Investigations of Methanobrevibacter spp. Culture to Enable Archaea Virus Propagation, and Comparison of the Efficacy of Individual Bacteriophages and their Cocktails against Escherichia coli O157
Date
2022-01-06
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Abstract
Viruses that target prokaryotes are a growing area of interest for control of pathogens and other detrimental microorganisms. Archaeal viruses were identified as an option for the mitigation of methane produced by Methanobrevibacter ruminantium, which lives in ruminants such as cattle. In this study we tested three protocols to culture Methanobrevibacter spp. While a PCR was able to confirm the presence of M. ruminantium in these cultures, we could not achieve replicable growth. Unfortunately, an anti-methanogen virus could not be isolated from rumen fluid, and the culture of the archaea was found to be challenging.
Another microbial target, Escherichia coli, was also explored in this study. Phages are used to control pathogenic E. coli, however, while phages can be used as support or as an alternative to antibiotic therapy, pre-existing or induced anti-phage mutations in the targeted bacteria can reduce the efficacy of this approach. Phage cocktails have been used as a strategy to reduce the likelihood that phage resistance would develop. However, in some cases, individual phages were found to be more effective at lysing bacteria and at reducing the frequency of resistance development. Therefore, we tested individual (T1, T4, T5, and rV5) and cocktail phage treatments against six strains of E. coli O157, including EDL933, R508N, CO281-31N, E32511, H4420N, and E318N. EDL933 was selected for further experiments as this is a well characterized human pathogenic strains. Our results showed that T5 and cocktail T1+T4+T5+rV5 treatments were effectively lysing bacteria, however, we have strong indications that phage resistance developed in both individual and cocktail-exposed EDL933. Cocktail T1+T4+rV5 was more effective at lysing EDL933 compared to T1, T4, or rV5 treatments. Initially the concentration of T1 in T1+T4+rV5, and T5 in cocktail T1+T4+T5+rV5 decreased before increasing to the levels of replication observed in individual treatments. T1 in T1+T4+rV5 decreased over time until it was not detectable. Sequencing of exposed strains and the phages is needed to reveal the genetic basis of resistance. More research into strategic combinations of phages, based on receptor usage or lytic activity, is required to increase the susceptibility of pathogenic E. coli to phages or antibiotics.
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Keywords
Methane, Virus, Bacteriophage, Cattle, Escherichia coli, Cocktail
Citation
Fletcher, J. (2022). Investigations of Methanobrevibacter spp. Culture to Enable Archaea Virus Propagation, and Comparison of the Efficacy of Individual Bacteriophages and their Cocktails against Escherichia coli O157 (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.)