In Vitro Double and Triple Synergistic Activities of Polymyxin B, Imipenem, and Rifampin against Multidrug-Resistant Acinetobacter baumannii

Eight unrelated clinical Acinetobacter baumannii isolates resistantto all commonly used antibiotics were subjected to three-dimensionalcheckerboard microtiter plate dilution and time-kill studiesat one-fourth of their MICs of polymyxin B, imipenem, and rifampin.Synergy was demonstrated with combinations of polymyxin B andimipenem, polymyxin B and rifampin, and polymyxin B, imipenem,and rifampin. Double combinations of polymyxin B and imipenemand of polymyxin B and rifampin were bactericidal for sevenof eight isolates, and triple combinations were bactericidalfor all isolates within 24 h. Future clinical studies usingdouble and triple therapy with these antibacterials may providean effective option against potentially lethal infection dueto multiresistant Acinetobacter baumannii.

INTRODUCTION

Top

Introduction

In recent years, Acinetobacter baumannii has emerged as oneof the more ubiquitous antibiotic-resistant gram-negative nosocomialpathogens among critically ill patients (4, 23). Although thecarbapenems, ampicillin-sulbactam, and amikacin have retainedexcellent in vitro and clinical activities against susceptiblestrains of A. baumannii, a growing number of reports have documentedresistance to these antibacterials (1, 2, 10, 12-14, 16). Asa result nontraditional agents, including polymyxin B and colistin,have been used to treat patients infected with multiresistantA. baumannii (6, 9, 15). However, pulmonary infections havenot responded well to such monotherapy, and resistance has occurredamong strains that have persisted during treatment (6, 15, 24).

In this communication, we describe the in vitro double and tripleinteractions of polymyxin B, imipenem, and rifampin againsteight unique strains of multidrug-resistant A. baumannii usingcheckerboard microdilution and time-kill methods.

MATERIALS AND METHODS

Top

Materials and Methods

Eight multidrug-resistant clinical strains of A. baumannii wereobtained from the Clinical Microbiology Laboratory at New YorkHospital Queens. All isolates had different pulsed-field gelelectrophoresis patterns and were considered unrelated accordingto the criteria established by Tenover et al. (21). MICs ofselected individual antibiotics were then determined using microdilutionand E-test methods (AB Biodisk North America Inc., Piscataway,N.J.) by the Infectious Disease Research Laboratory. E-testmetallo-beta-lactamase (E-test MBL) strips were used to detectthe presence of molecular class B beta-lactamases (25). Rifampinand polymyxin B were obtained from Sigma (St. Louis, Mo.), andimipenem powder was supplied by Merck and Co. (Rahway, N.J.).MIC determinations were performed using checkerboard microdilutionmethods with Mueller-Hinton broth and a final inoculum of approximately5 x 10⁵ CFU/ml. Microdilution plates for evaluation of tripledrug combinations were performed in the following manner. Thefirst microtiter plate contained no imipenem and increasingconcentrations of rifampin ranging from 0 to 32 µg/mlon the x axis and increasing concentrations of polymyxin B rangingfrom 0 to 32 µg/ml on the y axis. Each of the subsequentsix plates contained a fixed concentration of imipenem rangingfrom 1 to 32 µg/ml with increasing concentrations of rifampinranging from 0 to 32 µg/ml on the x axis and increasingconcentrations of polymyxin B ranging from 0 to 32 µg/mlon the y axis. MICs and fractional inhibitory concentrations(FICs) were determined after 24 h of growth. The MIC was definedas the well in the microtiter plate with the lowest drug combinationat which no visible growth was observed. A bactericidal effectwas defined as $>=$ 99.9% killing of the starting inoculum.FICs of <1.0, 1.0, and >1.0 were used to define synergy,addition, and antagonism, respectively, according to previouslypublished methods (3, 27). Time-kill studies were also performedaccording to earlier described techniques (7, 17). Escherichiacoli strain ATCC 25922 was used as a control for all experiments.The limit of detection in these studies was 2 log₁₀ CFU/ml.Three-dimensional isobolograms were generated using Surfer 8(Golden Software, Inc., Golden, Colo.).

RESULTS

Top

Results

The MICs of the three antibiotics studied for all eight isolatesare shown in Table 1. All eight isolates with imipenem MICsof 32.0 µg/ml or >32.0 µg/ml did not demonstratean eightfold MIC reduction in the presence of EDTA using theE-test MBL procedure and were presumed not to possess metallo-beta-lactamases,according to the manufacturer's specifications. The resultsof time-kill experiments using 0.25 µg of polymyxin B/ml,0.5 µg of rifampin/ml, and 8.0 µg of imipenem/mlfor the eight unique clinical isolates of A. baumannii testedin this study are also summarized in Table 1. All isolates werekilled within 24 h using polymyxin B-imipenem-rifampin, oneisolate showed regrowth with imipenem plus polymyxin B (isolateE), and one isolate showed regrowth with polymyxin B plus rifampin(isolate A). Using three-dimensional checkerboard microdilutionwith polymyxin B, rifampin, and imipenem, the combined concentrationsof each antibiotic showing synergy and their sum FICs ( ${Sigma}$ FICs)of less than 1.0 against isolates A and B are presented in Table2. Double combinations of polymyxin B (0.5 µg/ml) andimipenem (2.0 to 8 µg/ml) were synergistic against isolateA. Double combinations of polymyxin B (0.25 to 0.5 µg/ml)and imipenem (0.25 to 8.0 µg/ml) were synergistic againstisolate B. Synergy was observed with a combination of polymyxinB and rifampin at concentrations of 0.5 µg/ml of eachdrug against isolate A. Double combinations of polymyxin B (0.25to 0.5 µg/ml) and rifampin (0.03 to 0.5 µg/ml) weresynergistic. Triple combinations of polymyxin B (0.12 to 0.5µg/ml), rifampin (0.03 to 1.0 µg/ml), and imipenem(1.0 to 16.0 µg/ml) were also synergistic against isolatesA and B. These results are also shown as three-dimensional isobologramsfor isolates A and B (Fig. 1). The areas of concavity correspondto the aforementioned concentration ranges of double and triplecombinations. The graphs of time-kill experiments using one-fourthMICs of each agent (0.25 µg of polymyxin B/ml, 0.5 µgof rifampin/ml, 8.0 µg of imipenem/ml) alone and in combinationare shown for four isolates (A, B, E, and H) in Fig. 2.

View this table:
[in this window]
[in a new window]
TABLE 1. MICs of each antibiotic alone and time-kill results for the eight clinical A. baumannii isolates

View this table:
[in this window]
[in a new window]
TABLE 2. Individual and sum total FICs of the three antibiotics

View larger version (42K):
[in this window]
[in a new window]
FIG. 1. (A) Three-dimensional isobologram for A. baumannii isolate A. The areas on the isobologram showing depressions (concave sinks) that approach zero represent the greatest synergy ( ${Sigma}$ FIC $<=$ 1.0). (B) Results for isolate B.

View larger version (39K):
[in this window]
[in a new window]
FIG. 2. Time-kill experiments using one-fourth the MIC of each agent (0.25 µg of polymyxin B/ml, 0.5 µg of rifampin/ml, and 8.0 µg of imipenem/ml) alone and in combination for isolates A, B, E, and H. The dotted lines indicate the lower limit of detection of 2 log₁₀ CFU/ml.

DISCUSSION

Top

Discussion

Several in vitro and animal model studies have demonstratedsynergy with polymyxin B plus rifampin or imipenem and withpolymyxin B plus meropenem against multidrug-resistant A. baumannii(23, 26; N. X. Chin, B. Scully, and P. Della-Latta, Abstr. 38thIntersci. Conf. Antimicrob. Agents Chemother., abstr. E-56,1998). Another study has shown synergy between polymyxin B andrifampin against Serratia marcescens isolates from infectedpatients, even though the isolates were resistant to each antibioticalone (19). In vitro triple synergistic studies with polymyxinB, trimethoprim, and sulfonamide have shown efficacy againstmultidrug-resistant Burkholderia cepacia, as has the combinationof polymyxin E (methane sulfonate, colistimethate), trimethoprim,and sulfamethoxazole against S. marcescens (20, 22).

In the present study, double combinations of polymyxin B plusimipenem and polymyxin B plus rifampin were bactericidal againstseven out of eight isolates tested. The triple combination ofpolymyxin B-rifampin-imipenem was synergistic against all isolatesby checkerboard and time-kill studies. In these studies FICs(concentrations or MICs of the antibiotics in the combinationdivided by the MIC of the antibiotic alone) were calculatedfor two- and three-drug combinations (Table 2). All MICs havebeen graphically represented in a three-dimensional isobologram(Fig. 1). Synergy for three antibiotics has been defined byseveral investigators as a fractional sum of <1.0 ( ${Sigma}$ FIC of<1.0) (3, 27). Plotted values showing concave surface areason the isobolograms correspond to synergy, and convex areasrepresent antagonism (3, 27). The probable role of polymyxinB in such synergy is its rapid permeabilization of the outermembrane, allowing enhanced penetration and activity of bothimipenem and rifampin. Synergy between polymyxin B or otherpeptide antibacterials and the carbapenems would be expectedwhen carbapenem resistance is due to porin protein defects.Synergy may not be evident if the organism possesses significantcarbapenemase activity due to class B beta-lactamases. Metallo-carbapenemaseswere most likely not contributing to imipenem resistance inthe eight isolates, since EDTA using MBL strips did not reverseresistance. Numerous mechanisms other than class B enzymes havebeen implicated in carbapenem resistance in Acinetobacter, includingother beta-lactamase classes, porin protein losses or theirreduced expression, and penicillin-binding protein changes (8,23).

Although the use of two or more agents is accepted as appropriatetreatment of patients with tuberculosis or human immunodeficiencyvirus infection, this approach has not been tested in controlledclinical trials against multidrug-resistant Acinetobacter. However,it is known that the selection and isolation of mutants fromexisting populations of cells depend on a number of variables,including the bacterial inoculum at the site of infection, antibacterialagent(s) used, and mechanisms of resistance associated withtargeted bacteria. It therefore seems reasonable to use at leasttwo agents for treatment of selected patients infected withmultidrug-resistant pathogens that have demonstrated mutation-relatedprogressive resistance during single-drug therapy. Others havealso suggested combination therapy as a tool to prevent theemergence of bacterial resistance and for improved outcomesin severely ill patients infected with gram-negative bacteriaknown to develop resistance on single therapy (5, 18).

Our studies showed that the combination of polymyxin B plusimipenem was as effective as polymyxin B plus rifampin and mayprovide an alternative for treatment even when Acinetobacterisolates are resistant to the carbapenems due to mechanismsother than metallo-carbapenemases. The addition of rifampinas a third component remains controversial. Synergy betweencolistin and rifampin has been reported previously in vitro(11). However, all except one of our eight isolates were killedwithin 24 h by imipenem and polymyxin B. Clinical trials willbe necessary to determine whether combination therapy with twoor three drugs is more effective than use of a single agent.

ACKNOWLEDGMENTS

This work was supported by the BMA Medical Foundation, the BeatriceSnyder Foundation, and Agnes Varis.

FOOTNOTES

* Corresponding author. Mailing address: New York Hospital Queens, 56-45 Main St., Flushing, NY 11355. Phone: (718) 670-1525. Fax: (718) 661-7899. E-mail: cmurban{at}nyp.org.

REFERENCES

Top