Ruminal Fusobacterium and their phages - Fundamentals and Applications

Date
2023-12-01
Journal Title
Journal ISSN
Volume Title
Publisher
Embargo
Abstract

In the ongoing quest for sustainable and effective antimicrobial solutions, bacteriophages are experiencing a resurgence as a bacterial control method especially in the context of mitigating the spread of antibiotic resistance. This thesis focuses on leveraging phage technology to replace or use in conjunction with antibiotics in the animal agricultural sector, particularly the feeding of tylosin to cattle to suppress the growth of bovine pathogen Fusobacterium necrophorum and, subsequently, prevent liver abscessation. This research sheds light on the composition of the bovine ruminal community, highlighting the isolation of a previously overlooked Fusobacterium species, Fusobacterium varium, in the bovine rumen. Furthermore, F. varium was found to be more abundant in the rumen fluid of cattle and under culture conditions tailored to enrich F. necrophorum. Using near-full length 16S rRNA sequencing, we demonstrate that F. varium grows under restrictive conditions commonly used to enumerate F. necrophorum, suggesting that previous F. necrophorum abundance assessment may have been inaccurate and that F. varium may be an underestimated member of the ruminal bacterial community. Fusobacterium varium isolates were found to be less susceptible than F. necrophorum to in-feed antibiotics conventionally used in feedlots, specifically, tylosin, the current gold standard for liver abscess reduction strategies in cattle. F. varium strains were totally or highly resistant (0 - 13% reduction in maximum yield, P < 0.05). Genomic analyses of F. varium isolates highlighted intrinsic antibiotic resistance patterns and potential virulence traits, thus underlining its significance in the broader microbial ecosystem and perhaps its pathogenicity in cattle, which has been previously established in humans. Furthermore, whole genome sequencing of bovine isolates revealed a collection of antibiotic resistance genes, notably to tylosin, in strains of F. necrophorum. Tylosin has been used as a liver abscess prophylactic for the last 45 years within the feedlot cattle industry, largely to great success. The discovery of the ermb gene within two strains of F. necrophorum combined with the finding that the biochemical fingerprinting methods historically used to identify F. necrophorum may be imprecise signals a potential paradigm shift in liver abscess prevention strategies. Previously discounted strategies should be revisited, and new techniques will be required to combat this pathogen as tylosin resistance spreads. To this end, phages capable of killing F. necrophorum were isolated as an alternative or complementary biocontrol strategy. Six novel phages were isolated from rumen fluid or ruminal F. necrophorum isolates via prophage induction and subjected to host range testing on both F. necrophorum subspecies necrophorum and funduliforme. Four F. necrophorum subspecies necrophorum phages were tested for cross resistance and host growth inhibition individually and in pairs. Additionally, genomic sequencing, annotation and analysis were performed. Though all F. necrophorum phages isolated contained sequences bearing similarities to genes implicated in lysogeny, four selected for use in cocktails showed potential in inhibiting host growth, with several demonstrating promising attributes for biocontrol and therapeutic applications, indicating the potential of some lysogenic phages to be adapted for biocontrol or therapeutic purposes when lytic phages are difficult to obtain. Six phages infecting F. varium were also isolated and genetically characterized, all of which were identified as lytic via lifestyle prediction algorithms. The typically broad host range of these phages underscores their potential utility in bacterial control and perhaps suitability as an “off the shelf” product for use in repressing F. varium growth, though it is important to note that two isolates bore genetic similarity to a phage plasmid found within deposited strain ATCC 27725. Interestingly, three of the phages had high genetic similarity (>95%) but demonstrated different host ranges, indicating that the small differences between the genomes may have a large impact on behavior and host range. Furthermore, the variability of phage response to two types of phage training to improve longitudinal growth repression performance did not follow a clear pattern – similar phages responded quite differently to the same treatment. The primary objective of these techniques was to prolong the period during which bacterial growth was suppressed by over 50%, which was not observed under either treatment schema. Overall, the results of this study throw into stark relief the need for more comprehensive genome annotation techniques and further phage genome sequencing to better understand the diversity and complexity of phages infecting Fusobacterium species. Lastly, a small pilot study in vivo hinted at the promise of phages as an antimicrobial alternative, showing a significant inhibition of challenge strain 8L1 when exposed to a high dose (10E10 – 10E11) of phage, although larger and more comprehensive studies are needed. As a whole, this thesis underscores the importance of accurate microbial characterization, the need for more comprehensive metagenomic and sequencing efforts, and demonstrates the proof of concept of use of phages as an antibiotic alternative within in modern agriculture.

Description
EMBARGO NOTE: This item is embargoed until 2024-12-01
Degree
Doctor of Philosophy
Type
Thesis
Keywords
fusobacterium, phage, antibiotic resistance, ruminal, microbiology
Citation

Schwarz, Cory Elizabeth. "Ruminal Fusobacterium and their phages - Fundamentals and Applications." (2023). PhD diss., Rice University. https://hdl.handle.net/1911/115411

Has part(s)
Forms part of
Published Version
Rights
Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.
Link to license
Citable link to this page