The Application of Bacteriophage Host Recognition Binding Proteins for the Isolation of Yersinia enterocolitica in Foods
The isolation of Yersinia enterocolitica, an important zoonotic agent causing yersiniosis in humans poses a major challenge due to the poor sensitivity of culture methods. The objective of this research was to investigate the application of bacteriophage derived host recognition binding proteins (RBPs) for selective capture and isolation of the epidemiologically significant Y. enterocolitica serotypes O:3, O:5,27, O:8 and O:9 from contaminated foods. To this aim, study of previously reported Y. enterocolitica phages and characterization of novel phages isolated from pig manure and raw sewage based on morphology, host range, genome sequence, and host cell receptor specificity, led to the selection of three candidate RBPs. RBP Gp47 derived from the previously reported Yersinia phage φ80-18 was identified as a suitable ligand with specificity for the O-specific polysaccharide (O-PS) of serotype O:8 strains. RBP Gp17 derived from the novel Podovirus phage vB_YenP_AP5 was identified as a ligand with specificity for the O-PS of serotype O:3 strains. The distal long tail fiber protein, RBP Gp37, derived from the novel Myovirus phage vB_YenM_TG1 was identified as a ligand for the outer membrane protein OmpF of serotype O:3, O:5,27 and O:9 strains. Confocal laser immunofluorescent microscopy determined that recombinant forms of these RBPs produced by expression in E. coli bound specifically to host cell surfaces and demonstrated a similar binding spectrum to the host range of the phages from which they were derived. Additionally, Localized Surface Plasmon Resonance analyses indicated equilibrium association constant (KA) values in the nanomolar range, which indicate an overall high binding affinity of the RBPs to host cells. Collectively, these observations suggested the recombinant RBPs could be considered structurally equivalent to the native proteins since they retained their biological function. Selective capture of Y. enterocolitica O:3, the most significant serotype causing yersiniosis, was achieved from cell suspensions by use of magnetic microparticles functionalized with RBP Gp17. Notably, simultaneous capture of Y. enterocolitica O:3, O:5,27, O:8 and O:9 was also attained utilizing a mixture of magnetic microparticles coated with RBP Gp47 and RBP Gp37. This last approach (RBP-MS) in combination with CIN agar, the most widely used isolation medium for Y. enterocolitica applied to cell suspensions of 160 Yersinia spp. and 20 other non-Yersina spp. strains achieved a specificity of 85% for the isolation of Y. enterocolitica O:3, O:5,27, O:8 and O:9 irrespective of the pathogenic potential of the strains. In contrast, when applied in combination with CAY agar, a chromogenic medium selective for virulent Y. enterocolitica, the specificity was increased to 95.7%. Moreover, only potentially pathogenic strains were captured and grew as typical mauve coloured colonies on this agar. RBP-MS applied to artificially inoculated ground pork samples, established a 2 log10 CFU/ml improvement in sensitivity compared to direct plating on CIN or CAY agars. In addition, RBP-MS in combination with CAY agar was able to detect a higher number of samples inoculated at low levels (0.1 CFU/g, 1 CFU/g, and 10 CFU/25g) after 8 h and 24 h of enrichment at 25°C in non-selective TSB accompanied with reduced background microbiota, compared to samples that did not undergo RBP-MS. Collectively this research demonstrates that use of phage RBPs as capture molecules represent an alternative approach for bacterial concentration as a preparative step to improve the culture isolation of pathogenic Y. enterocolitica from foods. Additionally, due to their lytic nature, the phages obtained can be considered prospective biotechnological tools for further diagnostic, therapeutic and or bio-control uses given that they target the predominant serotypes involved in yersiniosis.