Name: Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry
Uploaded: 2024-05-10T14:30:00
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry at JoVE.com

The authors acknowledge the Rehovot-Hohenheim partnership program and Deutsche Forschungsgemeinschaft (DFG) SE 2059/7-2. This project was developed as part of the research unit P-FOWL (FOR 2601).

The protocol is divided into four parts: sampling, bacterial isolation, identification, and preservation of the obtained microorganisms. Approved permissions on the use of animals were issued by the ethical commission of Regierungspr&#228;sidium T&#252;bingen, Germany with the approval numbers HOH50/17 TE and HOH67-21TE.
1. Obtaining samples for the cultivation of anaerobic bacteria
<ol>
	<li>Maintenance of animals
	<ol>
		<li>Maintain animals on an ad libitum comme...

Cultivation Strategies for Uncultured Microbial Diversity in the Chicken GI Tract

<ol>
	<li>Wylensek, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+collection+of+bacterial+isolates+from+the+pig+intestine+reveals+functional+and+taxonomic+diversity.">A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity.</a> Nat Comm. 11 (1), 6389 (2020).</li><li>Medvecky, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+genome+sequencing+and+function+prediction+of+133+gut+anaerobes+isolated+from+chicken+caecum+in+pure+cultures.">Whole genome sequencing and function prediction of 133 gut anaerobes isolated from chicken caecum in pure cultures.</a> BMC Geno. 19 (1), 561 (2018).</li><li>Zenner, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early-life+immune+system+maturation+in+chickens+using+a+synthetic+community+of+cultured+gut+bacteria.">Early-life immune system maturation in chickens using a synthetic community of cultured gut bacteria.</a> mSystems. 6 (3), 1110-1128 (2021).</li><li>Borda-Molina, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+on+the+ileal+microbiota+of+phosphorus+and+calcium+utilization,+bird+performance,+and+gender+in+japanese+quail.">Effects on the ileal microbiota of phosphorus and calcium utilization, bird performance, and gender in japanese quail.</a> Animals. 10 (5), 885 (2020).</li><li>Bonnet, M., Lagier, J. 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I., Ohtani, K., Okumura, K., Hayashi, H., Shimizu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+transcriptional+regulation+of+myo-inositol+operon+in+Clostridium+perfringens.">Organization and transcriptional regulation of myo-inositol operon in Clostridium perfringens.</a> FEMS Microbiol Lett. 235 (2), 289-295 (2004).</li><li>Hellinckx, J., Heermann, R., Felsl, A., Fuchs, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+binding+affinity+of+repressor+IolR+avoids+costs+of+untimely+induction+of+myo-inositol+utilization+by+Salmonella+Typhimurium.">High binding affinity of repressor IolR avoids costs of untimely induction of myo-inositol utilization by Salmonella Typhimurium.</a> Sci Rep. 7 (1), 44362 (2017).</li><li>Yebra, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+gene+cluster+enabling+Lactobacillus+casei+BL23+to+utilize+myo-inositol.">Identification of a gene cluster enabling Lactobacillus casei BL23 to utilize myo-inositol.</a> Appl Environ Microbiol. 73 (12), 3850-3858 (2007).</li><li>Zhang, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+iol+clusters+in+Lactobacillus+casei+strains.">Comparative analysis of iol clusters in Lactobacillus casei strains.</a> World J Microbiol Biotechnol. 26, 1949-1955 (2010).</li><li>Labeda, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bergey's manual of systematic bacteriology. , (2001).</li><li>Peterson, L. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+culturomics:+Paradigm+shift+in+the+human+gut+microbiome+study.">Microbial culturomics: Paradigm shift in the human gut microbiome study.</a> Clin Microbiol Infect. 18 (12), 1185-1193 (2012).</li><li>Lagkouvardos, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+intestinal+bacterial+collection+(mibc)+provides+host-specific+insight+into+cultured+diversity+and+functional+potential+of+the+gut+microbiota.">The mouse intestinal bacterial collection (mibc) provides host-specific insight into cultured diversity and functional potential of the gut microbiota.</a> Nat Microbiol. 1 (10), 1-15 (2016).</li><li>Gilroy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+microbial+diversity+within+the+chicken+gut+microbiome+revealed+by+metagenomics+and+culture.">Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture.</a> PeerJ. 9, e10941 (2021).</li><li>Huang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+gut+metagenome+and+the+modulatory+effects+of+plant-derived+benzylisoquinoline+alkaloids.">The chicken gut metagenome and the modulatory effects of plant-derived benzylisoquinoline alkaloids.</a> Microbiome. 6 (1), 1-17 (2018).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+microbial+genomes+and+gene+catalog+of+the+chicken+gut+from+metagenomic+sequencing+of+high-fidelity+long+reads.">Improved microbial genomes and gene catalog of the chicken gut from metagenomic sequencing of high-fidelity long reads.</a> GigaScience. 11, 116 (2022).</li><li>Segura-Wang, M., Grabner, N., Koestelbauer, A., Klose, V., Ghanbari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-resolved+metagenomics+of+the+chicken+gut+microbiome.">Genome-resolved metagenomics of the chicken gut microbiome.</a> Front Microbiol. 12, 726923 (2021).</li><li>Borrelli, R. C., Fogliano, V., Monti, S. M., Ames, J. 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The purpose of this methodology is to enhance the cultivation of anaerobic intestinal bacteria by improving the quality of sampling conditions, sample processing, and media formulation and preparation. The physicochemical conditions of samples (pH, the availability of carbon, nitrogen, and cofactors) must be taken into consideration when formulating the culture media. Compared to bacterial culture collections obtained from pigs, humans, or mice1,26,<su...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>VWR</td>
			<td>20104.334</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>VWR</td>
			<td>97064-332</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Carl Roth</td>
			<td>P726.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerobic station</td>
			<td>Don Whitley Scientific</td>
			<td>A35 HEPA</td>
			<td></td>
		</tr>
		<tr>
			<td>Butyric acid</td>
			<td>Merck</td>
			<td>8.0045.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>VWR</td>
			<td>97061-904</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken lysozyme (Muramidase)</td>
			<td>VWR</td>
			<td>1.05281.0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteine</td>
			<td>VWR</td>
			<td>97061-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>VWR</td>
			<td>90000-908</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-potassium hydrogen phosphate</td>
			<td>Carl Roth</td>
			<td>P749.1</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Legehennen/ Junghennenfutter</td>
			<td>Deutsche Tiernahrung Cremer GmbH &#38; Co. KG, D&#252;sseldorf, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>MagAttract HMW DNA Kit</td>
			<td>Qiagen</td>
			<td>67563</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Carl Roth</td>
			<td>2189.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixed gas (80% N2 (quality level 5.0), 15% CO2 (quality level 3.0) and 5% H2 (quality level 5.0))</td>
			<td>Westfalen Gase GmbH, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Mutanolysin, recombinant (lyophilisate)</td>
			<td>A&#38;A Biotechnology</td>
			<td>1017-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>Myo-inositol</td>
			<td>Carl Roth</td>
			<td>4191.2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>ChemSolute</td>
			<td>8418.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Carl Roth</td>
			<td>6026.1</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiFluor dsDNA System</td>
			<td>Promega</td>
			<td>E2671</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse A&#160;</td>
			<td>QIAGEN Ribonuclease A (RNase A)&#160;</td>
			<td>19101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>27800.291</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium resazurin</td>
			<td>VWR</td>
			<td>85019-296</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thioglycolate</td>
			<td>Sigma-Aldrich</td>
			<td>102933</td>
			<td></td>
		</tr>
		<tr>
			<td>Soy Peptone, GMO-Free, Animal-Free</td>
			<td>VWR</td>
			<td>97064-186</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Carl Roth</td>
			<td>8952.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween80</td>
			<td>Carl Roth</td>
			<td>9139.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin mix (supplement)</td>
			<td>VWR</td>
			<td>968290NL</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Star Lab</td>
			<td>07127/92930</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Carl Roth</td>
			<td>9257.05</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-D-Fructose</td>
			<td>VWR</td>
			<td>53188-23-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

strategies to enhance cultivation of anaerobic bacteria from gastrointestinal tract of chicken

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, digestion, nutrient absorption, immune system maturation, and prevention of pathogen intrusion. For optimal animal health and productivity, it is imperative to characterize these microorganisms and comprehend their role. While the GIT of poultry holds a reservoir of microorganisms with potential probiotic applications, most of the diversity remains unexplored. To enhance our understanding of uncultured microbial diversity, concerted efforts are required to bring these microorganisms into culture. Isolation and cultivation of GIT-colonizing microorganisms yield reproducible material, including cells, DNA, and metabolites, offering new insights into metabolic processes in the environment. Without cultivation, the role of these organisms in their natural settings remains unclear and limited to a descriptive level. Our objective is to implement cultivation strategies aimed at improving the isolation of a diverse range of anaerobic microbes from the chicken&#39;s GIT, leveraging multidisciplinary knowledge from animal physiology, animal nutrition, metagenomics, feed biochemistry, and modern cultivation strategies. Additionally, we aim to implement the use of proper practices for sampling, transportation, and media preparation, which are known to influence isolation success. Appropriate methodologies should ensure a consistent oxygen-free environment, optimal atmospheric conditions, appropriate host incubation temperature, and provision for specific nutritional requirements in alignment with their distinctive needs. By following these methodologies, cultivation will not only yield reproducible results for isolation but will also facilitate isolation procedures, thus fostering a comprehensive understanding of the intricate microbial ecosystem within the chicken&#39;s GIT.

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.

Monitoring of anaerobic conditions during transportation 
Due to addition of sodium resazurin, the change in color of transport solution to pink before the transfer of sample into the tube indicates a disruption or failure in maintenance of anaerobic conditions. Hence, the tube showing color change were refrained from being used during transport and only the tubes showing no color change were used, as can be seen in Figure 2.
Analysis...

Read this Scientific Article Protocol about Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry at JoVE.com

The protocol presents two methodologies to improve the isolation of anaerobic intestinal bacteria. The first focuses on the isolation of a diverse range of bacteria using different culture media. The second focuses on the cultivation steps of a specific microbial group, possibly assimilating myo-inositol, to fully comprehend its ecological significance.

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, ...

strategies-to-enhance-cultivation-anaerobic-bacteria-from

Research

JoVE Journal

Biology

Anaerobic Bacterial Isolation from Chicken Digesta as a Cultivation Improvement

To begin, combine 250 milliliters of each carbohydrate, protein, agar and mineral solutions in an autoclave glass bottle. Seal the bottle with a cap having a bore and a rubber stopper. After degassing the mixture with nitrogen, pour it into sterile Petri dishes inside an anaerobic station.Next, transfer the chicken intestinal samples to the anaerobic station containing a bottled gas mixture of nitrogen, carbon dioxide, and hydrogen. With a pair of sterile forceps, transfer the intestinal samples from the Hungate tube to the sterile Petri dish. Use sterile scissors to carefully cut the open section.Then use a sterile spatula to extract one gram of the digested content. Transfer the digested content into a tube containing a sterile physiological solution, and perform tenfold serial dilution. Pipette 0.1 milliliter of the sample from dilutions 10 to the negative fourth to 10 to the negative seven into sterile and properly-labeled media plates.Spread the sample with a sterile Drigalski spatula. Finally incubate the Petri dishes inside the anaerobic station in an inverted position for 24 to 48 hours at 39 degrees Celsius. 15 different genera of bacteria were isolated.The diversity of isolates includes eight strains, demonstrating novel taxonomic species related to members of the families Clostridiaceae, Lactobacillaceae, and Oscillospiraceae.

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.