Molecular Characterization of WCK 5222 (Cefepime/Zidebactam)-Resistant Mutants Developed from a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate

ABSTRACT WCK 5222 (cefepime/zidebactam) is a β-lactam/β-lactamase inhibitor combination that is effective against a broad range of highly drug-resistant bacterial pathogens, including those producing metallo-β-lactamase. In this study, we isolated a multidrug-resistant Pseudomonas aeruginosa clinical strain that is resistant to a variety of β-lactam antibiotics and the ceftazidime-avibactam combination. A metallo-β-lactamase gene blaDIM-2 was identified on a self-transmissible megaplasmid in the strain, which confers the resistance to β-lactam antibiotics, leaving WCK 5222 potentially one of the last treatment resorts. In vitro passaging assay combined with whole-genome sequencing revealed mutations in the pbpA gene (encoding the zidebactam target protein PBP2) in the evolved resistant mutants. Among the mutations, a V516M mutation increased the bacterial virulence in a murine acute pneumonia model. Reconstitution of the mutations in the reference strain PAO1 verified their roles in the resistance to zidebactam and revealed their influences on cell morphology in the absence and presence of zidebactam. Microscale thermophoresis (MST) assays demonstrated that the mutations reduced the affinity between PBP2 and zidebactam to various extents. Overall, our results revealed that mutations in the pbpA gene might be a major cause of evolved resistance to WCK 5222 in clinical settings. IMPORTANCE Antibiotic resistance imposes a severe threat on human health. WCK 5222 is a β-lactam/β-lactamase inhibitor combination that is composed of cefepime and zidebactam. It is one of the few antibiotics in clinical trials that are effective against multidrug-resistant Pseudomonas aeruginosa, including those producing metallo-β-lactamases. Understanding the mechanisms and development of bacterial resistance to WCK 5222 may provide clues for the development of strategies to suppress resistant evolvement. In this study, we performed an in vitro passaging assay by using a multidrug-resistant P. aeruginosa clinical isolate. Our results revealed that mutations in the zidebactam target protein PBP2 play a major role in the bacterial resistance to WCK 5222. We further demonstrated that the mutations reduced the affinities between PBP2 and zidebactam and resulted in functional resistance of PBP2 to zidebactam.

1. I found that the authors did not present more discussion about the results that mutations of PBP2 caused different effect on bacterial growth as well as competition index (CI) in competition assay, what they thought might be the physiological significance.

A number of grammatical errors need corrected (a few examples below):
Line 20: bacteria-> bacterial Line 22 and 86: resistance-> resistant Line 78:mutation-> mutations Line 98：and-> to Line 195：of that of-> of Reviewer #2 (Comments for the Author): The authors have addressed previous comments.
Staff Comments:

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Review comments
In this manuscript, Pan et.al., isolated a multidrug resistant P. aeruginosa clinical strain called NKPa-71, which is resistant to a variety of β-lactam antibiotics and ceftazidime-avibactam combination. A self-transmissible megaplasmid was identified in this strain, which harbored a metallo-β-lactamase gene blaDIM-2, confered this strain resistance to β-lactam antibiotics, except WCK 5222. By in vitro passaging assay and whole genome sequencing the authors characterized substantial mutations in zidebactam target protein PBP2 encoding gene pbpA, and the authors verified the the roles of these mutations in reference strain PAO1, revealed that mutations in the pbpA gene might be a major cause of evolved resistance to WCK 5222 in clinical settings. The study is well designed and conducted, with a large number of solid experiments. The authors uncovered significant information about the underlying resistance mechanisms.

Minor Comments
1. I found that the authors did not present more discussion about the results that mutations of PBP2 caused different effect on bacterial growth as well as competition index (CI) in competition assay, what they thought might be the physiological significance. Reviewer #1 (Comments for the Author):

A number of grammatical errors need corrected (a few examples below
In this manuscript, Pan et.al., isolated a multidrug resistant P. aeruginosa clinical strain called NKPa-71, which is resistant to a variety of β-lactam antibiotics and ceftazidime-avibactam combination. A self-transmissible megaplasmid was identified in this strain, which harbored a metallo-β-lactamase gene blaDIM-2, confered this strain resistance to β-lactam antibiotics, except WCK 5222. By in vitro passaging assay and whole genome sequencing the authors characterized substantial mutations in zidebactam target protein PBP2 encoding gene pbpA, and the authors verified the the roles of these mutations in reference strain PAO1, revealed that mutations in the pbpA gene might be a major cause of evolved resistance to WCK 5222 in clinical settings. The study is well designed and conducted, with a large number of solid experiments. The authors uncovered significant information about the underlying resistance mechanisms. Minor Comments 1. I found that the authors did not present more discussion about the results that mutations of PBP2 caused different effect on bacterial growth as well as competition index (CI) in competition assay, what they thought might be the physiological significance.
Response: We added the discussion about the effects of the PBP2 mutations on bacterial growth and competition index (lines 232-239).

A number of grammatical errors need corrected (a few examples below):
Line 20: bacteria-> bacterial Line 22 and 86: resistance-> resistant Line 78: mutation-> mutations Line 98: and-> to Line 195: of that of-> of Response: The errors have been corrected. We also double checked the whole manuscript to correct grammatical errors.
Reviewer #2 (Comments for the Author): 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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