Acinetobacter baumannii and Cefiderocol, between Cidality and Adaptability

ABSTRACT Among the bacterial species included in the ESKAPE group, Acinetobacter baumannii is of great interest due to its intrinsic and acquired resistance to many antibiotics and its ability to infect different body regions. Cefiderocol (FDC) is a novel cephalosporin that is active against Gram-negative bacteria, with promising efficacy for A. baumannii infections, but some studies have reported therapeutic failures even in the presence of susceptible strains. This study aims to investigate the interactions between FDC and 10 A. baumannii strains with different susceptibilities to this drug. We confirmed diverse susceptibility profiles, with resistance values close to the EUCAST-proposed breakpoints. The minimal bactericidal concentration (MBC)/MIC ratios demonstrated bactericidal activity of the drug, with ratio values of ≤4 for all of the strains except ATCC 19606; however, bacterial regrowth was evident after exposure to FDC, as were changes in the shapes of colonies and bacterial cells. A switch to a nonsusceptible phenotype in the presence of high FDC concentrations was found in 1 strain as an adaptation mechanism implemented to overcome the cidal activity of this antibiotic, which was confirmed by the presence of heteroresistant, unstable subpopulations in 8/10 samples. Genomic analyses revealed the presence of mutations in penicillin-binding protein 3 (PBP3) and TonB3 that were shared by all of the strains regardless of their resistance phenotype. Because our isolates harbored β-lactamase genes, β-lactamase inhibitors showed the ability to restore the antimicrobial activity of FDC despite the different nonsusceptibility levels of the tested strains. These in vitro results support the concept of using combination therapy to eliminate drug-adapted subpopulations and regain full FDC activity in this difficult-to-treat species. IMPORTANCE This work demonstrates the underrated presence of Acinetobacter baumannii heteroresistant subpopulations after exposure of A. baumannii strains to FDC and its instability. Both A. baumannii and FDC are of great interest for the scientific community, as well as for clinicians; the former represents a major threat to public health due to its resistance to antibiotics, with related costs of prolonged hospitalization, and the latter is a novel, promising cephalosporin currently under the magnifying glass.

reads with difficulty; please indicate the meaning of boxed values in footnotes. Moreover, it is impressive to see that many strains yield colonies on 32 mg/L FDC but no colonies (zero value) on much lower concentrations of the drug (2-16 mg/L). Unless I am missing some relevant aspects of the experiment, this result deserves more in depth discussion and better clarification along the text.
L. 284-5: It is reported that SAM and AMP strips did not show any difference in plates with or without FDC, however Fig. 3 shows only a plate with FDC 1 mg/L and a clear inhibition zone in the e-test with SAM. Authors conclude that SAM has a poor activity in restoring the efficacy of FDC in the induced isolate. This conclusion should be corroborated by illustrations of the result obtained with SAM on FDC-free plates.
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Thank you for submitting your paper to Microbiology Spectrum.
Dear Paolo Visca, we are grateful for the interest you expressed about our work and we really appreciate all your comments.
After the improvements you asked us to make to the manuscript, we now hope that the updated version of our paper will meet the standard of quality needed for the publication on Microbiology Spectrum.  used as a genetic marker of the species (also included in the MLST scheme). This resistance gene should be included in the Table. According to literature analyses, OXA66, OXA82, OXA95, OXA98, and OXA116 (harbored by our strains) are OXA51-like enzymes. Unfortunately, there is still confusion in the oxa gene nomenclature and even the presence of a single SNP results in a new oxa number. We added a sentence in the text (lines 202-203) and a reference [24], specifying that all these oxa are of the same group of OXA51. L. 202, 219 and 242: Table 1 only (I can't see sections, a, b in the revised Table 1)  impressive to see that many strains yield colonies on 32 mg/L FDC but no colonies (zero value) on much lower concentrations of the drug (2-16 mg/L). Unless I am missing some relevant aspects of the experiment, this result deserves more in depth discussion and better clarification along the text.
We added the sentence "boxed squares indicate MIC values" in the footnotes and discussed the results at lines 336-340 . This behavior was surprising for us too, but both MBC/MIC and PAP experiments showed a higher number of colonies at a concentration of cefiderocol equal to 16-32 mg/L. We can just hypothesize something similar to the paradox-effect due to the activation of regulatory circuitries activate by the drug high concentration.
L. 284-5: It is reported that SAM and AMP strips did not show any difference in plates with or without FDC, however Fig. 3 shows only a plate with FDC 1 mg/L and a clear inhibition zone in the e-test with SAM.
Authors conclude that SAM has a poor activity in restoring the efficacy of FDC in the induced isolate. This conclusion should be corroborated by illustrations of the result obtained with SAM on FDC-free plates. Done.
You will find all our modifications highlighted in yellow in the PDF file.
Once again, thank you for all your suggestions,

Stefania Stefani
Prof. Stefania Stefani University of Catania, Catania, Italy Catania Italy Re: Spectrum02347-22R1 (Acinetobacter baumannii and cefiderocol between cidality and adaptability) Dear Prof. Stefani, cara Stefania: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.