The use of bacteriophages as natural biocontrol agents against bacterial pathogens

摘要

Bacteriophages are viruses that specifically infect bacteria. The bactericidal natureof lytic bacteriophages has been exploited by scientists for decades with the hope to utilisethem in the fight against bacterial infections and antibiotic resistant bacteria in medicalsettings. More recently, the potential applications of bacteriophages for biocontrol in theagrifood and environmental sectors have been investigated in an attempt to develop‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantagesover conventional methods of controlling pathogenic bacteria, such as high hostspecificity, the ability to self-replicate, and the ability to evolve with their hosts. However,more research is needed to optimise the parameters for phage applications, including theimpact of environmental conditions on lysis efficiency, multiplicity of infection, and tosignificantly minimise the emergence of bacterial resistance to phages.Temperature plays a key role in every biological activity in nature. It is alsoassumed that temperature has an effect on phage lysis efficiency. A comprehensive studyof it and how it affects both the host cells and their corresponding phages is crucial toensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectivenessof the three different phages belonging to the family of the Myoviridea that were isolatedand purified from a single water sample taken from a brook receiving treated wastewater.We used the multiplicity of infection of 1 in all of our study in this project. Temperaturewas found to have a significant impact on phage-mediated lysis efficiency. Both thetemperature of incubation of the phage-bacteria mixture (incubation temperature) and thetemperature history of bacterial hosts were found to have profound effects on plaque sizesas well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°Ccompared to 30 or 37°C. Phages were suggested to be well adapted to the environmentwhere they were isolated from with general implications for use in biological disinfection.Furthermore, the temperature history of the bacteria (prior to phage encounter) was foundto have a modulating effect on their susceptibility to lysis.A second part of this study compared the performance of the three phages inregard to bacterial resistance. The emergence of bacterial resistance is a major obstacleto the success of bacteriophages applications. The use of multiple phages is typicallyrecommended and has proven better than the use of a single phage. However, the bestwayto perform phage treatment is still very unclear. This study therefore comparedsimultaneous addition of multiple phages (in form of a cocktail) with the sequentialaddition of the individual phages at different time points in trying to delay the emergenceof bacterial resistance. The data obtained from this work suggest that lysis effectivenesscan be adjusted to optimize any treatment goal. For fast initial bacterial clearance the useof a single phage with short time maximal lysis efficiency proved most efficient, whilethe simultaneous addition of phages in the form of a cocktail was most successful strategyin our study. Addition of selected phages sequentially can be normalized in such a waythat is just as effective as a cocktail.A third part of this thesis looked into the susceptibility of bacteria that hadundergone sublethal disinfection. We addressed the question whether bacteria subjectedto sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. Thechlorine treatments indicated the development of a phage-insensitive phenotype for acritical chlorine dose in the transition zone between live and dead. The remaining live(and culturable) bacteria were shown insensitive to the selected phage. The lowest UV exposure at 2.8 mJ/cm2 eliminated bacteria susceptibility to the phages. This phage-resistant phenotype may have serious consequences for the application of phages on foodsor water that have previously undergone a weak disinfection regime.