In vitro selection of resistance in Pseudomonas aeruginosa and Acinetobacter spp. by levofloxacin and ciprofloxacin alone and in combination with ß-lactams and amikacin

Lorenzo Drago*, Elena De Vecchi, Lucia Nicola, Loredana Tocalli and Maria Rita Gismondo

Laboratory of Clinical Microbiology, Department of Clinical Sciences, L. Sacco Teaching Hospital, University of Milan, Via GB Grassi 74, 20157 Milan, Italy


* Corresponding author. Tel: +39-02-390-42469; Fax: +39-02-503-19651; E-mail: lorenzo.drago{at}unimi.it

Received 11 February 2005; returned 21 March 2005; revised 27 April 2005; accepted 24 May 2005


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Objectives: The aim of this study was to evaluate the ability of levofloxacin and ciprofloxacin alone and in combination with either ceftazidime, cefepime, imipenem, piperacillin–tazobactam or amikacin to select for antibiotic-resistant mutants of Pseudomonas aeruginosa and Acinetobacter spp.

Methods: Clinical strains of P. aeruginosa (n = 5) and Acinetobacter spp. (n = 5) susceptible to all the drugs used in the study were assayed. Development of resistance was determined by multi-step and single-step methodologies. For multi-step studies, MICs were determined after five serial passages on antibiotic-gradient plates containing each antibiotic alone or in combination with levofloxacin or ciprofloxacin. Acquisition of resistance was defined as an increase of ≥4-fold from the starting MIC. In single-step studies, the frequency of spontaneous mutations was calculated after a passage on plates containing antibiotics alone and in combinations at concentrations equal to the highest NCCLS breakpoints.

Results: Serial passages on medium containing single antibiotics resulted in increased MICs for each antibiotic; MIC increases were limited by antibiotics in combination. A decrease in the number of strains with MICs above the NCCLS breakpoints occurred when fluoroquinolones were combined with a second antibiotic for both P. aeruginosa and Acinetobacter spp. isolates. Frequencies of mutation were higher for antibiotics alone than for combinations.

Conclusions: Use of combinations of fluoroquinolones with ß-lactams and amikacin reduces the risk for in vitro selection of resistant P. aeruginosa and Acinetobacter spp.

Keywords: mutation frequencies , ß-lactams , fluoroquinolones


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa is a clinically relevant pathogen, being a frequent cause of nosocomial infections, particularly in high-risk populations. Acinetobacter species, particularly Acinetobacter baumannii, are emerging as a major cause of nosocomial infections, especially in intensive care and burn units, where antimicrobial use is greatest. Both of these bacteria are characterized by intrinsic resistance due to the limited permeability of the outer membrane along with several efflux pump mechanisms and by the impressive ability to rapidly acquire resistance to a wide variety of structurally unrelated antimicrobial agents, even during the course of therapy.14 Therefore, selection of a proper antibiotic regimen is essential for the successful treatment of serious infections caused by P. aeruginosa and Acinetobacter spp., since development of resistance is often associated with therapy failure.57

Recommended treatments for systemic P. aeruginosa and Acinteobacter spp. infections involve combination therapy, as this increases the chance of effective initial therapy before receiving susceptibility results. Combination therapy may minimize the risk of developing resistance and has the potential for synergic activity.8 Therapy involving an aminoglycoside plus a ß-lactam with anti-pseudomonal activity has for many years represented the therapy of choice for treatment of infections caused by P. aeruginosa and related pathogens.911 However, this combination has not always been effective in preventing the emergence of resistance during therapy.12 Nowadays, anti-pseudomonal fluoroquinolones are often considered a valid alternative to aminoglycosides because of their limited nephrotoxicity and better tissue penetration, and several studies have shown in vitro synergy between ciprofloxacin or levofloxacin and anti-pseudomonal ß-lactams.1316 However, most studies on the effects of drug combinations on P. aeruginosa have investigated bactericidal and inhibitory activities by means of time–kill studies, chequerboard assays or by determination of post-antibiotic effects,11,1321 and only a few have evaluated the ability of such combinations to limit development of resistance.13,22

The aim of this study was to evaluate the tendency of clinical isolates of P. aeruginosa and Acinetobacter spp. to develop phenotypic resistance to levofloxacin, ciprofloxacin, cefepime, ceftazidime, imipenem, piperacillin–tazobactam and amikacin after single and sequential exposures to these drugs singly or in combination (levofloxacin or ciprofloxacin with amikacin, cefepime, ceftazidime, imipenem and piperacillin–tazobactam).


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Bacterial strains

Five non-duplicate clinical isolates of P. aeruginosa and five of Acinetobacter spp. (four Acinetobacter baumannii and one Acinetobacter calcoaceticus) obtained from patients with pneumonia or septicaemia at the intensive care unit of L. Sacco Teaching Hospital, Milan, Italy were included in the study. The isolates were chosen based on their proven susceptibility to all the antibiotics evaluated in the study, as shown in Table 1.


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Table 1. Starting MIC of the strains used in the study

 
Antibiotics

Levofloxacin (Aventis Pharma Spa, Lainate, Italy), ciprofloxacin (Bayer Italia, Milan, Italy), amikacin (Sigma, St Louis, MO, USA), cefepime (Bristol Myers-Squibb, Latina, Italy), ceftazidime (GlaxoSmithKline, Verona, Italy), imipenem (Merck Sharp & Dohme, Rome, Italy) and piperacillin–tazobactam (Wyeth Lederle, Latina, Italy), as powders of stated potency, were used to prepare stock solutions at concentrations of 5120 mg/L as recommended by the NCCLS.23

Determination of MIC

Determination of antibiotic susceptibility was performed by the broth microdilution method according to NCCLS standards.24 NCCLS breakpoints used to define resistance were: ≥8 mg/L for levofloxacin, ≥4 mg/L for ciprofloxacin, ≥32 mg/L for cefepime and ceftazidime, ≥16 mg/L for imipenem, ≥128/4 mg/L for piperacillin–tazobactam and ≥64 mg/L for amikacin.

Selection of resistant bacteria (multi-step)

The ability to select resistant bacteria was evaluated by serially subculturing the chosen strains onto agar plates containing a linear gradient of the antibiotics singly or in combination, as described previously.22,25 Gradients were prepared in Petri dishes, which were poured with two layers of agar. The bottom layer consisted of Mueller–Hinton agar, allowed to harden with the plate slanted sufficiently to cover the entire bottom. The top layer, added to the dish in the normal position, generally contained antibiotics (alone or in combination) at concentrations of ~4–8 x MIC. An inoculum of 109 cfu (sufficiently high to be representative of bacterial strain) was homogeneously spread on each plate, and incubated for 48 h at 37°C. Colonies growing at the highest antibiotic concentration were sampled, checked for purity, grown overnight into antibiotic-free broth, and replated on new antibiotic gradient plates. Bacteria were exposed to a maximum of five consecutive passages on the antibiotic gradients. MICs were determined after the first, the second and the fifth passage by the broth microdilution technique as described above.

Acquisition of resistance was defined as ≥4-fold increase in MIC with respect to the initial value, according to other authors.22

Mutational frequency (single-step)

The frequency of spontaneous single-step mutations was determined on the chosen strains by spreading 0.1 mL from a bacterial suspension of ~1010 cfu/mL on antibiotic-free agar plates (after proper dilution) and on antibiotic-containing agar plates (undiluted inoculum).22 NCCLS resistance breakpoints reported in the above section were used to calculate defined antibiotic concentrations. Colonies grown after 48 h of incubation at 37°C were counted. Frequency of mutation was calculated as the number of resistant colonies per inoculum.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Multi-step selection of resistant bacteria

Results of gradient plates resistance selection for single and combined antibiotics are summarized in Tables 2GoGo5, which report changes in MICs and number of strains showing increment in MICs of at least four times the initial values.


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Table 2. Susceptibilities of P. aeruginosa strains before and after five serial passages on plates containing an antibiotic gradient

 

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Table 3. Susceptibilities of P. aeruginosa strains before and after five serial passages on plates containing an antibiotic gradient of levofloxacin or ciprofloxacin in combination

 

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Table 4. Susceptibilities of Acinetobacter spp. strains before and after five serial passages on plates containing an antibiotic gradient

 

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Table 5. Susceptibilities of Acinetobacter spp. strains before and after five serial passages on plates containing an antibiotic gradient of levofloxacin or ciprofloxacin in combination

 
Incubation of P. aeruginosa with single antibiotics generally produced a significant increase in MICs after only one passage in all the tested strains. According to NCCLS criteria, at the same step all strains shifted from susceptible to resistant to amikacin and imipenem, while zero, zero, one, four and zero strains became resistant to levofloxacin, ciprofloxacin, cefepime, ceftazidime and piperacillin–tazobactam, respectively. For the other antibiotics, a similar change was observed in the following passages and, after the last one, almost all strains became resistant to the studied drugs. Combinations of fluoroquinolones with amikacin or cefepime and piperacillin–tazobactam limited 4-fold MIC increments after the initial passages, but, more interestingly, notably reduced the rate of strains shifting from susceptible to resistant. Whereas all combinations seemed to protect the fluoroquinolones from the emergence of resistance, development of resistance continued to occur with ceftazidime and imipenem when they were combined with a fluoroquinolone.

Overall, Acinetobacter showed less development of resistance than Pseudomonas. Emergence of resistance in Acinetobacter spp. appeared slower for other agents than for fluoroquinolones after two passages with single antibiotics, while, after the last passage, three strains shifted to resistant to levofloxacin and to ceftazidime, four to amikacin, cefepime and piperacillin–tazobactam, five to ciprofloxacin and none to imipenem (Table 4). After the last passage in the combinations, most of the acinetobacters showed a ≥4-fold MIC increment (Table 5). Levofloxacin was more protective than ciprofloxacin in limiting changes of susceptibility, with only one strain resistant to levofloxacin when assessed in combination with imipenem. In contrast, a total of nine strains were found to be resistant after passages in combinations with ciprofloxacin: seven were resistant to ciprofloxacin (two recovered from amikacin, cefepime and ceftazidime agar plates, and one from imipenem plates) and two were resistant to piperacillin–tazobactam (Table 4).

Single-step selection of resistant bacteria

The highest frequencies of mutations for antibiotics alone were found for cefepime, imipenem and piperacillin–tazobactam in P. aeruginosa (~10–7) and piperacillin–tazobactam (~10–6) in Acinetobacter spp. Levofloxacin and ciprofloxacin showed a similar rate in P. aeruginosa, while a difference was found in Acinetobacter spp., where levofloxacin showed a lower frequency than ciprofloxacin (<10–12 versus 10–9). Combinations of fluoroquinolones with ß-lactams or amikacin reduced the occurrence of mutations favouring bacterial growth to a rate of <10–12, as shown in Tables 6 and 7.


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Table 6. Mutational frequencies of P. aeruginosa after 48 h incubation with levofloxacin, ciprofloxacin, amikacin, cefepime, ceftazidime, imipenem and piperacillin–tazobactam alone and in combination

 

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Table 7. Mutational frequencies of Acinetobacter spp. after 48 h incubation with levofloxacin, ciprofloxacin, amikacin, cefepime, ceftazidime, imipenem and piperacillin–tazobactam alone and in combination

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
P. aeruginosa and Acinetobacter spp. can cause a multitude of hospital infections, including nosocomial pneumonia, that are associated with a high mortality rate.26,27 Since the probability of selecting colonies resistant to both drugs in a combination is approximately equal to the product of the probabilities of resistance to individual agents, use of antibiotics in combination to prevent or minimize the likelihood of emergence of drug-resistant subpopulations should be considered, once synergic activity has been confirmed.28

Combinations of aminoglycosides with ß-lactams represent the classical treatment for severe infections due to Gram-negative bacilli, but several studies have failed to demonstrate any benefit when combinations of aminoglycosides with ß-lactams were compared with monotherapy, whereas in another study, a decrease in pharmacodynamic–pharmacokinetic parameters was observed for amikacin and imipenem when they were administered in combination compared with when they were administered separately.2931 Aminoglycoside toxicity is another factor that encourages the search for novel combinations in order to provide effective therapies.

Fluoroquinolones have proven to have a high rate of synergy with ß-lactams in in vitro evaluations, while ability to limit resistance has been rarely evaluated.1321,32

The results obtained in this study indicate the high potential of P. aeruginosa and Acinetobacter spp., although at different rates, to acquire phenotypic resistance to fluoroquinolones, ß-lactams and amikacin. The use of antibiotic combinations notably reduced the rate and the extent of MIC increase, as already reported for ciprofloxacin by other authors.22 Differences observed between mutational frequencies of levofloxacin and ciprofloxacin in Acinetobacter and between P. aeruginosa and Acinetobacter strains confirmed that the propensity of fluoroquinolones to select resistance varies regarding the agent concerned, its antibiotic concentrations relative to the MIC and the microorganisms involved, as shown in other studies.22,33,34

In the same way, differences observed among combinations are suggestive of differences in the ability of ß-lactams to select resistance. Therefore, the choice of antibiotics involved in combination should take this into account, in addition to antimicrobial activity in terms of synergy shown by antibiotics themselves.

Recent studies have identified use of fluoroquinolones as a risk factor for infection or colonization resulting from multidrug-resistant P. aeruginosa and A. baumannii, while in another study, use of fluoroquinolones in combinations and longer duration of therapy have been independently associated with emergence of multidrug resistance.3537 However, these studies are small and to date, no large-scale prospective randomized clinical trials have specifically evaluated the relationship between fluoroquinolone use and the emergence of resistance during monotherapy and combination therapy.

Determination of whether in vitro results obtained in this study may correlate with clinical efficacy remains difficult, since, even in in vitro pharmacokinetic models that allow treatment of bacteria with the same pharmacokinetically changing concentrations of antibiotic occurring in vivo, many factors involved in the host, such as immunity, are absent. A recent in vitro pharmacokinetic study on emergence of resistance in P. aeruginosa, simulating treatment with a combination of levofloxacin plus imipenem, demonstrated superiority of combinations over antibiotics alone in limiting the emergence of resistance.32 These data contrast with those obtained in the present study, where resistance to imipenem also developed in the combinations. The different experimental models employed in the two studies are likely to be responsible for this difference.

The principal aim of this study was to compare the abilities of ciprofloxacin and levofloxacin alone and in combinations with other antibiotics to select phenotypic resistance, without any molecular characterization of the resistant isolates. However, it is well known that mutations or overexpression of specific efflux pumps are essential mechanisms of P. aeruginosa resistance to several antibiotics. Topoisomerase mutations that confer higher levels of resistance seem to be a secondary event in resistance to fluoroquinolones, and chromosomal and plasmid-carried ß-lactamase concurred in resistance to ß-lactams.14,38,39 In Acinetobacter spp., gyrA and parC mutations and upregulation of the adeB efflux pump seem to be the major factors responsible for the development of multidrug resistance.40,41 Any hypothesis on the mechanisms in our strains is rather difficult, and all of the above-mentioned mechanisms (particularly efflux pumps) could be involved, as suggested by others.32

Although our study has several limitations, such as the limited number of strains and passages evaluated, and the absence of molecular typing of resistant isolates, results indicate that use of fluoroquinolones in combination with ß-lactams or amikacin may limit the decrease of susceptibility and development of resistance in P. aeruginosa and Acinetobacter spp. during therapy. However, further studies, including kinetic evaluations, are needed to better investigate the issue.


    Acknowledgements
 
This work was supported by Sanofi-Aventis SpA (Lainate, Italy).


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 Introduction
 Methods
 Results
 Discussion
 References
 
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