In vitro activity of cefiderocol in Pseudomonas aeruginosa isolates from people with cystic fibrosis recovered during three multicentre studies in Spain Free

Abstract

Objectives

Despite the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators, Pseudomonas aeruginosa is still a major pathogen in people with cystic fibrosis (pwCF). We determine the activity of cefiderocol and comparators in a collection of 154 P. aeruginosa isolates recovered from pwCF during three multicentre studies performed in 17 Spanish hospitals in 2013, 2017 and 2021.

Methods

ISO broth microdilution was performed and MICs were interpreted with CLSI and EUCAST criteria. Mutation frequency and WGS were also performed.

Results

Overall, 21.4% were MDR, 20.8% XDR and 1.3% pandrug-resistant (PDR). Up to 17% of the isolates showed a hypermutator phenotype. Cefiderocol demonstrated excellent activity; only 13 isolates (8.4%) were cefiderocol resistant by EUCAST (none using CLSI). A high proportion of the isolates resistant to ceftolozane/tazobactam (71.4%), meropenem/vaborbactam (70.0%), imipenem/relebactam (68.0%) and ceftazidime/avibactam (55.6%) were susceptible to cefiderocol. Nine out of 13 cefiderocol-resistant isolates were hypermutators (P < 0.001). Eighty-three STs were detected, with ST98 being the most frequent. Only one isolate belonging to the ST175 high-risk clone carried bla_VIM-2. Exclusive mutations affecting genes involved in membrane permeability, AmpC overexpression (L320P-AmpC) and efflux pump up-regulation were found in cefiderocol-resistant isolates (MIC = 4–8 mg/L). Cefiderocol resistance could also be associated with mutations in genes related to iron uptake (tonB-dependent receptors and pyochelin/pyoverdine biosynthesis).

Conclusions

Our results position cefiderocol as a therapeutic option in pwCF infected with P. aeruginosa resistant to most recent β-lactam/β-lactamase inhibitor combinations.

Introduction

The emergence of antimicrobial resistance is a significant public health issue worldwide.¹ The WHO has listed carbapenem-resistant Pseudomonas aeruginosa as a pathogen for which there is a critical need to develop new antibiotics.² Additionally, P. aeruginosa is one of the main pathogens causing serious infections in people with cystic fibrosis (pwCF).

CF is a recessive genetic disease caused by a series of mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR). This defect leads to dehydration and impaired mucus clearance, reduced bacterial internalization, and inhibition of the natural antimicrobial peptides in the lungs, determining a favourable environment for bacterial growth and pathogenic colonization.³ The presence of P. aeruginosa in the airways is associated with lung function deterioration and increased morbidity in affected pwCF. That is why the control and clinical treatment of this pathogenic colonization is essential.⁴

P. aeruginosa has a remarkable variety of antibiotic resistance mechanisms, including acquired resistance genes that can be transferred horizontally, but also multiple chromosomal determinants associated with complex regulatory pathways involved in intrinsic resistance, such as membrane permeability, efflux systems and production of enzymes that inactivate antibiotics. Additionally, this species exhibits adaptive resistance, involving temporary changes in bacterial physiology or gene expression in response to environmental signals or antibiotic exposure.^5–7 Resistance can be accelerated in pwCF by the high prevalence of hypermutable P. aeruginosa, which is between 30% and 60% of the isolates.⁸ Moreover, the spread among pwCF of the so-called high-risk clones of P. aeruginosa has also contributed to antimicrobial resistance and a worse prognosis of chronic infection with few options for treatment.⁹

Cefiderocol (S-649266, Shionogi & Co. Ltd) is a novel siderophore cephalosporin targeting Gram-negative bacteria, including isolates resistant to carbapenems. Cefiderocol has demonstrated structural stability against hydrolysis by both serine-β-lactamases and MBLs. Several studies have demonstrated excellent in vitro activity against P. aeruginosa from different infection sites, but specific data on isolates recovered from pwCF are still scarce.^10–15 In this work, we determine the in vitro activity of cefiderocol and commercially available comparator antibiotics against a well-characterized collection of P. aeruginosa recovered from pwCF obtained during three Spanish multicentre studies (2013, 2017 and 2021). We also characterized the population structure and the resistome, focusing on the resistance mechanisms possibly affecting cefiderocol.

Materials and methods

Study of isolates

A total of 154 clinical P. aeruginosa isolates were collected from 126 patients from CF units of 17 hospitals as a part of three multicentre studies performed in 2013 (n = 79), 2017 (n = 63) and 2021 (n = 12) in Spain.¹⁶ All isolates were recovered as part of the routine management of the CF patients and were stored at −80°C for further analysis. Different morphotypes [mucoid, metallic, rough, small colony variants (SCVs) or regular] were defined by visual inspection as previously described.¹⁷ One or more than one isolate per patient, if they expressed different morphotypes, were included in the study.¹⁶ Bacterial identification was performed using MALDI-TOF (Bruker, Germany).

Antimicrobial susceptibility testing of cefiderocol and comparators

MICs of cefiderocol (range 0.003–64 mg/L) and other antipseudomonal agents were determined following the standard broth microdilution method according to ISO 20776-1:2019.¹⁸ Antimicrobials most commonly used in pwCF and new ones potentially useful in the treatment of P. aeruginosa infections were included: piperacillin/tazobactam (MIC range 0.5–64 mg/L); ceftazidime (0.03–32 mg/L); ceftazidime/avibactam (0.125–8 mg/L); ceftolozane/tazobactam (0.25–8 mg/L); imipenem/relebactam (0.03–8 mg/L); meropenem (0.06–16 mg/L); meropenem/vaborbactam (0.06–8 mg/L); ciprofloxacin (0.002–4 mg/L); and colistin (0.12–4 mg/L). We used dehydrated 96-well microtitre plates supplied by IHMA Inc. (Schaumburg, IL, USA). MICs of cefiderocol were assessed in iron-depleted CAMHB (ID-CAMHB) according to CLSI methodology.¹⁹ MIC results were interpreted following both EUCAST 2024 and CLSI 2024 guidelines.^19,20P. aeruginosa ATCC 27853 and PAO1 P. aeruginosa were used as control isolates.

Antimicrobial susceptibility patterns were established based on previous guidelines: MDR, no susceptibility to at least one agent in three or more antimicrobial categories; XDR, no susceptibility to at least one agent in all but two or fewer antimicrobial categories; and pandrug resistant (PDR), no susceptibility to all agents in all antimicrobial categories.²¹

Mutation frequency determination

Mutation frequency was defined as the ratio of mutant colonies to total viable cells. The classification criteria were hypomutators (5 × 10⁻¹⁰–5 × 10⁻⁹), normomutators (7.5 × 10⁻⁹–7.5 × 10⁻⁸) and hypermutators (1 × 10⁻⁷–5 × 10⁻⁶) as previously described.²²

Statistical analysis

Categorical variables were described using frequencies and percentages and were compared using Pearson’s chi-squared test or Fisher test, as appropriate. All tests were performed using R software (RStudio, Boston, MA, http://www.rstudio.com/), and P < 0.05 was considered statistically significant. The statistical analysis included each episode in which a P. aeruginosa isolate was detected.

WGS and bioinformatics analysis

Genomic DNA extraction was carried out using the commercial chemagic DNA Bacterial External Lysis Kit (PerkinElmer, US). WGS was performed on all 154 P. aeruginosa isolates using the Illumina NovaSeq 6000 platform (Oxford Genomics Centre, Oxford, UK), with 2 × 150 bp paired-end reads. Five low-quality sequences were excluded and a total of 149 sequences were included in the subsequent analysis. Sequencing processing, molecular typing, and screening for acquired resistance genes were carried out as previously described.²³ Variant calling analysis was performed in 40 chromosomal genes related to antimicrobial resistance in P. aeruginosa as previously defined.²³ Additionally, a subset of 120 genes involved in iron uptake and with a potential impact on cefiderocol resistance were also analysed.¹⁰ The presence of mutations in genes involved in the mutator phenotype (mutS/mutL) and in alginate biosynthesis was also studied. MASH and iTOL applications were used to generate and visualize a similarity tree, respectively.

The complete genomes were deposited at DDBJ/ENA/GenBank under the project number PRJNA975709 (JASKOW000000000–JASKUM000000000).

Ethics

The study was approved by the ethical committee of the Hospital Ramón y Cajal (Ref. 19-056).

Results

In vitro activity of cefiderocol and comparators

Overall, 98.0% (151/154) of P. aeruginosa were susceptible (S) to cefiderocol by CLSI (MIC_50/90 = 0.12/2 mg/L) and 91.6% (141/154) by EUCAST (MIC_50/90 = 0.12/2 mg/L) (Table 1). Activity of cefiderocol by year was: 2013 (89.9% S cefiderocol), 2017 (93.7% S cefiderocol) and 2021 (91.7% S cefiderocol) by EUCAST. Overall, the activity of cefiderocol was comparable to that of colistin (99.4% intermediate by CLSI and 99.4% S by EUCAST), ceftazidime/avibactam (94.1% S by CLSI and EUCAST) and meropenem/vaborbactam (93.5% S by EUCAST) and higher than ceftolozane/tazobactam (86.4% S by CLSI and EUCAST) and imipenem/relebactam (83.8% S by CLSI and EUCAST) (Table 1). Using the MIC₅₀ value, cefiderocol was also the most active antibiotic (MIC₅₀= 0.12 mg/L) (Table 1 and Table S1, available as Supplementary data at JAC Online).

According to the morphotypes, P. aeruginosa isolates were classified as follows: 41.5% (64/154) regular type, 22.2% (34/154) mucoid, 12.9% (20/154) metallic, 10.5% (16/154) rough, and 12.9% (20/154) SCVs. Up to 56.5% of the isolates were classified as non-MDR (87/154), 21.4% (33/154) as MDR, 20.8% (32/154) as XDR, and 1.3% (2/154) as PDR.

No differences were observed in the activity of cefiderocol regarding colony morphotype or the origin of the CF centre (Figure 1). Overall, cefiderocol showed good activity against MDR (96.9%, 32/33), XDR (68.8%, 22/32) and PDR (50%, 1/2) isolates (EUCAST criteria) (Table 2). Nevertheless, activity of cefiderocol was higher than that of the comparators against the subsets of isolates resistant to other antimicrobials, including ceftolozane/tazobactam (71.0% susceptible), meropenem (70.6%), meropenem/vaborbactam (70.0% susceptible), imipenem/relebactam (68.0% susceptible) and ceftazidime/avibactam (55.6% susceptible). Additionally, cefiderocol also demonstrated effectiveness against isolates resistant to piperacillin/tazobactam plus ceftazidime/avibactam plus meropenem (69.2%) (Table 2).

Cefiderocol-resistant isolates

Only 13 isolates (8.4%) from 12 patients in the studied collection were resistant to cefiderocol (MIC range: 4–8 mg/L) (Table 3) when considering EUCAST breakpoints (resistant MIC >2 mg/L). No isolate was resistant when considering CLSI breakpoints (MIC ≥16 mg/L) (Table 1), but three isolates were categorized as intermediate (MIC = 8 mg/L). Note that 76.9% (10/13) of the cefiderocol-resistant isolates presented an XDR profile and 53% (7/13) a regular morphotype (Table 3, Table S2, Figure 1). Overall, cefiderocol-resistant isolates showed high percentages of resistance to other antimicrobials: ciprofloxacin (92.0%); piperacillin/tazobactam (84.0%); ceftazidime (76.9%); imipenem/relebactam (61.5%); ceftolozane/tazobactam (46.2%); meropenem (38.5%); ceftazidime/avibactam (30.7%); meropenem/vaborbactam (23.0%) and colistin (15.3%) (Table 3).

Hypermutation screening

A total of 28 (18.2%) isolates showed a hypomutator phenotype (5 × 10⁻¹⁰–5 × 10⁻⁹), 100 (64.9%) a normomutator phenotype (7.5 × 10⁻⁹–7.5 × 10⁻⁸) and 26 (16.9%) a hypermutator phenotype (1 × 10⁻⁷–5 × 10⁻⁶). Among the hypermutators, XDR was the most frequent resistant phenotype (61.5%), followed by non-MDR (19.2%), MDR (11.5%) and PDR (7.7%). According to EUCAST criteria, nine isolates resistant to cefiderocol were hypermutators (69.2%), while only 12% of susceptible strains showed this phenotype (P < 0.001). The most effective antibiotic within hypermutators was colistin, with 88.9% of susceptibility.

Molecular typing and resistome

A total of 149 genomes were analysed by WGS. MLST analysis revealed up to 83 different STs, with ST198 (n = 9), ST792 (n = 8), ST1068 (n = 7) and ST3350-2LV (n = 6) being the most frequent clones (Figure 1, Table S3). Interestingly, 6/9 (66.6%) ST198, 5/7 (71.4%) ST1068 and 5/6 (83%) ST3350-2LV were recovered in the same CF Unit in the study performed in 2017. Thirteen non-previously described STs, differing in one or two alleles were identified. Different STs were observed in different samples isolated from the same patient in five cases (Table S3). Diversity of ST clones found among the cefiderocol-resistant isolates is shown in Table 3.

Isolates belonging to high-risk clones ST175 (n = 2) and ST235 (n = 1) were found in different centres, all of them susceptible to cefiderocol (MIC range: ≤0.12–0.5 mg/L). One of the ST175 P. aeruginosa (cefiderocol MIC = 0.25 mg/L) isolates carried the resistance gene bla_VIM-2. Three isolates assigned as ST244 clones (n = 3, MIC ≤ 0.12 mg/L) but showing different morphotypes were also identified in one patient.

The aminoglycoside [aph(3′)-IIb] and fosfomycin (fosA) resistance genes were detected in all isolates. Furthermore, the chloramphenicol-resistance gene catB was present in 96% of the isolates, while the fluoroquinolone-resistance gene crpP was identified in 23.5%.

A total of 36 PDC variants associated with different STs were detected among the 149 P. aeruginosa isolates. The most frequent variant was bla_PDC-3 (23%), followed by bla_PDC-5 (17.5%) and bla_PDC-8 (8.7%). Among the cefiderocol-resistant isolates, we found eight PDC variants, including PDC-3 (n = 4) and PDC-5 (n = 2) (Table S3). Overall, no association between PDC mutations and cefiderocol susceptibility was observed.

Mutational resistome characterization

Mutations (non-synonymous SNPs and insertions/deletions) detected in chromosomal genes known to be related to antimicrobial resistance and in genes involved in iron uptake are summarized in Tables S4 and S5, respectively. Mutations affecting genes involved in the up-regulation of efflux pumps (93.9%, 139/149), AmpC overexpression (67.1%, 100/149), membrane permeability (51.7%, 77/149) and PBP3 (18.1%, 27/149) were identified (Table S4). Eleven out of the 13 isolates with resistant cefiderocol MIC values encountered exclusive mutations in these genes (mainly affecting AmpC overproduction and efflux pump regulation) and in 7 of them inactivating mutations in OprD were detected. A mutation in AmpC (L320P) previously described to be involved in cefiderocol resistance was found in one resistant isolate (isolate S99, MIC = 4 mg/L). Other mutations in AmpC (T96I, G183D, G216S, E247K, F533L and R540C) and AmpD (T139M) previously related to increased cefiderocol MIC values were not found.²⁴ Exclusive mutations were also found in other genes previously associated with reduced susceptibility to cefiderocol, such as pmrAB, fusA1 and ftsI (PBP3) (Table 3, Table S4).

On the other hand, in 11 out of 13 cefiderocol-resistant isolates, a range of 13–41 genes involved in iron uptake and previously identified as cefiderocol transporters in P. aeruginosa were also mutated.²⁵ Exclusive mutations were also found in TonB and TonB-dependent receptors (piuC and pirA) related to increased cefiderocol MICs (Table 3, Table S5).

In addition, mutations in genes involved in the mutator phenotype were found in 56% of the isolates. Up to 37.6% (n = 56) and 41.6% (n = 62) of the isolates presented mutations in mutS and mutL, respectively. Moreover, 31 out of 149 (20.8%) carried mutations in both genes. A total of 22/26 (84.6%) of the hypermutator isolates had mutations in mutS (n = 4), mutL (n = 8) or both mutS/mutL (n = 9). Ten out of 13 cefiderocol-resistant isolates (76.9%, P = 0.08) had mutations in mutS (n = 2), mutL (n = 3) or both mutS/mutL (n = 5), and in six of them, inactivating mutations were found (Table 3, Table S4). Mutations in mutS/mutL were also found in cefiderocol-susceptible isolates, but in a lower proportion (56.6%) (Table S4).

WGS analysis also revealed that the gene encoding the ferric pyoverdine receptor fpvA was absent in 53.7% of the isolates, including 6/13 isolates with resistant cefiderocol MIC values. Furthermore, exclusive mutations in the pyoverdine FpvA (2/13) and the pyochelin FptA (2/13) receptors were found in two cefiderocol-resistant isolates each (Table 3, Table S5).

On the other hand, no specific mutations in the alginate biosynthetic gene cluster were found in the isolates with a mucous phenotype compared with those with other phenotypes (Table S6).

Discussion

P. aeruginosa is one of the most important pathogens in the context of CF since chronic bronchopulmonary infection with this organism leads to a rapid decline in lung function and higher morbidity and mortality.²⁶ In Spain, the 2021 European patient registry indicates that about 35% of the Spanish CF adult population is chronically colonized with P. aeruginosa.²⁷ In addition, this species is considered an ESKAPE pathogen with high rates of antimicrobial resistance reported worldwide. In 2021, ECDC stated that 12.6% of P. aeruginosa isolates were resistant to at least three groups of antimicrobials and 3.5% of them to all five groups of antimicrobials under surveillance.²⁸ In P. aeruginosa from pwCF, these figures might be higher due to high antimicrobial exposure and genetic adaptations accelerated by the high prevalence of hypermutable traits.⁸ That is why the introduction of new antibiotics against infections caused by P. aeruginosa is one of the WHO’s priorities.²⁹ The genomic diversity of P. aeruginosa and its mutational capacity, along with its ability to produce chronic infection in pwCF, provides a unique situation for the investigation of new antibiotic therapies.

Cefiderocol has been approved for the treatment of complicated urinary tract infections and respiratory infections. Several studies have reported excellent activity of cefiderocol against P. aeruginosa isolates (95%–99% susceptible).^30,31 However, there are few studies evaluating the in vitro activity of cefiderocol in P. aeruginosa isolates recovered exclusively from pwCF.^32,33 Coinciding with these studies, in our collection cefiderocol was one of the most active agents (>91%), along with colistin (>99%). IV colistin has historically been considered in the treatment of pulmonary infections; however, its utility has become increasingly ambiguous due to concerns about resistance, toxicity, limited clinical data, availability of alternative treatments, and variations in guideline recommendations. Nevertheless, colistin is included as inhaled therapy in CF guidelines.³⁴ In our collection, no changes in the rate of resistance to cefiderocol were observed over time. In clinical studies, despite the complicated nature of CF not being represented, cefiderocol was generally well tolerated for treating infections by MDR P. aeruginosa, in contrast to the significant complications and frequent adverse effects reported with colistin.³⁵

Overall, cefiderocol was also highly active against MDR, XDR and PDR isolates, positioning this antibiotic as a therapeutic option against P. aeruginosa isolates from pwCF. The study of Lasarte-Monterrubio et al.²⁴ showed good activity of cefiderocol against P. aeruginosa CF isolates, including isolates with acquired carbapenemases or PDC variants that caused resistance to other new antibiotics such as β-lactam/β-lactamase inhibitor combinations. Another recent study, performed in Spain with MDR P. aeruginosa from pwCF, reported that cefiderocol was more active (70% susceptible) than ceftolozane/tazobactam (52% susceptible) and ceftazidime/avibactam (47% susceptible).¹¹ In our collection, as in other studies with pwCF, cefiderocol was the most active agent against isolates resistant to meropenem (70%) but also against those resistant to the novel β-lactam/β-lactamase inhibitor combinations such as meropenem/vaborbactam (70%), imipenem/relebactam (68%) and ceftazidime/avibactam (56%).³⁶ Although vaborbactam is a β-lactamase inhibitor used to enhance the activity of meropenem against KPC-producing isolates, its specific effect on P. aeruginosa is limited or variable. Therefore, the difference in susceptibility of P. aeruginosa isolates to meropenem and meropenem/vaborbactam could be due to higher breakpoints.

Epidemiological studies have shown that pwCF are most frequently infected by environmentally acquired P. aeruginosa isolates, but there are also epidemic clones commonly associated with large groups of pwCF.^16,37 In fact, we found possible clonal dissemination (ST198 and ST3350-2LV) in one CF unit in 2017. In our study, a large variety of undescribed STs was detected, but high-risk clones such as ST244, ST175 and ST235 were also present, as well as the CF-associated epidemic clones ST146, ST253 and ST274, all of them susceptible to cefiderocol. Note that VIM is the carbapenemase most frequently detected in P. aeruginosa in Spain^38,39 and that we found one VIM-2-ST175 P. aeruginosa strain in our collection.

According to our results, acquired β-lactamases do not appear to be involved in antimicrobial resistance in P. aeruginosa from pwCF. Overall, mutations in chromosomal genes affecting the up-regulation of efflux pumps, AmpC overexpression, membrane permeability, and mutations in PBP3 were frequently found. Exclusive mutations in chromosomal genes that have previously been associated with reduced susceptibility to cefiderocol (ampC, pmrAB, fusA1 and ftsI) were detected in most of the cefiderocol-resistant isolates.¹⁰ Note that one resistant isolate encountered the L320P-AmpC mutation, previously described to have a major impact on cefiderocol MICs, but not on ceftolozane/tazobactam or ceftazidime/avibactam.^40,41 The R504C mutation in PBP3, a target of cephalosporins, has also been associated with resistance to cefiderocol but was absent in our collection.²⁴ Nevertheless, inactivating mutations in OprD and mutations affecting AmpC and efflux pumps and their regulators could also have contributed to the increase in the resistance to cefiderocol.

In the subset of cefiderocol-resistant isolates, exclusive mutations were also found in genes involved in the biosynthesis of pyoverdine and pyochelin (fpvA and fptA receptors, respectively) and in the uptake and transport of iron (piuC, pirR). Mutations in these genes have already been described to be related to increased cefiderocol MICs.⁴⁰ Furthermore, we also detected exclusive mutations in other genes previously identified as cefiderocol transporters, including tonB and the tonB-dependent receptors (pirA, optJ and optE).^10,40

Remarkably, the percentage of hypermutators was significantly higher in the subset of cefiderocol-resistant (69%) isolates than among cefiderocol-susceptible (12%) isolates. In this sense, the hypermutator phenotype is frequently found in P. aeruginosa isolates from pwCF and is associated with a higher mutation rate and thus a greater ability to develop antibiotic resistance, particularly during treatment.⁴⁰ Note that pwCF included in our study had previously received several courses of various inhaled, oral and IV antibiotics, but never cefiderocol (data not shown).

On the other hand, recent studies have recently analysed a correlation between mutations in the chromosomal PDC enzyme and the susceptibility to novel β-lactam antibiotics.⁴² In our study, unlike other ones that showed high resistance to ceftolozane/tazobactam and ceftazidime/avibactam in isolates carrying different PDC variants, we did not observe a direct association with susceptibility to cefiderocol or other antimicrobials.^42,43

Our study presents some limitations that should be considered when interpreting the results. Firstly, the local epidemiological data obtained are applicable only to the specific study centres, potentially limiting their generalizability to other geographic areas. Additionally, another limitation is the relatively small sample size of isolates, which may affect the representativeness of cefiderocol susceptibility and its consideration as a therapeutic option in pwCF. In CF, antimicrobial susceptibility has not always been correlated with clinical outcomes, underscoring the need to consider other factors in treatment selection and management of these infections. Note that, patients’ prior exposure to cefiderocol has not been registered, and that there are no data available to correlate the findings of this study with patient outcomes.

In conclusion, cefiderocol demonstrated excellent activity in P. aeruginosa isolates from pwCF in Spain. This activity was maintained regardless of the resistance phenotypes, including MDR, XDR and PDR, and in the subsets of isolates resistant to novel β-lactam/β-lactamase inhibitor combinations. Cefiderocol resistance was mainly associated with mutations affecting membrane permeability, AmpC overexpression and efflux pump up-regulation, but mutations in genes involved in iron uptake could also contribute. Although further analysis is needed, our results position cefiderocol as a therapeutic option in pwCF infected with P. aeruginosa.

Funding

This study was supported by Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC) (CB21/13/00084 and CB21/13/00099) co-financed by the European Development Regional Fund ‘A way to achieve Europe’ (ERDF), PI19/01043 project, Instituto de Salud Carlos III, Madrid, Spain, and by a research grant from Shionogi (Ref. 2020/0352). A.M.-A. is supported by a pre-doctoral contract associated with PI19/01043 project. M.H.-G. is supported by a postdoctoral contract by CIBERINFEC (CB21/13/00084).

Transparency declarations

R.C. has participated in educational programmes organized by MSD, Pfizer and Shionogi, and has projects funded by Shionogi. Other authors declare no conflict of interest with the content of this article.

Supplementary data

Tables S1 to S6 are available as Supplementary data at JAC Online.

References