MOLECULAR GATED STRATEGY FOR RAPID DETECTION OF KLEBSIELLA PNEUMONIAE AND CARBAPENEM-RESISTANT GENES IN RESPIRATORY AND HOSPITAL-ACQUIRED INFECTIONS [COMUNICACIÓN PREMIADA]

Klebsiella pneumoniae is a major pathogen in healthcare-associated infections. This bacterium plays a critical role in severe respiratory infections such as hospital-acquired pneumonia, which can have mortality rates exceeding 50%, especially when multidrug-resistant strains are involved. K. pneumoniae increasing resistance to antibiotics, including carbapenems, makes it one of the most pressing pathogens in clinical environments. Rapid and accurate identification of K. pneumoniae is crucial for effective treatment. As current diagnostic methods based on culture and phenotypic assays can take up to 48 hours, new tools for faster diagnosis are urgently needed. This work focuses on two main objectives: developing biosensors for the detection and identification of i) K. pneumoniae and ii) resistance genes encoding carbapenemases. To achieve this, we have applied molecular gated systems consisting of nanoporous anodic alumina (NAA) films loaded with rhodamine B (RhB), a fluorescent dye and capped with specific recognition motifs. In a first example, after surface functionalization, these supports are capped with a peptide that specifically binds to K. pneumoniae lipopolysaccharide (LPS). This peptide is anchored to the materials surface by a short oligonucleotide acting as the molecular gate that blocks the pores. Upon recognition of the pathogen, the molecular gates open, releasing RhB and producing a fluorescence signal. Additionally, a separate detection system was designed to identify carbapenem-resistance genes. Therefore, the integration of both biosensors into a multiplex system would allow simultaneous identification of K. pneumoniae and its resistance associated with the selected gene. Both systems demonstrated strong responses, high sensitivity and selectivity in buffered media containing either LPS or the bla-OXA-48-like gene target. Validation in bacterial cultures further confirmed robust performance. Current efforts focus on validating both biosensors in patient-derived samples and developing a multiplexed system capable of simultaneously identifying K. pneumoniae and determining its resistance profile. These results highlight this technology's potential to detect K. pneumoniae and its resistance providing a powerful clinical tool for guiding appropriate antimicrobial therapy.