In this study, we identified specific carbapenemase-producing isolates applying an easy and rapid protocol for the detection of mature KPC-2 β-lactamase by MALDI-TOF MS from colony and positive blood culture bottles. In addition, we evaluated the correlation of the ~11,109 Da signal as a biomarker associated with KPC-2 production. A collection of 126 well-characterized clinical isolates were evaluated (including 60 KPC-2-producing strains).Presence of KPC-2 was assessed by MALDI-TOF MS on protein extracts. Samples were prepared using the double layer sinapinic acid technique. In order to identify mature KPC-2, raw spectra were analyzed focusing on the range between m/z 25,000-30,000 Da. A single distinctive peak, at approximately m/z 28,544 Da was found in all clinical and control KPC-2-producing strains, and consistently absent in the control groups (ESBL producers and susceptible strains). This peak was detected in all species independently of where the gene bla KPC-2 was embedded. Statistical results showed 100% sensitivity, CI95%: [94.0%; 100%] and 100% specificity, CI95%: [94.6%; 100%], indicating a promising test with a high discriminative power. KPC-2 β-lactamase could be directly detected from both colonies and blood culture bottles. On the other hand, the m/z 11,109 Da signal determinant was only associated with 32% of Klebsiella pneumoniae and Escherichia coli KPC positive isolates. This MALDI-TOF MS methodology has the potential to detect directly the widespread and clinically relevant carbapenemase, KPC-2, in Enterobacterales with a straightforward, low cost process, assuming MALDI-TOF MS is already adopted as the main identification tool, with clear clinical implications on antibiotic stewardship for early infection treatment.

New Delhi metallo-β-lactamase (NDM)-producing isolates are usually resistant to most β-lactams and other antibiotics as a result of the coexistence of several resistance markers, and they cause a variety of infections associated to high mortality rates. Although NDM-1 is the most prevalent one, other variants are increasing their frequency worldwide. In this study we describe the first clinical isolate of NDM-5- and RmtB-producing Escherichia coli in Latin America. E. coli (Ec265) was recovered from a urine sample of a female outpatient. Phenotypical and genotypical characterization of resistance markers and conjugation assays were performed. Genetic analysis of Ec265 was achieved by whole genome sequencing. Ec265 belonging to ST9693 (CC354), displayed resistance to most β-lactams (including carbapenems), aminoglycosides (gentamicin and amikacin), and quinolones. Several resistance genes were found, including blaNDM-5 and rmtB, located on a conjugative plasmid. blaNDM-5 genetic context is similar to others found around the world. Co-transfer of multiple antimicrobial resistance genes represents a particular challenge for treatment in clinical settings, whereas the spread of pathogens resistant to last resort antibiotics should raise an alarm in the healthcare system worldwide.

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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 blaKPC 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.