In vitro activity of cefiderocol against European Pseudomonas aeruginosa and Acinetobacter spp., including isolates resistant to meropenem and recent β-lactam/β-lactamase inhibitor combinations

| Clinical Microbiology | Research Article In vitro activity of cefiderocol against European Pseudomonas aeruginosa and Acinetobacter spp., including isolates resistant to meropenem and recent β-lactam/β-lactamase inhibitor combinations Anne Santerre Henriksen,1 Katy Jeannot,2 Antonio Oliver,3 John D. Perry,4 Mathias W. Pletz,5 Stefania Stefani,6 Ian Morrissey,7 Christopher Longshaw,1 ARTEMIS Study Investigators AUTHOR AFFILIATIONS See affiliation list on p. 15. ABSTRACT Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter spp. represent major threats and have few approved therapeutic options. Non-ferment ing Gram-negative isolates were collected from hospitalized inpatients from 49 sites in 6 European countries between 01 January 2020 and 31 December 2020 and underwent susceptibility testing against cefiderocol and β-lactam/β-lactamase inhibitor combinations. Meropenem-resistant (MIC >8 mg/L), cefiderocol-susceptible isolates were analyzed by PCR, and cefiderocol-resistant isolates were analyzed by whole-genome sequencing to identify resistance mechanisms. Overall, 1,451 (950 P. aeruginosa; 501 Acinetobacter spp.) isolates were collected, commonly from the respiratory tract (42.0% and 39.3%, respectively). Cefiderocol susceptibility was higher than β-lactam/β-lactamase inhibitor combinations against P. aeruginosa (98.9% vs 83.3%–91.4%), and P. aeruginosa resistant to meropenem (n = 139; 97.8% vs 12.2%– 59.7%), β-lactam/β-lactamase inhibitor combinations (93.6%–98.1% vs 10.7%–71.8%), and both meropenem and ceftazidime-avibactam (96.7% vs 5.0%–45.0%) or ceftolo zane-tazobactam (98.4% vs 8.1%–54.8%), respectively. Cefiderocol and sulbactam-dur lobactam susceptibilities were high against Acinetobacter spp. (92.4% and 97.0%) and meropenem-resistant Acinetobacter spp. (n = 227; 85.0% and 93.8%) but lower against sulbactam-durlobactam- (n = 15; 13.3%) and cefiderocol- (n = 38; 65.8%) resistant isolates, respectively. Among meropenem-resistant P. aeruginosa and Acineto bacter spp., the most common β-lactamase genes were metallo-β-lactamases [30/139; blaVIM-2 (15/139)] and oxacillinases [215/227; blaOXA-23 (194/227)], respectively. Acquired β-lactamase genes were identified in 1/10 and 32/38 of cefiderocol-resistant P. aeru ginosa and Acinetobacter spp., and pirA-like or piuA mutations in 10/10 and 37/38, respectively. Conclusion: cefiderocol susceptibility was high against P. aeruginosa and Acinetobacter spp., including meropenem-resistant isolates and those resistant to recent β-lactam/β-lactamase inhibitor combinations common in first-line treatment of European non-fermenters. IMPORTANCE This was the first study in which the in vitro activity of cefiderocol and non-licensed β-lactam/β-lactamase inhibitor combinations were directly compared against Pseudomonas aeruginosa and Acinetobacter spp., including meropenem- and β-lactam/β-lactamase inhibitor combination-resistant isolates. A notably large number of European isolates were collected. Meropenem resistance was defined according to the MIC breakpoint for high-dose meropenem, ensuring that data reflect antibiotic activity against isolates that would remain meropenem resistant in the clinic. Cefiderocol susceptibility was high against non-fermenters, and there was no apparent cross April 2024 Volume 12 Issue 4 10.1128/spectrum.03836-23 1 Editor Rolf Müller, Helmholtz-Institut fur Pharmazeutische Forschung Saarland, Saarbrücken, Germany Address correspondence to Anne Santerre Henriksen, [email protected]. A.S.H. is an employee of Maxel Consulting ApS, Jyllinge, Denmark and is a contract employee of Shionogi B.V. Both K.J. and A.O. declare honoraria and grants from Shionogi, Pfizer, and MSD. I.M. is an employee of Antimicrobial Focus Ltd and was involved with the reporting of the study data by IHMA. C.L. is an employee of Shionogi B.V. J.D.P., M.W.P., and S.S. have no relevant conflicts of interest to disclose. Received 6 November 2023 Accepted 5 February 2024 Published 14 March 2024 Copyright © 2024 Santerre Henriksen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Downloaded from https://journals.asm.org/journal/spectrum on 03 April 2025 by 193.147.173.206. resistance between cefiderocol and β-lactam/β-lactamase inhibitor combinations, with the exception of sulbactam-durlobactam. These results provide insights into therapeutic options for infections due to resistant P. aeruginosa and Acinetobacter spp. and indicate how early susceptibility testing of cefiderocol in parallel with β-lactam/β-lactamase inhibitor combinations will allow clinicians to choose the effective treatment(s) from all available options. This is particularly important as current treatment options against non-fermenters are limited. KEYWORDS cefiderocol, Pseudomonas aeruginosa, Acinetobacter spp., β-lactam/β-lactamase inhibitor combinations, meropenem, ceftazidime-avibac tam, ceftolozane-tazobactam, meropenem-vaborbactam, imipenem-relebactam, aztreonam-avibactam, cefepime-taniborbactam, sulbactam-durlobactam, resistance, meropenem-resistant, β-lactamases, Europe, in vitro Antimicrobial resistance is widespread throughout Europe (1). In particular, the high rates of carbapenem resistance observed in Pseudomonas aeruginosa and Acinetobacter spp. (19% and 48%, respectively, in 2021) represent a major threat, considering few approved therapeutic options are available (1, 2). The World Health Organization has, therefore, recognized carbapenem-resistant (CR) P. aeruginosa and CR Acinetobacter baumannii (CRAB) as critical priority pathogens (2). Major mechanisms of carbapenem resistance in P. aeruginosa include loss of outer membrane porin D function, overexpression of efflux pumps, and carbapenemases [including Class B metallo-β-lactamases (MBLs) such as the Verona integron-borne MBL (VIM) and emerging class A carbapenemases such as Guiana extended-spectrum β-lactamase (GES) and Klebsiella pneumoniae carbapenemase (KPC)] (3–9). Carbapene mases most commonly found in CR Acinetobacter spp. include OXA-23 and OXA-24/40 oxacillinases; with the exception of New Delhi MBL (NDM)-1, MBLs are less common, although their potency makes them problematic for treatment (5, 10–12). Efflux pumps and reduced membrane permeability due to CarO porin loss are also associated with carbapenem resistance in Acinetobacter spp. (13, 14). Cefiderocol is a unique catechol-siderophore cephalosporin approved in Europe for the treatment of infections due to aerobic Gram-negative organisms in adults with limited treatment options (15). The mechanism of action of cefiderocol is the disruption of peptidoglycan cell-wall synthesis via inhibition of penicillin-binding proteins (PBPs) (16, 17). The structure of cefiderocol and “Trojan Horse” mechanism of bacterial cell entry provide enhanced stability to a wide range of β-lactamases, and allow for the activity of cefiderocol to be broadly unaffected by efflux pump overexpression and porin channel modifications observed in CR non-fermenters (17–21). Various β-lactam/β-lactamase inhibitor (BLBLI) combinations have been approved (ceftazidime-avibactam, ceftolozane-tazobactam, imipenem-relebactam, and meropenem-vaborbactam) or are in development (aztreonam-avibactam, cefepime-taniborbactam, and sulbactam-durlobactam) for clinical use in Europe to treat infections caused by CR P. aeruginosa and/or Acinetobacter spp. (22–27). However, several of these combinations are affected or rendered inactive by one or more of the known mechanisms of resistance in CR non-fermenters; these BLBLI combinations remain unable to inhibit certain β-lactamases, MBLs in particular, and are still affected by porin channel modifications (28–32). There is notable variation in the European Society of Clinical Microbiology and Infectious Diseases and Infectious Diseases Society of America guidance for treatment of infections due to CR P. aeruginosa and Acinetobacter spp., although both gener ally recognize that in vitro activity of antimicrobials is an important consideration for treatment decision-making (33, 34). The longitudinal surveillance studies SENTRY and SIDERO generated data on the susceptibilities of cefiderocol and approved BLBLI combinations against non-fermenters, including CR and BLBLI combination-resistant isolates (35, 36). Although, despite the current and anticipated use of cefiderocol and Research Article Microbiology Spectrum April 2024 Volume 12 Issue 4 10.1128/spectrum.03836-23 2 Downloaded from https://journals.asm.org/journal/spectrum on 03 April 2025 by 193.147.173.206. BLBLI combinations against CR Gram-negative infections, there remains limited data comparing these antimicrobials and developmental BLBLI combinations against CR non-fermenter isolates, including those also resistant to a comparator antimicrobial. The aim of this study was to evaluate the in vitro activity of cefiderocol, merope nem, BLBLI combinations (approved and in development), and colistin against clinical Gram-negative isolates collected between 01 January 2020 and 31 December 2020, across six countries in Europe. Here, we report the results for P. aeruginosa and Acinetobacter spp. isolates. Results for Enterobacterales isolates collected in this study are reported elsewhere. RESULTS Epidemiology In total, 1,451 isolates were collected from European hospitals, of which 950 (65.5%) were P. aeruginosa and 501 (34.5%) were Acinetobacter spp. [including 458 (91.4%) A. baumannii complex] (see Table S1 for isolates by country). P. aeruginosa and Acinetobacter spp. isolates were collected from a range of infection sources, the most common for both species being respiratory tract [42.0% (399/950); 39.3% (197/501)], bloodstream [25.4% (241/950); 26.3% (132/501)], and skin [21.7% (206/950); 20.0% (100/501)], respectively (see Fig. S1 for all infection sources). Susceptibility profiles of isolates Susceptibility to cefiderocol was 98.9% against P. aeruginosa isolates and 92.4% against Acinetobacter spp. isolates [Table 1; see Table S2 for susceptibility rates using Clini cal and Laboratory Standards Institute (CLSI) breakpoints or United States Food and Drug Administration (FDA) breakpoints where CLSI breakpoints were not available]. By individual country, susceptibility to cefiderocol was similar against P. aeruginosa, ranging from 98.7% in France to 99.3% in the United Kingdom, but showed more variability against Acinetobacter spp. (88.7% in Italy to 98.0% in Spain) (Table S3A through E). Against P. aeruginosa isolates, rates of susceptibility to BLBLI combinations were lower than cefiderocol, ranging from 83.3% for imipenem-relebactam to 91.4% for cefepime-taniborbactam (Table 1). Against Acinetobacter spp. isolates, rates of suscepti bility were 92.4% to cefiderocol and 97.0% to sulbactam-durlobactam (FDA breakpoint) (Table 1) (37, 38). Using epidemiological cut-off (ECOFF) values, susceptibility to colistin was 99.7% and 98.4% for P. aeruginosa and Acinetobacter spp., respectively (Table 1). Susceptibility profiles of isolates with antibiotic-resistant phenotypes In total, 14.6% (139/950) P. aeruginosa isolates were classified as meropenem resist ant (meropenem MIC >8 mg/L) (Table 2; see Fig. 1A for MIC distributions). Suscept ibility of meropenem-resistant P. aeruginosa to cefiderocol was 97.8%, which was higher than susceptibility rates of <60% to all tested BLBLI combinations (Table 2). Meropenem-vaborbactam and imipenem-relebactam had the lowest susceptibility (12.2%), followed by aztreonam-avibactam (41.7%) and then cefepime-taniborbactam, ceftazidime-avibactam, and ceftolozane-tazobactam (55.4%–59.7%) (Table 2). This trend was detected across all countries (Table S4A through D). Susceptibility to cefiderocol remained high against P. aeruginosa isolates resistant to BLBLI combinations, ranging from 93.6% against ceftazidime-avibactam-resistant isolates to 98.1% against imipenem-relebactam-resistant isolates (Table 2). On the other hand, BLBLI combinations did not show high levels of activity against BLBLI combinationresistant isolates, with the highest susceptibility being 71.8% for ceftolozane-tazobactam against aztreonam-avibactam-resistant isolates. Colistin showed 100% susceptibility against BLBLI combination-resistant phenotypes. The activity of antimicrobials against cefiderocol-resistant P. aeruginosa was not analyzed due to the low number of isolates (n = 10). Research Article Microbiology Spectrum April 2024 Volume 12 Issue 4 10.1128/spectrum.03836-23 3 Downloaded from https://journals.asm.org/journal/spectrum on 03 April 2025 by 193.147.173.206. TABLE 1 In vitro activity of cefiderocol, BLBLI combinations, and other relevant antibiotics against P. aeruginosa and Acinetobacter spp. isolatesa,b FDC MEM CZA C-T MVB I-R ATM-AVI FEP-TAN SUL-DUR (CST) Isolates (n) MIC90 (mg/L) S (%) MIC90 (mg/L) S/I (%) MIC90 (mg/L) S (%) MIC90 (mg/L) S (%) MIC90 (mg/L) S/I (%) MIC90 (mg/L) S (%) MIC90 (mg/L) S/I (%) MIC90 (mg/L) S/I (%) MIC90 (mg/L) S (%) MIC90 (mg/L) S (%) P. aeruginosa (950) 1 98.9 16 85.4 8 90.1 8 89.1 16 87.2 4 83.3 32 86.2 8 91.4 N/A N/A (1) (99.7) Acinetobacter spp. (501)c 2 92.4 >16 54.7 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 4 97.0 (0.5) (98.4) aATM-AVI, aztreonam-avibactam; BLBLI, β-lactam/β-lactamase inhibitor; CST, colistin; C-T, ceftolozane-tazobactam; CZA, ceftazidime-avibactam; ECOFF, epidemiological cut-off; EUCAST, European Committee on Antimicrobial Susceptibility Testing; FDA, Food and Drug Administration; FDC, cefiderocol; FEP-TAN, cefepime-taniborbactam; I, susceptible, increased exposure; I-R, imipenem-relebactam; MEM, meropenem; MVB, meropenem-vaborbactam; N/A, not applicable; PD, pharmacodynamic; PK, pharmacokinetic; S, susceptibility; SUL-DUR, sulbactam-durlobactam. bAntibiotics were tested against P. aeruginosa and/or Acinetobacter spp. based on expected use in a real-world setting. Susceptibility was assessed according to EUCAST breakpoints (including non-species-specific PK/PD breakpoints, high-dosage breakpoints, and breakpoints for the agent without inhibitor, where applicable), except for sulbactam-durlobactam and colistin where FDA breakpoints and ECOFF values were used, respectively. Data on susceptibility to colistin are shown in parentheses as colistin is not recommended for monotherapy and is not associated with a clinical monotherapy breakpoint (as per EUCAST v.14.0 guidance). cIncludes 458 A. baumannii complex isolates. Research Article Microbiology Spectrum April 2024 Volume 12 Issue 4 10.1128/spectrum.03836-23 4 Downloaded from https://journals.asm.org/journal/spectrum on 03 April 2025 by 193.147.173.206. Against P. aeruginosa isolates that were resistant to both meropenem and ceftazidime-avibactam, susceptibility to cefiderocol was 96.7%, which was higher than any BLBLI combination (≤45.0% susceptibility) (Table 2). Similarly, against meropenem- and ceftolozane-tazobactam-resistant isolates, susceptibility to cefiderocol was higher than all tested BLBLI combinations (98.4% vs <55% susceptibility, respectively). Suscepti bility rates for antibiotics against antibiotic-resistant P. aeruginosa using CLSI breakpoints are reported in Table S2. In total, 45.3% (227/501) Acinetobacter spp. isolates were classified as merope nem resistant (MIC >8 mg/L) (Table 2; see Fig. 1B for MIC distributions). Susceptibil ity of meropenem-resistant Acinetobacter spp. was 85.0% to cefiderocol [European Committee on Antimicrobial Susceptibility Testing (EUCAST) non-species-specific pharmacokinetic/pharmacodynamic (PK/PD) breakpoint] and 93.8% to sulbactam-dur lobactam (FDA breakpoint) (Table 2) (37, 38). The MIC90 was lower for sulbactamdurlobactam (4 mg/L) than cefiderocol (8 mg/L). Cefiderocol susceptibility against sulbactam-durlobactam-resistant isolates (n = 15) was 13.3%, while sulbactam-durlo bactam susceptibility against cefiderocol-resistant isolates (n = 38) was 65.8%. Colistin showed high susceptibility (>92.1%) against all resistant phenotypes. Antimicrobial susceptibility rates in antibiotic-resistant Acinetobacter spp. using CLSI or FDA break points are reported in Table S2. β-Lactamase genes in meropenem-resistant pathogens Of the 139 meropenem-resistant P. aeruginosa isolates, 2.9% (4/139) were cefiderocol resistant and were analyzed by whole-genome sequencing (WGS), while the remain ing isolates were analyzed by PCR for the presence of specific β-lactamase genes. Antimicrobial susceptibility rates in meropenem-resistant P. aeruginosa according to β-lactamase genes identified are reported in Table S5A. The most common β-lactamase TABLE 2 In vitro activity of cefiderocol, BLBLI combinations, and other relevant antibiotics against P. aeruginosa and Acinetobacter spp. isolates with resistant phenotypesa,b Isolates n Susceptibilityc (%) FDC MEM CZA C-T MVB I-R ATM-AVI FEP-TAN SUL-DUR (CST) P. aeruginosa 950 98.9 85.4 90.1 89.1 87.2 83.3 86.2 91.4 N/A (99.7) MEM-R 139 97.8 56.8 55.4 12.2 12.2 41.7 59.7 N/A (100) CZA-R 94 93.6 36.2 36.2 39.4 37.2 54.3 55.3 N/A (100) C-T-R 104 94.2 40.4 42.3 45.2 36.5 64.4 59.6 N/A (100) MVB-R 122 97.5 0 53.3 53.3 10.7 38.5 59.0 N/A (100) I-R-R 159 98.1 23.3 62.9 58.5 31.4 54.1 64.8 N/A (100) ATM-AVI-R 131 95.4 38.2 67.2 71.8 42.7 44.3 61.8 N/A (100) FEP-TAN-R 82 95.1 31.7 48.8 48.8 39.0 31.7 39.0 N/A (100) MEM-R and CZA-R 60 96.7 28.3 5.0 8.3 45.0 43.3 N/A (100) MEM-R and C-T-R 62 98.4 30.6 8.1 9.7 54.8 50.0 N/A (100) Acinetobacter spp. 501d92.4 54.7 N/A N/A N/A N/A N/A N/A 97.0 (98.4) FDC-R 38 10.5 N/A N/A N/A N/A N/A N/A 65.8 (92.1) MEM-R 227 85.0 N/A N/A N/A N/A N/A N/A 93.8 (97.4) SUL-DUR-Re15 13.3 6.7 N/A N/A N/A N/A N/A N/A (100) aATM-AVI, aztreonam-avibactam; BLBLI, β-lactam/β-lactamase inhibitor; CST, colistin; C-T, ceftolozane-tazobactam; CZA, ceftazidime-avibactam; ECOFF, epidemiological cut-off; EUCAST, European Committee on Antimicrobial Susceptibility Testing; FDA, Food and Drug Administration; FDC, cefiderocol; FEP-TAN, cefepime-taniborbactam; I-R, imipenem-relebactam; MEM, meropenem; MVB, meropenem-vaborbactam; N/A, not applicable; PD, T, ceftolozane-tazobactam; CZA, ceftazidime-avibactam; ECOFF, epidemiological cut-off; EUCAST, European Committee on Antimicrobial Susceptibility Testing; FDA, Food and Drug Administration; FDC, cefiderocol; FEP-TAN, cefepimetaniborbactam; I-R, imipenem-relebactam; MEM, meropenem; MVB, meropenem-vaborbactam; N/A, not applicable; PD, pharmacodynamic; PK, pharmacokinetic; R, resistant; SUL-DUR, sulbactam-durlobactam. bAntimicrobials were tested against P. aeruginosa and/or Acinetobacter spp. based on expected use in a real-world setting. Results are not reported for isolates tested against antibiotics to which they had an expected resistance phenotype. Susceptibility was assessed according to EUCAST breakpoints (including non-species-specific PK/PD breakpoints, high dosage breakpoints, and breakpoints for the agent without inhibitor, where applicable), except for sulbactam-durlobactam and colistin where FDA breakpoints and ECOFF values were used, respectively. Data are shown where n ≥ 20 isolates were available. Data on susceptibility to colistin are shown in parentheses as colistin is not recommended for monotherapy and is not associated with a clinical monotherapy breakpoint (as per EUCAST v.14.0 guidance). cRefers to susceptibility, or susceptibility with increased exposure for meropenem, meropenem-vaborbactam, aztreonam-avibactam, and cefepime-taniborbactam. dIncludes 458 A. baumannii complex isolates. eSulbactam-durlobactam-resistant isolates were included irrespective of the number of isolates due to the scarcity of published data. Research Article Microbiology Spectrum April 2024 Volume 12 Issue 4 10.1128/spectrum.03836-23 5 Downloaded from https://journals.asm.org/journal/spectrum on 03 April 2025 by 193.147.173.206. genes identified in isolates were MBLs [21.6% (30/139)], including 27 isolates with only MBL genes and 3 isolates that each co-harbored 1 KPC, extended-spectrum β-lacta mases (ESBL), or acquired OXA gene (Table 3). This trend was similar across all Euro pean countries (Table S6A). A small proportion of meropenem-resistant P. aeruginosa isolates harbored only ESBL genes [5.8% (8/139)] (Table 3). The most common MBL gene in meropenem-resistant P. aeruginosa isolates was VIM [17.3% (24/139), including blaVIM-2 (15/139) and blaVIM-1 (7/139)], while 2.9% (4/139) of the isolates harbored IMP (blaIMP-13: 3/139) and 1.4% (2/139) harbored NDM (blaNDM-1) genes (Table S6A). Overall, 5.0% (7/139) of isolates harbored GES genes (blaGES-5: 6/139), 1.4% (2/139) harbored Vietnamese extended-spectrum β-lactamase (VEB) genes (blaVEB-9), and 0.7% (1/139) harbored blaKPC-2 or blaOXA-10 (Table S6A). FIG 1 Cumulative MIC distributions of cefiderocol, BLBLI combinations, and colistin against (A) meropenem-resistant P. aeruginosa (n = 139) and (B) merope nem-resistant Acinetobacter spp. (n = 227). Antimicrobials were tested against P. aeruginosa and/or Acinetobacter spp. based on expected use in a real-world setting. Data on susceptibility to colistin are shown as dashed lines as colistin is not recommended for monotherapy and is not associated with a clinical monotherapy breakpoint (as per EUCAST v.14.0 guidance). Resistance to meropenem was defined using a breakpoint of MIC >8 mg/L, relating to high-dose, extended-infusion (2 g, 3-h infusion) meropenem (as per EUCAST v14.0 guidance). ATM-AVI, aztreonam-avibactam; BLBLI, β-lactam/β-lactamase inhibitor; CST, colistin; C-T, ceftolozane-tazobactam; CZA, ceftazidime-avibactam; EUCAST, European Committee on Antimicrobial Susceptibility Testing; FDC, cefiderocol; FEP-TAN, cefepime-taniborbactam; I-R, imipenem-relebactam; MVB, meropenem-vaborbactam; SUL-DUR, sulbactam-durlobactam. TABLE 3 Acquired β-lactamase genes identified in meropenem-resistant P. aeruginosa isolates (n = 139)a,b Isolate β-Lactamase group TotalMBL MBL + KPC MBL + ESBL MBL + OXA ESBL Negative P. aeruginosa 27 1 1 1 8 101 139 aESBL, extended-spectrum β-lactamase; KPC, Klebsiella pneumoniae carbapenemase; MBL, metallo-β-lactamase; OXA, oxacillinase; PCR, polymerase chain reaction; WGS, whole-genome sequencing. bData were generated by WGS if meropenem-resistant isolates were resistant to cefiderocol (4/139) or PCR if isolates were susceptible to cefiderocol (135/139). Isolates are grouped as “negative” if only intrinsic β-lactamase genes were present. Research Article Microbiology Spectrum April 2024 Volume 12 Issue 4 10.1128/spectrum.03836-23 6 Downloaded from https://journals.asm.org/journal/spectrum on 03 April 2025 by 193.147.173.206. Of the 227 meropenem-resistant Acinetobacter spp. isolates, 16.7% (38/227) were cefiderocol resistant and were analyzed by WGS, while the remaining isolates were analyzed by PCR for the presence of specific β-lactamase genes. Antimicrobial suscepti bility rates in meropenem-resistant Acinetobacter spp. according to β-lactamase genes identified are reported in Table S5B. The majority of meropenem-resistant Acinetobacter spp. isolates harbored acquired OXA genes [94.7% (215/227)], including 202 isolates with only acquired OXA genes, 9 that co-harbored MBL genes, and 4 that co-harbored ESBL genes (Table 4). This trend was similar across all European countries (Table S6B). A low proportion of isolates harbored only MBL [1.3% (3/227)] genes or co-harbored ESBL and KPC genes [0.4% (1/227)] (Table 4). Overall, 85.5% (194/227) of meropenem-resistant Acinetobacter spp. isolates harbored blaOXA-23 or genes of the OXA-23 group, 14.5% (33/227) harbored blaOXA-24 or genes of the OXA-24 group (including 16/227 isolates that co-harbored blaOXA-23-24), and 3.1% (7/227) harbored blaOXA-72; 5.3% (12/227) of isolates harbored blaNDM-1, and 0.4% (1/227) harbored blaKPC-3 (Table S6B). β-Lactamase genes and other potential resistance mechanisms identified in cefiderocol-resistant pathogens In total, 10 P. aeruginosa and 38 Acinetobacter spp. (37 A. baumannii; 1 Acinetobacter calcoaceticus) isolates were cefiderocol resistant and were analyzed by WGS. Four cefiderocol-resistant P. aeruginosa isolates were of a sequence type (ST) previously reported (ST235, ST298, ST708, and ST773), while six novel STs were identified (ST4283, ST4290, ST4291, ST4304, ST4305, and ST4306) for isolates collected in France, Germany, Italy, Spain, and the United Kingdom (Table 5). Only one isolate harbored an acquired β-lactamase gene (blaNDM-1). All cefiderocol-resistant isolates had either mutations in piuA and pirA-like genes encoding siderophore uptake receptors or these genes were not detected; the majority also had piuC (9/10) and pvdS (6/10) alterations. Only 20% (2/10) had ftsl and oprD mutations. Half (19/38) of the cefiderocol-resistant Acinetobacter spp. isolates were found to be ST2, collected in all six European countries, while the other half consisted of isolates of other STs, including two novel STs from France and Germany (Table 6). A total of 84.2% (32/38) of isolates harbored acquired β-lactamase genes. Of the ST2 isolates, 73.7% (14/19) harbored blaOXA-23, 26.3% (5/19) harbored blaOXA-72, and 5.3% (1/19) harbored blaNDM-1 or the blaPER-7 carbapenemase gene. All ST2 isolates had piuA mutations and 42.1% (8/19) had ftsl mutations, but no piuC, pirA-like, or carO gene mutations were observed. Of the non-ST2 isolates, 52.6% (10/19) harbored blaOXA-23 and 57.9% (11/19) harbored blaNDM-1. All non-ST2 isolates had either piuA mutations or this gene was not detected; 57.9% (11/19) had ftsI mutations, and 47.4% (9/19) had pirA-like gene mutations, or this gene was not detected, but no carO mutations were observed. TABLE 4 Acquired β-lactamase genes identified in meropenem-resistant Acinetobacter spp. isolates (n = 227)a,b Isolate β-Lactamase group TotalOXA OXA+ MBL OXA+ ESBL MBL ESBL+ KPC Negative A. baumannii 156 8 3 3 1 6 177 A. baumannii complex 32 – – – – 2 34 A. bereziniae 1 – – – – – 1 A. nosocomialis 1 – – – – – 1 Other Acinetobacter spp. 12 1 1 – – – 14 Total 202 9 4 3 1 8 227 aESBL, extended-spectrum β-lactamase; KPC, Klebsiella pneumoniae carbapenemase; MBL, metallo-β-lactamase; OXA, oxacillinase; PCR, polymerase chain reaction; WGS, whole-genome sequencing. bData were generated by WGS if meropenem-resistant isolates were resistant to cefiderocol (174/227, including 1 A. baumannii complex and 4 other Acinetobacter spp.) or PCR if isolates were susceptible to cefiderocol (53/227, including 33 A. baumannii complex and 10 other Acinetobacter spp.). Isolates are grouped as “negative” if only intrinsic β-lactamase genes were present (n = 1) or if no screened β-lactamase gene was present (n = 7). Research Article Microbiology Spectrum April 2024 Volume 12 Issue 4 10.1128/spectrum.03836-23 7 Downloaded from https://journals.asm.org/journal/spectrum on 03 April 2025 by 193.147.173.206. DISCUSSION This study provides additional data on the in vitro susceptibilities of cefiderocol and BLBLI combinations, including those still in development, against a large collection of European isolates of glucose non-fermenting Gram-negative bacteria. The data collected in this study are from a greater number of sites per European country compared with the longitudinal SENTRY and SIDERO surveillance programs, which are more geographically spread (35, 36). The susceptibility rate for cefiderocol was higher than BLBLI combinations (including those still in development, such as aztreonam-avibactam and cefepime-taniborbactam) against P. aeruginosa overall (98.9% vs 83.3%–91.4%, respectively) and meropenemresistant isolates (97.8% vs ≤59.7%). These observations are consistent with similar previous in vitro studies (5, 26, 39–45). It is important to note that meropenemresistant P. aeruginosa isolates in this study were defined according to the EUCAST MIC resistance breakpoint for high-dose (2 g), extended (3-h)-infusion meropenem (>8 mg/L), to represent isolates that are meropenem resistant even when treated with the highest meropenem dose available to patients. As some previous studies have defined meropenem-resistant/non-susceptible P. aeruginosa according to the EUCAST MIC resistance breakpoint for standard-dose meropenem (>2 mg/L), susceptibility rates for BLBLI combinations tested in previous studies may be higher (39, 43). Of the β-lactamase genes observed in meropenem-resistant P. aeruginosa isolates, most were MBLs (most commonly VIM), against which cefiderocol retains high activ ity, in contrast to most BLBLI combinations (46). Low frequencies (≤5%) of IMP, NDM (blaNDM-1), and GES were observed, lower than previously published data (6). The 71.2% of meropenem-resistant P. aeruginosa isolates which did not harbor β-lactamase genes of interest likely exhibited non-β-lactamase mechanisms of resistance, such as increased expression of efflux systems, chromosomal cephalosporinase activity, or reduced porin expression (3–6, 47). TABLE 5 β-Lactamase genes and other potential resistance mechanisms identified in cefiderocol-resistant P. aeruginosa isolates (n = 10)a,b Isolate Country FDC MIC (mg/L) ST Potential resistance mechanism(s) identified β-Lactamasecftsl oprD piuA piuC pirA-like pvdS 1 France 8 4291dOXA-396; PDC-543 WT No gross disruption Gene not found Gene not found A370T WT 2 France 4 773 OXA-395; PDC-16; NDM-1 WT No gross disruption Q34H R201H A370T V180L; H182N 3 France 8 235 OXA-19; OXA-488; PDC-35 WT No gross disruption piuD R201H S20N; T235I; A370T V180L; H182N 4 Germany 4 298 OXA-848; PDC-219 A244T Gross disruption piuD R201H Gross disruption V26A 5 Germany 8 4290dOXA-1124; PDC-86 WT No gross disruption Q34H T10S; R201H A13V; A370T V180L; H182N 6 Italy 8 708 OXA-50; PDC-446 WT No gross disruption Q34H WT R7H; A370T; Q620K V180L; T181A; H182N 7 Italy 16 4305dOXA-488; PDC-337 WT No gross disruption Q34H R201H A370T WT 8 Spain 4 4304dOXA-1022; PDC-46 V137M Gross disruption Q34H R201H Y2S; A370T WT 9 Spain 4 4306dOXA-1188; PDC-5 WT No gross disruption piuD V104I A370T V180L; T181A; H182N; R186P 10 UK 4 4283dOXA-1135; PDC-63 WT No gross disruption Gene not found D49E; E125A; R198Q; V203I T144S; A208T; A370T; T480A WT aFDC, cefiderocol; MBL, metallo-β-lactamase; NDM, New Delhi MBL; OXA, oxacillinase; PDC, Pseudomonas-derived cephalosporinase; ST, sequence type; UK, United Kingdom; WT, wild type. bData were generated by whole-genome sequencing. A gene was considered to have a gross disruption if the coding sequence carried a nonsense mutation, frameshift, indels of >20 codons, or ablation of the canonical stop or start codons without a replacement immediately adjacent and in-frame. Genes were listed to be not found if a BLAST search with the reference gene yielded no hit with E-value <1E-25. Non-β-lactamase genes that were either found to have gross disruptions or mutations or were not found are shown in gray. cData shown are a curated summary. dIndicates a novel ST. Research Article Microbiology Spectrum April 2024 Volume 12 Issue 4 10.1128/spectrum.03836-23 8 Downloaded from https://journals.asm.org/journal/spectrum on 03 April 2025 by 193.147.173.206. TABLE 6 β-Lactamase genes and other potential resistance mechanisms identified in cefiderocol-resistant Acinetobacter spp.a isolates (n = 38)b,c Isolate Country FDC MIC (mg/L) ST Potential resistance mechanism(s) identified β-LactamasedftsI carO piuA piuC pirA-like 1 Austria 8 2 ADC-74; OXA-66; OXA-72; NDM-1 WT No gross disruption G216V; N489D; K658R WT WT 2 Austria 4 2 ADC-73; OXA-66; OXA-23 A515V No gross disruption Gross disruption WT WT 3 France 8 2 ADC-85; OXA-66; OXA-23 A515V No gross disruption Gross disruption WT WT 4 France 8 2 ADC-73; OXA-66; OXA-23 A515V No gross disruption Gross disruption WT WT 5 France 4 2 ADC-73; OXA-66; OXA-23 A515V No gross disruption Gross disruption WT WT 6 France >32 1 ADC-191; OXA-69; NDM-1; OXA-23 L480I; T511S No gross disruption Gross disruption WT Gross disruption 7 France 32 1 ADC-191; OXA-69; NDM-1; OXA-23 L480I; T511S No gross disruption Gross disruption WT Y479N; K543R 8 France 16 85 ADC-165; OXA-94; NDM-1 WT No gross disruption Gross disruption (~56% ident to reference) WT K475R; H566N 9 France >32 2163eADC-2; OXA-132 A512T No gross disruption I10F; N489D WT H566N 10 France 8 1 ADC-204; OXA-69; TEM-Trunc; OXA-23 WT No gross disruption L76F; N489D WT Y479N; K543R 11 France 32 85 ADC-80; OXA-94; NDM-1 WT No gross disruption Gross disruption (~56% ident to reference) WT K475R; H566N 12 France 4 203 ADC-Trunc; OXA-65 WT No gross disruption S10T; N19D; R42Q; L52I; N58T; A63S; T66Q; H67Q; Q90N; L119V; E125D; I164V; E185D; N206Q; S208E WT T340I; H566N 13 Germany >32 600 ADC-73; OXA-66; TEM-1D; OXA-23 A515V No gross disruption Gross disruption WT WT 14 Germany >32 2 ADC-30; OXA-66; PER-7; OXA-23 WT No gross disruption G216V; N489D; K658R WT WT 15 Germany 16 85 ADC-176; OXA-94; NDM-1 WT No gross disruption Gross disruption (and~56% ident to reference) WT K475R; H566N 16 Germany 4 636 ADC-74; OXA-66; OXA-72 WT No gross disruption Gross disruption WT WT 17 Germany >32 2262eADC-11; OXA-66; OXA-72; PER-1 WT No gross disruption G216V; N489D; K658R WT WT 18 Italy 16 2 ADC-33; OXA-82; OXA-23 H370Y Gross disruption G216V; N489D; K658R WT WT 19 Italy 4 2 ADC-73; OXA-66; OXA-23 A515V No gross disruption G216V; N489D; K658R WT WT 20 Italy 4 2 ADC-33; OXA-82; OXA-23 WT No gross disruption G216V; N489D; K658R WT WT 21 Italy 16 2 ADC-33; OXA-82; OXA-23 WT No gross disruption G216V; N489D; K658R WT WT 22 Italy 4 2 ADC-33; OXA-82; OXA-23 WT No gross disruption G216V; N489D; K658R WT WT 23 Italy >32 2 ADC-30; OXA-66; OXA-72 WT No gross disruption G216V; N489D; K658R WT WT 24 Italy 16 2 ADC-30; OXA-66; OXA-72 WT No gross disruption G216V; N489D; K658R WT WT (Continued on next page) Research Article Microbiology Spectrum April 2024 Volume 12 Issue 4 10.1128/spectrum.03836-23 9 Downloaded from https://journals.asm.org/journal/spectrum on 03 April 2025 by 193.147.173.206. 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