Cell wall hydrolase as a surface-associated protein target for the specific detection of Lactobacillus rhamnosus using flow cytometry

Abstract

Abstract Flow cytometry (FC) is a very popular technique for to the study of different eukaryotic cell phenotypes. In addition, FC has many applications in the field of food microbiology, although the few number of specific molecules that can be potentially targeted with fluorescence-conjugated antibodies limits its potential applications. The present work represents a step forward in the application of FC for detection of specific foodborne or probiotic bacteria by using fluorescent antibodies binding specific surface-associated protein. To illustrate this novel approach, we have used a monoclonal and polyclonal antibodies targeting cell-wall hydrolase (CWH), one of the major surface-associated proteins of the probiotic Lactobacillus rhamnosus GG. In order to verify the specific union of the antibodies to this surface protein, ten different bacterial strains belonging to different bacteria species taxonomically related and no related to L. rhamnosus, were tested. The optimal conditions for bacteria detection corresponded to a final suspension of 5E107 bacteria in exponential phase of growth using FC buffer, without freezing, without adding extra BSA to the FC buffer and without fixing bacteria. Our results showed that GG strain was detected using the polyclonal anti-CWH serum in a higher extent (91.62%) than the rest of strains (L. casei 393: 64.49%; L. amylovorus: 46.13%, E. coli: 45.13%; L. acidophilus DSM20079T: 35.83%; B. longum NCIMB8809: 33.68%; L. gasseri BM7/10: 32.41%; L. reuteri DSM21016: 29.26%; L. plantarum NCIMB8826: 26.33% and L. delbruekii: 22.52%). The percentage of L. rhamnosus labeled with the monoclonal antibody was 53.06, which contrasted with the 91.62% labeled with the polyclonal antibody. The low affinity of the monoclonal antibody was perhaps due to the lack of immunogenicity of the single epitope chosen for antibody generation (CWHp). The high efficiency observed for the polyclonal anti-CWH serum is maybe a consequence of being constituted by a mixture of antibodies that recognizes different epitopes in the L. rhamnosus GG protein. With this proof of concept, we have shown for the first time that foodborne or probiotic bacteria can be specifically detected using antibodies targeting adequate molecules present on their surface.