The growing threat of NDM-producing E. coli with penicillin-binding protein 3 mutations in the United States - is there a potential role for durlobactam?

We report identification of 5 patients with infections caused by NDM-5-producing E. coli harboring PBP3 mutations that showed reduced susceptibility to aztreonam-avibactam and cefiderocol. Durlobactam, a novel diazabicyclooctane ß-lactamase inhibitor, demonstrated minimum inhibitory concentrations ranging from 0.5 to 2 µg/mL supporting future investigations into a potential role in clinical management.

Rates of resistance to ceftazidime-avibactam and ceftolozane-tazobactam among patients treated for multidrug-resistant Pseudomonas aeruginosa bacteremia or pneumonia.

Among consecutive patients with multidrug-resistant Pseudomonas aeruginosa bacteremia or pneumonia we found those treated with ceftazidime-avibactam were more likely to develop resistance (defined as ≥4-fold increased MIC) than those treated with ceftolozane-tazobactam (40% vs. 10%; P=0.002). Ceftazidime-avibactam resistance was associated with new mutations in ampC and efflux regulatory pathways.

Differential in vitro susceptibility to ampicillin/ceftriaxone combination therapy among Enterococcus faecalis infective endocarditis clinical isolates.

OBJECTIVES: To investigate the genomic diversity and ß-lactam susceptibilities of Enterococcus faecalis collected from patients with infective endocarditis (IE). METHODS: We collected 60 contemporary E. faecalis isolates from definite or probable IE cases identified between 2018 and 2021 at the University of Pittsburgh Medical Center. We used whole-genome sequencing to study bacterial genomic diversity and employed antibiotic checkerboard assays and a one-compartment pharmacokinetic-pharmacodynamic (PK/PD) model to investigate bacterial susceptibility to ampicillin and ceftriaxone both alone and in combination. RESULTS: Genetically diverse E. faecalis were collected, however, isolates belonging to two STs, ST6 and ST179, were collected from 21/60 (35%) IE patients. All ST6 isolates encoded a previously described mutation upstream of penicillin-binding protein 4 (pbp4) that is associated with pbp4 overexpression. ST6 isolates had higher ceftriaxone MICs and higher fractional inhibitory concentration index values for ampicillin and ceftriaxone (AC) compared to other isolates, suggesting diminished in vitro AC synergy against this lineage. Introduction of the pbp4 upstream mutation found among ST6 isolates caused increased ceftriaxone resistance in a laboratory E. faecalis isolate. PK/PD testing showed that a representative ST6 isolate exhibited attenuated efficacy of AC combination therapy at humanized antibiotic exposures. CONCLUSIONS: We find evidence for diminished in vitro AC activity among a subset of E. faecalis IE isolates with increased pbp4 expression. These findings suggest that alternate antibiotic combinations against diverse contemporary E. faecalis IE isolates should be evaluated.

Evolution and Transmission of Cefiderocol-Resistant Acinetobacter baumannii During an Outbreak in the Burn Intensive Care Unit.

We report on 11 critically ill burn patients treated with cefiderocol for carbapenem-resistant Acinetobacter baumannii infections. Clinical success was achieved in 36% and complicated by treatment-emergent resistance and interpatient transmission of cefiderocol-resistant A. baumannii. Resistant isolates harbored disrupted pirA and piuA genes that were not disrupted among susceptible isolates.

Early initiation of three-drug combinations for the treatment of carbapenem-resistant A. baumannii among COVID-19 patients.

OBJECTIVES: We evaluated the clinical characteristics and outcomes of patients with COVID-19 who received three-drug combination regimens for treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) infections during a single-centre outbreak. Our objective was to describe the clinical outcomes and molecular characteristics and in vitro synergy of antibiotics against CRAB isolates. MATERIALS AND METHODS: Patients with severe COVID-19 admitted between April and July 2020 with CRAB infections were retrospectively evaluated. Clinical success was defined as resolution of signs/symptoms of infection without need for additional antibiotics. Representative isolates underwent whole-genome sequencing (WGS) and in vitro synergy of two- or three-drug combinations was assessed by checkerboard and time-kill assays, respectively. RESULTS: Eighteen patients with CRAB pneumonia or bacteraemia were included. Treatment regimens included high-dose ampicillin-sulbactam, meropenem, plus polymyxin B (SUL/MEM/PMB; 72%), SUL/PMB plus minocycline (MIN; 17%) or other combinations (12%). Clinical resolution was achieved in 50% of patients and 30-day mortality was 22% (4/18). Seven patients had recurrent infections, during which further antimicrobial resistance to SUL or PMB was not evident. PMB/SUL was the most active two-drug combination by checkerboard. Paired isolates collected before and after treatment with SUL/MEM/PMB did not demonstrate new gene mutations or differences in the activity of two- or three-drug combinations. CONCLUSIONS: Use of three-drug regimens for severe CRAB infections among COVID-19 resulted in high rates of clinical response and low mortality relative to previous studies. The emergence of further antibiotic resistance was not detected phenotypically or through WGS analysis. Additional studies are needed to elucidate preferred antibiotic combinations linked to the molecular characteristics of infecting strains.

Phage therapy in a lung transplant recipient with cystic fibrosis infected with multidrug-resistant Burkholderia multivorans.

BACKGROUND: There is increased interest in bacteriophage (phage) therapy to treat infections caused by antibiotic-resistant bacteria. A lung transplant recipient with cystic fibrosis and Burkholderia multivorans infection was treated with inhaled phage therapy for 7 days before she died. METHODS: Phages were given via nebulization through the mechanical ventilation circuit. Remnant respiratory specimens and serum were collected. We quantified phage and bacterial deoxyribonucleic acid (DNA) using quantitative polymerase chain reaction, and tested phage neutralization in the presence of patient serum. We performed whole genome sequencing and antibiotic and phage susceptibility testing on 15 B. multivorans isolates. Finally, we extracted lipopolysaccharide (LPS) from two isolates and visualized their LPS using gel electrophoresis. RESULTS: Phage therapy was temporally followed by a temporary improvement in leukocytosis and hemodynamics, followed by worsening leukocytosis on day 5, deterioration on day 7, and death on day 8. We detected phage DNA in respiratory samples after 6 days of nebulized phage therapy. Bacterial DNA in respiratory samples decreased over time, and no serum neutralization was detected. Isolates collected between 2001 and 2020 were closely related but differed in their antibiotic and phage susceptibility profiles. Early isolates were not susceptible to the phage used for therapy, while later isolates, including two isolates collected during phage therapy, were susceptible. Susceptibility to the phage used for therapy was correlated with differences in O-antigen profiles of an early versus a late isolate. CONCLUSIONS: This case of clinical failure of nebulized phage therapy highlights the limitations, unknowns, and challenges of phage therapy for resistant infections.

Evolution of Imipenem-Relebactam Resistance Following Treatment of Multidrug-Resistant Pseudomonas aeruginosa Pneumonia.

We report the emergence of imipenem-relebactam nonsusceptible Pseudomonas aeruginosa in 5 patients treated for nosocomial pneumonia for 10-28 days. Genome sequence analysis identified treatment-emergent mutations in MexAB-OprM and/or MexEF-OprN efflux operons that arose independently in each patient across distinct P. aeruginosa sequence types. Testing with efflux-inhibitor PAßN restored imipenem-relebactam susceptibility.

Molecular Epidemiology, Natural History, and Long-Term Outcomes of Multidrug-Resistant Enterobacterales Colonization and Infections Among Solid Organ Transplant Recipients.

BACKGROUND: Multidrug-resistant Enterobacterales (MDR-E), including carbapenem-resistant and third-generation cephalosporin-resistant Enterobacterales (CRE, CefR-E), are major pathogens following solid organ transplantation (SOT). METHODS: We prospectively studied patients who underwent lung, liver, and small bowel transplant from February 2015 through March 2017. Weekly perirectal swabs (up to 100 days post-transplant) were cultured for MDR-E. Whole-genome sequencing (WGS) was performed on gastrointestinal (GI) tract-colonizing and disease-causing isolates. RESULTS: Twenty-five percent (40 of 162) of patients were MDR-E GI-colonized. Klebsiella pneumoniae was the most common CRE and CefR-E. Klebsiella pneumoniae carbapenemases and CTX-M were leading causes of CR and CefR, respectively. Thirty-five percent of GI colonizers developed MDR-E infection vs 2% of noncolonizers (P < .0001). The attack rate was higher among CRE colonizers than CefR-E colonizers (53% vs 21%, P = .049). GI colonization and high body mass index were independent risk factors for MDR-E infection (P ≤ .004). Thirty-day mortality among infected patients was 6%. However, 44% of survivors developed recurrent infections; 43% of recurrences were late (285 days to 3.9 years after the initial infection). Long-term survival (median, 4.3 years post-transplant) did not differ significantly between MDR-E-infected and MDR-E-noninfected patients (71% vs 77%, P = .56). WGS phylogenetic analyses revealed that infections were caused by GI-colonizing strains and suggested unrecognized transmission of novel clonal group-258 sublineage CR-K. pneumoniae and horizontal transfer of resistance genes. CONCLUSIONS: MDR-E GI colonization was common following SOT and predisposed patients to infections by colonizing strains. MDR-E infections were associated with low short- and long-term mortality, but recurrences were frequent and often occurred years after initial infections. Findings provide support for MDR-E surveillance in our SOT program.

Optimal ceftazidime/avibactam dosing exposure against KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Infections due to carbapenem-resistant Enterobacterales are considered urgent public health threats and often treated with a ß-lactam/ß-lactamase inhibitor combination. However, clinical treatment failure and resistance emergence have been attributed to inadequate dosing. We used a novel framework to provide insights of optimal dosing exposure of ceftazidime/avibactam. METHODS: Seven clinical isolates of Klebsiella pneumoniae producing different KPC variants were examined. Ceftazidime susceptibility (MIC) was determined by broth dilution using escalating concentrations of avibactam. The observed MICs were characterized as response to avibactam concentrations using an inhibitory sigmoid Emax model. Using the best-fit parameter values, %fT>MICi was estimated for various dosing regimens of ceftazidime/avibactam. A hollow-fibre infection model (HFIM) was subsequently used to ascertain the effectiveness of selected regimens over 120 h. The drug exposure threshold associated with bacterial suppression was identified by recursive partitioning. RESULTS: In all scenarios, ceftazidime MIC reductions were well characterized with increasing avibactam concentrations. In HFIM, bacterial regrowth over time correlated with emergence of resistance. Overall, suppression of bacterial regrowth was associated with %fT>MICi ≥ 76.1% (100% versus 18.2%; P < 0.001). Using our framework, the optimal drug exposure could be achieved with ceftazidime/avibactam 2.5 g every 12 h in 5 out of 7 isolates. Furthermore, ceftazidime/avibactam 2.5 g every 8 h can suppress an isolate deemed resistant based on conventional susceptibility testing method. CONCLUSIONS: An optimal drug exposure to suppress KPC-producing bacteria was identified. The novel framework is informative and may be used to guide optimal dosing of other ß-lactam/ß-lactamase inhibitor combinations. Further in vivo investigations are warranted.

Ceftolozane/tazobactam for refractory P. aeruginosa endocarditis: A case report and pharmacokinetic analysis.

We describe a case of a 48 years old male with left sided endocarditis and septic emboli secondary to a Pseudomonas aeruginosa strain that developed resistance to other ß-lactam antibiotics during therapy resulting in prolonged bacteremia. Blood cultures sterilized within 1 day of initiating ceftolozane/tazobactam 3 g every 8 hours in combination with ciprofloxacin. Steady state free ceftolozane plasma Cmax and Cmin concentrations were calculated to be 122.2µg/mL and 24.3µg/mL, respectively. The multidrug-resistant strain harbored chromosomal ß-lactamases OXA-486 and PDC-3, mutations in ampD and dacB predicted to lead to ampC over-expression, and mutations in OprD predicted to decrease outer membrane permeability. Following completion of a 42 day course and aortic valve replacement, the patient was deemed clinically cured without recurrence of infection at follow up 2 years later. To our knowledge, this is the first reported case to measure ceftolozane concentrations during the treatment of endocarditis which supports dose optimization approaches of severe endovascular disease due to multidrug resistant pathogens.

In Vitro Susceptibility of Multidrug-Resistant Pseudomonas aeruginosa following Treatment-Emergent Resistance to Ceftolozane-Tazobactam.

We compared the in vitro susceptibility of multidrug-resistant Pseudomonas aeruginosa isolates collected before and after treatment-emergent resistance to ceftolozane-tazobactam. Median baseline and postexposure ceftolozane-tazobactam MICs were 2 and 64 µg/ml, respectively. Whole-genome sequencing identified treatment-emergent mutations in ampC among 79% (11/14) of paired isolates. AmpC mutations were associated with cross-resistance to ceftazidime-avibactam but increased susceptibility to piperacillin-tazobactam and imipenem. A total of 81% (12/16) of ceftolozane-tazobactam-resistant isolates with ampC mutations were susceptible to imipenem-relebactam.

Clinical Evolution of AmpC-Mediated Ceftazidime-Avibactam and Cefiderocol Resistance in Enterobacter cloacae Complex Following Exposure to Cefepime.

We report 2 independent patients from whom carbapenem and ceftazidime-avibactam-resistant Enterobacter cloacae complex strains were identified. The ceftazidime-avibactam resistance was attributed to a 2-amino acid deletion in the R2 loop of AmpC ß-lactamase, which concurrently caused resistance to cefepime and reduced susceptibility to cefiderocol, a novel siderophore cephalosporin.

Early Experience With Meropenem-Vaborbactam for Treatment of Carbapenem-resistant Enterobacteriaceae Infections.

Twenty patients with carbapenem-resistant Enterobacteriaceae infections were treated with meropenem-vaborbactam. Thirty-day clinical success and survival rates were 65% (13/20) and 90% (18/20), respectively. Thirty-five percent of patients had microbiologic failures within 90 days. One patient developed a recurrent infection due to meropenem-vaborbactam-nonsusceptible, ompK36 porin mutant Klebsiella pneumoniae.

Structural Basis of Reduced Susceptibility to Ceftazidime-Avibactam and Cefiderocol in Enterobacter cloacae Due to AmpC R2 Loop Deletion.

Ceftazidime-avibactam and cefiderocol are two of the latest generation ß-lactam agents that possess expanded activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales Here, we show that structural changes in AmpC ß-lactamases can confer reduced susceptibility to both agents. A multidrug-resistant Enterobacter cloacae clinical strain (Ent385) was found to be resistant to ceftazidime-avibactam and cefiderocol without prior exposure to either agent. The AmpC ß-lactamase of Ent385 (AmpCEnt385) contained an alanine-proline deletion at positions 294 and 295 (A294_P295del) in the R2 loop. AmpCEnt385 conferred reduced susceptibility to ceftazidime-avibactam and cefiderocol when cloned into Escherichia coli TOP10. Purified AmpCEnt385 showed increased hydrolysis of ceftazidime and cefiderocol compared to AmpCEnt385Rev, in which the deletion was reverted. Comparisons of crystal structures of AmpCEnt385 and AmpCP99, the canonical AmpC of E. cloacae complex, revealed that the two-residue deletion in AmpCEnt385 induced drastic structural changes of the H-9 and H-10 helices and the R2 loop, which accounted for the increased hydrolysis of ceftazidime and cefiderocol. The potential for a single mutation in ampC to confer reduced susceptibility to both ceftazidime-avibactam and cefiderocol requires close monitoring.

Spontaneous Mutational Frequency and FKS Mutation Rates Vary by Echinocandin Agent against Candida glabrata.

Echinocandins are front-line agents for treatment of invasive candidiasis. There are no reported agent-specific differences in Candida mutational frequency of resistance or propensity to develop FKS mutations. The objective of this study was to measure spontaneous and FKS mutation rates among Candida glabrata strains. Twenty bloodstream isolates from patients with or without prior echinocandin exposure were included. Minimum inhibitory concentrations (MICs), minimum fungicidal concentrations (MFCs), and mutation prevention concentrations were higher for caspofungin than for anidulafungin (P < 0.0001) and micafungin (P < 0.0001). Mutational frequencies of resistance at 3× the baseline MIC were highest for caspofungin and lowest for micafungin. A total of 247 isolates were recovered at or above the MFC for caspofungin (n = 159), anidulafungin (n = 74), or micafungin (n = 14). Agent-specific MIC increases were noted for anidulafungin and caspofungin, but not micafungin. Thirty-three percent of isolates harbored hot spot mutations in FKS1 (n = 6) or FKS2 (n = 76). Mutations at the Ser629 (Fks1) or Ser663 (Fks2) loci were more common after selection with anidulafungin or micafungin than with caspofungin (P = 0.003). Four isolates demonstrated >4-fold increases in MICs without FKS hot spot mutations; three of these harbored Fks2 mutations upstream of hot spot 1. The final isolate was FKS1 and FKS2 wild-type, but the 50% inhibitory concentrations of caspofungin and micafungin were increased 2.7- and 8-fold, respectively. In conclusion, micafungin may be superior in vitro to the other agents in limiting the emergence of resistance among C. glabrata Caspofungin exposure may be most likely to promote resistance development. These data provide a foundation for future investigations of newly developed echinocandin agents.

Effects of KPC Variant and Porin Genotype on the In Vitro Activity of Meropenem-Vaborbactam against Carbapenem-Resistant Enterobacteriaceae.

Meropenem-vaborbactam is a new agent with the potential to treat carbapenem-resistant Enterobacteriaceae (CRE) infections. We describe the in vitro activity of meropenem-vaborbactam against representative CRE genotypes and laboratory-engineered Escherichia coli isolates harboring mutant blaKPC genes associated with ceftazidime-avibactam resistance. We also compared disk diffusion and gradient strip testing methods to standard broth microdilution methods. Against 120 CRE isolates, median ceftazidime-avibactam and meropenem-vaborbactam MICs were 1 and 0.03 µg/ml, respectively. Ninety-eight percent (117/120) of isolates were susceptible to meropenem-vaborbactam (MICs ≤ 4 µg/ml). Against Klebsiella pneumoniae isolates harboring mutant blaKPC, the addition of vaborbactam lowered the meropenem MICs in 78% of isolates (14/18); 100% were susceptible to meropenem-vaborbactam. Median meropenem-vaborbactam MICs were higher against K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates with mutant ompK36 porin genes (n = 26) than against those with wild-type ompK36 porin genes (n = 54) (0.25 versus 0.03 µg/ml; P < 0.0001). Against E. coli TOP10 isolates with plasmid constructs containing wild-type blaKPC or mutant blaKPC, the addition of vaborbactam at 8 µg/ml lowered the meropenem MICs 2- to 512-fold, resulting in meropenem-vaborbactam MICs of 0.03 µg/ml. The rates of categorical agreement with broth microdilution for disk diffusion or gradient strips ranged from 90 to 95%. Essential agreement rates were higher for research-use-only (RUO) gradient strips manufactured by bioMérieux (82%) than for those manufactured by Liofilchem (48%) (P < 0.0001). Taken together, our data highlight the potent in vitro activity of meropenem-vaborbactam against CRE, including isolates resistant to ceftazidime-avibactam. Vaborbactam inhibited both wild-type and variant KPC enzymes. On the other hand, KPC-producing K. pneumoniae isolates with ompK36 mutations displayed higher meropenem-vaborbactam MICs than isolates with wild-type ompK36 The results of susceptibility testing with RUO bioMérieux gradient strips most closely aligned with those of broth microdilution methods.

Colistin Does Not Potentiate Ceftazidime-Avibactam Killing of Carbapenem-Resistant Enterobacteriaceae In Vitro or Suppress Emergence of Ceftazidime-Avibactam Resistance.

We tested ceftazidime-avibactam and colistin against 24 carbapenem-resistant Enterobacteriaceae (CRE) isolates by time-kill studies. Ceftazidime-avibactam at 0.25×, 1×, and 4× the MIC was bactericidal against 8%, 21%, and 88% of the isolates, respectively. Colistin (2 µg/ml) was bactericidal against 83% (12 h) and 42% (24 h) of the isolates. In combination, synergy and antagonism were identified against 13% and 46% of the isolates, respectively. The combination did not suppress ceftazidime-avibactam resistance. Colistin plus ceftazidime-avibactam did not provide a benefit over ceftazidime-avibactam against most CRE isolates.

Emergence of high-level aztreonam-avibactam and cefiderocol resistance following treatment of an NDM-producing Escherichia coli bloodstream isolate exhibiting reduced susceptibility to both agents at baseline.

Background: Cefiderocol (FDC) or ceftazidime-avibactam with aztreonam (CZA-ATM) are frontline agents for New Delhi metallo-ß-lactamase (NDM)-producing Enterobacterales; however, clinical data are scarce, and mechanisms of treatment-emergent resistance are ill-defined. Our objectives were to characterize serial isolates and stool microbiota from a liver transplant recipient with NDM-producing Escherichia coli bacteraemia. Methods: Isolates collected pre- and post-CZA-ATM treatment underwent broth microdilution susceptibility testing and whole-genome sequencing. Longitudinal stool collected during CZA-ATM therapy underwent metagenomic sequencing (Nanopore MinION). Results: The baseline isolate exhibited elevated MICs for ATM-AVI (16/4 µg/mL) and FDC (8 µg/mL). Posttreatment, a rectal surveillance isolate exhibited high-level resistance to ATM-AVI (> 128/4 µg/mL) and FDC (32 µg/mL). Both isolates belonged to ST361 and harboured WT bla NDM-5. The baseline isolate contained wild type (WT) bla CMY-145 and mutations in ftsI (which encodes PBP3), including a YRIN insertion at residue 338 and the non-synonymous substitutions Q227H, E353K and I536L. The posttreatment isolate harboured new mutations in ftsI (A417 V) and bla CMY-145 (L139R and N366Y). Analysis of four stool samples collected during CZA-ATM treatment revealed high E. coli abundance. E. coli relative abundance increased from 34.5% (first sample) to 61.9% (last sample). Conclusions: Baseline mutations in ftsI were associated with reduced susceptibility to ATM-AVI and FDC in an ST361 NDM-5-producing E. coli bloodstream isolate. High-level resistance was selected after CZA-ATM treatment, resulting in new ftsl and bla CMY-145 mutations. These findings underscore the need for ATM-AVI susceptibility testing for NDM producers, and the potential for PBP3 mutations to confer cross-resistance to ATM-AVI and FDC, which can emerge after CZA-ATM treatment.

Frequency of cefiderocol heteroresistance among patients treated with cefiderocol for carbapenem-resistant Acinetobacter baumannii infections.

Background: Cefiderocol exhibits potent in vitro activity against carbapenem-resistant Acinetobacter baumannii (CRAb), but this activity has not consistently translated to improved outcomes among patients. Cefiderocol heteroresistance, or the presence of a resistant subpopulation, has been proposed as one possible explanation. The objective of this study was to explore associations between heteroresistance and outcomes of patients with CRAb infections. Methods: Baseline CRAb isolates were collected from 27 consecutive patients in the USA and Italy. Cefiderocol susceptibility was tested by broth microdilutions in triplicate. Heteroresistance was defined by population analysis profiling in duplicate. Resistance mechanisms and strain relatedness were evaluated through comparative genomic analysis. Results: Overall, 59% of infecting CRAb isolates were identified as cefiderocol-heteroresistant; rates were higher among isolates from Italy (79%) than the USA (38%). The median Charlson Comorbidity and SOFA scores were 4 and 5, respectively; 44% of patients had pneumonia, which was the most common infection type. Rates of 28-day clinical success and survival were 30% and 73%, respectively. By broth microdilution, cefiderocol MICs ≥1 mg/L were associated with higher failure rates than MICs ≤0.5 mg/L (81% versus 55%). Rates of clinical failure were numerically higher among patients infected by cefiderocol-heteroresistant compared with susceptible CRAb (81% versus 55%). Whole-genome sequencing identified a premature stop codon in the TonB-dependent receptor gene piuA in six isolates, all of which were heteroresistant. Conclusions: This pilot study supports the hypothesis that cefiderocol treatment failure may be associated with higher MICs and/or the presence of heteroresistance. Further studies are needed to confirm these findings.

In vitro activity of cefiderocol against Pseudomonas aeruginosa demonstrating evolved resistance to novel ß-lactam/ß-lactamase inhibitors.

Background: Cefiderocol demonstrates excellent activity against MDR Pseudomonas aeruginosa; however, the activity against isolates from patients previously treated with ß-lactam agents is unknown. We aimed to determine the activity of cefiderocol against P. aeruginosa collected before and after treatment with traditional ß-lactams and new ß-lactam/ß-lactamase inhibitors. Methods: Cefiderocol MICs were determined in triplicate in iron-depleted cation-adjusted Mueller-Hinton broth and compared with ß-lactam MICs tested by standard methods. All isolates underwent WGS analysis to identify mutations associated with resistance. Results: One hundred and seventy-eight P. aeruginosa isolates were evaluated; 48% (86/178) were non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam. The cefiderocol MIC50 and MIC90 were 0.12 and 1 mg/L, respectively. Median cefiderocol MICs did not vary against isolates classified as MDR, XDR, or those non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam when compared with non-MDR isolates. Against isolates collected from patients previously treated with ceftolozane/tazobactam, cefiderocol MICs were increased 4-fold compared with baseline. Cross-resistance to cefiderocol was identified in 21% (3/14) of patients who developed treatment-emergent resistance to ceftolozane/tazobactam. Overall, 6% (11/178) of isolates demonstrated cefiderocol MICs ≥2 mg/L, which were disproportionately collected from patients previously treated with ceftolozane/tazobactam (73%; 8/11). Isolates with reduced cefiderocol susceptibility harboured mutations in ampC, tonB-dependent receptors, the response regulator pirR and ftsI. Conclusions: Cefiderocol demonstrates excellent in vitro activity against P. aeruginosa isolates exposed to other novel ß-lactam agents; however, some exceptions were identified. Cross-resistance between cefiderocol and ceftolozane/tazobactam was evident, but not with ceftazidime/avibactam or imipenem/relebactam. Reduced cefiderocol susceptibility was mediated by mutations in ampC and tonB-dependent receptors.