targeted isolation of specific myo-inositol utilizing bacterial groups from chicken digesta

Cultivation Strategies for Uncultured Microbial Diversity in the Chicken GI Tract

The resurgence of cultivation in studying microorganisms has complemented insights from metagenomic studies by providing material to test metabolic hypotheses that were previously only partially described and quantified. Cultivation of intestinal bacteria provides material to sustain future research on microbial-host interactions, facilitate targeted colonization studies, and improve molecular interaction studies1,2,3. The knowledge gained about gastrointestinal microorganisms has improved animal nutrition and welfare by influencing diet formulations and enhancing nutrient availability4. This understanding has contributed to performance improvements in utilizing prebiotic and probiotic interaction. However, in-depth research is required to gain a complete understanding of how biochemical and physicochemical conditions interact and impact the microbial profile and its structure. To achieve this objective, cultivation remains imperative, serving as a crucial tool to delve into the intricate dynamics of microbial communities within the gastrointestinal environment.
In contrast to the extensive research on microbes associated with the human gut and clinical cultivation studies5, reports on microorganisms from livestock have predominantly utilized a limited range of media for isolation, potentially constraining the diversity of isolates2,3. Furthermore, improvements in the formulation of media and studies on the interaction of phosphate and salts with agar, as elucidated by Tanaka et al. and Kawasaki et al., have not yet been implemented for gut-microbiome studies6,7,8,9.
Considered a semi-essential substance, myo-inositol (MI) has been reported to play a pivotal role in diverse metabolic, physiological, and regulatory processes10,11. These include involvement in bone mineralization, breast muscle development, cellular signaling, promotion of ovulation and fertility, modulation of neuronal signaling, and acting as a regulator of glucose homeostasis and insulin regulation in poultry10,11. MI plays a role as a precursor through its interconversion within pivotal biochemical processes, including the glycolysis/gluconeogenesis process, the citric acid cycle, and the pentose phosphate pathway. Additionally, it also serves as a precursor of phosphatidylinositol (PI), which is further involved in glycerophospholipid metabolism12. Few investigations have reported that the metabolization of MI leads to alterations in bone stability and animal performance. This includes enhancements in feed conversion rate and body weight gain, demonstrating its impact after absorption and utilization within the animal13,14. However, the pathway for MI metabolization and its impact on poultry metabolism remains elusive15. Furthermore, few studies propose a potential role of bacteria in MI utilization, particularly in regions of high metabolic activity such as the ileum16,17,18,19.
Efforts on cultivating bacteria from the GIT of animals aim to enhance genomic databases and expand research, verify genome-based hypothesis, and understand the ecological importance of these resources20. The objective of this work is to improve strategies for bacterial cultivation from the GIT of chicken to enhance the isolation diversity and the targeted isolation of an ecological group of interest that assimilate and metabolize myo-inositol.

Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken

We aim to isolate and cultivate bacteria from the intestine of chickens to improve our knowledge of microbial networks and interactions with the host. The characterization of these anaerobic bacteria supports the knowledge of intestinal processes and enrich bacteria databases for future research. One of the main challenges is the prevention of oxygen exposition and oxygen-reactive species along the sampling and cultivation process to prevent bacterial inactivation.To our experience, this has improved the diversity recovery of anaerobic bacteria. Our protocol addresses the gap in understanding myo-inositol metabolism in poultry. By isolating potential myo-inositol-metabolizing bacteria from chicken intestine, our goal is to shed light on the specific bacterial community and metabolic pathways involved, advancing knowledge in this area.Our primary goal is understanding how microbial communities interact with a host, especially in the gut. This is where diet is broken down, nutrients are absorbed, and significant interactions occurs with these microorganisms.

Targeted Isolation of Specific Myo-Inositol Utilizing Bacterial Groups from Chicken Digesta

To begin, homogenized chicken digest are harvested from an extracted chicken intestine on a vortex for 10 to 15 seconds. Transfer the homogenized sample into the tube containing enrichment medium. After incubation, use a vortex mixer to mix the enriched tube thoroughly for 10 to 15 seconds.Then transfer the mixed sample to sterile minimal broth medium before incubating as before. Next, serially dilute the samples in sterile saline solution, starting with a one to 10 dilution and continuing until one to 1000 dilution. Homogenize the sample thoroughly on a vortex mixer after each dilution.Transfer one milliliter of the diluted sample to a Petri dish under sterile and anaerobic conditions. Then pour 15 to 20 milliliters of melted minimal agar medium into the Petri dish. Swirl the dish gently to ensure uniform mixing.When the agar has solidified, incubate the dishes in an inverted position in an anaerobic station for 24 hours. at 39 degrees Celsius. The colonies should appear as distinct, small embedded dots at the end of incubation.Now use a sterile inoculation loop to carefully pick each individual bacterial colony. Transfer the colonies into separate tubes containing five milliliters of minimal broth medium. The increased turbidity of the end of incubation is indicative of bacterial growth.Bacterial colonies capable of utilizing myo-inositol were observed on minimal agar medium.

Research

JoVE Journal

Biology

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, ...