Xpert carba r

Clinical isolates were identified by MALDI-TOF MS assay (Bruker Daltonics, Germany) and antimicrobial susceptibility testing (AST) was performed using the MicroScan Walkaway system.^{13 (link)} MIC results for novel βL-βLICs were confirmed by test strip on regular non-supplemented Mueller–Hinton agar (Liofilchem, Italy) and for cefiderocol using the microdilution reference method utilizing ID-CAMHB.^{14 (link)} MICs were interpreted following EUCAST clinical breakpoints v12.0 (https://www.eucast.org/clinical_breakpoints/). Carbapenemase production was detected by the NG-Test CARBA 5 (NG Biotech, France) and confirmed with a molecular assay (Xpert Carba-R, Cepheid, USA).

Clinical CRKP isolates were worked up in the UCLA Clinical Microbiology Laboratory, from 4 patients admitted to UCLA Medical Center, and 2 patients from outside hospitals. The specimens included blood, hematoma, penile, peritoneal cavity fluid, expectorated sputum, tissue (abdominal and parotid), and urine. Table 1 provides a summary of the demographics and clinical presentation of the cohort. Routine identification (culture and MALDI-TOF) and broth microdilution-based susceptibility testing identified CRKP, and Xpert Carba-R (Cepheid, Sunnyvale, CA, USA) detected the co-production of NDM-1 and OXA-48-like carbapenemase.

The growth of K. pneumoniae as green colonies on a chromogenic medium (ChromIDCarba, bioMérieux, France) indicated the strains’ ability to produce carbapenemases. The presence of genes encoding carbapenemases was confirmed using Real-Time PCR XpertCarbaR (Cepheid, Solna, Sweden).

Various clinical samples (including urine, blood, peritoneal fluid, wounds, tissue, and endotracheal tube sample) were collected from patients hospitalized at the medical center and sent to the laboratory. As part of the routine practice of the microbiology laboratory, each sample was inoculated on several media. Following incubation, suspected colonies were identified using the MALDI-TOF technology (Bruker Daltonics, Bremen, Germany) and then antibiotic susceptibility testing was performed according to the bacterial and infection type, using the disk diffusion method (Kirby Bauer) in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2014–2017 guidelines. An isolate belonging to the Enterobacterales family and with a decreased susceptibility to carbapenems, was tested with the β CARBA kit (Bio-Rad Laboratories Ltd., Rishon Lezion, Israel) and the Xpert® Carba-R (Cepheid, Sunnyvale, CA, USA), as described in the previous section.

Carbapenem-resistant Enterobacterales and P. aeruginosa from clinical cultures were forwarded to the CDC and the BPHL for carbapenem resistance mechanism testing. Colonization screenings were conducted by testing rectal swabs for carbapenemase genes using the Cepheid Xpert CarbaR (Cepheid, Sunnyvale, CA).¹⁶
when carbapenemase genes were detected, a swab was cultured to recover carbapenem-resistant organisms (Supplementary File 1 online).

Presence of CRE and associated MICs were identified using the Vitek^®2 Microbial Identification System (bioMérieux) in the University of Florida (UF) Health Shands Hospital Clinical Microbiology Laboratory. MICs were subsequently confirmed via Etest (bioMérieux). Organisms were further analysed via the Xpert^® Carba-R (Cepheid) to detect the presence of carbapenemase genes.¹⁶ As part of standard daily hospital infection control activities, all Gram-negative bacterial isolates found by the hospital microbiology laboratory to have resistance to carbapenem were identified through an automated infection control/laboratory surveillance system, and basic data on patient location and movement during hospitalization were collected. This resulted in identification of 11 NDM-carrying strains across a 1 year time period; sequence data were obtained for all 11 strains, as described below. A subset of patients infected with NDM strains were enrolled in an institutional review board-approved study at the University of Florida that permitted collection of additional clinical and epidemiological data from patients infected with antimicrobial-resistant bacterial strains. Informed consent was not required for the use of de-identified samples.