Multiplexed real-time PCR for the detection and differentiation of O-antigen serotypes.

has emerged as a global health threat due to its role in the spread of antimicrobial resistance and because it is a frequent cause of hospital-acquired infections and neonatal sepsis. Capsular and lipopolysaccharide (LPS) O-antigen polysaccharide surface antigens are major immunogens that are useful for strain classification and are candidates for vaccine development. We have developed real-time PCR reagents for molecular serotyping, subtyping, and quantitation of the most prevalent LPS O-antigen types (i.e., O1, O2, O3, and O5) of . We describe two applications for this O-typing assay: for screening culture isolates and for direct typing of present in stool samples. We find 100% concordance between the results of the O-typing assay and whole-genome sequencing of 81 culture isolates, and >90% agreement in O-typing performed directly on specimens of human stool, with disagreement arising primarily from a lack of sensitivity of the culture-based comparator method. Additionally, we find evidence for mixed O-type populations at varying levels of abundance in direct tests of stool from a hospitalized patient population. Taken together, these results demonstrate that this novel O-typing assay can be a useful tool for epidemiologic and vaccine studies.IMPORTANCE is an important opportunistic pathogen. The gastrointestinal (GI) tract is the primary reservoir of in humans, and GI carriage is believed to be a prerequisite for invasive infection. Knowledge about the dynamics and duration of GI carriage has been hampered by the lack of tools suitable for detection and strain discrimination. Real-time PCR is particularly suited to the higher-throughput workflows used in population-based studies, which are needed to improve our understanding of carriage dynamics and the factors influencing colonization.