with the Animals (Scientific Procedures) Act 1986 and were carried out under Home Office licence.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sequencing of the rpoB gene from the four parent strains, 38–15, 40–4, 40–8 and 343–3 revealed open reading frames that encode proteins of 1208 amino acids. RpoB shares 95% identity with the RpoB of E. faecalis, its closest relative. However, the RRDR of the two species is identical. There is significant divergence among the rpoB sequences of the isolates, which differ by 8–10%, except isolates 40–4 and 40–8, which differ only by 1%.

Rifampicin-resistant mutants were selected relatively easily from all four parent strains, with mutation frequencies of approximately 10–6. The DNA sequences of the rpoB genes of the rifampicin-resistant derivatives were examined for differences in relation to the rpoB gene of the respective parent strains. In all cases, a point mutation resulting in an amino acid substitution was found in the so-called rifampicin resistance cluster I, covering residues 480 to 494, corresponding to residues 517–531 in E.coli2 (Table 2 and Figure 1). In addition, the mutant 40–4 Rif E had acquired an additional mutation, A536G, which was silent, i.e. it did not generate an amino acid substitution. Two of the amino acid substitutions in RRDR, Q480H and G485D, have not been identified previously in other organisms, whereas the remainder have been identified in other bacteria. Three isolates contained an additional amino acid substitution located outside the known RRDR, namely, V224I. Curiously, these three mutants had higher MICs of rifampicin (>256 mg/L) than the other mutants suggesting this mutation might influence the level of rifampicin resistance. Interestingly, although the mutants were isolated independently, the H489Y/Q substitution was seen in half the mutants (6/12).


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Table 2. Rifampicin susceptibility and substitutions in RpoB of rifampicin-resistant mutants
 


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Figure 1. Alignment of amino acids in rifampicin resistance cluster I of RpoB from E.coli,2,8,19 M. tuberculosis,20,21 S. pyogenes16 and E. faecium. Arrows indicate substitutions observed in E. faecium. Residues associated with rifampicin resistance in other organisms are in bold-face type.

 
The fitness cost of mutation to rifampicin resistance in E. faecium appears to be relatively low, with mutants exhibiting per generation fitness costs of 10% or less (Table 3). The double mutants, although most highly resistant to rifampicin were the least fit with per generation fitness costs of 6.6–10%. Mutants with the substitution R592H were relatively unfit, with fitness costs of 9.5% and 6.9%. The mutant with the novel Q480H substitution was relatively fit, with a fitness cost of 2.9%. Mutants with the substitution H489Y/Q were the most fit with per generation fitness cost of –2.5% to 4.2% (Table 3). There was some correlation between fitness cost and the level of rifampicin resistance, with isolates with higher MICs tending to have higher fitness costs (Figure 2).


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Table 3. Fitness cost of rifampicin resistance in E. faecium mutants
 


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Figure 2. The relationship between rifampicin MIC (mg/L) and percentage per generation fitness cost in E. faecium rifampicin-resistant mutants. The data points indicate individual mutants.

 
Curiously, the Q480H mutant appeared initially to do better than its parent in competition experiments. After day three however, the parent out-competed the mutant (Figure 3).



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Figure 3. Competition dynamic of 343–3 Rif C (Q480H) versus parent strain.

 
The three most fit mutants of different parentage, 38–15 Rif C, 40–4 Rif B and 343–3 Rif A were taken and inoculated as an equal mixture into four pigs. Faecal samples were collected on days 3, 5, 7, 10 and 12 post-inoculation and screened for the presence of rifampicin-resistant faecal streptococci. Towards the end of the experiment, these were found at concentrations of 2 x 103 to 2 x 104 cfu per gram of faeces, depending on the animal. On the final day of sampling, 25 colonies were taken and identified using API-20Strep strips. Because all three mutants that were used in the inoculum have different biochemical profiles we were able to distinguish one from the other. All 25 colonies had the same profile as 38–15 Rif C.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rifampicin resistance among Enterococcus faecium is associated with mutations in the rpoB gene, as is the case with numerous other bacteria. In E. faecium, all rifampicin-resistant mutants examined had mutations in resistance cluster I (Table 2). We report for the first time the substitutions Q480H and G485D, which have not been identified in another organism. Three of the mutants examined carry an additional substitution, V224I, outside known rifampicin resistance clusters. These isolates were more resistant to rifampicin than the other rifampicin-resistant mutants examined. It appears therefore that this second substitution may increase the level of rifampicin resistance in E. faecium. The cluster I substitutions seen in the double mutants, namely S494L and G485D, were not obtained independently of the V224I substitution and therefore we were unable to determine the contribution of each mutation to the elevated level of resistance. The occurrence of double mutations has also been reported in S. pneumoniae,15 Streptococcus pyogenes16 and M. tuberculosis.17 However, in these cases, the second mutation did not appear to increase the level of rifampicin resistance. In S. pneumoniae, both mutations occurred within known resistance clusters whereas in S. pyogenes the second mutation was also located outside the known resistance clusters at position 722 (E. coli numbering). Although all the E. faecium mutants were independently isolated, the H489Y/Q substitution was obtained in 6/12 mutants. Substitution at this position has also been found to be prominent in other bacteria such S. pneumoniae,15 M. tuberculosis17 and S. aureus.11,18 This prominence may reflect the low fitness cost imposed by amino acid substitutions at this position.

In this study, we examined the effects of these mutations in the ß subunit of RNA polymerase on the fitness of the organisms concerned. Contrary to what has previously been found in E. coli,8 there seemed to be a correlation between the level of rifampicin resistance and the magnitude of the fitness cost (Figure 2). Intuitively, this can be anticipated as a mutation that renders the polymerase less susceptible to rifampicin might be expected also to have a reduced transcription efficiency.

Competition experiments were carried out using peptone water as the growth medium because the E. faecium strains did not grow in DM25 medium, which is commonly used for competition experiments to estimate fitness cost.14 It appeared to be a suitable medium for carrying out competition experiments with E. faecium as growth rates and cell densities were similar to those obtained for E. coli in DM25 medium (unpublished observations). The fitness cost of rifampicin resistance in E. faecium as measured by determining growth rate in nutrient broth did not differ significantly from those obtained by growth competition in peptone water. However, the absolute values as well as the standard deviations determined by growth rates were higher than those determined by growth competition (Table 3). However, determining growth rate can be useful as a rapid method for estimating fitness cost in this organism.

In general, the fitness costs tended to be lower than those that have been reported previously for rifampicin resistance in other organisms. In E. coli, fitness costs of 10% or more were detected in four out of nine mutants as opposed to one out of 12 mutants of E. faecium in this study.8 In S. typhimurium, generation times of two RifR mutants were 69% and 123% greater than those of the parent.9 In M. tuberculosis, 7/9 mutants had fitness costs greater than 10%,10 whereas in S. aureus 8/18 mutants had a fitness cost greater than 10%.11 It would be interesting to know whether this phenomenon of lower fitness cost is unique to rifampicin resistance in E. faecium or whether it applies to other resistance mechanisms in this organism. It could provide an explanation, at least in part, as to why antibiotic-resistant strains of this organism are so common. Although the fitness costs of many of the mutants were low in the test systems, it may be that they carry more significant costs under different conditions. In the pig gut, mutant 38–15 Rif C persisted for at least 12 days whereas mutants 40–4 Rif B and 343–3 Rif A were not detected on day 12 when identities of rifampicin-resistant colonies were determined, indicating that they were at least 25 times less frequent than 38–15 Rif C. This, despite the fact that all three strains showed similar in vitro fitness costs and that all three carried a substitution at histidine 489, although in 38–15 Rif C a glutamine is substituted at this position whereas in 40–4 Rif B and 343–3 Rif A the substitution is a tyrosine residue. This suggests that the characteristics of the original parent strain are likely to influence the ability to survive in vivo as well as mutations in rpoB.

The genetic background in which a mutation in rpoB occurred did not appear to influence fitness costs significantly. In cases where the same mutation was found in different parent strains, fitness costs as determined in the laboratory were approximately the same (Table 3). To the best of our knowledge, the fitness cost of a given mutation has not previously been compared in different strains and therefore we do not know whether this is a general phenomenon. The genetic background in which the mutation occurred did, however, appear to influence MIC. In mutants 343–3 Rif A and B, the H489Y substitution resulted in a rifampicin MIC of 32 mg/L, whereas in 40–8 Rif D the same mutation resulted in a rifampicin MIC of 96 mg/L. The RpoB proteins of the parent strains differ from each other by 1%.

The competition experiment with mutant 343–3 Rif C containing the substitution Q480H gave intriguing results. Initially, until day 3 of the competition experiment the mutant appeared to out-compete the parent. Then, after day three, the parent started to out-compete the mutant (Figure 3). This was not a random event as it was observed in all six independent replicate experiments preformed. We offer no explanation for this result. However, we suggest this mutant must be competitive during short-term growth but deficient in long-term survival, but can offer no explanation as to why this may be.

This study demonstrates that mutation to antibiotic resistance, in this case rifampicin resistance, may involve a fitness cost, as reflected by poorer growth on laboratory culture, both in pure culture and in competition with its parent organism. However, this disadvantage may not be evident beyond the laboratory as demonstrated by the survival of E. faecium 38–15 Rif C for several days in the pig intestinal tract. That this organism was recovered in relatively large numbers 12 days after feeding to the pigs attests to its fitness and its ability to grow in this natural state. The failure to recover the other mutants, despite them showing similar fitness in the laboratory emphasizes the importance of testing a number of isolates before choosing one for in vivo studies. Although mutations to rifampicin resistance impose a fitness cost, some mutants are clearly minimally afflicted. Furthermore, because rifampicin resistance is rare, it serves as an ideal marker for in vivo studies


    Acknowledgements
 
This study was supported by a grant from the Department for Environment, Food and Rural Affairs (DEFRA). The authors would like to acknowledge Matthew Avison for assistance with locating the E. faecium rpoB sequence.


    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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