In Vitro Activity of Cefiderocol Against Meropenem-Nonsusceptible Gram-Negative Bacilli with Defined β-Lactamase Carriage: SIDERO-WT Surveillance Studies, 2014–2019

We examined the in vitro susceptibility of meropenem-nonsusceptible Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii complex isolates from five consecutive annual SIDERO-WT surveillance studies (2014–2019) to cefiderocol and comparator agents in the context of their carbapenemase carriage. 1,003 Enterobacterales, 1,758 P. aeruginosa, and 2,809 A. baumannii complex isolates from North America and Europe that were meropenem nonsusceptible (CLSI M100, 2022) were molecularly characterized for β-lactamase content by PCR followed by Sanger sequencing or by whole genome sequencing. Among Enterobacterales, 91.5% of metallo-β-lactamase (MBL)–producing, 98.4% of KPC-producing, 97.3% of OXA-48 group–producing, and 98.7% of carbapenemase-negative, meropenem-nonsusceptible isolates were cefiderocol susceptible (MIC ≤4 mg/L). Among P. aeruginosa, 100% of MBL-producing, 100% of GES carbapenemase-producing, and 99.8% of carbapenemase-negative, meropenem-nonsusceptible isolates were cefiderocol susceptible (MIC ≤4 mg/L). Among A. baumannii complex, 60.0% of MBL-producing, 95.6% of OXA-23 group-producing, 89.5% of OXA-24 group-producing, 100% of OXA-58 group-producing, and 95.5% of carbapenemase-negative, meropenem-nonsusceptible isolates were cefiderocol susceptible (MIC ≤4 mg/L). Cefiderocol was inactive against A. baumannii complex isolates carrying a PER or VEB β-lactamase (n = 103; 15.5% susceptible). Ceftazidime–avibactam and ceftolozane–tazobactam were inactive against MBL-carrying and A. baumannii complex isolates; ceftolozane–tazobactam was also inactive against serine carbapenemase–carrying Enterobacterales and P. aeruginosa. In summary, cefiderocol was highly active in vitro against Gram-negative isolates carrying MBLs and serine carbapenemases, as well as carbapenemase-negative, meropenem-nonsusceptible isolates.


Introduction
C efiderocol is a parenteral siderophore-cephalosporin conjugate whose current use is primarily as therapy for patients infected with carbapenem-resistant, multidrugresistant (MDR), and difficult-to-treat resistant (DTR) Gramnegative bacilli when there are limited treatment options. [1][2][3] In the United States, cefiderocol is approved for the treatment of adults with complicated urinary tract infections, including pyelonephritis, caused by susceptible Gramnegative bacilli (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae complex, and Pseudomonas aeruginosa) for the treatment of hospitalacquired bacterial pneumonia and ventilator-associated bacterial pneumonia caused by Enterobacterales (E. coli, K. pneumoniae, E. cloacae complex, and Serratia marcescens), P. aeruginosa, and Acinetobacter baumannii complex. 4 In Europe, cefiderocol is licensed for the treatment of infections due to aerobic Gram-negative organisms in adults with limited treatment options. 5 As the prevalence of carbapenem-resistant, MDR, and DTR Gram-negative bacilli increases, and therapeutic options become increasingly limited, greater reliance will be placed on newer therapies such as cefiderocol to treat patients with recalcitrant infections. [6][7][8][9][10] Previously we reported phenotypic in vitro susceptibility data from five consecutive annual surveillance studies (SIDERO-WT) that examined the activity of cefiderocol and comparator agents against 47,276 Gram-negative bacilli collected from clinical laboratories in North America and Europe from 2014 through 2019. 11 That study showed that cefiderocol was highly active against antimicrobial nonsusceptible phenotypic subsets, including meropenemnonsusceptible, ceftazidime-avibactam-nonsusceptible, and ceftolozane-tazobactam-nonsusceptible Enterobacterales and P. aeruginosa, as well as meropenem-nonsusceptible A. baumannii complex isolates. To further our understanding of cefiderocol's ability to overcome b-lactamase mediated resistance, we characterized the meropenemnonsusceptible subsets of Enterobacterales, P. aeruginosa, and A. baumannii complex from the 2014 to 2019 SIDERO-WT surveillance studies for b-lactamase carriage.

Materials and Methods
IRB approval and informed consent were not required because all isolates received into the study followed multiple subcultures and were completely de-identified. The secondary research use of de-identified isolates is considered exempt research according to the Regulations for the Protection of Human Subjects in Research of the U.S. Department of Health and Human Services, Office for Human Research Protections (45 CFR 46).

Antimicrobial susceptibility testing
The CLSI broth microdilution method was used to determine isolate MICs, 12  Most meropenem-nonsusceptible Enterobacterales (n = 943), P. aeruginosa (n = 1,755), and A. baumannii complex (n = 2,726) isolates were interrogated by PCR for b-lactamase genes followed by Sanger sequencing. Isolate genomic DNA was obtained using the QIAamp Ò DNA Mini protocol for the QiaCube (Qiagen, Gaithersburg, MD) following manufacturer's recommendations. Meropenemnonsusceptible Enterobacterales, P. aeruginosa, and A. baumannii complex isolates were screened for the presence of bla encoding extended-spectrum b-lactamases baumannii complex and P. aeruginosa], and OXA-48 group [Enterobacterales and A. baumannii complex]) by multiplex PCR using published primers. [16][17][18] Genes encoding the following enzymes were amplified with extragenic primers and sequenced: KPC, OXA-48-like, IMP, VIM, NDM, GES, VEB, PER, TEM, and SHV. bla TEM and bla SHV were first screened by limited sequencing to identify genes encoding enzymes containing amino acid substitutions common to TEM-type (amino acid positions 104, 164, 238, and 240) and SHV-type (amino acid positions 146, 179, 238, and 240) ESBLs. Only bla SHV and bla TEM that encoded ESBLs were completely sequenced. For all b-lactamase genes sequenced, the deduced amino acid sequence was compared to available databases maintained by the NCBI (www.ncbi.nlm.nih.gov) to identify enzyme variants.

Whole genome sequencing
One hundred and forty-six meropenem-nonsusceptible isolates that also tested with elevated cefiderocol MICs ( ‡4 mg/L [in 2018] and ‡8 mg/L [in 2019]), including 60 Enterobacterales, three P. aeruginosa, and 83 A. baumannii complex isolates, were subjected to WGS (in lieu of PCR and Sanger sequencing). Cells were pelleted from 3 mL liquid cultures grown overnight from one colony in Brain Heart Infusion broth (Sigma-Aldrich, St. Louis, MO) at 37°C with shaking. DNA was subsequently extracted using the DNeasy Ultraclean Microbial Extraction Kit (Qiagen). Sequencing libraries were prepared using the Illumina DNA Prep Library Preparation Kit (Illumina, San Diego, CA). Sequencing was performed on an Illumina HiSeq system using 2 · 150 bp paired-end reads with a target coverage depth of 100 times.
All analyses were carried out using the CLC Genomics Workbench, version 20 (Qiagen). For resistance gene identification, de novo assemblies of each genome were queried using the ''find resistance'' module, which interrogates the CGE database for resistance genes. b-lactamase genes with less than 100% sequence identity to a known nucleotide reference were translated to their deduced amino acid sequence and BLASTP searched against the RefSeq database in GenBank dedicated to b-lactamase nomenclature (Bio-Project 313047) to assign the enzyme variant. Table 1 summarizes the identities and distribution of carbapenemases identified in the meropenem-nonsusceptible Enterobacterales, P. aeruginosa, and A. baumannii complex isolates stratified by geographic region (North America, Europe) and by year of collection (2014-2019). Among Enterobacterales, VIM was the most common MBL encountered (53.6% of MBL-positive isolates; 113/211), followed by NDM (45.5%; 96/211) and IMP (0.9%; 2/211). Geographically, MBL producers were more likely to be isolated in Europe, as 24.9% (198/794) of the meropenemnonsusceptible Enterobacterales carried MBLs, compared to 6.2% (13/209) of North American isolates. In Europe, MBL-producing isolates were most numerous among those collected in Greece (n = 55), Russia (n = 38), Italy (n = 36), Turkey (n = 29), and Spain (n = 21). KPC was identified in 387 Enterobacterales isolates, including four isolates co-carrying VIM and one isolate cocarrying NDM. Proportionally, KPC producers were more commonly identified in meropenem-nonsusceptible North American Enterobacterales (58.4%; 122/209) than in isolates from Europe (33.4%; 265/794).
PER and VEB ESBLs are known to contribute to reduced cefiderocol susceptibility in A. baumannii. 19,20 Ninety-nine A. baumannii complex isolates (3.5% of all meropenem nonsusceptible A. baumannii complex isolates tested) from the collection were determined to harbor PER; 54 of the 99 isolates co-carried an OXA-24 group enzyme, 40 isolates co-carried an OXA-23 group enzyme, and five isolates did not carry a carbapenemase. Four A. baumannii complex isolates were identified carrying VEB, each also carrying an OXA-24 group enzyme. Cefiderocol was largely inactive versus against the 103 PER/VEB-positive isolates with only 15.5% of isolates testing as susceptible (MIC 90 ‡256 mg/L). The MIC 90 values for all comparators against this isolate subset were greater than the highest concentration tested, except for ceftazidime-avibactam (MIC 90 64 mg/L) and colistin (MIC 90 1 mg/L).

Discussion
Cefiderocol is noteworthy for its unique mechanism of Gram-negative bacterial cell entry. The chloro-catechol moiety of its C-3 side chain forms chelated complexes with ferric iron and promotes its transport across the outer membrane of Gram-negative bacilli using constitutive iron transport systems. 21 Once in the periplasmic space, the iron dissociates and cefiderocol binds primarily to penicillin binding protein (PBP) 3, similar to other cephalosporins, and inhibits peptidoglycan synthesis. 21 Cefiderocol is not hydrolyzed by most clinically important b-lactamases, including both serine b-lactamases (Ambler class A, class C, and class D) and MBLs, and is minimally affected by effluxmediated resistance and porin deletions (two-to four-fold increases in cefiderocol MIC). [21][22][23][24][25][26][27] We previously reported that the vast majority of Enterobacterales (99.8%), P. aeruginosa (99.9%), and A.  28 Cross-resistance between cefiderocol and other classes of antimicrobial agents has only rarely been reported. [29][30][31] Isolates of Gram-negative bacilli resistant to other antimicrobial agents are generally susceptible to cefiderocol. 11,28 Most isolates resistant to newer b-lactam/b-lactamase inhibitor combinations, including ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, and ceftolozane-tazobactam, remain susceptible to cefiderocol. 11,28 The newer b-lactam/b-lactamase inhibitor combinations were developed specifically to treat infections arising from serine carbapenemase-producing (KPC, OXA-48) Gram-negative bacilli. Unlike cefiderocol, none of the newer b-lactam/b-lactamase inhibitor combinations has activity against MBL-producing Gram-negative bacilli or carbapenem-resistant A. baumannii complex. 11,28 Among MBL-producing isolates in this study, a greater percentage of VIM carriers (96.5%; MIC 90 , 4 mg/L) than NDM carriers (85.4%; MIC 90 , 8 mg/L) were cefiderocol susceptible. A similar observation has been reported by others. 10,23,25,27,32 However, NDM production alone is likely not sufficient to cause cefiderocol resistance as elevated cefiderocol MICs in NDM producers were observed even in the presence of dipicolinic acid, an MBL inhibitor, and MICs were substantially reduced only when dipicolinic acid was used in combination with avibactam. 32 Many isolates of NDMproducing Enterobacterales demonstrate cefiderocol MICs of £4 mg/L, 10,23,25,32 and infections caused by NDM-producing Enterobacterales have been successfully treated with cefiderocol. 33 In some isolates, simultaneous production of MBLs (NDM) and serine b-lactamases may lead to cefiderocol resistance that can be reversed in vitro by the addition of blactamase inhibitors, suggesting that resistance is likely mediated by combination of resistance mechanisms. 10 In the current study, meropenem-nonsusceptible P. aeruginosa were highly susceptible to cefiderocol; 100% of MBL-producing, 100% of GES carbapenemase-producing, and 99.8% of carbapenemase-negative isolates were cefiderocol susceptible. Although it is unsurprising that newer approved b-lactam/b-lactamase-inhibitor combinations like ceftazidime-avibactam and ceftolozane-tazobactam were not active against MBL-carrying P. aeruginosa, the MIC 50 value of >8 mg/L observed for aztreonam-avibactam suggests that it would also not be an effective therapeutic agent for this pathogen.
Among meropenem-nonsusceptible A. baumannii complex isolates, 60.0% of MBL-producing, 95.6% of OXA-23 group-producing, 89.5% of OXA-24 group-producing, 100% of OXA-58 group-producing, and 95.5% of carbapenemase-negative isolates were cefiderocol susceptible. Our report confirms previously published results. 10 The b-lactamase inhibitor sulbactam has been shown to potentiate b-lactam antimicrobials against A. baumannii. 34 Although ampicillin-sulbactam was tested on a limited number of organisms in this study solely from 2019 (n = 538), it exhibited poor in vitro activity against these meropenem nonsusceptible A. baumannii complex isolates as only 16.9% of this population was susceptible (MIC 90 , 64 mg/L).
PER and VEB ESBLs have been reported to contribute to reduced susceptibility in A. baumannii 10,19,20 ; in the current study, 76 of 103 (73.8%) isolates of A. baumannii complex isolates that harbored PER or VEB were cefiderocol resistant and 11 (10.7%) were cefiderocol intermediate (cefiderocol MIC 90 , ‡256 mg/L). PER and VEB ESBLs were not identified in MBL-producing A. baumannii complex. Cefiderocol resistance due to PER in A. baumannii complex can be reversed by avibactam. 10 Of interest, the eight P. aeruginosa isolates carrying PER, as well as the five carrying VEB, were all susceptible to cefiderocol, as were the two K. pneumoniae isolates identified carrying VEB.
Our study has three important limitations. First, we only investigated b-lactamases and no other potential mechanisms of resistance in meropenem-nonsusceptible isolates. However, current data suggest that efflux pump hyperexpression and porin channel mutations/loss, that can be responsible for carbapenem resistance, do not significantly influence the in vitro activity of cefiderocol. 21 Second, evaluation of the mechanism(s) potentially responsible for the few cefiderocol-resistant, meropenem-nonsusceptible isolates identified was beyond the scope of this report. Other investigators have reported a limited number of isolates with increased cefiderocol MICs due to mutations (expression/ function) in outer membrane TonB-dependent siderophore receptors (CirA and Fiu in Enterobacterales; PiuA and PiuD in P. aeruginosa; PirA and PiuA in A. baumannii) and siderophore expression-related genes (FecIRA operon), TonB-dependent iron transporter system energy generation component mutants/loss (TonB-ExbB-ExbD), PBP mutations, AmpC mutations (R2 loop mutations also associated with cross-resistance to ceftazidime-avibactam and ceftolozane-tazobactam), KPC variants, SHV-12, OXA-427, and the development of heteroresistance (primarily in A. baumannii complex isolates) on monotherapy in carbapenem-resistant isolates. 2,31,[35][36][37][38] A thorough review of cefiderocol resistance mechanisms was recently published. 39 Third, we did not collect patient treatment or outcome information and do not know whether cefiderocol is in use in the hospitals that submitted isolates to SIDERO-WT. Given that cefiderocol was approved in the United States in November 2019 and in Europe in September 2020, this is unlikely.
We conclude that, based upon in vitro susceptibility testing data, cefiderocol may benefit the treatment of patients infected with carbapenem-nonsusceptible isolates of Enterobacterales, P. aeruginosa, and A. baumannii complex. In the current study, cefiderocol was highly active against Gram-negative isolates carrying MBLs and serine carbapenemases. The newer b-lactam/b-lactamase inhibitor combinations were inactive against isolates carrying serine carbapenemases (ceftolozane-tazobactam only), MBLs (ceftazidime-avibactam and ceftolozane-tazobactam), and A. baumannii complex (ceftazidime-avibactam and ceftolozane-tazobactam). Cefiderocol was also more active than ceftazidime-avibactam and ceftolozane-tazobactam against carbapenemase-negative, meropenem-nonsusceptible isolates of Enterobacterales and P. aeruginosa. Given that cefiderocol was only approved in both the United States and European countries in 2020, continued global surveillance will be important as clinical use of cefiderocol increases.