Activity of imipenem/relebactam and comparators against KPC-producing Klebsiella pneumoniae and imipenem-resistant Pseudomonas aeruginosa

Vol.:(0123456789) 1 3 European Journal of Clinical Microbiology & Infectious Diseases https://doi.org/10.1007/s10096-023-04735-1 ORIGINAL ARTICLE Activity ofimipenem/relebactam andcomparators againstKPC‑producing Klebsiella pneumoniae andimipenem‑resistant Pseudomonas aeruginosa MercedesDelgado‑Valverde1,2 · InésPortillo‑Calderón1,2 · ManuelAlcalde‑Rico2,3 · M.CarmenConejo4 · CarmenHidalgo1 · CarlosdelToroEsperón4· ÁlvaroPascual1,2,4 Received: 23 May 2023 / Accepted: 5 December 2023 © The Author(s) 2023 Abstract Purpose Relebactam is a novel β-lactamase inhibitor, which, when combined with imipenem/cilastatin, is active against both class A and class C β-lactamases. To evaluate invitro antimicrobial activity of imipenem/relebactam against a collection of recent clinical isolates of carbapenem-non-susceptible P. aeruginosa and K. pneumoniae ST258 and ST512 KPC producers belonging to different lineages from hospitals in Southern Spain. Methods Six hundred and seventy-eight isolates were tested: 265 K. pneumoniae (230 ST512/KPC-3 and 35 ST258/KPC-3) and 413 carbapenem-non-susceptible P. aeruginosa. Imipenem, piperacillin/tazobactam, ceftazidime, cefepime, aztreonam, ceftolozane/tazobactam, meropenem, amikacin, ciprofloxacin, colistin, and ceftazidime/avibactam were used as comparators against P. aeruginosa. Against K. pneumoniae ceftazidime, cefepime, aztreonam, and ceftolozane/tazobactam were not tested, and tigecycline was studied instead. MICs were determined in duplicate by broth microdilution according to EUCAST guidelines. Results Imipenem/relebactam displayed potent invitro activity against both sequence types of KPC-3-producing K. pneumoniae. MIC50 and MIC90 values were 0.25 mg/L and 1 mg/L, respectively, with percent of susceptible isolates >97%. Only three K. pneumoniae ST512/KPC-3 isolates and one ST258/KPC-3 were resistant to imipenem/relebactam. Relebactam sensitized 98.5% of K. pneumoniae isolates resistant to imipenem. The activity of imipenem/relebactam against P. aeruginosa was moderate (susceptibility rate: 62.7%). Analysis of the acquired and mutational resistome of isolates with high levels of resistance to imipenem/relebactam has not shown a clear association between them. Conclusion Imipenem/relebactam showed excellent activity against K. pneumoniae KPC-3. The activity of imipenem/ relebactam against imipenem-resistant P. aeruginosa was moderate. Keywords Imipenem/relebactam· P. aeruginosa· K. pneumoniae· Carbapenem-resistant Introduction Carbapenem-resistant Gram-negative bacilli (CRGNB) are a major public health problem, recently described by WHO as a global crisis [1]. Since nosocomial and healthcare-asso- ciated infections caused by CRGNB organisms significantly increase morbidity and mortality, length of hospital stay, and medical costs [2], the development of new antimicrobials or new combinations of β-lactam-β-lactamase inhibitors active against these pathogens has become a priority [3, 4]. Pseudomonas aeruginosa and Klebsiella pneumoniae are common pathogens in hospitals, have a high propensity to develop antibiotic resistance, and also a high capacity for dissemination in the nosocomial environment [5–7]. P. * Mercedes Delgado-Valverde [email protected] 1 UGC Enfermedades Infecciosas y Microbiología Clínica, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen Macarena/CSIC/Universidad de Sevilla, Sevilla, Spain 2 Centro de Investigación Biomédica en Red en Enfermedades Infecciosas (CIBERINFEC), Sevilla, Spain 3 Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen Macarena, CSIC, Universidad de Sevilla, Sevilla, Spain 4 Departamento de Microbiología, Universidad de Sevilla, Sevilla, Spain European Journal of Clinical Microbiology & Infectious Diseases 1 3 aeruginosa carbapenem resistance is driven by different resistance mechanisms, which often act synergistically. The most common mechanisms of imipenem resistance in this microorganism include repression or inactivation of the carbapenem porin OprD coupled with hyperexpression of the chromosomal cephalosporinase AmpC and/or overexpression of efflux pumps, as well as carbapenemase production [5, 8, 9]. In K. pneumoniae, the most frequent resistance mechanism to carbapenem is carbapenemase production, mainly of classes A (KPC), B (MBL), and D (OXA-48-like) β-lactamases [10]. In 2017, the WHO published a priority list of pathogens for which the development of new antibiotics was urgently required. Carbapenem-resistant Enterobacterales are included on this list, as well as carbapenem-resist- ant P. aeruginosa and carbapenem-resistant Acinetobacter baumannii [11]. Relebactam is a novel diazabicyclooctane β-lactamase inhibitor, which in combination with imipenem/cilastatin is active against class A and class C β-lactamase-producing microorganisms [12, 13]. Imipenem/relebactam was approved by the EMA and FDA in 2020 for the treatment of complicated urinary tract infections, complicated intraabdominal infections, and hospital-acquired and ventilatorassociated bacterial pneumonia in adult patients with limited or no alternative therapeutic options [14, 15]. The purpose of this study was to provide data on the comparative invitro antimicrobial activity of imipenem/ relebactam against a collection of recent clinical isolates of carbapenem-non-susceptible P. aeruginosa and K. pneumoniae ST258 and ST512 KPC producers belonging to different lineages from hospitals in Southern Spain. Methods Bacterial strain Isolates of K. pneumoniae (n = 265, 230 ST512/KPC-3 and 35 ST258/KPC-3) and P. aeruginosa (n = 399) tested in this study (n = 664) were selected from a well-charac- terized collection held in the Reference Laboratory of the Andalusian program for the surveillance and control of healthcare-associated infections and antibiotic stewardship (PIRASOA Program), based in the Hospital Universitario Virgen Macarena, Seville, Spain [16, 17]. The isolate source was 77.9% clinical (61.1% K. pneumoniae, 88.5% P. aeruginosa), 14.9% colonization (24.9% K. pneumoniae, 8.5% P. aeruginosa), 1% environmental (2.6% K. pneumoniae, 0% P. aeruginosa), and 6.2% non-specified (11.3% K. pneumoniae, 3% P. aeruginosa). Two isolates of K. pneumoniae ST512 were KPC-31 producers. The selected isolates came from 18 hospitals located in the eight provinces of Andalusia. Thirty-three of the isolates (2 P. aeruginosa and 31 K. pneumoniae) selected were from 2014, 68 (6 P. aeruginosa and 62 K. pneumoniae) from 2015, 63 (6 P. aeruginosa and 57 K. pneumoniae) from 2016, 234 (187 P. aeruginosa and 47 K. pneumoniae) from 2017, 208 (175 P. aeruginosa and 33 K. pneumoniae) from 2018, 26 (23 P. aeruginosa and 3 K. pneumoniae) from 2019, and 32 K. pneumoniae from 2020. The inclusion criteria for isolate selection were KPC-3 production in K. pneumoniae and imipenem resistance in P. aeruginosa non-MBL-producers. Bacterial identification, molecular epidemiology, andgenomic characterization Identification of the isolates was confirmed in the reference laboratory by MALDI-TOF MS (MALDI-TOF Biotyper 3.1; Microflex Bruker, Madrid, Spain). PFGE analysis of XbaI (Enterobacterales)- and SpeI (P. aeruginosa)-digested DNA was used to determine the degree of genetic relatedness between isolates. Isolates differing by one or more bands in PFGE assays were assigned to different pulsotypes. A dendrogram was created with Bionumerics 8.0 software (BioMérieux), using the Dice coefficient with optimization set at 1% and position tolerance at 1.2% (data not shown). In-house Miseq sequencing (Illumina, San Diego, CA, USA) was performed on one isolate of each K. pneumoniae pulsetype and in resistant imipenem/relebactam P. aeruginosa with MIC >4 mg/L. Libraries were prepared with the Nextera XT DNA library preparation kit (Illumina) and sequencing with a reagent cartridge, V3 600 cycles (Illumina). CLC Genomic Workbench software (Qiagen, Netherlands) was used for de novo assembly of Illumina reads. Genomes were analyzed in the Center for Genomic Epidemiology resistance and MLST databases from https:// www. genom icepi demio logy. org/. All pulsotypes assigned to the same MLST were considered to belong to the same clone. Whenever possible, isolates from the same clone with different pulsotypes were selected. The total antimicrobial resistance gene content of the K. pneumoniae sequenced was analyzed in silico using ResFinder v4.1 (https:// cge. food. dtu. dk/ servi ces/ ResFi nder/) and the Comprehensive Antibiotic Resistance Database (CARD) (https:// card. mcmas ter. ca/). K. pneumoniae ATCC10031 was used as a reference to compare porin, PBPs, and efflux bomb aminoacid sequences. In addition, one susceptible isolate was selected for each MIC value for the analysis of mutations related to ß-lactam resistance. For P. aeruginosa genome analysis, raw reads were trimmed with Trimmomatic v0.39 excluding those reads exhibiting a Phred quality score <30, and a subsequent analysis of raw read quality was determined by FASTQC v0.11.9 (https:// www. bioin forma tics. babra ham. ac. uk/ proje cts/ fastqc/) and MultiQC v1.10.1. [18]. The genomes were European Journal of Clinical Microbiology & Infectious Diseases 1 3 de novo assembled with SPAdes v3.13.0 [19], and the quality of the assemblies was evaluated with QUAST v5.0.2 [20]. The identification of antibiotic resistance genes was performed by AMRFinderPlus [21]. The non-synonymous polymorphisms of genes previously described in P. aeruginosa as part of the ß-lactam mutational resistome [22] were identified by calling SNP with Snippy v4.6.0 software (https:// github. com/ tseem ann/ snippy), mapping the trimmed raw reads of each bacterial isolate with respect the PAO1 reference genome (NC_002516.2). Data availability The genomes were published in the NCBI database under accession no. PRJNA1048341 (K. pneumoniae) and PRJNA1048411 (P. aeruginosa). Antimicrobial susceptibility testing Susceptibility testing was performed in duplicate by broth microdilution assay, according to international standard ISO 20776-1.[23] Broth microdilution panels for P. aeruginosa included the following antimicrobial agents in doubling dilution concentration ranges (mg/L): imipenem/relebactam (0.03/4–64/4), imipenem (0.03–64), piperacillin/tazobactam (0.06/4–64/4), ceftazidime (0.03–32), cefepime (0.25-32), aztreonam (0.5–64), ceftolozane/tazobactam (0.125/4–16/4), meropenem (0.03–64), amikacin (0.5–32), ciprofloxacin (0.06–2), colistin (0.125–8), and ceftazidime/avibactam (0.015/4–16/4). For K. pneumoniae, the activities of piperacillin/tazobactam, ceftazidime, cefepime, aztreonam, and ceftolozane/tazobactam were not tested, and tigecycline activity (concentration range 0.015–1 mg/L) was studied instead. Discrepancies between both replicates were verified using the same method. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as control strains on each day of testing, checking that all MIC values were within the specified EUCAST ranges [24]. EUCAST interpretive criteria were used to interpret MIC values of all antimicrobials tested [25]. Results Antimicrobial activity againstKPC‑producing K. pneumoniae Figure1 shows the MIC distributions of imipenem alone or in combination with relebactam for all K. pneumoniae included in the study. The susceptibility rate to imipenem/ relebactam was 98.5% (three K. pneumoniae ST512/KPC-3 producers and one K. pneumoniae ST258/KPC-3 were resistant). With respect to imipenem, only 1.5% of isolates were susceptible. Overall, relebactam sensitized 98.5% of imipenem-resistant isolates (256/260), improving the activity of imipenem by 4 to 8 two-fold dilutions. The MIC range, MIC50, MIC90 values and percentages of susceptibility and resistance for all isolates are shown in Table1. Imipenem/relebactam and ceftazidime/avibactam displayed similar activity (98.5% susceptibility, MIC50 = 0.25/4 mg/L, MIC90 = 0.5/4 mg/L compared to 97.7% susceptibility, MIC50 = 4/4 mg/L, MIC90 = 8/4 mg/L, respectively). Analysis of MIC90 values revealed that imipenem/relebactam was four times more active than ceftazidime/avibactam. All ceftazidime/avibactam-resistant isolates were susceptible to imipenem/relebactam. Two of the five isolates resistant to ceftazidime/avibactam produced KPC-31. There were no differences in imipenem/relebactam MIC values between ST512 and ST258 isolates (MIC90 of 1/4 mg/L for both; p = 0.8). The next most active antimicrobial was colistin (46.2% susceptibility, MIC50 = 16 mg/L, MIC90 = >16 mg/L). In general, all antimicrobials tested showed similar percentages of susceptibility regardless of MLST, except for colistin, with ST258 isolates being more susceptible than ST512 isolates (88.6% vs 39.7%; p < 0.01). All isolates were also resistant to ciprofloxacin and tigecycline. Analysis of chromosomal mutations in the porin genes and PBPs showed no differences between isolates susceptible and resistant to imipenem/relebactam (Table2). All isolates had wild-type OmpK36 and AcrAB-TolC regulator (RamR). Mutations in OmpK35 and OmpK37 were detected in two isolates resistant to imipenem/relebactam, although the same mutations were detected in susceptible isolates. Antimicrobial activity againstimipenem‑resistant P. aeruginosa Overall, the new antimicrobials imipenem/relebactam, ceftazidime/avibactam, and ceftolozane/tazobactam showed moderate activity (% susceptibility): imipenem/relebactam (62.7%), ceftazidime/avibactam (73.3%), and ceftolozane/ tazobactam (78%). Amikacin was the most active antimicrobial (91.1% susceptibility), followed by colistin (86.7%) (Table3). The MIC range, MIC50, MIC90 values, and percentages of susceptibility and resistance for all antimicrobials tested are shown in Table2. Analysis of MIC90 and MIC50 values revealed that imipenem/relebactam was two to four-fold more active than imipenem. The MIC distributions of imipenem/relebactam and imipenem are shown in Fig.2. Among the ceftolozane/tazobactam-resistant isolates, 32.6% were susceptible to imipenem/relebactam, while 70.4% of isolates were susceptible to both antimicrobials. Forty-three imipenem/relebactam-susceptible strains were resistant to ceftazidime/avibactam (39.4%). Both antimicrobials were active against 213 isolates (70.1%). In addition, European Journal of Clinical Microbiology & Infectious Diseases 1 3 13.6% of isolates were resistant to ceftazidime/avibactam plus ceftolozane/tazobactam. In this group, the imipenem/ relebactam susceptibility rate was 35.7% (20/56 isolates). MIC distribution of imipenem/relebactam according to ceftolozane/tazobactam and ceftazidime/avibactam clinical category is shown in Fig.3. Among the colistin-resistant isolates, 51.8% were found to be susceptible to ceftolozane/tazobactam and ceftazidime/ avibactam, and up to 58.9% were susceptible to imipenem/ relebactam. On the other hand, one-third (32.4%) of the amikacin-resistant isolates were susceptible to relebactam activity. Regarding WGS results, of the 51 isolates sequenced, only 46 could be analyzed due to technical problems. MLST analysis showed a high variability in our collection. Twentysix different sequences were detected, with two major ST: ST175 (n = 9) and ST274 (n = 4). The remaining STs were represented by 3 or less isolates, and one isolate could not be characterized by this method. The 88.8% of isolates from ST175 had an imipenem/relebactam MIC = 8 mg/L, but among isolates with this MIC value, up to 20 different STs were detected, so there does not show a clear association between clone and MIC in isolates with this MIC value. The analysis of genes involved in ß-lactam resistance is shown in the supplementary table. With respect to chromosomal ß-lactamases analysis: 19 OXA-50 variants were detected among the isolates, with the wild-type variant being the predominant one (n = 12), followed by the OXA-488 (n = 6) and OXA-486 (n = 4) variants, and 18 AmpC variants (PDC-type). The most frequent PDC variants were PDC-1 (n = 12) and PDC-3 (n = 5). The most frequent polymorphism found in the PDC variants was T105A (71.7%), detected in all PDC alleles except in PDC-1. In general, an association between OXA-50 and PDC variants and ST was detected. The presence of acquired ß-lactamases was detected in only 11 isolates, mostly OXA-10 or OXA-10-like (n = 8). For the mutational analysis of ß-lactam resistance, the polymorphisms present in at least 50% of the P. aeruginosa collection were selected, and the results are shown in Table4. The genes were grouped into 8 functional categories (Table4 and Supplementary table). Polymorphisms were detected in genes of all categories analyzed. Fig. 1 Distribution of imipenem and imipenem/relebactam MICs against K. pneumoniae KPC-3 isolates by clone. Vertical dashed line represents the EUCAST resistance breakpoint for imipenem/relebactam (>2/4 mg/L for Enterobacterales) and vertical continuous line represents the EUCAST resistance breakpoints for imipenem (>4 mg/L for Enterobacterales) European Journal of Clinical Microbiology & Infectious Diseases 1 3 The functional categories and the prevalence of polymorphisms detected were MexXY-OprM and its regulators (30.1%), OprD (18.5%), AmpC and its regulators (13.9%), other PBPs (9.6%), MexEF-OprN and its regulators (8.4%), other ß-lactamases (8.2%), MexAB-OprM and its regulators (7.6%), and LPS modification and RND efflux system regulator (3.9%). In the functional categories AmpC and its regulators, OprD, and other PBPs, none of the polymorphisms was present in more than 90% of the isolates. All isolates presented the mutations K329Q, W358R in MexX of the MexXY-OprM system. Other mutations detected with a high frequency (>95%) were NalC (G71E), MexT (F172I), ParS (H398R), MexY (T543A), ArmZ (L88P), and PIB-1 (I106V) (Table4). Discussion One of the results of the increase in MDR-GNB infections worldwide is that the approved antimicrobials provide few treatment options for systemic infections. There is an urgent need for new antimicrobials active against MDR-GNB, as well as sufficient information to facilitate their use in severe infections. The collection of bacterial strains selected for this study includes KPC-3-producing K. pneumoniae isolates from the two most prevalent clones worldwide, as well as the most representative imipenem-resistant non-MBL producer P. aeruginosa isolates, both of which cause Table 1 MIC range, MIC50, and MIC90 of the tested antibiotics against K. pneumoniae KPC-3 by clone and resistance rates *EUCAST breakpoints for E. coli. 1 For susceptibility testing purposes, the concentrations of relebactam and avibactam were fixed at 4 mg/L 2 I—susceptible, increased exposure Species/clone/antibiotic MIC (mg/L) Resistance (%) MIC range MIC50 MIC90 S I2R K. pneumoniae (n = 264) Imipenem/relebactam10.06–4 0.25 0.5 98.5 NA 1.5 Imipenem 0.125–>64 64 >64 1.5 0 98.5 Meropenem 0.5–>64 >64 >64 1.1 3.0 95.9 Ceftazidime/avibactam10.5–>16 4 8 97.7 NA 2.3 Amikacin 4–>32 >32 >32 1.9 NA 98.1 Ciprofloxacin 2–>2 >2 >2 0 0 100 Colistin 0.125–>16 16 >16 46.2 NA 53.8 Tigecycline* 1–>2 2 >2 0 NA 100 K. pneumoniae ST512 (n = 229) Imipenem/relebactam10.06–4 0.25 1 98.7 NA 1.3 Imipenem 0.125–>64 >64 >64 1.7 0 98.3 Meropenem 0.5–>64 >64 >64 1.3 0.9 97.8 Ceftazidime/avibactam10.5–>16 4 8 97.8 NA 2.2 Amikacin 4–>32 >32 >32 1.7 NA 98.3 Ciprofloxacin 2–>2 >2 >2 0 0 100 Colistin 0.125–>16 16 >16 39.7 NA 60.3 Tigecycline* 1–>2 2 >2 0 NA 100 K. pneumoniae ST258 (n = 35) Imipenem/relebactam10.06–4 0.25 1 97.1 NA 2.9 Imipenem 0.125–>64 >64 >64 0 0 100 Meropenem 0.5–>64 >64 >64 0 17.1 82.9 Ceftazidime/avibactam10.5–>16 4 8 97.1 NA 2.9 Amikacin 4–>32 >32 >32 2.9 NA 97.1 Ciprofloxacin 2–>2 >2 >2 0 0 100 Colistin 0.125–>16 16 >16 88.6 NA 11.4 Tigecycline* 1–>2 2 >2 0 NA 100 European Journal of Clinical Microbiology & Infectious Diseases 1 3 Table 2 ST, carbapenemase, antimicrobial susceptibility testing, and mutational ß-lactam resistance genotype of imipenem/relebactam-resistant and susceptible K. pneumoniae MEM meropenem, AK amikacin, CIP ciprofloxacin, COL colistin, TIG tigecycline, IMP imipenem, IMR imipenem/relebactam, CZA ceftazidime/avibactam, PBP penicillin binding protein, mut mutation detected, wt wild type *One susceptible isolate has been included for each MIC value obtained †K. pneumoniae ATCC10031 was used as reference to compared porin, PBPs, and efflux bomb aminoacid sequences 1 M2_Y36del, G37E, M39W, V40S 2 N230G, M233Q, M233_T234insHYTH, Q235E, T236R, N238Y, R240K, E244D, N247S, D275T, D275_G276insSSTNGG, V277I 3 I17V, V296_P297insPAQ, V297P, H657R, S662R 4 D98G, T348S, G515S Strain no.* ST CP MIC (mg/L) ß-lactam resistance genotype† MEM AK CIP COL TIG IPM IMR CZA ß-lactalactamase OmpK351OmpK36 OmpK372AcrAB- TolC (RamR) PBP13PBP24 2015351 258 KPC-3 >64 >32 >2 0.25 1 >64 4 4 SHV-11 mut wt mut wt mut mut 20171161 512 KPC-3 16 16 >2 2 1 8 4 0.5 SHV-11 mut wt mut wt mut mut 20190807 512 KPC-3 >64 >32 >2 >16 >2 >64 4 8 SHV-11 wt wt mut wt mut mut 20190822 512 KPC-3 >64 >32 >2 >16 2 >64 4 4 SHV-11, TEM-1 wt wt mut wt mut mut 2016080 258 KPC-3 8 >32 >2 0.5 1 8 0.06 1 SHV-11 mut wt mut wt mut mut 2017964 512 KPC-3 >64 >32 >2 1 2 64 0.125 8 SHV-11 mut wt mut wt mut mut 20180393 512 KPC-3 >64 >32 >2 >16 1 64 0.25 2 SHV-11 mut wt mut wt mut mut 20180260 512 KPC-3 >64 >32 >2 >16 2 >64 0.5 4 SHV-11 mut wt mut wt mut mut 20180214 512 KPC-3 >64 >32 >2 >16 2 >64 1 8 SHV-11 mut wt mut wt mut mut 2016252 512 KPC-23 >64 >32 >2 1 2 64 2 8 SHV-11, TEM-1 mut wt mut wt mut mut European Journal of Clinical Microbiology & Infectious Diseases 1 3 healthcare-associated infections in Southern Spain (Andalusia has a population of more than 8 million people) and very similar to those causing infections in other neighboring countries. The results obtained in the current study showed that the invitro activity of imipenem/relebactam was superior to that of comparators against recent high-risk clone isolates of K. pneumoniae KPC-3 producers. Imipenem/relebactam showed potent antimicrobial activity, with MIC90 values of ≤1 mg/L against K. pneumoniae. MIC90 values showed no differences according to the ST tested, as in previous studies [26]. To date, a small number of imipenem/relebactam-resist- ant K. pneumoniae KPC-3 producers have been reported. In our study, 98.5% of KPC-3-producing K. pneumoniae were susceptible to imipenem/relebactam. Our results are consistent with those of previous studies. Hernández- García etal. evaluated the invitro activity of imipenem/ relebactam against 14 K. pneumoniae KPC-3 producers, all of which were susceptible to imipenem/relebactam [27]. Galani etal. analyzed imipenem/relebactam activity against 314 non-MBL carbapenemase-producing K. pneumoniae. Among KPC-producing isolates, 98% were inhibited by this combination, and relebactam effectively restored the invitro activity of imipenem, with MIC50 and MIC90 values decreasing from 32/4 to 0.25/4 mg/L, and from >64/4 to 1/4 mg/L, respectively [28]. In a recent study in Spain, 91 KPC-producing isolates were analyzed. The percentage of Table 3 MIC range, MIC50, and MIC90 of the tested antibiotics against non-carbapenem- susceptible P. aeruginosa and resistance rates 1 For susceptibility testing purposes, the concentrations of relebactam, tazobactam, and avibactam were fixed at 4 mg/L 2 I—susceptible, increased exposure Antimicrobial MIC (mg/L) Resistance (%) MIC range MIC50 MIC90 S I2R Imipenem/relebactam10.125–>64 2 8 64.1 NA 35.9 Imipenem 8–>64 16 32 0 0 100 Piperacillin/tazobactam11–>32 32 >32 0 39.3 60.7 Ceftazidime 1–>32 16 >32 0 35.6 64.4 Ceftazidime/avibactam10.5–>32 8 32 76.2 NA 23.8 Ceftolozane/tazobactam10.25–>16 2 16 80.5 NA 19.5 Cefepime 1–>32 16 >32 0 37.6 62.4 Aztreonam 0.5–>64 32 >64 0 48.9 51.1 Meropenem 1–>64 16 32 4.0 38.1 57.9 Amikacin 0.125–>32 4 16 92.7 NA 7.3 Ciprofloxacin 0.03–>2 2 >2 0 43.1 56.9 Colistin 0.125–>16 1 4 86.0 NA 14.0 Fig. 2 MIC distribution of imipenem and imipenem/relebactam against P. aeruginosa. Vertical dashed line represents the EUCAST resistance breakpoint for imipenem/relebactam (>2/4 mg/L for P. aeruginosa) European Journal of Clinical Microbiology & Infectious Diseases 1 3 susceptibility to imipenem was 15.5%, and 100% of the isolates were susceptible to imipenem/relebactam [29]. Ceftazidime/avibactam has been positioned as an alternative for the treatment of infections caused by high-risk clones of KPC-producing K. pneumoniae, although the emergence of KPC enzyme variants resistant to this combination has been described, mainly selected after exposure during treatment [30, 31]. Our results agree with those obtained in previous studies, which show that imipenem/relebactam has excellent invitro activity and clinical efficacy against KPC-producing isolates, even against variants resistant to ceftazidime/avibactam [27, 29, 32]. In our collection, 2.3% of isolates were resistant to ceftazidime/avibactam and all of them were susceptible to imipenem/relebactam. Moreover, MIC50/MIC90 values were significantly lower than those of ceftazidime/avibactam. Vázquez-Ucha etal. reported similar results, with MIC50/MIC90 values for imipenem/relebactam and ceftazidime/avibactam of ≤0.25/1 mg/L and 1/8 mg/L, respectively [29]. Several studies have previously shown that reduced porin expression decreases the invitro activity of imipenem/relebactam. Imipenem/relebactam resistance has been associated with mutations resulting in non-functional OmpK35 and OmpK36 porins in KPC-producing K. pneumoniae strains [28, 33, 34]. In our case, we detected four resistant isolates but did not find the mutations associated with resistance to imipenem/relebactam. Since the mutations detected in the porin genes were also present in isolates susceptible to imipenem/relebactam, they cannot explain the resistance to imipenem/relebactam in these four isolates. To our knowledge, there is still very limited data on the clinical efficacy of imipenem/relebactam in patients with severe infections caused by carbapenemase-producing Enterobacterales. The results of the RESTORE-IMI 1 and RESTORE-IMI 2 clinical trials, evaluating the clinical efficacy of imipenem/relebactam for the treatment of infections caused by imipenem-non-susceptible isolates, as well as for treatment of hospital-acquired/ventilator-associated bacterial pneumonia, concluded that imipenem/relebactam was an appropriate treatment option. It should be noted however that the number of carbapenemase-producing isolates was very low [14, 15]. With respect to P. aeruginosa, we analyzed a large number of imipenem-resistant non-MBL producer isolates. In our P. aeruginosa collection, a susceptibility rate of 62.7% was detected for imipenem/relebactam. Previous studies have reported similar results. The SUPERIOR and STEP studies found a susceptibility rate of 75.7% [35]. Zhang etal. analyzed a collection of 835 non-imipenem-susceptible P. aeruginosa isolates from the global SMART surveillance program, and the susceptibility rates to imipenem/relebactam were 64.4%, and the MIC50 and MIC90 values were 2/4 mg/L and >32/4 mg/L, respectively. Compared with our data, the susceptibility percentages were very similar, but the MIC90 value was 2-fold higher [36]. In our study, imipenem/ Fig. 3 MIC distribution of imipenem/relebactam according to clinical category of ceftolozane/ tazobactam (A) and ceftazidime/avibactam (B) against P. aeruginosa. Vertical dashed line represents the EUCAST resistance breakpoint for imipenem/ relebactam (>2/4 mg/L for P. aeruginosa) European Journal of Clinical Microbiology & Infectious Diseases 1 3 Table 4 Prevalence, function, and variability of mutated genes and polymorphisms detected in more than 50% of imipenem/relebactam-resistant P. aeruginosa isolates Gene† V of mutated genes (%) Pv of mutated gene (%) Antibiotic resistance association Polymorphism Pv of polymorphisms (%) Total IMR MIC 8 mg/L IMR MIC ≥16 mg/L AmpC and its regulators PA0807(ampDh3) 1.7 67.4 B, C A219T 56.5 51.6 66.7 PA4020(mpl) 2.0 65.2 M297V 56.5 51.6 66.7 PA4110(ampC) 4.7 73.9 T105A 71.7 77.4 60.0 PA4522(ampD) 4.0 80.4 G148A 76.1 74.2 80.0 MexAB-OprM regulator PA3721(nalC) 2.0 95.7 B, C, Q G71E 95.7 100 86.7 S209R 63.0 67.7 53.3 MexEF-OprN regulators PA2491(mexS) 2.7 91.3 C, Q D249N 91.3 90.3 93.3 PA2492(mexT) 1.7 95.7 Q80fs 89.1 87.1 93.3 F172I 95.7 96.8 93.3 LPS modifications and RND efflux system regulation PA1798(parS) 2.7 95.7 A, B, C, P, Q H398R 95.7 96.8 93.3 MexXY-OprM and its regulators PA0018(fmt) 3.0 100 A, B, C, Q I181V 80.4 77.4 86.7 PA2018(mexY) 6.0 100 T543A 97.8 100 93.3 Q840E 47.8 41.9 60.0 PA2019(mexX) 3.0 100 K329Q 100 100 100 L331V 76.1 74.2 80.0 W358R 100 100 100 PA5471(armZ) 4.2 100 L88P 97.8 96.8 100 D161G 65.2 64.5 66.7 H182Q 65.2 64.5 66.7 V243A 89.1 90.3 86.7 OprD PA0958(oprD) 12.4 100 C D43N 52.2 61.3 33.3 SGS57EGR 58.7 64.5 46.7 E202Q 63.0 64.5 60.0 I210A 69.6 74.2 60.0 E230K 63.0 67.7 53.3 S240T 65.2 67.7 60.0 N262T 54.3 54.8 53.3 A281G 50.0 51.6 46.7 K296Q 56.5 64.5 40.0 Q301E 54.3 61.3 40.0 R310G 45.7 51.6 33.3 D43N 52.2 61.3 33.3 SGS57EGR 58.7 64.5 46.7 E202Q 63.0 64.5 60.0 I210A 69.6 74.2 60.0 E230K 63.0 67.7 53.3