RESUMEN
Whole cell MALDI is regularly used for the identification of bacteria to species level in clinical Microbiology laboratories. However, there remains a need to rapidly characterize and differentiate isolates below the species level to support outbreak management. We describe the implementation of a modified preparative approach for MALDI-MS combined with a custom analytical computational pipeline as a rapid procedure for subtyping Shigatoxigenic E. coli (STEC) and accurately identifying strain-specifying biomarkers. The technique was able to differentiate E. coli O157:H7 from other STEC. Within O157 serotype O157:H7 isolates were readily distinguishable from Sorbitol Fermenting O157 isolates. Overall, nine homogeneous groups of isolates were distinguished, each exhibiting distinct profiles of defining mass spectra features. This offers a robust analytical tool useable in reference/diagnostic public health scenarios.
Asunto(s)
Técnicas de Tipificación Bacteriana/estadística & datos numéricos , Escherichia coli O157/aislamiento & purificación , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Técnicas de Tipificación Bacteriana/métodos , Análisis de Componente Principal , Serogrupo , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/estadística & datos numéricos , Factores de TiempoRESUMEN
Malignant catarrhal fever (MCF) is a fatal disease of cattle and other ungulates caused by certain gamma-herpesviruses including alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2). An attenuated virus vaccine based on AlHV-1 has been shown to induce virus-neutralising antibodies in plasma and nasal secretions of protected cattle but the targets of virus-specific antibodies are unknown. Proteomic analysis and western blotting of virus extracts allowed the identification of eight candidate AlHV-1 virion antigens. Recombinant expression of selected candidates and their OvHV-2 orthologues confirmed that two polypeptides, the products of the ORF17.5 and ORF65 genes, were antigens recognised by antibodies from natural MCF cases or from AlHV-1 vaccinated cattle. These proteins have potential as diagnostic and/or vaccine antigens.