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ABSTRACTCeftazidime-avibactam and cefiderocol represent two of the few alternatives for infections by KPC-producing Enterobacterales. We reported the emergence of resistance to both ceftazidime-avibactam and cefiderocol in a KPC-producing ST131-Escherichia coli (KPC-ST131-Ec) clinical isolate. Antimicrobial susceptibility testing, Fourier-transform infrared (FTIR) spectroscopy, whole-genome sequencing, and cloning experiments were performed. A KPC-49-Ec isolate resistant to ceftazidime-avibactam (MICCZA > 16/4 mg/L) and susceptible to cefiderocol (MICFDC: 2 mg/L) was recovered in a blood sample from an oncologic patient hospitalized in the medical ICU (June 2019) during ceftazidime-avibactam treatment. After 44 days, a KPC-31-Ec resistant to both ceftazidime-avibactam and cefiderocol (MICCZA > 16/4 mg/L, MICFDC: 8 mg/L) was found in a rectal sample during a second cycle of ceftazidime-avibactam treatment. Both KPC-49 (R163S) and KPC-31 (D179Y) were detected in the epidemic ST131-H30R1-Ec high-risk clone and showed a phenotype resembling that of ESBL producers. FTIR spectroscopy managed to differentiate cefiderocol-susceptible and resistant ST131-Ec isolates, and these from others belonging to different clones. After cloning and transformation experiments, KPC-49 and KPC-31 were responsible for ceftazidime-avibactam resistance (MICCZA > 16/4 mg/L) and decreased carbapenem MICs (MICMER ≤ 0.12 mg/L, MICIMI ≤ 1 mg/L). KPC-31 was also shown to be associated with increased MICs of cefiderocol (twofold and threefold dilutions over KPC-3 and KPC-49, respectively). However, mutations in proteins participating in outer membrane stability and integrity, such as TolR, could have a more relevant role in cefiderocol resistance. The effects of ceftazidime-avibactam and cefiderocol co-resistance in clinical isolates of Enterobacterales producing KPC mutants make their identification challenging for clinical laboratories.IMPORTANCEThroughout four admissions in our hospital of a single patient, different KPC-3 variants (KPC-3, KPC-49, and KPC-31) were found in surveillance and clinical ST131-Escherichia coli isolates, after prolonged therapies with meropenem and ceftazidime-avibactam. Different patterns of resistance to cefiderocol and ceftazidime-avibactam emerged, accompanied by restored carbapenem susceptibility. The inability to detect these variants with some phenotypic methods, especially KPC-31 by immunochromatography, and the expression of a phenotype similar to that of ESBL producers, posed challenge to identify these variants in the clinical microbiology laboratory. Molecular methods and whole-genome sequencing are necessary and new techniques able to cluster or differentiate related isolates could also be helpful; this is the case of Fourier-transform infrared spectroscopy, which managed in our study to discriminate isolates by cefiderocol susceptibility within ST131, and those from the non-ST131 ones.

Min Jiang,* Bin Sun,* Yong Huang, Chengyang Liu, Yan Wang, Yanli Ren, Yuhong Zhang, Yunying Wang, Di Mu Department of Laboratory Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Di Mu; Yunying Wang, Department of Laboratory Medicine, The Second Affiliated Hospital of Chongqing Medical University, No. 74 Linjiang Road, Yuzhong District, Chongqing, People’s Republic of China, Tel/Fax +86 23-62887386, Email 303921@hospital.cqmu.edu.cn; Wangyunying@hospital.cqmu.edu.cnPurpose: The aim of this study was to understand the resistance mechanism of ceftazidime/avibactam (CZA) in carbapenem-resistant Klebsiella pneumoniae under antibiotic selection pressure.Patients and Methods: Four CZA-resistant Klebsiella pneumoniae strains were isolated from two patients, and six CZA-resistant strains that were produced in vitro were screened from 25 carbapenem-resistant Klebsiella pneumoniae strains. The mechanisms of resistance to CZA of these strains were characterized by PCR and Sanger sequencing.Results: CZA-resistant Klebsiella pneumoniae with different resistance mechanisms (including upregulation of the expression of efflux pumps and KPC variants (KPC-14, KPC-44)) were isolated from the same patient (patient 1). In patient 2, the resistance mechanism of CZA-resistant Klebsiella pneumoniae was the mutation of KPC-2 to KPC-33. In addition, among the CZA-resistant Klebsiella pneumoniae that were produced in vitro, we found 3 new KPC variants: KPC-86 (D179G), KPC-87 (GT241A) and KPC-88 (G523T).Conclusion: In this study, although the CZA-resistant bacteria originated from only two clinical patients, four different mechanisms of CZA resistance were detected. In the in vitro induction experiment, the mechanisms of resistance to CZA in strains from different patients were also different. The above result implies that the mechanisms of resistance to CZA are generally random and diverse. Therefore, elucidating the mechanism of resistance to CZA can provide a certain theoretical basis for the effective response of CZA-resistant strains and the selection of antibiotics.Keywords: ceftazidime/avibactam-resistance, KPC mutants, antibiotic pressure, Klebsiella pneumoniae, resistance mechanisms

Ceftazidime-avibactam and cefiderocol represent two of the few alternatives for infections by KPC-producing Enterobacterales. We reported the emergence of resistance to both ceftazidime-avibactam and cefiderocol in a KPC-producing ST131- Escherichia coli (KPC-ST131- Ec ) clinical isolate. Antimicrobial susceptibility testing, Fourier-transform infrared (FTIR) spectroscopy, whole-genome sequencing, and cloning experiments were performed. A KPC-49- Ec isolate resistant to ceftazidime-avibactam (MIC CZA > 16/4 mg/L) and susceptible to cefiderocol (MIC FDC : 2 mg/L) was recovered in a blood sample from an oncologic patient hospitalized in the medical ICU (June 2019) during ceftazidime-avibactam treatment. After 44 days, a KPC-31- Ec resistant to both ceftazidime-avibactam and cefiderocol (MIC CZA > 16/4 mg/L, MIC FDC : 8 mg/L) was found in a rectal sample during a second cycle of ceftazidime-avibactam treatment. Both KPC-49 (R163S) and KPC-31 (D179Y) were detected in the epidemic ST131-H30R1- Ec high-risk clone and showed a phenotype resembling that of ESBL producers. FTIR spectroscopy managed to differentiate cefiderocol-susceptible and resistant ST131- Ec isolates, and these from others belonging to different clones. After cloning and transformation experiments, KPC-49 and KPC-31 were responsible for ceftazidime-avibactam resistance (MIC CZA > 16/4 mg/L) and decreased carbapenem MICs (MIC MER ≤ 0.12 mg/L, MIC IMI ≤ 1 mg/L). KPC-31 was also shown to be associated with increased MICs of cefiderocol (twofold and threefold dilutions over KPC-3 and KPC-49, respectively). However, mutations in proteins participating in outer membrane stability and integrity, such as TolR, could have a more relevant role in cefiderocol resistance. The effects of ceftazidime-avibactam and cefiderocol co-resistance in clinical isolates of Enterobacterales producing KPC mutants make their identification challenging for clinical laboratories.IMPORTANCEThroughout four admissions in our hospital of a single patient, different KPC-3 variants (KPC-3, KPC-49, and KPC-31) were found in surveillance and clinical ST131- Escherichia coli isolates, after prolonged therapies with meropenem and ceftazidime-avibactam. Different patterns of resistance to cefiderocol and ceftazidime-avibactam emerged, accompanied by restored carbapenem susceptibility. The inability to detect these variants with some phenotypic methods, especially KPC-31 by immunochromatography, and the expression of a phenotype similar to that of ESBL producers, posed challenge to identify these variants in the clinical microbiology laboratory. Molecular methods and whole-genome sequencing are necessary and new techniques able to cluster or differentiate related isolates could also be helpful; this is the case of Fourier-transform infrared spectroscopy, which managed in our study to discriminate isolates by cefiderocol susceptibility within ST131, and those from the non-ST131 ones., Competing Interests: The authors declare no conflict of interest.