MALDI-TOF MS-Based KPC Direct Detection from Patients' Positive Blood Culture Bottles, Short-Term Cultures, and Colonies at the Hospital

Abstract

Carbapenemase resistance in Enterobacterales is a global public health problem and rapid and effective methods for detecting these resistance mechanisms are needed urgently. Our aim was to evaluate the performance of a MALDI-TOF MS-based "Klebsiella pneumoniae carbapenemase" (KPC) detection protocol from patients' positive blood cultures, short-term cultures, and colonies in healthcare settings. Bacterial identification and KPC detection were achieved after protein extraction with organic solvents and target spot loading with suitable organic matrices. The confirmation of KPC production was performed using susceptibility tests and bla_KPC amplification using PCR and sequencing. The KPC direct detection (KPC peak at approximately 28.681 Da) from patients' positive blood cultures, short-term cultures, and colonies, once bacterial identification was achieved, showed an overall sensibility and specificity of 100% (CI95: [95%, 100%] and CI95: [99%, 100%], respectively). The concordance between hospital routine bacterial identification protocol and identification using this new methodology from the same extract used for KPC detection was ≥92%. This study represents the pioneering effort to directly detect KPC using MALDI-TOF MS technology, conducted on patient-derived samples obtained from hospitals for validation purposes, in a multi-resistance global context that requires concrete actions to preserve the available therapeutic options and reduce the spread of antibiotic resistance markers.

Keywords: KPC; MALDI-TOF MS; blood culture; short-term culture.

Figure 1

Control strains spectra. Recombinant strains spectra expressing (a) KPC-2 or (b) KPC-3 are shown in red and receptor strains spectra are shown in blue (E. coli TOP10) and green (E. coli TOP10+pK19).

Figure 2

Spectra obtained from (a) patients’ positive BC bottles; (b) STC; and (c) COL. KPC peaks on KPC producers’ spectra are shown in red and spectra from samples containing non-KPC-producing bacteria are shown in blue. The KPC m/z value of one spectrum is displayed as an example.

Figure 3

Box plots showing median and interquartile range 95% for spectra intensities at KPC m/z obtained from (a) BC, (b) STC, and (c) COL. Intensities for KPC m/z of spectra obtained from samples containing KPC producers and non-KPC producers are shown in red and blue boxes, respectively. KPC m/z shown value corresponds to the median calculated for every type of sample.

Figure 4

Comparison of spectra after target spot loading with (a) SA and (b) FA. KPC-producers’ spectra are shown in red (red) and non-KPC producers’ spectra are shown in blue. The KPC m/z value of one spectrum is displayed as an example.

Figure 5

Comparison of turnaround time for phenotypic KPC confirmation methodologies (black lines) and MALDI-TOF MS detection with FA-ISO extraction method (blue lines) from positive blood culture bottles, short-term cultures, and colonies.