Synergistic potential of ceftazidime plus amikacin or levofloxacin against Pseudomonas aeruginosa as determined using a checkerboard and a disk diffusion technique

Synergistic potential of ceftazidime plus amikacin or levofloxacin against Pseudomonas aeruginosa as determined using a checkerboard and a disk diffusion technique

Diagnostic Microbiology and Infectious Disease 53 (2005) 157 – 160 www.elsevier.com/locate/diagmicrobio Note Synergistic potential of ceftazidime pl...

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Diagnostic Microbiology and Infectious Disease 53 (2005) 157 – 160 www.elsevier.com/locate/diagmicrobio

Note

Synergistic potential of ceftazidime plus amikacin or levofloxacin against Pseudomonas aeruginosa as determined using a checkerboard and a disk diffusion technique Maria Pia Montanaria, Laura Piccolib, Marina Mingoiaa, Federico Marchettib, Pietro Emanuele Varaldoa,T a

Institute of Microbiology and Biomedical Sciences, Marche Polytechnic University Medical School, 60131 Ancona, Italy b Medical Department and Medicines Research Center, GlaxoSmithKline, 37135 Verona, Italy Received 28 February 2005; accepted 3 June 2005

Abstract The synergistic potential of ceftazidime plus amikacin or levofloxacin was assessed against 61 Pseudomonas aeruginosa isolates with variable susceptibility patterns to the 3 antibiotics. A checkerboard broth method and a disk diffusion method were used and compared. The latter, also easy to perform as a triple-disk assay, could be a helpful laboratory screening tool for drug synergism to drive possible combination treatments. D 2005 Elsevier Inc. All rights reserved. Keywords: Pseudomonas aeruginosa; Screening for synergism; Ceftazidime; Amikacin; Levofloxacin

Pseudomonas aeruginosa is a ubiquitous pathogen capable of infecting virtually all tissues. It is commonly encountered as a versatile opportunist in a variety of infections, especially in nosocomial settings and in highrisk populations such as oncology, transplant, cystic fibrosis, and critically ill patients (Lyczak et al., 2000). Like other nonfermentative Gram-negative bacteria, P. aeruginosa is inherently resistant to many drugs; moreover, its low outer membrane permeability coupled with additional resistance mechanisms such as inducible h-lactamase and efflux pumps allows it to easily become resistant to other antibiotics at concentrations that can be achieved in vivo (Hancock, 1998). The use of a combination of antipseudomonal antibiotics for serious P. aeruginosa infections is widely accepted in clinical practice to overcome intrinsic and acquired resistance, although it is controversial whether adequate combination therapy is associated with a better outcome than adequate monotherapy (Chamot et al., 2003; Chow and Yu, 1999; Giamarellou, 2002; Hancock and Speert, 1996; Lynch, 2001). Recently, a meta-analysis has provided evidence of a 50% relative reduction in mortality from P. aeruginosa T Corresponding author. Tel.: +39-71-2204694; fax: +39-71-2204693. E-mail address: [email protected] (P.E. Varaldo). 0732-8893/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2005.06.002

bacteremia with combination therapy (Safdar et al., 2004). A combination of an antipseudomonal h-lactam with an aminoglycoside is the standard regimen for these infections due to the well-documented synergism between these agents. Fluoroquinolones with antipseudomonal activity, reported by a recent meta-analysis to be effective against nosocomial pneumonia (Shorr et al., 2005), may be a less nephrotoxic alternative to aminoglycosides in combination treatment (Fish et al., 2002; Lynch, 2001), also considering that comparable degrees of antipseudomonal synergism between a h-lactam plus an aminoglycoside and a h-lactam plus a fluoroquinolone have been documented in vitro (Burgess and Nathisuwan, 2002). Although ciprofloxacin is considered the most active quinolone against P. aeruginosa, levofloxacin has lately been assigned by the National Committee for Clinical Laboratory Standards (NCCLS, 2004) to the same test/report group against P. aeruginosa, and the 2 drugs are reported as equivalent antipseudomonal fluoroquinolones in an evidence-based guideline for the management of adults with hospital-acquired pneumonia (American Thoracic Society Documents, 2005). In vitro assessment of synergism is commonly performed using either the checkerboard or the time-kill technique. However, both methods have a number of limitations, due

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Table 1 Ceftazidime plus amikacin and ceftazidime plus levofloxacin interactions against 61 test strains of P. aeruginosa a Interaction

Checkerboard

Synergism Additivityb Indifferenceb Antagonism

Disk diffusion (distance between disks)

CAZ + AMK

CAZ + LVX

CAZ + AMK (12 mm)

CAZ + AMK (14 mm)

CAZ + LVX (12 mm)

CAZ + LVX (14 mm)

44 (72.1) 12 (19.7) 5 (8.2) 0

41 (67.2) 13 (21.3) 7 (11.5) 0

43 (70.5) 18 (29.5)

42 (68.9) 19 (31.1)

30 (49.2) 31 (50.8)

24 (39.3) 37 (60.7)

0

0

0

0

CAZ = ceftazidime; AMK = amikacin; LVX = levofloxacin. a Data are presented as number (%) of isolates. b Additivity and indifference cannot be differentiated using the disk diffusion test.

especially to correlation and reproducibility problems, particularly when resistant strains of P. aeruginosa are tested (Cappelletty and Rybak, 1996). Moreover, being laborintensive and time-consuming, they are infrequently performed in diagnostic laboratories; in particular, research or reference laboratory settings are explicitly recommended for synergism testing of P. aeruginosa isolates (Kiska and Gilligan, 2003). A simpler screening for synergism can be obtained using a disk diffusion technique, by analyzing the zones of inhibition around 2 disks containing the 2 antibiotics approximated on the surface of an agar plate streaked with the test organism. Although this test only provides qualitative information, simplicity and use of readily available materials (commercial disks and agar plates) represent major advantages for routine use (Eliopoulos and Moellering, 1996). In this study, the synergistic potential of ceftazidime (GlaxoSmithKline, Verona, Italy) plus amikacin (SigmaAldrich, Milan, Italy) or levofloxacin (GlaxoSmithKline) was investigated using a checkerboard and a disk diffusion technique against 61 clinical isolates of P. aeruginosa. The isolates, recovered from a variety of clinical specimens in different Italian hospitals during the last 2 years, were identified by conventional methods (Kiska and Gilligan,

2003) and maintained at 70 8C in Microbank vials (Pro-Lab Diagnostics, Richmond Hill, Ontario, Canada) before testing. The isolates exhibited variable susceptibility patterns to the 3 antibiotics, with broth microdilution MICs (NCCLS, 2003a) ranging between 4 and 256 Ag/mL for ceftazidime, 0.25 and 256 Ag/mL for amikacin, and 1 and 128 Ag/mL for levofloxacin. Standard checkerboard titration experiments were performed in microtiter trays with Mueller–Hinton II broth (BBL Microbiology Systems, Cockeysville, MD), synergism being determined from the fractional inhibitory concentration (FIC) index (Eliopoulos and Moellering, 1996). FICs were calculated as the MIC of drug A or B in combination/the MIC of drug A or B alone, and the FIC index was obtained by summing the FICs of each drug. FIC indices were interpreted as indicating synergism if the values were V 0.5, additivity if they were N 0.5 to 1, indifference if they were N1 to 4, and antagonism if they were N 4. Concentrations within the FIC panel were such that the MIC of each antibiotic was in the middle of the range of the concentrations tested. The final inoculum was approximately 5  105 CFU/mL. Trays were incubated aerobically overnight. Qualitative screening for synergism using the diffusion method was performed according to a standard double-disk technique (Eliopoulos and Moellering, 1996).

Table 2 Ceftazidime plus amikacin and ceftazidime plus levofloxacin interactions against 61 test strains of P. aeruginosa distributed according to their susceptibility patternsa Susceptibility pattern CAZ

AMK

No. of isolates

CAZ + AMK Syn

R R R R R R I I S S S S

R R R S S S R S R S S S

R I S R I S R R R R I S

CAZ + LVX Disk diffusiona

Checkerboard

Ind

Syn

Add/Ind

Syn

Add

Ind

Syn

Add/Ind

5

12 1

6 1 1

12 2

1

5

2 2 1 5 7 2 1 2 1 5 2

16

Checkerboard

LVX

18 2 1 10 8 2 1 2 1 7 2 7

13 1 10 7 1 2 1 4 1 4

Add 1 1 1 1 1

3 1 3

10 7 1 2 1 4 1 4

1 1 1

3 1 3

9 8 1 1 2 1 4 1

Disk diffusiona

1 1 1

3 1 6

1

S = susceptible; I = intermediate; R = resistant; Syn = synergism; Add = additivity; Ind = indifference; Add/Ind = additivity or indifference. a With disks placed 12 cm apart.

5 1

2 7

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Commercial disks (ceftazidime [30 Ag], amikacin [30 Ag], and levofloxacin [5 Ag], all from Oxoid [Basingstoke, UK]), test medium (Mueller–Hinton agar [BBL]), inoculum preparation, and inoculation of test plates were as for a routine Bauer–Kirby susceptibility test (NCCLS, 2003b). Antibiotic-impregnated disks were assayed at 2 different distances (12 and 14 mm apart), established based on the zone diameter break points and taking into account the possibility to deal with strains resistant to one or both antibiotics (Eliopoulos and Moellering, 1996). After overnight incubation at 37 8C, drug interactions were determined from the shape of the zones of inhibition. While 2 independent circles denoted additive or indifferent combinations, enhancement or bridging at or near the junction of the 2 circles denoted synergism and truncation antagonism. The results of the checkerboard method and the diffusion technique with disks 12 and 14 mm apart are compared in Table 1. Antagonism was never observed with either method. With the disk diffusion assay, the 12-mm distance between disks appeared to be more suitable than the 14-mm distance in recognizing synergism, particularly with the ceftazidime/levofloxacin combination. The distribution of results according to the isolates’ susceptibility patterns is summarized in Table 2. Consistently with previous in vitro studies (Fish et al., 2002; Visalli et al., 1998), a greater frequency of synergism was observed in strains resistant to one or both drugs in combination. All interactions indicated as synergistic by the disk diffusion technique were indicated as such also by the checkerboard method. The latter, however, also showed as synergistic some interactions that were additive/indifferent according to the disk diffusion technique, particularly when isolates were resistant to all 3 antibiotics. Remarkably, 4 isolates with different susceptibility patterns, which in ceftazidime plus levofloxacin checkerboard tests yielded FIC indices of 0.56—that is, a value barely exceeding the 0.5 synergism threshold and just falling in the additivity range, demonstrated synergistic interactions in disk diffusion tests. The results obtained using the double-disk diffusion method (Fig. 1A) were consistently reproducible using a triple-disk diffusion assay with the ceftazidime disk placed at the center and the amikacin and levofloxacin disks placed 12 mm apart on either side (Fig. 1B). Although antibiotic combinations are extensively used therapeutically, laboratory tests of antibiotic interactions, besides being impractical, are generally unnecessary barring some circumstances, when combinations are used in an attempt to attain synergistic interactions (Eliopoulos and Eliopoulos, 1988). On the other hand, synergism itself, despite the wide use and fortune enjoyed by the term in medical literature, has been long regarded as an elusive concept (Moellering, 1979). Furthermore, there are relatively few circumstances in which laboratory documentation of synergism is highly predictive of superior clinical efficacy (Eliopoulos and Eliopoulos, 1988; Eliopoulos and Moellering, 1996). Serious P. aeruginosa infections are likely to be just such cases, although it is uncertain

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Fig. 1. Examples of drug interactions against clinical isolates of P. aeruginosa detected using the disk diffusion technique. (A) Double-disk method (the ceftazidime disk is placed on the left side and the levofloxacin disk on the right side). (B) Triple-disk method (the ceftazidime disk is placed at the center and the amikacin and levofloxacin disks are placed on its left and right, respectively). In both panels, the left plate shows indifference/additivity and the right plate shows synergism. The susceptibility profiles of the strains used (expressed as antibiotic MICs) were as follows. (A, left plate) Ceftazidime, 32 Ag/mL; amikacin, 64 Ag/mL; levofloxacin, 128 Ag/mL. (A, right plate) Ceftazidime, 2 Ag/mL; amikacin, 16 Ag/mL; levofloxacin, 16 Ag/mL. (B, left plate) Ceftazidime, 256 Ag/mL; amikacin, 64 Ag/mL; levofloxacin, 128 Ag/mL. (B, right plate) Ceftazidime, 4 Ag/mL; amikacin, 16 Ag/mL; levofloxacin, 16 Ag/mL.

whether the benefit actually arises from synergistic interactions (Eliopoulos and Moellering, 1996). In this context, in line with the concept that, in some cases, screening methods can be used as substitutes for formal synergism testing (Eliopoulos and Eliopoulos, 1988), a diffusion test using commercial disks could be as easy to perform as helpful to screen for synergism. Because the synergism interactions detected using this method are fewer than those detected by the checkerboard method, the disk diffusion technique might be more stringent than the other in recognizing those P. aeruginosa infections that could respond to a combination treatment using a less nephrotoxic fluoroquinolone, instead of an aminoglycoside, together with an antipseudomonal h-lactam. Trials with larger numbers of strains, and possibly with different antibiotic combinations, are warranted. Acknowledgments This study was partly supported by a grant from GlaxoSmithKline, Verona, Italy. References American Thoracic Society Documents (2005) Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 171: 388 – 416. Burgess DS, Nathisuwan S (2002) Cefepime, piperacillin/tazobactam, gentamicin, ciprofloxacin, and levofloxacin alone and in combination against Pseudomonas aeruginosa. Diagn Microbiol Infect Dis 44:35 – 41.

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