Comparative in vitro bactericidal activity between cefepime and ceftazidime, alone and associated with amikacin, against carbapenem-resistant Pseudomonas aeruginosa strains

Comparative in vitro bactericidal activity between cefepime and ceftazidime, alone and associated with amikacin, against carbapenem-resistant Pseudomonas aeruginosa strains

Diagnostic Microbiology and Infectious Disease 37 (2000) 41– 44 www.elsevier.com/locate/diagmicrobio Antimicrobial susceptibility studies Comparati...

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Diagnostic Microbiology and Infectious Disease 37 (2000) 41– 44

www.elsevier.com/locate/diagmicrobio

Antimicrobial susceptibility studies

Comparative in vitro bactericidal activity between cefepime and ceftazidime, alone and associated with amikacin, against carbapenem-resistant Pseudomonas aeruginosa strains Carlos Bantar*, Marcela Di Chiara, Federico Nicola, Silvia Relloso, Jorgelina Smayevsky Department of Microbiology, Centro de Educacio´n Me´dica e Investigaciones Clı´nicas “Dr. Norberto Quirno”, Billingurst 2447, (1425) Buenos Aires, Argentina Received 18 October 1999; revised and accepted 18 December 1999

Abstract Fifteen unique isolates of carbapenem-resistant Pseudomonas aeruginosa were selected for time-kill studies to assess the bactericidal activity of cefepime (CFP) and ceftazidime (CZD) (at 4 and 16 ␮g/mL), alone and associated with amikacin (AMK) (4 ␮g/mL). CFP proved more active than CZD (p ⬍ 0.05, Student‘s t test). Bactericidal activity after 24-h incubation was only achieved by the combination of CFP (16 ␮g/mL) plus AMK. The higher in vitro activity of cefepime over that of ceftazidime against imipenem-resistant P. aeruginosa strains highlights the differences of these drugs beyond Enterobacter spp. and Staphylococcus aureus. © 2000 Elsevier Science Inc. All rights reserved.

1. Introduction Nosocomial infections caused by Pseudomonas aeruginosa strains resistant to multiply drugs are becoming a worrisome therapeutic problem (Jarvis and Martone 1992). Although improved outcome of bacteremia caused by this organism has been associated with the advent of carbapenems (Kuikka and Valtonen 1998), a number of studies describing different mechanisms of resistance displayed by P. aeruginosa against these antibiotics have already been published (Ko¨hler et al. 1999; Nakae et al. 1999; Troillet et al. 1997). Thus, efforts to search for additional therapeutic options are warranted. Cefepime is a relatively new cephalosporin antibiotic showing a low affinity for major chromosomally mediated ␤-lactamases, enhanced penetration into Gram-negative bacteria and the ability to be less prone to select resistant P. aeruginosa strains, compared with the current antipseudo-

This work was presented in part at the 36th Infectious Diseases Society of America Annual Meeting, held on 12–15 November, 1998, in Denver, Colorado. * Corresponding author. Tel.: ⫹1-5411-4804-9312; fax: ⫹1-54114805[3233]. E-mail address: [email protected] (C. Bantar)

monal third-generation cephalosporin, ceftazidime (Gradelski et al. 1993). Both clinical trials and in vitro susceptibility patterns comparing such drugs have been reported. However, comparative time-kill studies against P. aeruginosa are scarce and they were neither focused on carbapenemresistant strains nor evaluated the synergism of their combination with an aminoglycoside (Johnson et al. 1995). Thus, we have undertaken a comparative study to assess, by time-kill studies, the bactericidal activity of cefepime and ceftazidime, alone and associated with amikacin, against several carbapenem-resistant P. aeruginosa strains. Fifteen unique isolates recovered from clinical specimens were selected because of their resistance to imipenem. MICs of meropenem (Zeneca Farma, Buenos Aires, Argentina), imipenem (Merck Sharp & Dohme Argentina Inc.), ceftazidime (Glaxo, Buenos Aires, Argentina), cefepime and amikacin (Bristol-Myers Squibb Argentina S.A.) were determined by the agar dilution method following the National Committee for Clinical Laboratory Standards guidelines (NCCLS 1997). P. aeruginosa ATCC 27853 was used as the control. Time-kill studies were performed with ceftazidime and cefepime at concentrations of 4 ␮g/mL and 16 ␮g/mL, alone and associated with amikacin (4 ␮g/mL), as described previously (Bantar et al. 1993). Briefly, tubes containing

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Table 1 MIC inhibiting 50% and 90% of the strains (MIC50 and MIC90, respectively) and MIC range of five antibiotics against 15 strains of P. aeruginosa Antibiotic

Imipenem Meropenem Amikacin Ceftazidime Cefepime

Values (␮g/mL) corresponding to: MIC50

MIC90

Range

32 8 8 64 8

32 8 64 64 16

8–32 ⱕ0.5–32 4–128 2–128 2–16

cation-supplemented Mueller-Hinton broth (Difco Laboratories, Detroit, MI) with and without (growth control) antibiotics were seeded with a log-phase inoculum of roughly 5 ⫻ 106 (CFU/mL). Viable cell counts were performed after a 0-, 4-, 8- and 24-h incubation at 35°C in room atmosphere and a 24-h killing curve was established for each strain. Mean and standard deviation (SD) were calculated for the viable cell counts from the 15 strains and the differences in the bactericidal extent were analyzed by the Student‘s t test. A p value of ⱕ0.05 was considered as significant. Synergy was defined as a ⱖ100-fold increase in killing by the drug combination over the killing accomplished by the most active of the two cephalosporins when tested separately. Bactericidal activity was defined as a ⱖ3 log10 CFU/mL decrease from the initial inoculum. MICs inhibiting 50% and 90% of the strains (MIC50 and MIC90, respectively), as well as the MIC ranges, for the different antibiotics are given in Table 1. Most of the strains proved more susceptible to meropenem than to imipenem. Similar feature was observed with cefepime compared with ceftazidime. Results of the time-kill studies are shown in Table 2. Cefepime at 16 ␮g/mL proved more active than cefepime at

4 ␮g/mL and ceftazidime (both at 4 and 16 ␮g/mL) (p ⬍ 0.05). Likewise, although synergism between amikacin and both ceftazidime and cefepime could be demonstrated, the association with cefepime at 16 ␮g/mL showed the highest decrease in the viable cell count (p ⬍ 0.05), being the only combination able to display bactericidal activity (i.e., a ⱖ3 log10 CFU/mL decrease from the initial inoculum after 24 h of incubation). Furthermore, the combination cefepime (16 ␮g/mL)-amikacin (4 ␮g/mL) avoided any overgrowth in all of the strains, whereas this phenomenon was observed in 40% of the strains tested against ceftazidime (16 ␮g/mL) plus amikacin. An illustrative comparative time-kill curve with the mean and the SD values is depicted in Figure 1. Reports on P. aeruginosa isolates resistant to cephalosporins and carbapenems are increasing (Bouza et al. 1999; Jones et al. 1998). Apart from some authors (Johnson et al. 1995), who suggest that cefepime proves active against ceftazidime-resistant P. aeruginosa strains, most of the studies show similar resistant rates for both cephalosporins (Bouza et al. 1999; Jones et al. 1998). However, none of them discriminated between carbapenem-resistant and -susceptible strains to undertake an exhaustive comparative analysis between ceftazidime and cefepime MICs. In addition, time-kill and synergy tests were not performed. This fact seems to be important, because it has recently been described that the interplay between the expression of OprD, the presence of the MexAB-OprM efflux system and the overexpression of the cromosomally encoded ␤-lactamase associated with carbapenem-resistant P. aeruginosa strains, may distinctively influence the in vitro susceptibility to either the carbapenems or the antipseudomonal cephalosporins (Ko¨hler et al. 1999; Nakae et al. 1999). Thus, the higher permeability and the decreased hydrolysis displayed by the cromosomally encoded ␤-lactamase against cefepime compared with ceftazidime might be better evidenced in this kind of isolates rather than among strains lacking these

Table 2 Comparative time-kill studies between cefepime and ceftazidime, alone and associated with amikacin, against 15 carbapenem-resistant P. aeruginosa strains Antibiotica (concentration)

Viable Cell Count (log10 CFU/mL) After the Time of Incubationb: 0h

4h

8h

24 h

Control AMK (4 ␮g/mL) CZD4 (4 ␮g/mL) CFP4 (␮g/mL) CZD16 (16 ␮g/mL) CFP16 (16 ␮g/mL) CZD4 ⫹ AMK CFP4 ⫹ AMK CZD16 ⫹ AMK CFP16 ⫹ AMK

6.6 ⫾ 0.3 6.6 ⫾ 0.3 6.6 ⫾ 0.3 6.6 ⫾ 0.3 6.6 ⫾ 0.3 6.6 ⫾ 0.3 6.6 ⫾ 0.3 6.6 ⫾ 0.3 6.6 ⫾ 0.3 6.6 ⫾ 0.3

7.7 ⫾ 0.5 5.6 ⫾ 2.3 6.6 ⫾ 1.1 5.7 ⫾ 0.9 5.5 ⫾ 1.0 4.2 ⫾ 1.0 4.5 ⫾ 2.5 4.1 ⫾ 2.0 3.5 ⫾ 2.3 2.7 ⫾ 1.4

8.9 ⫾ 1 6.1 ⫾ 2.8 7.3 ⫾ 1.6 6.4 ⫾ 2 6.0 ⫾ 1.9 4.0 ⫾ 1.7c 4.7 ⫾ 3.1 3.9 ⫾ 2.7 3.7 ⫾ 2.6 1.8 ⫾ 1.0d

11.4 ⫾ 0.5 6.6 ⫾ 3.2 10 ⫾ 24 9.3 ⫾ 2.9 8.7 ⫾ 3.5 5.8 ⫾ 3.1c 5.7 ⫾ 4.9 5.2 ⫾ 4.8 4.8 ⫾ 4.0 2.4 ⫾ 2.2d

a

AMK, amikacin; CZD, ceftazidime; CFP, cefepime. Values are given as mean ⫾ SD. c p ⬍ 0.05 versus CZD16 and CFP4. d p ⬍ 0.05 versus CZD16 ⫹ AMK and CFP4 ⫹ AMK. b

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Fig. 1. Comparative bactericidal activity between ceftazidime (CZD) and cefepime (CFP), alone and associated with amikacin (AMK) against carbapenemresistant Pseudomonas aeruginosa. Values and mean ⫾ SD from 15 strains.

mechanisms of resistance to carbapenems. Indeed, we have observed in our strains lower MICs of both meropenem and cefepime, compared with those of imipenem and ceftazidime, respectively. In addition, differences between the cephalosporins were confirmed by time-kill studies, both alone and associated with amikacin. Despite synergy could be observed with both ceftazidime and cefepime even at the lower cephalosporin concentration (i.e., 4 ␮g/mL), bactericidal activity after 24-h incubation was only achieved by the combination of cefepime (16 ␮g/mL)-amikacin (4 ␮g/mL). This feature seems to be closely related with the cefepime MIC (MIC90, 16 ␮g/mL) and this concentration may be usually attainable in serum with the current dosage recommended for the treatment of most of the infections (Barradell and Bryson 1994). It is noteworthy that 6 of 15 strains whose ceftazidime MICs were ⬎ 32 ␮g/mL (i.e., resistant) were intermediate to cefepime (MIC, 16 ␮g/mL). Cefepime alone proved more active than ceftazidime by time-kill studies against the six strains. When the cephalosporins were combined with amikacin, bactericidal synergy was observed only against the combination with cefepime (data not shown). This fact may be of clinical relevance, since, although clinicians would not use ceftazidime against these isolates, may also reject cefepime because it should be reported as intermediate by the routine tests. However, bactericidal activity would have been obtained against the combination with amikacin in all the cases. Indeed, enhanced synergistic activity, both in vitro and in vivo, of cefepime against ceftazidime-resistant isolates has been described recently with an isogenic pair of Enterobacter cloacae (Mimoz et al. 1998) In summary, cefepime, especially associated with amikacin, displayed bactericidal properties against carbapenem-resistant P. aeruginosa strains that makes it a suitable

choice for treating infections caused by these organisms. Furthermore, the higher in vitro activity of cefepime over that of ceftazidime against these isolates highlights the differences of these drugs beyond Enterobacter spp. and Staphylococcus aureus.

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