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'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.
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.
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
2. Isolation of anaerobic bacteria
3. Identification of anaerobic bacteria
4. Preservation of pure bacterial cultures
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 of sequencing results
DNA extraction of isolates having chromatogram data with excessive background noise or disruption in baseline, peak with low signals intensity, presence of multiple or overlapping peaks at single position, and inconsistent peak heights were repeated using fresh and pure culture as they indicate contamination of the sample, uneven amplification, or problem during sequencing process. On performing BLAST search in NCBI, the strain showing highest percent identity and high query cover was the related strain to our isolate leading to the identification of the isolate.
Cultivation of a broad spectrum of anaerobic bacteria
The isolation of anaerobic bacteria resulted in more than 600 isolates obtained in five different media prepared and sterilized in separate bottles. The identification using the 16S rRNA gene resulted in 15 different genera of bacteria (Figure 4). Among the total number of isolates, more than half represent strict anaerobic bacteria and the rest are facultative anaerobic.
The diversity of isolates includes eight strains, demonstrating novel taxonomic species related to members of the families Clostridiaceae, Lactobacillaceae and Oscillospiraceae (Table 5)25. These results highlight the significance of adhering to good practices in culture media preparation and use of diverse range of media for the success of cultivation studies.
Targeted isolation
The observation of colonies on minimal agar medium suggests the potential presence of anaerobic bacteria capable of utilizing MI as a carbon source (Figure 5). To conclusively confirm these findings at genetic level, comprehensive analysis through whole genome sequencing can be conducted. This sequencing approach enables the identification of specific genes associated with or contributing to MI metabolism, including iolA, iolB, iolC, iolD, iolE, iolF, iolG, iolH, iolI, iolJ, iolT, and iolW.
Figure 1: Process of sampling and transporting GIT sections for anaerobic bacteria cultivation. 1) Sections are clamped closed before cutting. The dotted lines indicate the recommended cut position. 2) Transfer of the sampled section into a Hungate tube filled with transportation solution. 3) Replacement of the air atmosphere by injecting transportation solution to maintain anaerobic conditions. Please click here to view a larger version of this figure.
Figure 2: Illustration of Hungate tubes containing samples obtained from ileum section of chicken. The image demonstrates proper filling of the Hungate tubes with transport solution, ensuring the absence of air space and maintenance of consistent color compared to the control having no sample. Noticeable turbidity in sample tubes is attributed to the dissolution of digesta. Please click here to view a larger version of this figure.
Figure 3: Sequential steps in the preservation of anaerobic bacteria. 1) Biomass harvest from 24 h to 48 h axenic culture in an isotonic solution. 2) Washing of bacterial cells by the process of centrifugation and supernatant removal to get rid of leftover media. 3) Repetition of washing process. 4) Pellet dissolution in culture media and 50% glycerol solution followed by storage at -80 °C in sterile cryovials. Please click here to view a larger version of this figure.
Figure 4: Taxonomic distribution of isolated bacteria at genus level. The stacked bar chart shows the diversity of bacteria isolated from five distinct media formulations, emphasizing the beneficial impact of using diverse media formulations in this study. The media formulation is listed in Table 1. Please click here to view a larger version of this figure.
Figure 5: Anaerobic bacterial colonies on minimal agar medium. Anaerobic bacterial colonies thriving on minimal agar media after 24 h of incubation at 39 °C. The development of colonies suggests the potential assimilation of myo-inositol as a primary carbon source, among other carbon sources. Two dilutions are presented, (A) 10-1, revealing a colony count surpassing a practical enumeration due to their abundance; and (B) 10-2, displaying distinguishable, countable colonies, facilitating selective analysis. Please click here to view a larger version of this figure.
Media | 1 | 2 | 3 | 4 | 5 |
Peptones media | Polysaccharides media | Short fatty acids media | Carbohydrates media | Sulphato-reducing bacteria (SRB) media | |
Source of C | Dextrose 0.2% | Poultry feed (2%) | Acetic acid 0.17%, Isovaleric acid 0.01%, Propionic acid 0.2%, Butyric acid 0.2% | Dextrose 5% | Na-DL-lactate, Ascorbic acid |
Starch 0.05% | |||||
Source of N | Amino acids from soybean (1%) | – | Amino acids from Tryptone and meat extract (0.1%) | Amino acids from soy peptones (0.1%), (NH4)3 citrate (0.1%) | NH4Cl (0.1%) |
Source of minerals | K2HPO4 (0.05%) | – | K2HPO4 (0.05%) | K2HPO4 (0.05%) | K2HPO4 (0.05%) |
Agar | 1.50% | 1.50% | 1.50% | 1.50% | 1.50% |
Cofactor (Added after sterilization) | Yeast extract (0.1%) | – | Vitamin mix (0.01%) | Vitamin mix (0.01%) | Yeast extract (0.01%) |
Inhibitors | none | none | Tween 80 (1%) | Menadione (0.05%) | – |
Table 1: Composition of the culture media for broad range of bacterial isolation. Composition of five agar media, each uniquely formulated based on variations in carbon source, nitrogen source, source of mineral, and the incorporation of co-factors and inhibitors.
Composition | Amount (g/L) |
Casein peptone | 17 |
Soya peptone | 3 |
Sodium chloride | 5 |
Dipotassium hydrogen phosphate | 2.5 |
Myo-inositol | 2.5 |
Table 2: Enrichment medium composition. Formulation of medium designed with a blend of complex ingredients to support the growth of a broad spectrum of bacteria. The quantity of each ingredient is specified in g/L.
Composition | Minimal Agar Media (g/mL)/ L | Minimal Broth Media (g/mL)/ L |
L-cystine | 0.5 | 0.5 |
Sodium chloride | 0.3 | 0.3 |
Sodium thioglycolate | 0.5 | 0.5 |
Sodium hydrogen phosphate dihydrate | 0.53 | 0.53 |
Potassium dihydrogen phosphate | 0.41 | 0.41 |
Ammonium chloride | 0.3 | 0.3 |
Calcium chloride | 0.11 | 0.11 |
Magnesium chloride | 0.1 | 0.1 |
Tryptone | 10 | 10 |
EDTA | 0.00773 | 0.00773 |
Trace element solution SL7 | 1 | 1 |
Vitamin mix | 0.2 | 0.2 |
Myo-inositol (20 mM) | 3.6 | 3.6 |
Agar | 15 | — |
pH (using 1 mM HCl) | 6 ± 0.2 | 6 ± 0.2 |
Table 3: Minimal medium composition. The medium is formulated with myo-inositol as the primary carbon source.
Initial Denaturation | 95 °C | 4 min | 1x |
Denaturation | 95 °C | 30 s | |
Annealing | 52 °C | 30 s | 30x |
Extension | 68 °C | 90 s | |
Final Extension | 68 °C | 10 min | 1x |
4 °C | ∞ |
Table 4: Optimal conditions for 16S rRNA gene amplification. The ideal temperature, duration, and cycling parameters to be employed in the thermocycler for efficient amplification of the target gene.
Novel taxonomy assignation | Genome size | GC content | Gut region | 16S accession |
Limosilactobacillus galli | 1861581 | 50.43 | crop | OM760982 |
Limosilactobacillus avium | 2158112 | 48.49 | ileum | OM760983 |
Limosilactobacillus pulli | 1924125 | 45.87 | ileum | OQ831033 |
Clostridium butanoliproducens | 3507657 | 28.34 | crop | OQ831034 |
Faecalispora anaeroviscerum | 2921858 | 50.36 | ileum | OM760984 |
Limosilactobacillus viscerum | 1993658 | 44.9 | ileum | OM760986 |
Ligilactobacillus hohenheimensis | 1381484 | 50.47 | jejunum | OM760988 |
Limosilactobacillus difficilis | 1998683 | 47.38 | ileum | OQ832131 |
Table 5. Taxonomic identification of novel bacterial species. The novel bacterial species isolated and identified from the gastrointestinal tract of chickens in this study, are detailed alongside their genome size, GC content, section, and 16S rRNA gene accession. This table has been modified from25.
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 mice1,26,27 report on selection of media for chicken is rather narrow and possibly underestimated even for sections of high interest such as feces and caeca2,28. In this sense, the formulation and preparation of media used in this work offered 80 different conditions that intend to cover the metabolic demands of microorganisms reported by sequencing approaches in other works29,30,31.
Along with the selection of media and supplementation, the formulation of culture media and interaction of the ingredients during preparation and sterilization is observed. The combination of phosphates and agar, reported to generate H2O2, was avoided by preparing the phosphate and agar solution separately. This modification improved the spectrum of isolates diversity by enhancing the viability of catalase negative bacteria. To avoid the formation of Maillard reaction products (MRP) during sterilization, carbohydrates were prepared in a separate solution and sterilized under different conditions. These two modifications increase the demand of material and worktime but provide the advantage to mix and formulate an exponential number of different media combinations.
The preparation of separate solutions in smaller volumes might represent a challenge during dissolution of the ingredients and finding adequate sterilization conditions to avoid precipitations. But culture media formulations normally do not reach saturation. We prefer filtered-sterilized mineral solutions dissolved at 60 °C to prevent precipitations. For solutions sterilized by heat and pressure in the autoclave, special attention was paid to carbohydrate solutions' oxidation at high temperatures and pressure. To avoid any chemical modification, all carbohydrate solutions must be sterilized at 110 °C for 30 min at 5 psi. These precautions improve the availability of nutrients such as essential amino acids, cofactors like copper, zinc, or iron, as well as the reduction on the hydrolytic activity of some proteins modified by the generated carbonyl compounds32.
Across studies, very few authors discuss the direct influence of culture media on the cultivation results. Common treatments applied in human cultivation studies (pre-incubation in blood culture, rumen fluid, or mucin) have been repeated indistinctively along gut culturomics studies on other species with different intestinal conditions2,28,33. In this work, the plant-based diet given to chicken supported the modification on the formulation of media by using major components of plant-based nutrients (peptides and carbohydrates) that mimic the GIT environment found in chicken and considered nutrient availability.
Most of the studies have a cultivation approach on the lower digestive regions of chicken (caeca and feces)2,3,28,34. Few cultivation approaches have isolated members of the family Lactobacillaceae, Clostridiaceae and Enterobacteriaceae35,36 from the upper digestive tract. However, none of them used the modern protocols for culturomics. In 2021, Zenner et al., reported the sole isolation of Ligilactobacillus aviarius obtained from ileum of chicken while the rest of the cultures originated from caeca. Contrastingly, the diversity recovered in this work reported a redundant isolation of L. aviarius, together with other species from the genera Lactobacillus, Ligilactobacillus and Limosilactobacillus25. This difference illustrates the advantage of using separately prepared media over commercially available ready-to-use formulations.
Maintaining anaerobic conditions throughout the isolation and cultivation process is a very crucial step, as even a small amount of oxygen can adversely affect the growth of anaerobes. To monitor and sustain anaerobic conditions during sample transportation, sodium resazurin is added to the transport solution. This chemical compound undergoes reduction when exposed to oxygen, resulting in a color transition from blue to pink. At low concentration, it does not impact bacterial viability. However, it is essential to avoid excessive concentrations, as they may lead to unintended impact on bacteria. Careful control of sodium resazurin levels ensures accurate monitoring of anaerobic conditions without compromising the integrity of the bacterial samples. For the targeted isolation, the pour plate method was employed using the spread plate method due to the advantage of maintaining a homogeneous distribution of bacterial colonies, facilitating the precise selection of individual bacteria. Furthermore, this method exhibits reduced susceptibility to airborne or external contamination. Additionally, it offers a consistent growth environment for diverse bacterial types, both fast- and slow-growing species allowing each bacterial strain to multiply at its own individual pace without interference from other microbial species.
For preservation, pellet-based glycerol stock was chosen over broth-based glycerol stock as it facilitates a higher bacterial concentration and lower susceptibility of contamination. This method proves to be an efficient method for preserving the bacterial culture at -80 °C, ensuring long-term storage stability.
The initial part of this methodology outlines an approach for isolating novel anaerobic cultures, with the primary goal to explore the diversity of previously uncultured strains within the chicken intestine. Cultivating and identifying anaerobic bacteria serves as a valuable tool for better understanding of poultry health, animal welfare, microbe-host interaction, and the potential isolation of probiotic strains. Additionally, it can also provide a deeper understanding of genetic makeup and metabolite capabilities, paving the way for advancements in manipulating the gut microbiota to achieve desired outcomes in poultry production. The subsequent part of the methodology focuses on the potential isolation of MI metabolizing anaerobes. This endeavor holds significant promise for elucidating the MI metabolization pathway, a process that has remained unclear to date. Notably, the versatility of these methodologies extends beyond chickens, offering an isolation technique applicable to various sample types. Therefore, the method presented in this study is one of the efficient approaches for isolation of anerobic cultures.
The authors have nothing to disclose.
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).
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 |
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