The LAMP-CRISPR-Cas13a technique for detecting the CBASS mechanism of phage resistance in bacteria
IntroductionAntimicrobial 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...
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Frontiers Media S.A.
2025-03-01
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| author | Concha Ortiz-Cartagena Concha Ortiz-Cartagena Patricia Fernández-Grela Patricia Fernández-Grela Lucia Armán Lucia Armán Lucía Blasco Lucía Blasco Daniel Pablo-Marcos Daniel Pablo-Marcos Inés Bleriot Inés Bleriot Laura Fernández-García Laura Fernández-García Clara Ibarguren-Quiles Clara Ibarguren-Quiles Felipe Fernández-Cuenca Felipe Fernández-Cuenca Felipe Fernández-Cuenca Antonio Barrio-Pujante Antonio Barrio-Pujante Belén Aracil Belén Aracil Belén Aracil Jorge Calvo-Montes Jorge Calvo-Montes María Tomás María Tomás María Tomás |
| author_facet | Concha Ortiz-Cartagena Concha Ortiz-Cartagena Patricia Fernández-Grela Patricia Fernández-Grela Lucia Armán Lucia Armán Lucía Blasco Lucía Blasco Daniel Pablo-Marcos Daniel Pablo-Marcos Inés Bleriot Inés Bleriot Laura Fernández-García Laura Fernández-García Clara Ibarguren-Quiles Clara Ibarguren-Quiles Felipe Fernández-Cuenca Felipe Fernández-Cuenca Felipe Fernández-Cuenca Antonio Barrio-Pujante Antonio Barrio-Pujante Belén Aracil Belén Aracil Belén Aracil Jorge Calvo-Montes Jorge Calvo-Montes María Tomás María Tomás María Tomás |
| author_sort | Concha Ortiz-Cartagena |
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| description | IntroductionAntimicrobial 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. Understanding these resistance mechanisms is essential for optimizing phage therapy.MethodsThis study analyzed 48 Klebsiella pneumoniae strains to identify pathogenicity islands (PAIs) containing anti-phage defense (APD) proteins. We constructed a knockout strain lacking the cyclase gene from the type II CBASS defense 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.ResultsA total of 309 pathogenicity islands (PAIs), containing 22.1% of anti-phage defense (APD) proteins, were identified. 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. Alluding to these defense mechanisms, the QS revealed to be involved in the regulation of the type II CBASS systems contained in PAIs. Finally, the LAMP-CRISPR-Cas13a technology successfully detected the main cyclases habored in type I and II CBASS systems, respectively.DiscussionThe study findings highlight the regulatory role of the QS network in APD systems. Notably, this is the first study to develop an innovative biotechnological application for the LAMP-CRISPR-Cas13a rapid-technique (<2 h), thereby helping to optimize phage therapy by detecting bacterial resistance mechanisms and predicting the potential inefficacy of therapeutic phages and thus improving patient prognosis. |
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| issn | 1664-302X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj-art-84abbce32ebe4cf2b93991dbc474d7ac2025-08-20T02:40:43ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-03-011610.3389/fmicb.2025.15505341550534The LAMP-CRISPR-Cas13a technique for detecting the CBASS mechanism of phage resistance in bacteriaConcha Ortiz-Cartagena0Concha Ortiz-Cartagena1Patricia Fernández-Grela2Patricia Fernández-Grela3Lucia Armán4Lucia Armán5Lucía Blasco6Lucía Blasco7Daniel Pablo-Marcos8Daniel Pablo-Marcos9Inés Bleriot10Inés Bleriot11Laura Fernández-García12Laura Fernández-García13Clara Ibarguren-Quiles14Clara Ibarguren-Quiles15Felipe Fernández-Cuenca16Felipe Fernández-Cuenca17Felipe Fernández-Cuenca18Antonio Barrio-Pujante19Antonio Barrio-Pujante20Belén Aracil21Belén Aracil22Belén Aracil23Jorge Calvo-Montes24Jorge Calvo-Montes25María Tomás26María Tomás27María Tomás28Departamento de Microbiología-Hospital A Coruña (HUAC), Grupo de Microbiología Traslacional y Multidisciplinar (Micro-TM), A Coruña, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainDepartamento de Microbiología-Hospital A Coruña (HUAC), Grupo de Microbiología Traslacional y Multidisciplinar (Micro-TM), A Coruña, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainDepartamento de Microbiología-Hospital A Coruña (HUAC), Grupo de Microbiología Traslacional y Multidisciplinar (Micro-TM), A Coruña, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainDepartamento de Microbiología-Hospital A Coruña (HUAC), Grupo de Microbiología Traslacional y Multidisciplinar (Micro-TM), A Coruña, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainMicrobiology Service, University Hospital Marqués de Valdecilla—IDIVAL, Santander, SpainDepartamento de Microbiología-Hospital A Coruña (HUAC), Grupo de Microbiología Traslacional y Multidisciplinar (Micro-TM), A Coruña, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainDepartamento de Microbiología-Hospital A Coruña (HUAC), Grupo de Microbiología Traslacional y Multidisciplinar (Micro-TM), A Coruña, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainDepartamento de Microbiología-Hospital A Coruña (HUAC), Grupo de Microbiología Traslacional y Multidisciplinar (Micro-TM), A Coruña, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainMicrobiology Service, University Hospital Virgen Macarena-IBIS, Seville, SpainCIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), Madrid, SpainDepartamento de Microbiología-Hospital A Coruña (HUAC), Grupo de Microbiología Traslacional y Multidisciplinar (Micro-TM), A Coruña, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainCIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), Madrid, SpainLaboratorio de Referencia e Investigación de Resistencias Antibióticas e Infecciones Sanitarias, Centro Nacional de Microbiología, ISCIII, Majadahonda, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainMicrobiology Service, University Hospital Marqués de Valdecilla—IDIVAL, Santander, SpainDepartamento de Microbiología-Hospital A Coruña (HUAC), Grupo de Microbiología Traslacional y Multidisciplinar (Micro-TM), A Coruña, SpainGrupo de Estudio de Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) en nombre de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Madrid, SpainMEPRAM, Proyecto de Medicina de Precisión contra las resistencias Antimicrobianas, ISCIII, Majadahonda, SpainIntroductionAntimicrobial 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. Understanding these resistance mechanisms is essential for optimizing phage therapy.MethodsThis study analyzed 48 Klebsiella pneumoniae strains to identify pathogenicity islands (PAIs) containing anti-phage defense (APD) proteins. We constructed a knockout strain lacking the cyclase gene from the type II CBASS defense 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.ResultsA total of 309 pathogenicity islands (PAIs), containing 22.1% of anti-phage defense (APD) proteins, were identified. 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. Alluding to these defense mechanisms, the QS revealed to be involved in the regulation of the type II CBASS systems contained in PAIs. Finally, the LAMP-CRISPR-Cas13a technology successfully detected the main cyclases habored in type I and II CBASS systems, respectively.DiscussionThe study findings highlight the regulatory role of the QS network in APD systems. Notably, this is the first study to develop an innovative biotechnological application for the LAMP-CRISPR-Cas13a rapid-technique (<2 h), thereby helping to optimize phage therapy by detecting bacterial resistance mechanisms and predicting the potential inefficacy of therapeutic phages and thus improving patient prognosis.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1550534/fullphage resistanceCBASS systemsCRISPRCas13aPurification-free |
| spellingShingle | Concha Ortiz-Cartagena Concha Ortiz-Cartagena Patricia Fernández-Grela Patricia Fernández-Grela Lucia Armán Lucia Armán Lucía Blasco Lucía Blasco Daniel Pablo-Marcos Daniel Pablo-Marcos Inés Bleriot Inés Bleriot Laura Fernández-García Laura Fernández-García Clara Ibarguren-Quiles Clara Ibarguren-Quiles Felipe Fernández-Cuenca Felipe Fernández-Cuenca Felipe Fernández-Cuenca Antonio Barrio-Pujante Antonio Barrio-Pujante Belén Aracil Belén Aracil Belén Aracil Jorge Calvo-Montes Jorge Calvo-Montes María Tomás María Tomás María Tomás The LAMP-CRISPR-Cas13a technique for detecting the CBASS mechanism of phage resistance in bacteria Frontiers in Microbiology phage resistance CBASS systems CRISPR Cas13a Purification-free |
| title | The LAMP-CRISPR-Cas13a technique for detecting the CBASS mechanism of phage resistance in bacteria |
| title_full | The LAMP-CRISPR-Cas13a technique for detecting the CBASS mechanism of phage resistance in bacteria |
| title_fullStr | The LAMP-CRISPR-Cas13a technique for detecting the CBASS mechanism of phage resistance in bacteria |
| title_full_unstemmed | The LAMP-CRISPR-Cas13a technique for detecting the CBASS mechanism of phage resistance in bacteria |
| title_short | The LAMP-CRISPR-Cas13a technique for detecting the CBASS mechanism of phage resistance in bacteria |
| title_sort | lamp crispr cas13a technique for detecting the cbass mechanism of phage resistance in bacteria |
| topic | phage resistance CBASS systems CRISPR Cas13a Purification-free |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1550534/full |
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