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In vitro double and triple bactericidal activities of doripenem, polymyxin B, and rifampin were assessed against 20 carbapenem-resistant clinical isolates with different mechanisms of carbapenem resistance. Bactericidal activity was achieved in 90% of all bacteria assayed using combinations of polymyxin B, doripenem, and rifampin against five each of the carbapenem-resistant Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli isolates studied. Combinations with these antibacterials may provide a strategy for treatment of patients infected with such organisms.
Carbapenem resistance in Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli is acknowledged worldwide (1, 2, 4, 13, 15, 16). Mechanisms of carbapenem resistance in these bacteria can be due to a variety of carbapenemases alone and/or β-lactamases with porin protein mutations as well as other contributory strategies (13, 15). The latest carbapenem approved in the United States, doripenem, has demonstrated in vitro activity against a variety of multidrug-resistant (MDR) Gram-negative organisms, which produce well-characterized β-lactamases, and delayed development of resistance to doripenem has been demonstrated (6, 7, 8, 12). Combination therapy with several classes of antibiotics against multidrug-resistant pathogens has revealed increased activity over single agents and delayed development of resistance (14). This investigation studied the in vitro bactericidal activities of double and triple antibiotic combinations using doripenem (D), polymyxin B (PB), and rifampin (R) against Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae because of their progressive resistance to all available agents.
Twenty carbapenem-resistant clinical isolates with different mechanisms of carbapenem resistance and nonrelated by pulsed-field gel electrophoresis were studied, including five imipenem-resistant K. pneumoniae isolates (two with KPC and three with ACT-1 [AMPC-type] β-lactamases), five A. baumannii isolates (non-MBL or KPC β-lactamases), five P. aeruginosa isolates (one KPC and four non-MBL or KPC β-lactamases), and five E. coli isolates (one KPC-3 and four KPC-2 β-lactamases). Susceptibility of the isolates was initially determined by our clinical microbiology laboratory using the Phoenix system, and the results were confirmed in the infectious disease research laboratory using Etest methodology according to the manufacturer's specifications (bioMérieux North America). E. coli ATCC 25922 was tested as the control strain.
Bactericidal experiments were performed using double and triple antibiotic combinations of polymyxin B plus doripenem, polymyxin B plus rifampin, doripenem plus rifampin, and polymyxin B plus doripenem and rifampin as previously described (17). Time-kill studies were performed at concentrations at 1/4 of their MICs. Doripenem, polymyxin B, and rifampin alone were also tested at 1/4 MIC against each isolate. For bactericidal assays, samples were taken at time zero and 2, 4, 8, and 24 h. Aliquots were serially diluted, and a 10-μl aliquot was transferred onto plates, spread with a loop to minimize carryover to quantify bacterial counts, and incubated at 35°C for 24 h. Bactericidal activity was defined as a ≥3-log CFU/ml decrease in 24 h.
Genotypic and phenotypic characteristics for the carbapenem-resistant isolates used in this study are displayed in Table Table1.1. All 20 isolates had the following MICs (μg/ml): for rifampin, ranging from 8 to >32; for ertapenem, >32; for doripenem, 1.5 to >32; for imipenem; 6 to >32; for meropenem, 2 to >32; and for polymyxin B, 0.5 to 12. The results of in vitro bactericidal activities and quantitative fold changes with double and triple antibiotic combinations are shown in Table Table2.2. Combinations of polymyxin B-doripenem-rifampin at 1/4 MICs for each antibiotic were bactericidal for 4/5 K. pneumoniae, 3/5 A. baumannii, 5/5 P. aeruginosa, and 5/5 E. coli isolates. Combinations of polymyxin B-doripenem at 1/4 MICs for each antibiotic were bactericidal for 1/5 K. pneumoniae, 1/5 A. baumannii, 1/5 P. aeruginosa, and 4/5 E. coli isolates. Combinations of polymyxin B-rifampin at 1/4 MICs for each antibiotic were bactericidal for 1/5 K. pneumoniae, 2/5 A. baumannii, 1/5 P. aeruginosa, and 2/5 E. coli isolates. Combinations of doripenem-rifampin at 1/4 MICs for each antibiotic were bactericidal for 2/5 K. pneumoniae, 2/5 A. baumannii, 1/5 P. aeruginosa, and 1/5 E. coli isolates. Bactericidal activity was achieved in 85% of all bacteria assayed using combinations of polymyxin B-doripenem-rifampin, 30% with polymyxin B-doripenem, 30% with doripenem-rifampin, and 25% with polymyxin B-rifampin at 1/4 MICs. Doripenem, polymyxin B, and rifampin tested alone, at 1/4 MIC, were not bactericidal.
Extreme drug resistance (XDR) and pan resistance in Gram-negative bacteria is being reported with increasing frequency (5, 10). Although combination therapy has been widely accepted for management of patients infected with Mycobacterium tuberculosis and human immunodeficiency virus, it has not been widely accepted for infections caused by multidrug resistant Gram-negative bacteria. Studies performed in vitro or in animal models often demonstrate synergy with antibiotic combinations, but few translate this success to the clinical arena because of the lack of well-controlled studies (3, 9, 11).
Our study showed that combinations of polymyxin B-doripenem-rifampin achieved 100% bactericidal activity, defined as a ≥3-log-CFU/ml decrease in 24 h at 1/4 MICs for P. aeruginosa and E. coli, 80% for K. pneumoniae, and 60% for A. baumannii despite resistance to the carbapenems and rifampin alone. A previous study using similar methodology demonstrated that a combination of polymyxin B at 0.5 times the MIC plus rifampin had synergistic activity against 15/16 KPC-producing Klebsiella pneumoniae isolates and synergistic bactericidal activity against 10/16 of the isolates using a combination of polymyxin B plus imipenem (2). While in vitro studies do not always correlate with in vivo efficacy, our study showed that bactericidal activity was achieved in 85% of our multidrug resistant isolates at 1/4 their MICs. Administration of approved doses of each of the antibiotics would be in excess of the concentrations used in this in vitro study. In an era of burgeoning multidrug resistance, including that against carbapenems, triple combinations with these antibacterials may provide a strategy for treatment of patients infected with such organisms.
This study was funded by a grant from Johnson & Johnson Pharmaceutical Research and Development and by BMA Medical Foundation, Inc.
Published ahead of print on 5 April 2010.