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1.
mBio ; 12(4): e0127721, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34425705

RESUMO

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.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Escherichia coli/genética , Microbioma Gastrointestinal/genética , Plasmídeos/genética , Alcinos/administração & dosagem , Ração Animal , Animais , Escherichia coli/efeitos dos fármacos , Ácidos Graxos Insaturados/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Técnicas de Transferência de Genes , Transferência Genética Horizontal , Camundongos , Camundongos Endogâmicos C57BL , Rios/microbiologia
2.
Methods Mol Biol ; 2075: 99-110, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31584157

RESUMO

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.


Assuntos
Bactérias/genética , Conjugação Genética , Genes Reporter , Imagem Óptica/métodos , Plasmídeos/genética , Bactérias/metabolismo , Citometria de Fluxo , Imunofluorescência , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos
3.
Front Microbiol ; 8: 2260, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29201021

RESUMO

Bacteria display a variety of mechanisms to control plasmid conjugation. Among them, fertility inhibition (FI) systems prevent conjugation of co-resident plasmids within donor cells. Analysis of the mechanisms of inhibition between conjugative plasmids could provide new alternatives to fight antibiotic resistance dissemination. In this work, inhibition of conjugation of broad host range IncW plasmids was analyzed in the presence of a set of co-resident plasmids. Strong FI systems against plasmid R388 conjugation were found in IncF/MOBF12 as well as in IncI/MOBP12 plasmids, represented by plasmids F and R64, respectively. In both cases, the responsible gene was pifC, known also to be involved in FI of IncP plasmids and Agrobacterium T-DNA transfer to plant cells. It was also discovered that the R388 gene osa, which affects T-DNA transfer, also prevented conjugation of IncP-1/MOBP11 plasmids represented by plasmids RP4 and R751. Conjugation experiments of different mobilizable plasmids, helped by either FI-susceptible or FI-resistant transfer systems, demonstrated that the conjugative component affected by both PifC and Osa was the type IV conjugative coupling protein. In addition, in silico analysis of FI proteins suggests that they represent recent acquisitions of conjugative plasmids, i.e., are not shared by members of the same plasmid species. This implies that FI are rapidly-moving accessory genes, possibly acting on evolutionary fights between plasmids for the colonization of specific hosts.

4.
PLoS One ; 11(1): e0148098, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26812051

RESUMO

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.


Assuntos
Produtos Biológicos/farmacologia , Conjugação Genética/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Naftalenos/farmacologia , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/genética , Produtos Biológicos/síntese química , Produtos Biológicos/química , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácidos Graxos Insaturados/síntese química , Ácidos Graxos Insaturados/química , Células HCT116 , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Naftalenos/síntese química , Naftalenos/química , Plasmídeos/genética , Plasmídeos/metabolismo
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