In Vitro and In Vivo Activities of Amikacin, Cefepime, Amikacin plus Cefepime, and Imipenem against an SHV-5 Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Strain

Abstract

The in vitro and in vivo effectiveness of amikacin, cefepime, and imipenem was studied using a high inoculum of an extended-spectrum β-lactamase-producing Klebsiella pneumoniae strain. An in vitro susceptibility test at the standard inoculum predicted the in vivo outcome of amikacin or imipenem while it did not do so for cefepime due to the inoculum effect.

Extended-spectrum β-lactamase (ESBL) production is one of the main mechanisms of resistance to β-lactam antibiotics among the strains of the family Enterobacteriaceae (14). The therapeutic choices in infections caused by such strains remain limited because of cross-resistance (4). Attempts have been made to compare the activities of β-lactams with β-lactamase inhibitors, showing inconsistent results (5, 20, 24, 27). Conflicting reports have been published concerning the activities of the broad-spectrum and “fourth generation” cephalosporins with an explanation of the inoculum effect (5, 15, 27).

Our aim was to compare the activities of amikacin, cefepime, amikacin plus cefepime, and imipenem in septic mice infected by an SHV-5 ESBL-producing Klebsiella pneumoniae strain using a high initial inoculum.

The SHV-5 ESBL-producing K. pneumoniae strain originated in a premature intensive-care unit (26). Amikacin and cefepime (Bristol-Myers Squibb), imipenem (Merck, Sharp & Dohme), and cisplatin (Ebewe) were used according to the manufacturers' instructions. The MICs and the minimal bactericidal concentrations (MBCs) were determined by the microdilution method with inocula of 10⁵ and 10⁷ CFU/ml, respectively (20, 21). For the killing curve, the initial bacterial concentration was 8 log₁₀ CFU/ml. The concentrations of antibiotics chosen were close to the in vivo mean levels in serum: amikacin, 4 μg/ml; cefepime, 40 μg/ml; amikacin plus cefepime in the same concentrations listed above; and imipenem, 16 μg/ml. Synergy was defined as a ≥2-log₁₀ decrease in the number of CFU per milliliter between the combination and its most active constituent after 24 h (17).

Randomly selected male CD-1 mice (30 to 35 g) were used for the pharmacokinetic study, for the determination of blood bacterial counts, and for survival analysis. Each group contained 15 mice. Cisplatin (18 mg/kg of body weight as determined in a pilot study) had been administered by intraperitoneal (i.p.) injection 3 days before infection in order to cause renal impairment (18). The mice were infected i.p. with 10⁷ CFU/g; the uninfected group received only cisplatin. Three groups of uninfected mice were used for pharmacokinetic analysis, and they were followed up for 48 h (3, 6, 9, 10, 16, 19, 28). Single doses of amikacin (7.5 mg/kg), cefepime (80 mg/kg), and imipenem (40 mg/kg) were administered i.p. Blood samples were taken 15 and 30 min and 1, 2, and 3 h after drug administration. Antibiotic levels in sera were determined by a paper disk method for imipenem and cefepime with Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 25922, respectively, as indicator organisms on antibiotic medium 1 (Becton Dickinson) (1). The amikacin serum levels were detected by the fluorescence polarization immunoassay (Abbott TDx system) (30). The lower limits of detection were 0.05 μg/ml for amikacin, 8 μg/ml for cefepime, and 1 μg/ml for imipenem. The coefficient of variability was ≤5% for each antibiotic assay. The pharmacokinetic values half-life (T_1/2), peak concentration (C_max), first occurrence of C_max (T_max), area under the concentration-time curve extrapolated to infinity (AUC), and area under the concentration-time curve from 0 to 24 h (AUC_0–24) were calculated by a noncompartmental method (2).

Four infected treated groups and one infected untreated group were used for the determination of blood bacterial counts. The treatment started 3 h after infection and lasted for 24 h. Two doses of amikacin (7.5 mg/kg every 10.5 h), three doses of cefepime (80 mg/kg every 7 h), the same doses of amikacin plus cefepime as in monotherapy, or four doses of imipenem (40 mg/kg every 5.25 h) were given i.p. Blood samples were taken 3 and 9 h after the beginning of antibiotic therapy. The Kruskal-Wallis test followed by the Mann-Whitney U test were used for statistical analysis, taking a P value of <0.05 as significant. Four infected treated groups and two control groups (uninfected treated and infected untreated) were used for the survival analysis after up to 48 h, with death as the end point. Survival was assessed at 24 and 48 h for calculating lethality, and 24-h survival was assessed by estimating the cumulative probability using the Kaplan-Meier survival curve. The relative risk between groups was estimated by the hazard ratio with the appropriate 95% confidence interval comparing the observed deaths with expected deaths. The log rank test was used for statistical analysis, accepting a P value of <0.05 as significant.

The MIC and MBC of amikacin were 0.5 μg/ml at both 10⁵ and 10⁷ CFU/ml. The MIC and MBC of cefepime were 1 μg/ml at 10⁵ CFU/ml, and the MIC was >256 μg/ml at 10⁷ CFU/ml. The MIC and MBC of imipenem were 0.125 μg/ml at 10⁵ CFU/ml, and the MIC and MBC were 0.5 and 1 μg/ml at 10⁷ CFU/ml, respectively. The killing curve study is shown in Fig. 1A. Synergy was not detected due to the limits of detection of bacterial counts.

FIG. 1.

(A) Killing curves of ESBL-producing K. pneumoniae incubated without antibiotic, with cefepime, with amikacin, with imipenem, and with amikacin plus cefepime. (B) Mean bacterial counts observed in untreated mice and in those receiving amikacin, amikacin plus cefepime, cefepime, and imipenem after infection with ESBL-producing K. pneumoniae. Error bars indicate standard deviations. The difference was significant when all groups were compared (P < 0.0001) and between the infected untreated group and all treated groups (P < 0.01 in each pair compared). The cefepime-treated group differed statistically from the other treated groups (P < 0.05 in each pair compared). There was no difference between groups receiving amikacin, amikacin plus cefepime, and imipenem (P > 0.37 in each pair compared).

Data from the pharmacokinetic analysis are given in Table 1, except for amikacin plus cefepime because they do not affect the elimination of each other (2).

TABLE 1.

Pharmacokinetic parameters of antibiotics following an i.p. injection in noninfected mice with impaired renal function^a

Antibiotic (dose)	T1/2 (h)	Cmax (μg/ml)	Tmax (min)	AUC (μg · h/ml)	T > MIC (h/%)		AUC0–24 MIC (h)		Cmax/MICbc
					105b	107c	105b	107c
Amikacin (7.5 mg/kg)	0.86 ± 0.14	20.18 ± 4.30	15	35.34 ± 4.90	4.81 ± 0.95/46.00 ± 9.05	4.81 ± 0.95/46.00 ± 9.05	161.54 ± 22.40	161.54 ± 22.40	40.36 ± 8.60
Cefepime (80 mg/kg)	1.01 ± 0.07	195.00 ± 45.00	15	260.97 ± 12.67	7.40 ± 0.36/105.72 ± 5.20	—d	894.75 ± 43.44	NCe	NC
Imipenem (40 mg/kg)	0.45 ± 0.03	45.00 ± 3.00	30	47.83 ± 2.63	4.31 ± 0.24/82.09 ± 4.65	3.37 ± 0.18/64.19 ± 3.51	1,749.20 ± 96.18	437.30 ± 24.04	NC

^aAll figures except T_max are means ± standard deviations. 

^bMIC at 10⁵ CFU/ml. 

^cMIC at 10⁷ CFU/ml. 

^dC _max did not reach MIC. 

^eNC, not calculated. 

The blood bacterial count increased persistently in the untreated group; cefepime initially decreased it, but an increase occurred after 6 h, while it decreased persistently in the other treated groups (Fig. 1B).

There were no deaths in the uninfected group. After 24 h, the lethality was 14 of 15 in the infected untreated and cefepime-treated groups, 6 of 15 in the amikacin- and amikacin-plus-cefepime-treated groups, and 7 of 15 in the imipenem-treated group (Fig. 2).

FIG. 2.

Survival curve of mice treated with amikacin, cefepime, cefepime plus amikacin, and imipenem and previously infected with ESBL-producing K. pneumoniae. The hazard ratios (95% confidence interval) were as follows: infected, untreated group versus uninfected group, 26.47 (7.21 to 70.42), P< 0.001; cefepime versus infected untreated group, 0.65 (0.29 to 1.37), P > 0.2; amikacin versus infected untreated group, 0.17 (0.03 to 0.26), P < 0.001; amikacin plus cefepime versus infected untreated group, 0.16 (0.03 to 0.24), P < 0.001; imipenem versus infected untreated group, 0.19 (0.04 to 0.33), P < 0.001; amikacin versus cefepime, 0.18 (0.04 to 0.34), P < 0.001; amikacin plus cefepime versus cefepime, 0.16 (0.03 to 0.24), P < 0.001; imipenem versus cefepime, 0.21 (0.05 to 0.37), P < 0.001; amikacin plus cefepime versus amikacin, 0.98 (0.31 to 3.08), P > 0.2; amikacin plus cefepime versus imipenem, 0.71 (0.24 to 2.11), P > 0.2; amikacin versus imipenem, 0.77 (0.26 to 2.30), P > 0.2. p₂₄, probability of survival at 24 h postinfection.

The in vitro susceptibilities of ESBL-producing strains to cefepime have been found to be 52 or 90% (15, 25). Cefepime was recommended for the treatment based on this in vitro susceptibility (12). Others did not recommend it, despite its in vivo effectiveness, arguing on the basis of the inoculum effect, dose dependence, and other factors (15, 27). In our study, we saw an inoculum effect for cefepime, and it was ineffective in vitro and in vivo with a high inoculum.

Carbapenems have been reported to be stable against ESBL enzymes and are considered to be the treatment of choice (14, 24). In our study, imipenem showed a slight inoculum dependence, but the organisms remained susceptible. It persistently decreased the in vitro and in vivo bacterial counts, and it was biologically effective in the survival analysis.

Amikacin has good in vitro activity against ESBL-producing K. pneumoniae strains (15, 22). In our study, it did not show an inoculum effect, which can be an advantage compared to β-lactams. Synergy between amikacin and cefepime was not detected in vitro, and the combination was not more effective in vivo than amikacin alone.

Overall, the observed biological differences could not be explained by the pharmacokinetics because they were appropriate for all antibiotics, taking into account the susceptibility at the standard inoculum (7, 8, 11, 13, 23, 29). The in vitro susceptibility test and the percentage of T greater than the MIC calculated with the standard inoculum were not predictive for the biological effectiveness of cefepime due to a possible in vivo inoculum effect.

Based on our results, imipenem or amikacin can be the therapy of choice against ESBL-producing K. pneumoniae strains showing susceptibility at the standard inoculum, while cefepime cannot be recommended even though the organisms show susceptibility.