Binding of temocillin to plasma proteins in vitro and in vivo: the importance of plasma protein levels in different populations and of co-medications.

BACKGROUND: Temocillin plasma protein binding (PPB) in healthy individuals is reported to be ∼85% but had not been studied in patients. OBJECTIVES: To obtain normative data on temocillin PPB in patients in relation to infection and impact of co-medications widely used in ICU. METHODS: Plasma was obtained from healthy individuals (Group #1), non-ICU patients with UTI (Group #2), ICU patients with suspected/confirmed ventriculitis (Group #3) or with sepsis/septic shock (Group #4). Total and unbound temocillin concentrations were measured in spiked samples from temocillin-naive donors (in vitro) or in plasma from temocillin-treated subjects (in vivo). The impact of diluting plasma, using pharmaceutical albumin, or adding drugs potentially competing for PPB was tested in spiked samples. Data were analysed using a modified Hill-Langmuir equation taking ligand depletion into account. RESULTS: Temocillin PPB was saturable in all groups, both in vitro and in vivo. Maximal binding capacity (Bmax) was 1.2-2-fold lower in patients. At 20 and 200 mg/L (total concentrations), the unbound fraction reached 12%-29%, 23%-42% and 32%-52% in Groups #2, #3, #4. The unbound fraction was inversely correlated with albumin and C-reactive protein concentrations. Binding to albumin was 2-3-fold lower than in plasma and non-saturable. Drugs with high PPB but active at lower molar concentrations than temocillin caused minimal displacement, while fluconazole (low PPB but similar plasma concentrations to temocillin) increased up to 2-fold its unbound fraction. CONCLUSIONS: Temocillin PPB is saturable, 2-4-fold lowered in infected patients in relation to disease severity (ICU admission, hypoalbuminaemia, inflammation) and only partially reproducible with albumin. Competition with other drugs must be considered for therapeutic concentrations to be meaningful.

Large-scale WGS of carbapenem-resistant Acinetobacter baumannii isolates reveals patterns of dissemination of ST clades associated with antibiotic resistance.

OBJECTIVES: To describe the population genetics and antibiotic resistance gene distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates causing infections in three Mediterranean countries. METHODS: Isolates were collected during the 2013-17 AIDA clinical trial in six hospitals in Israel, Greece and Italy. WGS, bioinformatic characterization and antibiotic resistance profiling were performed. RESULTS: In the 247 CRAB isolates characterized in this study, ST distribution varied by country: 29/31 (93.5%) Greek isolates, 34/41 (82.9%) Italian isolates and 70/175 (40.0%) Israeli isolates belonged to ST2. The identified ST2 isolates included eight distinct clades: 2C, 2D and 2H were significantly more common in Italy, while 2F was unique to Greece. The uncommon ST3 was not present among Greek isolates and constituted only 5/41 (12%) Italian isolates. On the other hand, it was much more common among Israeli isolates: 78/175 (44.6%) belonged to ST3. The vast majority of isolates, 240/247 (97.2%), were found to harbour acquired carbapenemases, primarily blaOXA-23. The chromosomal oxaAb (blaOXA-51-like) and ampC genes characteristic of this organism were also ubiquitous. Most (96.4%) ST3 isolates carried a broad-host-range plasmid IncP1α. CONCLUSIONS: The geographical differences in CRAB populations support the theory that clonal spread of CRAB leads to endemicity in hospitals and regions. The close association between antibiotic resistance genes and clades, and between plasmids and STs, suggest that de novo creation of MDR A. baumannii is rare. The clustering of antibiotic resistance genes and plasmids that is unique to each clade/ST, and nearly uniform within clades/STs, suggests that horizontal transmission is rare but crucial to the clade's/ST's success.

Activity of Cefepime in Combination with the Novel ß-Lactamase Inhibitor Taniborbactam (VNRX-5133) against Extended-Spectrum-ß-Lactamase-Producing Isolates in In Vitro Checkerboard Assays.

Extended-spectrum-ß-lactamase (ESBL)-producing strains are increasing worldwide, limiting therapeutic options. Taniborbactam (VNRX-5133) is a newly developed ß-lactamase inhibitor with a wide spectrum of activity covering both serine and metallo enzymes. We therefore evaluated cefepime-taniborbactam activity against ESBL-producing isolates and determined the concentrations to be used in MIC determinations in the clinical laboratory. The in vitro activity of cefepime (0.06 to 256 mg liter-1) combined with taniborbactam (0.03 to 32 mg liter-1) against 129 clinically and molecularly well-documented ESBL-producing isolates (42 Escherichia coli, 39 Klebsiella pneumoniae, 28 Pseudomonas aeruginosa, 16 Enterobacter cloacae, 2 Citrobacter freundii, and 2 Enterobacter aerogenes) was tested with a broth microdilution checkerboard method based on the ISO standard. The MICs of cefepime alone and in combination, together with percentage resistance at different concentrations of taniborbactam, were calculated for each species and resistance mechanism. The median (range)/MIC90 of cefepime was 32 (0.125 to 256)/256 mg liter-1 for all Enterobacterales isolates (n = 101), with 72% being resistant, and 32 (8 to 256)/128 mg liter-1 for the 28 P. aeruginosa isolates, with 86% being resistant. The median (range)/90th percentile concentration of taniborbactam required to restore Enterobacterales susceptibility to cefepime (MIC ≤1 mg liter-1) was 0.06 (≤0.03 to 32)/4 mg liter-1 and P. aeruginosa susceptibility to increased exposure to cefepime (MIC ≤8 mg liter-1) 1 (≤0.032 to 32)/32 mg liter-1 At a fixed concentration of 4 mg liter-1 of taniborbactam, cefepime median (range)/MIC90 were reduced to 0.125 (0.06 to 4)/1 mg liter-1 for Enterobacterales with no resistant isolates found, and to 8 (2 to 64)/16 mg liter-1 for P. aeruginosa isolates, where 36% remained resistant. The combination cefepime-taniborbactam demonstrated a potent activity against ESBL isolates, restoring susceptibility of all Enterobacterales and two-thirds of P. aeruginosa isolates.

The Role of New Posaconazole Formulations in the Treatment of Candida albicans Infections: Data from an In Vitro Pharmacokinetic-Pharmacodynamic Model.

Posaconazole is more active than fluconazole against Candida albicansin vitro and is approved for the treatment of oropharyngeal candidiasis but not for that of invasive candidiasis (IC). Here, we explored the efficacy of posaconazole against C. albicans in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model of IC and determined the probability of pharmacodynamic target attainment for the oral solution and intravenous (i.v.)/tablet formulations. Three clinical C. albicans isolates (posaconazole MICs, 0.008 to 0.25 mg/liter) were studied in the in vitro PK/PD dilution model simulating steady-state posaconazole PK. The in vitro exposure-effect relationship, area under the 24-h free drug concentration curve (fAUC0-24)/MIC, was described and compared with in vivo outcome in animals with IC. PK/PD susceptibility breakpoints and trough levels required for optimal treatment were determined for EUCAST and CLSI 24-h/48-h (CLSI24h/CLSI48h) methods using the fAUC0-24/MIC associated with half-maximal activity (EI50) and Monte Carlo simulation analysis for oral solution (400 mg every 12 hours [q12h]) and i.v./tablet formulations (300 mg q24h). The in vitro mean (95% confidence interval [CI]) EI50 was 330 (183 to 597) fAUC0-24/MIC for CLSI24h and 169 (92 to 310) for EUCAST/CLSI48h methods, which are close to the near-stasis in vivo effect. The probability of target attainment for EI50 was estimated; for the wild-type isolates (MIC ≤ 0.06 mg/liter), it was low for the oral solution and higher than 95% for the i.v./tablet formulations for the EUCAST/CLSI48h methods but not for the CLSI 24-h method. Non-wild-type isolates with EUCAST/CLSI48h MICs of 0.125 and 0.25 mg/liter would require trough levels of >1.2 and >2.4 mg/liter, respectively. Posaconazole i.v./tablet formulations may have a role in the therapy of invasive infections by wild-type C. albicans isolates, provided that a steady state is reached quickly. A PK/PD susceptibility breakpoint at the epidemiological cutoff (ECV/ECOFF) of 0.06 mg/liter was determined.

Pharmacokinetic/pharmacodynamic analysis of oral fosfomycin against Enterobacterales, Pseudomonas aeruginosa and Enterococcus spp. in an in vitro bladder infection model: impact on clinical breakpoints.

OBJECTIVES: Fosfomycin is an established treatment for uncomplicated urinary tract infections (UTIs), yet evidence supporting susceptibility breakpoints is limited. We examine the UTI susceptibility criteria. METHODS: Fosfomycin susceptibility, heteroresistance and in vitro growth in a bladder infection model, after a single 3 g dose of oral fosfomycin, were bridged to human pharmacokinetics with pharmacokinetic/pharmacodynamic and Monte Carlo analyses. Data from common uropathogens (24 Escherichia coli, 20 Klebsiella pneumoniae, 4 Enterobacter cloacae, 14 Pseudomonas aeruginosa, 8 Enterococcus faecalis and 8 Enterococcus faecium) were compared and analysed to ascertain species-specific PTA. RESULTS: Glucose-6-phosphate (G6P) increased MICs of E. coli, K. pneumoniae and E. cloacae (median 2-fold dilutions 3-5), but not of P. aeruginosa and Enterococcus. Atypical E. coli lacking G6P potentiation were killed in the bladder infection model despite high MICs (32-128 mg/L). Fosfomycin heteroresistance was uncommon in E. coli (MIC > 2 mg/L) but was detected in the majority of K. pneumoniae (MIC > 1 mg/L) and P. aeruginosa (MIC >8 mg/L). For these species, baseline heteroresistance was a strong predictor for treatment failure in the model. No heteroresistance was found in Enterococcus. The fAUC/MIC targets for stasis were 1935, 3393, 9968, 2738 and 283 for typical E. coli, K. pneumoniae, E. cloacae, P. aeruginosa and E. faecalis, respectively (synthetic human urine medium alone promoted a 1 log10 kill in E. faecium). A >95% PTA for stasis was only found at MIC ≤ epidemiological cut-off (ECOFF) for E. coli (4 mg/L). For other species, PTAs were low for WT populations. CONCLUSIONS: With the exception of E. coli, fosfomycin is a poor target for other uropathogen species. A reduction in oral fosfomycin UTI breakpoints is supported.

Excluded versus included patients in a randomized controlled trial of infections caused by carbapenem-resistant Gram-negative bacteria: relevance to external validity.

BACKGROUND: Population external validity is the extent to which an experimental study results can be generalized from a specific sample to a defined population. In order to apply the results of a study, we should be able to assess its population external validity. We performed an investigator-initiated randomized controlled trial (RCT) (AIDA study), which compared colistin-meropenem combination therapy to colistin monotherapy in the treatment of patients infected with carbapenem-resistant Gram-negative bacteria. In order to examine the study's population external validity and to substantiate the use of AIDA study results in clinical practice, we performed a concomitant observational trial. METHODS: The study was conducted between October 1st, 2013 and January 31st, 2017 (during the RCTs recruitment period) in Greece, Israel and Italy. Patients included in the observational arm of the study have fulfilled clinical and microbiological inclusion criteria but were excluded from the RCT due to receipt of colistin for > 96 h, refusal to participate, or prior inclusion in the RCT. Non-randomized cases were compared to randomized patients. The primary outcome was clinical failure at 14 days of infection onset. RESULTS: Analysis included 701 patients. Patients were infected mainly with Acinetobacter baumannii [78.2% (548/701)]. The most common reason for exclusion was refusal to participate [62% (183/295)]. Non-randomized and randomized patients were similar in most of the demographic and background parameters, though randomized patients showed minor differences towards a more severe infection. Combination therapy was less common in non-randomized patients [31.9% (53/166) vs. 51.2% (208/406), p = 0.000]. Randomized patients received longer treatment of colistin [13 days (IQR 10-16) vs. 8.5 days (IQR 0-15), p = 0.000]. Univariate analysis showed that non-randomized patients were more inclined to clinical failure on day 14 from infection onset [82% (242/295) vs. 75.5% (307/406), p = 0.042]. After adjusting for other variables, non-inclusion was not an independent risk factor for clinical failure at day 14. CONCLUSION: The similarity between the observational arm and RCT patients has strengthened our confidence in the population external validity of the AIDA trial. Adding an observational arm to intervention studies can help increase the population external validity and improve implementation of study results in clinical practice. TRIAL REGISTRATION: The trial was registered with ClinicalTrials.gov, number NCT01732250 on November 22, 2012.

Colistin Resistance Development Following Colistin-Meropenem Combination Therapy Versus Colistin Monotherapy in Patients With Infections Caused by Carbapenem-Resistant Organisms.

BACKGROUND: We evaluated whether carbapenem-colistin combination therapy reduces the emergence of colistin resistance, compared to colistin monotherapy, when given to patients with infections due to carbapenem-resistant Gram-negative organisms. METHODS: This is a pre-planned analysis of a secondary outcome from a randomized, controlled trial comparing colistin monotherapy with colistin-meropenem combination for the treatment of severe infections caused by carbapenem-resistant, colistin-susceptible Gram-negative bacteria. We evaluated rectal swabs taken on Day 7 or later for the presence of new colistin-resistant (ColR) isolates. We evaluated the emergence of any ColR isolate and the emergence of ColR Enterobacteriaceae (ColR-E). RESULTS: Data were available for 214 patients for the primary analysis; emergent ColR organisms were detected in 22 (10.3%). No difference was observed between patients randomized to treatment with colistin monotherapy (10/106, 9.4%) versus patients randomized to colistin-meropenem combination therapy (12/108, 11.1%; P = .669). ColR-E organisms were detected in 18/249 (7.2%) patients available for analysis. No difference was observed between the 2 treatment arms (colistin monotherapy 6/128 [4.7%] vs combination therapy 12/121 [9.9%]; P = .111). Enterobacteriaceae, as the index isolate, was found to be associated with development of ColR-E (hazard ratio, 3.875; 95% confidence interval, 1.475-10.184; P = .006). CONCLUSIONS: Carbapenem-colistin combination therapy did not reduce the incidence of colistin resistance emergence in patients with infections due to carbapenem-resistant organisms. Further studies are necessary to elucidate the development of colistin resistance and methods for its prevention.

Toward Harmonization of Voriconazole CLSI and EUCAST Breakpoints for Candida albicans Using a Validated In Vitro Pharmacokinetic/Pharmacodynamic Model.

CLSI and EUCAST susceptibility breakpoints for voriconazole and Candida albicans differ by one dilution (≤0.125 and ≤0.06 mg/liter, respectively) whereas the epidemiological cutoff values for EUCAST (ECOFF) and CLSI (ECV) are the same (0.03 mg/liter). We therefore determined the pharmacokinetic/pharmacodynamic (PK/PD) breakpoints of voriconazole against C. albicans for both methodologies with an in vitro PK/PD model, which was validated using existing animal PK/PD data. Four clinical wild-type and non-wild-type C. albicans isolates (voriconazole MICs, 0.008 to 0.125 mg/liter) were tested in an in vitro PK/PD model. For validation purposes, mouse PK were simulated and in vitro PD were compared with in vivo outcomes. Human PK were simulated, and the exposure-effect relationship area under the concentration-time curve for the free, unbound fraction of a drug from 0 to 24 h (fAUC0-24)/MIC was described for EUCAST and CLSI 24/48-h methods. PK/PD breakpoints were determined using the fAUC0-24/MIC associated with half-maximal activity (EI50) and Monte Carlo simulation analysis. The in vitro 24-h PD EI50 values of voriconazole against C. albicans were 2.5 to 5 (1.5 to 17) fAUC/MIC. However, the 72-h PD were higher at 133 (51 to 347) fAUC/MIC for EUCAST and 94 (35 to 252) fAUC/MIC for CLSI. The mean (95% confidence interval) probability of target attainment (PTA) was 100% (95 to 100%), 97% (72 to 100%), 83% (35 to 99%), and 49% (8 to 91%) for EUCAST and 100% (97 to 100%), 99% (85 to 100%), 91% (52 to 100%), and 68% (17 to 96%) for CLSI for MICs of 0.03, 0.06, 0.125, and 0.25 mg/liter, respectively. Significantly, >95% PTA values were found for EUCAST/CLSI MICs of ≤0.03 mg/liter. For MICs of 0.06 to 0.125 mg/liter, trough levels 1 to 4 mg/liter would be required to attain the PK/PD target. A PK/PD breakpoint of C. albicans voriconazole at the ECOFF/ECV of 0.03 mg/liter was determined for both the EUCAST and CLSI methods, indicating the need for breakpoint harmonization for the reference methodologies.

Oral Fosfomycin Efficacy with Variable Urinary Exposures following Single and Multiple Doses against Enterobacterales: the Importance of Heteroresistance for Growth Outcome.

Oral fosfomycin trometamol is licensed as a single oral dose for the treatment of uncomplicated urinary tract infections, with activity against multidrug-resistant uropathogens. The impact of interindividual variability in urinary concentrations on antimicrobial efficacy, and any benefit of giving multiple doses, is uncertain. We therefore performed pharmacodynamic profiling of oral fosfomycin, using a dynamic bladder infection in vitro model, to assess high and low urinary exposures following a single oral dose and three repeat doses given every 72 h, 48 h, and 24 h against 16 clinical isolates with various MICs of fosfomycin (8 Escherichia coli, 4 Enterobacter cloacae, and 4 Klebsiella pneumoniae isolates). Baseline fosfomycin high-level-resistant (HLR) subpopulations were detected prior to drug exposure in half of the isolates (2 E. coli, 2 E. cloacae, and 4 K. pneumoniae isolates; proportion, 1 × 10-5 to 5 × 10-4% of the total population). Fosfomycin exposures were accurately reproduced compared to mathematical modeling (linear regression slope, 1.1; R2, 0.99), with a bias of 3.8% ± 5.7%. All 5/5 isolates with MICs of ≤1 µg/ml had no HLR and were killed, whereas 8/11 isolates with higher MICs regrew regardless of exposure to high or low urinary concentrations. A disk diffusion zone of <24 mm was a better predictor for baseline HLR and regrowth. Administering 3 doses with average exposures provided very limited additional kill. These results suggest that baseline heteroresistance is important for treatment response, while increased drug exposure and administering multiple doses may not be better than standard single-dose fosfomycin therapy.

Oral Fosfomycin Treatment for Enterococcal Urinary Tract Infections in a Dynamic In Vitro Model.

There are limited treatment options for enterococcal urinary tract infections, especially vancomycin-resistant Enterococcus (VRE). Oral fosfomycin is a potential option, although limited data are available guiding dosing and susceptibility. We undertook pharmacodynamic profiling of fosfomycin against E. faecalis and E. faecium isolates using a dynamic in vitro bladder infection model. Eighty-four isolates underwent fosfomycin agar dilution susceptibility testing (E. faecalis MIC50/90 32/64 µg/ml; E. faecium MIC50/90 64/128 µg/ml). Sixteen isolates (including E. faecalis ATCC 29212 and E. faecium ATCC 35667) were chosen to reflect the MIC range and tested in the bladder infection model with synthetic human urine (SHU). Under drug-free conditions, E. faecium demonstrated greater growth restriction in SHU compared to E. faecalis (E. faecium maximal growth 5.8 ± 0.6 log10 CFU/ml; E. faecalis 8.0 ± 1.0 log10 CFU/ml). Isolates were exposed to high and low fosfomycin urinary concentrations after a single dose, and after two doses given over two days with low urinary concentration exposure. Simulated concentrations closely matched the target (bias 2.3%). E. faecalis isolates required greater fosfomycin exposure for 3 log10 kill from the starting inoculum compared with E. faecium The ƒAUC0-72/MIC and ƒ%T > MIC0-72 for E. faecalis were 672 and 70%, compared to 216 and 51% for E. faecium, respectively. There was no rise in fosfomycin MIC postexposure. Two doses of fosfomycin with low urinary concentrations resulted in equivalent growth inhibition to a single dose with high urinary concentrations. With this urinary exposure, fosfomycin was effective in promoting suppression of regrowth (>3 log10 kill) in the majority of isolates.

Trends, seasonality and the association between outpatient antibiotic use and antimicrobial resistance among urinary bacteria in the Netherlands.

OBJECTIVES: To determine trends, seasonality and the association between community antibiotic use and antimicrobial resistance (AMR) in Escherichia coli and Klebsiella pneumoniae in urinary tract infections. METHODS: We analysed Dutch national databases from January 2008 to December 2016 regarding antibiotic use and AMR for nitrofurantoin, trimethoprim, fosfomycin and ciprofloxacin. Antibiotic use was expressed as DDD/1000 inhabitant-days (DID) and AMR was expressed as the percentage of resistance from total tested isolates. Temporal trends and seasonality were analysed with autoregressive integrated moving average (ARIMA) models. Each antibiotic use-resistance combination was cross-correlated with a linear regression of the ARIMA residuals. RESULTS: The trends of DID increased for ciprofloxacin, fosfomycin and nitrofurantoin, but decreased for trimethoprim. Similar trends were found in E. coli and K. pneumoniae resistance to the same antibiotics, except for K. pneumoniae resistance to ciprofloxacin, which decreased. Resistance levels peaked in winter/spring, whereas antibiotic use peaked in summer/autumn. In univariate analysis, the strongest and most significant cross-correlations were approximately 0.20, and had a time delay of 3-6 months between changes in antibiotic use and changes in resistance. In multivariate analysis, significant effects of nitrofurantoin use and ciprofloxacin use on resistance to these antibiotics were found in E. coli and K. pneumoniae, respectively. There was a significant association of nitrofurantoin use with trimethoprim resistance in K. pneumoniae after adjusting for trimethoprim use. CONCLUSIONS: We found a relatively low use of antibiotics and resistance levels over a 9 year period. Although the correlations were weak, variations in antibiotic use for these four antibiotics were associated with subsequent variations in AMR in urinary pathogens.

Efficacy of single and multiple oral doses of fosfomycin against Pseudomonas aeruginosa urinary tract infections in a dynamic in vitro bladder infection model.

OBJECTIVES: We used a dynamic bladder infection in vitro model with synthetic human urine (SHU) to examine fosfomycin exposures to effectively kill, or prevent emergence of resistance, among Pseudomonas aeruginosa isolates. METHODS: Dynamic urinary fosfomycin concentrations after 3 g oral fosfomycin were simulated, comparing single and multiple (daily for 7 days) doses. Pharmacodynamic response of 16 P. aeruginosa (MIC range 1 to >1024 mg/L) were examined. Baseline disc diffusion susceptibility, broth microdilution MIC and detection of heteroresistance were assessed. Pathogen kill and emergence of resistance over 72 h following a single dose, and over 216 h following daily dosing for 7 days, were investigated. The fAUC0-24/MIC associated with stasis and 1, 2 and 3 log10 kill were determined. RESULTS: Pre-exposure high-level resistant (HLR) subpopulations were detected in 11/16 isolates after drug-free incubation in the bladder infection model. Five of 16 isolates had >2 log10 kill after single dose, reducing to 2/16 after seven doses. Post-exposure HLR amplification occurred in 8/16 isolates following a single dose and in 11/16 isolates after seven doses. Baseline MIC ≥8 mg/L with an HLR subpopulation predicted post-exposure emergence of resistance following the multiple doses. A PK/PD target of fAUC0-24/MIC >5000 was associated with 3 log10 kill at 72 h and 7 day-stasis. CONCLUSIONS: Simulated treatment of P. aeruginosa urinary tract infections with oral fosfomycin was ineffective, despite exposure to high urinary concentrations and repeated daily doses for 7 days. Emergence of resistance was observed in the majority of isolates and worsened following prolonged therapy. Detection of a baseline resistant subpopulation predicted treatment failure.

A multicentre study to optimize echinocandin susceptibility testing of Aspergillus species with the EUCAST methodology and a broth microdilution colorimetric method.

BACKGROUND: The determination of the minimal effective concentration (MEC) of echinocandins against Aspergillus species is subjective, time consuming and has been associated with very major errors. METHODS: The MECs/MICs of 40 WT [10 each of Aspergillus fumigatus species complex (SC), Aspergillus flavus SC, Aspergillus terreus SC and Aspergillus niger SC] and 4 non-WT A. fumigatus isolates were determined with EUCAST E.Def 9.3.1 read microscopically, macroscopically, spectrophotometrically and colorimetrically in three centres. The optimal conditions for spectrophotometric (single- versus multi-point readings) and colorimetric (XTT/menadione concentration and stability, incubation time) methods were evaluated in preliminary studies using different cut-offs for the determination of macroscopic, spectrophotometric and colorimetric MIC endpoints compared with the microscopically determined MEC. Inter-centre and inter-method essential (within one 2-fold dilution) agreement (EA) and categorical agreement (CA) were determined. RESULTS: Both macroscopic and spectrophotometric endpoint readings showed poor inter-centre EA (53%-66%) and low CA (41%-88%) in distinguishing WT from non-WT A. fumigatus SC isolates, while significant differences compared with the microscopic MECs were observed for all echinocandins (EA 6%-54%). For the colorimetric method, the optimal conditions were 400 mg/L XTT/6.25 µΜ menadione, incubation for 1-2 h until the drug-free control reached an absorbance at 450/630 nm of >0.8 and use of 50% inhibition of XTT conversion as a cut-off for all species and echinocandins. All non-WT isolates had high XTT MICs >1 mg/L, whereas the overall inter-centre EA and CA were 72%-89% and 100%, respectively. CONCLUSIONS: The XTT colorimetric assay improved the antifungal susceptibility testing of echinocandins against Aspergillus spp., reliably detecting non-WT isolates.

Voriconazole efficacy against Candida glabrata and Candida krusei: preclinical data using a validated in vitro pharmacokinetic/pharmacodynamic model.

BACKGROUND: Voriconazole exhibits in vitro activity against Candida glabrata and Candida krusei (EUCAST/CLSI epidemiological cut-off values 1/0.25 and 1/0.5 mg/L, respectively). Yet, EUCAST found insufficient evidence to set breakpoints for these species. We explored voriconazole pharmacodynamics (PD) in an in vitro dynamic model simulating human pharmacokinetics (PK). METHODS: Four C. glabrata and three C. krusei isolates (voriconazole EUCAST and CLSI MICs of 0.03-2 mg/L) were tested in the PK/PD model simulating voriconazole exposures (t½ ∼6 h q12h dosing for 3 days). PK/PD breakpoints were determined calculating the PTA for exposure indices fAUC0-24/MIC associated with half-maximal activity (EI50) using Monte Carlo simulation analysis. RESULTS: Fungal load increased from 3.60±0.35 to 8.41±0.24 log10 cfu/mL in the drug-free control, with a maximum effect of ∼1 log10 kill of C. glabrata and C. krusei isolates with MICs of 0.06 and 0.25 mg/L, respectively, at high drug exposures. The 72 h log10 cfu/mL change versus fAUC0-24/MIC relationship followed a sigmoid curve for C. glabrata (R2=0.85-0.87) and C. krusei (R2=0.56-0.76) with EI50 of 49 (32-76) and 52 (33-78) fAUC/MIC for EUCAST and 55 (31-96) and 80 (42-152) fAUC/MIC for CLSI, respectively. The PTAs for C. glabrata and C. krusei isolates with EUCAST/CLSI MICs ≤0.125/≤0.06 mg/L were >95%. Isolates with EUCAST/CLSI MICs of 0.25-1/0.125-0.5 would require trough levels 1-4 mg/L; isolates with higher MICs would not attain the corresponding PK/PD targets without reaching toxicity. CONCLUSIONS: The in vitro PK/PD breakpoints for C. glabrata and C. krusei for EUCAST (0.125 mg/L) and CLSI (0.06 mg/L) bisected the WT populations. Trough levels of >4 mg/L, which are not clinically feasible, are necessary for efficacy against WT isolates.

Methodological features of clinical pharmacokinetic-pharmacodynamic studies of antibacterials and antifungals: a systematic review.

BACKGROUND: Pharmacokinetic (PK)-pharmacodynamic (PD) indices relate measures of drug exposure to antibacterial effect. Clinical PK-PD studies aim to correlate PK-PD indices with outcomes in patients. Optimization of dosing based on pre-clinical studies means that PK-PD relationships are difficult to establish; therefore studies need to be designed and reported carefully to validate pre-clinical findings. OBJECTIVES: To describe the methodological features of clinical antibacterial and antifungal PK-PD studies that reported the relationship between PK-PD indices and clinical or microbiological responses. METHODS: Studies published between 1980 and 2015 were identified through systematic searches. Methodological features of eligible studies were extracted. RESULTS: We identified 85 publications containing 97 PK-PD analyses. Most studies were small, with fewer than 100 patients. Around a quarter were performed on patients with infections due to a single specific pathogen. In approximately one-third of studies, patients received concurrent antibiotics/antifungals and in some other studies patients received other treatments that may confound the PK-PD-outcome relationship. Most studies measured antimicrobial concentrations in blood/serum and only four measured free concentrations. Most performed some form of regression, time-to-event analysis or used the Hill/Emax equation to examine the association between PK-PD index and outcome. Target values of PK-PD indices that predict outcomes were investigated in 52% of studies. Target identification was most commonly done using recursive partitioning or logistic regression. CONCLUSIONS: Given the variability in conduct and reporting, we suggest that an agreed set of standards for the conduct and reporting of studies should be developed.

Impact of bacterial species and baseline resistance on fosfomycin efficacy in urinary tract infections.

OBJECTIVES: To assess the antibacterial effects of a single 3 g oral fosfomycin dose on Escherichia coli and Klebsiella pneumoniae clinical isolates within a dynamic bladder infection model. METHODS: An in vitro model simulating dynamic urinary fosfomycin concentrations was used. Target fosfomycin exposure (Cmax = 1984 mg/L and Tmax = 7.5 h) was validated by LC-MS/MS. Pharmacodynamic responses of 24 E. coli and 20 K. pneumoniae clinical isolates were examined (fosfomycin MIC ≤0.25-128 mg/L). Mutant prevention concentration (MPC), fosfomycin heteroresistance, fosfomycin resistance genes and fosA expression were examined. Pathogen kill and emergence of high-level resistance (HLR; MIC >1024 mg/L) were quantified. RESULTS: Following fosfomycin exposure, 20 of 24 E. coli exhibited reductions in bacterial counts below the lower limit of quantification without regrowth, despite baseline fosfomycin MICs up to 128 mg/L. Four E. coli regrew (MIC = 4-32 mg/L) with HLR population replacement. At baseline, these isolates had detectable HLR subpopulations and MPC >1024 mg/L. All E. coli isolates were fosA negative. In contrast, 17 of 20 K. pneumoniae regrew post exposure, 6 with emergence of HLR (proportion = 0.01%-100%). The three isolates without regrowth did not have a detectable HLR subpopulation after dynamic drug-free incubation. All K. pneumoniae had MPC >1024 mg/L and were fosA positive. WGS analysis and fosA expression failed to predict fosfomycin efficacy. CONCLUSIONS: E. coli and K. pneumoniae isolates demonstrate discrepant responses to a single fosfomycin dose in a dynamic bladder infection in vitro model. Treatment failure against E. coli was related to an HLR subpopulation, not identified by standard MIC testing. Activity against K. pneumoniae appeared limited, regardless of MIC testing, due to universal baseline heteroresistance.

Single-dose pharmacokinetics of temocillin in plasma and soft tissues of healthy volunteers after intravenous and subcutaneous administration: a randomized crossover microdialysis trial.

BACKGROUND: The antibiotic temocillin has recently been rediscovered as a promising therapeutic option against MDR Gram-negative bacteria. However, some aspects of the pharmacokinetic (PK) profile of the drug are still to be elucidated: subcutaneous administration of temocillin might be of interest as an alternative to the intravenous route in selected patients. Similarly, information on the penetration of temocillin into human soft tissues is lacking. OBJECTIVES: To investigate the feasibility and plasma PK of subcutaneous dosing as well as soft tissue PK of temocillin after intravenous administration to healthy volunteers. METHODS: Eight healthy volunteers received 2 g of temocillin both as intravenous and subcutaneous infusion in a randomized two-period crossover study. Concentration-time profiles of total temocillin in plasma (after both routes) and of unbound temocillin in plasma, muscle and subcutis (only after intravenous dosing) were determined up to 12 h post-dose. RESULTS: Subcutaneous dosing caused some infusion site discomfort but resulted in sustained drug concentrations over time with only slightly decreased overall exposure compared with intravenous dosing. Plasma protein binding of temocillin showed concentration-dependent behaviour and was higher than previously reported. Still, unbound drug concentrations in muscle and subcutis determined by microdialysis markedly exceeded those in plasma, suggesting good tissue penetration of temocillin. CONCLUSIONS: The subcutaneous administration of temocillin is a valid and feasible alternative to intravenous dosing. With the description of plasma protein binding and soft tissue PK of temocillin in healthy volunteers, this study provides important information that adds to the ongoing characterization of the PK profile of temocillin and might serve as input for PK/PD considerations.

Population pharmacokinetics of vancomycin in obesity: Finding the optimal dose for (morbidly) obese individuals.

AIMS: For vancomycin treatment in obese patients, there is no consensus on the optimal dose that will lead to the pharmacodynamic target (area under the curve 400-700 mg h L-1 ). This prospective study quantifies vancomycin pharmacokinetics in morbidly obese and nonobese individuals, in order to guide vancomycin dosing in the obese. METHODS: Morbidly obese individuals (n = 20) undergoing bariatric surgery and nonobese healthy volunteers (n = 8; total body weight [TBW] 60.0-234.6 kg) received a single vancomycin dose (obese: 12.5 mg kg-1 , maximum 2500 mg; nonobese: 1000 mg) with plasma concentrations measured over 48 h (11-13 samples per individual). Modelling, internal validation, external validation using previously published data and simulations (n = 10.000 individuals, TBW 60-230 kg) were performed using NONMEM. RESULTS: In a 3-compartment model, peripheral volume of distribution and clearance increased with TBW (both p < 0.001), which was confirmed in the external validation. A dose of 35 mg kg-1 day-1 (maximum 5500 mg/day) resulted in a > 90% target attainment (area under the curve > 400 mg h L-1 ) in individuals up to 200 kg, with corresponding trough concentrations of 5.7-14.6 mg L-1 (twice daily dosing). For continuous infusion, a loading dose of 1500 mg is required for steady state on day 1. CONCLUSION: In this prospective, rich sampling pharmacokinetic study, vancomycin clearance was well predicted using TBW. We recommend that in obese individuals without renal impairment, vancomycin should be dosed as 35 mg kg-1 day-1 (maximized at 5500 mg/day). When given over 2 daily doses, trough concentrations of 5.7-14.6 mg L-1 correspond to the target exposure in obese individuals.

Zinc-Impregnated Mesh for Abdominal Wall Repair Reduces Infection in a Rat Model of Peritonitis.

BACKGROUND: The objective of this study was to assess whether a zinc-impregnated polypropylene mesh (ZnMesh) has better antibacterial properties in a contaminated environment compared with a regular polypropylene mesh. MATERIALS AND METHODS: Thirty-eight Wistar Han rats underwent cecal ligation and puncture to induce peritonitis 24 h before implantation of an intraperitoneal ZnMesh or a regular polypropylene mesh. Primary outcome was the number of colony forming units (CFU) per sample (mesh and abdominal wall). Secondary outcomes were macroscopic (incorporation of mesh, abscesses, and adhesions on mesh surface) and histological (inflammatory cell reaction, mesh-specific parameters, and collagen deposition) parameters. All outcomes were evaluated after 30 and 90 d. RESULTS: After 30 d, no significant difference in CFU per sample was present between the ZnMesh and control groups. After 90 d, a lower number of CFU per sample was present in the ZnMesh group compared with the control group (trypticase soy agar with 5% sheep blood: 0 log10 CFU/sample IQR: 0-1.40 versus 1.58 log10 CFU/sample IQR: 0-4.30, P = 0.012; MacConkey: 0 log10 CFU/sample IQR: 0-2.65 versus 1.18 log10 CFU/sample IQR: 0-4.04, P = 0.438). After 90 d, the percentage of adhesions on mesh surface was significantly higher in the ZnMesh group (95% IQR: 60%-100% versus 50% IQR: 23%-75%, P = 0.029). No differences were seen in other macroscopic outcomes or histology. CONCLUSIONS: A significantly lower number of CFU per sample was found in the ZnMesh group after 90 d. After 30 d, no statistically significant differences in CFU per sample were seen. This result suggests that the ZnMesh group has better antibacterial properties in a contaminated environment. However, this is at the cost of a significantly higher percentage of adhesions.

Diagnostic and medical needs for therapeutic drug monitoring of antibiotics.

Therapeutic drug monitoring (TDM) of antibiotics has been practiced for more than half a century, but it is still not widely applied for infected patients. It has a traditional focus on limiting toxicity of specific classes of antibiotics such as aminoglycosides and vancomycin. With more patients in critical care with higher levels of sickness severity and immunosuppression as well as an increasingly obese and ageing population, an increasing risk of suboptimal antibiotic exposure continues to escalate. As such, the value of TDM continues to expand, especially for beta-lactams which constitute the most frequently used antibiotic class. To date, the minimum inhibitory concentration (MIC) of infectious microbes rather than classification in terms of susceptible and resistant can be reported. In parallel, increasingly sophisticated TDM technology is becoming available ensuring that TDM is feasible and can deliver personalized antibiotic dosing schemes. There is an obvious need for extensive studies that will quantify the improvements in clinical outcome of individual TDM-guided dosing. We suggest that a broad diagnostic and medical investigation of the TDM arena, including market analyses and analytical technology assessment, is a current priority.