In vitro effects of combinations of antipseudomonal agents against seven strains of multidrug-resistant Pseudomonas aeruginosa

Shigeharu Oie1, Toshinari Uematsu1, Akihiro Sawa1, Hidekazu Mizuno2, Masaaki Tomita3, Shiro Ishida4, Yoshiro Okano4 and Akira Kamiya1,*

1 Department of Pharmacy and 2 Clinical Laboratory, Yamaguchi University Hospital, 1-1-1 Minamikogushi, Ube 755-8505; 3 Yamaguchi Prefectural Research Institute of Public Health, 2-5-67 Aoi, Yamaguchi 753-0821; 4 Department of Pharmaceutical Care and Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima 770-8514, Japan

Received 10 June 2003; returned 16 July 2003; revised 16 September 2003; accepted 20 September 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The aim of this study was to evaluate the combined effects of antibiotic combinations by agar incorporation inhibitory tests and by time–kill tests on seven geographically and epidemiologically distinct isolates of multidrug-resistant Pseudomonas aeruginosa. All seven strains were resistant to piperacillin, meropenem, ceftazidime, cefoperazone-sulbactam, aztreonam, amikacin and ciprofloxacin.

Methods: Strains were distinguished by pulsed-field gel electrophoresis after DNA extraction and restriction with SpeI. MICs of the seven antibiotics listed above were determined by agar dilution. The effect of combinations of these agents was determined by agar incorporation tests and by time–kill studies.

Results: Among the two-drug combinations, the combination aztreonam and amikacin was the most effective, inhibiting proliferation in five of the seven strains. Among the three-drug combinations, the combinations of piperacillin, ceftazidime and amikacin, and that of ceftazidime, aztreonam and amikacin were the most effective, inhibiting proliferation in all seven strains. In the killing tests, the three-drug combination of ceftazidime, aztreonam and amikacin was the most effective. This three-drug combination had bacteriostatic effects on all seven strains 2, 4, 6 and 24 h after drug addition, synergic effects on 2–3 strains and bactericidal effects on 1–2 strains after 4, 6 and 24 h.

Conclusions: The three-drug combination of ceftazidime, aztreonam and amikacin may be effective against P. aeruginosa resistant to all commonly used antipseudomonal drugs, and deserves further study.

Keywords: P. aeruginosa, antimicrobial agents, combined effects, ceftazidime, aztreonam, amikacin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ß-Lactam antibiotics, such as ceftazidime and aztreonam, aminoglycoside antibiotics, such as amikacin and gentamicin, and fluoroquinolones, such as ciprofloxacin and levofloxacin, are usually effective against Pseudomonas aeruginosa.13 In addition, combinations of ß-lactam antibiotics and aminoglycosides are more effective.415 However, based on our experience in seven hospitals over 3–4 years, clinically significant P. aeruginosa strains resistant to each of seven antibiotics (piperacillin, meropenem, ceftazidime, cefoperazone-sulbactam, aztreonam, amikacin and ciprofloxacin) were isolated with low frequency. For some of these strains, the combination of ß-lactam antibiotics and aminoglycoside antibiotics was completely ineffective. Therapeutic options for infection with such multi-resistant P. aeruginosa are limited because there is little choice among available agents. Development of multidrug resistance in clinical strains has been associated with the production of acquired ß-lactamases, constitutive overexpression of the cephalosporinase AmpC or non-enzymic mechanisms such as drug efflux or outer membrane permeability.16 The drug efflux of P. aeruginosa is attributed to expression of a number of broad specificity multidrug efflux (Mex) systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM, that promote acquired multidrug resistance as a consequence of hyper-expression of the efflux genes caused by mutational events.17,18 It is also speculated that the prevalence of such multi-resistant P. aeruginosa will increase in the future. Therefore, we evaluated the effects of drug combinations on such multi-resistant P. aeruginosa.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

From a total of 10 800 P. aeruginosa strains isolated at seven hospitals in Yamaguchi Prefecture between June 1998 and December 2001, seven were resistant to the ß-lactam antibiotics piperacillin, meropenem, ceftazidime, cefoperazone–sulbactam and aztreonam, the aminoglycoside amikacin, and the quinolone antibiotic ciprofloxacin. The seven strains were isolated at seven different hospitals. The sources of the seven strains were bronchial secretion (two strains), blood (two), pus (two) and urine (one).

Pulsed-field gel electrophoresis

The preparation of high molecular weight chromosomal DNA was carried out according to Murray et al.19 Digestion was carried out by placing a small slice of an agarose plug in 200 µL of reaction buffer with 30 U SpeI (New England Bio Labs, USA). Pulsed-field gel electrophoresis was carried out with the Bio-Rad Gene Path system in a 1% agarose gel in 0.5x TBE buffer at 14°C with a linear ramp time of 1 to 23 s over a period of 18.5 h. Thereafter, the gels were stained with ethidium bromide and photographed.

Susceptibility tests and combined drug effects by agar dilution methods

MICs were determined after 18 h of incubation at 37°C by dilution on Sensitivity Disc Agar-N (Nissui Pharmaceuticals, Tokyo, Japan). The following antimicrobial agents were tested: piperacillin (Toyama Chemicals, Tokyo, Japan), meropenem (Sumitomo Pharmaceuticals, Tokyo), ceftazidime (Glaxo Japan Co., Tokyo), cefoperazone-sulbactam (Pfizer Pharmaceuticals Inc., USA), aztreonam (Eisai Co., Tokyo), amikacin (Banyu Pharmaceuticals, Tokyo) and ciprofloxacin (Bayer Japan Co., Tokyo). These antibiotics except for ciprofloxacin were provided in the form of a freeze-dried amorphous powder. The inocula (104 cfu/spot) were plated using a multipoint inoculator (Sakuma Co., Tokyo). The MIC was defined as the lowest drug concentration that inhibited visible growth. P. aeruginosa IFO 3919 was used as a reference strain.

The effects of the combination of two or three drugs among the above seven types of drug were evaluated. The drug concentrations (breakpoints) were set as follows: piperacillin, 64 mg/L; meropenem, 8 mg/L; ceftazidime, 16 mg/L; cefoperazone–sulbactam, 16 mg/L; aztreonam, 16 mg/L; amikacin, 4 mg/L; ciprofloxacin, 2 mg/L. Breakpoints used for all agents tested except amikacin were according to National Committee for Clinical Laboratory Standards (NCCLS) criteria.20 The concentration of amikacin was 4 mg/L, which is lower than the criteria of the NCCLS. This was because in Japan, the routine dose of amikacin is lower (200–400 mg/day in one to two divided doses) than that in Western countries. The presence or absence of growth on Sensitivity Disc Agar-N was determined by a method similar to that used for the measurement of MIC.

Drug combined effects by killing tests

Killing experiments were carried out to evaluate the bactericidal activities of two-drug and three-drug combinations. As two-drug combinations, ceftazidime plus aztreonam, and a ß-lactam antibiotic (piperacillin, meropenem, ceftazidime or aztreonam) combined with amikacin were evaluated. As a three-drug combination, ceftazidime was combined with aztreonam and amikacin. The concentrations of the five drugs were the same as those in the previous section. Sensitivity Test Broth (Nissui Pharm) was used as medium. The final concentration of the log-phase inocula was approximately 5 x 105 cfu/mL.6,7,15,21 Viability was determined from bacterial counts carried out at 2, 4, 6 and 24 h after incubation at 37°C by plating 500 µL of serial dilutions from each tube onto trypticase soy agar plates. The numbers of viable bacteria were counted after 24 and 48 h of incubation of the plates at 37°C. In preliminary experiments, drug carryover was ruled out by plating samples of a bacterial suspension containing 2–4 x 102 cfu/mL in the presence or absence of antimicrobial agents alone or in combination. Bactericidal activity was defined as a >=3 log10 cfu/mL decrease in the starting inoculum. Synergy was defined as a >=2 log10 cfu/mL decrease between the combination and its most active single component, and the number of surviving organisms in the presence of the combination had to be >=2 log10 cfu/mL below the starting inoculum. A bacteriostatic effect was defined as any decrease in viable count from the starting inoculum. Antagonism was defined as a >=2 log10 cfu/mL increase between the combination and the most active single antimicrobial agent.21

Data analysis

In the agar plate method, the effects on the seven P. aeruginosa strains were compared among the two-drug combinations and among the three-drug combinations by the {chi}2 test, using the number of growth-inhibited strains as a variable.

In the killing tests, the effects on the seven P. aeruginosa strains were compared with the antibiotic-free control, single drugs, the two-drug combinations, and the three-drug combination by the Kruskal–Wallis test using the decrease in viable count from the initial count 2, 4, 6 and 24 h after drug addition.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of P. aeruginosa showing resistance to piperacillin, meropenem, ceftazidime, cefoperazone–sulbactam, aztreonam, amikacin and ciprofloxacin was about 0.06%. All seven strains tested were confirmed to differ in DNA pattern by pulsed-field gel electrophoresis. Table 1 shows the MICs of the seven drugs against the seven strains of multidrug-resistant P. aeruginosa.


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Table 1. MICs (mg/L) of seven antipseudomonal agents against seven strains of multidrug-resistant P. aeruginosa
 
Table 2 shows the effects of two-drug or three-drug combinations among the seven drugs on the seven multi-resistant P. aeruginosa strains by the agar plate incorporation method. The two-drug combination of ceftazidime and aztreonam, or aztreonam and amikacin significantly inhibited growth compared with the other two-drug combinations (P < 0.05). The three-drug combinations of ceftazidime, aztreonam and amikacin, or piperacillin, ceftazidime and amikacin, significantly inhibited the growth of all seven strains compared with the other three-drug combinations (P < 0.05).


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Table 2. Combined effects of antimicrobial drugs on seven multi-resistant P. aeruginosa strains (agar plate method)
 
Table 3 shows the effects of single drugs and drug combinations on multi-resistant P. aeruginosa by killing tests. Pairwise comparison of the viable counts 2, 4, 6 and 24 h after drug addition by the Kruskal–Wallis test among the control (without drugs), single drugs and drug combinations showed significant decreases 4, 6 and 24 h after addition of the three-drug combination of ceftazidime, aztreonam and amikacin compared with the control (P < 0.01 after 4 h, P < 0.05 after 6 and 24 h) and 4 h after addition of the three-drug combination of ceftazidime, aztreonam and amikacin compared with amikacin alone (P < 0.05). However, no other significant differences were observed by pairwise comparison in the viable count each hour after drug addition among the control, single drugs and drug combinations. Antagonism was not observed in any drug combination.


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Table 3. Effects of antimicrobial drugs on seven strains of multi-resistant P. aeruginosa (killing test)
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The three-drug combination of ceftazidime, aztreonam and amikacin was effective against multi-resistant P. aeruginosa. P. aeruginosa infection has been conventionally treated with the combination of ß-lactam antibiotics, such as ceftazidime and aztreonam, and aminoglycosides, such as amikacin.9,11,14,15 However, even compared with such two-drug combinations, the three-drug combination of ceftazi-dime, aztreonam and amikacin was effective. The combination of ceftazidime, aztreonam and amikacin is worthy of further evaluation against multidrug-resistant P. aeruginosa. This study suggested only slight effects of the combination of ciprofloxacin or meropenem with other drugs on multi-resistant P. aeruginosa.

The incidence of multi-resistant P. aeruginosa was found to be low (0.06%). However, there is a possibility that multi-resistant P. aeruginosa will spread, assuming that the multidrug resistance observed in this study is attributed to a consequence of hyper-expression of the efflux genes by a mutational event.17,18 Therefore, it is necessary to minimize the development of multi-resistance in P. aeruginosa by preventing abuse of antibiotics and to establish effective drug combinations for multi-resistant P. aeruginosa. Further work is required to validate the clinical efficacy of drug combinations such as these against multidrug-resistant P. aeruginosa.


    Acknowledgements
 
We thank Teruko Nakazawa, an emeritus professor at Yamaguchi University, for useful suggestions. We are also grateful to Clinical Laboratories, Shimonoseki Kosei Hospital, Nagato General Hospital, Tsushimi Hospital, Saiseikai Yamaguchi General Hospital, Yamaguchi Rosai Hospital and Onoda Municipal Hospital for the kind donation of P. aeruginosa strains.


    Footnotes
 
* Corresponding author. Tel: +81-836-22-2665; Fax: +81-836-22-2673; E-mail: akira-ygc{at}umin.ac.jp Back


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 Introduction
 Materials and methods
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 Discussion
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