Immobilization of Reporter Bacteriophage PP01 on Electrospun Polyhydroxybutyrate Fiber for Escherichia coli O157:H7 detection

  • Author / Creator
    Chen, Sim Yee
  • Food pathogens are the main sources of food poisoning. Among the pathogens, Escherichia coli (E. coli) O157:H7 is a Shiga toxin-producing bacterium that leads to thousand of illnesses. Most infection cases are related to consuming undercooked contaminated food products. People of any age can be infected. Symptoms of infections includes severe stomach cramps, diarrheas and may lead to kidney failure.
    The current detection methods of E.coli O157:H7 provides a sensitive tool to detect low number of cells, however, they are time consuming (48 to 72 h), labor intensive and require professionals in handling and interpreting the results. Therefore, a portable biosensor that can report results rapidly are desired. Here we propose a bacteriophage (phage)-based detection method using engineered NanoLuc reporter phages, namely PP01, immobilized on electrospun poly-3-hydroxybutyrate (PHB). The engineered reporter phages express a luminescent protein when E. coli O157:H7 cells are infected, indicating the presence of target pathogens during enrichment period. To implement phages in this application, it is desirable to immobilize them on a substrate to serve as a swab. For the swab application, high density of immobilized phages are required to generate detectable signal. Previous work has looked at phage immobilization on PHB substrate and found that plasma treated polymer thin film enables immobilization of phages on the polymer substrate. In this work, it is hypothesized that higher phage loading on the polymer substrate could be achieved from porous electrospun mats, increasing the sensitivity of the biosensor.
    Infectivity of immobilized phages on 4 different substrates: 2 solvent casted and 2 electrospun from 1 and 5 % w/v PHB in chloroform, were studied. Porous nonwoven microfibers PHB mats were produced from electrospinning. Flat PHB films were solvent-casted and plasma treated. Phages were immobilized on PHB substrates by immersing substrates in 108 PFU/ml of phage suspension at 4 oC overnight. To study the infectivity of immobilized phages, E. coli infection dynamic assays were carried out. A bioluminescence assay was conducted with the most promising material samples to validate the bacterial sensing property of the phage-immobilized PHB substrates in detecting E. coli O157:H7. We found that immobilized phages on electrospun fibers from 5 % w/v PHB solution displayed higher infectivity than that of phages on plasma-treated flat film in the E. coli infection dynamic assays. In the bioluminescence assay, immobilized phages on electrospun fibers detected E. coli cells at a concentration of 103 CFU/ml within 3 hours in milk, a complex medium.
    We anticipate our phage-immobilized PHB to be a starting point for more sophisticated biosensors. For example, phage cocktails, which is to combine two or more phages, could be immobilized on the substrates for diverse host range. Furthermore, the phage-immobilized PHB substrate also has a potential use as antimicrobial packaging. In conclusion, microstructures of the electrospun PHB increased infectivity of immobilized phages on the substrate and may play a key role in supporting other phage-immobilized applications.

  • Subjects / Keywords
  • Graduation date
    Spring 2020
  • Type of Item
  • Degree
    Master of Science
  • DOI
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