Comparative isolation of bacteria from inflamed sites to identify inflammation-tailored bacteria

• Author / Creator

  Moote, Paul E

• Microbial culture collections are fundamental to microbiology research. However, with the advent of next generation sequencing research, the generation and maintenance of comprehensive culture collections has become deprioritized. As such a need exists for the improved isolation of diverse bacterial communities enabling bacterial communities to be archived for future investigations. This need in particular is highlighted in animal agriculture, since modern production practises such as the ingestion of a consistent diet can lead to loss of autochthonous members of the microbiota. Culture collections can be improved through understanding how to better select isolation methods and how to better evaluate both previously-cultured and -uncultured bacteria, as well as evaluating mechanistic processes involved in bacteria colonization of enteric habitats such as the inflamed tissues of the pig gastro-intestinal tract. Therefore, the goals of my research were to: 1) provide a better understanding of distinct differences between the effectiveness of direct plating and enrichment strategies to isolate diverse communities of bacteria within a chicken model; 2) expand evaluation of these comparisons to include the Ichip and endospore selection methods to isolate diverse communities of bacteria in a pig model; and finally, 3) identify candidate bacteria that are able to selectively colonize inflamed tissues. In the first study, 899 bacteria were isolated from chickens, which included the isolation of 75 unique bacterial taxa using various direct plating and enrichment methods. Differences between isolation methods were observed, with direct plating onto De Man, Rogosa and Sharpe agar (MRS) medium representing the lowest diversity of medium evaluated. In contrast, no differences were observed when comparing the diversity of bacterial communities isolated between enteric locations. These traditional isolation methods were further compared against more novel methods including the Ichip, as well as the use of ethanol or heat (i.e. tyndallization) to select for endospore forming bacteria. With these methods, 1,523 bacteria were evaluated spanning 80 genera and 7 phyla. No differences were observed when comparing the diversity of specific isolation strategies; in addition, it was determined that the use of heat to select for bacteria, generated collections that were less diverse than those obtained by enrichment broths, Ichips, or direct plating methods. The ability of these methods to isolate previously uncultured bacteria was also evaluated and demonstrated that enrichment and Ichip methods more frequently isolated previously uncultured bacteria than other isolation methods. In contrast, endospore selective methods were found to isolate novel bacteria less frequently than other methods; however, ethanol was more effective than heat at isolating novel bacteria. In the final study, these isolation methods were used to isolate bacteria from inflamed tissues of pigs. In this investigation 24 Landrace x Duroc piglets were inoculated with S. enterica var Typhimurium phage type DT104 or Columbia broth to generate inflammation (ST+; n = 12) or as a control (ST-; n = 12) respectively. The pigs challenged with Salmonella (ST+), had a reduction in feed consumption, and body weight, as well as increased body temperature, tissue inflammation with increased expression of mediators of inflammation and immune function. In this study it was also determined that the culturable diversity of bacteria differed from evaluations using culture-independent methods. This was most obvious as culture-dependent methods were predominantly populated by Bacillus spp., whereas culture-independent analyses were dominated by Prevotella spp. Culture-independent analyses determined that Prevoteallaceae and Enterobacteriaceae were more abundant within ST+ animals, whereas culturomics determined that Bacteroides spp. (B. uniformis, B. fragilis), Streptococcus spp. (S. gallolyticus), were associated with ST+ piglets. Ruminococcacea was associated with the microbiota of ST- piglets through both culture-dependant and -independent analyses. In conclusion, this research has evaluated various traditional and modern isolation strategies to provide researchers with a better understanding of the best methods to employ in future culturomics strategies, and identified members of the autochthonous microbiota that can be used as beneficial bacteria for host health or drug delivery strategies within the habitats of inflamed tissues.

• Subjects / Keywords

  • Anaerobic Microbiology
  • Inflammation
  • Pigs
  • Chickens

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-h1c0-6z64

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.