4 research outputs found
The use of bacteriophages as natural biocontrol agents against bacterial pathogens
Bacteriophages are viruses that specifically infect bacteria. The bactericidal nature
of lytic bacteriophages has been exploited by scientists for decades with the hope to utilise
them in the fight against bacterial infections and antibiotic resistant bacteria in medical
settings. More recently, the potential applications of bacteriophages for biocontrol in the
agrifood and environmental sectors have been investigated in an attempt to develop
‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantages
over conventional methods of controlling pathogenic bacteria, such as high host
specificity, 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 the
impact of environmental conditions on lysis efficiency, multiplicity of infection, and to
significantly minimise the emergence of bacterial resistance to phages.
Temperature plays a key role in every biological activity in nature. It is also
assumed that temperature has an effect on phage lysis efficiency. A comprehensive study
of it and how it affects both the host cells and their corresponding phages is crucial to
ensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectiveness
of the three different phages belonging to the family of the Myoviridea that were isolated
and 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. Temperature
was found to have a significant impact on phage-mediated lysis efficiency. Both the
temperature of incubation of the phage-bacteria mixture (incubation temperature) and the
temperature history of bacterial hosts were found to have profound effects on plaque sizes
as well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°C
compared to 30 or 37°C. Phages were suggested to be well adapted to the environment
where they were isolated from with general implications for use in biological disinfection.
Furthermore, the temperature history of the bacteria (prior to phage encounter) was found
to have a modulating effect on their susceptibility to lysis.
A second part of this study compared the performance of the three phages in
regard to bacterial resistance. The emergence of bacterial resistance is a major obstacle
to the success of bacteriophages applications. The use of multiple phages is typically
recommended and has proven better than the use of a single phage. However, the bestway
to perform phage treatment is still very unclear. This study therefore compared
simultaneous addition of multiple phages (in form of a cocktail) with the sequential
addition of the individual phages at different time points in trying to delay the emergence
of bacterial resistance. The data obtained from this work suggest that lysis effectiveness
can be adjusted to optimize any treatment goal. For fast initial bacterial clearance the use
of a single phage with short time maximal lysis efficiency proved most efficient, while
the simultaneous addition of phages in the form of a cocktail was most successful strategy
in our study. Addition of selected phages sequentially can be normalized in such a way
that is just as effective as a cocktail.
A third part of this thesis looked into the susceptibility of bacteria that had
undergone sublethal disinfection. We addressed the question whether bacteria subjected
to sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. The
chlorine treatments indicated the development of a phage-insensitive phenotype for a
critical 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 foods
or water that have previously undergone a weak disinfection regime
Impact of treated sewage effluent on the microbiology of a small brook using flow cytometry as a diagnostic tool
Flow cytometry was applied to assess the microbiological impact of treated sewage effluent discharge into a small brook carrying surface runoff water. Increases in dissolved organic carbon and soluble reactive phosphorous were accompanied by increases in counts of intact bacteria by up to eightfold. Effluent ingress furthermore resulted in a pronounced shift of bacterial clusters. Whereas brook water upstream of the discharge point was characterised by a bacterial cluster with low nucleic acid (LNA) content, downstream water showed a shift to bacteria with high nucleic acid (HNA) content. Changes in the LNA/HNA ratio were largely maintained along the course of the brook. Results suggest that the LNA/HNA ratio can under certain conditions serve as an indicator of anthropogenic nutrient impact. Measuring impact on this low trophic level might be more sensitive and straightforward than measuring macroindicators. More evidence will however be required to assess the usefulness of LNA/HNA measurements to assess the ecological nutrient status of natural waters and the impact of nutrient pollution
Lysis performance of bacteriophages with different plaque sizes and comparison of lysis kinetics after simultaneous and sequential phage addition
Background: Although bacteriophages see a revival for specifically removing undesired bacteria, there is still much uncertainty about how to achieve the most rapid and long-lasting clearance.
Materials and Methods: This study investigated the lysis kinetics of three distinct environmental coliphages, reproducibly forming different plaque sizes (big, medium, and small). Lysis performance by individual phages was compared with the one obtained after simultaneous or sequential addition of all three phages. Kinetics was monitored by density absorbance or by flow cytometry, with the latter having the advantage of providing higher sensitivity.
Results: Plaque size happened to correlate with lysis kinetics in liquid suspensions, with phages producing big (phage B), medium (phage M), and small (phage S) plaques showing maximal bacterial clearance under the chosen conditions within ∼6, 12, and 18 h, respectively. Use of a phage cocktail (all three phages added simultaneously) resulted in slower initial lysis compared with the fastest lysing phage with the greatest plaque size alone, but it showed longer efficacy in suppression. When adding phages sequentially, overall lysis kinetics could be influenced by administering phages at different time points. The lowest bacterial concentration after 36 h was obtained when administering phages in the sequence S, M, and B although this combination initially took the longest to achieve bacterial clearance.
Conclusions: Results support that timing and order of phage addition can modulate strength and duration of bacterial suppression and, thus, influence the overall success of phage treatment