JAC Advance Access originally published online on July 26, 2006
Journal of Antimicrobial Chemotherapy 2006 58(3):689-692; doi:10.1093/jac/dkl303
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Prevention of rifampicin resistance in Acinetobacter baumannii in an experimental pneumonia murine model, using rifampicin associated with imipenem or sulbactam
1 Service of Infectious Diseases, Hospitales Universitarios Virgen del Rocío Avda. Manuel Siurot s/n, 41013, Sevilla, Spain 2 Service of Microbiology, Hospital Universitario Virgen Macarena Avda. Dr Fedriani s/n, 41009, Sevilla, Spain 3 Department of Medicine, University of Sevilla Avda. Dr Fedriani s/n, 41009, Sevilla, Spain 4 Department of Microbiology, University of Sevilla Avda. Sánchez Pizjuan s/n, 41009, Sevilla, Spain
*Correspondence address. Servicio de Enfermedades Infecciosas, Hospitales Universitarios Virgen del Rocío, Avda. Manuel Siurot s/n, 41013, Sevilla, Spain. Tel/Fax: +34-955012376; E-mail: jeronimopachon{at}telefonica.net
Received 21 April 2006; returned 29 June 2006; revised 29 June 2006; accepted 5 July 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Objectives: To examine the development of rifampicin resistance in multidrug-resistant Acinetobacter baumannii exposed to rifampicin and the prevention of the appearance of rifampicin-resistant mutants when rifampicin is used in association with imipenem or sulbactam.
Methods: A clinical strain of multidrug-resistant A. baumannii was used to examine the frequency of resistance to rifampicin in vivo, in a pneumonia model in immunocompetent C57BL/6 mice. The in vitro and in vivo prevention of the development of resistance to rifampicin was analysed using rifampicin alone or in association with imipenem or sulbactam, in time–kill studies and in the experimental murine pneumonia, respectively.
Results: Rifampicin-resistant mutants were found at 48 and 72 h, both in vitro and in vivo, when rifampicin was used alone, with the MIC increasing from 4 to 
128 mg/L. The in vivo frequency of rifampicin-resistant mutants was 3 x 10–6. On the contrary, no resistant mutants appeared after 72 h, in vitro or in vivo, when rifampicin was employed in association with imipenem or sulbactam. After six daily passages in rifampicin-free agar plates the resistant mutants maintained the high resistance to rifampicin (128 mg/L).
Conclusions: These results suggest that rifampicin must not be used alone in the treatment of infections caused by multidrug-resistant A. baumannii. In these cases, rifampicin may be used in combination with imipenem or sulbactam, which prevent the development of resistance to rifampicin.
Keywords: resistant mutant , combined treatment , in vivo resistance
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Acinetobacter baumannii is an important cause of nosocomial infections worldwide. Most nosocomial isolates of A. baumannii are resistant to a wide variety of antimicrobials, including imipenem and rifampicin.1 Studies on experimental murine pneumonia have reported that rifampicin is active in vitro and in vivo against multidrug-resistant A. baumannii, including those with intermediate resistance to rifampicin.2 Consequently, rifampicin has been used successfully, combined with colistin, for the therapy of selected A. baumannii infections in humans.3
It is well known that, in infections caused by other bacteria, rifampicin must not be used as monotherapy because of the rapid development of resistance in vitro and in vivo. However, the development of resistance to rifampicin can be avoided with the use of combination treatment. A classic example is the treatment of tuberculosis. Monotherapy with rifampicin induces the development of resistance within 3 months. On the contrary, the association of isoniazid with rifampicin prevents the development of resistance.
The aim of the present study was to examine the frequency of resistance in multidrug-resistant A. baumannii exposed to rifampicin in vitro and in vivo and the prevention of the development of resistance when rifampicin is used in association with other antimicrobials.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Antimicrobials
The antimicrobials for the in vitro experiments were obtained as laboratory standard powders: rifampicin (Sigma Chemical Co., Madrid, Spain), imipenem (Merck Sharp & Dohme, Madrid, Spain) and sulbactam (Pfizer, Orsay, France). The anaesthetic for the in vivo experiments was 5% sodium thiopental (B. Braun Medical S.A., Rubi, Barcelona, Spain).
Bacterial strain
A clinical strain of A. baumannii causing nosocomial bacteraemia (HUVR 1327) was used that was collected and identified in a previous study of A. baumannii bacteraemia.4 This strain was multidrug-resistant, including to imipenem and sulbactam, and was susceptible to rifampicin and colistin.
Determination of MICs and MBCs
MICs of rifampicin, imipenem and sulbactam were determined by the standard microdilution method.5 Concentrations of antimicrobials from 128 to 0.125 mg/L were tested. MBCs were determined by subculturing 0.1 mL samples from MIC broth cultures on Mueller–Hinton agar plates.6 Escherichia coli ATCC 25922 was used as a reference strain. Breakpoints for susceptibility and resistance of A. baumannii to rifampicin were those from the French Society for Microbiology.7
In vitro selection of rifampicin-resistant mutants
Time–kill curves were used. A. baumannii was incubated with rifampicin, imipenem or sulbactam at concentrations of 1x MIC, 2 x MIC and 4x MIC. Furthermore, the 18 possible combinations of rifampicin plus imipenem or sulbactam at these concentrations were tested. Tubes with 20 mL of MHB with an inoculum of 5 x 105 cfu/mL of the strain HUVR 1327 were used. Tubes with the bacterial inoculum and without antimicrobials were used as growth controls. The bacterial growth was counted at 0, 24, 48 and 72 h after incubation at 37°C. Ten-fold dilutions were made and 100 µL was plated on sheep blood agar and incubated for 24 h at 37°C. For the detection of rifampicin-resistant mutants, the MIC of rifampicin was carried out in triplicate for a maximum of five colonies at each time-point.
Stability of rifampicin-resistant mutants
The stability of mutants of A. baumannii resistant to rifampicin was tested by giving them six daily passages in rifampicin-free agar plates. After these passages, we selected a maximum of five colonies to determine the MIC in triplicate.
Laboratory animals
Immunocompetent specific-pathogen-free C57BL/6 young female mice, weighing 16–20 g, were used. They were supplied by Universidad de Sevilla's facility. Animals were housed in regulation cages and given free access to food and water. The use of mice for these experiments was approved by the Ethics Committee of the University Hospitals Virgen del Rocío (Document 4/2000).
In vivo selection of rifampicin-resistant mutants
An experimental murine pneumonia model8 was used to evaluate the selection of rifampicin-resistant mutants of A. baumannii. An inoculum of 
108 cfu/mL was used. Therapy was started 4 h after the inoculation.
A total of 30 mice were randomly included in the following treatment groups for 72 h, 6 mice for each treatment group: rifampicin 100 mg/kg/day, imipenem 120 mg/kg/day, sulbactam 240 mg/kg/day, rifampicin plus imipenem, and rifampicin plus sulbactam. In each case the total daily dose was divided in three administrations. Rifampicin was administered intraperitoneally and the others intramuscularly. Two mice of each group were sequentially sacrificed every 24 h. The lungs were aseptically removed, weighed and homogenized for 2 min in 2 mL of sterile saline solution (Stomacher 8, Tekmar Co., Cincinnati, OH, USA). Thereafter, they were vortexed for 1 min and centrifuged at 800 rpm for 10 min at 4°C. Next, the supernatant was placed into a new tube and vortexed for 1 min and centrifuged at 4000 rpm for 15 min at the same temperature. The supernatant was taken out and mixed in 600 µL of sterile saline solution. Finally, we plated 100 µL on agar and incubated for 24 h at 37°C; determination of the MIC of rifampicin was carried out in triplicate for a maximum of five colonies from these plates.
Frequency of rifampicin-resistant mutants
Two mice were inoculated with A. baumannii HUVR 1327. One of them was used as control (without treatment). The other was treated with rifampicin 100 mg/kg/day as previously detailed. Both were sacrificed 24 h after the inoculation. The lungs were removed and homogenized for 2 min in 2 mL of sterile saline solution. After 10-fold dilutions, 100 µL was plated on sheep blood agar for 24 h at 37°C and the counts were expressed as log10 cfu/g of tissue. To calculate the frequency of appearance of rifampicin-resistant mutants we divided the log10 cfu/g of the treated mouse by the same figure from the control mouse. These experiments were carried out twice.
Statistical analysis
2 and Student t-tests were performed. A value of P < 0.05 was considered significant. The statistical package SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
MICs and MBCs
The MICs/MBCs of imipenem, sulbactam and rifampicin for the HUVR 1327 strain were 32/32, 32/32 and 4/8 mg/L, respectively.
In vitro selection of rifampicin-resistant mutants
The bactericidal activity of the drugs at different concentrations is shown in Figure 1. The susceptibility to rifampicin did not change in the tubes containing imipenem or sulbactam alone, at 24, 48 and 72 h of incubation. In the tubes with rifampicin alone the MIC of rifampicin remained 4 mg/L at 24 h and changed to 128 mg/L after 48 and 72 h of incubation.
|
In the same way, at 24 h with the combinations of rifampicin plus imipenem or sulbactam, the MIC of rifampicin did not change from 4 mg/L. No bacteria were recovered from the tubes in the experiments made with the different combinations of rifampicin plus imipenem or sulbactam at 48 and 72 h of incubation.
Stability of rifampicin-resistant mutants
The rifampicin-resistant mutants from HUVR 1327 A. baumannii maintained their resistance (MIC 128 mg/L) over six daily passages in fresh rifampicin-free agar plates.
In vivo selection of rifampicin-resistant mutants
Susceptibilities of the strains of A. baumannii recovered from the lungs of the infected mice treated with the different monotherapies or combinations are shown in Table 1.
|
Frequency of rifampicin-resistant mutants
The number of colonies was 2 x 108 cfu/g of lung in the untreated mice and 0.66 x 102 cfu/g in the mice treated with rifampicin. The MIC of rifampicin for the isolates from treated mice increased to 128 mg/L. Thus, the frequency of appearance of rifampicin-resistant mutants was 3 x 10–6.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
These results show that A. baumannii develops resistance to rifampicin when this drug is used alone, both in vitro and in vivo. The association of imipenem or sulbactam with rifampicin prevents the development of resistance to rifampicin. We used a strain susceptible to rifampicin, because in A. baumannii experimental pneumonia rifampicin was efficacious as monotherapy using strains showing MICs of rifampicin between 4 and 8 mg/L.9
The appearance of rifampicin resistance in staphylococcal infections can be avoided with the use of rifampicin in association with other antimicrobials,10 like happened in our experiments when we used rifampicin in combination with imipenem or sulbactam. The association of ciprofloxacin or vancomycin with rifampicin in the treatment of methicillin-resistant S. aureus experimental osteomyelitis in rats prevented the emergence of resistance to rifampicin, whereas rifampicin-resistant mutants appeared in the animals treated with rifampicin alone.10
In our experiments, the in vivo mutation rate in A. baumannii was 10–6, which is higher than that found in other bacteria, which ranges from 10–7 to 10–8.11 We also found that the rifampicin-resistant mutants, both in the in vitro and in vivo experiments, were stable and maintained their resistance through six daily transfers in rifampicin-free agar.
In summary, as occurred in the treatment of infections by other bacteria, our results suggest that rifampicin must not be used alone in the treatment of infections caused by A. baumannii. In these cases, rifampicin may be used in association with imipenem or sulbactam, which prevents the development of resistance to rifampicin.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
This study has been supported in part by research grants from the Consejeria de Salud de la Junta de Andalucia (172/00), Spain, and from the Spanish Network for the Research in Infectious Diseases (REIPI—ISCIII—C03/14).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
1 Appleman MD, Belzberg H, Citron DM, et al. (2000) In vitro activities of nontraditional antimicrobials against multiresistant Acinetobacter baumannii strains isolated in an intensive care unit outbreak. Antimicrob Agents Chemother 44:1035–40.
2
Montero A, Ariza J, Corbella X, et al. (2004) Antibiotic combinations for serious infections caused by carbapenem-resistant A. baumannii in a mouse pneumonia model. J Antimicrob Chemother 54:1085–91.
3 Motaouakkil S, Charra B, Hachimi A, et al. (2006) Colistin and rifampicin in the treatment of nosocomial infections from multiresistant Acinetobacter baumannii. J Infect doi:10.1016/j.jinf.2005.11.019.
4
Martín-Lozano D, Cisneros JM, Becerril B, et al. (2002) Comparison of a repetitive extragenic palindromic sequence-based PCR method and clinical and microbiological methods for determining strain sources in cases of nosocomial Acinetobacter baumannii bacteremia. J Clin Microbiol 40:4571–5.
5 Clinical and Laboratory Standards Institute. (2005) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Sixth Edition: Approved Standard M7-A6. (CLSI, Wayne, PA, USA).
6 National Committee for Clinical Laboratory Standards. (1999) Methods for Determining Bactericidal Activity of Antimicrobial Agents: Approved Standard M26-A. (NCCLS, Wayne, PA, USA).
7 Comité de l'antibiogramme de la Société Française de Microbiologie. Communiqué 2006. http://www.sfm.asso.fr (26 June 2006, date last accessed).
8
Rodríguez-Hernández MJ, Pachón J, Pichardo C, et al. (2000) Imipenem, doxycycline and amikacin in monotherapy and in combination in Acinetobacter baumannii experimental pneumonia. J Antimicrob Chemother 45:493–501.
9
Wolff M, Joly-Guillou ML, Farinotti R, et al. (1999) In vivo efficacies of combinations of ß-lactams, ß-lactamase inhibitors, and rifampicin against Acinetobacter baumannii in a mouse pneumonia model. Antimicrob Agents Chemother 43:1406–11.
10 Henry NK, Rouse MS, Whitesell AL, et al. (1987) Treatment of methicillin-resistant Staphylococcus aureus experimental osteomyelitis with ciprofloxacin or vancomycin alone or in combination with rifampicin. Am J Med 82:73–5.[Web of Science][Medline]
11 Farr BM and Mandell GL. (1982) Rifampicin. Med Clin North Am 66:157–68.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. A. Russo, J. M. Beanan, R. Olson, U. MacDonald, N. R. Luke, S. R. Gill, and A. A. Campagnari Rat Pneumonia and Soft-Tissue Infection Models for the Study of Acinetobacter baumannii Biology Infect. Immun., August 1, 2008; 76(8): 3577 - 3586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Y. Peleg, H. Seifert, and D. L. Paterson Acinetobacter baumannii: Emergence of a Successful Pathogen Clin. Microbiol. Rev., July 1, 2008; 21(3): 538 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Perez, A. M. Hujer, K. M. Hujer, B. K. Decker, P. N. Rather, and R. A. Bonomo Global Challenge of Multidrug-Resistant Acinetobacter baumannii Antimicrob. Agents Chemother., October 1, 2007; 51(10): 3471 - 3484. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||