RESUMO
Bacterial transduction particles were critical to early advances in molecular biology and are currently experiencing a resurgence in interest within the diagnostic and therapeutic fields. The difficulty of developing a robust and specific transduction reagent capable of delivering a genetic payload to the diversity of strains constituting a given bacterial species or genus is a major impediment to their expanded utility as commercial products. While recent advances in engineering the reactivity of these reagents have made them more attractive for product development, considerable improvements are still needed. Here, we demonstrate a synthetic biology platform derived from bacteriophage P1 as a chassis to target transduction reagents against four clinically prevalent species within the Enterobacterales order. Bacteriophage P1 requires only a single receptor binding protein to enable attachment and injection into a target bacterium. By engineering and screening particles displaying a diverse array of chimeric receptor binding proteins, we generated a potential transduction reagent for a future rapid phenotypic carbapenem-resistant Enterobacterales diagnostic assay.
Assuntos
Bacteriófago P1/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Infecções por Enterobacteriaceae/diagnóstico , Engenharia Genética/métodos , Proteínas da Cauda Viral/genética , Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Infecções por Enterobacteriaceae/microbiologia , Ertapenem/farmacologia , Testes de Sensibilidade Microbiana/métodos , Fenótipo , Biologia Sintética/métodos , Transdução Genética/métodos , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genéticaRESUMO
OBJECTIVES: To identify the molecular mechanism of colistin resistance in an MDR Acinetobacter baumannii clinical strain isolated in 2008 from a meningitis case in Brazil. METHODS: Long- and short-read WGS was used to identify colistin resistance genes in A. baumannii strain 597A with a colistin MIC of 64 mg/L. MS was used to analyse lipid A content. mcr was cloned into pET-26b (+) and transformed into Escherichia coli BL21(λDE3)pLysS for analysis. RESULTS: A novel plasmid (pAb-MCR4.3) harbouring mcr-4.3 within a Tn3-like transposon was identified. The A. baumannii 597A lipid A MS spectra showed a main molecular ion peak at m/z=2034, which indicated the addition of phosphoethanolamine to the lipid A structure. E. coli BL21 transformed with pET-26b-mcr-4.3 gained colistin resistance with a colistin MIC of 8 mg/L. CONCLUSIONS: Colistin resistance in A. baumannii 597A was correlated with the presence of a novel plasmid-encoded mcr-4.3 gene.
