«The LAMP-CRISPR-Cas13a technique for detecting the CBASS-mechanism of phage resistance in bacteria»

Concha Ortiz Cartagena ¹ Patricia Fernandez-Grela ¹ Lucia Arman ¹ Clara Ibarguren-Quiles ¹ Lucia Blasco ¹ Daniel Pablo-Marcos ² Ines Bleriot ¹ Laura Fernandez-Garcia ¹ Felipe Fernández Cuenca ³ Antonio Barrio Pujante ¹ BELÉN ARACIL ⁴ Maria Tomas ^1*

• ¹ INIBIC-A Coruña Hospital, A Coruña, Spain, A Coruña, Spain
• ² Marqués de Valdecilla Health Research Institute (IDIVAL), Santander, Cantabria, Spain
• ³ IBIS-Virgen Macarena Hospital, Seville, Spain
• ⁴ Centro Nacional de Microbiologia, ISCIII, Madrid, Spain

Antimicrobial resistance (AMR) is a major public health threat, driving the need for alternative treatments such as phage therapy. However, bacterial defense mechanisms, often regulated by the quorum sensing (QS) network and encoded in genomic islands (GIs), can generate phage-resistant mutants. This study analyzed 48 K. pneumoniae strains, identifying 309 pathogenicity islands (PAIs) containing 22.1% of anti-phage defense (APD) proteins. Type I and II CBASS APD systems were also detected in the genome of the 48 K. pneumoniae strains, and only two type II CBASS systems were located in PAIs. We constructed a knockout strain lacking the cyclase gene from the type II CBASS systems present in PAIs to investigate QS regulation and its role in cell viability. The LAMP-CRISPR-Cas13a technique was used to confirm gene knockout and to detect the main cyclase in type I CBASS systems, i.e. APECO1. The study findings also highlight the regulatory role of the QS network in APD systems.This is the first study to develop an innovative biotechnological application for the LAMP-CRISPR-Cas13a rapid-technique (< 2 hours), thereby helping to optimize phage therapy by detecting bacterial resistance mechanisms and predicting the potential inefficacy of therapeutic phages and thus improving patient prognosis.