ABSTRACT
Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae are classified as critical priority pathogens, with extended-spectrum β-lactamases (ESBLs) as principal resistance determinants. Enmetazobactam (formerly AAI101) is a novel ESBL inhibitor developed in combination with cefepime for empirical treatment of serious Gram-negative infections in settings where ESBLs are prevalent. Cefepime-enmetazobactam has been investigated in a phase 3 trial in patients with complicated urinary tract infections or acute pyelonephritis. This study examined pharmacokinetic-pharmacodynamic (PK-PD) relationships of enmetazobactam, in combination with cefepime, for ESBL-producing isolates of Klebsiella pneumoniae in 26-h murine neutropenic thigh infection models. Enmetazobactam dose fractionation identified the time above a free threshold concentration (fT > CT) as the PK-PD index predictive of efficacy. Nine ESBL-producing isolates of K. pneumoniae, resistant to cefepime and piperacillin-tazobactam, were included in enmetazobactam dose-ranging studies. The isolates encoded CTX-M-type, SHV-12, DHA-1, and OXA-48 β-lactamases and covered a cefepime-enmetazobactam MIC range from 0.06 to 2 μg/ml. Enmetazobactam restored the efficacy of cefepime against all isolates tested. Sigmoid curve fitting across the combined set of isolates identified enmetazobactam PK-PD targets for stasis and for a 1-log10 bioburden reduction of 8% and 44% fT > 2 μg/ml, respectively, with a concomitant cefepime PK-PD target of 40 to 60% fT > cefepime-enmetazobactam MIC. These findings support clinical dose selection and breakpoint setting for cefepime-enmetazobactam.
INTRODUCTION
Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae have been classified as critical priority pathogens by the WHO, with an estimated 50 million serious infections occurring annually worldwide due to Escherichia coli and Klebsiella pneumoniae (1, 2). Extended-spectrum β-lactamases (ESBLs) are the main determinants of 3GC resistance, and the occurrence of ESBLs in clinical isolates of Enterobacteriaceae increased from 10% to 24% between 1997 and 2017 (3, 4). The adoption of carbapenems as definitive therapy for infections caused by ESBL-producing Enterobacteriaceae (5) has been accompanied by an increase in infections with carbapenem-resistant Enterobacteriaceae (CRE) from 0.6 to 2.9% over the corresponding period, with an estimated 3 million cases reported annually worldwide (1).
Novel carbapenem-sparing therapies for empirical treatment of 3GC-resistant Enterobacteriaceae are needed urgently to limit the spread of carbapenem resistance (6–8). Although piperacillin-tazobactam has been proposed as a carbapenem-sparing option for infections by 3GC-resistant Enterobacteriaceae, this β-lactam/β-lactamase inhibitor combination continues to lose activity against contemporary ESBL-producing Enterobacteriaceae (9), and its role in treating bloodstream infections by such pathogens is controversial and likely diminishing (7, 10, 11).
Enmetazobactam, formerly AAI101, is a novel extended-spectrum β-lactamase inhibitor with a unique mechanism that overcomes tazobactam-resistant variants of class A β-lactamases (12, 13) (Fig. 1). The combination of enmetazobactam with the 4th-generation cephalosporin cefepime was as potent as meropenem and outperformed piperacillin-tazobactam when tested in vitro against a collection of recent clinical isolates of Enterobacteriaceae (9), and it also was more potent than cefepime-tazobactam against a subset of ESBL-producing isolates of K. pneumoniae (14). Cefepime-enmetazobactam is intended as an empirical carbapenem-sparing therapy for serious Gram-negative infections in settings where ESBL-producing Enterobacteriaceae are expected. The intrinsic activity of cefepime against isolates expressing AmpC and OXA-48 β-lactamases also makes the combination suitable for the treatment of organisms that coproduce such β-lactamases.
Structures of enmetazobactam and tazobactam. The charged moieties of enmetazobactam are highlighted in red and blue.
Pharmacokinetic-pharmacodynamic (PK-PD) models of infection are routinely applied to investigate the efficacy of novel agents against pathogens and resistance mechanisms of interest, supporting clinical dose selection and breakpoint setting by Monte Carlo simulation (15–17). In this study, the PK-PD relationship of enmetazobactam combined with cefepime was investigated in neutropenic mouse thigh models infected with clinical isolates of ESBL-producing K. pneumoniae resistant to cefepime and piperacillin-tazobactam.
(Parts of the data were presented at the 2019 ASM/ESCMID Conference on Drug Development in Boston, MA.)
RESULTS
In vitro activity of cefepime-enmetazobactam against ESBL-producing isolates of K. pneumoniae.Nine clinical isolates of K. pneumoniae, with cefepime MICs of >32 μg/ml and piperacillin-tazobactam MICs ranging from 16 to >128 μg/ml, were used in this study (Table 1). The isolates carried different β-lactamase genes, including CTX-M-2, CTX-M-14, CTX-M-15, CTX-M-28, and SHV-12, with or without the plasmid-encoded class C cephalosporinase DHA-1 or the class D carbapenemase OXA-48, and belonged to seven different sequence types (18). Mutations in the OmpK36 porin were detected in five isolates. Enmetazobactam, fixed at a concentration of 8 μg/ml, restored the activity of cefepime against each isolate, reducing their MICs to within the CLSI susceptible category, with values ranging from 0.06 to 2 μg/ml.
Cefepime-resistant, ESBL-producing isolates of K. pneumoniae used in this study
The PKs of enmetazobactam and cefepime are adequately described by linear, two-compartmental models.Four different cefepime-enmetazobactam dose combinations were administered intravenously (i.v.) to infected animals every 8 h (q8h). Enmetazobactam and cefepime concentration profiles showed a two-phase decline. Consecutive profiles from day 1 and day 2 were similar, indicating no time-dependent effects, and dose-normalized profiles collapsed, indicating no concentration-dependent effects (see Fig. S1 in the supplemental material). Enmetazobactam and cefepime plasma concentrations were well described in a two-compartment model (Fig. S2), with linear clearance from the central compartment. The model estimated plasma clearances of 41 ml/h for both enmetazobactam and cefepime. PK parameters are summarized in Table 2.
Estimates of plasma enmetazobactam and cefepime PK model parameters from mice infected with CTX-M-15-producing K. pneumoniae isolate 1280740
The fraction of the dosing interval during which enmetazobactam is above a free threshold concentration best describes the exposure-response relationship of enmetazobactam.The PK-PD relationship of enmetazobactam, combined with cefepime, was investigated by dose fractionation in a 26-h neutropenic murine thigh infection model challenged with the cefepime-resistant, CTX-M-15-producing isolate K. pneumoniae 1077711 (Table 1). Test articles were administered by i.v. infusion 2 h postinfection in a matrix design with four animals per group. Total enmetazobactam dosages included 6, 20, 60, 200, and 600 mg/kg of body weight/day, each fractionated at q4h, q8h, q12h, and q24h. Cefepime was administered concomitantly at a fixed dose of 100 mg/kg q4h to achieve a PK-PD target of 44% fT > cefepime-enmetazobactam MIC (MIC of 1 μg/ml), an exposure that is readily achieved in patients dosed with 2 g cefepime q8h (see Discussion). The terminal bioburden was expressed as the log10(CFU/g) difference between the pretreatment group at 2 h postinfection and treatment groups at 26 h postinfection [Δlog10(CFU/g)].
A robust infection with K. pneumoniae 1077711 was established, exhibiting a bioburden increase of 2.3 Δlog10(CFU/g). Treatment with cefepime alone administered at 600 mg/kg/day was ineffective, with a bioburden increase of 1.9 Δlog10(CFU/g). Combining cefepime with increasing total daily doses of enmetazobactam was associated with greater reductions in bioburden for each of the different dosing frequencies, and increasing the enmetazobactam dosing frequency was associated with greater reductions in bioburden for each of the different total daily enmetazobactam doses (Table 3).
Terminal thigh bioburden obtained in enmetazobactam dose fractionation studies combined with a fixed dose of cefepime
For each treatment arm, changes in bioburden were plotted against the respective enmetazobactam exposure, expressed as (i) fraction of the dosing interval above a free threshold concentration (fT > CT, with CT = 2 μg/ml; see below), (ii) area under the free concentration-time profile (fAUC), and (iii) maximum free concentration (fCmax), and sigmoid curves were fitted by regression analysis. The PK-PD relationship of enmetazobactam was best described by fT > CT, followed by fAUC and fCmax (Fig. 2). Enmetazobactam exposures required for stasis and bioburden reductions of 1 and 2 Δlog10(CFU/g) were 12%, 27%, and 50% fT > 2 μg/ml, respectively.
Enmetazobactam exposure-response relationship resulting from a dose fractionation study of different total enmetazobactam doses, combined with a fixed dose of cefepime, in a 26-h murine thigh infection model. The cefepime-resistant, CTX-M-15-producing K. pneumoniae isolate 1077711, with a cefepime-enmetazobactam MIC of 1 μg/ml, was the infecting organism. y axes show the bioburden difference between pretreatment and treatment groups. x axes show enmetazobactam exposures as fT > 2 μg/ml (a), fAUC (b), and fCmax (c). Each dot corresponds to one dosage group. EMT, enmetazobactam; S, standard error of regression; R2, coefficient of determination.
An enmetazobactam PK-PD target of 44% fT > 2 μg/ml is sufficient for a 1-log10 reduction in thigh bioburden.Enmetazobactam dose-response studies combined with fixed doses of cefepime were conducted for additional clinical isolates of ESBL-producing K. pneumoniae to quantify PK-PD targets required to achieve stasis and reductions in bioburden relative to pretreatment. Nine clinical isolates of K. pneumoniae encoding different ESBLs, with or without an AmpC or OXA-48, were included in this study. All isolates were resistant to cefepime, with eight of them exhibiting resistance to piperacillin-tazobactam (Table 1). Enmetazobactam was administered to groups of five animals at doses of 1, 3.16, 10, 31.6, and 100 mg/kg on a q4h schedule. Cefepime was administered concomitantly at a fixed dose between 25 and 200 mg/kg q4h depending on the cefepime-enmetazobactam MIC of the respective isolates to achieve exposures of 43 to 53% fT > cefepime-enmetazobactam MIC (see Table 5). Exposures of 79% were obtained for each of isolates 1280740 and 1237221 due to the very low MIC (0.06 μg/ml) and, in the latter case, due to a correction of the modal MIC value postefficacy study.
A robust infection was achieved in the vehicle group with all isolates, resulting in bioburden increases ranging from 0.8 to 4.0 Δlog10(CFU/g) (Table 4). Enmetazobactam, in combination with fixed doses of cefepime, exerted a strong dose-response against all isolates examined, with bioburden reductions of ≥1 Δlog10(CFU/g) at a dose of 31.6 mg/kg q4h, with the exception of 900679, for which enmetazobactam at 100 mg/kg q4h was required (Table 4).
Terminal thigh bioburden obtained in enmetazobactam dose-response studies combined with a fixed dose of cefepime
Enmetazobactam exposure, expressed as fT > CT, was plotted against the corresponding bioburden, and sigmoid curves were fitted by regression analysis for each isolate. Isolate-specific targets for stasis and for a bioburden reduction of 1 Δlog10(CFU/g) are reported in Table 5. Exposure-response data for all isolates were pooled for combined regression analysis (Fig. 3). The global fit simulated across data points from all 9 isolates identified 2% and 16% fT > 2 μg/ml enmetazobactam required for stasis and a bioburden reduction of 1 Δlog10(CFU/g), respectively. For the 75th percentile of the global fit, 8% and 44% fT > 2 μg/ml enmetazobactam were identified for stasis and a bioburden reduction of 1 Δlog10(CFU/g), respectively (Table 5).
Individual and combined enmetazobactam PK-PD targetsa
Enmetazobactam exposure-response relationship in a 26-h murine thigh infection model comprising nine cefepime-resistant, ESBL-producing isolates of K. pneumoniae. The y axis shows the bioburden as log10(CFU/g) difference between pretreatment and treatment groups. The x axis shows the enmetazobactam exposure as fT > 2 μg/ml. The lines represent the global fit and the 75th and 90th percentiles.
Modeling the exposure-response relationship for the combined set of isolates was largely independent of the applied concentration used to express the fT exposure. Similar goodness-of-fit values were obtained with either constant CT values between 0.125 and 4 μg/ml or when isolate-specific cefepime-enmetazobactam MICs were used (Fig. S3). However, only CT values of 1 and 2 μg/ml prevented enmetazobactam data points from being shifted to the far left (0% fT > CT) or the far right (100% fT > CT) of the exposure-response plots. Magnitudes required for 1-log10 bioburden reduction resulting from simulations of the global fit using CTs of 0.5, 1, or 2 μg/ml were interchangeable, i.e., the corresponding exposures were achieved with the same dose of enmetazobactam.
DISCUSSION
Enmetazobactam is an extended-spectrum β-lactamase inhibitor belonging to the penicillanic acid sulfone class. This study identified fT > CT as the PK-PD index best describing the exposure-response relationship of enmetazobactam against ESBL-producing isolates of K. pneumoniae. These findings are concordant with results from a hollow-fiber infection model that also identified fT > CT as the PK-PD index (19). A study assessing the PK-PD relationship of enmetazobactam in a mouse pneumonia model concluded that either fT > CT or fAUC could account for the experimental data (20). For the penicillanic acid sulfone tazobactam, fT > CT has consistently been identified as the PK-PD index (21), implying a class effect for penicillanic acid sulfones.
Isolates included in this study were resistant to cefepime and piperacillin-tazobactam with no bias in the cefepime-enmetazobactam MIC, covering a range of 0.06 to 2 μg/ml. Increased cefepime-enmetazobactam MICs were observed for isolates expressing either multiple ESBLs, an ESBL in combination with the OXA-48 carbapenemase, isolates bearing a two-amino-acid insertion at position 134 of porin OmpK36, or an N-terminal truncation of OmpK36. The exposure-response modeling employed the more stringent 75th percentile compared to the global fit, with PK-PD targets estimated at the stricter endpoint of 1-log10 bioburden reduction rather than net stasis (22, 23). The molecular diversity and unbiased MIC range of the isolates included, together with the adaptive cefepime dosing and the conservative modeling approach, makes the suggested enmetazobactam preclinical target of 44% fT > 2 μg/ml a conservative estimate for use in clinical dose justification and breakpoint setting.
Cefepime doses employed in enmetazobactam dose-response studies were selected to achieve exposures ranging from 40% to 60% fT > cefepime-enmetazobactam MICs. In clinical settings, cefepime administered as 2 g q8h by 2-h i.v. infusion achieved 60% fT > MIC (MIC of 8 μg/ml) with a 98.5% probability of target attainment in complicated urinary tract infection patients (Allecra; references 24 and 25 and unpublished results). Therefore, the average cefepime exposure of 53% fT > cefepime-enmetazobactam MIC achieved in this study (Table 5) is conservative, and an artificially lower enmetazobactam PK-PD target due to cefepime overexposure for two isolates (1280740 and 1237221) is well compensated by an underexposure obtained for the other isolates. Cefepime-enmetazobactam MICs used for modeling and simulation were determined using enmetazobactam at a fixed concentration of 8 μg/ml, as approved in a CLSI M23 tier 2 study design.
In the hollow-fiber model, an enmetazobactam PK-PD target of 31 to 46% fT > 2 μg/ml was required for stasis to a 1-log10 bioburden reduction when combined with cefepime (19). In the mouse pneumonia model, fT > CT was chosen as the enmetazobactam PK-PD index because it was potentially a more conservative measure than fAUC. Enmetazobactam plasma or epithelial lining fluid exposures of ≥20% fT > 2 μg/ml were required for a ≥2-log10 reduction in lung bioburden with a concomitant cefepime exposure of ≥20% fT > MIC (20). These findings, together with the presented data, support a clinical enmetazobactam PK-PD target of 45% fT > 2 μg/ml when administered in combination with high-dose cefepime.
When tazobactam was combined with cefepime or ceftolozane, mean magnitudes of 39.7% fT > 0.25 μg/ml and 44.0% fT > 0.5 μg/ml, respectively, were required for a 1-log10 bioburden reduction against ESBL-producing isolates of Enterobacteriaceae in murine thigh models (26, 27). In vitro PK-PD models identified magnitudes of 26.6 to 52.8% fT > threshold concentrations required for a 1-log10 reduction from baseline when combined with cefepime or ceftolozane (28–30).
A minimum cefepime exposure of 40% fT > cefepime-enmetazobactam MIC was necessary to achieve an enmetazobactam dose-response. Since the maximum tolerated dose of cefepime in mice was 200 mg/kg q8h, corresponding to a cefepime exposure of 43% fT > 2 μg/ml, the model was limited to isolates with a cefepime-enmetazobactam MIC of 2 μg/ml. Therefore, isolates with cefepime-enmetazobactam MICs of 4 and 8 μg/ml within the CLSI-susceptible dose-dependent interpretive category for cefepime need to be evaluated in hollow-fiber infection models simulating human exposures of enmetazobactam and cefepime.
K. pneumoniae is recognized as a heterogenous pathogen with a diverse armamentarium of virulence factors and antimicrobial resistance genes, causing a wide range of difficult-to-treat infections, including pneumonia, urinary tract infections, bacteremia, and liver abscesses (31–33). Given enmetazobactam also restores the activity of cefepime against ESBL-producing isolates of Escherichia coli and Enterobacter cloacae (12, 34, 35) and that the MIC90 of cefepime-enmetazobactam against K. pneumoniae is at the higher end of the spectrum relative to other genera of Enterobacteriaceae, the proposed target may well be generalized to ESBL-producing Enterobacteriaceae other than K. pneumoniae.
The PK-PD relationship developed in this study supported a dosing regimen of 2 g cefepime combined with 0.5 g enmetazobactam for adults with complicated urinary tract infections or acute pyelonephritis in a randomized, double-blind, noninferiority phase 3 study against 4 g piperacillin combined with 0.5 g tazobactam. The primary efficacy evaluation was performed in the microbiologically modified intent-to-treat population. Overall success, defined as clinical cure and microbiological eradication, was 79.1% for cefepime-enmetazobactam and 58.9% for piperacillin-tazobactam (difference, 21.2% [95% confidence interval, 14.3% to 27.9%] by stratified Newcombe confidence intervals [36, 37]).
MATERIALS AND METHODS
MIC testing and molecular analyses.Isolates were obtained from IHMA Europe Sàrl (Monthey, Switzerland). MICs were determined by broth microdilution by following CLSI guidelines (38, 39). Enmetazobactam was fixed at 8 μg/ml for cefepime-enmetazobactam MIC determination. Isolates were genotyped by multiplex PCR for β-lactamase genes encoding class A ESBLs (CTX-M, SHV, and TEM) and KPCs, MBLs (IMP, VIM, NDM, and SPM), AmpCs (ACC, CMY, DHA, FOX, and ACT), and class D ESBLs (OXA-48-like β-lactamases), followed by sequencing using methods described previously (40). OmpK36 porin protein sequences were extracted from whole-genome next-generation sequences and alignments made using Clustal Omega, with sequence variants reported against the K. pneumoniae isolate HS11286 as a reference sequence (41–44).
Preparation of pathogens and test articles.Bacterial isolates were grown in cation-adjusted Mueller-Hinton broth in a shaking incubator at 37°C. Overnight cultures were washed twice in phosphate-buffered saline (PBS) and adjusted to a concentration of ca. 1.2 × 109 CFU/ml by optical density measurement and then further diluted to the required inoculum. Inoculum concentrations were confirmed by plating a sample onto drug-free agar. Enmetazobactam (provided by Allecra Therapeutics SAS, Saint-Louis, France), cefepime hydrochloride monohydrate (no. J66237; Alfa Aesar), and meropenem (clinical grade; AstraZeneca, Ltd.) were reconstituted in saline for injection (SFI), 5% dimethyl sulfoxide–95% SFI, and water for injection, respectively, at twice the highest final concentration required for a 5-ml/kg dosing formulation.
Animals and housing.All studies were performed under UK Home Office Licenses with local ethical committee clearance. All animal experiments were performed by experienced technicians that had completed the UK Home Office Personal License course and held current personal licenses. Specific-pathogen-free male ICR mice (ca. 4 weeks old, 11 to 15 g; Charles River UK, Ltd., Margate, UK) were housed on sterile aspen chip bedding with free access to food and water in individual ventilated cages, exposed to a 12-h light/dark cycle, HEPA-filtered air at 22°C, and a relative humidity of 60%. Mice were acclimatized for at least 7 days prior to the start of studies. During infection, mice had additional access to wet food. For studies using mice with jugular vein cannula (JVC) implants, surgery was performed at Charles River, and mice were allowed 48 h to recover from surgery before transportation to the experimental site, where they were allowed another 48-h recovery time. Lines were flushed as required with heparin in 5% glucose. Access to the JVC was via pinports in the nape of the neck. JVC animals were single housed in metabolic cages attached to syringe pumps through a swivel and tether during the treatment phase of the models.
Neutropenic murine thigh infection model.Mice were immunosuppressed using cyclophosphamide at 150 mg/kg on day −4 and at 100 mg/kg on day −1. Prior to infection on day 1, mice were anaesthetized with 2.5% isofluorane–97.5% oxygen. Once anesthesia was confirmed by the absence of pedal reflex, both thighs were infected by intramuscular injection with 50 μl of bacterial suspension containing ∼5 × 105 CFU. While still under anesthesia, mice were administered 0.03 mg/kg buprenorphine for pain relief (this was readministered after 8 and 16 h postinfection). Mice were returned to cages in a warming cabinet with frequent observations until full recovery from anesthesia. Treatment was initiated 2 h postinfection by i.v. injection of a 5-ml/kg test article via the lateral caudal vein for doses of 150 to 600 cefepime mg/kg/day combined with 6 to 600 enmetazobactam mg/kg/day and via an indwelling JVC using programmed syringe pumps for doses of 1,200 mg/kg/day cefepime combined with 6 to 600 mg/kg/day enmetazobactam. Animals in pretreatment groups were euthanized 2 h postinfection, and animals of treatment groups were euthanized 26 h postinfection, both by a pentobarbitone overdose followed by cervical dislocation. Immediately following confirmation of death, thighs were removed and weighed. Thighs were placed individually in bead-beating tubes containing 2 ml of PBS plus 10% glycerol and subjected to mechanical disruption. Organ homogenates were diluted in PBS and cultured quantitatively on drug-free agar, resulting in two data points per animal (right and left thighs). Numbers of CFU per gram of tissue from each treatment group were converted to the log10 of the group geometric mean [log10(CFU/g)]. The terminal bioburden resulting from a specific treatment regimen was expressed as the log10(CFU/g) difference between pretreatment and treatment groups [Δlog10(CFU/g)]. Results from the dose fractionation (q4/q4 fractionation) and dose-response study employing isolate 1077711 confirmed the reproducibility of the in vivo data.
PK sampling and mathematical PK modeling.Animals were infected with K. pneumoniae isolate 1280740. Cefepime/enmetazobactam was administered q8h at doses of 25/6.25, 50/25, 100/75 and 200/100 mg/kg by i.v. bolus injection into the lateral caudal vein. Blood samples were collected in triplicate into heparinized vials at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24.083, 24.25, 24.5, 25, 26, 28, and 32 h by cardiac puncture and placed immediately on ice pending centrifugation. Plasma samples were stored at –80°C prior to analysis by a qualified liquid chromatography-tandem mass spectrometry method. Individual cefepime and enmetazobactam observations were pooled and fitted to a linear, two-compartment model using least squares, minimizing the difference between the log-transformed predicted and observed values. For the least-squares fitting, the method L-BFGS-B, implemented in the R function optim(), was used (45). Free drug concentrations were calculated based on reported mouse protein binding of 0% for cefepime and 0% for enmetazobactam (34, 46).
Exposure-response modeling.The terminal bioburden as Δlog10(CFU/g) was modeled from the enmetazobactam exposure (fEx), expressed as fT > CT, fAUC, or Cmax, by fitting a sigmoid curve defined by the baseline effect, Emin, the maximal effect, Emax, the potency, EC50, and the steepness of the sigmoid curve γ using Equation 1.
The sigmoid curve was fit to the data using least squares, minimizing the difference between the log-transformed predicted and observed values. For the least-squares fitting, the method L-BFGS-B, implemented in the R function optim(), was used (45). Percentiles in the global fit simulations were calculated by vertically shifting the exposure-response curve to capture the required number of data points.
ACKNOWLEDGMENTS
We thank Stuart Shapiro for critically reviewing the manuscript.
M.M., P.W., and P.K. designed the study. R.O., S.S., and C.C. supervised the work. F.B., M.M., P.W., A.B., and P.K. analyzed the data. A.B. and P.K. wrote the manuscript.
F.B. and M.M. are full-time employees of LYO-X GmbH, Switzerland. P.W., R.O., and S.S. are full-time employees of Evotec, UK. C.C. is a full-time employee of IHMA Europe Sàrl, Switzerland. A.B. and P.K. are full-time employees of Allecra Therapeutics SAS, France. R.C. and S.F. are full-time employees of Aptuit (Verona) Srl, Verona, Italy. Aptuit (Verona) Srl is an Evotec Company.
FOOTNOTES
- Received 24 January 2020.
- Returned for modification 25 February 2020.
- Accepted 29 March 2020.
- Accepted manuscript posted online 6 April 2020.
Supplemental material is available online only.
For a companion article on this topic, see https://doi.org/10.1128/AAC.00180-20.
- Copyright © 2020 American Society for Microbiology.
