ORIGINAL RESEARCH published: 30 June 2022 doi: 10.3389/fmicb.2022.918362 CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3 Javier E. Cañada-García 1† , Zaira Moure 1† , Pedro J. Sola-Campoy 1 , Mercedes Delgado-Valverde 2,3 , María E. Cano 4 , Desirèe Gijón 3,5 , Mónica González 3,6 , Irene Gracia-Ahufinger 3,7 , Nieves Larrosa 3,8 , Xavier Mulet 3,9 , Cristina Pitart 10 , Alba Rivera 11 , Germán Bou 3,6 , Jorge Calvo 3,4 , Rafael Cantón 3,5 , Juan José González-López 3,8 , Luis Martínez-Martínez 3,7,12 , Ferran Navarro 11 , Antonio Oliver 3,9 , Zaira R. Palacios-Baena 2,3,13 , Álvaro Pascual 2,3,13 , Guillermo Ruiz-Carrascoso 14 , Jordi Vila 3,10 , Belén Aracil 1,3 , María Pérez-Vázquez1,3‡ , Jesús Oteo-Iglesias1,3* ‡ and Edited by: the GEMARA/GEIRAS-SEIMC/REIPI CARB-ES-19 Study Group§ Costas C. Papagiannitsis, 1 University of Thessaly, Greece Laboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain, 2 Unidad de Enfermedades Reviewed by: Infecciosas y Microbiología, Hospital Universitario Virgen Macarena, Instituto de Biomedicina de Sevilla (Hospital Marc Finianos, Universitario Virgen Macarena/CSIC/Universidad de Sevilla), Seville, Spain, 3 CIBER de Enfermedades Infecciosas Charles University, Czechia (CIBERINFEC), REIPI, Instituto de Salud Carlos III, Madrid, Spain, 4 Servicio de Microbiología, Hospital Universitario Marqués Tamara Salloum, de Valdecilla, IDIVAL, Santander, Spain, 5 Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Brigham and Women’s Hospital and Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain, 6 Servicio Microbiología, Hospital Universitario A Coruña, Instituto Harvard Medical School, Investigación Biomédica A Coruña (INIBIC), A Coruña, Spain, 7 Microbiology Unit, Reina Sofia University Hospital, United States Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Córdoba, Spain, 8 Departament de Genetica i Microbiologia, *Correspondence: Servei de Microbiologia, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain, 9 Servicio Jesús Oteo-Iglesias de Microbiología, Hospital Universitario Son Espases, Instituto de investigación sanitaria Illes Balears (IdISBa), Palma jesus.oteo@isciii.es de Mallorca, Spain, 10 Servicio de Microbiología, Hospital Clínic de Barcelona, ISGlobal Barcelona Institute for Global Health, † These authors have contributed Barcelona, Spain, 11 Microbiology Department, Hospital de la Santa Creu i Sant Pau, Universitat Autónoma de Barcelona equally to this work (UAB), Sant Pau Biomedical Research Institute (IIB Sant Pau), Barcelona, Spain, 12 Department of Agricultural Chemistry, Soil Science and Microbiology, University of Córdoba, Córdoba, Spain, 13 Departamento de Microbiología, Universidad ‡ These authors have contributed de Sevilla, Seville, Spain, 14 Servicio de Microbiología Clínica, Hospital Universitario La Paz (IdiPAz), Madrid, Spain equally to this work and share senior authorship § Members of the Group are listed in Objectives: CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating Acknowledgments whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical Specialty section: This article was submitted to distribution, phylogeny, and resistance mechanisms in Spain. Antimicrobials, Resistance and Chemotherapy, Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the a section of the journal first 10 isolates per hospital (February to May 2019); CPE isolates were first identified Frontiers in Microbiology according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, Received: 12 April 2022 Accepted: 23 May 2022 colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI- Published: 30 June 2022 TOF). Prevalence and incidence were calculated according to population denominators. Frontiers in Microbiology | www.frontiersin.org 1 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single- nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA−48 (263/377), blaKPC−3 (62/377), blaVIM−1 (28/377), and blaNDM−1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5). Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3. Keywords: CARB-ES-19 study, carbapenemases, whole genome sequencing, Klebsiella pneumoniae, high-risk clones INTRODUCTION The CARB-ES-19 project utilized the national antibiotic- resistance surveillance framework and was designed to provide The rapid spread of carbapenemase-producing Enterobacterales continuity with previous national, multicenter studies (Miró (CPE) is a threat to individual and public health worldwide; et al., 2013; Oteo et al., 2015). This large-scale, nationwide, infections caused by CPE significantly increase morbidity structured survey integrated whole-genome sequencing and mortality (Barrasa-Villar et al., 2017). The World (WGS) analyses of two CPE species with high clinical and Health Organization has included CPE as a critical priority epidemiological impacts, Klebsiella pneumoniae (CP-Kpn) and issue (Tacconelli et al., 2018). The carbapenemases most Escherichia coli (CP-Eco), to (i) determine the prevalence and frequently found in Enterobacterales are KPC, OXA- incidence of these microorganisms, (ii) describe their inter- 48, VIM, IMP, and NDM, although prevalence rates regional distribution and molecular epidemiology, and (iii) differ according to the geographical area considered describe their resistance mechanisms and susceptibility profiles. (Grundmann et al., 2017). Previous studies performed in Spain revealed the rapid evolution of CPEs from isolated cases in 2009 (Miró et al., MATERIALS AND METHODS 2013) to interregional dissemination in 2013 (Oteo et al., 2015). Recent European studies have indicated epidemiological changes Study Design and Isolates in CPE infections (Haller et al., 2019; Ludden et al., 2020; Oteo- CARB-ES-19 is a prospective, multicenter study designed to Iglesias et al., 2020; Di Pilato et al., 2021). According to the identify clinical cases associated with CP-Kpn and CP-Eco. In first structured survey on the occurrence of carbapenemase- total, 71 hospitals, representing all 50 Spanish provinces, collected producing K. pneumoniae and E. coli in European hospitals the first 10 non-duplicate consecutive isolates of carbapenem (Grundmann et al., 2017), Spain had the fourth highest non-susceptible CP-Eco or CP-Kpn isolated from clinical samples incidence in Europe (0.04 cases per 100 patients) after Italy, from individual consecutive patients between February and May Greece, and Montenegro. 2019. The geographical distribution of participating hospitals is However, recent multicenter studies with adequate available in the free and interactive online access tool Microreact.1 geographical representation are scarce, since regions with Not all participating hospitals had 10 CP-Eco or CP-Kpn low prevalence are often underrepresented. isolates during the study period. Isolates from rectal exudates for Accurate data collection at the national level is required for the successful implementation of CPE control measures. 1 https://microreact.org/project/vXbqq3eF5WXY5qZe9HBUJL-carb-es-2019 Frontiers in Microbiology | www.frontiersin.org 2 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain the detection of EPC carriers were not included. All provinces 2 × 150 bp paired-end reads (Illumina Inc.) Raw sequence (NUTS-3 regions in Spain) were represented by at least one data were submitted to the European Nucleotide Archive hospital; seven of the provinces with the largest population (PRJEB50822). The quality of short reads was assessed using were represented by more than one hospital (range 2–6). FASTQC, and assembly into contigs was performed with A similar study design was used by the European Centre for Unicycler 0.4.8 (Wick et al., 2017). The quality of the assembly Disease Prevention and Control (ECDC) in the European survey was assessed with QUAST.2 Prokka v1.14-beta (Seemann, 2014) of carbapenemase-producing Enterobacteriaceae (EuSCAPE; was used for automatic de novo assembly annotation. Grundmann et al., 2017). Initial assays were performed at each participating hospital Phylogenetic Analysis using standard microbiological methods. CPE isolates Assembly contigs were used as input for Roary version 3.13.0 were identified according to the European Committee on (Page et al., 2015). An alignment of 2,645 core genes (present Antimicrobial Susceptibility Testing (EUCAST) established in > 99% of isolates), comprising 2,415,034 bp, was generated for meropenem cutoff value for CPE (meropenem MIC > 0.12 mg/L; K. pneumoniae. Variable positions were extracted (85,696 single- European Society of Clinical Microbiology and Infectious nucleotide polymorphisms [SNPs]), and a maximum-likelihood Diseases [EUCAST], 2017). Confirmation of carbapenemase phylogenetic tree of SNPs was constructed using RAxML version production was verified using at least one EUCAST- 7.0.4 (Stamatakis, 2006) with a general time-reversible model recommended method (European Society of Clinical and gamma correction for site variation. The phylogenetic tree Microbiology and Infectious Diseases [EUCAST], 2017), and associated metadata were visualized using Microreact and such as immunochromatography, biochemical (colorimetric) iTOL.3 tests, carbapenem inactivation, or detection of carbapenem Sequence types (STs) were calculated according to multilocus hydrolysis with MALDI-TOF. sequence typing (MLST) schemes of the Institut Pasteur and In total, 10 hospitals and the Spanish National Centre the University of Warwick for K. pneumoniae and E. coli, of Microbiology (CNM) acted as node centers, performing respectively, using Ariba version 2.6.2 (Hunt et al., 2017). molecular confirmation of standard carbapenemase genes using A simple diversity index (SDI; Gastmeier et al., 2006) was PCR (Supplementary Table 1). Confirmed CPE isolates were applied to analyze population diversity. Core genome MLST submitted to the Antibiotic Reference Laboratory of the CNM (cgMLST) was performed, consisting of 2,538 K. pneumoniae for WGS. Prevalence was estimated as the proportion of targets provided by SeqSphere + version 3.5.0 (Ridom, Münsten, CP-Kpn and CP-Eco isolates relative to the total collected Germany). clinical K. pneumoniae and E. coli isolates, respectively. Overall cumulative incidence and incidence density estimates are Analysis of Antimicrobial Resistance, reported as the number of admitted patients diagnosed with either CP-Kpn or CP-Eco per 100 admitted patients and per 1,000 Virulence Genes, and Capsular Locus patient-days, respectively. The denominators for the incidence Antibiotic resistance genes were analyzed by SRST2 (Inouye and prevalence estimates were adjusted to the date on which the et al., 2014) using the ARG-ANNOT database (Gupta et al., last isolate included in the study was collected in each hospital. 2014) and ResFinder (CGE server4 ), with ID thresholds of 100% for β-lactamase variants and 98% for other genes. The K-locus and virulence genes were characterized using Kleborate.5 The Drug Susceptibility Testing presence of ybt, clb, and iuc was used to assign a virulence score, Antibiotic susceptibility testing was performed using the as described by Lam et al. (2021). broth microdilution susceptibility method (DKYMGN SensititreTM panels, Thermo Fisher Scientific, United States) Characterization of Plasmids Carrying (International Organization for Standardization [ISO], 2006). Antibiotic gradient strips were used to study susceptibility to Carbapenemase Genes meropenem/vaborbactam and cefepime (bioMérieux, Marcy- The plasmids carrying the carbapenemase genes in six l’Étoile, France) and to imipenem/relebactam, plazomicin, and representative K. pneumoniae isolates (ST307/KPC-3, cefiderocol (Liofilchem, Roseto degli Abruzzi, Italy) in Mueller ST512/KPC-3, ST512/KPC-23, ST147/NDM-1, ST307/OXA- Hinton agar (bioMérieux, Marcy-l’Étoile, France). EUCAST 48, and ST11/VIM-1) were reconstructed by the in-house v12.0 clinical breakpoints and guidelines for Enterobacterales script (PlasmidID6 ). PlasmidID was used to (i) map reads were used for interpretation. An FDA-approved susceptibility over a plasmid curated database to find those with the higher breakpoint of ≤ 2 mg/L was used for plazomicin. coverage and de novo assembly of these reads and (ii) make local alignments to localize resistance and replicative genes (Pérez-Vázquez et al., 2019a). Genomic Library Preparation and Sequence Analysis 2 http://quast.sourceforge.net/ Genomic DNA paired-end libraries were generated using the 3 https://iTOL.embl.de/ Nextera XT DNA Sample Preparation Kit (Illumina Inc., San 4 http://www.genomicepidemiology.org/? Diego, CA, United States). These libraries were sequenced 5 https://github.com/katholt/Kleborate using the Illumina HiSeq 500 next-generation sequencer with 6 https://github.com/BU-ISCIII/plasmidID Frontiers in Microbiology | www.frontiersin.org 3 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain Ethics Statement The most active antibiotics in vitro were cefiderocol This study was authorized by the Spanish Agency for Medicines (93.9% susceptibility), plazomicin (93.4%), colistin (90.5%), and Health Products (code JOI-AVI-2019-01). The Research meropenem/vaborbactam (89.4%), ceftazidime/avibactam Ethics Committee of the University Hospitals Virgen Macarena (84.1%), and imipenem/relebactam (78%; Table 1A). and Virgen del Rocio (Sevilla, Spain) approved this study. However, these numbers varied significantly depending on the carbapenemase type (Table 1B). In general, CP-Eco isolates were more susceptible to antibiotics (Supplementary Table 3) than CP-Kpn isolates, with RESULTS the greatest differences observed for tigecycline (84.6% in CP-Eco vs. 29.2% in CP-Kpn). Bacterial Isolates, Prevalence, Incidence, and Carbapenemase Types Of 403 CPE cases, 377 (93.5%) were identified as CP-Kpn and High-Risk Clones of 26 (6.5%) as CP-Eco. Patients were mainly men (211, 52.4%) Carbapenemase-Producing Klebsiella older than 65 years (280, 69.5%). CPE isolates were collected pneumoniae from urine (215, 53.3%), wounds and abscesses (61, 15.1%), CP-Kpn isolates were grouped into 48 STs (SDI: 12.7; mean: blood (52, 12.9%), respiratory samples (47, 11.7%), and other 7.8 isolates per ST; range: 1–82). A new ST was detected in an locations (28, 6.9%). OXA-48-producing K. pneumoniae strain. The most prevalent At least one case was identified in 62 (87.3%) of participating STs (≥10 isolates) were ST307 (82, 21.7%), ST11 (68, 18%), hospitals and in 46 (92%) of 50 Spanish provinces. Participating ST258/512 complex (4/48, 13.8%), ST15 (48, 12.7%), ST147 (36, hospitals isolated a total of 15,100 and 70,760 isolates of 9.5%), and ST392 (15, 4%), accounting for 79.7% of all CP-Kpn K. pneumoniae and E. coli, respectively. The average CP- isolates. ST307 and ST11 expressed four different carbapenemase Kpn prevalence was 2.5% (377/15100; interprovincial range: types (OXA-48, KPC, VIM-1, and NDM-1), but ST258/512 0–17.3%), with 16 (22.5%) hospitals reporting prevalences only expressed KPC. Among CP-Eco isolates, 21 different STs greater than 5%. The average prevalence of CP-Eco was were identified (SDI: 80.8; mean: 1.2 isolates per ST; range: 1– 0.04% (26/70,760; interprovincial range: 0–0.5%; Supplementary 4), with only ST131 (4 isolates, 15.4%) expressing more than Table 2). The prevalence distribution by province is detailed two carbapenemases. in Figure 1 and Supplementary Table 2. CP isolates were Considering ST/carbapenemase combinations, eight high-risk more prevalent in blood (CP-Kpn: 5.8%, 50/853; CP-Eco: 0.06%, CP-Kpn clones (≥ 7 isolates) were detected, with ST307/OXA-48 2/3,353) than in urine (CP-Kpn: 1.4%, 201/14,464; CP-Eco: (16.4%), ST11/OXA-48 (16.4%), ST512-ST258/KPC (13.8%), and 0.02%, 14/56,848). ST15/OXA-48-like (11.4%) as the most common combinations Overall, cumulative incidence and incidence density estimates (Table 2). These high-risk clones were detected in at least were 0.05 per 100 admitted patients (interprovincial range: 0– four hospitals and two autonomous communities, suggesting 0.34) and 0.08 per 1,000 patient-days (interprovincial range: interregional dissemination (Table 2). ST512-258/KPC and 0–0.58), respectively (Supplementary Table 2). ST15/OXA-48-like were the most frequent bacteremia-producing The carbapenemase genes detected in CP-Kpn were clones, responsible for 24 and 16% of CP-Kpn–induced blaOXA−48 (263 isolates, 69.8%), blaKPC−3 (62, 16.4%), blaVIM−1 bacteremia, respectively (Table 2). (28, 7.4%), blaNDM−1 (12, 3.2%), blaKPC−23 (7, 1.9%), blaKPC−2 (5, 1.3%), blaOXA−245 (3, 0.8%), with blaIMP−8 , blaGES−2 , blaNDM−3 , blaNDM−23 , and blaOXA−505 in one isolate each Phylogenetic Analysis of (0.3%). In CP-Eco, blaOXA−48 (19, 73.1%) was predominant, Carbapenemase-Producing Klebsiella followed by blaVIM−1 (5, 19.2%), blaKPC−3 (2, 7.7%), and pneumoniae blaNDM−5 (1, 3.8%). Eight CP-Kpn (two OXA-48 + VIM-1, A total of 92,608 high-quality SNPs, identified by referencing the two OXA-48 + KPC, and one each of OXA-48 + NDM-1, sequence of K. pneumoniae strain NTUH-K2044, were used to VIM-1 + NDM-1, NDM-1 + GES-2, and VIM-1 + NDM-3) construct a maximum-likelihood phylogenetic tree (Figure 2). and one CP-Eco (OXA-48 + VIM-1) isolates co-produced two The median pairwise distance between isolates was 9,678 SNPs carbapenemase types. (range: 0–13,254 SNPs). CP-Kpn isolates were grouped into six The Canary Islands and northern Spain had more OXA-48- major clusters (clusters 1–6; Figure 2). The main characteristics producing CP-Kpn, whereas southern Spain had more KPC- of these six clusters are detailed in Supplementary Table 4. producing CP-Kpn (Figure 1, Supplementary Table 2). The average difference between isolates from different clusters was 10,580 SNPs. Antibiotic Susceptibility Testing Genome assemblies of all CP-Kpn isolates, analyzed using the The antibiotic susceptibilities of CP-Kpn isolates are listed in gene-by-gene approach and allelic distance from cgMLST, are Tables 1 and 2. The overall rates of susceptibility to carbapenems reflected in a minimum-spanning tree (Figure 3). The average were 54.6% for imipenem, 52.3% for meropenem, and 3.4% for allelic distances in pairwise comparisons of isolates were 45 alleles ertapenem (Table 1A), with all isolates non-susceptible to at in cluster 1 (range: 0–235 alleles), 68 alleles in cluster 2 (range: 0– least one carbapenem. 49), 64 alleles in cluster 3 (range: 0–139), 21 alleles in cluster 4 Frontiers in Microbiology | www.frontiersin.org 4 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain FIGURE 1 | Geographical distribution of the prevalence of carbapenemase-producing Klebsiella pneumoniae according to carbapenemase groups. (range: 0–43), 30 alleles in cluster 5 (range: 0–106), and 39 alleles quinolone resistance qnr-like determinants were detected in 202 in cluster 6 (range: 0–64). (50.1%) isolates, with qnrB1-like (37.7%) as the most frequent. Applying a threshold of both 5 alleles and 10 SNPs, 12 Seven isolates contained colistin ARGs; mcr-9 in four CP- groups with more than four closely related isolates were Kpn and two CP-Eco and mcr-1 in one CP-Eco. However, all six detected, with ST512 (4), ST11 (4), and ST307 (3) commonly isolates harboring mcr-9 were susceptible to colistin, as previously identified (Figure 3). described (Macesic et al., 2021). There were 36 colistin-resistant CP-Kpn isolates, all of them showing amino acid substitution in both proteins of the PhoQ/PhoP regulatory system: D1509G and Identification and Distribution of H406Y in PhoQ protein and R114A and L26Q in PhoP (Elias Resistance and Virulence Genes et al., 2021). Additional MgrB mutations were detected in four An average of 7.9 acquired resistance genes (ARGs) were isolates: stop codon in L4 (3 ST512/KPC-3 isolates) and W47C (1 detected in CP-Kpn isolates (range: 1–20 ARGs), and 5.5 ARGs ST307/OXA-48 isolates). were detected in CP-Eco isolates (range: 1–18 ARGs). The The most prevalent ARGs associated with high-risk CP-Kpn most frequent extended-spectrum β-lactamase (ESBL) gene was clones are detailed in Table 2. blaSHV−28 allele was detected in blaCTX−M−15 (247 overall, 61.3%), detected in 240 CP-Kpn 96.3% of ST307 but only in 21.1% of other clones. Additionally, (63.7%) and 7 CP-Eco (26.9%). Other ESBL genes identified were the association of the rmtF and arr genes with ST147 was detected blaCTX−M−9 (7, 1.7%), blaSHV−12 (4, 1%), blaCTX−M−3 (2, 0.5%), in 52.8 and 66.7%, respectively, compared with 0 and 6.2% blaCTX−M−88 (2, 0.5%), and blaCTX−M−65 (1, 0.25%). in other clones. The predominant aminoglycoside resistance genes encoded CP-Kpn isolates belonged to 46 capsular polysaccharide loci N-acetyltransferases aac(6’)-Ib-cr (189, 46.9%), aac(3)-IIa (K-loci); 24 K-loci contained more than one isolate, and six (141, 35%), and aac(6’)-Ib (70, 17.4%). Acquired 16S rRNA included 78.3% of isolates, namely, KL102 (80), KL24 (75), KL107 methyltransferase rmtF1 was detected in 19 CP-Kpn (5%) (56), KL64 (39), KL112 (29), and KL27 (17). High correlations isolates, all belonging to ST147 and encoding blaOXA−48 (14) or between K-loci and STs were observed: all KL102 isolates were blaNDM−1 (5). ST307, all KL112 isolates were ST15, 92% of KL24 isolates were ARGs encoding resistance to chloramphenicol, sulfonamides, ST11, 87% of KL64 isolates were ST147, and 94.5% of KL107 trimethoprim, and tetracyclines were detected in 261 [64.7%; isolates were ST512-258. mainly catB3 (46.2%)], 280 [69.5%; sul1 (36.2%) and sul2 The yersiniabactin-encoding locus (ybt) was detected in (40.9%)], 252 [62%; mainly dfrA14 (42.7%)], and 106 [26.3%, 43% of CP-Kpn isolates, particularly ST11 (40.2%), ST147 mainly tetA (23.1%)] isolates, respectively. Plasmid-mediated (15.9%), and ST15 (14.6%). Nine different ybt lineages Frontiers in Microbiology | www.frontiersin.org 5 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain were identified, with ybt10, associated with ICEKp4 and detected in two isolates each (Supplementary Figure 1). One ST11, being the most frequent (58.5%; Supplementary ST147/NDM-1 isolate contained the rmpA2 gene associated with Figure 1). Colibactin (clb) and aerobactin (iuc) loci were a hypermucoid/hypervirulent phenotype. TABLE 1 | Antibiotic susceptibility of 377 carbapenemase-producing Klebsiella pneumoniae isolates as determined by the microdilution method and antibiotic gradient strips (antibiotics with *) according to EUCAST clinical breakpoints: (A) General results. (B) Results according to carbapenemase groups. A Antibiotics S (%) R (%) MIC50 a MIC90 a Rangea Cefiderocol* 93.9 6.1 0.12 1 ≤0.015–16 Plazomicin* 93.4 6.1 1 2 0.25 to >256 Colistin 90.5 9.5 1 2 0.5 to >8 Meropenem/vaborbactam* 89.4 10.6 0.5 16 ≤0.015 to >128 Ceftazidime/avibactam 84.1 15.9 2 >16 ≤0.5 to >16 Imipenem/relebactam* 77.4 22 1 8 0.12 to >32 Amikacin 72.9 27.1 8 >32 ≤4 to >32 Imipenem 54.6 36.9 2 >16 ≤0.5 to >16 Meropenem 52.3 28.4 2 >16 0.25 to >16 Gentamicin 45.6 54.4 4 >8 ≤0.5 to >8 Trimethoprim/sulfamethoxazole 26.8 71.1 >8 >8 ≤1 to >8 Tobramycin 25.7 74.3 >8 >8 ≤1 to >8 Aztreonam 14.9 83.3 >32 >32 ≤0.5 to >32 Cefepime* 11.9 84.1 32 >256 0.12 to >256 Ceftazidime 10.6 85.9 >16 >16 ≤0.5 to >16 Ceftolozane/tazobactam 9.8 90.2 >32 >32 ≤0.5 to >32 Cefotaxime 6.1 90.5 >8 >8 ≤0.5 to >8 Ciprofloxacin 5.6 94.2 >2 >2 ≤0.06 to >2 Ertapenem 3.4 96.6 >2 >2 0.25 to >2 B Antibiotic Susceptibility (%) OXA-48-group- KPC-group- MBL-group- producing isolates producing isolates producing isolates (n = 262) (n = 72) (n = 37) Cefiderocol* 95.8 86.1 94.6 Plazomicin* 93.5 98.6 86.5 Colistin 92.4 81.9 91.9 Meropenem/vaborbactam* 89.3 100 73 Ceftazidime/avibactam 95.4 90.3 0 Imipenem/relebactam* 75.2 100 56.7 Amikacin 86.6 33.3 56.8 Imipenem 68.3 13.9 43.2 Meropenem 64.9 13.9 45.9 Gentamicin 47.3 48.6 32.4 Trimethoprim/sulfamethoxazole 34.7 6.9 13.5 Tobramycin 32.8 9.7 8.1 Aztreonam 17.6 0 27 Cefepime* 16.8 0 0 Ceftazidime 15.3 0 0 Ceftolozane/tazobactam 14.1 0 0 Cefotaxime 8.4 0 0 Ciprofloxacin 7.6 1.4 0 Ertapenem 2.3 0 16.2 S, susceptible, standard dosing regimen; R, resistant; MIC, minimum inhibitory concentrations. a Expressed in mg/L. Isolates with two carbapenemases of different groups are excluded. Frontiers in Microbiology | www.frontiersin.org 6 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain TABLE 2 | High-risk carbapenemase-producing Klebsiella pneumoniae clones defined according to combinations of sequence type/carbapenemase. High-risk clones Hospitals Geographical Evolution trends Representation in Carbapenemase Other prevalent acquired resistance (n) (n) distribution* (%) bacteremia (%) genes (%) genes (%) 2013→2019** ST307/OXA-48 (62) 23 Andalucía, Castilla La 1.4→16.4 14 blaOXA−48 (100) blaSHV−28 (95.1), blaCTX−M−15 (88.5), Mancha, Castilla y León, aac(6’)-Ib-cr (78.7), qnrB1 (78.7), ant(3’)-Ia Extremadura, Cataluña, (75.4), sul2 (73.8), blaOXA−1 (73.8), Galicia, Canarias, País aph(3”)-Ib (73.8), aph(6)-Id (73.8), catB3 Vasco, Comunidad (73.8%), dfrA14 (73.8), aac(3)-IIa (72.1), Valenciana, Madrid blaTEM−1b (70.5). ST11/OXA-48 (62) 22 Andalucía, Castilla La 24.1→16.4 12 blaOXA−48 (100), blaCTX−M−15 (96.7), blaSHV−182 (95.1), Mancha, Castilla y León, blaOXA−1 (60.7), aac(6’)-Ib-cr (63.9), catB3 Cantabria, Cataluña, La (63.9), aac(3)-IIa (55.7), qnrB1-like (57.4). Rioja, Canarias, País Vasco, Madrid ST512-ST258/KPC 10 Andalucía, Castilla La 0→13.8 24 blaKPC−3 (86.5) blaSHV−182 (97.9), aph(3”)-Ia (95.8), (52) Mancha, Comunidad blaKPC−23 (13.5) blaOXA−9 (95.8), aac(6’)-Ib (93.8), ant(3’)-Ia Valenciana, Cataluña, (93.8), sul1 (93.8), dfrA12 (93.3), catA1 Madrid (91.7), dfrA12 (91.7), mphA (91.7), blaTEM−187 (64.6). ST15/OXA-48 like 16 Andalucía, Castilla La 8.2→11.4 16 blaOXA−48 (93) blaSHV−28 (100), ant(3’)-Ia (83.7), blaOXA−1 (43) Mancha, Madrid, Aragón, blaOXA−245 (7) (74.4%), blaCTX−M−15 (72.1), aac(6’)-Ib-cr Cataluña, Canarias, País (76.7), catB3 (74.4), dfrA14 (74.4), Vasco, Galicia aph(3”)-Ib (60.5), aph(6)-Id (58.1), sul2 (58.1), blaTEM−1b (53.5). ST147/OXA-48 (22) 10 Andalucía, Cataluña, 2.5→5.8 0 blaOXA−48 (95.5) ant(3’)-Ia (90.9), blaCTX−M−15 (86.4), Galicia, País Vasco, blaOXA−505 (4.5) blaSHV−67 (86.4), aac(6’)-Ib (72.7), AAR-2 Asturias, Navarra (72.7), rmtF (63.6), qnrB1 (47.6), dfrA14 (68.2), mphA (63.6), dfrA12 (59.1), sul1 (59.1). ST307/KPC-3 (15) 4 Madrid, Extremadura, 0→4 4 blaKPC−3 (100) blaCTX−M−15 (100), blaOXA−1 (100), Castilla La Mancha blaOXA−9 (100), aac(6’)-Ib-cr (100), aac(3)-IIa (100), ant(3’)-Ia (100), aph(3”)-Ib (100), aph(6)-Id (100), catB3 (100), dfrA14 (100), blaSHV−67 (100), sul2 (100), qnrB1-like (71.4). ST392/OXA-48 (14) 11 Andalucía, Castilla La 0→3.7 4 blaOXA−48 (100). blaSHV−67 (100), aph(3”)-Ib (92.9), aph(6)-Ib Mancha, Madrid, Cataluña, (92.9), sul2 (86.7), blaTEM1−b (85.7), Comunidad Valenciana, blaCTX−M−15 (78.6), dfrA14 (50), qnrB1 Galicia (40), aac(6’)-Ib-cr (57.1), blaOXA−1 (50), catB3 (50). ST147/NDM-1 (7) 4 Cataluña, Galicia 0→1.9 2 blaNDM−1 (100) aadA1(100), ARR(100), blaCTX−M−1−group (85.7), blaSHV−67 (100), sul1 (100), aac(6’)-Ib (71.4), dfrA12 (71.4), dfrA14 (71.4), qnrB1 (71.4), rmtF1 (71.4), aph(3”)-lb (57.1), aph(6)-ld (57.1). *Autonomous Communities in which the high-risk clones were detected. **Evolution trends detected based on the results of a previous Spanish study conducted in 2013 (Oteo et al., 2015) relative to the study in 2019. Characterization of Plasmids Harboring was 5,943 bp, with the sequence groL-groS-nagA-trpF-ble- Carbapenemases Genes blaNDM−1 -(trun)ISAba, which is similar to the previously described pNDM-11_IncFIB_KPN_Spain (Pérez-Vázquez et al., The plasmidID mapping tool reconstructed three IncFIB plasmids carrying blaKPC genes, all of them highly similar to 2019a). NZ_CP027056 of ∼90,000 bp (average identity > 95 and 99.9% The plasmids detected carrying blaOXA−48 (ST307/OXA-48 coverage). In these three plasmids, blaKPC genes were carried in isolate) and blaVIM−1 (ST11/VIM-1 isolate) were IncL. The the transposon Tn4401b, which was modified by the insertion of first one was highly similar to NZ_CP023251 (∼63,000 bp, Tn5403, similar to that previously described (Rada et al., 2020). average identity > 95% and 100% coverage) and the other to In the ST147/NDM-1 isolate, blaNDM−1 was carried NZ_CP023419 (∼70,000 bp, average identity > 95% and 100% by an IncFIB plasmid of ∼105,000 bp (highly similar to coverage). The blaOXA−48 gene was located in a Tn1999 in NZ_CM008884, average identity > 95% and 99.82% coverage). which lysR and blaOXA−48 were flanked by two copies of IS1999 The largest genetic environment constructed for this gene (Mairi et al., 2018). The blaVIM−1 gene was located in the class Frontiers in Microbiology | www.frontiersin.org 7 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain FIGURE 2 | Population structure of carbapenemase-producing Klebsiella pneumoniae isolates: maximum-likelihood tree showing the relationship between isolates, with more frequent STs displayed in the inner ring and labeled. The inner ring colors indicate the carbapenemase type (CP type); the outer ring colors indicate geographical distribution (CCAA: autonomous communities); and branch lengths are indicative of the number of SNPs. 1 integron In624, showing the sequence Int1-blaVIM−1 -aacA4- The overall incidence of CP-Kpn and CP-Eco increased by dfrB1-aadA1-catB2-qacEdelta1-sul1 (Villa et al., 2014). 25%, from 0.04 cases per 100 patients in 2014 (Grundmann et al., 2017), with 13 provinces reporting incidences at least 2.5- fold higher in this study than the general one found in 2014. DISCUSSION Compared with the 2.5% CP-Kpn prevalence reported here, the prevalences detected in 2009 and 2013 in Spain were significantly We conducted a comprehensive analysis using WGS in CP- lower, at 0.2% (Miró et al., 2013) and 1.7% (Oteo et al., 2015), Kpn and CP-Eco isolates in Spain, revealing an overall respectively. Increased prevalence was accompanied by a wide cumulative incidence of 0.05 per 100 admitted patients and geographical spread, with strains detected in 92% of the 50 the wide interregional dissemination of high-risk clones. The Spanish provinces and the presence of seven “high-risk clones” in study’s strengths include representation of all Spanish provinces, at least three provinces. In a recent Italian study (Di Pilato et al., application of population denominators for prevalence and 2021), 80% of 30 participating hospitals reported CP-Kpn cases. incidence calculations, and the possibility of establishing The large difference in CP-Kpn prevalences between provinces evolutionary trends compared with previous studies using a detected in this study (four provinces had prevalences > 10%, similar methodology conducted by the same research team (Miró Supplementary Table 2) highlights the importance of designing et al., 2013; Oteo et al., 2015; Grundmann et al., 2017). Although a study with representation from all geographical regions and the use of only 10 strains per hospital might be a limitation, this with a design that minimizes the possible overrepresentation of approach aligns with ECDC strategies (Grundmann et al., 2017), a specific clone or a region due to the existence of an outbreak. In avoiding the overrepresentation of geographical regions/clones this sense, our study provides a realistic and representative view observed in other studies. of the Spanish situation, minimizing possible biases. Frontiers in Microbiology | www.frontiersin.org 8 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain FIGURE 3 | Population structure of carbapenemase-producing Klebsiella pneumoniae: minimum-spanning tree. Distances shown are based on cgMLST of 2,358 genes using the parameter “pairwise ignoring missing values.” Colors in each circle indicate carbapenemase type. Red ovals represent clusters. Circles can correspond to more than one isolate, indicating that they are identical. Gray shadows represent clusters of strains, applying a threshold of both 5 SNPs and 10 alleles. Our results confirm previous reports of the preponderance of in 2013 (Oteo et al., 2015) to 16.4%, making it the predominant OXA-48 carbapenemase in Spain (detected in 82% of Spanish clone with a wide geographical distribution. Other recent studies provinces; Oteo et al., 2015; Grundmann et al., 2017; Vázquez- also have identified that ST307 is an emerging clone worldwide Ucha et al., 2021). The blaOXA−505 gene (OXA-48 family, (Wyres et al., 2019; Di Pilato et al., 2021), and the hyperepidemic GenBank number: NG_049783) was detected in a carbapenem clonal complex ST258/ST512 is widely predominant in Italy non-susceptible isolate with a positive colorimetric lacking and Greece (David et al., 2019; Di Pilato et al., 2021). This other genes encoding carbapenemases, so it was included in epidemiological shift is associated with the increased CP-Kpn the study despite the absence of published evidence on its population diversity, from an SDI of 10.6 in 2013 (Oteo et al., carbapenemase activity. 2015) to 12.7 in this study. Regarding the NDM-producers, the New to this study was the observation of significant main high-risk K. pneumoniae clone ST147/NDM-1 detected in epidemiological evolution among CP-Kpn strains, from the this study (Table 2) was the cause of one of the great NDM- predominance of the OXA-48–producing ST11 and ST405 1-producing K. pneumoniae outbreaks previously described in (Oteo et al., 2015) to the emergence of ST307/OXA-48 and Spain (Pérez-Vázquez et al., 2019b). the dispersion of ST512/KPC (detected in seven provinces in CPE bacteremia is associated with high mortality (Tamma Southern Spain). Compared with a multicenter study conducted et al., 2017), mainly associated with delays in adequate in 2013 (Oteo et al., 2015), our study shows a significant increase treatment (Gutiérrez-Gutiérrez et al., 2017). This study showed in all high-risk clones, except ST11/OXA-48, which decreased a significantly higher prevalence of CP-Kpn in bacteremia in frequency from 24.1 to 16.4%, and ST15, which remained than in the total infections considered altogether (more than stable (Table 2). The frequency of ST307 increased from 1.4% double) or urinary tract infections (more than fourfold), with Frontiers in Microbiology | www.frontiersin.org 9 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain ST512/KPC and ST15/OXA-48 causing 40% of CP-Kpn–induced DATA AVAILABILITY STATEMENT bacteremia. Accurate and timely diagnosis could be critical in providing effective care. The datasets presented in this study can be found in online Regarding the level of resistance to carbapenem antibiotics repositories. The names of the repository/repositories as the main target of carbapenemases, it should be noted and accession number(s) can be found in the that the susceptibility profile of meropenem and/or imipenem article/Supplementary Material. susceptibility with ertapenem resistance was frequently detected in this study. This profile was mainly due to the high prevalence of OXA-48 isolates, although it can also be found in VIM-1 ETHICS STATEMENT producers (Oteo et al., 2015; Vázquez-Ucha et al., 2021). Vaborbactam and relebactam do not inhibit metallo- The studies involving human participants were reviewed and β-lactamases; however, differences in susceptibility were approved by the Spanish Agency for Medicines and Health observed between imipenem/imipenem-relebactam and Products (code JOI-AVI-2019-01) and the Research Ethics meropenem/meropenem-vaborbactam in this collection Committee of the University Hospitals Virgen Macarena and (Table 1B). These discrepancies were due to the different cutoff Virgen del Rocio (Sevilla, Spain). Written informed consent from points established by EUCAST in the case of meropenem- the participants’ legal guardian/next of kin was not required vaborbactam and to the five isolates that had MICs of 4 mg/L and to participate in this study in accordance with the national 2 mg/L for imipenem and imipenem/relebactam, respectively. legislation and the institutional requirements. New antibiotics (cefiderocol, plazomicin, meropenem/vaborbactam, and imipenem/relebactam) have significantly improved the treatment options for CPE infections GEMARA/GEIRAS-SEIMC/REIPI (Doi, 2019). In our study, all CP-Kpn isolates showed > 75% CARB-ES-19 STUDY GROUP susceptibility to these antibiotics. In accordance with previous studies (Doi, 2019), plazomicin was not active against ST147 Members of the GEMARA/GEIRAS-SEIMC/REIPI CARB-ES-19 isolates carrying rmtF1 (OXA-48 or NDM-1 producers), and Collaborating Group are as follows: meropenem/vaborbactam and imipenem/relebactam activities Mariela Martínez Ramírez (H. Universitario de Guadalajara, were higher in KPC-producers compared with OXA-48 and Guadalajara); Pilar Zamarrón (Hospital Virgen de la Salud, metallo-β-lactamases producers. As previously described Toledo); Miriam Albert Hernández (Hospital Virgen de la (Yamano, 2019), cefiderocol showed good activity in vitro against Concha Complejo Asistencial de Zamora, Zamora); M. Pilar CP-Kpn, irrespective of the carbapenemase types. The seven Ortega Lafont (Complejo Asistencial Universitario de Burgos, KPC-23-producing isolates were resistant (CMI > 16 mg/L) Burgos); Emilia Cercenado (H. General Universitario Gregorio to ceftazidime/avibactam. This carbapenemase was previously Marañón, CIBERES Madrid); Cristobal del Rosario and Jose associated with a decrease in ceftazidime/avibactam susceptibility Luis Perez Arellano (Hospital Universitario Insular Materno (Galani et al., 2019). Overall susceptibility to colistin and Infantil de Gran Canaria, Las Palmas); María Lecuona (Hospital meropenem decreased from 95.5 to 81.3%, respectively, in 2013 Universitario de Canarias, La Laguna, Sta. Cruz Tenerife); (Oteo et al., 2015) to 90.5% and 52.3% in this study, due primarily Luis López-Urrutia Lorente (H. Universitario Río Hortega, to the rise in KPC and NDM. The rate of colistin resistance was Valladolid); José Leiva and José Luis del Pozo (Clínica lower than previously reported, associated with KPC-producers Universidad de Navarra, Navarra); Salvador Giner and Juan (Di Pilato et al., 2021) but was consistent with the rate reported Frasquet (H. Universitario La Fe de Valencia, Valencia); Lidia in a recent Spanish study (Vázquez-Ucha et al., 2021). Garcia Agudo and Soledad Illescas (H General Universitario The increasing worldwide dispersion of carbapenemases de Ciudad Real, Ciudad Real); Pedro de la Iglesia (Hospital is due to a mixed spread: i) clonal, with the existence of de Cabueñes, Asturias); Rosario Sánchez Benito (Hospital San high-risk clones predominant in the carbapenemase-producing Pedro de Alcántara, Cáceres); Eugenio Garduño (Hospital K. pneumoniae population, and ii) polyclonal, with the spread of Universitario Badajoz, Badajoz); Ma Isabel Fernández Natal conjugative epidemic plasmids capable of carrying the different and Marta Arias (Complejo Asistencial Universitario de León, carbapenemases genes (Villa et al., 2014; Mairi et al., 2018; Pérez- León); Marta Lamata Subero (Fundación Hospital de Calahorra Vázquez et al., 2019a; Kraftova et al., 2021). Megalab, La Rioja); Mar Olga Pérez Moreno (H. Verge de Our study elucidates the epidemiology of CP-Kpn and CP- la Cinta de Tortosa, Tarragona); Ana Isabel López-Calleja (H. Eco in Spain using WGS in 403 clinical isolates, representing a Universitario Miguel Servet, Zaragoza); Luis Torres Sopena (H. first step toward the integration of WGS in CPE surveillance in San Jorge de Huesca, Huesca); José Manuel Azcona (H. San Spain, compliant with the high-priority recommendations of the Pedro, La Rioja); Alba Belles and Mercè García González (H. ECDC (ECDC, 2019). Our findings will aid in the development Universitario Arnau de Vilanova, Lleida); Miriam Valverde Troya of the Network of Laboratories for the Surveillance of Resistant and Begoña Palop (Hospital Regional Universitario de Málaga, Microorganisms (RedLabRA; Cañada-García et al., 2021). The Málaga); Fernando García Garrote (Hospital Universitario data generated by this study may serve as a reliable benchmark Lucus Augusti, Lugo); Jose Luis Barrios Andrés and Leyre for CP-Kpn status in Spain in the year before the COVID-19 López Soria (Hospital Universitario Cruces, Vizcaya); Adelina pandemic, facilitating implementation of control measures. Gimeno (H. General Universitario de Alicante, Alicante); Frontiers in Microbiology | www.frontiersin.org 10 June 2022 | Volume 13 | Article 918362 Cañada-García et al. Carbapenemase-Producing K. pneumoniae and E. coli, Spain Susana Sabater (Hospital General de Castellón, Castellón); Ester AUTHOR CONTRIBUTIONS Clapés Sanchez (Hospital Dr. Josep Trueta de Girona, Girona); Jennifer Villa (H. Universitario 12 de Octubre, Madrid); Nuria JO-I and MP-V conceived and designed the study. JC-G, Iglesias Nuñez and Rafael Sánchez Arroyo (Hospital Nuestra ZM, ÁP, ZP-B, AO, JV, RC, GB, JG-L, FN, LM-M, Señora de Sonsoles Ávila); Inmaculada García García (Complejo GR-C, and JO-I coordinated the study. JC-G, PS-C, Hospitalario de Salamanca, Salamanca); Susana Hernando (Real MD-V, MC, DG, MG, IG-A, NL, XM, CP, AR, BA, and Hospital General de Segovia, Segovia); Cristina Seral and Javier GEMARA-SEIMC/REIPI CARB-ES-19 Study Group performed Castillo (H. Clínico Universitario Lozano Blesa, Zaragoza); Eva the experiments. ZM, JC, MP-V, and JO-I wrote the Riquelme Bravo, Caridad Sainz de Baranda, and Oscar Esparcia manuscript. All authors have read, edited, and approved the Rodríguez (C. Hospitalario Universitario de Albacete, Albacete); final manuscript. Jorge Gaitán, María Huertas (Hospital General La Mancha Centro Alcazar, Ciudad Real); M.a José Rodríguez Escudero (Hospital General Virgen de La Luz, Cuenca); Carmen Aldea FUNDING and Nerea Sanchez (Complejo Asistencial de Soria, Soria); Antonio Casabella Pernas (Hospital Universitari Parc Taulí, This research was supported by grants from the Instituto Institut d’Investigació i Innovació Parc Taulí I3PT, Universitat de Salud Carlos III (numbers PI18CIII/00030 and Autònoma de Barcelona, Sabadell, Barcelona); Ma Dolores PI21CIII/00039). It was also supported by Plan Nacional Quesada (H. Germans Trias I Pujol, Badalona Barcelona); Maria de I + D + i 2013–2016, Instituto de Salud Carlos III, Pilar Chocarro and Francisco Javier Ramos (Hospital Obispo Subdirección General de Redes y Centros de Investigación Polanco, Teruel); Carmina Martí Sala (H. General de Granollers, Cooperativa, Ministerio de Economía y Competitividad, Barcelona); Laura Mora, Encarnación Clavijo (H. Virgen de Spanish Network for Research in Infectious Diseases (grants la Victoria, Málaga); Natalia Chueca and Federico García RD16CIII/0004/0002, RD16/0016/0001, RD16/0016/0003, (H. Clínico San Cecilio, Granada); José Gutierrez Fernández RD16/0016/0004, RD16/0016/0006, RD16/0016/0007, (Hospital Virgen de las Nieves, Granada); Juan Manuel Sánchez RD16/0016/0008, RD16/0016/0010, and RD16/0016/0011). Hospital de Jérez (Jerez de la Frontera, Cádiz); Fátima Galán Cofinanced by the European Development Regional Fund “A way Sánchez (H. Puerta del Mar, Cádiz); Carmen Liébana and to achieve Europe,” Operative Program Intelligent Growth 2014– Carolina Roldán (Hospital Universitario de Jaén, Jaén); Ma 2020. CIBER – Consorcio Centro de Investigación Biomédica Isabel Cabeza (Hospital de Poniente, Almería); José María en Red (CB21/13/00095, CB21/13/00012, CB21/13/00049, Saavedra (Hospital Juan Ramón Jimenez Huelva); Ma Teresa CB21/13/00054, CB21/13/00055, CB21/13/00068, CB21/13/00081, Cabezas Fernández (Complejo Hospitalario de Torrecárdenas, CB21/13/00084, and CB21/13/00099) (CIBERINFEC) and Almería); Lucía Martínez Lamas and Sonia Rey Cao (Complejo Instituto de Salud Carlos III, Ministerio de Ciencia e Hospitalario Universitario de Vigo, Pontevedra); Ma Isabel Innovación and Unión Europea – NextGenerationEU also Paz Vidal (Complejo Hospitalario de Orense, Orense); Raquel supported this work. Elisa Rodríguez Tarazona (Hospital Santos Reyes, Aranda de Duero, Burgos); Amparo Coira Nieto and Ma Luisa Pérez del Molino Bernal (Hospital Clínico Universitario de ACKNOWLEDGMENTS Santiago de Compostela A Coruña); María Gomáriz Díaz (H. Universitario de Donostia, Guipuzcoa); Matxalen Vidal- We thank all participating hospitals and the Genomics García and Jose Luis Díaz de Tuesta (Hospital Universitario Unit of the Centro Nacional de Microbiología for support de Basurto, IIS Biocruces, Bizkaia); Moises García Bravo and with DNA sequencing. Almudena Tinajas (H. General Río Carrión, Palencia); Andrés Canut Blasco and Ma Luz Albina Cordón Rodriguez (H. Universitario de Álava, Álava); Nieves Gonzalo Jiménez (H. SUPPLEMENTARY MATERIAL General Universitario de Elche, Alicante); Genoveva Yagüe The Supplementary Material for this article can be found Guirao (Hospital Virgen de la Arrixaca de Murcia, Murcia); Fe online at: https://www.frontiersin.org/articles/10.3389/fmicb. Tubau Quintano (Hospital Universitario de Bellvitge, Barcelona); 2022.918362/full#supplementary-material Carmen Aspiroz (H. Royo Villanova Zaragoza); Nuria Prim (H. del Mar, Barcelona); Jesús Rodríguez-Baño (H. 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In Vitro Activity of Cefiderocol Against a Broad Range of doi: 10.3389/fmicb.2022.918362 Clinically Important Gram-negative Bacteria. Clin. Infect. Dis. 69, S544–S551. doi: 10.1093/cid/ciz827 Copyright © 2022 Cañada-García, Moure, Sola-Campoy, Delgado-Valverde, Cano, Gijón, González, Gracia-Ahufinger, Larrosa, Mulet, Pitart, Rivera, Bou, Calvo, Conflict of Interest: The authors declare that the research was conducted in the Cantón, González-López, Martínez-Martínez, Navarro, Oliver, Palacios-Baena, absence of any commercial or financial relationships that could be construed as a Pascual, Ruiz-Carrascoso, Vila, Aracil, Pérez-Vázquez, Oteo-Iglesias and the potential conflict of interest. GEMARA/GEIRAS-SEIMC/REIPI CARB-ES-19 Study Group. This is an open-access article distributed under the terms of the Creative Commons Attribution License Publisher’s Note: All claims expressed in this article are solely those of the authors (CC BY). 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