Activity of Cefiderocol Against Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii Endemic to Medical Centers in New York City

Therapeutic options for the treatment of infections owing to multidrug-resistant Gram-negative pathogens are often limited. Cefiderocol is a novel siderophore cephalosporin with activity against Gram-negative pathogens, including many multidrug-resistant strains. The activity of cefiderocol was examined against Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii that included (1) a recent surveillance collection of clinical isolates, (2) a collection of carbapenem-resistant isolates from a previous surveillance study, and (3) a collection of well-characterized isolates. Susceptibility testing for cefiderocol was performed with iron-depleted cation-adjusted Mueller–Hinton broth. Cefiderocol minimum inhibitory concentrations (MICs) were correlated with resistance mechanisms in the well-characterized isolates. For the Enterobacterales, including a collection of KPC-possessing Klebsiella pneumoniae, cefiderocol MICs were all ≤4 mg/L. Cefiderocol MICs were two- to fourfold higher in cephalosporin-resistant isolates. For K. pneumoniae, MICs did not correlate with expression of genes encoding porins or efflux systems. For P. aeruginosa, >99% of isolates were inhibited by ≤4 mg/L, including the collection of carbapenem-resistant isolates. For P. aeruginosa, cefiderocol activity was not affected by expression of ampC, oprD, or several efflux systems. All the surveillance isolates of A. baumannii, and 88% of the collection of carbapenem-resistant isolates, had cefiderocol MICs ≤4 mg/L. MICs were twofold higher in A. baummannii isolates with proven extended-spectrum beta-lactamases, and cefiderocol activity did not correlate with expression of efflux systems. Cefiderocol demonstrated potent activity against important nosocomial pathogens. Continued development of this agent as a therapeutic option against multidrug-resistant bacteria should be encouraged.


Introduction
T he World Health Organization has declared antimicrobial resistance a global emergency and the development of new antimicrobials as a priority. 1 Carbapenemresistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii are considered ''critical'' pathogens. 1 The spread of multidrug-resistant Gram-negative pathogens has spurred the development of novel and potentially therapeutic agents. Next-generation aminoglycosides and b-lactamase inhibitors that have been recently brought into clinical practice are welcomed additions to our therapeutic armamentarium; however, resistance to these agents may limit their utility. For example, the diazabicyclooctanes are a new class of b-lactamase inhibitors that are active against many pathogens carrying serine blactamases. 2 However, the currently available agents are not therapeutic options for isolates possessing metallo-blactamases. Clearly, alternative approaches are needed.
Cefiderocol (previously S-649266) is novel siderophore cephalosporin with a catechol moiety at position 3 of the cephalosporin side chain. This moiety facilitates formation of chelated complexes with iron and crosses the outer membrane of Gram-negative bacilli through active iron transporters. 3 Several studies have shown potent in vitro activity of cefiderocol against Enterobacterales, P. aeruginosa, and A. baumannii, including isolates resistant to carbapenems. [4][5][6][7][8] The spectrum of activity of cefiderocol includes isolates harboring a wide range of carbapenemases, including serine (KPC, OXAtype) and metallo (VIM, IMP, NDM) b-lactamases. 7 In this report we examine the in vitro activity of cefiderocol against Enterobacterales, P. aeruginosa, and A. baumannii endemic to New York City.

Isolates
Clinical isolates of Enterobacterales, P. aeruginosa, and A. baumannii underwent susceptibility testing. Three groups of Enterobacterales were analyzed, including (1) 2558 singlepatient isolates of Escherichia coli, Enterobacter spp. (included in this group was Klebsiella aerogenes), and Klebsiella pneumoniae gathered during a 3-month surveillance study involving seven hospitals in Brooklyn, New York in 2017; (2) 111 carbapenem-resistant (and KPC-possessing) isolates of K. pneumoniae gathered from a similar surveillance study performed in 2013-2014; and (3) 34 well-characterized isolates of K. pneumoniae. [15][16][17] For the last group, the presence of b-lactamases and genetic expression of bla KPC , ompK35, ompK36, and acrB were previously determined. 17 Multilocus sequence typing was performed on select isolates of K. pneumoniae according to established protocols. 18 For P. aeruginosa and A. baumannii, a similar three groups of isolates were analyzed. These groups included (1) 269 single-patient isolates of P. aeruginosa and 46 isolates of A. baumannii collected during the 2017 surveillance study, (2) carbapenem-resistant isolates of P. aeruginosa (n = 130) and A. baumannii (n = 78) gathered during a 2013-2014 surveillance study, and (3) 33 isolates of P. aeruginosa and 34 isolates of A. baumannii that were previously characterized for mechanisms of antimicrobial resistance. [19][20][21] For P. aeruginosa, the presence of b-lactamases and genetic expression of ampC, oprD, mexA, mexC, mexE, and mexX were analyzed. 20 For the A. baumanii isolates, the presence of b-lactamases and genetic expression of ampC, oxa51, adeB, and abeM were determined, as previously described. 21

Susceptibility testing
Cefiderocol minimum inhibitory concentrations (MICs) were performed in iron-depleted cation-adjusted Mueller-Hinton broth. 22 MICs for the remaining antibiotics were performed by the agar dilution method with Mueller-Hinton agar according to established CLSI methods. 23 Susceptibility rates were determined using CLSI criteria; for cefiderocol, the provisional breakpoint of 4 mg/L was used. 24 Control strains included E. coli ATCC 25922 and 35218 and P. aeruginosa ATCC 27853.
Statistical analysis using the two-tailed Student's t-test and multiple linear regression were used to compare MICs with characterized mechanisms of resistance. A value of p < 0.05 was considered significant.
All the 2017 surveillance isolates of K. pneumoniae (n = 517) had cefiderocol MICs of £2 mg/L, including 19 isolates with bla KPC . Of the 19 bla KPC -possessing isolates, 12 belonged to ST258, two belonged to ST340, and one each belonged to ST45, ST327, ST584, ST3359, and ST3369. Compared with the ceftazidime-susceptible isolates, the ceftazidime-resistant isolates (but lacking bla KPC ) had greater mean cefiderocol MICs  Table S1). The mean cefiderocol MIC for isolates (n = 10) that did not have an extended-spectrum beta-lactamase (ESBL) or KPC b-lactamase was 0.24 -0.18 mg/L (range = 0.06-0.5 mg/L). The mean cefiderocol MIC for isolates (n = 10) that possessed only an ESBL (bla SHV ) was 1.1 mg/L -1.09 mg/L (range = 0.25-4 mg/L; p = 0.04 compared with isolates lacking an ESBL). The one isolate in this group with an MIC = 4 mg/L also possessed an AmpC-type (ACT-1) enzyme. Four isolates with bla KPC but without an ESBL had cefiderocol MICs of 0.5-1 mg/L (mean 0.875 mg/L). The remaining 10 isolates possessed both an ESBL and KPC, with a mean cefiderocol MIC of 1.07 -1.19 mg/L ( p = 0.07 compared with isolates lacking an ESBL, range = 0.06-4 mg/L). There was no correlation between cefiderocol MICs and expression of bla KPC , the efflux-related genes marA, ramA, soxS, and acrB, and the porin-related genes ompK35 or ompK36. Isolates with a frameshift mutation involving ompK35 had similar MICs as isolates without this mutation.

P. aeruginosa and A. baumannii
Pseudomonas aeruginosa. There were 269 isolates of P. aeruginosa gathered in the 2017 surveillance study (Table 2), and 99.6% had a cefiderocol MIC £4 mg/mL.  Table S2). Isolates with increased expression of ampC (>10 times control) had similar cefiderocol MICs  Acinetobacter baumannii. There were 46 isolates of A. baumannii gathered in 2017, and all had cefiderocol MICs £4 mg/L. Compared with the isolates susceptible to ceftazidime, the MIC 50 value for cefiderocol was twofold higher in the 18 ceftazidime-nonsusceptible isolates (0.25 vs. 0.12 mg/L). There were 78 carbapenem-resistant isolates gathered in 2013-2014, including 47 with bla OXA-23-type blactamases. Overall, the MIC 50 /MIC 90 values were 0.5/ 8 mg/L, which was identical to the subset of isolates with bla OXA-23-type b-lactamases.
There were 34 isolates characterized of A. baumannii (Supplementary Table S3). Isolates with an SHV ESBL had significantly higher cefiderocol MICs than isolates without ESBLs (8.04 -11.58 vs. 0.86 -1.08 mg/L, p = 0.02). By regression analysis, there was no correlation between cefiderocol MICs and expression of genes encoding ampC, bla OXA-51 , and the efflux systems adeB and abeM.

Discussion
The progressive and global spread of multidrug-resistant Gram-negative pathogens has led to increasingly difficultto-treat nosocomial infections. Given the extremely limited treatment options currently available, novel therapeutic agents are urgently needed. Cefiderocol is a novel catecholsubstituted siderophore cephalosporin with a broad spectrum of antimicrobial activity. Against Enterobacterales, MIC 90 values of *0.5-1 mg/L have been reported, with >95% being inhibited by £4 mg/L (the proposed breakpoint for cefiderocol). 4,6,9,10 Consistent with these reports, the Enterobacterales in our study had overall MIC 90 values of 0.5 mg/L, and all were inhibited by £4 mg/L. In a study by Jacobs et al., cefiderocol MICs were higher in Enterobacterales possessing an ESBL, carbapenemase, or AmpC-type b-lactamase compared with isolates lacking these enzymes. 12 Our study also documented greater cefiderocol MICs in isolates of E. coli and K. pneumoniae that were cephalosporin resistant. The activity of cefiderocol has been reported to be maintained in Enterobacterales possessing a wide variety of carbapenemases, including KPC, NDM VIM, IMP, and OXA-48. 5,9,10,12 The activity of cefiderocol was maintained in all our KPC-producing K. pneumoniae, with 90% of the isolates being inhibited by £2 mg/L, consistent with other reports. 10,12 Cefiderocol has also been reported to have considerable activity against other Gram-negative pathogens. For P. aeruginosa, including carbapenem-resistant isolates, MIC 90 values of 0.5-1 mg/L have been reported. [4][5][6][7][8]10,12 In our study, 99.6% of our surveillance isolates had cefiderocol MICs £4 mg/L. MICs did not appear to be significantly affected by the development of cephalosporin or carbapenem resistance, consistent with findings reported elsewhere for P. aeruginosa. 12 Similarly, for A. baumannii overall MIC 90 values of 1-2 mg/L, and 8 mg/L for carbapenem-resistant isolates, have been reported. [4][5][6][7][8]10,12 All our 2017 surveillance isolates, and 88% of our previous collection of carbapenem-resistant A. baumannii, were inhibited by £4 mg/L. For A. baumannii, cefiderocol MICs were unaffected by the presence of OXA-23, consistent with findings reported elsewhere. 10 However, as with the Enterobacterales, we noted cefiderocol MICs were significantly higher in ESBL-possessing isolates of A. baumannii.
The catechol moiety in cefiderocol chelates free iron, enabling entry into bacteria through their iron transport system. 25 As such, cefiderocol may evade porin-related bacterial mechanisms of antimicrobial resistance. Consistent with other reports, when our collection of characterized isolates of K. pneumoniae were examined, cefiderocol MICs were not significantly affected by alterations in the porins OmpK35 and OmpK36. 3,10 In addition, we found no correlation between cefiderocol MICs and expression of several efflux-related genes. For P. aeruginosa, overexpression of the MexAB-OprM efflux system had no effect on the activity of cefiderocol. 3 Similarly, in our collection of characterized isolates of P. aeruginosa, we found no correlation between expression of several efflux systems or in expression of the porin oprD. Finally, when tested against our collection of characterized isolates of A. baumannii, no correlation was found between cefiderocol MICs and expression of the efflux genes adeB and abeM.
Our study reaffirms the activity of cefiderocol against a large number of Gram-negative pathogens, including multidrug-resistant isolates. However, our findings may not be generalized to other multidrug-resistant pathogens, because only a limited variety of carbapenemases was identified (bla KPC in Enterobacterales and bla OXA-type in A. baumannii). Given the limited options available for many resistant nosocomial pathogens, our findings support the continued development of this agent.

Disclosure Statement
No competing financial interests exist.

Funding Information
Shionogi & Co., Ltd, Osaka, Japan provided financial support for these studies.

Supplementary Material
Supplementary Table S1  Supplementary Table S2  Supplementary Table S3