Conjugation inhibitors effectively prevent plasmid transmission in natural environments

Plasmid conjugation is a major route for the spread of antibiotic resistance genes. Inhibiting conjugation has been proposed as a feasible strategy to stop or delay the propagation of antibiotic resistance genes. Several compounds have been shown to be conjugation inhibitors in vitro, specifically targeting the plasmid horizontal transfer machinery. However, the in vivo efficiency and the applicability of these compounds to clinical and environmental settings remained untested. Here we show that the synthetic fatty acid 2-hexadecynoic acid (2-HDA), when used as a fish food supplement, lowers the conjugation frequency of model plasmids up to 10-fold in controlled water microcosms. When added to the food for mice, 2-HDA diminished the conjugation efficiency 50-fold in controlled plasmid transfer assays carried out in the mouse gut. These results demonstrate the in vivo efficiency of conjugation inhibitors, paving the way for their potential application in clinical and environmental settings. IMPORTANCE The spread of antibiotic resistance is considered one of the major threats for global health in the immediate future. A key reason for the speed at which antibiotic resistance spread is the ability of bacteria to share genes with each other. Antibiotic resistance genes harbored in plasmids can be easily transferred to commensal and pathogenic bacteria through a process known as bacterial conjugation. Blocking conjugation is thus a potentially useful strategy to curtail the propagation of antibiotic resistance. Conjugation inhibitors (COINS) are a series of compounds that block conjugation in vitro. Here we show that COINS efficiently block plasmid transmission in two controlled natural environments, water microcosms and the mouse gut. These observations indicate that COIN therapy can be used to prevent the spread of antibiotic resistance.Acknowledgments: The work performed by the de la Cruz research group was supported by the European Union Seventh Framework Program (FP7-HEALTH-2011-single-stage) “Evolution and Transfer of Antibiotic Resistance” (EvoTAR), grant agreement number 282004. The work performed by C.P.-G. and M.G. was supported by Ph.D. fellowships funded by the University of Cantabria. The work performed by the B.G.-Z. laboratory was supported by The EFFORT project (www.effort-against-amr.eu) FP7-KBBE-2013-7, grant agreement 613754

Conjugation inhibitors compete with palmitic acid for binding to the conjugative traffic ATPaseTrwD, providing a mechanism to inhibit bacterial conjugation

Bacterial conjugation is a key mechanism by which bacteria acquire antibiotic resistance. Therefore, conjugation inhibitors (COINs) are promising compounds in the fight against the spread of antibiotic resistance genes among bacteria. Unsaturated fatty acids (uFAs) and alkynoic fatty acid derivatives, such as 2-hexadecanoic acid (2-HDA), have been reported previously as being effective COINs. The traffic ATPase TrwD, a VirB11 homolog in plasmid R388, is the molecular target of these compounds, which likely affect binding of TrwD to bacterial membranes. In this work, we demonstrate that COINs are abundantly incorporated into Escherichia coli membranes, replacing palmitic acid as the major component of the membrane. We also show that TrwD binds palmitic acid, thus facilitating its interaction with the membrane. Our findings also suggest that COINs bind TrwD at a site that is otherwise occupied by palmitic acid. Accordingly, molecular docking predictions with palmitic acid indicated that it shares the same binding site as uFAs and 2-HDA, although it differs in the contacts involved in this interaction. We also identified 2-bromopalmitic acid, a palmitate analog that inhibits many membrane-associated enzymes, as a compound that effectively reduces TrwD ATPase activity and bacterial conjugation. Moreover, we demonstrate that 2-bromopalmitic and palmitic acids both compete for the same binding site in TrwD. Altogether, these detailed findings open up a new avenue in the search for effective synthetic inhibitors of bacterial conjugation, which may be pivotal for combating multidrug-resistant bacteria.This work was supported by Spanish Ministerio de Economia y Competitividad (MINECO) Grants BFU2016-78521-R (to E. C. and I. A.) and BFU2014-55534 (to F. d. l. C.) and by Grant P20GM103475-16 from the National Center for Research Resources and NIGMS, National Institutes of Health (to D. S. R.). The authors declare that they have no conflicts of interest withthe contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

A search for new mechanisms to inhibit plasmid conjugation

Infections due to antibiotic-resistant (AbR) bacteria are a major cause of morbidity and mortality throughout the world. In addition, the number of new antibiotics being developed has plummeted. Although resistance genes can disseminate by any horizontal gene transfer mechanism, the vast majority of reports of bacterial gene transfer in the environment involve conjugation. Our group developed a method for high-throughput analysis of conjugation. This method was used to check for host genes in the recipient cell involved in conjugation, concluding that lab strains of Escherichia coli like DH5[alfa] have no non-essential genes that play an essential role in conjugation. In recipient cells, conjugation can be inhibited by different mechanisms: restriction systems, CRISPRs, entry exclusion systems or incompatibility, among others. Our aim is to screen a set of natural plasmids to look for conjugation broad-range inhibitor genes in enterobacterial plasmids, in order to hopefully identify new mechanisms of inhibition that could be used to control AbR propagation.Máster U. en Biología Molecular y Biomedicin

Control of bacterial conjugation

RESUMEN: La conjugación bacteriana es el principal mecanismo de diseminación de resistencias a antibióticos que ha sido objeto de control en este trabajo. Se han descubierto funciones bacterianas relacionadas con la síntesis de ATP y la composición de la membrana, cuya mutación en el donador inhibe la conjugación. Se encontraron mecanismos que previenen la entrada de plásmidos en enterobacterias clínicas, como los sistemas de restricción o la producción de bacteriocinas. Los plásmidos conjugativos poseen sistemas de inhibición de la fertilidad capaces de impedir la conjugación de plásmidos co-residentes, como los descubiertos en los plásmidos pOX38 y R64. También se han hallado nuevos grupos de inhibidores de la conjugación, los ácidos grasos 2-alquinoicos y los ácidos tanzawaicos, que principalmente inhiben la transferencia de plásmidos IncW, IncF y aquellos movilizados por ellos. El control conjugativo que producen se ha demostrado suficiente para que el plásmido multi-resistente R1drd19 sea desplazado de una población bacteriana.ABSTRACT: Bacterial conjugation is the main mechanism involved in antibiotic resistance spread that has been under control in this work. Bacterial functions related to ATP synthesis and membrane composition were discovered, whose mutation in donors inhibits conjugation. Mechanisms to prevent the entry of plasmids in clinical enterobacteria were found, such as restriction systems or bacteriocin production. Conjugative plasmids display fertility inhibition systems that block conjugation of co-resident plasmids, such as those found in plasmids pOX38 and R64. Novel groups of conjugation inhibitors were also found, 2-alkynoic fatty acids and tanzawaic acids, which mainly inhibit transfer of plasmids IncW, IncF, and those mobilized by them. Conjugation control produced by conjugation inhibitors was proven sufficient for multi-resistant plasmid R1drd19 to be displaced from a bacterial population

Prevención del consumo de alcohol en la población escolar

Incluye memoria de actividades de formación y el seguimiento del proyecto por parte del CPR, los materiales realizados, encuestas y hojas de evaluación.El proyecto surge al detectar consumo de alcohol en la población escolar. Los objetivos son aportar la base teórica necesaria para comprender las conductas de los chicos y chicas en relación al consumo de alcohol; analizar modelos teóricos de intervención preventiva; proporcionar los recursos necesarios para enfrentarse a situaciones propicias al consumo de alcohol; promover hábitos de salud y utilización positiva del tiempo de ocio; retrasar la edad de inicio en el consumo de alcohol; y prevenir factores de riesgo. Mediante encuestas se analiza la situación para, en pequeños grupos, elaborar unidades didácticas. Se realizan juegos de afirmación, de resolución de conflictos y de autorregulación; se ilustran cuentos; se elaboran murales con los efectos del alcohol y el tabaco; y se analiza la publicidad. También se organizan ponencias para los padres. Se evalúa la organización del equipo, los recursos, el papel de las instituciones y la participación..Madrid (Comunidad Autónoma). Consejería de SanidadMadridNo disponibleES