AimsPiliated Lactobacillus rhamnosus (pLR) strains have attracted much attention owing to their excellent mucus adhering capacity and immunomodulatory effects. Here, we aimed to develop a rapid, sensitive method for isolating pLR strains in complex ecosystems using immunomagnetic separation (IMS) with colony immunoblotting (CIB). Methods and ResultsMagnetic nanobeads (diameter: 180nm) conjugated with anti-pLR SpaA pilin antibodies (anti-SpaA) were prepared and used to preconcentrate pLR strains in samples, followed by confirmation with anti-SpaA-based CIB analysis. Under optimized experimental conditions, IMS-CIB selectively recovered pLR strains from 10(7)CFUml(-1) of faecal microbiota samples spiked with 29x10(1) to 24x10(6)CFUml(-1) of pLR strains. No positive colonies were detected in samples without addition of pLR strains. The detection limit of IMS-CIB was 29CFUpLRml(-1) of faecal microbiota, which is much lower than that of CIB without IMS preconcentration (20x10(4)CFUml(-1)). ConclusionsIMS-CIB allowed selective preconcentration of pLR strains in highly heterogeneous bacterial suspensions and direct detection of pLR colonies, which remained readily available for subsequent isolation. Significance and Impact of the StudyOur findings established an effective method for selective enrichment and detection of pLR strains.