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

Tanzawaic acids, a chemically novel set of bacterial conjugation inhibitors

Bacterial conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the conjugation process. A search for conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in bacterial, fungal or human cells, when compared to synthetic conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination.Work in the FDLC group was supported by grants BFU2014-55534-C2-1-P from the Spanish Ministry of Economy and Competitiveness (http://www.mineco.gob.es) and 612146/FP7-ICT-2013-10 and 282004/FP7-HEALTH-2011-2.3.1-2 from the European Seventh Framework Programme (https://ec.europa.eu/research/fp7). MG was supported by a PhD fellowship from the University of Cantabria (http://www.unican.es). JCG was supported by a postdoctoral fellowship ASTF 402-2010 from the European Molecular Biology Organization (http://www.embo.org). Biomar Microbial Technologies was supported by grant 282004/FP7-HEALTH-2011-2.3.1-2 from the European Seventh Framework Programme (https://ec.europa.eu/research/fp7).USD 1,495 APC fee funded by the EC FP7 Post-Grant Open Access PilotPeer reviewe

Measuring Plasmid Conjugation Using Fluorescent Reporters

Fluorescence-based methods are increasingly popular because they (1) offer a faster alternative to labor-intensive traditional methods, (2) enable the development of automated high-throughput screening procedures, and (3) allow direct visualization of biological processes. Here we describe three fluorescence-based methods applicable for the detection and quantitation of plasmid conjugation. The first method uses flow cytometry as a fast and reliable alternative to traditional plating methods. A second one employs fluorescence expression for high-throughput analysis of plasmid conjugation. Finally we review a third method that enables direct visualization of plasmid transfer under the microscope

Fertility inhibition between conjugative plasmids

Resumen del trabajo presentado al Plasmid Biology Meeting, celebrado en Cambridge (UK) del 18 al 23 de septiembre de 2016.Infections due to antibiotic-resistant enterobacteria are a worldwide cause of morbidity and mortality. Antibiotic resistance genes disseminate mostly by conjugative plasmids. Plasmid conjugation could be controlled by mechanisms naturally displayed by bacteria. Among them, fertility inhibition systems prevent conjugation of co-resident plasmids in donor cells. The study of interactions between conjugative systems could provide useful information to fight against antibiotic resistance dissemination. In this work, conjugation ability of IncW broad host range plasmids was analyzed in the presence of a representative set of co-resident plasmids. Two novel fertility inhibition systems against R388 conjugation were discovered in IncFI plasmid pOX38 and IncI1 plasmid R64. The precise mechanism of inhibition is being investigated. Interestingly, when R388 was substituted by a reduced synthetic version, these effects diminished two orders of magnitude, whereas the effects caused by other fertility inhibition systems encoded by IncP1α and IncX2 plasmids were maintained. This kind of knowledge could also be valuable for constructing bacterial computing devices employing conjugative plasmids as wires.Peer reviewe

Effect of conjugation inhibitors in natural environments

Resumen del trabajo presentado en el 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Santander (España) del 10 al 13 de septiembre de 2018.Bacterial conjugation is one of the main mechanisms of antibiotic resistances (AbR) spread among bacterial species1. The constant exposition of environmental bacteria to antibiotics, either added in food industry or derived from wastewater, can trigger the appearance of AbR. AbR are usually encoded in mobile genetic elements such as plasmids, which could get to animal or human pathogens, thus causing serious health problems2. Conjugation Inhibitors (COINs) are compounds that inhibit bacterial conjugation by targeting the conjugative machinery in a specific manner. 2-Hexadecynoic acid (2-HDA) is a synthetic unsaturated fatty acid (UFA) that has shown strong inhibitory activity against a number of conjugative plasmids3. Here, we show the effect of 2-HDA on conjugation in a natural microcosms. We set up an aquatic microcosm as model environment for AbR emergence and transfer, and populated it with zebrafish. We used the conjugative plasmid pOX38, a derrepressed derivative from F plasmid, contained in Escherichia coli, as subject for the experiments. Aquarium water (AqW) was not suitable for conjugation, but when amended with LB broth to simulate a polluted environment, conjugation occurred at high frequencies. In non-amended AqW, Zebrafish gut resulted as a concentrator for bacterial cells, facilitating mating, and conjugation frequency arose in comparison with results in AqW. When 2-HDA was added either to AqW or fed to zebrafish, conjugation frequencies were significantly diminished. COINs effect applied in mammals, tests carried out in mice gut gave similar results. The effect of 2-HDA on conjugation in an aquatic microcosm and in mammalian¿s gut shows the potential use of COINs as a mean to control the spread of AbR in natural systems, or as adjuvants of antibiotics

Extent and control of plasmid conjugation: dynamics, interactions and barriers

Resumen del trabajo presentado al Plasmid Biology Meeting, celebrado en Cambridge (UK) del 18 al 23 de septiembre de 2016.Plasmid conjugation is the preferred route of dissemination of important adaptive traits in bacteria, such as antibiotic resistance genes. If conjugation could be tamed and controlled at will, we will have a strong hand to control antibiotic resistance acquisition by human pathogens. To achieve this objective, we determined the dynamics of plasmid conjugation using various plasmid prototypes. We could distinguish between density-dependent and frequency-dependent conjugation dynamics. Broad-host range plasmids, which show inherently high conjugation rates (R0 >1), are able to invade recipient populations. This is in contrast to repressed plasmids, such as members of the MOBF12 / IncF complex, which show R0 <1. Comparative genomics analysis shows that the transfer systems of MOBF12 / IncF are adapted to their host bacteria and show interesting variations in their regulatory systems. Besides, and using plasmid R388 as a model, we determined how interactions with other plasmids and host chromosomes greatly affect the plasmid rate of transfer. Additionally, we screened a large series of compounds as conjugation inhibitors and investigated the mechanisms by which they affect conjugation. These inhibitors are able to eliminate plasmid invasion of recipient populations. Finally, we developed a genome analysis tool, called PLACNET, which helps in reconstructing plasmids from whole genome sequences. PLACNET has now been updated as a web tool that can be run from a PC. Using PLACNET, we were able to show that E. coli ST131, sometimes considered as an E. coli clone, contains nearly as much plasmid diversity as the whole enterobacteria. All these results taken together suggest new approaches to control plasmid conjugation and, thus, the dissemination of antibiotic resistance to human pathogens.Peer Reviewe

CF of prototype plasmids in the presence of TZA-B.

<p>CF in the presence of 0.4 mM TZA-B (TZA-B, blue squares) or in its absence (C+, orange circles) using a representative set of conjugative plasmids. Each point represents the result of one independent experiment in logarithmic scale measured by plate-conjugation assay. Horizontal and vertical bars represent the mean ± SD of each group of data (* p < 0.05, ** p < 0.01, *** p < 0.001). Inc, incompatibility group [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148098#pone.0148098.ref025" target="_blank">25</a>].</p

TZAs A, B and E structure and activity.

<p>(A) Chemical structure of TZAs A, B, and E. (B) CF of plasmid R388, measured by plate-conjugation assay and represented in logarithmic scale in the presence of 1 mM TZAs A, B, or E. C+, control in the absence of added compound. Bars represent the mean CF + SD of at least three independent experiments (*** p < 0.001).</p

Structural elucidation of TZA-B.

<p>(A) Chemical structure of TZA-B, indicating carbon positions. (B) ¹H and ¹³C NMR spectral data of TZA-B [δ (ppm), J_HH (Hz); CDCl₃].</p

Mobilization frequency (MF) in the presence of TZA-B.

<p>MF of three mobilizable plasmids in the presence of 0.4 mM TZA-B (TZA-B, blue squares) or in its absence (C+, orange circles), using four different helper plasmids. Each point represents the result of one independent experiment in logarithmic scale measured by plate-conjugation assay. Horizontal and vertical bars represent the mean ± SD of each group of data (** p < 0.01, *** p < 0.001). ^<i>a</i> CFs of helper plasmids in the presence of mobilizable plasmids were similar to that obtained alone (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148098#pone.0148098.g004" target="_blank">Fig 4</a>).</p