RESUMEN
OBJECTIVES: This study analysed the novel carbapenem-hydrolysing class D ß-lactamase OXA-822 identified in the clinical Acinetobacter calcoaceticus isolate AC_2117. METHODS: WGS was employed for identification of ß-lactamases. Micro-broth dilution was used for evaluation of antibiotic susceptibility of AC_2117 and transformants containing blaOXA-822. After heterologous purification of OXA-822, OXA-359 and OXA-213, enzyme kinetics were determined using spectrometry. The effect of OXA-822 upon meropenem treatment was analysed in the Galleria mellonella in vivo infection model. RESULTS: OXA-822 is a member of the intrinsic OXA-213-like family found in A. calcoaceticus and Acinetobacter pittii. Amino acid sequence similarity to the nearest related OXA-359 was 97%. Production of OXA-822, OXA-359 and OXA-213 in Acinetobacter baumannii ATCC® 19606T resulted in elevated MICs for carbapenems (up to 16-fold). Penicillinase activity of the purified OXA-822 revealed high KM values, in the millimolar range, combined with high turnover numbers. OXA-822 showed the highest affinity to carbapenems, but affinity to imipenem was â¼10-fold lower compared with other carbapenems. Molecular modelling revealed that imipenem does not interact with a negatively charged side chain of OXA-822, as doripenem does, leading to the lower affinity. Presence of OXA-822 decreased survival of infected Galleria mellonella larvae after treatment with meropenem. Only 52.7%â±â7.7% of the larvae survived after 24 h compared with 90.9%â±â3.7% survival in the control group. CONCLUSIONS: The novel OXA-822 from a clinical A. calcoaceticus isolate displayed penicillinase and carbapenemase activity in vitro, elevated MICs in different species and decreased carbapenem susceptibility in A. baumannii in vivo.
Asunto(s)
Acinetobacter calcoaceticus , Proteínas Bacterianas , beta-Lactamasas , Acinetobacter , Acinetobacter calcoaceticus/enzimología , Acinetobacter calcoaceticus/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Carbapenémicos/farmacología , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/genéticaRESUMEN
Acinetobacter baumannii is an antibiotic-resistant, Gram-negative pathogen that causes a multitude of nosocomial infections. However, pathogenicity mechanisms and the host cell response during infection remain unclear. In this study, we determined virulence traits of A. baumannii clinical isolates belonging to the most widely disseminated international clonal lineage, international cluster 2 (IC2), in vitro and in vivo. Complexome profiling of primary human endothelial cells with A. baumannii revealed that mitochondria, and in particular complexes of the electron transport chain, are important host cell targets. Infection with highly virulent A. baumannii remodelled assembly of mitochondrial protein complexes and led to metabolic adaptation. These were characterized by reduced mitochondrial respiration and glycolysis in contrast to those observed in infection with low-pathogenicity A. baumannii. Perturbation of oxidative phosphorylation, destabilization of mitochondrial ribosomes, and interference with mitochondrial metabolic pathways were identified as important pathogenicity mechanisms. Understanding the interaction of human host cells with the current global A. baumannii clone is the basis to identify novel therapeutic targets. IMPORTANCE Virulence traits of Acinetobacter baumannii isolates of the worldwide most prevalent international clonal lineage, IC2, remain largely unknown. In our study, multidrug-resistant IC2 clinical isolates differed substantially in their virulence potential despite their close genetic relatedness. Our data suggest that, at least for some isolates, mitochondria are important target organelles during infection of primary human endothelial cells. Complexes of the respiratory chain were extensively remodelled after infection with a highly virulent A. baumannii strain, leading to metabolic adaptation characterized by severely reduced respiration and glycolysis. Perturbations of both mitochondrial morphology and mitoribosomes were identified as important pathogenicity mechanisms. Our data might help to further decipher the molecular mechanisms of A. baumannii and host mitochondrial interaction during infection.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Humanos , Acinetobacter baumannii/genética , Células Endoteliales , Infecciones por Acinetobacter/tratamiento farmacológico , Farmacorresistencia Bacteriana Múltiple/genética , Antibacterianos/farmacología , Proteínas Mitocondriales/uso terapéuticoRESUMEN
Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.