RESUMO
Despite the threat posed by antibiotic resistance in infectious bacteria, little is known about the diversity, distribution and origins of resistance genes, particularly among the as yet unculturable environmental bacteria. One potentially rich but largely unstudied environmental reservoir is soil. The complexity of its microbial community coupled with its high density of antibiotic-producing bacteria makes the soil a likely origin for diverse antibiotic resistance determinants. To investigate antibiotic resistance genes among uncultured bacteria in an undisturbed soil environment, we undertook a functional metagenomic analysis of a remote Alaskan soil. We report that this soil is a reservoir for beta-lactamases that function in Escherichia coli, including divergent beta-lactamases and the first bifunctional beta-lactamase. Our findings suggest that even in the absence of selective pressure imposed by anthropogenic activity, the soil microbial community in an unpolluted site harbors unique and ancient beta-lactam resistance determinants. Moreover, despite their evolutionary distance from previously known genes, the Alaskan beta-lactamases confer resistance on E. coli without manipulating its gene expression machinery, demonstrating the potential for soil resistance genes to compromise human health, if transferred to pathogens.
Assuntos
Bactérias/enzimologia , Bactérias/genética , Microbiologia do Solo , beta-Lactamases/genética , Alaska , Antibacterianos/farmacologia , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de Sequência , Resistência beta-Lactâmica , beta-Lactamas/farmacologiaAssuntos
DNA/genética , Metagenoma/genética , Metagenômica/métodos , Microbiologia do Solo , Bactérias/genética , Bactérias/isolamento & purificação , Cromossomos Artificiais Bacterianos/genética , DNA/química , DNA/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Biblioteca Genômica , Peso Molecular , Plasmídeos/genéticaRESUMO
We describe here an improved method for isolating, purifying, and cloning DNA from diverse soil microbiota. Soil microorganisms were extracted from soils and embedded and lysed within an agarose plug. Nucleases that copurified with the metagenomic DNA were removed by incubating plugs with a high-salt and -formamide solution. This method was used to construct large-insert soil metagenomic libraries.
