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

Summary

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.

Abstract

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host'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'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's GIT.

Introduction

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 studies^1,2,3. 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 isolates^2,3. 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 studies^6,7,8,9.

Considered a semi-essential substance, myo-inositol (MI) has been reported to play a pivotal role in diverse metabolic, physiological, and regulatory processes^10,11. 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 poultry^10,11. 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 animal^13,14. 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 ileum^16,17,18,19.

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.

Protocol

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 Regierungspräsidium Tübingen, Germany with the approval numbers HOH50/17 TE and HOH67-21TE.

1. Obtaining samples for the cultivation of anaerobic bacteria

1. Maintenance of animals
   1. Maintain animals on an ad libitum commercial corn-based diet (Legehennen/ Junghennenfutter; see Table of Materials) and house at the experimental station of the University of Hohenheim.
2. Preparation of transport solution
   1. At 1-2 days before sample collection, prepare the transport solution containing 1.00 g/L sodium thioglycolate, 0.10 g/L calcium chloride dihydrate (CaCl₂.2H₂O), and 0.5% of cysteine²¹ and 0.1% sodium resazurin solution.
   2. Adjust the pH of the medium to 6.0 ± 0.2 at 25 °C and autoclave at 121 °C for 15 min at 15 psi pressure.
   3. After the medium has cooled to approximately 50 °C, replace the cap of the medium bottle with sterile screw cap with a bore and a rubber stopper. Insert two sterile syringe needles into the rubber stopper at different positions and affix a hydrophobic syringe filter (0.22 µm) onto one of the needles.
      NOTE: Replacement of caps and insertion of needles and filter should be carried out inside a laminar air flow (LAF).
   4. Attach the 100% nitrogen (N₂) tube to the syringe filter, and sparge N₂ gas into the bottle for 10 min at 1 psi.
   5. Subsequently, transfer the medium bottle and sterile Hungate tubes into the anaerobic station and aseptically transfer the transport medium into the Hungate tubes. Completely fill the Hungate tubes to the top, ensuring absence of any air inside.
3. Slaughtering and sample collection
   1. During the slaughtering process, maintain axenic conditions to avoid contamination by using gloves, mask, plastic lab apron, and sterile surgical instruments throughout the entire procedure. Moreover, properly sanitize the working bench using ethanol and tissues to uphold a sterile environment.
   2. In the experimental station, anesthetize 10, 50-week-old Lohmann laying hens using a bottled gas mixture consisting of 35% N₂, 35% CO₂, and 30% O₂ and immediately decapitate them.
      NOTE: The anesthetization and sacrificing should be conducted by a laboratory animal technician well-versed in animal welfare and scientific procedures. This ensures that the sacrificing procedure adheres to the ethical standards, legal regulations, and safety guidelines.
   3. Before proceeding with the dissection of the GIT section, secure the required intestinal segment, namely the crop, ileum, and jejunum, using sterile Kelly hemostat (also known as a Kelly clamp; Figure 1).
      NOTE: The dissection of the animal should be performed by either a veterinarian or a well-trained person who is familiar with the anatomy of chickens.
   4. Transfer each individual section into a separate Hungate tube filled with transport medium and close the lid properly. If required, add extra transport medium to the tube to displace any remaining air.
      NOTE: The transfer of the section is a crucial step, and it is essential to minimize any delay or opening of the section to prevent prolonged exposure to oxygen
4. Transportation of sample
   1. Handle the transportation of the Hungate tubes with care, placing them inside a Styrofoam box to prevent any risk of breakage. In case the outside temperature is lower than 15 °C, maintain the temperature of the sample by using warm packs. Ensure the time of transportation does not exceed 4 h.
   2. Transfer the tubes carefully inside the anaerobic station for further process.

2. Isolation of anaerobic bacteria

1. Culture media preparation
   NOTE: The outlined method presents various considerations for culture media preparation. Table 1 illustrates an example of how solutions are divided for the diverse isolation of microorganisms.
   1. Divide the ingredients of the media formulation to be used in four solution-groups: carbon source, nitrogen source, agar, and mineral source. For rich media like Brain-Heart infusion (BHI), separation is not always possible (see example of medium 2 in Table 1). In such cases, ensure separate preparation and autoclaving of agar or additional solutions.
   2. Prepare the carbohydrate solution in 1/4^th of the final volume of media required, e.g., in 250 mL for a total volume of 1 L of media. Following the example of medium 1 in Table 1, dissolve 2 g of dextrose and 0.5 g of starch in 250 mL of distilled water.
   3. Prepare the protein solution in 1/4^th of the final volume of media required, e.g., in 250 mL for a total volume of 1 L of media. Following the example of medium 1 in Table 1, dissolve 10 g of peptone in 250 mL of distilled water.
   4. Prepare agar solution in 45% to 48% of the final volume of media, e.g., in 450 to 480 mL for a total volume of 1 L of media. Following the example of mediu 1 in Table 1, dissolve 15 g of agar in 450 to 480 mL of distilled water.
   5. Prepare the mineral solution in the left 20 to 50 mL volume of media when the final volume of media is 1 L. Following the example of medium 1 in Table 1, dissolve 0.5 g of K₂HPO₄ in 20 to 50 mL of distilled water.
   6. Autoclave the protein solution, agar solution, and mineral solutions at 121 °C for 15 min at 21 psi pressure. Autoclave the carbohydrate solution at 110 °C for 30 min at 5 psi.
   7. Once all solutions are sterile, cool them down to 50 °C and mix under sterile conditions into an appropriate autoclaved glass bottle with a cap having a bore and a rubber stopper. After this, follow steps 1.2.3 and 1.2.4.
      NOTE: The mixing of solutions should be done inside a LAF.
   8. Once the media has been mixed and degassed, pour into sterile Petri dishes or tubes inside the anaerobic station.
2. Isolation of anaerobic bacteria
   NOTE: The following methodology explains an isolation strategy for cultivating a broad range of anaerobic bacteria that inhabit the digestive tract of chicken.
   1. Transfer the samples to an anaerobic station containing a bottled gas mixture composed of 80% N₂ (quality level 5.0), 15% CO₂ (quality level 3.0) and 5% H₂ (quality level 5.0) provided by a commercial supplier (see Table of Materials).
   2. Inside the anaerobic station, transfer the section from the hungate tube to the sterile Petri dish using autoclaved forceps.
   3. Carefully cut open the section using a sterile pair of scissors and extract approximately 1 g of digesta content from the intestinal segment using a sterile spatula.
      NOTE: After taking 1 g of digesta content, scrap and transfer the remaining digesta to its respective transport media tube for the targeted isolation.
   4. Perform a 10-fold serial dilution using a sterile physiological solution (0.85% NaCl).
   5. Dispense 0.1 mL of the sample from dilutions 10−4 to 10−7 into sterile and properly labeled Petri dishes media plates and spread with a sterile Drigalski spatel.
   6. Incubate the plates inside the anaerobic station for 24–48 h at 39 °C.
      NOTE: When incubating inside an incubator, transfer the Petri dishes into an airtight box before taking the Petri dishes out of the anaerobic station.
3. Targeted isolation of a specific bacterial group.
   NOTE: The following methodology explains an isolation strategy for anaerobic bacteria that can potentially assimilate MI.
   1. For the preparation of enrichment medium, minimal agar medium, and minimal broth medium, follow the composition specified in Table 2 and Table 3, adhering to the guidelines outlined in step 2.1. Add 0.2 mL/L of filter-sterile vitamin mixture (as outlined in the composition) into minimal agar media and minimal broth media after the autoclaving process.
   2. Homogenize the initial sample using a vortex mixer for 10-15 s. Subsequently, transfer the homogenized sample into the tube containing enrichment medium at a rate of 5% (e.g. if the volume of enrichment media is 10 mL, inoculate 0.5 mL of homogenized sample) using a pipette. Incubate the freshly inoculated sample for 24 h at 39 °C inside the anaerobic station.
   3. Following incubation, mix the enriched tube thoroughly by either using a vortex mixer for 10-15 s or by pipetting. Subsequently, transfer the mixed sample to sterile minimal broth medium at a rate of 5% and then incubate for 24 h at 39 °C.
   4. Serially dilute the samples in sterile saline solution (i.e., 0.85% NaCl), starting with a 1:10 dilution and continuing till 1:1000 dilution. Following each dilution, homogenize the sample thoroughly at room temperature by using a vortex mixer for 10-15 s or by pipetting.
   5. Under sterile and anaerobic conditions, transfer 1 mL of diluted sample to a Petri dish. Pour approximately 15-20 mL of melted minimal agar medium into the Petri dish and gently swirl horizontally for uniform mixing.
      NOTE: The temperature of agar should be around 45 °C so that no lumps are present and to prevent the sample from being killed by heat. Medium pouring should be done under anaerobic conditions.
   6. Upon agar solidification, incubate the Petri dishes for 24 h at 39 °C in inverted position inside an anaerobic station or in incubator. After 24 h of incubation, bacterial colonies appear as distinct, small dots embedded in the media.
      NOTE: When incubating the Petri dish inside an incubator, transfer the plates to an airtight box before taking them out of anaerobic station.
   7. Using a sterile inoculating loop, carefully pick individual bacterial colony and transfer it into separate tubes of minimal broth medium.
   8. After transferring the colonies to individual tubes, anaerobically incubate the inoculated tubes at 39 °C for 24 h. After incubation, increased turbidity in the media will indicate bacterial growth.
      NOTE: Every time before transferring the media bottles inside the anaerobic station, they should be degassed as mentioned earlier in step 2.1.7. Steps 2-8 should be carried out inside the anaerobic station.

3. Identification of anaerobic bacteria

1. Extract DNA from cultures of 24 h to 48 h incubation time, following an enzymatic lysis protocol²².
2. Centrifuge the culture at 3000 x g for 10 min at 4 °C and discard the supernatant using a pipette.
3. Wash the pellet 2x with phosphate buffered saline (PBS) by suspending in 2 mL of PBS. Homogenize the sample by using a vortex mixer for 10-15 s. Repeat the centrifugation step at 3000 x g for 10 min at 4 °C and discard the supernatant carefully.
4. Suspend the cell pellet in 0.2 mL of PBS pH 7.4 and incubate at 50 °C with 1 U of lysozyme and 1 U of recombinant mutanolysin for 30 min, followed by an incubation of 30 min at 56 °C with 5 µL of proteinase K.
5. Add 4 µL unit of RNAseA to each tube and incubate at room temperature for 10 min. Centrifuge tubes at 21168 x g for 1 min. Recover the supernatant and purify using magnetic beads.
6. Perform quantification of DNA with a fluorometric method according to manufacturer instructions for dsDNA quantification kit (mentioned in Table of Materials).
   NOTE: Extracted DNA can be stored at -20 °C.
7. Use DNA samples as templates for PCR amplification using the primers 27f and 1492r²³ following the conditions shown in Table 4. To ensure that the PCR reaction was successful, perform agarose gel electrophoresis according to standard agarose gel electrophoresis method using 1X TAE buffer and 1% agarose.
8. Purify the PCR product using a commercial PCR purification kit to get rid of primers, nucleotides, and other contaminants.
9. Send the PCR product for Sanger sequencing to the suitable sequencing service provider.
   NOTE: Along with sequencing, service providers can also provide services including purification of the PCR product with extra fees. PCR products can be stored at 4 °C or at -20°C.
10. After getting the sequencing results in FASTA format along with chromatogram file (commonly in ABI or SCF format), analyze sequences using bioinformatics tools. Use sequence analysis software to open and visualize the chromatogram file.
    NOTE: The overall quality of chromatogram was checked based on clear and distinct peaks without excessive noise
11. Compare amplicons and align to the closely related species at the non-redundant GenBank 16S ribosomal RNA database from the National Center for Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) tool²⁴ and the Refseq genome database.
    NOTE: When using NCBI BLAST, copy the whole FASTA sequence in the search bar.

4. Preservation of pure bacterial cultures

1. Harvest bacterial cells from a well-grown culture of 24 h to 48 h either by centrifugation (3000 x g for 10 min) or biomass collection from an axenic culture on a plate.
2. Suspend the bacterial culture in a suitable isotonic sterile solution (NaCl 0.85%) and wash by centrifuging the bacterial solution at 3000 x g for 10 min at 4 °C.
3. Discard the supernatant and resuspend the bacterial pellet in a small volume of sterile isotonic solution (0.5 mL). To ensure the removal of any growth medium residue, repeat 1x.
4. Resuspend the pellet in 0.8 mL to 1 mL of sterile culture medium concentrate and homogenize either with a vortex mixer for 5-10 s or by pipetting gently.
5. Transfer 0.5 mL of cell suspension to a sterile and properly labelled 1 mL cryovial and add 0.5 mL of autoclaved 50% glycerol solution. Homogenize the cryovial using a vortex mixer, and store in a freezer rack at -80 °C (Figure 3).
   NOTE: All steps before transferring the cryovials to -80 °C should be done inside the anaerobic station.

Results

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...

Discussion

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 mice^1,26,

Materials

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