Emergence of Klebsiella pneumoniae ( Kp ) isolates carrying novel bla KPC variants conferring ceftazidime-avibactam (CAZ/AVI) resistance is being increasingly reported. We evaluated the accuracy of phenotypic methods commonly used in routine clinical laboratories in the detection of novel KPC enzymes. Additionally, we characterized by WGS the KPC-ST307- Kp isolates recovered in our hospital before and after CAZ/AVI therapy. Rectal colonization or infection by carbapenem-resistant KPC-3- Kp isolates (imipenem MIC 16 mg/L, meropenem MIC 8->16 mg/L) and CAZ/AVI-susceptible (CAZ/AVI MIC 1-2 mg/L) were first detected in three ICU patients admitted between March-2020 and July-2020. KPC- Kp isolates with increased CAZ/AVI MICs (8-32 mg/L) and carbapenem susceptibility (imipenem and meropenem MIC <1 mg/L) were recovered within 6-24 days after CAZ/AVI treatment. WGS confirmed that all KPC- Kp isolates belonged to the ST307 high-risk clone and carried identical antimicrobial resistance genes and virulence factors. The presence of the novel bla KPC-46 , bla KPC-66 and bla KPC-92 genes was confirmed in the Kp isolates with increased CAZ/AVI MICs and restored carbapenem activity. KPC production was confirmed by immunochromatography, the eazyplex®-Superbug-CRE system and the Xpert® Carba-R assay in all KPC- Kp isolates, but not in any isolate using chromogenic agar plates for carbapenemase producers (ChromID-CARBA), the KPC/MBL/OXA-48 Confirm Kit and the β-CARBA test. Nevertheless, all grew in chromogenic agar plates for ESBL producers (ChromID-ESBL). We report the failure of the most common phenotypic methods used for the detection of novel KPC carbapenemases but not of rapid molecular or immunochromatography assays thus highlighting their relevance in microbiology laboratories.

Objectives Ceftazidime/avibactam and cefiderocol are two of the latest antibiotics with activity against a wide variety of Gram-negatives, including carbapenem-resistant Enterobacterales. We sought to describe the phenotypic and genotypic characteristics of ceftazidime/avibactam- and cefiderocol-resistant KPC-Klebsiella pneumoniae (KPC-Kp) detected during an outbreak in 2020 in the medical ICU of our hospital. Methods We collected 11 KPC-Kp isolates (6 clinical; 5 surveillance samples) resistant to ceftazidime/avibactam and cefiderocol from four ICU patients (November 2020 to January 2021), without prior exposure to these agents. All patients had a decontamination regimen as part of the standard ICU infection prevention protocol. Additionally, one ceftazidime/avibactam- and cefiderocol-resistant KPC-Kp (June 2019) was retrospectively recovered. Antibiotic susceptibility was determined by broth microdilution. β-Lactamases were characterized and confirmed. WGS was also performed. Results All KPC-Kp isolates (ceftazidime/avibactam MIC  ≥16/4 mg/L; cefiderocol MIC ≥4 mg/L) were KPC + CTX-M-15 producers and belonged to the ST307 high-risk-clone (ST307-HRC). KPC-62 (L168Q) was detected in all isolates involved in the 2020 outbreak, contained in January 2021. KPC-31 (D179Y) was identified in the KPC-Kp from 2019. Cloning experiments demonstrated that both blaKPC-62 and blaKPC-31 were responsible for ceftazidime/avibactam resistance (MIC &gt;16 mg/L) and an increased cefiderocol MIC. Additionally, mutations in OmpA and EnvZ/OmpR porin proteins (in KPC-62-Kp) and in PBP2 (in KPC-31-Kp) were found and may be involved in cefiderocol resistance. Conclusions The emergence of resistance to both ceftazidime/avibactam and cefiderocol in KPC-Kp-HRCs, together with the diversification of novel KPC enzymes displaying different antibiotic resistance phenotypes, is an epidemiological and clinical risk.