Evaluation of the post-antibiotic effect in vivo for the combination of a ß-lactam antibiotic and a ß-lactamase inhibitor: ceftazidime-avibactam in neutropenic mouse thigh and lung infections.

The post-antibiotic effect (PAE) of ceftazidime-avibactam in vivo was evaluated using models of thigh- and lung-infection with Pseudomonas aeruginosa in neutropenic mice. In thigh-infected mice, the PAE was negative (-2.18 to -0.11 h) for three of four strains: caused by a 'burst' of rapid bacterial growth after the drug concentrations had fallen below their pre-specified target values. With lung infection, PAE was positive, and longer for target drug concentrations in ELF (>2 h) than plasma (1.69-1.88 h). The time to the start of regrowth was quantified as a new parameter, PAER, which was positive (0.35-1.00 h) in both thigh- and lung-infected mice. In the context that measurements of the PAE of ß-lactam/ß-lactamase inhibitor combinations in vivo have not previously been reported, it is noted that the negative values were consistent with previous measurements of the PAE of ceftazidime-avibactam in vitro and of ceftazidime alone in vivo.

Pharmacodynamics of fosfomycin against ESBL- and/or carbapenemase-producing Enterobacteriaceae.

BACKGROUND: The increase in antibiotic resistance in Gram-negative bacteria and the limited therapeutic options due to the shortage of new antibiotics have increased the interest of the 'old' antibiotic fosfomycin in the treatment of infections. However, there are contradictory reports on the pharmacodynamics of and emergence of resistance to fosfomycin. METHODS: Time-kill assays were performed with 11 ESBL-positive and 3 ESBL-negative strains, exposing the bacteria to 2-fold static concentrations from 0.125× to 32× MIC. The sigmoid maximum effect (Emax) model was fitted to the time-kill curve data. Amplification of resistance over time was evaluated under various conditions of selective pressure by plating on 16× MIC plates. RESULTS: Fosfomycin was bactericidal for all strains within 8 h. Using the Emax model, no significant differences between strains were observed for the pharmacodynamic parameters. However, the large variation in Hill slope factors for Escherichia coli of 0.87 up to 4.02 indicates that the killing behaviour appears to be more time dependent for some strains but concentration dependent for others. In the fosfomycin-exposed cultures under low and high selective pressure (≥2× MIC) the median resistance proportions between the resistant and total population increased from ≤2 × 10-6 (T = 0 h) to 0.652-0.899 (T = 24 h). Resistance appeared stable after repeated subculturing. CONCLUSIONS: Killing behaviour of fosfomycin does not only differ between species but also within species and may have an impact on the design of optimal dosing regimens. Although fosfomycin was bactericidal against all strains (re)growth of resistant subpopulations occurred relatively fast. This may limit the use of fosfomycin as a single drug therapy.

Pharmacodynamics of nitrofurantoin at different pH levels against pathogens involved in urinary tract infections.

BACKGROUND: Urinary tract infections are among the most common human infections. Due to the progressive increase in ESBL-producing bacteria and the unavailability of new antibiotics, re-evaluation of 'old' antibiotics is needed. However, the pharmacodynamics of nitrofurantoin under variable pH conditions are poorly understood. We determined the pharmacodynamic properties of nitrofurantoin at different pH levels using time-kill assays. METHODS: Time-kill assays were performed at four pH levels (5.5, 6.5, 7.5 and 8.5), exposing the bacteria to 2-fold increasing concentrations from 0.125 to 32 times the MIC. Seven ESBL-positive and two ESBL-negative strains (MICs 8-32 mg/L) were used. The Δlog10 cfu/mL values at 6 and 24 h were plotted against each log10-transformed concentration and analysed with non-linear regression analysis using the sigmoid maximum effect model with variable slope. Geometric means normalized by the MIC of the EC50, stasis and 1 and 3 log10 cfu/mL kill were calculated. RESULTS: Minimum bactericidal effects differed significantly by species and pH level. At pH 5.5-6.5 bactericidal effects were observed at ≥ 0.5 × MIC for Escherichia coli and Enterobacter cloacae. At pH 8.5 only the two highest concentrations were considered bactericidal. Strong pH-dependent pharmacodynamic output parameters were observed in 6 h and especially 24 h modelling. At 24 h, pH 5.5-6.5 for E. coli and Klebsiella pneumoniae required significantly lower nitrofurantoin concentrations compared with pH 7.5 or 8.5. Although for E. cloacae similar strong decreasing trends were visible with decreasing pH, none of the tested pharmacodynamic parameters was significant. CONCLUSIONS: Nitrofurantoin bactericidal activity against Enterobacteriaceae significantly increases at lower pH levels. Bactericidal activity of nitrofurantoin may be overestimated or underestimated, which may have implications for therapy and the interpretation of clinical breakpoints.

Pharmacodynamics of Cefepime Combined with Tazobactam against Clinically Relevant Enterobacteriaceae in a Neutropenic Mouse Thigh Model.

The lack of new antibiotics has prompted investigation of the combination of two existing agents-cefepime, a broad-spectrum cephalosporin, and tazobactam-to broaden their efficacy against extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae We determined the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the combination in a murine neutropenic thigh model in order to establish its exposure-response relationships (ERRs). The PK of cefepime were determined for five doses; that of tazobactam was determined in earlier studies (Melchers et al., Antimicrob Agents Chemother 59:3373-3376, 2015, https://doi.org/10.1128/AAC.04402-14). The PK were linear for both compounds. The estimated mean (standard deviation [SD]) half-life of cefepime was 0.33 (0.12) h, and that of tazobactam was 0.176 (0.026) h; the volumes of distribution (V) were 0.73 liters/kg and 1.14 liters/kg, respectively. PD studies of cefepime administered every 2 h (q2h) with or without tazobactam, including dose fractionation studies of tazobactam, were performed against six ESBL-producing isolates. A sigmoidal maximum-effect (Emax) model was fitted to the data. In the dose fractionation study, the q2h regimen was more efficacious than the q4h and q6h regimens, indicating time-dependent activity of tazobactam. The threshold concentration (CT ) best correlating with tazobactam efficacy was 0.25 mg/liter, as evidenced by the best fit of the percentage of time above the threshold concentration (%fT>CT ) and response. A mean %fT>CT of 24.6% (range, 11.4 to 36.3%) for a CT of 0.25 mg/liter was required to obtain a bacteriostatic effect. We conclude that tazobactam enhanced the effect of cefepime in otherwise resistant isolates of Enterobacteriaceae and that the %fT>CT of 0.25 mg/liter best correlated with efficacy. These studies provide the basis for the development of human dosing regimens for this combination.

Pharmacodynamics and differential activity of nitrofurantoin against ESBL-positive pathogens involved in urinary tract infections.

BACKGROUND: Although nitrofurantoin has been used for >60 years for the treatment of uncomplicated urinary tract infections, its pharmacodynamic properties are not fully explored. Use is increasing because of increasing resistance to other antimicrobials due to ESBLs. METHODS: We tested nine ESBL+ and two ESBL- strains in time-kill assays. Bactericidal activity and regrowth were assessed for all species and concentrations. Early-phase pharmacodynamics was analysed with a sigmoidal Emax model and the maximal killing rate, slope and EC50/MIC ratio were determined for each species. RESULTS: A bactericidal effect was found at ≥2× MIC for Enterobacter cloacae after 4-8 h, for Klebsiella pneumoniae after 8-10 h and for Escherichia coli after 12-16 h. Overall, no killing was observed at low sub-MIC concentrations, whereas regrowth was found at 0.5-1× MIC after a short decline in cfu. The lowest maximal killing rates were observed for E. coli (0.21 ±â€Š0.05 h(-1)), followed by K. pneumoniae (0.37 ±â€Š0.09 h(-1)) and E. cloacae (0.87 ±â€Š0.01 h(-1)). Surprisingly, the Hill slopes for these three species were significantly different (10.45 ±â€Š9.37, 2.68 ±â€Š0.64 and 1.01 ±â€Š0.06, respectively), indicating a strong concentration-dependent early-phase antibacterial activity against E. cloacae. EC50/MIC ratios were significantly lower for E. coli (0.24 ±â€Š0.08 mg/L) and K. pneumoniae (0.27 ±â€Š0.03 mg/L) as compared with E. cloacae (0.77 ±â€Š0.18 mg/L). CONCLUSIONS: Nitrofurantoin was bactericidal against all species, demonstrating an unusual differential pattern of activity with concentration-dependent-type killing behaviour against E. cloacae and time-dependent killing behaviour against E. coli, which may have significant consequences on species-dependent dosing regimens. The results also demonstrate that the pharmacodynamic properties of some drugs cannot be generalized within a family, here the Enterobacteriaceae.

Pharmacodynamics of Ceftazidime and Avibactam in Neutropenic Mice with Thigh or Lung Infection.

Avibactam is a new non-ß-lactam ß-lactamase inhibitor that shows promising restoration of ceftazidime activity against microorganisms producing Ambler class A extended-spectrum ß-lactamases (ESBLs) and carbapenemases such as KPCs, class C ß-lactamases (AmpC), and some class D enzymes. To determine optimal dosing combinations of ceftazidime-avibactam for treating infections with ceftazidime-resistant Pseudomonas aeruginosa, pharmacodynamic responses were explored in murine neutropenic thigh and lung infection models. Exposure-response relationships for ceftazidime monotherapy were determined first. Subsequently, the efficacy of adding avibactam every 2 h (q2h) or q8h to a fixed q2h dose of ceftazidime was determined in lung infection for two strains. Dosing avibactam q2h was significantly more efficacious, reducing the avibactam daily dose for static effect by factors of 2.7 and 10.1, whereas the mean percentage of the dosing interval that free drug concentrations remain above the threshold concentration of 1 mg/liter (%fT>C(T) 1 mg/liter) yielding bacteriostasis was similar for both regimens, with mean values of 21.6 (q2h) and 18.5 (q8h). Dose fractionation studies of avibactam in both the thigh and lung models indicated that the effect of avibactam correlated well with %fT>C(T) 1 mg/liter. This parameter of avibactam was further explored for four P. aeruginosa strains in the lung model and six in the thigh model. Parameter estimates of %fT>C(T) 1 mg/liter for avibactam ranged from 0 to 21.4% in the lung model and from 14.1 to 62.5% in the thigh model to achieve stasis. In conclusion, addition of avibactam enhanced the effect of ceftazidime, which was more pronounced at frequent dosing and well related with %fT>C(T) 1 mg/liter. The thigh model appeared more stringent, with higher values, ranging up to 62.5% fT>C(T) 1 mg/liter, required for a static effect.

In Vitro Activity of Ceftolozane Alone and in Combination with Tazobactam against Extended-Spectrum-ß-Lactamase-Harboring Enterobacteriaceae.

Ceftolozane, formally CXA-101, is a new antipseudomonal cephalosporin that is also active in vitro against Enterobacteriaceae but is vulnerable to extended-spectrum ß-lactamases (ESBLs). The addition of tazobactam is intended to broaden coverage to most ESBL-producing Escherichia coli and Klebsiella pneumonia as well as other Enterobacteriaceae. The in vitro activities of ceftolozane-tazobactam combinations against 67 clinically and molecularly characterized ESBL-producing isolates were examined by checkerboard MIC testing to evaluate their potential clinical feasibility and to assess the optimal tazobactam concentrations to be used in MIC determinations of ceftolozane. Isolates included those from E. coli (n = 32), K. pneumoniae (n = 19), Enterobacter cloacae (n = 15), and Citrobacter freundii (n = 1). Checkerboard experiments were performed to study interactions over the range of 0.008 to 64 mg/liter ceftolozane and 0.063 to 32 mg/liter tazobactam using 2-fold-dilution series. The MIC50 and MIC90 of ceftolozane alone for all isolates were 16 and ≥64 mg/liter, respectively. Increasing concentrations of tazobactam resulted in decreasing MICs of ceftolozane. The 50th and 90th percentile concentrations of tazobactam required to reduce the MIC of ceftolozane to 8 mg/liter for all organisms in this ESBL collection were 0.5 and 4 mg/liter, respectively. For E. coli, K. pneumoniae, and E. cloacae, these values were 0.5 and 2, 1 and 16, and 0.5 and 4 mg/liter, respectively. When combined with a fixed amount of 4 mg/liter tazobactam (current CLSI concentration used for susceptibility testing), 90% of the isolates would have an MIC of ≤4 mg/liter. The combination ceftolozane-tazobactam is a promising alternative option for treating infections due to ESBL-harboring isolates.

Pharmacokinetics and penetration of ceftazidime and avibactam into epithelial lining fluid in thigh- and lung-infected mice.

Ceftazidime and the ß-lactamase inhibitor avibactam constitute a new, potentially highly active combination in the battle against extended-spectrum-ß-lactamase (ESBL)-producing bacteria. To determine possible clinical use, it is important to know the pharmacokinetic profiles of the compounds related to each other in plasma and the different compartments of infection in experimentally infected animals and in humans. We used a neutropenic murine thigh infection model and lung infection model to study pharmacokinetics in plasma and epithelial lining fluid (ELF). Mice were infected with ca. 10(6) CFU of Pseudomonas aeruginosa intramuscularly into the thigh or intranasally to cause pneumonia and were given 8 different (single) subcutaneous doses of ceftazidime and avibactam in various combined concentrations, ranging from 1 to 128 mg/kg of body weight in 2-fold increases. Concomitant samples of serum and bronchoalveolar lavage fluid were taken at up to 12 time points until 6 h after administration. Pharmacokinetics of both compounds were linear and dose proportional in plasma and ELF and were independent of the infection type, with estimated half-lives (standard deviations [SD]) in plasma of ceftazidime of 0.28 (0.02) h and of avibactam of 0.24 (0.04) h and volumes of distribution of 0.80 (0.14) and 1.18 (0.34) liters/kg. The ELF-plasma (area under the concentration-time curve [AUC]) ratios (standard errors [SE]) were 0.24 (0.03) for total ceftazidime and 0.27 (0.03) for unbound ceftazidime; for avibactam, the ratios were 0.20 (0.02) and 0.22 (0.02), respectively. No pharmacokinetic interaction between ceftazidime and avibactam was observed. Ceftazidime and avibactam showed linear plasma pharmacokinetics that were independent of the dose combinations used or the infection site in mice. Assuming pharmacokinetic similarity in humans, this indicates that similar dose ratios of ceftazidime and avibactam could be used for different types and sites of infection.

In vitro activity of ceftazidime-avibactam combination in in vitro checkerboard assays.

To evaluate the in vitro effects of the combination of ceftazidime and avibactam on the MICs of both compounds, checkerboard assays were performed for 81 clinical strains, including 55 Enterobacteriaceae strains (32 Klebsiella pneumoniae, 19 Escherichia coli, 1 Citrobacter freundii, and 3 Enterobacter cloacae) and 26 strains of Pseudomonas aeruginosa, all with known resistance mechanisms such as extended-spectrum ß-lactamases (ESBLs) and carbapenemases, phenotypically or molecularly determined. Phenotypically ceftazidime-resistant strains (n=69) were analyzed in more detail. For the Enterobacteriaceae strains, a concentration-dependent effect of avibactam was found for most strains with a maximum effect of avibactam at a concentration of 4 mg/liter, which decreased all ceftazidime MICs to ≤4 mg/liter. Avibactam alone also showed antibacterial activity (the MIC50 and MIC90 being 8 and 16 mg/liter, respectively). For the ceftazidime-resistant P. aeruginosa strains, considerable inhibition of ß-lactamases by avibactam was acquired at a concentration of 4 mg/liter, which decreased all ceftazidime MICs except one to ≤8 mg/liter (the CLSI and EUCAST susceptible breakpoint). Increasing the concentration of avibactam further decreased the MICs, resulting in a maximum effect for most strains at 8 to 16 mg/liter. In summary, for most strains, the tested addition of avibactam of 4 mg/liter restored the antibacterial activity of ceftazidime to a level comparable to that of wild-type strains, indicating full inhibition, and strains became susceptible according to the EUCAST and CLSI criteria. Based on these in vitro data, avibactam is a promising inhibitor of different ß-lactamases, including ESBLs and carbapenemases.