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Yen-Hsu Chen, Chien-Fang Peng,1 Po-Liang Lu, Jih-Jin Tsai, and Tyen-Po Chen
Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University Hospital, and 1School of Technology for Medical Sciences, Kaohsiung
Medical University, Kaohsiung, Taiwan.
Combination therapy has been recommended to treat Pseudomonas aeruginosa infections worldwide. The purpose of the present study was to determine the in vitro activities of piperacillin, cefepime, aztreonam, amikacin, and ciprofloxacin alone and in combination against 100 clinical isolates of P. aeruginosa from one medical center in southern Taiwan. The combination susceptibility assay was performed using the checkerboard technique. The percentage of resistance of P. aeruginosa to single agents in our study was relatively high for the Asia-Pacific area, except to aztreonam. Piperacillin plus amikacin exhibited the highest potential for synergy (59/100) in this study. Moreover, a high percentage of synergism was also noted with amikacin combined with cefepime (7/100) or aztreonam (16/100). The combination of two beta-lactams, such as cefepime with piperacillin, and aztreonam with cefepime or piperacillin, showed synergistic effects against some P. aeruginosa isolates. Although ciprofloxacin is a good anti-pseudomonal agent, a very low potential for synergy with other antibiotics was demonstrated in this study. No antagonism was exhibited by any combination in our study. Among piperacillin-resistant strains, there was synergy with a beta-lactam plus amikacin, including the combination of piperacillin and amikacin. However, the combination of two beta-lactams, such as piperacillin and cefepime or aztreonam, did not have any synergistic activity against these strains. In summary, the combinations of amikacin with the tested beta-lactams (piperacillin, aztreonam, cefepime) had a greater synergistic effect against P. aeruginosa, even piperacillin-resistant strains, than other combinations. Understanding the synergistic effect on clinical strains may help clinicians choose better empirical therapy in an area with high prevalence of multidrug-resistant P. aeruginosa.
Key Words: Pseudomonas aeruginosa, antimicrobial combination, in vitro susceptibility (Kaohsiung J Med Sci 2004;20:261–7)
Received: February 3, 2004 Accepted: April 14, 2004
Address correspondence and reprint requests to: Dr. Po-Liang Lu, Division of Infectious Diseases, Department of Internal Medicine,
Kaohsiung Medical University, 100 Tzyou 1st Road, Kaohsiung 807,
Taiwan.
E-mail: [email protected]
Pseudomonas aeruginosa, one of the leading pathogens of
nosocomial infections around the world, has been one of the most difficult to treat [1,2]. It contributes to major infections in high-risk populations, particularly those who
have cancer or are mechanically ventilated [3,4]. Despite the recent advances in antimicrobial therapy, P. aeruginosa infections cause high morbidity and mortality, especially among immunocompromised patients [5]. The development of antimicrobial resistance, a major challenge for physicians and hospitals worldwide, has been well documented with monotherapy [6]. Consequently, combination therapy has been generally recommended for P. aeruginosa infections, to prevent development of antimicrobial resistance and harness synergistic effects [7]. The combination of a beta-lactam and an aminoglycoside has been the standard empirical therapy
for P. aeruginosa for years [8]. Fluoroquinolones, especially ciprofloxacin, have emerged recently as an alternative due to their lower toxicity [9].
However, in the microbiology laboratory, susceptibility is only tested with single antibiotics, which is often inade-quate for multiresistant pathogens. Furthermore, the compa-risons of in vitro synergism or antagonism among different combinations against P. aeruginosa have not been reported in Taiwan. Without this in vitro information, the choice of a combination regimen can often only be made empirically, which may result in a suboptimal treatment outcome.
In response to these challenges, we report here on the first analyses of in vitro activities of various combinations of several newly developed anti-pseudomonal agents on clinical P. aeruginosa isolates from a medical center in southern Taiwan.
M
ATERIALSANDM
ETHODSBacterial strains
A total of 100 consecutive, non-repetitive, clinical P.
aeru-ginosa isolates were collected from the clinical
bacterio-logy laboratory of Kaohsiung Medical University Hos-pital, a 1,200-bed medical center in southern Taiwan, be-tween September 1 and 30, 2001. Forty isolates were from sputum specimens, 31 were from wounds, 16 were from urine samples, eight were from blood specimens, and five were from bile. Bacterial strains were stored at –70°C be-fore testing.
Antimicrobial susceptibility testing
Antimicrobial susceptibility was determined by both the agar dilution and disk diffusion tests, according to the National Committee for Clinical Laboratory Standards (NCCLS) [10,11]. For susceptibility testing by the agar dilution method, the following antimicrobial agents were obtained as standard reference powders of known potency for laboratory use: piperacillin (Lederle Laboratories, Pearl River, NY, USA), ciprofloxacin (Bayer Co, Leverkusen, Germany), and aztreonam, amikacin, and cefepime (Bristol-Myers Squibb Co, Princeton, NJ, USA). All drugs were incorporated into Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, MD, USA) in serial two-fold con-centrations from 0.03 to 128 µg/mL. Three control strains,
Escherichia coli ATCC 35218 and ATCC 25922 and Pseu-domonas aeruginosa ATCC 27853, were included in each
test run. Inoculated plates were incubated in ambient air at 35°C for 16 to 18 hours. The minimal inhibitory
con-centration (MIC) of each antimicrobial agent was defined as the lowest concentration that inhibited visible growth of the organism on a plate.
Checkerboard assay
The anti-pseudomonal properties of nine combinations of antimicrobial agents were determined using a two-dimensional checkerboard agar dilution method [12]. The nine combinations were: amikacin with ciprofloxacin, piperacillin, cefepime, or aztreonam; aztreonam with ciprofloxacin, cefepime, or piperacillin; and cefepime with ciprofloxacin or piperacillin. Serial dilutions of two different antimicrobial agents were mixed in Mueller-Hinton broth. Inocula were prepared from colonies grown on Mueller-Hinton agar plates after overnight culture. Bacterial suspensions with turbidities equivalent to that of a 0.5 Mc-Farland standard were prepared to yield a final inoculum of 5 × 105 colony-forming units (CFU)/mL. After incubation at 35°C for 24 hours, MICs were determined as the lowest concentration at which there was no visible growth in broth. The fractional inhibitory concentration (FIC) (the ratio of the MIC of antibiotic A in the combination and the MIC of antibiotic A alone) was calculated for each antibiotic in each combination. The FIC for the combination was the sum of the FICs for the two antibiotics. Of the FIC indices calculated for all rows in the checkerboard, the minimum value was the FIC index for that isolate. Synergism was defined as when the FIC index was 0.5 or less, and anta-gonism was defined as when the FIC index was more than 4. No interaction was defined as an FIC index between 0.5 and 4. The definition of the effect of the combination of two antimicrobial agents was according to the latest reported criteria [13]. Quality control strains were the same as those described above.
R
ESULTSThe percentages of non-susceptible isolates for various antimicrobial agents using the disk diffusion method were as follows: moxalactam (95%), ceftriaxone (66%), piperacil-lin (31%), ticarcilpiperacil-lin/clavulanate (22%), gentamicin (17%), ceftazidime (12%), aztreonam (12%), ciprofloxacin (9%), piperacillin/tazobactam (9%), cefepime (9%), amikacin (7%), and imipenem (5%). The MICs for, and susceptibilities to, aztreonam, amikacin, ciprofloxacin, piperacillin, and cefepime using the broth microdilution method are shown in Table 1.
The distribution of FICs of nine combinations of two antimicrobial agents are shown in Table 2. Synergism was
greatest with a combination of piperacillin and amikacin (59%), followed by aztreonam plus amikacin (16%), and cefepime with piperacillin (8%). No antagonism was seen with any of the nine combinations. All four amikacin-containing combinations had synergism in some isolates, especially when in combination with beta-lactams (pip-eracillin, cefepime, aztreonam).
Subgroup analysis for piperacillin-resistant
isolates
Twenty-two P. aeruginosa isolates were resistant to pip-eracillin in our study. Of these, 9.1% were not susceptible to amikacin, 36.4% to ciprofloxacin, 36.4% to cefepime, and 40.9% to aztreonam. These non-susceptible rates are significantly higher than those among piperacillin-susceptible isolates (Fisher’s exact test; p = 0.039, p = 0.01,
p < 0.001, p < 0.001, respectively). The effects of combinations
of two antimicrobial agents on the 22 piperacillin-resistant isolates are shown in Table 3. Amikacin showed synergy with piperacillin for five strains (22.7%), while piperacillin combined with cefepime or aztreonam did not reveal a synergistic effect. Amikacin also had a synergistic effect on
some piperacillin-resistant isolates when combined with the other two beta-lactams (cefepime or aztreonam).
D
ISCUSSIONAlthough combination therapy has been recommended for
P. aeruginosa infections for years, to date, no in vitro data
have been documented on its effect against clinical strains isolated in Taiwan.
During the past decade, the increasing prevalence of antimicrobial resistance among clinical isolates of P.
aeruginosa has been a critical issue in hospitals worldwide
[14–16]. Compared with the results of the worldwide SENTRY antimicrobial surveillance program published in 2002 [17], the prevalence of non-susceptible clinical iso-lates in our study was relatively high for the Asia-Pacific area, except for aztreonam (our study vs Asia-Pacific area in SENTRY, 13% vs 19.1%), cefepime (14% vs 6.5%), pipera-cillin (22% vs 14.5%), amikacin (7% vs 4.8%), and ciproflo-xacin (19% vs 11.6%) (Table 1). The high resistance rate to piperacillin in our single-agent susceptibility study might Table 1. In vitro susceptibilities of 100 Pseudomonas aeruginosa isolates to five antimicrobial agents
MIC (µg/mL) Number of isolates
Antibiotic Range MIC50 MIC90 Susceptible Intermediate Resistant
Aztreonam 0.25–128 4 32 87 4 9
Amikacin 0.5–64 4 16 93 4 3
Ciprofloxacin 1–64 1 8 81 5 14
Piperacillin 1–128 8 128 78 – 22
Cefepime 0.5–128 4 16 86 6 8
MIC = minimum inhibitory concentration.
Table 2. Fractional inhibitory concentrations (FICs) of combinations of two antimicrobial agents for 100 clinical Pseudomonas aeruginosa isolates
FIC Number of isolates
Range Median Mean SD FIC ≤ 0.5 0.5 < FIC ≤ 2 2 < FIC ≤ 4 FIC > 4
Amk-Cip 0.31–1.25 1.03 1.01 0.13 2 98 0 0 Amk-Pip 0.03–2.25 0.375 0.55 0.4 59 39 2 0 Amk-Fep 0.25–1.5 1 0.91 0.29 7 93 0 0 Fep-Pip 0.28–2 1.25 1.09 0.38 8 92 0 0 Fep-Cip 0.06–1.5 1.03 1.05 0.14 1 99 0 0 Atm-Amk 0.16–2 1.03 0.91 0.35 16 84 0 0 Atm-Cip 0.63–1.5 1.02 1.02 0.08 0 100 0 0 Atm-Fep 0.38–1.5 1.06 1.10 0.21 2 98 0 0 Atm-Pip 0.31–2.5 1.06 1.08 0.44 1 97 2 0
be attributable to the extended use of piperacillin plus an aminoglycoside as the initial empirical combination therapy against nosocomial infections (Annual Report of Infection Control Committee, Kaohsiung Medical University Hospital, unpublished data), mainly because piperacillin has been the only first-line antimicrobial agent available among the tested anti-pseudomonal agents in the reimbursement system of the National Health Insurance. The lower resistance rate to aztreonam might be attributed to the lower consumption in our hospital, compared to other antimicrobial agents (Annual Report of Infection Control Committee, Kaohsiung Medical University Hospital, unpublished data).
Against P. aeruginosa, the combination of a beta-lactam and an aminoglycoside has been reported to have a relative-ly high rate of synergy [18,19]. The combination of an aminoglycoside (amikacin) and a beta-lactam (cefepime, piperacillin, or aztreonam) exhibited synergy in a high percentage of isolates in the present study (Table 2), especially the combination of amikacin plus piperacillin.
Synergistic effects were found with combinations of two beta-lactams, such as aztreonam plus cefepime or piperacillin, in some P. aeruginosa isolates, although all beta-lactams work through the inhibition of bacterial cell wall synthesis. Although several animal models have demonstrated antagonism against P. aeruginosa with beta-lactam/beta-lactam combinations [20], Lister et al [21] and Sader et al [22] have reported that aztreonam enhances the antibacterial activity of cefepime, especially against derepressed strains of P. aeruginosa. The synergism might be partially attributed to the protection provided by az-treonam to other beta-lactams in the extracellular envi-ronment from extracellular beta-lactamase inactivation,
such as Bush group 1 chromosomal cephalosporinase [21]. With the diminished level of extracellular inactivation of cefepime, more active cefepime would gain access to the periplasmic space where aztreonam could provide pro-tection as well. However, this synergy between cefepime and aztreonam has not been consistently exhibited in other studies [23,24].
Combination therapy is more important for infections with piperacillin-resistant strains, which had a higher percentage of multidrug resistance in the present study. Therefore, we studied the synergistic potential of combi-nation therapy against these resistant strains. The com-bination of piperacillin and amikacin showed synergism in five of the 22 piperacillin-resistant isolates. However, no synergy was found with the combinations of piperacillin with cefepime or aztreonam.
Recently, fluoroquinolones have become important in the treatment of P. aeruginosa infections. Various anti-microbial interactions have been documented in combina-tion therapy including a fluoroquinolone. Synergism, in-difference, or antagonism of fluoroquinolones have been reported when combined with beta-lactams in different studies [25–27]. In our study, a very low potential of synergy was found with the combinations of ciprofloxacin and cefepime (1/100) or aztreonam (0/100). Furthermore, cipro-floxacin plus amikacin exhibited synergism in only two of 100 strains. Among piperacillin-resistant strains, we also found no synergistic effect with amikacin plus cipro-floxacin. Some newly developed fluoroquinolones, such as trovafloxacin and gatifloxacin, may exhibit synergy with a beta-lactam in P. aeruginosa [27,28].
To date, there have been different criteria upon which to interpret the antagonism of antimicrobial combinations Table 3. Fractional inhibitory concentrations (FICs) of combinations of two antimicrobial agents for 22 piperacillin-resistant Pseudomonas aeruginosa isolates
FIC Number of isolates
Range Median Mean SD FIC ≤ 0.5 0.5 < FIC ≤ 2 2 < FIC ≤ 4 FIC > 4
Amk-Cip 0.51–1.25 1.0 0.99 0.15 0 22 0 0 Amk-Pip 0.04–1.06 0.625 0.679 0.32 5 17 0 0 Amk-Fep 0.25–1.25 0.75 0.82 0.26 3 19 0 0 Fep-Pip 1.03–1.5 1.25 1.16 0.12 0 22 0 0 Fep-Cip 0.52–1.02 1.00 0.97 0.12 0 22 0 0 Atm-Amk 0.16–1.06 0.75 0.74 0.28 4 18 0 0 Atm-Cip 0.63–1.13 1.0 0.93 0.14 0 22 0 0 Atm-Fep 0.38–1.5 1.0 1.06 0.22 1 21 0 0 Atm-Pip 0.56–1.5 1.09 1.16 0.25 0 22 0 0
using the checkerboard method. Originally, synergism was defined as an FIC index of 0.5 or less, and antagonism as an FIC index of at least 2.0 [12]. However, several recent publications recommend a stricter criterion to redefine an-tagonism as an FIC index of more than 4.0 [13,29]. Using this strict criterion, no antagonism was found in any combi-nation tested in our study, although with amikacin plus piperacillin, two strains had FIC indices between 2.0 and 4.0, and with aztreonam plus piperacillin, two strains had an FIC index between 2.0 and 4.0.
Several methods are used to evaluate in vitro anti-microbial combination effects, including the checkerboard and time-kill methods [12]. The checkerboard method is the best known and simplest. Although it is not able to provide a more dynamic description of antimicrobial effect over time, its results can easily be compared with most published data. However, the results generated from these two meth-ods do not correlate well [30,31]. Burgess et al, using the time-kill method, demonstrated synergy more frequently when a beta-lactam was combined with an aminoglycoside than with a fluoroquinolone for P. aeruginosa [32].
In conclusion, combination therapy has been recom-mended for P. aeruginosa infections, especially as there is a high prevalence of drug resistance in Taiwan. Ami-kacin plus piperacillin was the antibiotic combination with a synergistic effect for most clinical P. aeruginosa isolates. According to the greatest potential for synergy in our study, amikacin plus cefepime and amikacin plus aztreo-nam were also favorable combination therapies for most
P. aeruginosa infections, even those caused by
piperacillin-resistant strains. Combinations of two beta-lactams also exhibited synergy for some P. aeruginosa isolates. Compared with other combinations, combinations with ciprofloxacin provide no further synergistic effect. Although there was no concomitant investigation on clinical efficacy in our study, these in vitro results might provide practical infor-mation for the optimal choice of empirical combination therapy against P. aeruginosa.
R
EFERENCES1. NNIS System. National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2003, issued in August 2003. Am J Infect Control 2003;31:481–98.
2. Lin CJ, Lu PL, Hwang PL, et al. Secular trends of nosocomial infections in a medical center from 1985 to 1996. Nosocomial Infect Control J 2000;10:301–12.
3. Francioli P, Chastre J, Langer M, et al. Ventilator-associated
pneumonia – understanding epidemiology and pathogenesis to guide prevention and empiric therapy. Clin Microbiol Infect 1997;3(Suppl 1):S61–76.
4. Spencer RC. Predominant pathogens found in the European Prevalence of Infection in Intensive Care Study. Eur J Clin Microbiol Infect Dis 1996;15:281–5.
5. Carmeli Y, Troillet N, Karchmer AW, et al. Health and economic outcome of antibiotic resistance in Pseudomonas aeruginosa. Arch Intern Med 1999;159:1127–32.
6. Troillet N, Samore MH, Carmeli Y. Imipenem-resistant Pseudomonas aeruginosa: risk factors and antibiotic susceptibility patterns. Clin Infect Dis 1997;25:1094–8.
7. Hilf M, Yu VL, Sharp J, et al. Antibiotic therapy for Pseudomonas aeruginosa bacteremia: outcome correlations in a prospective study of 200 patients. Am J Med 1989;87:540–6.
8. Arnoff SC, Klinger JD. In vitro activities of aztreonam, piperacillin, and ticarcillin combined with amikacin against amikacin-resistant Pseudomonas aeruginosa and Pseudomonas cepacia from children with cystic fibrosis. Antimicrob Agents Chemother 1984;25:279–80.
9. Bauernfeind A. Comparison of the antibacterial activities of the quinolones Bay 12-8039, gatifloxacin (AM 1155), trovaflo-xacin, clinaflotrovaflo-xacin, levofloxacin and ciprofloxacin. J Antimicrob Chemother 1997;40:639–51.
10. Performance Standards for Antimicrobial Susceptibility Testing, eighth informational supplement. Approved standard, document M100-S8. Wayne, PA: National Committee for Clinical Laboratory Standards, 1998.
11. Performance Standards for Antimicrobial Disk Susceptibility Tests, approved standard, document M2-A6. Villanova, PA: National Committee for Clinical Laboratory Standards, 1997. 12. Eliopoulos GM, Moelering RC Jr. Antimicrobial combinations.
In: Lorian V, ed. Antibiotics in Laboratory Medicine, 4th
edition. Baltimore: Williams and Wilkins, 1996:330–96.
13. Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 2003;52:1. 14. Hsueh PR, Liu CY, Luh KT. Current status of antimicrobial
resistance in Taiwan. Emerg Infect Dis 2002;8:132–7.
15. Gales AC, Jones RN, Turnidge J, et al. Characterization of Pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin Infect Dis 2001;32(Suppl 2):S146–155.
16. Hanberger H, Garcia-Rodriguez JA, Gobernado M, et al. Anti-biotic susceptibility among aerobic Gram-negative bacilli in intensive care units in 5 European countries. JAMA 1999;281: 1–5.
17. Jones RN, Kirby JT, Beach ML, et al. Geographic variations in activity of broad-spectrum beta-lactams against Pseudomonas aeruginosa: summary of the worldwide SENTRY antimicrobial surveillance program (1997–2000). Diagn Microbiol Infect Dis 2002;43:239–43.
18. Bosso JA, Saxon BA, Matsen JM. In vitro activities of combina-tions of aztreonam, ciprofloxacin, and ceftazidime against clinical isolates of Pseudomonas aeruginosa and Pseudomonas cepacia from patients with cystic fibrosis. Antimicrob Agents Chemother 1990;34:487–8.
19. Meyer RD, Liu S. In vitro synergy studies with ciprofloxacin and selected beta-lactam agents and aminoglycosides against multidrug-resistant Pseudomonas aeruginosa. Diagn Microbiol Infect Dis 1988;11:151–7.
20. Kuck NA, Testa RT, Forbes M. In vitro and in vivo antibacterial effects of combinations of beta-lactam antibiotics. Antimicrob Agents Chemother 1981;19:634–8.
21. Lister PD, Sanders WE Jr, Sanders CC. Cefepime-aztreonam: a unique double beta-lactam combination for Pseudomonas aeruginosa. Antimicrob Agents Chemother 1998;42:1610–9. 22. Sader HS, Huynh HK, Jones RN. Contemporary in vitro synergy
rates for aztreonam combined with newer fluoroquinolones and beta-lactams tested against Gram-negative bacilli. Diagn Microbiol Infect Dis 2003;47:547–50.
23. Bosso JA, Saxon BA, Maxtsen JM. Comparative activity of cefepime, alone and in combination, against clinical isolates of Pseudomonas aeruginosa and Pseudomonas cepacia from cystic fibrosis patients. Antimicrob Agents Chemother 1991;35:783–4. 24. McGrath BJ, Bailey EM, Lamp KC, et al. Pharmacodynamics of once-daily amikacin in various combinations with cefepime, aztreonam, and ceftazidime against Pseudomonas aeruginosa in an in vitro infection model. Antimicrob Agents Chemother 1992; 36:2741–6.
25. Fish DN, Choi MK, Jung R. Synergic activity of cephalosporins plus fluoroquinolones against Pseudomonas aeruginosa with resistance to one or both drugs. J Antimicrob Chemother 2002; 50:1045–9.
26. Pohlman JK, Knapp CC, Ludwig MD, et al. Timed killing kinetic studies of the interaction between ciprofloxacin and
beta-lactams against Gram-negative bacilli. Diagn Microbiol Infect Dis 1996;26:29–33.
27. Gradelski E, Valera L, Bonner D, Fung-Tomc J. Synergistic activities of gatifloxacin in combination with other antimicro-bial agents against Pseudomonas aeruginosa and related species. Antimicrob Agents Chemother 2001;45:3220–2.
28. Isenberg HD, Alperstein P, France K. In vitro activity of cipro-floxacin, levocipro-floxacin, and trovacipro-floxacin, alone and in com-bination with beta-lactams, against clinical isolates of Pseudo-monas aeruginosa, StenotrophoPseudo-monas maltophila, and Burkholderia cepacia. Diagn Microbiol Infect Dis 1999;33:81–6.
29. Song W, Woo HJ, Kim JS, et al. In vitro activity of beta-lactams in combination with other antimicrobial agents against resis-tant strains of Pseudomonas aeruginosa. Int J Antimicrob Agents 2003;21:8–12.
30. Cappelletty DM, Rybak MJ. Comparison of methodologies for synergism testing of drug combinations against resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1996;40:677–83.
31. Visalli MA, Jacobs MR, Appelbaum PC. Determination of activities of levofloxacin, alone and combined with gen-tamicin, ceftazidime, cefpirome, and meropenem, against 124 strains of Pseudomonas aeruginosa by checkerboard and time-kill methodology. Antimicrob Agents Chemother 1998;42: 953–5.
32. Burgess DS, Hastings RW. Activity of piperacillin/tazobactam in combination with amikacin, ciprofloxacin, and trovafloxacin against Pseudomonas aeruginosa by time-kill. Diagn Microbiol Infect Dis 2000;38:37–41.
