Molecular determinants associated with resistance to imipenem and imipenem–relebactam in clinical Pseudomonas aeruginosa isolates

Molecular determinants associated with resistance to imipenem and imipenem–relebactam in clinical Pseudomonas aeruginosa isolates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular determinants associated with resistance to imipenem and imipenem–relebactam in clinical Pseudomonas aeruginosa isolates André Bittencourt Lorusso, Joyce Souza, Larissa Bail, Carmen Antonia Sanches Ito, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7801871/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Background Pseudomonas aeruginosa accounts for 10–20% of hospital-acquired infections and is a major pathogen in immunocompromised patients. Combination therapies with beta-lactam antibiotics and beta-lactamase inhibitors, such as imipenem-relebactam have improved treatment options, yet resistant strains have already emerged, with mechanisms still not fully elucidated. Results We sequenced and analyzed 10 clinical P. aeruginosa isolates resistant to imipenem-relebactam (IMI/REL) and compared them with publicly available genomes of imipenem-resistant (IMI-R) and imipenem-susceptible (IMI-S) strains. Resistance genes were identified using the RGI CARD database, while amino acid variations in core-genome proteins were evaluated through Gene Ontology overrepresentation analysis (GO), followed by GWAS. In total, 15,758 ARGs were detected, 25.85% associated with carbapenem resistance, but only 568 classified as beta-lactamases. Among IMI/REL isolates, 36.36% carried Ambler class A and 54.54% class B beta-lactamases, contrasting with much lower frequencies in IMI-R (5.4% and 3.6%) and IMI-S (0% and 0.73%). Core-genome analysis revealed 1,106 proteins with resistance-associated variations. Comparative analyzes identified 1,618 proteins differing between IMI/REL and IMI-R genomes, and 1,015 differing between IMI/REL and all other strains. GWAS highlighted candidate genes with strong statistical associations, including those involved in metal ion transport (e.g., tonB , foxA , phuR , pfeA ) and efflux pumps (e.g., czcB ), as well as regulators such as mexT and biofilm-related proteins. Conclusions These findings suggest that, beyond classical beta-lactamases, resistance involves multifactorial contributions from periplasmic and outer membrane proteins, metal ion homeostasis, efflux regulation, and biofilm-associated pathways. Our results expand current knowledge of P. aeruginosa resistome and highlight novel genomic signatures potentially driving resistance to imipenem-relebactam. Pseudomonas aeruginosa imipenem-relebactam resistance genomic comparison metal ion Figures Figure 1 Figure 2 Figure 3 Figure 4 BACKGROUND Bacterial resistance to antimicrobial agents is a phenomenon observed since the 1940s ( 1 ), just over 10 years after the discovery of the first antibiotic ( 2 ), and it has been demonstrated that antibiotic resistance genes are part of the natural gene repertoire of microbial populations ( 3 ). In general, antimicrobial resistance is mediated by: a) inactivation or enzymatic alteration of the drug; b) modification of its binding site; c) reduction of intracellular accumulation of the drug; d) overexpression of the target; and e) formation of biofilms ( 4 ). In addition to the resistance mechanisms themselves, alterations in genes that encode proteins associated with DNA repair and recombination have been described, which lead to an increase in the mutation rates observed in organisms and, consequently, increase the variation in bacterial population structures (5). This phenomenon is probably associated with the emergence of resistance mechanisms, which can then spread to different species by horizontal gene transfer ( 6 ). The most important multidrug-resistant bacteria from a public health point of view belong to the ESKAPE group, which comprises Enterococcus spp ., Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , e Enterobacter spp. ( 7 , 8 ). Among the aforementioned pathogens, P. aeruginosa is one of the main etiological agents of nosocomial infections in intensive care units in Brazil ( 7 , 9 , 10 ), particularly important in patients with cystic fibrosis ( 11 , 12 ). Due to low cell wall permeability and a high capacity to acquire and express multiple resistance mechanisms, the prevalence of resistant strains of P. aeruginosa has increased dramatically in the last 20 years, as has the relative risk of mortality associated with infections by this pathogen ( 13 ). Several new antibiotic formulations have been developed in recent years, such as Ceftolozane/Tazobactam ( 14 ), Ceftazidime–avibactam ( 15 ) and Imipenem-Relebactam ( 16 ), which combine beta-lactam antibiotics with beta-lactamase inhibitors (a class of enzymes that degrade beta-lactam antibiotics). In general, beta-lactamase inhibitors interact durably with the active site of the enzyme, preventing its action in the degradation of the antibiotic ( 17 ). The Imipenem-Relebactam formulation was approved by the U.S. Food and Drug Administration in 2019 for the treatment of complicated intra-abdominal and urinary tract infections ( 18 ), and the approval was extended in 2020 for the treatment of hospital-acquired bacterial pneumonia and ventilator-associated pneumonia ( 19 ). In in vitro tests, approximately 90% of clinical isolates of P. aeruginosa demonstrate susceptibility to the new formulation ( 20 ). Genomic analyses of isolates resistant to imipenem-relebactam isolates of P. aeruginosa have already identified some genes and genetic variations associated with resistance, notably the presence of beta-lactamases of the GES, VIM and IMP classes, and mutations in porins, penicillin-binding proteins and proteins associated with the regulation of efflux pumps ( 21 – 24 ). The specific mechanisms related to decreased susceptibility to relebactam, however, have not yet been precisely elucidated. The epidemiological situation of multidrug-resistant P. aeruginosa infections, together with the limited amount of information on genomic variations related to imipenem-relebactam resistance in this pathogen, demonstrate the importance of continuous study of resistance mechanisms to new antimicrobials, as well as genetic alterations associated with the development of resistance mechanisms present in multidrug-resistant bacteria. The objective of this study was to perform genome sequencing of 10 imipenem-relebactam resistant (IMI/REL) P. aeruginosa strains and compare these genomes with those of imipenem-resistant (IMI-R) and imipenem sensitive (IMI-S) P. aeruginosa obtained from databases. Through resistance gene analysis, genomic comparison, GO enrichment studies and genome wide association studies (GWAS), we identified that variations in proteins associated with different metabolic functions probably linked to imipenem-relebactam resistance in P. aeruginosa . METHODOLOGY Bacterial strains The present study investigated 10 multidrug-resistant clinical isolates of P. aeruginosa from hospitals in the southern region of Brazil obtained from the Cajuru University Hospital (Curitiba, Brazil) in the period of January/2020 to December/2020. All isolates were obtained from clinical samples, including blood, respiratory, and urinary tract. Antimicrobial susceptibility tests to piperacillin/tazobactam, aztreonam, cefepime, ceftazidime, ceftazidime-avibactam, ceftolozane-tazobactam, imipenem, imipenem-relebactam, meropenem, ciprofloxacin, colistin, polymyxin B, gentamicin and amikacin were performed. Broth microdilution technique was used to determine the minimum inhibitory concentration (MIC) of each antibiotic for each isolate ( 25 ). The breakpoints according to EUCAST ( 26 ) were used, and the tests were performed in triplicate. Genome sequencing, assembly and annotation Genomic DNA from each isolate was extracted using the Wizard® HMW DNA Extraction Kit (Promega). DNA concentration was measured by the fluorometric method using the Qubit equipment (Thermo Fisher Scientific, Mississauga, ON, Canada). Sequencing libraries were prepared with Illumina DNA prep (Illumina, Inc., San Diego, CA, USA) following the manufacturer's instructions, and sequencing was performed on the Illumina NovaSeq 6000 platform (OGC, Oxford, UK) using NovaSeq SP reagent kits (300 cycles, paired-end reads) at the Instituto para Pesquisa do Câncer - IPEC (Guarapuava, Brazil). Genome assemblies were performed using SPAdes v3.15.5 ( 27 ), MeDuSa v1.6 ( 28 ) and FGAP 1.8.1 ( 29 ) tools, and the generated contigs were annotated using Prokka v1.14.5 software ( 30 ). The genomes have been deposited in the EMBL-EBI database (ebi.ac.uk/) under the accession numbers listed in Table S1 . Selection of genomes from databases P. aeruginosa genome sequences were obtained from the BV-BRC database ( 31 ), using the following parameters: Genome Status “WGS”; Genome Quality “Good”; Host Group “Human”. From this set, imipenem-resistant and -sensitive genomes were selected in the AMR Phenotypes category, with the filter: Evidence “Laboratory Method”, or indication of the methodology used (e.g. disk diffusion). Genomes sequenced exclusively by Oxford Nanopore technology were excluded. The BV-BRC IDs were listed in the Table S2 Identification of resistance genes To identify resistance genes, the Resistance Gene Identifier (RGI) software version 6.0.3 was used, with version 3.2.9 of the Comprehensive Antibiotic Resistance Database (CARD) ( 32 ). Only proteins identified in the “Perfect” category - which consists of sequences identical to those present in the database - and “Strict” - which detects previously unknown variants of antimicrobial resistance genes, using detection models with similarity cutoff lines curated by CARD - were considered. Subsequently, the result of the RGI analysis was filtered through Python scripts to identify proteins previously associated with resistance to β-lactams and, specifically, to carbapenems. The identified beta-lactamases were classified according to the Ambler system ( 33 ). Analysis of the oprD gene Initially, the oprD gene sequence (PA0958) was obtained from the genome of the reference strain PAO1, based on the Prokka annotation. The oprD gene sequence was aligned against all genomes to identify and extract the specific oprD sequence of each isolate using BLASTN ( 34 ). Subsequently, a global alignment was performed between the gene of each isolate and the reference gene, using the BioPython library ( 35 ). The alignments were analyzed to identify specific genetic variations in the oprD gene and the protein it encodes. The graphs generated from the results of this analysis were made with the Python library Matplotlib ( 36 ). Analysis of variations in proteins of the core genome We used CD-HIT software ( 37 ) to cluster orthologous proteins and identify variations in the core genome associated with resistance to imipenem and imipenem-relebactam. Only clusters containing proteins present in at least 95% of the isolates were considered part of the core genome, while the others were excluded from subsequent analyses. Functional annotation of the clusters was performed by comparing the sequence of the representative protein of each cluster with the proteins of the reference strain P. aeruginosa PAO1 ( 38 ) using the BLASTP algorithm ( 34 ). Functional information was obtained from the Pseudomonas Community Annotation Project (PseudoCAP) through the Pseudomonas Genome Database ( 39 ). Proteins with identity greater than 90% and minimum query coverage of 50% per subject were considered orthologous. The identifiers of the orthologous proteins in the PAO1 strain were used to assign Gene Ontology terms ( 40 ) to each cluster, by comparison with the PANTHER database ( 41 , 42 ). Next, for each cluster, a global alignment was performed between each protein and the protein of the reference strain using the BioPython library ( 35 ), and the alignments were analyzed to identify specific variations. At this stage, clusters that did not present one or more proteins of the reference strain were excluded. Statistical analysis In order to evaluate the statistical relationship between protein variations and imipenem and imipenem-relebactam resistance phenotypes, the odds ratio was calculated by comparing the number of resistant and susceptible isolates with and without each variation, and Fisher's exact test was used to assess whether there is a significant association between each variation and the resistance phenotype, through the SciPy library ( 43 ). For each variation, 2 distinct comparisons were made: imipenem-resistant isolates against imipenem-susceptible isolates (IMI-R vs. IMI-S) and imipenem-resistant isolates against imipenem-relebactam-resistant isolates (IMI-R vs. IMI/REL). Next, an analysis of the overrepresentation of GO terms of proteins that presented variations significantly associated with resistance (p < 0.05) with an odds ratio greater than 1 was performed, using the proteins of the core genome as a reference, through the PANTHER overrepresentation test. The significance of the overrepresented GO terms was calculated following the conventional parameters of the tool: Fisher's exact test to calculate the raw p-values and false discovery rate, using the Benjamini-Hochberg method, to calculate the corrected p-values. From this analysis, graphs were generated with the Python libraries Matplotlib ( 36 ) and seaborn ( 44 ). Genome-wide association study (GWAS) A genome-wide association study (GWAS) was conducted using Pyseer ( 45 ), comparing imipenem-resistant (IMI-R + IMI/REL) and imipenem-sensitive (IMI-S) groups. Because bacterial populations have a clonal structure, the samples cannot be considered independent. Therefore, it was necessary to adjust the association analyses for the effect of population structure using phylogenetic inference based on the core genome ( 46 ). The core genome was identified using the ROARY v.3.13.0 ( 47 ) from genomic annotation files in gff3 format. Subsequently, multiple alignment of the core genome was used in the FastTree program ( 48 ) to generate the phylogenetic tree. The GWAS was performed using the k-mer counting method, using the fms- lite tool, free of a reference genome. The effect of each identified variant on the resistance phenotype, adjusted for population structure, was estimated by fitting a linear mixed model (LMM), implemented in the Pyseer program. The p-value corresponding to each adjusted model, which will be corrected for the number of variants analyzed using the False Discovery Rate (FDR) method ( 49 ), were considered as associated with the resistance phenotype. RESULTS Evaluation of the resistance genotype and phenotype of P. aeruginosa isolates The determination of the minimum inhibitory concentration (MIC) by broth microdilution of the 10 sequenced isolates demonstrated that all isolates are resistant to imipenem (IMI), and that only one isolate (identified as 15_20783) is susceptible to the imipenem-relebactam combination (IMI/REL) (Table S3). Based on the resistance phenotypes observed, 8 isolates can be classified as extensively resistant (XDR) and 2 as multidrug resistant to antibiotics (MDR) ( 50 ). Isolate 15_20783, specifically, is also susceptible to two other combinations of beta-lactams with beta-lactamase inhibitors (ceftazidime-avibactam [CAZ/AVI] and ceftolozane-tazobactam [CTZ/TAZ]), and a similar phenotypic profile is observed in isolate 13_21675 - susceptible to CTZ/TAZ and with a four-fold lower MIC for IMI/REL compared to IMI. The other isolates do not show differences in MIC values between IMI and IMI/REL, but isolates 2_20589, 8_17683 and 12_18480 also show reduced MIC for ceftazidime in the presence of the inhibitor avibactam. Comparison of the annotated CDSs for each genome against the CARD database (Fig. 1 ) reveals the massive presence of efflux pumps and intrinsic resistance in all isolates. The consistent presence of genes linked to efflux systems is observed, notably the MexAB-OprM, MexXY, MexCD-OprJ, and MexEF-OprN pumps. Transcriptional regulators such as MexR, MexT, NalC, among others, regulate the expression of efflux pumps and are frequently associated with their overexpression in resistant isolates. In addition to efflux pumps, the analysis revealed the presence of the beta-lactamases PDC and OXA-50-like in all isolates. The beta-lactamases VIM-2, KPC-2, IMP-1, IMP-16, SPM-1, and NDM1 were also identified, but in different isolates. Only isolate R_2.20589 presents 2 acquired carbapenemases: NDM-1 and KPC-2. Identification of antimicrobial resistance genes (ARGs) To increase the number of genomes and the robustness of our analysis, we searched the BV-BRC database ( 31 ) for genomes of isolates resistant to imipinem-relebactam (IMI/REL), resistant to imipenem and without information regarding resistance to imipenem/relebactam (IMI-R) and sensitive to imipenem (IMI-S). In total, 110 genomes of IMI-R and 136 genomes of IMI-S isolates were obtained. Only 2 genomes had an indication of IMI/REL resistance. For the purposes of analysis, we considered the 110 IMI-R isolates resistant only to imipenem. All genomes retrieved from the BV-BRC database were reannotated to maintain analysis uniformity. Comparison with the CARD database, including both sequenced genomes and those retrieved from BV-BRC, identified 15,758 antibiotic resistance genes (ARGs) (Table S4), grouped into 192 distinct antibiotic resistance ontology (ARO) terms. Of these, 4,073 ARGs (25.85%) were linked to carbapenem resistance (Table S5), while 1,537 (9.75%) were associated with resistance to other beta-lactams (Table S6). Among the carbapenem resistance determinants, 3,505 (86.05%) correspond to efflux pump components (e.g., MexA, MexB, OprM) or regulators of efflux pump expression (e.g., ParR, ParS, MexR), classified under 16 ARO terms. The remaining 568 ARGs were beta-lactamases distributed across 88 distinct ARO terms. GES (Ambler class A) and NDM/SPM (Ambler class B) beta-lactamases were exclusively detected in IMI/REL genomes, whereas KPC (class A) and VIM (class B) beta-lactamases were identified in both IMI-R and IMI/REL genomes. Among IMI-S isolates, only one class B beta-lactamase, IMP-45, was detected. All P. aeruginosa genomes harbored the intrinsic beta-lactamases PDC (class C) and OXA (class D). Notably, multiple class D beta-lactamases were present in 3 IMI-S isolates (2.21%), 21 IMI-R isolates (18.92%), and 4 IMI/REL isolates (36.36%). In addition, all genomes carried the recently described PIB-1 (PA5542), an intrinsic carbapenemase of P. aeruginosa capable of hydrolyzing carbapenems broadly and resistant to inhibition by avibactam and clavulanic acid ( 51 , 52 ). Among the IMI/REL isolates, 36.36% presented Ambler class A beta-lactamases and 54.54% presented Ambler class B beta-lactamases. The frequency of class A and B beta-lactamases was much lower for IMI-R (5.4% and 3.6%, respectively) and IMI-S isolates (0% and 0.73%, respectively) (Table 1 ). Of the 4 IMI/REL isolates that do not have MBLs (Genome IDs: 13.21675, 287.14071, 287.16518 and 5.13697), only 287.14071 and 287.16518 present GES beta-lactamases resistant to relebactam inhibition ( 23 ). Isolate 8.17683 has a KPC-2 that, despite being inhibited by relebactam, confers resistance to imipenem-relebactam when overexpressed ( 53 ). The two remaining isolates (13.21675 and 5.13697), which do not have Ambler class A or B beta-lactamases, have OXA-486 and OXA-847 class D beta-lactamases, respectively. However, there are no records of specific activity of these enzymes against imipenem and they were identified in several IMI-S isolates among those analyzed in this study (Table S7). Table 1 Frequency of beta-lactamases of the four Amber classes identified in isolates with different resistance phenotypes to imipenem and imipenem-relebactam Phenotype Class A Class B Class C Class D IMI/REL 4 (36,36%) 6 (54,54%) 11 (100%) 11 (100%) IMI-R 6 (5,4%) 4 (3,6%) 111 (100%) 111 (100%) IMI-S 0 (0%) 1 (0,73%) 136 (100%) 136 (100%) Analysis of variations in the ARG oprD The oprD gene, responsible for encoding the OprD porin, was not identified in the comparison with the CARD database. Despite being part of the core genome of P. aeruginosa , the oprD gene frequently presents frameshifts or premature stop codons in carbapenem-intermediate and resistant strains ( 54 , 55 ), resulting in truncated proteins and making its annotation in the genomes difficult. To obtain the oprD gene sequences from the 258 genomes analyzed in this study, we conducted a BLASTN search using the oprD sequence from the PAO1 strain as a reference. One IMI-R genome (Genome ID 287.7811) was found to lack the oprD gene, while 12 IMI-R and 1 IMI-S isolates contained only partial sequences located at the ends of contigs. None of IMI/REL genomes were excluded. These isolates were excluded from further analysis, leaving 244 isolates (109 IMI-R + IMI/REL and 135 IMI-S), each carrying a single copy of the gene. The oprD sequences were compared to that of P. aeruginosa PAO1. Only 9 isolates encoded proteins identical to PAO1’s oprD , including one IMI-R isolate (Genome ID 6.10719) and 8 IMI-S isolates. Among the remaining IMI-R and IMI/REL isolates, 68 out of 109 (62.38%) carried insertions or deletions leading to frameshift mutations, 20 out of 109 (18.35%) had nonsense mutations, and 20 out of 109 (18.35%) exhibited nucleotide substitutions resulting in amino acid changes. In contrast, among the IMI-S isolates, 2 frameshift mutations, 2 nonsense mutations, and 123 non-synonymous substitutions were observed (Fig. 2 ). Considering only the 11 IMI/REL isolates, one had a partial sequence (ID 287.16518), one had a sequence identical to that of PAO1 (ID 6.10719) and the other 9 presented alterations in the reading frame of the gene. Analysis of variations in proteins of the core genome Considering that relebactam does not inhibit class B or class D beta-lactamases, and given that some IMI/REL resistant isolates lack these enzymes, we analyzed the core genome of the P. aeruginosa genome assembly to identify other factors possibly associated with resistance to imipenem and imipenem-relebactam. Cluster analysis performed with the CD-HIT tool resulted in 35,854 clusters of orthologous proteins among the 258 genomes evaluated. Of these, 5,010 clusters contained proteins present in at least 95% of the isolates and had an orthologous proteins described in the reference strain PAO1 were retained in the analysis. The analysis of amino acid variations was conducted for proteins within each cluster, comparing sequences between IMI-R and IMI-S (Table S8), as well as between IMI-R and IMI/REL (Table S9), to assess differences associated with distinct resistance phenotypes. Only variations with a p-value ≤ 0.05 and odds ratio greater than 1 were considered significant. Comparing isolates IMI-R with isolates IMI-S, 2,012 positions with significant variations were identified, present in 1,106 different proteins. In the comparison between IMI-R with IMI/REL isolates, 1,371 positions with significant variations were identified in 1,015 different proteins. Most of the identified variations consist of amino acid substitutions, followed by deletions and insertions. General information on the significant variations observed is available in Table 2 . Table 2 Significant variations identified in each comparison performed Proteins with significant variations* Positions with significant variations* Positions with substitutions Positions with deletions Positions with insertions IMI-R + IMI/REL vs. IMI-S 1 1106 2012 1799 184 29 IMI-R vs. IMI/REL 3 1015 1371 1307 41 23 * p ≤ 0,05 e odds ratio > 1 GO overrepresentation analysis To investigate whether proteins with significant amino acid variations were enriched in specific biological functions potentially associated with IMI/REL resistance, a GO overrepresentation analysis was performed. In this approach, the functional distribution of these proteins was compared to that of the core genome clusters. From the 5,010 clusters constituting the core genome, 124 were excluded: 114 due to representing duplicated orthologs in P. aeruginosa , and 10 due to lacking an associated GO term. Thus, a total of 4,886 clusters were retained for analysis. In the comparison between IMI-R vs. IMI-S (Fig. 3 a) and IMI-R vs. IMI/REL (Fig. 3 b) the same four overrepresented terms were identified: outer membrane (GO:0019867), cell outer membrane (GO:0009279), external encapsulating structure (GO:0030312) and cell envelope (GO:0030313). In total, 59 overrepresented proteins with variations associated with resistance to imipenem or imipenem-relebactam were identified among the 2 comparisons, with 17 proteins found in both comparisons (Table S10). However, the comparison between IMI-R vs. IMI-S (Table S11) and IMI-R vs. IMI/REL (Table S12) presented, respectively, 20 and 21 exclusive overrepresented proteins. Among the 17 overrepresented proteins common to both comparisons, 10 are related to iron/siderophore transport: TonB-dependent receptor-like (PA0434, PA2057, PA2089, PA2289, PA3268, PA4897), FecA (PA3902), HasR (PA3408), ChtA (PA4675) e NppA1 (PA1811). We can also highlight three other proteins: PA4545 (ComL), a component of the Bam machinery, whose dysfunction has been associated with increased susceptibility to antimicrobial agents, including the beta-lactam ampicillin ( 56 , 57 ); PA4423 (LpoA), a probable activator of penicillin-binding protein, previously associated with resistance to cefepime ( 58 ); and PA0740 (SdsA1), which has a domain characteristic of metallo-beta-lactamases ( 59 ). These results suggest that variations in these proteins may be associated with resistance to imipenem, but not necessarily to imipenem-relebactam. Regarding resistance to imipenem-relebactam, analyzing the proteins that appear exclusively in the IMI-R vs. IMI/REL comparison, among the 21 proteins with significant variations identified, 8 participate in the transport and retention of iron: TonB (PA0151, PA2070, PA2911), FoxA (PA2466), FemA (PA1910), PhuR (PA4710), PfeA (PA2688) and FepA (PA0931), in addition to a copper transport protein OprC (PA3790). However, the iron transporters identified were different between the two comparisons. Furthermore, 5 efflux pump components were identified: DppA5 (PA5317), nppA2 (PA1810), DguC (PA5082), OpuCC (PA3889) and CzcB (PA2521), part of the czcCBA efflux pump ( 60 ). Among the other proteins with significant variations associated with IMI/REL resistance, we can highlight MltF (PA3764) a murein lytic transglycosylase involved in the regulation of the AmpC beta-lactamase, whose loss was previously associated with increased susceptibility to beta-lactams ( 61 ), and 4 proteins associated with biofilm formation AlgO (PA3257), Alg44 (PA3542) and Mep72 (PA2070, PA2783). GWAS analysis Finally, we performed a k-mer based, reference-free GWAS using Pyseer, comparing IMI-S isolates with the combined group of IMI-R and IMI/REL isolates (Fig. 4 ). The combination of the IMI-R and IMI/REL groups was necessary due to the low number of samples available for the IMI/REL phenotype. The GWAS identified significant variants (p < 1 × 10⁻⁹) related to imipenem resistance in 148 genes, of which 94 encode hypothetical proteins. When these 148 genes were searched in the PAO1 genome reference, only 81 were identified, indicating that 67 genes associated with resistance to imipenem are either absent from the reference genome or highly divergent from their PAO1 counterparts. Among these 67 genes, 60 encode hypothetical proteins, while the remaining genes displayed less than 30% sequence identity to PAO1 genes. Most genes showing strong statistical significance had average effect size ranging from 0.4 to 0.5, suggesting that the presence of these variants confers a moderate to strong increase in the likelihood of imipenem resistance. The genes with the highest number of associated variants were folP (PA4750) and xerD (PA3738). The folP gene (dihydropteroate synthase) had 1,600 associated variants with a maximum -log10(p-value) (maxp) of 14.45, indicating that these variants are highly significant. The xerD gene (tyrosine recombinase) had 1,958 hits with a maxp of 15.8, also suggesting high significance. Furthermore, the effect size of 0.52 suggests a strong average effect of the variants on this gene. The genes with the highest maxp were rpmH (20.97) and gyrA (17.17). RmpH is a ribosomal protein (L34) that can affect translation and possibly resistance. The effect size value of 0.53 indicates a strong effect of these variations. GyrA is the A subunit of DNA gyrase, a common target of antibiotics. The effect size of 0.49 also suggests a strong population effect. Another relevant gene with multiple hits and good significance was the gene encoding the secreted protein Hcp (39 and 24 hits in both copies), maxp of 11.2, and with a high average allele frequency (avg_af) of 0.6, suggesting its frequent presence in resistant strains. In P. aeruginosa , the hemolysin-coregulated protein (Hcp) is a hallmark secreted protein of the Type VI Secretion System (T6SS) and acts as a structural component of its nanotubes. Hcp proteins, such as Hcp1, Hcp2, and Hcp3, are secreted as hexameric rings that stack to form tubes, serving as a channel for other effector proteins to be delivered into target cells, contributing to the bacterium's virulence and pathogenesis. DISCUSSION The combination of imipenem with the beta-lactamase inhibitor relebactam has expanded treatment options against P. aeruginosa , with in vitro activity against ~ 90% of clinical isolates. Nonetheless, resistance to imipenem-relebactam has been associated with metallo-beta-lactamases, porin mutations, alterations in penicillin-binding proteins, efflux pump and efflux pump regulators, although the specific mechanisms driving reduced susceptibility to relebactam remain unresolved. In this work, we investigated the molecular determinants associated with imipenem (IMI-R) and imipenem-relebactam (IMI/REL) in P. aeruginosa by integrating genomic comparisons between resistant and susceptible isolates. Our findings revealed significative amino acid variations in diverse proteins, suggesting that resistance is not driven by a single dominant mechanism but rather results from a multifactorial interplay. P. aeruginosa has intrinsic beta-lactamases of classes C ( bla PDC/AmpC ) and D ( bla OXA ), in addition to the bla PIB−1 gene, recently described as encoding a putative new class of beta-lactamase ( 51 , 52 , 62 ). bla PDC/AmpC expression is induced by imipenem, reducing susceptibility when combined with oprD inactivation, although its activity is inhibited by relebactam ( 63 , 64 ). bla OXA −50 , the predominant variant in P. aeruginosa , is not inhibited by relebactam but exhibits limited catalytic activity against imipenem ( 62 , 65 ). In our analysis, all isolates encoded intrinsic class C and D beta-lactamases, with no significant association with the resistance phenotype, and bla PIB−1 , whose clinical contribution to imipenem resistance remains unclear ( 66 ). On the other hand, beta-lactamases acquired through horizontal gene transfer (HGT) constitute central determinants of resistance, especially metallo-beta-lactamases (MBLs), which are not inhibited by relebactam ( 67 , 68 ). MBL genes were found in 54.5% of imipenem-relebactam-resistant isolates, but only in 3.6% of those resistant to imipenem alone. Among the resistant isolates without MBLs, three carried class A beta-lactamases with partial activity against the inhibitor (GES-2, GES-19/20, and KPC-2) ( 23 , 53 ). Two others lacked acquired beta-lactamases, and the bla OXA−486 and bla OXA−847 variants found in these genomes were also present in susceptible isolates, without clear evidence of activity against imipenem ( 69 ). The integrity of the outer membrane porin OprD is considered one of the main variables associated with imipenem resistance in this pathogen, being the main route of entry of the antibiotic into the periplasmic space ( 70 , 71 ). The oprD gene frequently presents disruptive alterations in its sequence in carbapenem-resistant P. aeruginosa isolates resulting in decreased intracellular antibiotic concentrations ( 23 , 55 ). In our analysis, 80.7% of the imipenem- and/or imipenem-relebactam-resistant isolates harboring the oprD gene (88/109) presented disruptive structural alterations in this gene, including 9 of the 10 imipenem-relebactam-resistant isolates, compared to only 2.96% (4/135) of the susceptible isolates. Among the remaining resistant isolates, 20 presented non-synonymous substitutions in oprD , and in isolate 6.10719, resistant to imipenem-relebactam and sequenced in our study, a sequence identical to that present in the reference strain PAO1 was observed. These data reinforce the central role of alterations in oprD in imipenem resistance, particularly to the imipenem-relebactam combination, but indicate that dysfunction of this gene is not essential to resistance. The absence of disruptive alterations in oprD in the IMI-R isolates analyzed and in one IMI-R isolate (ID 6.10719) suggests the existence of additional mechanisms associated with resistance. However, the possibility that resistant isolates without disruptive alterations in oprD present decreased expression of this gene cannot be ruled out, as previously observed ( 72 , 73 ). Increased expression of the MexAB-OprM efflux pump is directly related to meropenem resistance, but no direct correlation has been established with imipenem resistance ( 74 , 75 ). The use of the Phe-Arg efflux pump inhibitor beta-naphthylamide dihydrochloride (PAβN) causes an increase in the imipenem MIC in P. aeruginosa ( 76 ), however, this effect is likely due to the compound-induced alteration in outer membrane permeability rather than direct inhibition of efflux pumps ( 75 , 77 , 78 ). PA2521 ( czcB ), is associated with Ca2+-induced resistance to tobramycin ( 79 ), and that there is a relationship between the CzcR-CzcS two-component system, which regulates the expression of czcCBA , and resistance to carbapenems, but to our knowledge, no correlation has been established between the efflux pump itself and resistance ( 80 ). We identified 59 overrepresented proteins with variations associated with resistance to imipenem or imipenem-relebactam. Among these, 21 were exclusive to IMI/REL isolates, with 9 involved in metal ion transport and uptake. The increased metal ion influx may be associated with the enzymatic activity of metallo-beta-lactamases and the recently characterized carbapenem PIB-1, intrinsic to P. aeruginosa ( 52 ). Structural studies of PIB-1 revealed that the enzyme assumes a trimeric form in the presence of divalent metals (Zn, Co, and Ni) and that the trimeric form has increased activity relative to the monomeric form. Expression of PIB-1 in Escherichia coli reduces its susceptibility to carbapenems, even in the presence of the inhibitors avibactam and clavulanic acid. Iron is another important metal related to survival, virulence, pathogenicity and resistance ( 81 ). Of the 59 proteins identified with resistance-associated variations, 17 are shared between the two comparisons, 10 of which are directly related to iron and siderophore transport. These proteins include multiple TonB-dependent receptors (PA0434, PA2057, PA2089, PA2289, PA3268, PA4897), as well as FecA (Fe3 + dicitrate transport protein), HasR (heme uptake outer membrane receptor ) , ChtA (ferric aerobactin receptor), all traditionally involved in the capture and internalization of iron. Iron transport by siderophores is closely linked to bacterial virulence, as limited iron availability in the host is a natural defense strategy against infections ( 81 ). Furthermore, there is growing evidence in the literature linking the iron acquisition machinery with the modulation of resistance to antibiotics ( 82 – 84 ), including new drugs as cefiderocol ( 85 ). Studies have shown that iron transport systems, such as those dependent on TonB, can influence the expression and functionality of efflux pumps, beta-lactamase enzymes, and outer membrane permeability, mechanisms directly associated with resistance ( 86 ). Regarding specific resistance to imipenem-relebactam, 21 unique proteins were identified, eight of which are related to iron transport and retention, highlighting different variants of the TonB transporters (PA0151, PA2070, PA2911), FoxA, FemA, PhuR, PfeA, and FepA, as well as a copper transport protein, OprC (PA3790). The qualitative distinction of metal transport systems between resistance to imipenem alone and its combination with relebactam suggests differentiated adaptations that deserve further functional investigation, as metal transport can modulate metabolic pathways and mechanisms of response to antibiotic stress. The GWAS analysis revealed a set of genes with significant variations related to resistance to imipenem and/or imipenem-relebactam, different from the set of genes identified by the overrepresentation analysis. The folP and xerD genes stood out in the GWAS analysis, presenting the highest number of hits and high significance and effect in the population. FolP (dihydropteroate synthase) is an enzyme involved in folic acid metabolism, which may be related to sulfonamide resistance in many pathogens ( 87 ). The xerD gene, in turn, encodes a tyrosine recombinase involved in DNA recombination, which may promote genetic diversity and resistance ( 88 ). Only the cupC3 gene was common to both analyses. cupC3 encodes a protein associated with the cup system (chaperone-usher pathway), involved in the assembly of pili or fimbriae on the bacterial cell surface. Fimbriae influence adhesion, biofilm formation, and interaction with the environment, aspects that have been linked to both virulence and antimicrobial resistance through physical protection and permeability modulation ( 89 ). The unique presence of cupC3 at the intersection of the two analyses suggests that, although other genes related to iron transport and beta-lactamases are essential, resistance to imipenem and its combination with relebactam also involves changes in the structural architecture of the bacterial surface. CONCLUSION Our results are consistent with clinical and in vitro data showing that most P. aeruginosa isolates remain susceptible to imipenem-relebactam, while resistance emerges in a restricted subset of strains. Importantly, although our analyses identified candidate genes and mutations, the specific contribution of each alteration to relebactam resistance remains unclear. This highlights a critical gap in current knowledge, as the precise molecular mechanisms underlying decreased activity of relebactam are yet to be elucidated. Together, our study reinforces the concept that resistance to novel beta-lactam/beta-lactamase inhibitor combinations arise from the convergence of classical resistance determinants, while also pointing to the necessity of functional validation. Such investigations will be essential to define the relative weight of these genetic variations and to inform the rational use of imipenem-relebactam in clinical settings. Abbreviations GES Guiana extended-spectrum beta-lactamase IMP Imipenemase VIM Verona integron-encoded metallo-beta-lactamase IMI Imipenem OXA Oxacillinase PDC Pseudomonas cephalosporinase PIB 1-Pseudomonas aeruginosa chromosomally-encodedβ-lactamase KPC 2-Klebsiella pneumoniae carbapenemase 2 SPM 1 São Paulo Metallo-beta-lactamase NDM 1 New Delhi Metallo-β-lactamase REL relebactam IMI R-imipenem-resistant IMI S-impenem-sentitive IMI/REL imipenem/relebactam resitant FDA Food and Drug Administration GWAS Genome-Wide Association Study GO Gene Ontology MIC Minimum Inhibitory Concentration EUCAST European Committee on Antimicrobial Susceptibility Testing BV BRC-Bacterial and Viral Bioinformatics Resource Center WGS Whole genome sequencing CARD Comprehensive Antibiotic Resistance Database RGI Resistance Gene Identifier PAO1 Pseudomonas aeruginosa strain O1 LMM Linear Mixed Model FDR False Rate Discovery XDR Extensively Drug-Resistant MDR Multidrug-resistant CAZ/AVI ceftazidime-avibactam CTZ/TAZ ceftolozane-tazobactam CDS Coding sequence ARG Antimicrobial Resistance Gene ARO Antibiotic Resistance Ontology MBL Metallo-beta-lactamase HGT Horizontal Gene Transfer Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding information This research was funded by the National Council for Scientific and Technological Development (CNPq), CAPES, and Fiocruz. CNPq grant number 424410/2018-4; InovaFiocruz/Fundacão Oswaldo Cruz grant number VPPCB- 07-FIO-18-2-38. Author Contribution ABL: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing; JS: Data curation, Formal analysis; LB: Sample collection, Methodology, MIC determination; CASI: Sample collection, Methodology, MIC determination; JAC: Data curation, Formal analysis; FFBT: Conceptualization, Funding acquisition, Writing – review and editing; HF: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review and editing. 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As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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13:41:08","extension":"html","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":203112,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7801871/v1/0d3c8c4853e8e010ca035383.html"},{"id":95119238,"identity":"f6a4994c-4182-40b9-9ff2-d63af775da63","added_by":"auto","created_at":"2025-11-04 13:41:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1162042,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of resistance genes in the IMI/REL isolates. The predicted proteome of IMI/REL isolates was compared with the CARD database to identify antimicrobial resistance genes (ARGs). The isolates' genomes are identified by their IDs beginning with the prefix \"R_.\" The resistant antibiotics are shown next to the genomes. The color code defines the functional class of the ARGs.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7801871/v1/5ae2e2f0b0664f630e887afe.png"},{"id":95119237,"identity":"a5bd988b-1c9b-49b3-bf19-dc8576a49d27","added_by":"auto","created_at":"2025-11-04 13:41:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFrequency of insertions, deletions and premature stop codons in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eoprD\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene among all isolates analyzed.\u003c/strong\u003e Partial sequences were excluded from the analysis. (a) IMI-S isolates. (b) IMI-R+IMI/REL isolates.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7801871/v1/0c68467da2b31b7f9792e7a0.png"},{"id":95225576,"identity":"cba920dc-4df5-4680-a6f6-9d3c1c7241d4","added_by":"auto","created_at":"2025-11-05 16:25:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverrepresentation of GO terms.\u003c/strong\u003e Proteins with significant variations associated with resistance (p \u0026lt; 0.05, odds ratio \u0026gt; 1) analyzed through the PANTHER overrepresentation test using core genome proteins as a reference. The significance of the GO terms was assessed using Fisher's exact test for raw p-values and the Benjamini-Hochberg method for false discovery rate correction. (a) IMI-R X IMI-S, (b) IMI-R X IMI/REL\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7801871/v1/d77a7204cf96e646850ffd03.png"},{"id":95119334,"identity":"cbff265b-e0ae-4e91-936e-874dd13126cf","added_by":"auto","created_at":"2025-11-04 13:41:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGWAS results for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e resistant to imipenem and/or imipenem-relebactam.\u003c/strong\u003eEach point represents a locus significantly associated with resistance, plotted according to the average effect size (x-axis) and maximum –log10(p-value) (y-axis). The size of the circles indicates the number of k-mers supporting the association, while the color scale reflects the average minor allele frequency (MAF). Genes with the strongest associations include \u003cem\u003erpmH\u003c/em\u003e, \u003cem\u003egyrA\u003c/em\u003e, xerD, and others related to efflux systems, regulatory elements, and cell wall metabolism.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7801871/v1/c287580c7608a861d581da1e.png"},{"id":95312724,"identity":"9dbabf5e-6679-4263-a5ce-ab1fe10b381a","added_by":"auto","created_at":"2025-11-06 15:50:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2835043,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7801871/v1/1ad3222e-6074-42e6-99e9-2dc9b110c11c.pdf"},{"id":95119243,"identity":"59b5f9ab-0e7e-4121-8865-34b306405d0c","added_by":"auto","created_at":"2025-11-04 13:41:07","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3766880,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesS1S12.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7801871/v1/f754c00f9696e1b0bccc96a7.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular determinants associated with resistance to imipenem and imipenem–relebactam in clinical Pseudomonas aeruginosa isolates","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eBacterial resistance to antimicrobial agents is a phenomenon observed since the 1940s (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), just over 10 years after the discovery of the first antibiotic (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), and it has been demonstrated that antibiotic resistance genes are part of the natural gene repertoire of microbial populations (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In general, antimicrobial resistance is mediated by: a) inactivation or enzymatic alteration of the drug; b) modification of its binding site; c) reduction of intracellular accumulation of the drug; d) overexpression of the target; and e) formation of biofilms (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In addition to the resistance mechanisms themselves, alterations in genes that encode proteins associated with DNA repair and recombination have been described, which lead to an increase in the mutation rates observed in organisms and, consequently, increase the variation in bacterial population structures (5). This phenomenon is probably associated with the emergence of resistance mechanisms, which can then spread to different species by horizontal gene transfer (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe most important multidrug-resistant bacteria from a public health point of view belong to the ESKAPE group, which comprises \u003cem\u003eEnterococcus spp\u003c/em\u003e., \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, e \u003cem\u003eEnterobacter spp.\u003c/em\u003e (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Among the aforementioned pathogens, \u003cem\u003eP. aeruginosa\u003c/em\u003e is one of the main etiological agents of nosocomial infections in intensive care units in Brazil (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), particularly important in patients with cystic fibrosis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Due to low cell wall permeability and a high capacity to acquire and express multiple resistance mechanisms, the prevalence of resistant strains of \u003cem\u003eP. aeruginosa\u003c/em\u003e has increased dramatically in the last 20 years, as has the relative risk of mortality associated with infections by this pathogen (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral new antibiotic formulations have been developed in recent years, such as Ceftolozane/Tazobactam (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), Ceftazidime\u0026ndash;avibactam (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and Imipenem-Relebactam (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), which combine beta-lactam antibiotics with beta-lactamase inhibitors (a class of enzymes that degrade beta-lactam antibiotics). In general, beta-lactamase inhibitors interact durably with the active site of the enzyme, preventing its action in the degradation of the antibiotic (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Imipenem-Relebactam formulation was approved by the U.S. Food and Drug Administration in 2019 for the treatment of complicated intra-abdominal and urinary tract infections (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), and the approval was extended in 2020 for the treatment of hospital-acquired bacterial pneumonia and ventilator-associated pneumonia (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In in vitro tests, approximately 90% of clinical isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e demonstrate susceptibility to the new formulation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Genomic analyses of isolates resistant to imipenem-relebactam isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e have already identified some genes and genetic variations associated with resistance, notably the presence of beta-lactamases of the GES, VIM and IMP classes, and mutations in porins, penicillin-binding proteins and proteins associated with the regulation of efflux pumps (\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The specific mechanisms related to decreased susceptibility to relebactam, however, have not yet been precisely elucidated.\u003c/p\u003e\u003cp\u003eThe epidemiological situation of multidrug-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e infections, together with the limited amount of information on genomic variations related to imipenem-relebactam resistance in this pathogen, demonstrate the importance of continuous study of resistance mechanisms to new antimicrobials, as well as genetic alterations associated with the development of resistance mechanisms present in multidrug-resistant bacteria. The objective of this study was to perform genome sequencing of 10 imipenem-relebactam resistant (IMI/REL) \u003cem\u003eP. aeruginosa\u003c/em\u003e strains and compare these genomes with those of imipenem-resistant (IMI-R) and imipenem sensitive (IMI-S) \u003cem\u003eP. aeruginosa\u003c/em\u003e obtained from databases. Through resistance gene analysis, genomic comparison, GO enrichment studies and genome wide association studies (GWAS), we identified that variations in proteins associated with different metabolic functions probably linked to imipenem-relebactam resistance in \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e"},{"header":"METHODOLOGY","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial strains\u003c/h2\u003e\u003cp\u003eThe present study investigated 10 multidrug-resistant clinical isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e from hospitals in the southern region of Brazil obtained from the Cajuru University Hospital (Curitiba, Brazil) in the period of January/2020 to December/2020. All isolates were obtained from clinical samples, including blood, respiratory, and urinary tract. Antimicrobial susceptibility tests to piperacillin/tazobactam, aztreonam, cefepime, ceftazidime, ceftazidime-avibactam, ceftolozane-tazobactam, imipenem, imipenem-relebactam, meropenem, ciprofloxacin, colistin, polymyxin B, gentamicin and amikacin were performed. Broth microdilution technique was used to determine the minimum inhibitory concentration (MIC) of each antibiotic for each isolate (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The breakpoints according to EUCAST (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) were used, and the tests were performed in triplicate.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenome sequencing, assembly and annotation\u003c/h3\u003e\n\u003cp\u003eGenomic DNA from each isolate was extracted using the Wizard\u0026reg; HMW DNA Extraction Kit (Promega). DNA concentration was measured by the fluorometric method using the Qubit equipment (Thermo Fisher Scientific, Mississauga, ON, Canada). Sequencing libraries were prepared with Illumina DNA prep (Illumina, Inc., San Diego, CA, USA) following the manufacturer's instructions, and sequencing was performed on the Illumina NovaSeq 6000 platform (OGC, Oxford, UK) using NovaSeq SP reagent kits (300 cycles, paired-end reads) at the Instituto para Pesquisa do C\u0026acirc;ncer - IPEC (Guarapuava, Brazil). Genome assemblies were performed using SPAdes v3.15.5 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), MeDuSa v1.6 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and FGAP 1.8.1 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) tools, and the generated contigs were annotated using Prokka v1.14.5 software (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The genomes have been deposited in the EMBL-EBI database (ebi.ac.uk/) under the accession numbers listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eSelection of genomes from databases\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e genome sequences were obtained from the BV-BRC database (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), using the following parameters: Genome Status \u0026ldquo;WGS\u0026rdquo;; Genome Quality \u0026ldquo;Good\u0026rdquo;; Host Group \u0026ldquo;Human\u0026rdquo;. From this set, imipenem-resistant and -sensitive genomes were selected in the AMR Phenotypes category, with the filter: Evidence \u0026ldquo;Laboratory Method\u0026rdquo;, or indication of the methodology used (e.g. disk diffusion). Genomes sequenced exclusively by Oxford Nanopore technology were excluded. The BV-BRC IDs were listed in the Table S2\u003c/p\u003e\n\u003ch3\u003eIdentification of resistance genes\u003c/h3\u003e\n\u003cp\u003eTo identify resistance genes, the Resistance Gene Identifier (RGI) software version 6.0.3 was used, with version 3.2.9 of the Comprehensive Antibiotic Resistance Database (CARD) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Only proteins identified in the \u0026ldquo;Perfect\u0026rdquo; category - which consists of sequences identical to those present in the database - and \u0026ldquo;Strict\u0026rdquo; - which detects previously unknown variants of antimicrobial resistance genes, using detection models with similarity cutoff lines curated by CARD - were considered. Subsequently, the result of the RGI analysis was filtered through Python scripts to identify proteins previously associated with resistance to β-lactams and, specifically, to carbapenems. The identified beta-lactamases were classified according to the Ambler system (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of the\u003c/b\u003e \u003cb\u003eoprD\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInitially, the \u003cem\u003eoprD\u003c/em\u003e gene sequence (PA0958) was obtained from the genome of the reference strain PAO1, based on the Prokka annotation. The \u003cem\u003eoprD\u003c/em\u003e gene sequence was aligned against all genomes to identify and extract the specific \u003cem\u003eoprD\u003c/em\u003e sequence of each isolate using BLASTN (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Subsequently, a global alignment was performed between the gene of each isolate and the reference gene, using the BioPython library (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The alignments were analyzed to identify specific genetic variations in the \u003cem\u003eoprD\u003c/em\u003e gene and the protein it encodes. The graphs generated from the results of this analysis were made with the Python library Matplotlib (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eAnalysis of variations in proteins of the core genome\u003c/h3\u003e\n\u003cp\u003eWe used CD-HIT software (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) to cluster orthologous proteins and identify variations in the core genome associated with resistance to imipenem and imipenem-relebactam. Only clusters containing proteins present in at least 95% of the isolates were considered part of the core genome, while the others were excluded from subsequent analyses. Functional annotation of the clusters was performed by comparing the sequence of the representative protein of each cluster with the proteins of the reference strain \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) using the BLASTP algorithm (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Functional information was obtained from the Pseudomonas Community Annotation Project (PseudoCAP) through the Pseudomonas Genome Database (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Proteins with identity greater than 90% and minimum query coverage of 50% per subject were considered orthologous. The identifiers of the orthologous proteins in the PAO1 strain were used to assign Gene Ontology terms (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) to each cluster, by comparison with the PANTHER database (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Next, for each cluster, a global alignment was performed between each protein and the protein of the reference strain using the BioPython library (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), and the alignments were analyzed to identify specific variations. At this stage, clusters that did not present one or more proteins of the reference strain were excluded.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eIn order to evaluate the statistical relationship between protein variations and imipenem and imipenem-relebactam resistance phenotypes, the odds ratio was calculated by comparing the number of resistant and susceptible isolates with and without each variation, and Fisher's exact test was used to assess whether there is a significant association between each variation and the resistance phenotype, through the SciPy library (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). For each variation, 2 distinct comparisons were made: imipenem-resistant isolates against imipenem-susceptible isolates (IMI-R vs. IMI-S) and imipenem-resistant isolates against imipenem-relebactam-resistant isolates (IMI-R vs. IMI/REL). Next, an analysis of the overrepresentation of GO terms of proteins that presented variations significantly associated with resistance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with an odds ratio greater than 1 was performed, using the proteins of the core genome as a reference, through the PANTHER overrepresentation test. The significance of the overrepresented GO terms was calculated following the conventional parameters of the tool: Fisher's exact test to calculate the raw p-values and false discovery rate, using the Benjamini-Hochberg method, to calculate the corrected p-values. From this analysis, graphs were generated with the Python libraries Matplotlib (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and seaborn (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenome-wide association study (GWAS)\u003c/h3\u003e\n\u003cp\u003eA genome-wide association study (GWAS) was conducted using Pyseer (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), comparing imipenem-resistant (IMI-R\u0026thinsp;+\u0026thinsp;IMI/REL) and imipenem-sensitive (IMI-S) groups. Because bacterial populations have a clonal structure, the samples cannot be considered independent. Therefore, it was necessary to adjust the association analyses for the effect of population structure using phylogenetic inference based on the core genome (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The core genome was identified using the ROARY v.3.13.0 (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) from genomic annotation files in gff3 format. Subsequently, multiple alignment of the core genome was used in the FastTree program (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) to generate the phylogenetic tree. The GWAS was performed using the k-mer counting method, using the fms- lite tool, free of a reference genome. The effect of each identified variant on the resistance phenotype, adjusted for population structure, was estimated by fitting a linear mixed model (LMM), implemented in the Pyseer program. The p-value corresponding to each adjusted model, which will be corrected for the number of variants analyzed using the False Discovery Rate (FDR) method (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), were considered as associated with the resistance phenotype.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eEvaluation of the resistance genotype and phenotype of\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eisolates\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe determination of the minimum inhibitory concentration (MIC) by broth microdilution of the 10 sequenced isolates demonstrated that all isolates are resistant to imipenem (IMI), and that only one isolate (identified as 15_20783) is susceptible to the imipenem-relebactam combination (IMI/REL) (Table S3). Based on the resistance phenotypes observed, 8 isolates can be classified as extensively resistant (XDR) and 2 as multidrug resistant to antibiotics (MDR) (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Isolate 15_20783, specifically, is also susceptible to two other combinations of beta-lactams with beta-lactamase inhibitors (ceftazidime-avibactam [CAZ/AVI] and ceftolozane-tazobactam [CTZ/TAZ]), and a similar phenotypic profile is observed in isolate 13_21675 - susceptible to CTZ/TAZ and with a four-fold lower MIC for IMI/REL compared to IMI. The other isolates do not show differences in MIC values between IMI and IMI/REL, but isolates 2_20589, 8_17683 and 12_18480 also show reduced MIC for ceftazidime in the presence of the inhibitor avibactam.\u003c/p\u003e\u003cp\u003eComparison of the annotated CDSs for each genome against the CARD database (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) reveals the massive presence of efflux pumps and intrinsic resistance in all isolates. The consistent presence of genes linked to efflux systems is observed, notably the MexAB-OprM, MexXY, MexCD-OprJ, and MexEF-OprN pumps. Transcriptional regulators such as MexR, MexT, NalC, among others, regulate the expression of efflux pumps and are frequently associated with their overexpression in resistant isolates. In addition to efflux pumps, the analysis revealed the presence of the beta-lactamases PDC and OXA-50-like in all isolates. The beta-lactamases VIM-2, KPC-2, IMP-1, IMP-16, SPM-1, and NDM1 were also identified, but in different isolates. Only isolate R_2.20589 presents 2 acquired carbapenemases: NDM-1 and KPC-2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of antimicrobial resistance genes (ARGs)\u003c/h2\u003e\u003cp\u003eTo increase the number of genomes and the robustness of our analysis, we searched the BV-BRC database (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) for genomes of isolates resistant to imipinem-relebactam (IMI/REL), resistant to imipenem and without information regarding resistance to imipenem/relebactam (IMI-R) and sensitive to imipenem (IMI-S). In total, 110 genomes of IMI-R and 136 genomes of IMI-S isolates were obtained. Only 2 genomes had an indication of IMI/REL resistance. For the purposes of analysis, we considered the 110 IMI-R isolates resistant only to imipenem. All genomes retrieved from the BV-BRC database were reannotated to maintain analysis uniformity.\u003c/p\u003e\u003cp\u003eComparison with the CARD database, including both sequenced genomes and those retrieved from BV-BRC, identified 15,758 antibiotic resistance genes (ARGs) (Table S4), grouped into 192 distinct antibiotic resistance ontology (ARO) terms. Of these, 4,073 ARGs (25.85%) were linked to carbapenem resistance (Table S5), while 1,537 (9.75%) were associated with resistance to other beta-lactams (Table S6). Among the carbapenem resistance determinants, 3,505 (86.05%) correspond to efflux pump components (e.g., MexA, MexB, OprM) or regulators of efflux pump expression (e.g., ParR, ParS, MexR), classified under 16 ARO terms. The remaining 568 ARGs were beta-lactamases distributed across 88 distinct ARO terms.\u003c/p\u003e\u003cp\u003eGES (Ambler class A) and NDM/SPM (Ambler class B) beta-lactamases were exclusively detected in IMI/REL genomes, whereas KPC (class A) and VIM (class B) beta-lactamases were identified in both IMI-R and IMI/REL genomes. Among IMI-S isolates, only one class B beta-lactamase, IMP-45, was detected. All \u003cem\u003eP. aeruginosa\u003c/em\u003e genomes harbored the intrinsic beta-lactamases PDC (class C) and OXA (class D). Notably, multiple class D beta-lactamases were present in 3 IMI-S isolates (2.21%), 21 IMI-R isolates (18.92%), and 4 IMI/REL isolates (36.36%). In addition, all genomes carried the recently described PIB-1 (PA5542), an intrinsic carbapenemase of \u003cem\u003eP. aeruginosa\u003c/em\u003e capable of hydrolyzing carbapenems broadly and resistant to inhibition by avibactam and clavulanic acid (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the IMI/REL isolates, 36.36% presented Ambler class A beta-lactamases and 54.54% presented Ambler class B beta-lactamases. The frequency of class A and B beta-lactamases was much lower for IMI-R (5.4% and 3.6%, respectively) and IMI-S isolates (0% and 0.73%, respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of the 4 IMI/REL isolates that do not have MBLs (Genome IDs: 13.21675, 287.14071, 287.16518 and 5.13697), only 287.14071 and 287.16518 present GES beta-lactamases resistant to relebactam inhibition (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Isolate 8.17683 has a KPC-2 that, despite being inhibited by relebactam, confers resistance to imipenem-relebactam when overexpressed (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). The two remaining isolates (13.21675 and 5.13697), which do not have Ambler class A or B beta-lactamases, have OXA-486 and OXA-847 class D beta-lactamases, respectively. However, there are no records of specific activity of these enzymes against imipenem and they were identified in several IMI-S isolates among those analyzed in this study (Table S7).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFrequency of beta-lactamases of the four Amber classes identified in isolates with different resistance phenotypes to imipenem and imipenem-relebactam\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhenotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eClass A\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eClass B\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eClass C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eClass D\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIMI/REL\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4 (36,36%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6 (54,54%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11 (100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11 (100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIMI-R\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6 (5,4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (3,6%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e111 (100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e111 (100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIMI-S\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0 (0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (0,73%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e136 (100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e136 (100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of variations in the ARG\u003c/b\u003e \u003cb\u003eoprD\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eoprD\u003c/em\u003e gene, responsible for encoding the OprD porin, was not identified in the comparison with the CARD database. Despite being part of the core genome of \u003cem\u003eP. aeruginosa\u003c/em\u003e, the \u003cem\u003eoprD\u003c/em\u003e gene frequently presents frameshifts or premature stop codons in carbapenem-intermediate and resistant strains (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e), resulting in truncated proteins and making its annotation in the genomes difficult. To obtain the \u003cem\u003eoprD\u003c/em\u003e gene sequences from the 258 genomes analyzed in this study, we conducted a BLASTN search using the \u003cem\u003eoprD\u003c/em\u003e sequence from the PAO1 strain as a reference. One IMI-R genome (Genome ID 287.7811) was found to lack the \u003cem\u003eoprD\u003c/em\u003e gene, while 12 IMI-R and 1 IMI-S isolates contained only partial sequences located at the ends of contigs. None of IMI/REL genomes were excluded. These isolates were excluded from further analysis, leaving 244 isolates (109 IMI-R\u0026thinsp;+\u0026thinsp;IMI/REL and 135 IMI-S), each carrying a single copy of the gene. The \u003cem\u003eoprD\u003c/em\u003e sequences were compared to that of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1. Only 9 isolates encoded proteins identical to PAO1\u0026rsquo;s \u003cem\u003eoprD\u003c/em\u003e, including one IMI-R isolate (Genome ID 6.10719) and 8 IMI-S isolates. Among the remaining IMI-R and IMI/REL isolates, 68 out of 109 (62.38%) carried insertions or deletions leading to frameshift mutations, 20 out of 109 (18.35%) had nonsense mutations, and 20 out of 109 (18.35%) exhibited nucleotide substitutions resulting in amino acid changes. In contrast, among the IMI-S isolates, 2 frameshift mutations, 2 nonsense mutations, and 123 non-synonymous substitutions were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Considering only the 11 IMI/REL isolates, one had a partial sequence (ID 287.16518), one had a sequence identical to that of PAO1 (ID 6.10719) and the other 9 presented alterations in the reading frame of the gene.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of variations in proteins of the core genome\u003c/h2\u003e\u003cp\u003eConsidering that relebactam does not inhibit class B or class D beta-lactamases, and given that some IMI/REL resistant isolates lack these enzymes, we analyzed the core genome of the \u003cem\u003eP. aeruginosa\u003c/em\u003e genome assembly to identify other factors possibly associated with resistance to imipenem and imipenem-relebactam. Cluster analysis performed with the CD-HIT tool resulted in 35,854 clusters of orthologous proteins among the 258 genomes evaluated. Of these, 5,010 clusters contained proteins present in at least 95% of the isolates and had an orthologous proteins described in the reference strain PAO1 were retained in the analysis.\u003c/p\u003e\u003cp\u003eThe analysis of amino acid variations was conducted for proteins within each cluster, comparing sequences between IMI-R and IMI-S (Table S8), as well as between IMI-R and IMI/REL (Table S9), to assess differences associated with distinct resistance phenotypes. Only variations with a p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 and odds ratio greater than 1 were considered significant. Comparing isolates IMI-R with isolates IMI-S, 2,012 positions with significant variations were identified, present in 1,106 different proteins. In the comparison between IMI-R with IMI/REL isolates, 1,371 positions with significant variations were identified in 1,015 different proteins. Most of the identified variations consist of amino acid substitutions, followed by deletions and insertions. General information on the significant variations observed is available in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSignificant variations identified in each comparison performed\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteins with significant variations*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePositions with significant variations*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePositions with substitutions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePositions with deletions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePositions with insertions\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIMI-R\u0026thinsp;+\u0026thinsp;IMI/REL vs. IMI-S\u003c/b\u003e\u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1799\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e184\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIMI-R vs. IMI/REL\u003c/b\u003e\u003csup\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1371\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1307\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003e*\u003c/b\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0,05 e \u003cem\u003eodds ratio\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;1\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eGO overrepresentation analysis\u003c/h2\u003e\u003cp\u003eTo investigate whether proteins with significant amino acid variations were enriched in specific biological functions potentially associated with IMI/REL resistance, a GO overrepresentation analysis was performed. In this approach, the functional distribution of these proteins was compared to that of the core genome clusters. From the 5,010 clusters constituting the core genome, 124 were excluded: 114 due to representing duplicated orthologs in \u003cem\u003eP. aeruginosa\u003c/em\u003e, and 10 due to lacking an associated GO term. Thus, a total of 4,886 clusters were retained for analysis.\u003c/p\u003e\u003cp\u003eIn the comparison between IMI-R vs. IMI-S (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and IMI-R vs. IMI/REL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) the same four overrepresented terms were identified: outer membrane (GO:0019867), cell outer membrane (GO:0009279), external encapsulating structure (GO:0030312) and cell envelope (GO:0030313). In total, 59 overrepresented proteins with variations associated with resistance to imipenem or imipenem-relebactam were identified among the 2 comparisons, with 17 proteins found in both comparisons (Table S10). However, the comparison between IMI-R vs. IMI-S (Table S11) and IMI-R vs. IMI/REL (Table S12) presented, respectively, 20 and 21 exclusive overrepresented proteins. Among the 17 overrepresented proteins common to both comparisons, 10 are related to iron/siderophore transport: TonB-dependent receptor-like (PA0434, PA2057, PA2089, PA2289, PA3268, PA4897), FecA (PA3902), HasR (PA3408), ChtA (PA4675) e NppA1 (PA1811). We can also highlight three other proteins: PA4545 (ComL), a component of the Bam machinery, whose dysfunction has been associated with increased susceptibility to antimicrobial agents, including the beta-lactam ampicillin (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e); PA4423 (LpoA), a probable activator of penicillin-binding protein, previously associated with resistance to cefepime (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e); and PA0740 (SdsA1), which has a domain characteristic of metallo-beta-lactamases (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). These results suggest that variations in these proteins may be associated with resistance to imipenem, but not necessarily to imipenem-relebactam.\u003c/p\u003e\u003cp\u003eRegarding resistance to imipenem-relebactam, analyzing the proteins that appear exclusively in the IMI-R vs. IMI/REL comparison, among the 21 proteins with significant variations identified, 8 participate in the transport and retention of iron: TonB (PA0151, PA2070, PA2911), FoxA (PA2466), FemA (PA1910), PhuR (PA4710), PfeA (PA2688) and FepA (PA0931), in addition to a copper transport protein OprC (PA3790). However, the iron transporters identified were different between the two comparisons. Furthermore, 5 efflux pump components were identified: DppA5 (PA5317), nppA2 (PA1810), DguC (PA5082), OpuCC (PA3889) and CzcB (PA2521), part of the \u003cem\u003eczcCBA\u003c/em\u003e efflux pump (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the other proteins with significant variations associated with IMI/REL resistance, we can highlight MltF (PA3764) a murein lytic transglycosylase involved in the regulation of the AmpC beta-lactamase, whose loss was previously associated with increased susceptibility to beta-lactams (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e), and 4 proteins associated with biofilm formation AlgO (PA3257), Alg44 (PA3542) and Mep72 (PA2070, PA2783).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGWAS analysis\u003c/h2\u003e\u003cp\u003eFinally, we performed a k-mer based, reference-free GWAS using Pyseer, comparing IMI-S isolates with the combined group of IMI-R and IMI/REL isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The combination of the IMI-R and IMI/REL groups was necessary due to the low number of samples available for the IMI/REL phenotype. The GWAS identified significant variants (p\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026times; 10⁻⁹) related to imipenem resistance in 148 genes, of which 94 encode hypothetical proteins. When these 148 genes were searched in the PAO1 genome reference, only 81 were identified, indicating that 67 genes associated with resistance to imipenem are either absent from the reference genome or highly divergent from their PAO1 counterparts. Among these 67 genes, 60 encode hypothetical proteins, while the remaining genes displayed less than 30% sequence identity to PAO1 genes. Most genes showing strong statistical significance had average effect size ranging from 0.4 to 0.5, suggesting that the presence of these variants confers a moderate to strong increase in the likelihood of imipenem resistance.\u003c/p\u003e\u003cp\u003eThe genes with the highest number of associated variants were \u003cem\u003efolP\u003c/em\u003e (PA4750) and \u003cem\u003exerD\u003c/em\u003e (PA3738). The \u003cem\u003efolP\u003c/em\u003e gene (dihydropteroate synthase) had 1,600 associated variants with a maximum -log10(p-value) (maxp) of 14.45, indicating that these variants are highly significant. The \u003cem\u003exerD\u003c/em\u003e gene (tyrosine recombinase) had 1,958 hits with a maxp of 15.8, also suggesting high significance. Furthermore, the effect size of 0.52 suggests a strong average effect of the variants on this gene. The genes with the highest maxp were \u003cem\u003erpmH\u003c/em\u003e (20.97) and \u003cem\u003egyrA\u003c/em\u003e (17.17). RmpH is a ribosomal protein (L34) that can affect translation and possibly resistance. The effect size value of 0.53 indicates a strong effect of these variations. GyrA is the A subunit of DNA gyrase, a common target of antibiotics. The effect size of 0.49 also suggests a strong population effect. Another relevant gene with multiple hits and good significance was the gene encoding the secreted protein Hcp (39 and 24 hits in both copies), maxp of 11.2, and with a high average allele frequency (avg_af) of 0.6, suggesting its frequent presence in resistant strains. In \u003cem\u003eP. aeruginosa\u003c/em\u003e, the hemolysin-coregulated protein (Hcp) is a hallmark secreted protein of the Type VI Secretion System (T6SS) and acts as a structural component of its nanotubes. Hcp proteins, such as Hcp1, Hcp2, and Hcp3, are secreted as hexameric rings that stack to form tubes, serving as a channel for other effector proteins to be delivered into target cells, contributing to the bacterium's virulence and pathogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe combination of imipenem with the beta-lactamase inhibitor relebactam has expanded treatment options against \u003cem\u003eP. aeruginosa\u003c/em\u003e, with in vitro activity against ~\u0026thinsp;90% of clinical isolates. Nonetheless, resistance to imipenem-relebactam has been associated with metallo-beta-lactamases, porin mutations, alterations in penicillin-binding proteins, efflux pump and efflux pump regulators, although the specific mechanisms driving reduced susceptibility to relebactam remain unresolved. In this work, we investigated the molecular determinants associated with imipenem (IMI-R) and imipenem-relebactam (IMI/REL) in \u003cem\u003eP. aeruginosa\u003c/em\u003e by integrating genomic comparisons between resistant and susceptible isolates. Our findings revealed significative amino acid variations in diverse proteins, suggesting that resistance is not driven by a single dominant mechanism but rather results from a multifactorial interplay.\u003c/p\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e has intrinsic beta-lactamases of classes C (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003ePDC/AmpC\u003c/em\u003e\u003c/sub\u003e) and D (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eOXA\u003c/em\u003e\u003c/sub\u003e), in addition to the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003ePIB\u0026minus;1\u003c/em\u003e\u003c/sub\u003e gene, recently described as encoding a putative new class of beta-lactamase (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003ePDC/AmpC\u003c/em\u003e\u003c/sub\u003e expression is induced by imipenem, reducing susceptibility when combined with \u003cem\u003eoprD\u003c/em\u003e inactivation, although its activity is inhibited by relebactam (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eOXA\u003c/em\u003e\u0026minus;50\u003c/sub\u003e, the predominant variant in \u003cem\u003eP. aeruginosa\u003c/em\u003e, is not inhibited by relebactam but exhibits limited catalytic activity against imipenem (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). In our analysis, all isolates encoded intrinsic class C and D beta-lactamases, with no significant association with the resistance phenotype, and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003ePIB\u0026minus;1\u003c/em\u003e\u003c/sub\u003e, whose clinical contribution to imipenem resistance remains unclear (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). On the other hand, beta-lactamases acquired through horizontal gene transfer (HGT) constitute central determinants of resistance, especially metallo-beta-lactamases (MBLs), which are not inhibited by relebactam (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). MBL genes were found in 54.5% of imipenem-relebactam-resistant isolates, but only in 3.6% of those resistant to imipenem alone. Among the resistant isolates without MBLs, three carried class A beta-lactamases with partial activity against the inhibitor (GES-2, GES-19/20, and KPC-2) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Two others lacked acquired beta-lactamases, and the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eOXA\u0026minus;486\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eOXA\u0026minus;847\u003c/em\u003e\u003c/sub\u003e variants found in these genomes were also present in susceptible isolates, without clear evidence of activity against imipenem (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe integrity of the outer membrane porin OprD is considered one of the main variables associated with imipenem resistance in this pathogen, being the main route of entry of the antibiotic into the periplasmic space (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). The \u003cem\u003eoprD\u003c/em\u003e gene frequently presents disruptive alterations in its sequence in carbapenem-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates resulting in decreased intracellular antibiotic concentrations (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). In our analysis, 80.7% of the imipenem- and/or imipenem-relebactam-resistant isolates harboring the \u003cem\u003eoprD\u003c/em\u003e gene (88/109) presented disruptive structural alterations in this gene, including 9 of the 10 imipenem-relebactam-resistant isolates, compared to only 2.96% (4/135) of the susceptible isolates. Among the remaining resistant isolates, 20 presented non-synonymous substitutions in \u003cem\u003eoprD\u003c/em\u003e, and in isolate 6.10719, resistant to imipenem-relebactam and sequenced in our study, a sequence identical to that present in the reference strain PAO1 was observed. These data reinforce the central role of alterations in \u003cem\u003eoprD\u003c/em\u003e in imipenem resistance, particularly to the imipenem-relebactam combination, but indicate that dysfunction of this gene is not essential to resistance. The absence of disruptive alterations in \u003cem\u003eoprD\u003c/em\u003e in the IMI-R isolates analyzed and in one IMI-R isolate (ID 6.10719) suggests the existence of additional mechanisms associated with resistance. However, the possibility that resistant isolates without disruptive alterations in \u003cem\u003eoprD\u003c/em\u003e present decreased expression of this gene cannot be ruled out, as previously observed (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIncreased expression of the MexAB-OprM efflux pump is directly related to meropenem resistance, but no direct correlation has been established with imipenem resistance (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). The use of the Phe-Arg efflux pump inhibitor beta-naphthylamide dihydrochloride (PAβN) causes an increase in the imipenem MIC in \u003cem\u003eP. aeruginosa\u003c/em\u003e (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e), however, this effect is likely due to the compound-induced alteration in outer membrane permeability rather than direct inhibition of efflux pumps (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). PA2521 (\u003cem\u003eczcB\u003c/em\u003e), is associated with Ca2+-induced resistance to tobramycin (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e), and that there is a relationship between the CzcR-CzcS two-component system, which regulates the expression of \u003cem\u003eczcCBA\u003c/em\u003e, and resistance to carbapenems, but to our knowledge, no correlation has been established between the efflux pump itself and resistance (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe identified 59 overrepresented proteins with variations associated with resistance to imipenem or imipenem-relebactam. Among these, 21 were exclusive to IMI/REL isolates, with 9 involved in metal ion transport and uptake. The increased metal ion influx may be associated with the enzymatic activity of metallo-beta-lactamases and the recently characterized carbapenem PIB-1, intrinsic to \u003cem\u003eP. aeruginosa\u003c/em\u003e (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Structural studies of PIB-1 revealed that the enzyme assumes a trimeric form in the presence of divalent metals (Zn, Co, and Ni) and that the trimeric form has increased activity relative to the monomeric form. Expression of PIB-1 in \u003cem\u003eEscherichia coli\u003c/em\u003e reduces its susceptibility to carbapenems, even in the presence of the inhibitors avibactam and clavulanic acid. Iron is another important metal related to survival, virulence, pathogenicity and resistance (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e). Of the 59 proteins identified with resistance-associated variations, 17 are shared between the two comparisons, 10 of which are directly related to iron and siderophore transport. These proteins include multiple TonB-dependent receptors (PA0434, PA2057, PA2089, PA2289, PA3268, PA4897), as well as FecA (Fe3\u0026thinsp;+\u0026thinsp;dicitrate transport protein), HasR (heme uptake outer membrane receptor\u003cb\u003e)\u003c/b\u003e, ChtA (ferric aerobactin receptor), all traditionally involved in the capture and internalization of iron. Iron transport by siderophores is closely linked to bacterial virulence, as limited iron availability in the host is a natural defense strategy against infections (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e). Furthermore, there is growing evidence in the literature linking the iron acquisition machinery with the modulation of resistance to antibiotics (\u003cspan additionalcitationids=\"CR83\" citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e), including new drugs as cefiderocol (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e). Studies have shown that iron transport systems, such as those dependent on TonB, can influence the expression and functionality of efflux pumps, beta-lactamase enzymes, and outer membrane permeability, mechanisms directly associated with resistance (\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRegarding specific resistance to imipenem-relebactam, 21 unique proteins were identified, eight of which are related to iron transport and retention, highlighting different variants of the TonB transporters (PA0151, PA2070, PA2911), FoxA, FemA, PhuR, PfeA, and FepA, as well as a copper transport protein, OprC (PA3790). The qualitative distinction of metal transport systems between resistance to imipenem alone and its combination with relebactam suggests differentiated adaptations that deserve further functional investigation, as metal transport can modulate metabolic pathways and mechanisms of response to antibiotic stress.\u003c/p\u003e\u003cp\u003eThe GWAS analysis revealed a set of genes with significant variations related to resistance to imipenem and/or imipenem-relebactam, different from the set of genes identified by the overrepresentation analysis. The \u003cem\u003efolP\u003c/em\u003e and \u003cem\u003exerD\u003c/em\u003e genes stood out in the GWAS analysis, presenting the highest number of hits and high significance and effect in the population. FolP (dihydropteroate synthase) is an enzyme involved in folic acid metabolism, which may be related to sulfonamide resistance in many pathogens (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e). The \u003cem\u003exerD\u003c/em\u003e gene, in turn, encodes a tyrosine recombinase involved in DNA recombination, which may promote genetic diversity and resistance (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). Only the \u003cem\u003ecupC3\u003c/em\u003e gene was common to both analyses. \u003cem\u003ecupC3\u003c/em\u003e encodes a protein associated with the cup system (chaperone-usher pathway), involved in the assembly of pili or fimbriae on the bacterial cell surface. Fimbriae influence adhesion, biofilm formation, and interaction with the environment, aspects that have been linked to both virulence and antimicrobial resistance through physical protection and permeability modulation (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e). The unique presence of \u003cem\u003ecupC3\u003c/em\u003e at the intersection of the two analyses suggests that, although other genes related to iron transport and beta-lactamases are essential, resistance to imipenem and its combination with relebactam also involves changes in the structural architecture of the bacterial surface.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOur results are consistent with clinical and in vitro data showing that most \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates remain susceptible to imipenem-relebactam, while resistance emerges in a restricted subset of strains. Importantly, although our analyses identified candidate genes and mutations, the specific contribution of each alteration to relebactam resistance remains unclear. This highlights a critical gap in current knowledge, as the precise molecular mechanisms underlying decreased activity of relebactam are yet to be elucidated. Together, our study reinforces the concept that resistance to novel beta-lactam/beta-lactamase inhibitor combinations arise from the convergence of classical resistance determinants, while also pointing to the necessity of functional validation. Such investigations will be essential to define the relative weight of these genetic variations and to inform the rational use of imipenem-relebactam in clinical settings.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGES\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGuiana extended-spectrum beta-lactamase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIMP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImipenemase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVIM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eVerona integron-encoded metallo-beta-lactamase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIMI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImipenem\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOXA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOxacillinase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePDC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePseudomonas cephalosporinase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePIB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e1-Pseudomonas aeruginosa chromosomally-encodedβ-lactamase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKPC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e2-Klebsiella pneumoniae carbapenemase 2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSPM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e1 S\u0026atilde;o Paulo Metallo-beta-lactamase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNDM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e1 New Delhi Metallo-β-lactamase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eREL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003erelebactam\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIMI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eR-imipenem-resistant\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIMI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eS-impenem-sentitive\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIMI/REL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eimipenem/relebactam resitant\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFDA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFood and Drug Administration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGWAS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGenome-Wide Association Study\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMinimum Inhibitory Concentration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEUCAST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEuropean Committee on Antimicrobial Susceptibility Testing\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBRC-Bacterial and Viral Bioinformatics Resource Center\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWGS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eWhole genome sequencing\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCARD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eComprehensive Antibiotic Resistance Database\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRGI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eResistance Gene Identifier\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePAO1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePseudomonas aeruginosa strain O1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLMM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLinear Mixed Model\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFDR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFalse Rate Discovery\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eXDR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eExtensively Drug-Resistant\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMDR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMultidrug-resistant\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCAZ/AVI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eceftazidime-avibactam\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCTZ/TAZ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eceftolozane-tazobactam\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCDS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCoding sequence\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eARG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAntimicrobial Resistance Gene\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eARO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAntibiotic Resistance Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMBL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMetallo-beta-lactamase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHGT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHorizontal Gene Transfer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding information\u003c/h2\u003e\u003cp\u003eThis research was funded by the National Council for Scientific and Technological Development (CNPq), CAPES, and Fiocruz. CNPq grant number 424410/2018-4; InovaFiocruz/Fundac\u0026atilde;o Oswaldo Cruz grant number VPPCB- 07-FIO-18-2-38.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eABL: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing; JS: Data curation, Formal analysis; LB: Sample collection, Methodology, MIC determination; CASI: Sample collection, Methodology, MIC determination; JAC: Data curation, Formal analysis; FFBT: Conceptualization, Funding acquisition, Writing \u0026ndash; review and editing; HF: Conceptualization, Funding acquisition, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the Graduate Program in Biosciences and Biotechnology of the Carlos Chagas Institute, the Carlos Chagas Institute, Fiocruz and CNPq for their support.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbraham EP, Chain E. 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Microb Drug Resist. 2022;28(8):849\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDelgado-Valverde M, Portillo-Calder\u0026oacute;n I, Alcalde-Rico M, Conejo MC, Hidalgo C, Del Toro Esper\u0026oacute;n C, et al. Activity of imipenem/relebactam and comparators against KPC-producing Klebsiella pneumoniae and imipenem-resistant Pseudomonas aeruginosa. Eur J Clin Microbiol Infect Dis. 2024;43(3):445\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmeen N, Memon Z, Shaheen S, Fatima G, Ahmed F. Imipenem Resistant Pseudomonas: The fall of the final quarterback. Pak J Med Sci. 1969;31(3).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHirsch EB, Ledesma KR, Chang KT, Schwartz MS, Motyl MR, Tam VH. \u003cem\u003eVitro\u003c/em\u003e Activity of MK-7655, a Novel β-Lactamase Inhibitor, in Combination with Imipenem against Carbapenem-Resistant Gram-Negative Bacteria. Antimicrob Agents Chemother. 2012;56(7):3753\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoung K, Painter RE, Raghoobar SL, Hairston NN, Racine F, Wisniewski D, et al. In vitro studies evaluating the activity of imipenem in combination with relebactam against Pseudomonas aeruginosa. BMC Microbiol [Internet]. 2019;19(1):150.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi H, Luo YF, Williams BJ, Blackwell TS, Xie CM. Structure and function of OprD protein in Pseudomonas aeruginosa: From antibiotic resistance to novel therapies. Int J Med Microbiol. 2012;302(2):63\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNaenna P, Noisumdaeng P, Pongpech P, Tribuddharat C. Detection of outer membrane porin protein, an imipenem influx channel, in Pseudomonas aeruginosa clinical isolates. Southeast Asian J Trop Med Public Health. 2010;41(3):614\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAgah Terzi H, Kulah C, Riza Atasoy A, Hakki Ciftci I. Investigation of OprD Porin Protein Levels in Carbapenem-Resistant Pseudomonas aeruginosa Isolates. Jundishapur J Microbiol. 2015;8(12).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAzimi A, Naserpour T, Bazmi F, Peymani A, Aslanimehr M, Saadat S. Evaluation of oprD Gene Expression in Carbapenem-Resistant Pseudomonas aeruginosa Strains Isolated From Severe Burn Patients With Secondary Infection. Biotechnol Health Sci [Internet]. 2015;2(3).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoudhury D, Das Talukdar A, Dutta Choudhury M, Maurya AP, Paul D, Dhar Chanda D et al. Transcriptional Analysis of MexAB-OprM Efflux Pumps System of Pseudomonas aeruginosa and Its Role in Carbapenem Resistance in a Tertiary Referral Hospital in India. Chang YF, editor. PLOS ONE. 2015;10(7):e0133842.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Y, Li X, Sun L, Wang XK, Zhang YW, Pang J et al. High level non-carbapenemase carbapenem resistance by overlaying mutations of \u003cem\u003emexR\u003c/em\u003e, \u003cem\u003eoprD\u003c/em\u003e, and \u003cem\u003eftsI\u003c/em\u003e in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Kim M, editor. Microbiol Spectr. 2025;13(1):e01398-24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBialvaei AZ, Rahbar M, Hamidi-Farahani R, Asgari A, Esmailkhani A, Mardani Dashti Y, et al. Expression of RND efflux pumps mediated antibiotic resistance in Pseudomonas aeruginosa clinical strains. Microb Pathog. 2021;153:104789.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCompagne N, Vieira Da Cruz A, M\u0026uuml;ller RT, Hartkoorn RC, Flipo M, Pos KM. Update on the Discovery of Efflux Pump Inhibitors against Critical Priority Gram-Negative Bacteria. 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Structural basis of Zn(II) induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa. Dove SL. editor PLOS Pathog. 2017;13(7):e1006533.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchalk IJ. Bacterial siderophores: diversity, uptake pathways and applications. Nat Rev Microbiol. 2025;23(1):24\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi JS, Seok YJ, Cho YH, Roe JH. Iron-Induced Respiration Promotes Antibiotic Resistance in Actinomycete Bacteria. Whiteley M, editor. mBio. 2022;13(2):e00425-22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHolbein BE, Ang MTC, Allan DS, Chen W, Lehmann C. Iron-withdrawing anti-infectives for new host-directed therapies based on iron dependence, the Achilles\u0026rsquo; heel of antibiotic-resistant microbes. 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Sulfonamide drugs: structure, antibacterial property, toxicity, and biophysical interactions. Biophys Ver. 2021;13(2):259\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin DL, Traglia GM, Baker R, Sherratt DJ, Ramirez MS, Tolmasky ME. Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes. Antibiotics. 2020;9(7):405.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eB\u0026ouml;hning J, Dobbelstein AW, Sulkowski N, Eilers K, Von K\u0026uuml;gelgen A, Tarafder AK et al. Architecture of the biofilm-associated archaic Chaperone-Usher pilus CupE from Pseudomonas aeruginosa. Mulvey MA, editor. PLOS Pathog. 2023;19(4):e1011177.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pseudomonas aeruginosa, imipenem-relebactam resistance, genomic comparison, metal ion","lastPublishedDoi":"10.21203/rs.3.rs-7801871/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7801871/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e accounts for 10\u0026ndash;20% of hospital-acquired infections and is a major pathogen in immunocompromised patients. Combination therapies with beta-lactam antibiotics and beta-lactamase inhibitors, such as imipenem-relebactam have improved treatment options, yet resistant strains have already emerged, with mechanisms still not fully elucidated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe sequenced and analyzed 10 clinical \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates resistant to imipenem-relebactam (IMI/REL) and compared them with publicly available genomes of imipenem-resistant (IMI-R) and imipenem-susceptible (IMI-S) strains. Resistance genes were identified using the RGI CARD database, while amino acid variations in core-genome proteins were evaluated through Gene Ontology overrepresentation analysis (GO), followed by GWAS. In total, 15,758 ARGs were detected, 25.85% associated with carbapenem resistance, but only 568 classified as beta-lactamases. Among IMI/REL isolates, 36.36% carried Ambler class A and 54.54% class B beta-lactamases, contrasting with much lower frequencies in IMI-R (5.4% and 3.6%) and IMI-S (0% and 0.73%). Core-genome analysis revealed 1,106 proteins with resistance-associated variations. Comparative analyzes identified 1,618 proteins differing between IMI/REL and IMI-R genomes, and 1,015 differing between IMI/REL and all other strains. GWAS highlighted candidate genes with strong statistical associations, including those involved in metal ion transport (e.g., \u003cem\u003etonB\u003c/em\u003e, \u003cem\u003efoxA\u003c/em\u003e, \u003cem\u003ephuR\u003c/em\u003e, \u003cem\u003epfeA\u003c/em\u003e) and efflux pumps (e.g., \u003cem\u003eczcB\u003c/em\u003e), as well as regulators such as \u003cem\u003emexT\u003c/em\u003e and biofilm-related proteins.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThese findings suggest that, beyond classical beta-lactamases, resistance involves multifactorial contributions from periplasmic and outer membrane proteins, metal ion homeostasis, efflux regulation, and biofilm-associated pathways. Our results expand current knowledge of \u003cem\u003eP. aeruginosa\u003c/em\u003e resistome and highlight novel genomic signatures potentially driving resistance to imipenem-relebactam.\u003c/p\u003e","manuscriptTitle":"Molecular determinants associated with resistance to imipenem and imipenem–relebactam in clinical Pseudomonas aeruginosa isolates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 13:41:02","doi":"10.21203/rs.3.rs-7801871/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-24T17:01:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-30T13:29:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-30T08:50:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210142932371802687444917890595759173318","date":"2026-01-30T08:06:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338737023625436080231641656889506339103","date":"2026-01-23T23:48:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151247808278726305083317358271480068724","date":"2026-01-22T05:05:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T09:26:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12682823309826309299412343044329146017","date":"2025-10-28T08:32:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304448304173466232845570694565120210806","date":"2025-10-27T13:32:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-23T13:50:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-23T07:25:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-21T06:33:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-18T14:00:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-10-18T13:56:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"51f8f373-e4d1-45ad-aa95-77ea95a41a40","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T12:38:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-04 13:41:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7801871","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7801871","identity":"rs-7801871","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}