Evolution of ceftazidime-avibactam and cefiderocol resistance in ST131-H30R1- isolates with KPC-3 mutants and application of FTIR biotyping.

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- (KPC-ST131-) clinical isolate. Antimicrobial susceptibility testing, Fourier-transform infrared (FTIR) spectroscopy, whole-genome sequencing, and cloning experiments were performed. A KPC-49- isolate resistant to ceftazidime-avibactam (MIC > 16/4 mg/L) and susceptible to cefiderocol (MIC: 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- resistant to both ceftazidime-avibactam and cefiderocol (MIC > 16/4 mg/L, MIC: 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- high-risk clone and showed a phenotype resembling that of ESBL producers. FTIR spectroscopy managed to differentiate cefiderocol-susceptible and resistant ST131- 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 > 16/4 mg/L) and decreased carbapenem MICs (MIC ≤ 0.12 mg/L, MIC ≤ 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- 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.