Paper BI+MI-WeM1
Bacteriophage-Derived Surfaces for the Targeting of Pathogenic Bacteria

Wednesday, November 9, 2016, 8:00 am, Room 101A

Bacterial pathogens cause a high level of morbidity and mortality, specifically for infants, young children, elderly and immunocompromised individuals. Antibodies have been exploited as molecular probes in order to impart specificity to bacterial biosensing platforms. Antibodies however suffer from degradation and reliability issues. The high specificity of phages offers a potent alternative for the targetting of pathogens. More specifically, recombinant phage receptor binding proteins (RBPs) responsible for phage-host specificity can be used as biological probes and present numerous advantages over the use of whole phage.

We successfully coupled phage RBP Gp047 from phage NCTC12673 onto magnetic beads. These beads were then employed for the extraction of Campylobacter cells from food matrices. Recovery rates were greater than 80% in samples spiked with as low as 10² cfu mL⁻¹ of cells. Phage lysins have also been employed as capturing probes. We coupled recombinant lysin Gp10 from the mycobacteriophage L5 onto magnetic Dynabeads 280 for the capture of Mycobacterium avium subsp. paratuberculosis (MAP) cells from complex media. The study employed skim cow milk spiked with MAP cells, skim milk spiked with both MAP and Escherichia coli cells and Middlebrook 7H9 medium spiked with MAP cells as test matrices. The functionalized beads were incubated with the spiked sample, separated, cleaned, and subjected to DNA extraction. The resulting solution was analyzed by real time PCR. The entire test was completed within 24 hours. The capture process significantly increased the PCR sensitivity and demonstrated high specificity towards MAP cells.

Further, we demonstrated the use of cysteine-tagged P22 phage RBPs on gold surface for the specific SPR detection of Salmonella enterica serovar Typhimurium. These results demonstrate that N-teminus Cys tagged proteins capture bacteria efficiently compared to the C-terminus Cys tagged protein due to preferential orientations. Finally, micromechanical devices have also been proposed for the detection and enumeration of bacteria. We designed and developed a microresonator array optimized for such detection. This large array-based design offers a large total area for the capture of cells, while maintaining the ability to detect the attachment of a single cell anywhere on the array. The devices were functionalized with phage GST-Gp48 tail-spike proteins to impart specificity of detection. We successfully employed these arrays for the specific detection of Campylobacter jejuni from clean buffer. The devices did not show any sensitivity to Escherichia coli bacteria confirming the specificity of detection .