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Keywords:

  • experimental infections;
  • rifampicin;
  • Staphylococcus aureus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Statement
  9. References

This study was designed to investigate the role of hypermutability of Staphylococcus aureus on bacterial fitness and antibiotic resistance in a model of chronic bone infection. An isogenic pair of strains, S. aureus RN4220 and its mutator counterpart inactivated in the mutL gene were used in a rat model of osteomyelitis of the tibia. The effect of the mutator phenotype in the emergence of antibiotic resistance was assessed in rats infected by each strain separately and treated with rifampicin for 5 days. No difference between the two strains was found in bacterial growth in vitro and in bacterial survival in the animal model, indicating no fitness defect in the mutator strain. In competition studies performed in rats coinfected with the two strains at a same ratio and sacrificed at different times from day 3 to day 42 postinoculation, the mutator strain was clearly disadvantaged because it was found in all rats and at all study times at lower counts (P<0.05 from day 14 to day 42). Two of the 16 rats infected by the mutator strain and none of the 14 rats infected by the wild-type strain had acquired rifampicin-resistant mutants (P=0.4). Data suggest that the S. aureus mutator phenotype was associated with a decreased bacterial fitness in vivo and did not confer significant advantage in the acquisition of antibiotic resistance in a model of chronic bone infection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Statement
  9. References

Hypermutable derivatives of bacteria display an increased mutation rate compared with their wild-type counterparts. In the mutator phenotype, the mutation rate can be multiplied up to 10 000-fold (Marti et al., 2002). The mutator phenotype has been shown to play an important role in the adaptation of bacterial populations in changing and stressful environment (Taddei et al., 1997). It gives an advantage to the bacteria when submitted to various hostile conditions whether they result from extreme environmental conditions, host defences, or by administration of antibiotics. However, the primary benefit obtained from the frequent and versatile acquisition of adaptive mutations might be counterbalanced by acquisition of deleterious mutations leading to reduced fitness (Besier et al., 2005).

Mutator bacteria have been detected in both commensal and pathogen populations and they can represent a significant proportion of invasive clinical isolates. Mutator bacteria were first reported in gram-negative species including Escherichia coli (LeClerc et al., 1996; Denamur et al., 2002), Salmonella typhi (LeClerc et al., 1996), Pseudomonas aeruginosa (Oliver et al., 2000), and Neisseria meningitides (Richardson et al., 2002), and then in gram-positive species including Streptococcus pneumoniae and Staphylococcus aureus (Morosini et al., 2003; Prunier et al., 2003). The presence of a high proportion of hypermutable strains of P. aeruginosa, S. aureus, and Haemophilus influenzae has been particularly well documented in the context of cystic fibrosis (Oliver et al., 2000; Prunier et al., 2003; Watson et al., 2004). In addition to the facilitation of adaptation to the specific environment of cystic fibrosis lung, the mutator phenotype may also result in increased ability to acquire antibiotic resistance, as shown in P. aeruginosa and S. aureus (Oliver et al., 2000; Prunier et al., 2003).

The mutator phenotype has mostly been linked in several bacterial genera to defects in the methyl mismatch repair system (MMR). This system recruits several proteins including MutS, MutL, and MutH to recognize mismatched or unpaired nucleotides produced during replication and to correct them. In E. coli, MMR alterations induce an increased mutation rate and favour intra- and interspecies genetic exchanges (Marti et al., 2002). Recently, it has been shown that homologues of mutS and mutL exist in the chromosome of S. aureus and that inactivation of either gene yields a hypermutable phenotype (Prunier & Leclercq, 2005).

Both the theoretical notion that hypermutability favours long-term adaptation of bacteria, and the clinical observation of the importance of hypermutability in the context of cystic fibrosis, a model of chronic infection, suggested that hypermutability might have a major role in the development of chronic infections. In addition, it has been shown that natural clinical isolates of E. coli with a high mutation rate and several virulence determinants are significantly less virulent than strains with the same number of virulence determinants but with a low mutation rate, which is consistent with an involvement of hypermutators in chronic infections rather than in acute infection (Picard et al., 2001).

To obtain further insights into the role of the mutator phenotype in bacterial fitness and emergence of antibiotic resistance, an experimental model of tibia osteomyelitis was designed in rat. Indeed, chronic osteomyelitis, which is mainly due to S. aureus, is characterized by the progressive inflammatory destruction and new apposition of bone (Lew & Waldvogel, 1997). Once the microorganisms adhere to the bone, they express phenotypic resistance to antimicrobial treatment, which may explain the high failure rate of short-course therapy and the need for a prolonged antibiotic administration (Proctor et al., 2006). The behaviour of two strains of S. aureus, which differed only by their mutation rate, was studied in the experimental model.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Statement
  9. References

Bacterial strains

An isogenic pair of S. aureus strains was used. Staphylococcus aureus RN4220 is a laboratory strain susceptible to all usual antistaphylococcal antibiotics including penicillin, methicillin, and rifampicin. Staphylococcus aureus mutL is its mutator counterpart that was inactivated in the mutL gene by plasmid insertion (Prunier & Leclercq, 2005). Briefly, a 582-bp DNA fragment intragenic to mutL was cloned into the thermosensitive pBT1 plasmid mediating chloramphenicol resistance. The resulting plasmid was electroporated into S. aureus RN4220. By submitting the strains to successive subcultures at 42°C in broth medium containing chloramphenicol, plasmid integration in the bacterial chromosome was forced by homologous recombination.

Strains were kept as glycerol stock cultures at −80°C and streaked onto trypticase soy (TS) agar (Biorad, Marnes-la-Coquette, France) to provide working plates that were stored at 4°C. Staphylococcus aureus mutL was cultured in the presence of chloramphenicol (20 mg L−1). All cultures were performed in TS medium and were grown at 37°C.

Study of the stability of the inserted plasmid after 3 weeks of subcultures in TS broth without chloramphenicol indicated that <7% of the mutator strains lost their chloramphenicol resistance. In addition, study of the mutator phenotype was also performed for S. aureus RN4220 and S. aureus mutL after in vivo passage in several rats to check for the acquisition or loss of chloramphenicol resistance for the wild-type strain and for the mutator strain, respectively. None of these two events ever occurred.

Growth kinetics and in vitro competition

In order to determine if gross differences in fitness could exist between wild-type and mutant strains, growth kinetics of the two S. aureus strains were studied in vitro. Each strain was inoculated in 10 mL TS broth with c. 107 CFU mL−1 and incubated for 12 h at 37°C. At the beginning of the experiment and every 2 h, 0.1 mL samples were removed from the tubes, serially diluted, subcultured onto TS agar plates, and incubated for 48 h at 37°C before CFU were counted.

Antibiotic susceptibility and determination of mutation frequencies

Antibiotic susceptibility was determined by the broth microdilution technique (Comité de l'Antibiogramme de la Société Française de Microbiologie, 2006). For the determination of mutation frequencies, c. 108 cells from an overnight broth culture were plated onto TS agar plates supplemented with 100 mg L−1 of rifampicin. After 48 h of incubation at 37°C, colonies were counted and the mutation frequencies were determined relative to the total count of viable organisms plated. Experiments were performed six times and mean values were calculated. Staphylococcus aureus mutL displayed a 10- to 60-fold increase in mean frequencies of mutation to rifampicin or fusidic acid resistance (5.5 × 10−6 and 3.5 × 10−7, respectively) as compared with S. aureus RN4220 (8.6 × 10−8 and 3.5 × 10−8, respectively).

Mutant prevention concentration (MPC) was determined as described by Blondeau et al. (2001). MPC is the lowest concentration at which no mutant resistant to the tested antibiotic can be selected using a heavy inoculum of 1010 CFU, which is considered as the maximal quantity of bacteria present at an infection site. A 20 mL overnight culture was concentrated by centrifugation at 3000g for 30 min and suspended in 3 mL of TS medium. Aliquots of 200 μL, containing ≥1010 CFU, were spread onto TS agar plates containing increasing concentrations of antibiotics (fusidic acid, rifampin, and ciprofloxacin). After 48–72 h of incubation at 37°C, MPC was recorded as the lowest antibiotic concentration that prevented growth of any resistant colonies. Experiments were performed in triplicate for each strain, and mean values were calculated.

Detection of mutations in the rpoB gene

Mutants were also selected on broth media containing 0.2 and 2 mg L−1 of rifampicin and mutations in the rpoB gene were characterized in randomly selected mutants. Mutations in the rpoB gene were determined as described by Aubry-Damon et al. (1998). Total DNA from S. aureus was purified and used as a template for amplification by PCR. Two portions of the rpoB gene from S. aureus were amplified: a 702-bp fragment from nucleotide positions 441–673 (S. aureus coordinates) corresponding to the so-called rifampicin resistance-determining region in the centre of the E. coli rpoB gene, and a 158-bp fragment from nucleotide positions 94–144 in which a Val143Phe substitution conferring rifampicin resistance has been reported in E. coli. The 20-mer oligodeoxyribonucleotides used as primers were F3 (5′-AGTCTATCACACCTCAACAA) and F4 (5′-TAATAGCCGCACCAGAATCA) for the largest fragment and D1 (5′-GTGTAAAAGTGCGTCTAATC) and D2 (5′-ATAAACGGATGGTGAACGAA) for the smallest fragment.

Experimental protocol

The rat model of bacterial osteomyelitis of the tibia was used as described by O'Reilly & Mader (1999). Animal experiments were performed in accordance with the legal and ethical requirements of the Direction of Veterinary Department of Paris. Rats were anaesthetized i.m. with 0.57 mg of xylazine per 100 g of body weight in one leg (Rompum 2%) (Bayer, Puteaux, France) and with 2.8 mg of ketamine per 100 g of body weight in the other leg (Imalgene 1000) (Merial, Lyon, France). Legs were shaved and disinfected with polyvinylpyrrolidone-iodine (Betadine) (Viatis Manufacturing, Mérignac, France). The anterior tibial metaphysis of each leg was surgically exposed, and a hole was drilled through the cortex using a high-speed drill with a 0.5-mm diameter bit. Rats were inoculated into the proximal metaphysis of the tibia. The inoculum was injected slowly and the hole was closed by dental cement (Ciprospad) (Dentsply France, Montigny, France). Final inoculum injected to each animal was 107 CFU in a volume of 50 μL. The skin was closed with one clamp. Then, rat's sacrifice occurred at different times with pentobarbital (Abbott France, Rungis, France). Tibias were dislocated and stored at −80°C until they were crushed in a cryo-crusher (Fisher Bioblock, Illkirch, France). The crushed bone was suspended in 2 mL of saline solution and plated onto TS agar. Bacterial counts were enumerated after 72 h of incubation.

(1) Growth studies: In order to detect any difference in growth defect between the two strains, 20 rats were infected with each strain separately and were sacrificed at 7 days (n=10) and at 14 days (n=10). For each rat, the bacterial count in tibia was enumerated. In addition, in order to study the competitive growth of the two strains, 50 rats were coinfected with the two strains at the same proportion (ratio 1/1) and 10 rats were sacrificed at five different times: 3, 7, 14, 28, and 42 days after infection. For each rat the CFU number of mutL and RN4220 strains was determined by plating crush bone on TS agar with and without 20 mg L−1 of chloramphenicol. mutL distinction was established by the chloramphenicol resistance characteristic.

Finally, one clone of each strain that was previously passed in vivo, i.e. already adapted to the bone, was used to infect 20 rats. Sacrifice took place 7 and 21 days postinoculation and bacterial counts were determined for each strain.

(2) Resistance studies: To study a potential effect of the mutator phenotype in the emergence of antibiotic resistance, infected rats were treated with rifampicin and rifampicin-resistant mutant was detected at the end of the experiment.

Twenty rats were infected with RN4220 and 22 rats with mutL; within each group of infected animals, 14 and 16, respectively, were treated 10 days after infection with sub-cutaneous injection of rifampicin, 10 mg kg−1 twice daily for 5 days. The mean peak serum level (7 mg L−1) was comparable with those obtained in human (O'Reilly & Mader, 1999). Rats were sacrificed 48 h after the last injection. For each strain, three controls rats were sacrificed before treatment and three at the end of the experiment. The crushed bone was plated on TS agar to enumerate bacteria and on TS agar containing 1 mg L−1 rifampicin to detect rifampicin-resistant mutant(s).

Statistics

Results were expressed as mean±SD of the mean. Student's t-test was used to compare the mean CFUs from two groups of animals. A P-value of <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Statement
  9. References

Effect of mutL inactivation on fitness

No difference was observed between the growth curves of S. aureus RN4220 and S. aureus mutL in TS medium (data not shown). In addition, after infection of rats, bacterial counts were approximately equal to 4.5 log10 CFU bone−1 and were similar whether the rats were infected with S. aureus RN4220 (4.49±0.61 log10 CFU bone−1 at day 7 and 4.69±0.91 log10 CFU bone−1 at day 14) or with S. aureus mutL (4.65±0.5 log10 CFU bone−1 at day 7 and 4.58±0.74 log10 CFU bone−1 at day 14), confirming in vivo that mutL inactivation did not have any impact on bacterial fitness (Fig. 1a).

image

Figure 1.  Behaviour of the two study strains in experimental osteomyelitis in rat. Open circles, Staphylococcus aureus RN4220: wild strain; closed diamonds, S. aureus mutL: mutator strain;mean; *P<0.05. (a) separate infection; (b) coinfection with the two strains at the same ratio; (c) coinfection with the two strains previously passed in vivo.

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Effect of mutL inactivation on competition

To test the hypothesis that hypermutability might confer an advantage to the bacteria, bacterial competition assays were performed taking advantage of the persistence of infection in rats 6 weeks after the primary bacterial inoculation (Fig. 1b). After 3 days of infection, bacterial counts of the wild-type strain were 10-fold higher (c. 6 log10 CFU bone−1) than those for the hypermutable derivative (c. 5 log10 CFU bone−1). After 7 days of infection, while the global inoculum decreased by 10-fold, the difference in bacterial counts between the two strains were even more pronounced (4.8±0.54 log 10 CFU bone−1 for S. aureus RN4220 vs. 3.4±0.43 log10 CFU bone−1 for S. aureus mutL) and the difference became significant at day 14 (P<0.05). The difference was consistently observed for all the infected rats at all different times tested. After 14 days of infection, the bacterial inoculum in the bone remained stable and the difference between the two strains barely increased. However, three rats completely cleared the mutator strain after 42 days of infection.

To favour the adaptation of the S. aureus strains to the bone, rats with strains isolated after a first previous in vivo passage were inoculated. Again, the survival of the hypermutable derivative in the bone was disadvantaged (5.0±0.55 log10 CFU bone−1 at day 7 and 5.15±0.85 log10 CFU bone−1 at day 21 for the wild type vs. 4.29±1.0 log10 CFU bone−1 at day 7 and 3.20±1.2 log10 CFU bone−1 at day 21 for S.aureus mutL, P<0.05 at day 21) (Fig. 1c).

Effect of mutL inactivation on MPCs and in vitro selection of antibiotic-resistant mutants

The mutation rate of S. aureus RN4220 had a moderate impact on MPCs. mutL inactivation yielded an increase of two dilutions in MPCs for fusidic acid (from 32 to 128 mg L−1). However, MPCs of ciprofloxacin and rifampin were the same (2 and >1024 mg L−1, respectively) for S. aureus RN4220 and its mutL derivative.

Mutations in the rpoB gene were identified in independent mutants after selection for each strain on rifampicin containing media. In nine S. aureus RN4220 mutants, only substitutions at position 481 of the histidine for a tyrosine (six mutants), an asparagine (two mutants), or an arginine (one mutant) were found. In nine S. aureus mutL mutants, seven had substitution at position 481 (three His-481/Tyr and four His-481/Arg) and two had a substitution at position 527 (Phe-527/Ile).

Effect of hypermutability on in vivo selection of rifampicin-resistant mutants

At the end of treatment with rifampicin of rats infected separately with one of the two strains, there was no difference in the bacterial counts in the bone for the two strains (4.07±0.28 log10 CFU bone−1 for the wild type and 4.07±0.39 log10 CFU bone−1 for the mutator strain). Two of the 16 rats infected by the mutator strain and none of the 14 rats infected by the wild-type strain had acquired rifampicin-resistant mutants (P=0.4).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Statement
  9. References

In this animal model of chronic osteomyelitis, it was shown that the mutator phenotype did not confer any advantage in bacterial fitness in comparison with the nonmutator phenotype. When the two S. aureus strains were studied separately, they grew similarly both in vitro and in vivo. An unexpected result was the reduced survival of S. aureus mutL in the in vivo competition assays. The impaired ability of S. aureus mutL to compete with the isogenic strain expressing the MMR system might be related to a possible higher propensity to produce mutations that are lethal or deleterious for fitness. The model of separate infection by S. aureus mutL alone was unable to show this disadvantage because the deleterious mutants are probably generated at a low frequency and are rapidly replaced by S. aureus mutL cells efficient for infection. By contrast, this deleterious effect might be amplified in the competition assays because the mutant with impaired disadvantageous functions could be rapidly replaced either by a S. aureus RN4220 cell or a S. aureus mutL cell without deleterious mutations leading, therefore, to a progressive enrichment of the bacterial inoculum by S. aureus RN4220 cells.

It should be mentioned that although the animal model of this study can be considered as a model of chronic infection because the infection lasted for 6 weeks in rats, this duration is far lower than that observed for chronic osteomyelitis in humans and might be insufficient to observe the advantageous effects of the hypermutable status of Staphylococci. Nevertheless, the fact that the mutator strain was totally cleared from some animals after 6 weeks of infection and that the disadvantage of the mutator was obtained in all individuals and at all the times of sacrifice are strong arguments for the biological relevance of this observation.

Data did not show a clear impact of hypermutability on resistance in vitro both in terms of diversity of substitutions in rpoB in rifampicin-resistant mutants and on MPCs of several antistaphylococcal drugs, i.e. rifampicin, fluoroquinolones and fusidic acid for which mutation is the major mechanism of resistance. The data also did not confirm the recent observation that even a small increase in mutation frequencies may have an impact on the emergence of resistance to antibiotics (Denamur et al., 2005). Noticeably, it could be predicted on the basis of the in vitro frequencies of mutations for rifampicin resistance (7.5 × 10−8 for S. aureus mutL and 1.5 × 10−9 and S. aureus RN4220) that selection of mutants was unlikely to occur in vivo because the bacterial inoculum in the bone was weak (nearly 6 log10 CFU). Nevertheless, rifampicin-resistant mutants were detected in two rats infected by S. aureus mutL. These mutants were probably produced at each bacterial generation of the Staphylococci at the infection site. Although acquisition of resistance might result in an impairment of biological fitness for the bacteria and, as a consequence, to a possible loss of virulence, it has been recently shown for S. aureus and fusidic acid that compensatory mutations readily occur, which stabilize the mutant population (Aubry-Damon et al., 1998).

Conficting results have been reported concerning the relationship between S. aureus hypermutalibilty and the emergence of antibiotic resistance. Increased incidence of fluoroquinolone resistance in bacteria possessing a mutator phenotype has been demonstrated for S. aureus isolated from various infections and an elevated mutation frequency has been associated with the development of vancomycin resistance in vitro (Schaaff et al., 2003; Trong et al., 2005). Other reports on clinical isolates of S. aureus resistant to quinolones or glycopeptides failed to find any link between hypermutability and antibiotic resistance (Schmitz et al., 1999; O'Neill & Chopra, 2003). However, it is important to differenciate between in vitro and in vivo results, because compelling evidence has been provided that the process of adaptation to the costs of antibiotic resistance are different depending on whether the bacteria grows in vivo or in culture medium (Bjorkman et al., 2000).

In conclusion, this study suggests that hypermutability in S. aureus is associated with a significant decrease in bacterial fitness under specific conditions, i.e. the first weeks of bone infection, and therefore does not confer any advantage in pathogenesis. Under the same conditions, it is also not associated with a significant increased risk of selection of rifampicin-resistant mutants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Statement
  9. References

Claire Daurel was funded by ‘La Fondation pour la Recherche Médicale’. This work was funded in part by ‘Vaincre la Mucoviscidose’.

Statement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Statement
  9. References

This work was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, December 2005, Washington, DC.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Statement
  9. References
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