RESUMO
Gram-negative bacteria (GNB) that express CTX-M ß-lactamases have become a serious threat to the clinical management of GNB infections. While antibody-based platforms have been successfully used in research settings to study and detect other ß-lactamases-including SHV, CMY, and TEM enzymes-there is currently a lack of antibody-based tools to detect the CTX-M enzymes. Here we describe the development of an anti-CTX-M sandwich ELISA based on a pair of monoclonal antibodies (mAbs)-mAb 6101-33 and mAb 6101-19-used as the capture and detection antibody, respectively. This antibody pair detected CTX-M variants from group 1 (CTX-M-15), group 2 (CTX-M-2), group 8 (CTX-M-8), and group 9 (CTX-M-14) that were expressed by a training set of clinical GNB isolates. The limit of detection for this sandwich ELISA was 30ng of recombinant CTX-M-15, and CTX-Ms expressed by 106 lysed CFU of GNB. When tested against a blinded panel of 78 clinical isolates, the sandwich ELISA demonstrated a sensitivity of 96% and a specificity of 100%. The mAb pair did not cross-react with bacteria that contained other ß-lactamases, including TEM, SHV, OXA, KPC, NDM, CMY, and DHA. In conclusion, we developed a highly sensitive and specific sandwich ELISA, capable of detecting CTX-M enzyme production in GNB pathogens.
Assuntos
Anticorpos Monoclonais , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , beta-Lactamases/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , DNA Bacteriano/genética , Ensaio de Imunoadsorção Enzimática/métodos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Técnicas de Genotipagem/métodos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes , Sensibilidade e Especificidade , beta-Lactamases/genética , beta-Lactamases/imunologiaRESUMO
Embryonic stem cells (ESCs), which are derived from a peri-implantation embryo, are routinely cultured in medium containing diabetic glucose (Glc) concentrations. While pregnancy in women with pre-existing diabetes may result in small embryos, whether such high Glc levels affect ESC growth remains uncovered. We show here that long-term exposure of ESCs to diabetic Glc inhibits their proliferation, thereby mimicking in vivo findings. Molecularly, Glc exposure increased oxidative stress and activated Forkhead box O3a (FOXO3a), promoting increased expression and activity of the ROS-removal enzymes superoxide dismutase and catalase and the cell-cycle inhibitors p21(cip1) and p27(kip1). Diabetic Glc also promoted ß-catenin nuclear localization and the formation of a complex with FOXO3a that localized to the promoters of Sod2, p21(cip1), and potentially p27(kip1). Our results demonstrate an adaptive response to increases in oxidative stress induced by diabetic Glc conditions that promote ROS removal, but also result in a decrease in proliferation.