Articles Pseudomonas aeruginosa antibiotic susceptibility profiles, genomic epidemiology and resistance mechanisms: a nation-wide five-year time lapse analysis Miquel Àngel Sastre-Femenia,a Almudena Fernández-Muñoz,a María Antonia Gomis-Font,a Biel Taltavull,a Carla López-Causapé,a Jorge Arca-Suárez,b Luis Martínez-Martínez,c Rafael Cantón,d Nieves Larrosa,e Jesús Oteo-Iglesias,f Laura Zamorano,a and Antonio Oliver,a,∗ on behalf of the GEMARA-SEIMC/CIBERINFEC Pseudomonas study Group a Servicio de Microbiología, Hospital Son Espases, Instituto de Investigación Sanitaria Illes Balears (IdISBa), CIBERINFEC, Palma de Mallorca, España b Servicio de Microbiología, Complexo Hospitalario Universitario A Coruña, Instituto Investigación Biomédica A Coruña (INIBIC), CIBERINFEC, A Coruña, España c Unidad de Gestión Clínica de Microbiología, Hospital Universitario Reina Sofía, Departamento de Química Agrícola, Edafología y Microbiología, Universidad de Córdoba, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), CIBERINFEC, Córdoba, España d Servicio de Microbiología, Hospital Universitario Ramón y Cajal- IRYCIS, CIBERINFEC, Madrid, España e Servicio de Microbiología, Hospital Universitario Vall d`Hebron, Vall d’Hebron Institut de Recerca (VHIR), Departamento de Genética y Microbiología, Universitat Autònoma de Barcelona, CIBERINFEC, Barcelona, España f Centro Nacional de Microbiología, CIBERINFEC, Instituto de Salud Carlos III, Madrid, España Summary The Lancet Regional Health - Europe Background Pseudomonas aeruginosa healthcare-associated infections are one of the top antimicrobial resistance 2023;34: 100736 threats world-wide. In order to analyze the current trends, we performed a Spanish nation-wide high-resolution Published Online 19 analysis of the susceptibility profiles, the genomic epidemiology and the resistome of P. aeruginosa over a five-year September 2023 time lapse. https://doi.org/10. 1016/j.lanepe.2023. Methods A total of 3.180 nonduplicated P. aeruginosa clinical isolates from two Spanish nation-wide surveys 100736 performed in October 2017 and 2022 were analyzed. MICs of 13 antipseudomonals were determined by ISO-EUCAST. Multidrug resistance (MDR)/extensively drug resistance (XDR)/difficult to treat resistance (DTR)/ pandrug resistance (PDR) profiles were defined following established criteria. All XDR/DTR isolates were subjected to whole genome sequencing (WGS). Findings A decrease in resistance to all tested antibiotics, including older and newer antimicrobials, was observed in 2022 vs 2017. Likewise, a major reduction of XDR (15.2% vs 5.9%) and DTR (4.2 vs 2.1%) profiles was evidenced, and even more patent among ICU isolates [XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%)] (p < 0.001). The prevalence of Extended-spectrum β-lactamase/carbapenemase production was slightly lower in 2022 (2.1%. vs 3.1%, p = 0.064). However, there was a significant increase in the proportion of carbapenemase production among carbapenem- resistant strains (29.4% vs 18.1%, p = 0.0246). While ST175 was still the most frequent clone among XDR, a slight reduction in its prevalence was noted (35.9% vs 45.5%, p = 0.106) as opposed to ST235 which increased significantly (24.3% vs 12.3%, p = 0.0062). Interpretation While the generalized decrease in P. aeruginosa resistance, linked to a major reduction in the preva- lence of XDR strains, is encouraging, the negative counterpart is the increase in the proportion of XDR strains producing carbapenemases, associated to the significant advance of the concerning world-wide disseminated hypervirulent high-risk clone ST235. Continued high-resolution surveillance, integrating phenotypic and genomic data, is necessary for understanding resistance trends and analyzing the impact of national plans on antimicrobial resistance. Funding MSD and the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea— NextGenerationEU. DOI of original article: https://doi.org/10.1016/j.lanepe.2023.100740 *Corresponding author. E-mail address: antonio.oliver@ssib.es (A. Oliver). www.thelancet.com Vol 34 November, 2023 1 Articles Copyright © 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Pseudomonas aeruginosa; Antimicrobial resistance; Surveillance; Genomic epidemiology; Resistome Research in context Evidence before this study Added value of this study We searched PubMed, without language restrictions, for Taking advantage of two large-scale surveys with identical articles published before October 1st 2022 using the terms methodology, we performed a Spanish nationwide high- “Pseudomonas aeruginosa” and “surveillance” and “resistance” resolution analysis of the trends in the susceptibility profiles, and “MDR”, “XDR” or “DTR”. We identified multiple global the genomic epidemiology and resistance mechanisms of surveillance programs that analyzed P. aeruginosa resistance P. aeruginosa isolates from hospital-acquired infections over a trends, including concerning MDR (multidrug resistance), XDR five-year time lapse. Positive findings included a sharp (extensively drug resistance), and/or DTR (difficult to treat generalized decrease in resistance to older and newer resistance) phenotypes, and some of them used whole antipseudomonals, as well as a dramatic decrease in the genome sequencing (WGS) to identify globally disseminated prevalence of XDR P. aeruginosa. The negative counterpart clones and associated resistance mechanisms. We identified was a significant increase in the proportion of XDR and also some national surveys (including Spain) that analyzed the carbapenem resistant strains producing concerning prevalence of resistance that used as well WGS to decipher the horizontally-acquired carbapenemases, linked to a significant involved mechanisms and to identify widespread MDR/XDR/ progression of the world-wide disseminated hypervirulent DTR strains. All these studies have helped to settle the current high-risk clone ST235. knowledge on the global burden of P. aeruginosa antimicrobial Implications of all the available evidence resistance genomics and epidemiology, by integrating Continued high-resolution surveillance, integrating both, information from different time points and places. We did not phenotypic and genomic data, is necessary for understanding identify however any study that analyzed at a nationwide resistance trends and analyzing the impact of national and scale how resistance phenotypes, mechanisms and global plans on antimicrobial resistance. epidemiology may change over a time lapse. Introduction evidence of the existence of XDR/DTR global clones, Pseudomonas aeruginosa, is among the main causes of denominated high-risk clones, disseminated in hospi- hospital-acquired and chronic infections and is associ- tals worldwide.9–11 Moreover, beyond classical molecular ated with high antimicrobial resistance, morbidity and epidemiology analysis and phenotypic assessment of mortality.1 Indeed, antibiotic-resistant P. aeruginosa in- resistance mechanisms, Whole Genome Sequencing fections are estimated to be associated with over (WGS) studies are providing relevant information for 300,000 annual deaths and are at the top of the WHO building up the complex and evolving resistome of priority list for the need for research and development MDR/XDR P. aeruginosa high-risk clones.12–15 of new antibiotics.2,3 This growing threat results from The recent introduction of newer β-lactam/β-lacta- the extraordinary capacity of this pathogen for devel- mase inhibitors combinations (such as ceftolozane/ oping resistance through chromosomal mutations and tazobactam, ceftazidime/avibactam or imipenem/rele- from the increasing prevalence of transferable resis- bactam) has helped to mitigate, to some extent, the tance determinants, particularly those encoding car- problem of XDR/DTR P. aeruginosa.16,17 Indeed, these bapenemases or extended-spectrum β-lactamases agents show increased stability against the classic (ESBLs).4,5 Combinations of such mechanisms lead to P. aeruginosa chromosomally-encoded β-lactam resis- concerning and complex resistance profiles as defined tance mechanisms, such as the overexpression of the by the European Centre for Disease Prevention Control chromosomal β-lactamase AmpC and efflux pumps or (ECDC), [multidrug resistance (MDR), extensively drug OprD inactivation. However they are not exempt from resistance (XDR), and pandrug resistance (PDR)] or the resistance development through emerging mutational infectious Diseases Society of America (IDSA) [difficult mechanisms, such as the modification of the catalytic to treat resistance (DTR)].6,7 P. aeruginosa has a non- center of AmpC or the modification of efflux pumps clonal epidemic population structure, composed of a substrate specificity, evidenced right upon their intro- limited number of widespread clones, which are duction into clinical practice.18–21 Likewise, they are not selected from a background of a large quantity of rare active against most potent transferable carbapenemases, and unrelated genotypes that are recombining at high particularly the metallo-β-lactamases (MBLs), and thus frequency.8 Indeed, several surveys have provided their use may lead to the selection of such concerning 2 www.thelancet.com Vol 34 November, 2023 Articles mechanisms.22 Therefore, emerging resistance to older MIC differences between both periods, as shown in and newer antibiotics is of particular concern and Table S1. To confirm reproducibility as well for testing should be therefore closely monitored. Taking advan- resistant strains, 6 randomly selected XDR strains from tage of two large-scale (over 3.000 isolates from 66 the first (2017) study were retested in the second (2022) hospitals) surveys, we performed a Spanish nation-wide study yielding no significant MIC differences (Table S1). high-resolution analysis of the trends in the suscepti- Finally, to compare SIR data for both studies, MICs of bility profiles, the molecular epidemiology and the the first study were reinterpreted according to current resistome of P. aeruginosa over a five-year time lapse. EUCAST 2023 (v 13.0) clinical breakpoints. According to established recommendations by ECDC,6 Methods the MDR profile was defined as resistance to at least one P. aeruginosa strains and susceptibility testing agent in at least three of seven antibiotic classes including A total of 1735 P. aeruginosa isolates were studied in this antipseudomonal penicillins + β-lactamase inhibitor second Spanish nation-wide survey performed under the combinations (piperacillin/tazobactam), antipseudomonal auspices of the Spanish Society of Clinical Microbiology cephalosporins (ceftazidime and cefepime), mono- and Infectious Diseases (SEIMC) ensuring representa- bactams (aztreonam), antipseudomonal carbapenems tion of all 17 Spanish regions. This collection included (imipenem and meropenem), fluoroquinolones (cipro- up to 30 consecutive healthcare-associated non- floxacin), aminoglycosides (tobramycin and amikacin), duplicated (one per patient) P. aeruginosa clinical isolates and polymyxins (colistin) and the XDR profile as resis- collected during October 2022 from each of the 66 tance to at least one agent in all but one or two antibiotic participating hospitals (Figure S1). Species identification classes. Likewise, PDR profile was defined as resistance to was confirmed by matrix-assisted laser desorption/ioni- all agents in the seven antibiotic classes. The eight cate- zation time-of-flight (MALDI-TOF) mass spectrometry gory (fosfonic acids, fosfomycin) included in the ECDC (Bruker-Daltonics). Sample types [respiratory, urinary, recommendations was not considered given the lack of bloodstream, skin soft tissue and osteoarticular (SST), current EUCAST clinical breakpoints. On the other others] and sources [Intensive care unit (ICU), medical hand, the DTR (Difficult to Treat Resistance) profile was ward, surgical ward, emergency room, others] were defined according to IDSA recommendations as resis- recorded for each isolate. MICs of piperacillin/tazo- tance to all first line (classical) agents: antipseudomonal bactam (4/4–256/4 mg/L), ceftazidime (1–64 mg/L), penicillins + β-lactamase inhibitor combinations (piper- cefepime (1–64 mg/L), ceftolozane/tazobactam (0.5/ acillin/tazobactam), antipseudomonal cephalosporins 4–32/4 mg/L), ceftazidime/avibactam (0.5/4–32/4 mg/ (ceftazidime and cefepime), monobactams (aztreonam), L), aztreonam (2–128 mg/L), imipenem (0.5–64 mg/L), antipseudomonal carbapenems (imipenem and mer- imipenem/relebactam (0.12/4–64/4 mg/L), meropenem openem), and fluoroquinolones (ciprofloxacin).7 Thus, all (0.5–64 mg/L), ciprofloxacin (0.12–16 mg/L), tobramycin DTR isolates meet the XDR criteria, since they are resis- (0.25–32 mg/L), amikacin (2–128 mg/L), and colistin tant to at least five of seven classes. (0.5–16 mg/L) were determined by broth microdilution using Sensititre™ panels (Plate Code:FRCNRP2, WGS Thermo Fisher Diagnostics, S.LU), except for imipe- All XDR (and consequently all DTR) isolates, as well as nem/relebactam for which in house microdilution was those resistant to the any of the three newer β-lactam/ performed according to ISO-EUCAST guidelines (http:// β-lactamase inhibitor combinations herein evaluated www.eucast.org). EUCAST 2023 (v13.0) clinical break- (ceftolozane/tazobactam, ceftazidime/avibactam or points were used for interpretation of SIR categories. imipenem/relebactam), and/or producing an acquired Results were compared with those previously obtained in ESBL or carbapenemase from the 2022 study were fully the first Spanish nation-wide survey, which included sequenced (n = 138). Additionally, sequences from the 1445 isolates collected from 51 hospitals (all of them 185 XDR isolates available from the 2017 study were participating in the second survey) five years earlier included for comparison. It should be noted that a (October 2017) with exactly the same criteria (up to 30 number of these isolates (n = 28) are now reclassified consecutive healthcare-associated nonduplicated isolates as MDR when applying current EUCAST breakpoints. per hospital, same sample types and sources classifica- Strains from the 2022 study that would have been tion).15,23 Likewise, the same panel of antibiotics and classified as XDR in the 2017, but only as MDR with concentrations were tested by the same reference labo- the current criteria (n = 19) were also sequenced to ratory (Microbiology Department, Hospital Son Espases, have the complete picture. Table S2 collects the infor- Palma de Mallorca) using the same protocols in both mation from the 342 strains sequenced from 2017 to surveys. Moreover, to ensure interstudy reproducibility 2022 studies. of susceptibility results, reference strains ATCC27853 and PAO1 were included as controls. Each of the refer- Library preparation ence strains was tested in at least 6 independent occa- Genomic DNA was obtained with a commercially sions during each of the studies, yielding no significant available extraction kit (High Pure PCR Template www.thelancet.com Vol 34 November, 2023 3 Articles Preparation Kit, Roche Diagnostics). Indexed paired-end Phylogenic analysis libraries were generated by using the Illumina DNA With the aim to study the diversity of XDR ST175 and Prep library preparation kit (Illumina Inc, USA) and ST235, core genome phylogenetic reconstruction was then sequenced on either, an Illumina MiSeq® bench- performed with Parsnp from the Harvest Suite package top sequencer with MiSeq reagent kit v3 (600 cycles) or v1.2 with default parameters,35 forcing the inclusion of in an Illumina Novaseq 6000 system with NovaSeq 6000 all genomes (-c) and randomly selecting the reference SP Reagent Kit v1.5 (300 cycles). genome (-r!). Additionally, a minimum-spanning tree (MST) for XDR and reference strains PAO1, PA14 and Variant calling PA7 was inferred by using GrapeTree36 on the basis of Previously defined and validated protocols were used cgMLST scheme for P. aeruginosa37 created using the with slight modifications.24 The reads for each isolate open source ChewBBACCA algorithm.38 were mapped against the genome of the P. aeruginosa reference strain PAO1 (RefSeq accession number Acquired resistance determinants NC_002516.2) using Bowtie 2 software, v2.2.6 (http://bo To identify possible horizontally-acquired antimicrobial wtie-bio.sourceforge.net/bowtie2/index.shtml).25 Pileups resistance genes, we used the online tool ResFinder and raw files of the mapped reads were obtained by v3.1.0. with default options (https://cge.cbs.dtu.dk// using SAMtools, v0.1.16 (https://sourceforge.net/pro services/ResFinder/).39 Genomic information was com- jects/samtools/files/samtools/),26 and PicardTools, v1.140 plemented as needed by phenotypic (such as cloxacillin (https://github.com/broadinstitute/picard). Read align- and EDTA tests) and molecular (PCR + sanger ments surrounding all putative indels were realigned sequencing) methods for the detection of acquired using the Genome Analysis Toolkit (GATK), v3.4-46 β-lactamases.15 (https://www.broadinstitute.org/gatk/).27 The list of SNPs was compiled from the raw files that met the Statistical analysis following criteria: a quality score of ≥50, a root mean The data set analysed included a total of 3.180 non- square (RMS) mapping quality of ≥25 and a coverage duplicated P. aeruginosa clinical isolates from two depth of ≥3 reads. MicroIndels were extracted from the Spanish nation-wide surveys performed in October 2017 totalpileup files by use of the following criteria: a quality (1.445 isolates) and 2022 (1.735 isolates), as well as the score of ≥500, a RMS mapping quality of ≥25 and 342 genomes sequenced from 2017 (n = 185) and 2022 support from at least one-fifth of the covering reads. (n = 138) studies. The following variables were consid- SNPs and indels for each isolate were annotated by us- ered: Prevalence of resistance to each of 13 anti- ing SnpEff software v4.3 ().28 Finally, large chromosomal pseudomonals agents for all isolates and for ICU deletions were analyzed with Seqmonk v1.47.2 (https:// isolates. Prevalence of MDR, XDR and DTR resistance www.bioinformatics.babraham.ac.uk/projects/seqmonk/) profiles according to sample type [respiratory urinary, and R v4.2.3 within the RStudio v0.99.896 platform.29 bloodstream, skin soft tissue and osteoarticular, others], hospital wards [ICU, medical, surgical, emergency De novo assembly room, others], production of ESBLs and carbapene- Reads were de novo assembled using SPAdes v3.15 with mases and clonal types. We investigated how the default options. De novo assemblies were used to define different variables varied between the two periods (2017 the sequence type (ST) by using MLST v2.23.0 according and 2022) using Chi-square test. The Wilcoxon signed- to PubMLST typing schemes (https://pubmlst.org/) rank test was used to compare the proportion of developed by Keith Jolley.30 Additionally, assembled reads hospitals showing an increase or decrease of resistance were used to study the structural integrity of the OprD for the different antibiotics tested in 2022 vs 2017. In all porin. As different sequence variants of OprD have been cases, a p value of <0.05 was considered statistically described,31 the oprD gene was first classified according significant. All statistical analyses were performed using to their similarity to PAO1, LESB58, UCBP-PA14, GraphPad Prism 5 or IBM SPSS Statistics v22 software. MTB-1, FRD1 or F23197 reference sequences. Role of the funding source Analysis of the mutational resistome The funder of the study had no role in study design, data A total of 48 genes involved in mutational resistance collection, data analysis, data interpretation, or writing were selected according to findings of previous studies of the report. and analyzed.13,32,33 The complete list of the genes stud- ied, indicating their role in antibiotic resistance, is Data availability shown in Table S3. Nucleotide sequence variants located Genomic sequences from the 2022 and 2017 studies within these genes were filtered by using a list of natural have been deposited in the European Nucleotide polymorphisms that have been previously defined by Archive, under project numbers PRJEB61879 and our group.34 PRJEB31047, respectively. 4 www.thelancet.com Vol 34 November, 2023 Articles Fig. 1: a. Comparative analysis of the prevalence of resistance to 13 antipseudomonal agents in 2017 and 2022 Spanish nation-wide studies. b. Comparative analysis of the prevalence of resistance among ICU and nonICU isolates from the 2022 study. c. Comparative analysis of the prevalence of resistance in ICU isolates from 2017 to 2022 studies. In the case of meropenem, “I” (susceptible increased exposure) isolates were also represented (in a lighter tone and with numbers between brackets) in addition to resistant isolates for reference since these isolates show low level resistance. Statistical significance (Chi-square, X2) indicated (***p < 0.0001; **p < 0.01; *p < 0.05). Results origin was very similar for both studies (Fig. 3b). Antimicrobial susceptibility and resistance profiles Remarkably the highest decrease in the prevalence of A comparative analysis of resistance rates for a panel of XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%) in 2022 13 antipseudomonal agents is shown in Fig. 1a. A sta- vs 2017 was documented for ICU isolates. Moreover, tistically significant decrease in resistance for all anti- contrasting dramatically with 2017 results, XDR and biotics tested was documented in 2022 compared to DTR profiles were not more frequent in the ICU than in 2017. In both periods, the antibiotic showing lowest other hospital wards in 2022. The prevalence of MDR resistance rates was colistin followed by amikacin, imi- (nonXDR) profiles were, however, very similar for ICU penem/relebactam, ceftolozane/tazobactam and cefta- isolates in both study periods and much higher than zidime/avibactam. In contrast, the highest resistance those documented in other hospital wards. rates were documented for ciprofloxacin, followed by A paired analysis of the 51 hospitals participating in imipenem and piperacillin/tazobactam. Resistance rates both studies further emphasizes the generalized were higher for all agents in the ICU (Fig. 1b), except for decrease in resistance (Fig. 4). Particularly noteworthy, ciprofloxacin and amikacin, but again they were the percentage of XDR decreased in over 80% of the considerably lower in 2022 compared to 2017 (Fig. 1c). participating hospitals, while it increased in less than The distribution of resistance profiles is shown in 10% of them. Among individual agents, the prevalence Fig. 2a, revealing a major decrease as well in 2022 of of resistance to imipenem, meropenem, tobramycin, MDR (27.2% vs 14.8%), XDR (15.2% vs 5.9%) and DTR ciprofloxacin, cefepime and piperacillin/tazobactam was (4.2% vs 2.1%) profiles. PDR profiles were not detected decreased in >70% of the participating hospitals. in either period. The generalized decrease in the prev- alence of XDR profiles in nearly all Spanish regions is Horizontally-acquired β-lactamases and evidenced in Fig. 2b. distribution of high-risk clones As shown in Fig. 3a the distribution of clinical The nature and prevalence of horizontally-acquired sample types was nearly identical in both studied pe- ESBLs and carbapenemases is shown in Fig. 5a. The riods. Interestingly, while there was a generalized global prevalence of acquired enzymes tended to decrease reduction in the prevalence of MDR/XDR/DTR phe- from 3.1% in 2017 to 2.1% in 2022, but statistical sig- notypes, the decrease was highest for respiratory sam- nificance was not reached (p = 0.064). Regarding the ples and lowest for blood cultures. Likewise, the distribution of enzymes, a slight decrease in the preva- distribution of isolates according to the hospital ward of lence of VIM MBLs was documented in 2022, while GES www.thelancet.com Vol 34 November, 2023 5 Articles Fig. 2: a. Comparative analysis of the distribution of MDR XDR and DTR profiles in 2017 and 2022 Spanish nation-wide studies. As described in the material and methods section, XDR isolates are a fraction of MDR isolates and DTR isolates a fraction of XDR isolates. Statistical significance (Chi-square, X2) indicated (***p < 0.0001; **p < 0.01; *p < 0.05). b. Comparative analysis of the distribution of XDR profiles in the different Spanish region in 2017 and 2022 studies. enzymes slightly increased. However, the proportion of Phylogenetic reconstructions and association with carbapenemase producing isolates significantly increased horizontally acquired and mutational resistance are among XDR isolates (13.2% vs 22.3%) (Fig. 5b) and represented for XDR ST175 and ST235 clones in Fig. 7. among meropenem resistant isolates (18.1% vs 29.4%) As previously documented, XDR ST175 isolates were (Fig. 5c) in 2022 compared to 2017. characterized by a strong mutational resistance genomic The distribution of clonal types among XDR isolates signature which included nearly uniform QRDR and is shown in Fig. 6. While ST175 was still the most mexZ mutations (Fig. 7a). Additionally, the most frequent clone in 2022 a slight reduction (not statisti- frequent cluster of ST175 isolates both in 2017 and 2022 cally significant) in its prevalence was noted when showed the characteristic β-lactam mutational resistome comparing 2017 (45.5%) with 2022 (35.9%). However, described in isolates from nearly 15 years ago, including the most remarkable finding was the statistically sig- specific OprD (Q142*) and AmpR (G154R) mutations.13 nificant increase in the prevalence of ST235 in 2022 A second ST175 lineage, already documented in 2017, (12.3% vs 24.3%). produced the VIM-20 variant but seemed not to have 6 www.thelancet.com Vol 34 November, 2023 Articles Fig. 3: Distribution of isolates and resistance profiles according to sample type (a) and hospital wards (b) in 2017 and 2022 Spanish nation-wide studies. ER, emergency room. Statistical significance (Chi-square, X2) comparing both studies (***p < 0.0001; **p < 0.01; *p < 0.05) and between hospital wards in 2022 (###p < 0.0001; ##p < 0.01; #p < 0.05) are indicated. expanded in 2022. In sharp contrast, as shown in for other less frequent high-risk clones. For example, Fig. 7b, ST235 demonstrated an overwhelming associ- ST308 was less frequent in 2022, but accounted for the ation with the production of horizontally acquired single case of NDM production in the complete collec- resistance determinants, particularly noteworthy ESBLs tion. Likewise, while the overall prevalence of ST253 had and carbapenemases, and presented a more heteroge- slightly decreased 2022, an increased association with neous mutational resistome. Clonal expansion of a MBL-production was observed including a clonal ST235 epidemic lineage in multiple hospitals from the expansion of a VIM-1 lineage that was detected in a Madrid region was particularly evident in 2022 and was single hospital from Catalonia in 2017 but in three associated with GES enzymes production and specific (including the first) from the same region in 2022 mexZ and parS mutations. (Table S2). Moreover, an additional VIM-2-producing Figure S2 shows the cgMLST analysis of the com- ST253 lineage was first detected in a different region plete collection of XDR isolates from 2017 to 2022, in 2022. Globally expanding KPC enzymes were how- further evidencing interregional transmission and ever not detected in any of the two periods. Finally, persistence of several XDR clones, most noteworthy, but while the investigation of the transferable elements not only, ST175 and ST235. Increased association with (plasmids and transposons) harboring the carbapen- acquired carbapenemases in 2022 was also documented emase genes was beyond the scope of this work, the www.thelancet.com Vol 34 November, 2023 7 Articles Fig. 4: Percentage of hospitals showing lower (green), equal (orange) or higher (red) resistance rates in 2022 than in 2017. Only the 51 hospitals participating in both studies were included in the analysis. Statistical significance (Chi-square, X2) indicated (***p < 0.0001; **p < 0.01; *p < 0.05). BLAST analysis of their genetic context confirmed that β-lactamase inhibitor combinations was performed for they were in all cases linked to class 1 integrons. the 2022 study, and results are shown in Table 1. Pro- duction of horizontally acquired carbepenemases was Mechanisms of resistance to newerβ-lactam most frequent among imipenem/relebactam resistant β-lactamase inhibitors combinations strains (74.4%), but less frequent among ceftazidime/ A specific analysis of β-lactam resistance mechanisms avibactam resistant strains (25.0%). Particularly note- produced by strains resistant to the newer β-lactam/ worthy, none of the ceftazidime/avibactam resistant Fig. 5: a. Prevalence of ESBLs and carbapenemases for the complete collection of P. aeruginosa isolates from 2017 to 2022 studies. b. Prevalence of ESBLs and carbapenemases (carbapenemases specifically indicated in parenthesis) among XDR isolates. c. Prevalence of carbapenemases among meropenem resistant isolates. Statistical significance (Chi-square, X2) indicated (*p < 0.05). 8 www.thelancet.com Vol 34 November, 2023 Articles Fig. 6: Distribution of sequence types among XDR P. aeruginosa isolates recovered from the 2017 and 2022 studies. Statistical significance (Chi-square, X2) for the 2017 vs 2022 comparison indicated (**p < 0.01; *p < 0.05). STs accounting for ≥2% of the XDR isolates in any of the two studies are shown individually, while all those representing <2% of isolates are included in “Others”. strains produced a GES-5, in contrast to 33.3% of imi- mutational resistome, AmpC Ω-loop mutations were penem/relebactam resistant strains. Conversely, sus- exclusively seen in ceftolozane/tazobactam (15.1%) and ceptibility rates among the 16 GES-5 producers was ceftazidime/avibactam (12.5%) resistant isolates, but not 93.8% for ceftazidime/avibactam, 68.8% for ceftolo- in those resistant to imipenem/relebactam. Moreover, zane/tazobactam and 6.3% for imipenem/relebactam. ceftazidime/avibactam resistance was strongly associ- As expected, susceptibility rates for MBL producers were ated with mutations in regulators of the expression of below 10% for the three combinations. Regarding the the efflux pump MexAB-OprM (55.4%). Fig. 7: Core genome phylogenetic reconstruction of the 2017 and 2022 XDR ST175 (a) and ST235 (b)P. aeruginosa isolates. Year column labeled in gray and white corresponds with 2017 and 2022 isolates, respectively. Following columns correspond to β-lactamases and other acquired enzymes. Code description of changes in most commonly mutated genes (oprD, mexZ, mexR, ampR, ampC, parS, PBP3, parC and gyrA) is represented on hand-side. Each colour of each column corresponds to a single mutation. www.thelancet.com Vol 34 November, 2023 9 Articles Discussion associated with a specific resistome signature and that The May 2015 World Health Assembly (WHA) adopted had been already detected for over one decade in a global action plan on antimicrobial resistance, after Spanish hospitals, but that was quite uncommon in reaching the conclusion that it “threatens the very core other countries besides France.13,40 Fortunately, the of modern medicine and the sustainability of an effec- prevalence of the most concerning resistance mecha- tive, global public health response to the enduring threat nisms, inactivating even the newer β-lactam combina- from infectious diseases”. The five main objectives tions, the horizontally-acquired carbapenemases, was established included to (i) improve awareness and un- relatively low even among XDR strains. derstanding of antimicrobial resistance (ii) to strengthen In this work, we decided to repeat the survey under the the knowledge through surveillance and research (iii) to same conditions and using the same methodological reduce the incidence through prevention measures (iv) approach in order to determine for the first time how the to optimize the use of antibiotics and (v) to develop the antimicrobial susceptibility, the molecular epidemiology economic case for sustainable investment in new and the resistance genomics had evolved at a nation-wide medicines, diagnostic tools, vaccines and other in- level during a five-year period. To our initial surprise we terventions. Some years before (November 17th 2011), documented a major generalized reduction of antimicro- the EU commission requested member states to develop bial resistance, affecting nearly all older as well as newer national action plans on antimicrobial resistance, and in antipseudomonal agents. Particularly astonishing was the the case of Spain the first version was approved in 2014 significant reduction of the prevalence of XDR strains with objectives similar to those adopted in the WHA from over 15% to below 6%. The reduction was patent in (https://resistenciaantibioticos.es/es). Moreover, in all regions and was even more evident in the ICUs with 2018 the WHO published a priority list for the need for XDR profiles decreasing from 26% to 6%. A significant research and development of new antibiotics, in which reduction of DTR profiles was as well documented in carbapenem resistant P. aeruginosa, along with Enter- 2022, but should be noted that resistance to fluoroquinoles obacterales and Acinetobacter baumannii were in top was the highest of all tested agents in both periods, sup- (critical) position.3 porting the concern on the use of these antibiotics as first Within this scenario, in 2017 we performed a first line agents communicated by the European Medicines nation-wide survey of P. aeruginosa antimicrobial sus- Agency (EUPAS37856 report). ceptibility, resistance mechanisms and molecular While the factors underlying such a dramatic epidemiology using whole genome sequencing tools.15 reduction are likely multifactorial and complex, the ef- Overall resistance was found to be high, exceeding forts of the Spanish national plan of antibiotic resistance 20% for each available antipseudomonal except for toxic (PRAN) should be acknowledged, including the imple- polymyxins and the newer β-lactam β-lactamase inhibi- mentation of country-wide infection control initiatives tor combinations ceftolozane/tazobactam and ceftazi- such as the Zero resistance in ICUs and antimicrobial dime/avibactam that were being introduced into clinical stewardship programs.41,42 Another potential factor to practice at that time. Moreover, MDR profiles were consider is the role of the newer β-lactam β-lactamase documented in nearly 30% of the isolates and XDR inhibitor combinations that were active against the most patterns in over 15%. The molecular epidemiology prevalent XDR strains (ST175) and have been the first analysis performed revealed that close to half of the choice treatment for such infections in the last five XDR isolates belonged to a single clone, ST175, that was years.43 The COVID-19 pandemic and associated Resistance mechanism/mutations Ceftolozane/tazobactam resistant, Ceftazidime/avibactam resistant, Imipenem/relebactam resistant, n = 53 (%) n = 56 (%) n = 39 (%) ESBL/Carbapenemases 29 (54.7) 16 (28.6) 30 (76.9) ESBL GES-1 4 (7.5) 2 (3.6) 1 (2.6) Carbapenemases 25 (47.2) 14 (25.0) 29 (74.4) GES-5 9 (17.0) 0 13 (33.3) MBLs 16 (30.2) 14 (25.0) 16 (41.0) Narrow spectrum OXAs (only) 3 (5.7) 2 (3.6) 0 oprD 23 (43.4) 32 (57.1) 18 (46.2) ampC regulators (dacB, ampD, ampR) 29 (54.7) 29 (51.8) 19 (48.7) ampC (Ω-loop) 8 (15.1) 7 (12.5) 0 mexAB-OprM regulators (mexR, nalC, nalD) 16 (30.2) 31 (55.4) 13 (33.3) ftsI (PBP3) 1 (1.9) 4 (7.1) 2 (5.1) galU 1 (1.9) 3 (5.4) 2 (5.1) Table 1: Mechanisms detected among ceftolozane/tazobactam, ceftazidime/avibactam or imipenem/relebactam resistant strains from the 2022 study. 10 www.thelancet.com Vol 34 November, 2023 Articles measures, even if already highly relaxed by the last β-lactam β-lactamase-inhibitors combinations already trimester of 2022, could have had an impact as well on approved, the continuous introduction of new players resistance transmission.44 such as the recently commercialized siderophore- However, not all are positive findings in our survey, cephalosporin cefiderocol or next generation of β-lacta- and at least two highly concerning and related facts need mase inhibitors under investigation, including those to be considered. The first is a significant increase in the able to inhibit PBP2 (such as zidebactam) or MBLs proportion of carbapenemase-producing strains among (such as taniborbactam) need to be added in future XDR and carbapenem resistant isolates. The second is surveillance initiatives. Finally, given the large number the documentation of a major progression of the word- of statistical tests performed, some false positive results wide disseminated high-risk clone ST235. Indeed, this could have occurred with the 0.05 threshold, but the concerning clone is strongly associated with epidemic obtained p values, below 0.01 in most cases, support settings and the acquisition of horizontally-acquired their true significance. resistance determinants in hospitals world-wide, playing In summary, taking advantage of two large-scale a determinant role in the global variation of the preva- surveys, we performed a Spanish nation-wide high res- lence of carbapenemase-producing strains.45–47 Moreover, olution analysis of the trends in the susceptibility pro- in addition to its strong association with epidemic files, the molecular epidemiology and the resistome of dissemination and horizontally-acquired resistance, P. aeruginosa over a five-year time lapse. Positive find- ST235 is associated with a higher virulence, due to the ings included a sharp generalized decrease in resistance production of the ExoU toxin, unlike other MDR/XDR to older and newer antipseudomonals, as well as a dra- clones such as ST175.48–50 Therefore, findings of this matic decrease in the prevalence of XDR P. aeruginosa. study point towards a growing role of hypervirulent The negative counterpart was a significant increase in carbapenemase-producing ST235, and thus suggest that the proportion of XDR and carbapenem resistant strains this clone should be subjected to an specific surveyance producing concerning horizontally-acquired carbapene- and that efforts should be made for the implementation mases, linked to a significant progression of the major of rapid diagnostic techniques for such infections in the high-risk clone ST235. Continued high resolution sur- national health system. Moreover, from the antimicrobial veillance, integrating phenotypic and genomic data, is development perspective, efforts should likely be directed necessary for understanding resistance trends and towards alternatives providing an optimal coverage of analyzing the impact of National plans on antimicrobial carbapenemase-producing P. aeruginosa. resistance. The strengths of this work include the high number Contributors of hospitals participating in both surveys performed Members of GEMARA-SEIMC/CIBERINFEC Study Group carried under the same experimental conditions and methodo- out sample collection. SFMA, FMA, conducted laboratory assays logical approaches, providing robust data on how the with supervision and support from ZL and OA. GFMA, TB sequenced antimicrobial susceptibility, the molecular epidemiology and analyzed the genomic data with supervision and support and the resistance genomics had evolved at a nation- from LCC and OA. OA is the principal investigator and ASJ, MML, CR, LN, OIJ with OA were coordinating team members. All wide level during a five-year period. However, the authors contributed important intellectual content during manuscript study has also some relevant limitations. First, the drafting or revision. All authors had full access to all the data in the clinical characteristic of the P. aeruginosa infections study and had final responsibility for the decision to submit for were not analyzed, including risk factors, source of publication. bacteremia, management and outcomes, and therefore Data sharing statement conclusions related to these relevant aspects cannot be Genomic sequences from the 2022 and 2017 studies have been depos- reached. Second, while the study revealed a major ited in the European Nucleotide Archive, under project numbers change in the epidemiology of P. aeruginosa antibiotic PRJEB61879 and PRJEB31047, respectively. resistance at a nation-wide level, it was not designed to Editor note decipher the underlying causes of such findings that The Lancet Group takes a neutral position with respect to territorial need to be analyzed in subsequent studies. Likewise, claims in published maps and institutional affiliations. while the investigation of the transferable elements (plasmids and transposons) harboring the carbapen- Declaration of interests emase genes was beyond the scope of this work, it will This work was partially financed by a Grant from MSD to AO. The funders had no role in design, execution, analysis or reporting of the be useful to characterize them in future studies to un- research. AO has participated in educational programs organized by derstand their potential dissemination across strains. MSD, Pfizer and Shionogi and had conducted research studies financed Moreover, it will be of future interest to scale the find- by MSD and Shionogi. RC has participated in educational programs ings from this single nation initiative to the European organized by Menarini, MSD, Pfizer, Shionogi and had conducted and/or global levels. Moreover, although the study in- research studies financed by MSD and Shionogi. NL has participated in educational programs organized by Menarini, MSD, Pfizer and Shio- cludes the analysis of the resistance phenotypes and nogi. LM has participated in educational programs organized by MSD, genotypes for the most relevant classical anti- Pfizer and Shionogi and had conducted research studies financed by pseudomonals, and the three most relevant newer Janssen, MSD, Pfizer and Shionogi. www.thelancet.com Vol 34 November, 2023 11 Articles Acknowledgements de Octubre); Emilia Cercenado (H. Gregorio Marañón); Teresa Alar- This study was made under the auspices of the Study group of mech- cón, Paula Vargas y María Diez (Hospital La Princesa); Rafael Cantón anisms of antimicrobial action and resistance (GEMARA) from the y Patricia Ruiz (H. Ramón y Cajal); María Isabel Sánchez-Romero Spanish society of clinical microbiology and infectious diseases (SEIMC) (H. Puerta de Hierro); Felipe Pérez-García (H. Universitario Príncipe and the area of infectious diseases (CIBERINFEC) from the centers for de Asturias); Genoveva Yagüe-Guirao (H. Virgen de la Arrixaca); network biomedical research (CIBER) of the Spanish national institute Amaia Concepción Oteiza y José Leiva (Clínica Universidad de Nav- of health ISCIII. We are thankful to Dr Aina Millán from IdISBa for arra); María Eugenia Portillo (H. Universitario de Navarra); Andrés statistical support. Canut-Blasco (H. Universitario de Álava); Matxalen Vidal (H. Uni- versitario Basurto); Iker Alonso, Maider Zuriarrain y José Luis Bar- Funding: This work was supported by MSD and by the Instituto de rios-Andrés (H. Universitario de Cruces). Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea —NextGenerationEU through grants PI21/00017 and Personalized and Appendix A. Supplementary data precision medicine grant (MePRAM Project, PMP22/00092). Supplementary data related to this article can be found at https://doi. Members of GEMARA-SEIMC/CIBERINFEC Pseudomonas study Group org/10.1016/j.lanepe.2023.100736. Fátima Galán Sánchez (H. Universitario Puerta del Mar); Irene Gracia-Ahufinger y Luis Martínez Martínez (H. Universitario Reina Sofía); Carmen Liébana-Martos y Carolina Roldán (Complejo Hospi- References talario Ciudad de Jaén); Juan Manuel Sánchez-Calvo (H. Universitario 1 Horcajada JP, Montero M, Oliver A, et al. 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