Diagnostic Potential of Monoclonal Antibodies Specific to the Unique O-Antigen of Multidrug-Resistant Epidemic Escherichia coli Clone ST131-O25b:H4

Abstract

ABSTRACTTheEscherichia colilineage sequence type 131 (ST131)-O25b:H4 is a globally spread multidrug-resistant clone responsible for a great proportion of extraintestinal infections. Driven by the significant medical needs associated with this successful pathogenic lineage, we generated murine monoclonal antibodies (MAbs) against its lipopolysaccharide (LPS) O25b antigen in order to develop quick diagnostic tests. Murine monoclonal antibodies were generated by immunizing mice with whole killed nonencapsulated ST131-O25bE. colicells and screening hybridoma supernatants for binding to purified LPS molecules obtained from anE. coliST131-O25b clinical isolate. The MAbs selected for further study bound to the surface of liveE. coliO25b strains irrespective of the capsular type expressed, while they did not bind to bacteria or purified LPS from other serotypes, including the related classical O25 antigen (O25a). Using these specific MAbs, we developed a latex bead-based agglutination assay that has greater specificity and is quicker and simpler than the currently available typing methods. The high specificities of these MAbs can be explained by the novel structure of the O25b repeating unit elucidated in this article. Based on comparative analysis by nuclear magnetic resonance (NMR) and mass spectrometry, theN-acetyl-fucose in the O25a O-antigen had been replaced byO-acetyl-rhamnose in the O25b repeating unit. The genetic determinants responsible for this structural variation were identified by aligning the corresponding genetic loci and were confirmed bytrans-complementation of a rough mutant by the subserotype-specific fragments of therfboperons.