Monitoring in vivo fitness of rifampicin-resistant Staphylococcus aureus mutants in a mouse biofilm infection model

Jun Yu*, Jenny Wu, Kevin P. Francis, Tony F. Purchio and Jagath L. Kadurugamuwa

Xenogen Corporation, 860 Atlantic Avenue, Alameda, CA 94501, USA


* Corresponding author. Tel: +1-510-291-6220; Fax: +1-510-291-6232; Email: jun.yu{at}xenogen.com

Received 14 October 2004; returned 29 November 2004; revised 6 January 2005; accepted 7 January 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To investigate in vivo fitness of rifampicin-resistant Staphylococcus aureus mutants in a mouse biofilm model using bioluminescence imaging.

Materials and methods: S. aureus was engineered with a luciferase operon to emit bioluminescence that can be detected in vivo using an IVIS® imaging system. Two rifampicin-resistant strains of S. aureus that were previously isolated from animals undergoing rifampicin treatment, S464P (resistant to low concentrations of rifampicin) and H481Y (resistant to high concentrations of rifampicin), were characterized and then compared with their parental strain for in vivo fitness to form biofilm infections in the absence of rifampicin.

Results: The mutant S464P showed better adaptation to in vivo growth than either the parental strain or H481Y without selective pressure. Six days after implanting pre-colonized catheters, bioluminescent signals were seen from 100% of the catheters coated by the mutant S464P. In comparison, only 83% and 61% of the catheters coated by the parental strain and H481Y, respectively, maintained a signal in vivo. Rifampicin treatment of S464P biofilms in vivo resulted in a slight decline, but earlier rebound in bioluminescence from these catheters compared with the parental signal, whereas rifampicin had no affect on bioluminescence in mice infected with mutant H481Y.

Conclusions: The mutant with low-level rifampicin resistance appears to be better adapted to in vivo growth than the mutant that has high-level rifampicin resistance. Moreover, the former mutant may actually have a slight competitive advantage over the rifampicin-susceptible strain (parental), raising awareness for the occurrence of such strains in clinical environments.

Keywords: bioluminescence , animal models , imaging , IVIS


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prosthetic devices have found increased utility in modern medical practice. Unfortunately, once these devices are implanted they can allow bacteria to adhere and form microbial biofilms. Once such a biofilm infection is established, treatment becomes extremely difficult. Antibiotics that are highly effective against planktonic bacteria often do not prove satisfactory in eradicating biofilms.1 Once a biofilm is formed, the bacteria often become drug resistant and lead to chronic persistent infections. More often, the infected devices have to be surgically removed in order to eradicate the source of the infection.

Rifampicin is a semi-synthetic derivative of rifamycin B, an antibiotic produced by Streptomyces mediterranei. It is a broad-spectrum antibiotic that is readily distributed throughout the body, and is highly active against many Gram-negative and Gram-positive bacteria in vitro and in vivo. In some cases rifampicin is recommended for use prophylactically as a stand alone treatment, for example, to clear pharyngeal carriage of Haemophilus influenzae type b or Neisseria meningitidis;2 however, in most cases it is used in combination with other therapeutic agents, such as to treat infections caused by Mycobacterium,3 Streptococcus4,5 and fungi.6 Because of its excellent tissue penetration7 and strong activity against dormant or slow-growing bacteria, rifampicin is commonly used in combination with vancomycin to treat biofilm infections caused by Staphylococcus aureus and Staphylococcus epidermidis.8 Antibiotic lock in catheters to prevent staphylococcal biofilm infection usually involves the use of rifampicin and/or other antimicrobial agents.9,10 When rifampicin is used singly, drug-resistant bacteria develop rapidly.11

Rifampicin inhibits the bacterial DNA-dependent RNA polymerase (RNAP, subunit composition {alpha}2ßß'{sigma}) by binding to the ß-subunit deep within the DNA/RNA binding channel.12,13 Genetic analysis of rifampicin-resistant mutants obtained both from patients and from in vitro experiments has revealed that the mutations conferring rifampicin-resistant (RifR) phenotype map almost exclusively to regions on the rpoB gene that encodes the RNAP ß-subunit in all bacteria tested, including Mycobacterium tuberculosis,14,15 Escherichia coli,16 Helicobacter pylori,17,18 S. aureus,19,20 Streptococcus pneumoniae,21,22 Streptococcus pyogenes23 and Listeria monocytogenes.24 Most of the mutations are missense point mutations, clustering in two regions: cluster I at amino acid position 462–488 and cluster II at position 515–530. In E. coli, deletion or insertion mutations were also found in cluster I.16,25 Other mechanisms of action for rifampicin resistance include decreased membrane permeability,2628 and modification of rifampicin by ribosylation and glucosylation.2931

Recently, we have developed and validated an experimental biofilm infection model to monitor S. aureus biofilm formation on catheters in vivo in real time using bioluminescent bacteria.32 In previous studies of rifampicin antibiotic treatment of catheter-associated biofilm infections, we have shown that rifampicin monotherapy greatly reduces S. aureus biofilm infection in mice to undetectable levels, but that after the final treatment the infection relapses resulting in the development of resistant mutants. Two of the mutants were chosen for further investigation in this study. Re-administration of antibiotic was unsuccessful in eradicating the re-established infection.11,33 In the above studies we used biophotonic imaging to demonstrate the occurrence of in vivo resistance and disease relapse to therapeutic agents during treatment. Here, we describe the genetic and phenotypic characterization of such RifR mutants recovered from mice at different stages of biofilm infection, and investigate the in vivo fitness of such mutants in the catheter-associated biofilm infection model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strain and growth conditions

S. aureus strain Xen 29 was made bioluminescent through integration of a modified bacterial luciferase operon luxABCDE onto the host genome.32 S. aureus Xen 29 and its rifampicin-resistant mutants S464P and H481Y were grown in brain–heart infusion (BHI) broth in flasks or in 96-well microtitre plate, or on BHI agar plates supplemented with appropriate antibiotic.

Amplification of rpoB fragment by PCR

The clusters I and II of the rpoB gene on the S. aureus chromosome were amplified directly from bacterial suspension.34 In brief, colonies were suspended in 200 µL of a lysis buffer containing 10 mM Tris–HCl buffer (pH 8.0), 50 mM NaCl, lysostaphin (100 µg/mL), achromopeptidase (100 µg/mL) and RNase (100 µg/mL), incubated at 30°C for 45 min, boiled for 5 min, and then diluted by the addition of 400 µL of TE [10 mM Tris–HCl (pH 8.0), 1 mM EDTA]. For PCR, 1 µL of lysate was added as a template to 24 µL of a reaction mixture containing 10 mM Tris–HCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 0.2 mM each dNTPs and 0.75 U of Pfu DNA polymerase (Stratagene). The rpoB fragment was amplified with the primers rpoF (5'-GTT GTA CGT GAA AGA ATG-3') and rpoR (5'-ATG TGT ATC TAA ATC AAC-3'). Samples were subjected to 30 cycles consisting of 30 s at 95°C, 30 s at 55°C and 45 s at 72 °C in a GenAmp PCR Systems 9700 (Applied BioSystems). The PCR products were purified by Qiagen PCR purification kit and sequenced using rpoF as primer synthesized by Operon-Qiagen (Qiagen, Alameda, CA, USA).

Animal model of catheter-associated biofilm infection

The animal infection model was performed as described previously.11 Briefly, 1 cm Teflon catheter (14-gauge, Abbocath-T; Burns Vet Supply, Vancouver, WA, USA) carrying 104 cfu S. aureus, either the parental strain Xen 29 or the RifR mutants S464P or H481Y, were implanted subcutaneously in groups of nine mice per strain. One catheter segment was inserted on each side of each animal. Six days after the implantation of the catheters, five mice from each group were treated with rifampicin at 30 mg/kg intraperitoneally in 0.1 mL saline, twice daily for four consecutive days. The remaining four mice in each group were left untreated as controls. At various time points during the infection, the mice were anaesthetized using a constant flow of 1.5% isoflurane from the IVIS® manifold, and imaged using an IVIS® Image System 100 Series (Xenogen Corporation, Alameda, CA, USA). The bioluminescent signals (photons/s) emitted from the mice were analysed using LivingImage® software (Xenogen Corporation) and plotted over the course of infection. The mice were sacrificed 20 days after infection (11 days after final rifampicin treatment). The catheters were surgically removed and the bacteria were detached by sonication for determination of bacterial burdens on the catheters.

Screening for rifampicin-resistant mutants

Bacteria detached from the catheters were screened for the emergence of resistance to the antibiotic by plating undiluted or 10-fold dilutions on Mueller–Hinton agar plates supplemented with rifampicin at 0.05 or 40 mg/L. After incubation at 37 °C overnight, the bacterial colonies on rifampicin plates were enumerated. Randomly picked colonies from plates containing rifampicin were further analysed by the broth dilution method to determine MIC according to NCCLS procedures. The rpoB cluster regions of the mutants were sequenced by Bionexus (Oakland, CA, USA).

Determination of spontaneous mutation rate

Bacteria recovered from untreated mice that were implanted with rifampicin-susceptible S. aureus Xen 29 were detached from catheters at the end of the 20 day infection period and plated on BHI plates with or without rifampicin. To determine the in vitro mutation rate of planktonic bacteria, S. aureus Xen 29 was grown in BHI at 37 °C for 18 h with an agitation rate of 150 rpm. The bacteria were then concentrated to give ~1010 cfu/mL in BHI and serial dilutions were plated on BHI plates with or without rifampicin. To determine the in vitro mutation rate of catheter-attached S. aureus, Teflon catheters were incubated with Xen 29 as described previously;32 after 20 days the bacteria were detached from the catheters and plated on BHI plates with or without rifampicin. The spontaneous mutation rate was calculated by dividing the numbers of colonies detected on BHI containing rifampicin with the numbers of colonies on BHI with no antibiotic.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Characterization of RifR mutants

S. aureus recovered previously from implanted catheters of infected mice treated with rifampicin11 demonstrated that mutants resistant to both high and low rifampicin concentrations were present. No rifampicin-resistant staphylococci could be recovered from catheters populated by 104 cfu prior to implantation into mice, indicating that RifR mutants did not exist (at least not at this bacterial density) prior to rifampicin treatment in vivo. RifR mutants recovered from implanted catheters appeared as early as 3 days after rifampicin treatment (total of six doses). These early occurring mutants had rifampicin MICs < 20 mg/L, and were categorized as low MIC mutants. In contrast, mutants with rifampicin MICs > 640 mg/L, which were subsequently categorized as high MIC mutants, were isolated 9 days after the first regimen of rifampicin treatment. Notably, all the mutants isolated had either low or high MIC. No mutants with intermediate rifampicin MIC values (i.e. 20–640 mg/L) were isolated during the study.

Genetic analysis of the RifR mutants was focused on clusters I and II of the rpoB gene. Sequencing of the PCR products amplified using cluster I and II specific primers indicated that the majority of these mutants (either low or high MIC mutants) had point mutations in cluster I of rpoB. The mutant S464P had a point mutation TCT to CCT, resulting in an amino acid change at position 464 from serine to proline, while the mutant H481Y had a point mutation CAT to TAT changing histidine 481 to tyrosine. After administering rifampicin on re-established biofilm infection, second point mutations such as D471N (GAC to AAC) were often observed in addition to the original S464P mutation, resulting in a high MIC phenotype. In two cases, mutants with high MICs were isolated but no additional mutation was found within the clusters I and II region of the rpoB gene (data not shown), suggesting that mutation(s) could be present in other regions of the rpoB gene, or that other factors might be involved in the resistance to rifampicin.

The appearance of various mutants during the course of rifampicin treatment may be due to the difference in their growth rate. Two RifR mutants, S464P and H481Y, along with the Xen 29 parental strain, were grown in 96-well microtitre plate without agitation. Compared with a doubling time of 55 min for the parental strain Xen 29, the mutants S464P and H481Y had doubling times of 42 and 71 min, respectively (Figure 1). However, if the strains were grown in flasks with aeration, this difference in doubling time was not observed (data not shown).



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Figure 1. Growth curve of S. aureus strain Xen 29 and RifR mutants S464P and H481Y. Three strains were grown in 96-well microtitre plate at 37 °C and the optical densities (OD) were measured in TECAN. This is a representative graph of five independent experiments. Xen 29 is shown as open circles with solid line, S464 as open squares with dotted line, and H481 as open diamonds with dashed line.

 
Response to rifampicin by the RifR mutants

To assess the growth of the RifR mutants during drug treatment in vivo, pre-colonized catheters carrying 104 cfu of S. aureus Xen 29, S464P or H481Y were implanted in mice, treated with rifampicin and monitored using bioluminescence imaging. At day 6 post-infection, mice infected with each bacterial strain were divided into two groups. Five mice were treated twice daily with rifampicin 30 mg/kg intraperitoneally for four consecutive days, whilst the remaining four mice were treated with the vehicle control. Mice were monitored for bioluminescence over a 20 day period using an IVIS® Imaging System 100 Series (Xenogen Corporation). A rapid 10-fold decrease in bioluminescence was evident for Xen 29 after 1 day of treatment (Figure 2a), demonstrating susceptibility to rifampicin as shown before.11 A decrease in bioluminescence was also observed in mice infected with the RifR mutant S464P. However, this decrease was less prominent than Xen 29 and required two additional days of rifampicin treatment to reach a 10-fold reduction in bioluminescence. Also, the signals quickly recovered to the level observed in the groups of untreated mice (Figure 2b). In contrast, the mutant H481Y (high rifampicin MIC) showed no fluctuation in bioluminescence after the treatment (Figure 2c).



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Figure 2. Online monitoring of the responses of S. aureus Xen 29 and RifR mutants to rifampicin treatments in the catheter-associated biofilm infection in mice. (a) Rifampicin-susceptible strain Xen 29; (b) RifR mutant S464P; (c) RifR mutant H481Y. Each strain was used to infect nine mice: four as the untreated control group and five treated with rifampicin. Only the implanted catheters that emitted bioluminescence at day 6 (the beginning of the drug treatments) were included in the calculation for catheter signals (photons/s) in the graphs. The treatment days are indicated by arrows.

 
At the end of the 20 day study period, all S. aureus detached from rifampicin-treated catheters were found to be 100% rifampicin resistant. About 5% of the colonies from the mice infected with Xen 29 had high MIC, similar to the results from a previous study (data not shown). Rifampicin treatment on the low MIC RifR mutant S464P resulted in the appearance of a small percentage (0.1%) of high MIC mutants, most of which had the double mutation S464P and D471N. The mutant H481Y remained unchanged after the treatment and appeared to have no further mutations in cluster I of rpoB.

Establishment and survival of the RifR mutants without selective pressure

Based on bioluminescence detected from mice at day 6 post-catheter implantation (prior to antibiotic treatment), the mutant S464P had 100% infection rate (all 18 implanted catheters giving a signal) and appeared to form biofilms better than the parental strain Xen 29 (15 out of 18; 83%) (Table 1). Out of 18 catheters that were implanted with H481Y, only 11 (61%) were able to establish and maintain an infection, indicating this mutant had a decreased ability to establish a biofilm. In the untreated groups, continued surveillance to 20 days post-implantation of all the catheters that were pre-colonized with Xen 29 or S464P showed the bioluminescent signals to be maintained, indicating that once the biofilm infection had been established it was well maintained without selective pressure. However, in the group infected with mutant H481Y only two out of eight catheters implanted showed a bioluminescent signal (with one barely above detectable level) after 20 days, indicating this mutant to be unfit to maintain a biofilm without selective pressure. Furthermore, in the one catheter pre-colonized by H481Y that did maintain a strong bioluminescence signal, ~10% of the colonies were dark. These dark colonies became bioluminescent again once the decal aldehyde vapour was applied exogenously, suggesting that there could be a mutation(s) in the luxCDE genes coding the aldehyde synthesis complex. Sequence analysis and MIC testing confirmed that both dark and light colonies had original H481Y point mutations and a rifampicin MIC > 640 mg/L.


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Table 1. Bioluminescent signals emitted from catheters in mice at the beginning and the end of antibiotic treatment

 
Spontaneous mutation of S. aureus strain to rifampicin

The spontaneous RifR mutation rate of S. aureus Xen 29 grown overnight in broth as planktonic cells was determined to be 3.5 x 10–7, similar to previously published data estimating the naturally occurring mutation frequency in cells to be 10–6 to 10–8.35,36 However, when Xen 29 grew in biofilm on catheters in vitro for 20 days, the frequency of spontaneous RifR mutants increased ~10-fold to 5.0 x 10–6. Interestingly this spontaneous mutation rate increased even more in vivo, rising to 1.1 x 10–5 when the rifampicin-susceptible Xen 29 was grown on catheters in the untreated animals, an overall increase of > 100-fold (Table 2). The results suggest that bacteria grown in biofilms tend to have a higher mutation rate, this being most extreme when the bacteria grow on catheters in vivo. Noticeably, the codon GAC for amino acid Asp-471 appeared to be the hot-spot for spontaneous RifR mutations of in vitro-grown biofilm bacteria, while the point mutation resulting in mutant S464P was mostly associated with the RifR mutants isolated from in vivo biofilms. The mutant H481Y was isolated under all the conditions tested: spontaneous or drug-treated, in vitro or in vivo. All the spontaneous RifR mutants analysed had only a single point mutation. No mutants with double mutations were isolated, indicating that only a single step mutation was involved in naturally occurring RifR S. aureus.


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Table 2. Identification of RifR spontaneous mutations

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rifampicin and its derivatives have been widely used for treatment of infections for over 30 years. They are usually used in combination with other antimicrobial agents, such as flucloxacillin or vancomycin.8 The reason for this is to avoid the emergence of RifR mutants that inevitably occur during monotherapy. Our results show that during monotherapy in mice, RifR mutants emerge rapidly, as early as 3 days after treatment. The genetic determinants for the majority of RifR mutants identified previously are point mutations that have been mapped in clusters onto the rpoB gene coding for the RNA polymerase ß-subunit. Sequence analysis of the rpoB clusters of the RifR mutants isolated in this study suggested that most point mutations were located in cluster I (amino acid position 462–488, numbering in S. aureus rpoB) and cluster II (position 515–530).

Since the bacterial RNA polymerases are highly conserved, interaction between the antibiotic rifampicin and S. aureus RNAP is hypothesized based on the crystal structure of Thermus aquaticus RNA polymerase.12 Of the 12 residues that are in close contact with the rifampicin molecule, six residues (Q468, F469, D471, H481, H484 and S486) participate in hydrogen bond interaction with four critical hydroxyls of rifampicin. Substitutions of these residues, as a result of point mutations, often confer a RifR phenotype. Although mutations at position Asp-471 lead to high resistance to rifampicin in H. pylori,37 we found in this study that all the point mutations at Asp-471 confer low resistance to rifampicin in S. aureus, no matter whether the substitution leads to changes in hydrogen bond formation (changing to Y, V, A or G), loss of negative charge (D471N), or just a slightly larger residue (D471E). The correlation between rifampicin resistance and the point mutation at position His-481 depends on the resulting amino acid substitution. Larger residues in mutants H481Y (CAT->TAT) and H481R (CAT->CGT) lead to high rifampicin MICs, while mutants H481D (CAT->GAT) or H481N (CAT->AAT), with smaller residue substitutions, have low resistance to rifampicin.38

One of the two RifR mutants studied in detail in this study was S464P, which has an amino acid change from serine to proline at position 464. The other mutant that we chose to study was H481Y, which has an amino acid substitution from histidine to tyrosine at position 481. Although both mutants were the result of a single point mutation, the mutant S464P appeared to develop earlier in the study (only 3 days after the start of antibiotic treatment), while the mutant H481Y was isolated 6 days later. Comparison of the in vitro growth rates of two mutants with their parental strain Xen 29 indicated that the mutant S464P grew at a faster rate (42 min doubling time) than the parental strain (55 min), while the mutant H481Y had a considerably longer doubling time (71 min) than either of the other strains (Xen 29 and S464P). Moreover, not only did the mutant S464P grow faster, it was also found to be better at establishing/maintaining a biofilm on catheters in animals. An in vivo animal infection experiment indicated that the mutant S464P had 100% infection rate in mice after the pre-colonized catheters were implanted for 6 days, compared with 83% for parental Xen 29 and 61% for H481Y.

Previously, it has been shown that antibiotic-resistant mutants, such as H481Y, incur some biological cost, for example growth defects and in some cases reduced virulence in vivo.24,39 In the study by Wichelhaus et al.,38 the mutation S464P, isolated from an in vitro experiment, was shown to have a slight biological cost for being a RifR mutant. However, in our study this same mutant (S464P), with a similar MIC as that reported by Wichelhaus et al.,38 was shown to have a faster growth rate and better biofilm forming ability on catheters. Because serine at position 464 is one of the residues involved in forming an antibiotic binding pocket,12 it is likely that an amino acid change from serine to proline could affect the folding or packing of the RNA polymerase, resulting in weaker binding of rifampicin to the protein. However this mutation does not explain why this strain should grow faster, and possibly, as a result, cause greater infectivity and persistence on catheters in vivo. Since this mutation was isolated separately on numerous occasions, it is unlikely that an additional mutation would occur simultaneously on all occasions to account for this beneficial phenotype, although we cannot rule this possibility out at present.

The in vivo efficacy of antibiotic depends on its serum concentration and availability in the infected tissues, organs or catheters in our case. Rifampicin is well known for its quick absorption and wide distribution throughout the body. Previous pharmacokinetics studies have determined that the peak serum concentrations of rifampicin were 6.4 ± 3.0 mg/L in rats,40 24.3 ± 10.3 mg/L in mice41 and 8.3 ± 6.3 mg/L in guinea pigs1 when rifampicin was administrated at 25 mg/kg. Similarly, in clinical studies the average concentration of this drug in human serum was found to be 7 mg/L following the oral administration of a 600 mg dose (www.rxmed.com). In this study, we used a slightly higher rifampicin concentration (30 mg/kg) to treat S. aureus biofilm infections, where the peak serum concentration of rifampicin was expected to be > 5 mg/L. This might explain why rifampicin treatment reduced the bioluminescence significantly in mice infected with Xen 29 (peak serum concentration is 100-fold higher than the MIC, and ~10-fold higher than minimum biofilm eliminating concentration of Xen 29),33 but only slightly in mice infected with the mutant S464P, which has a rifampicin MIC of 5 mg/L. At the predicted serum concentration, rifampicin was not expected to have any efficacy on catheter infection (monitored by bioluminescence imaging) in mice inoculated with the mutant H481Y, which has a rifampicin MIC > 640 mg/L.

We also explored the possibility that a rifampicin-susceptible population arose while the mutants were grown in the absence of antibiotic in animals. However, we were not able to isolate such wild-type revertants from catheters recovered from animals that were infected but left untreated over a 20 day period. Sequence analysis of a limited numbers of colonies isolated from sacrificed animals showed that the original point mutation, either S464P or H481Y, was genetically stable. Interestingly, ~10% of the H481Y mutants recovered from the untreated animals (no rifampicin administered) were dark. The loss of bioluminescence appeared to be caused by a mutation(s) in the luxCDE genes (encoding the proteins for the aldehyde substrate), since exogenously added aldehyde could restore the light emission. Again, the original mutation H481Y and high MIC phenotype were not changed in such dark variants. In the untreated groups, Xen 29 and S464P established and maintained the biofilm infection without antibiotic selective pressure. In addition, we found that the majority of the catheters colonized with H481Y did not have infection in the control group. These results suggest that the mutant S464P had a better fitness for in vivo biofilm infection than the mutant H481Y, despite S464P having a low level of rifampicin resistance.

One aim of this study was to investigate the kinetics of resistance development during repeated antibiotic treatments. We found that all the spontaneous mutants isolated either in vivo or in vitro had only single point mutations in the region sequenced. Rifampicin treatment on catheters colonized with the low MIC mutant S464P gave rise to a secondary mutation. Most of these mutants with double mutations were resistant to a higher level of rifampicin. Similarly, other reports have demonstrated the recovery of such highly resistant RifR mutants isolated in vitro or from patients treated with rifampicin.38,42 In some cases, a total of nine point mutations were accumulated in one single clinical isolate of S. pneumoniae during rifampicin treatment.21 We found that certain S464P mutants with high-level rifampicin resistance had only one point mutation (TCT->CCT at 464) in clusters I and II on the rpoB gene after a second regimen of rifampicin treatment. This suggests that the genetic determinant for rifampicin resistance is not limited to the point mutations in the well-studied cluster I and II region, and may involve mutations in other regions, such as cluster III25 or cluster N,18,43 although no such point mutations have been reported in S. aureus. Other mechanisms such as decreased membrane permeability or modification to rifampicin by the host bacteria might also be involved.

In summary, we have successfully applied non-invasive in vivo biophotonic imaging technology to investigate the resistance development of S. aureus to the antibiotic rifampicin and in vivo fitness of these RifR mutants in an experimental biofilm-related infection in mice. This technology can readily serve as an excellent tool for screening of novel antibiotics and evaluating in vivo efficacies of antimicrobial compounds against bacteria in biofilm or other chronic diseases.


    Acknowledgements
 
We thank L. Zhang and L. Sin for their technical support in this study.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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