1 PLA General Hospital, 28 Fuxing Road, Beijing 100853, P. R. China; 2 Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA
Received 17 June 2005; returned 13 August 2005; revised 12 September 2005; accepted 13 September 2005
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Abstract |
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Methods: Tuberculosis patients (n = 372) were sampled for nasal colonization by Staphylococcus aureus at the beginning of anti-tuberculosis therapy with rifampicin-containing regimens and again after 2 and 4 weeks. Rifampicin susceptibility of S. aureus was determined, and S. aureus isolates from patients developing acquired resistance were examined by molecular strain typing. Diabetes patients (n = 200) served as untreated controls.
Results: Nasal colonization was 17% and 20% for the tuberculosis and diabetes patients, respectively. Four patients were initially colonized with rifampicin-resistant S. aureus and were excluded from further sampling. Initiation of anti-tuberculosis therapy eradicated S. aureus nasal colonization in 53/58 tuberculosis patients while allowing acquisition of rifampicin resistance in 5/58. Pulsed-field gel electrophoresis (PFGE) band patterns and protein A repeat sequence determination differed in S. aureus isolated from different patients but was identical in isolates obtained from the same patient before and after acquisition of resistance. No resistance was acquired in untreated control patients, which differed statistically from treated patients (P = 0.025).
Conclusions: Acquired resistance and eradication of susceptible bacteria can occur concurrently; restricting acquired resistance may require direct suppression of mutant growth and viability in addition to elimination of susceptible bacteria.
Keywords: mutant selection window , antimicrobial resistance , S. aureus , TB
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Introduction |
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One prediction of the selection window hypothesis is that eradication of susceptible cells and acquisition of resistance can occur concurrently when antimicrobial concentrations fall inside the selection window. Such concurrence is most likely to be observed when the antimicrobial is very potent against susceptible bacteria and ineffective with single-step resistant mutants. Treatment of Staphylococcus aureus with rifampicin may be particularly suitable for observing concurrence, since MIC is low with susceptible cells and high with mutants.3 A direct experiment is difficult because rifampicin is generally not used as monotherapy for S. aureus infections. However, nasal colonization by S. aureus is common, tuberculosis patients are often treated with rifampicin as the only agent having activity against S. aureus, and rifampicin resistance is observed in nasal isolates of S. aureus.4 To document acquired resistance, it is necessary to follow patients whose isolates are known to be rifampicin-susceptible at the beginning of therapy, a procedure not previously performed with nasal colonization.4 Below we describe rifampicin susceptibility and genetic features of S. aureus at the beginning and after 4 weeks of anti-tuberculosis therapy to determine whether bacterial eradication and acquisition of resistance can occur concurrently.
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Patients and methods |
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Tuberculosis patients (253 males, 119 females) had an average age of 40.2 years (range 1886 years); control diabetes patients (119 males, 81 females) had an average age of 54.6 years (range 1885 years). All tuberculosis patients were treated with rifampicin and isoniazid plus additional compounds in heterogeneous protocols established independent of this study. Additional compounds used included pyrazinamide, ethambutol, para-aminosalicylic acid, streptomycin and levofloxacin in various combinations. The control group was untreated with antimicrobial. Patients in both study and control groups were hospitalized throughout the study period, which minimized non-compliance to treatment.
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Results |
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No rifampicin-susceptible S. aureus was recovered after 2 and 4 weeks of treatment; by 2 weeks of treatment, four patients tested positive for rifampicin-resistant S. aureus. One additional resistant isolate was recovered in week 4. At that point, the trial was stopped. Four cases of acquired rifampicin resistance arose from 34 patients receiving rifampicin as the only agent to which S. aureus was susceptible (patients #1, #2, #4, #5, Figure 1). An additional 19 cases were also treated with streptomycin. Among these, streptomycin-susceptible S. aureus (zone of inhibition > 15 mm with a paper disc containing 10 µg streptomycin) was recovered from 15 patients at the beginning of therapy; rifampicin resistance was not observed with these patients. Four other cases treated with streptomycin had streptomycin-resistant S. aureus (zone of inhibition < 12 mm) at initiation of therapy. They were treated with rifampicin, isoniazid, pyrazinamide and streptomycin. In one of these four cases, rifampicin resistance was acquired (patient #3, Figure 1). No rifampicin-resistant case was found among the eight patients treated with levofloxacin-containing regimens.
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Of the 200 patients in the untreated control group, 41 were colonized by S. aureus; two carried rifampicin-resistant S. aureus at the start of the study and were excluded from further sampling. No rifampicin resistance was observed among the 39 remaining cases after 4 weeks of observation. Using Fisher's exact test, P = 0.025 for the acquisition of rifampicin resistance when study and control groups were compared, indicating that study size was sufficient for statistical significance.
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Discussion |
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Eradication of susceptible bacteria is explained by drug concentrations in plasma and nasal secretions being maintained above MIC4,10 and by drug exposure (i.e. plasma AUC24/MIC) for susceptible S. aureus (MIC 0.01 mg/L) being high11 (AUC24/MIC > 7000 h, 700 h when corrected for protein binding). The five resistant cases are most readily explained by assuming that resistant subpopulations were present in the initial colonizing population, since no population expansion of susceptible cells was expected and since even extremely high rifampicin concentrations allow growth of mutant subpopulations.3 If so, bacterial load in at least some of the colonized patients must have reached 108 cells or the spontaneous mutation frequency in patients must have been higher than 1 in 108, the value observed in vitro.12,13 An alternative that cannot be ruled out by the present work is rifampicin-induced mutation during treatment. Regardless of how resistant mutant subpopulations were generated, they can be selectively enriched to where they dominate nasal populations at the same time that susceptible bacteria are eradicated. Concurrent eradication of susceptible bacteria and acquisition of resistance are predicted by the mutant selection window hypothesis.
In vitro data suggest that acquired resistance will be observed more readily with the S. aureusrifampicin combination than with many other combinations,3 in part because rifampicin resistance occurs in an all-or-none, rather than a stepwise, fashion. Nevertheless, the distinction between eradicating susceptible bacteria and restricting resistance shown with this model system is likely to be general, since pre-existing mutant subpopulations are not expected to be eliminated by antimicrobial concentrations used to kill susceptible cells. For example, with Streptococcus pneumoniae the concentration of levofloxacin must be eight times higher to kill mutants than wild-type cells,14 and examples of acquired resistance have been reported.15 However, determining whether eradication of susceptible pathogens and acquisition of resistance occur concurrently for a particular combination may require examining large numbers of patients when mutants tend to be eliminated by the combination of host defence and antimicrobial treatment.
The present work is limited in three ways. First, we were unable to identify tuberculosis patients to serve as untreated controls, since rifampicin is a first-line anti-tuberculosis agent and since most cases of tuberculosis in China are treated with rifampicin. Second, we could not treat tuberculosis patients with very low rifampicin concentrations to determine whether even more resistant mutants would be selected below MIC than above it.3 Third, rifampicin-resistance mutations reduce the susceptibility of S. aureus so much that rifampicin concentrations could not be attained that would be high enough to prevent the amplification of resistant mutants and define an upper boundary of the selection window. Studies are in progress to address these limitations.
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Acknowledgements |
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References |
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