Identification and Characterization of Probiotics Isolates from Indigenous Chicken (Gallus domesticus) of Nepal

Introduction

An increasing global population and food security needs have imposed a great pressure on poultry and livestock sectors to increase their production utilizing limited resources (1).Consequently, sub-therapeutic doses of antibiotics are being used widely in poultry as animal growth promoters and for prophylaxis (2). This is particularly true in low and middle income countries where poultry industries are playing an important role in their national economy (3, 4), and where regulations on the use of antimicrobials are often weak (5). Such routine and irrational use of antimicrobials has increased the risk of emergence and spread of antimicrobial resistance (AMR) among poultry associated bacterial population, endangering both poultry and human health (6, 7).

Probiotics are both live microorganisms and their metabolites, which when taken orally, provide health benefits to the receiving host by preventing enteric diseases and/or enhancing health performance and productivity (8, 9). There are numerous ongoing efforts to discover effective probiotics strains for poultry health with similar beneficial effects as antibiotic growth promoters (AGPs). A successful probiotics candidate must be non-pathogenic and produce desirable in vivo benefits while surviving hosts’ gastro-intestinal environment, such as tolerance to acidic pH and high bile salt concentrations. Additionally, such probiotics must have a good adherence to the intestinal epithelium and symbiotic colonization with the natural gut microbiota (10). Lactic acid bacteria (LAB) including different species and strains of Lactobacillus are one of the most common types of bacterial microorganisms fulfilling these characteristics, and hence are increasingly used as probiotics (11, 12).

Several formulations of probiotics based on different strains of bacterial and non-bacterial agents are widely used in poultry sectors in high income countries. However, none are either produced, or used in developing countries like Nepal. Compared to commercial formulations composed of non-native strains, the use of probiotics bacterial strains isolated from indigenous chicken (Gallus domesticus) breeds may offer unique host-specific advantages of improved gut adherence and survival. In this study, we isolated LAB strains from cloacal swabs of various indigenous chicken breeds from Nepal that survived on natural foraging (without any known exposure to antibiotics), and selected potential probiotics candidates based on in vitro challenge tests for survival in presence of varying bile concentrations and acidic pH, percentage yield of lactic acid and inhibition of selected bacterial pathogens-thereby identifying bacterial isolates with unique and improved probiotics characteristics.

Materials and methods

Specimen collection and preparation

Free-ranging backyard chicken primarily belonging to various indigenous breeds reared in rural outskirt hills of the Kathmandu valley were selected for sampling (Supplemental table 1, Figure 1). The selected birds lacked exposure to commercial and/or antibiotics supplemented feed. Cloacal samples (n=41) were collected from each chicken using sterile swabs and preserved in cryo-vials containing 50% sterile glycerol. The samples were then transported in cold chain box to the BIOVAC laboratory in Kathmandu and stored at -20 °C.

Figure 1: The map of sampling districts (yellow colored) of Nepal showing the sampling locations indicated as numbered circles. The sample locations are as follows: 1) Lakuribhanjyang, 2) Parthali-Bethankchowk, 3) Chalnakhel-Bosandanda and 4) Jhor-Dhakalchaur. The diameter of sampling locations are shown as varying proportionately with the number of samples collected from that location as indicated in figure legend.

Selective isolation of LAB isolates

Glycerol stock of cloacal swabs was thawed in room temperature. After vortexing, 50 µl of each sample was inoculated into 5 ml of DeMan, Rogosa and Sharpe (MRS) Broth (HiMedia, India) with 0.5% calcium carbonate (CaCO₃). The inoculated broth was incubated at 37 °C for 72 hours in carbon-dioxide enriched environment using candle jar incubation method.

After incubation, 50 µL of inoculated broth was streaked on MRS agar (HiMedia, India) with 0.5% calcium carbonate. The agar plates were incubated at 37 °C for 24 hours in carbon-dioxide enriched environment. White to cream colored round colonies (3-4 mm) producing clear hydrolyzing halos were selected as potential LAB isolates, which were further re-plated on 0.5% CaCO₃ supplemented MRS agar for pure culture isolation. The overnight MRS broth culture of each selected LAB isolates were prepared, and subjected to further in-vitro screening and challenge tests as below.

Test for anti-bacterial activity

The anti-bacterial activity of selected LAB isolates were tested against a panel of bacterial pathogens using agar well diffusion method (13). Surface of Muller Hinton Agar (MHA) plates were lawn cultured with 0.5 O.D₆₃₀ adjusted bacterial suspension of six selected pathogens (Salmonella enterica spp., Escherichia coli, Shigella spp., Klebsiella pneumonia, Citrobacter freundii, and Staphylococcus aureus). After 15-20 minutes, six wells (≥ 30 mm apart) were bored with the broader end of a sterile pipette tip on the inoculated MHA agar. A 100 μl of the overnight MRS broth culture of each selected LAB isolate (adjusted to 0.5 O.D₆₃₀) was then inoculated into the wells. The broth was allowed to diffuse into the agar for approximately an hour by incubating the covered plates at 4°C in an upright position. After complete diffusion, the plates were incubated in an inverted position at 37°C for 24 hours. Development of clear zone of bacterial inhibition around each well was noted as positive anti-bacterial activity, and sizes of inhibition zone were recorded.

Phenotypic bacterial identification

Presumptive phenotypic bacterial identification of the selected LAB isolates demonstrating anti-bacterial activity was performed by using standard microbiological methods, which included Gram staining and biochemical tests for catalase, oxidase, sulfur and indole production (14). The Gram positive rod shaped bacteria that were negative for catalase, oxidase, sulphur, and indole production were phenotypically identified as Lactobacillus spp.

Test for production of total lactic acid

Lactic acid production by presumptive Lactobacillus spp. isolates was quantified. For this, overnight MRS broth culture of each Lactobacillus spp. was subjected to 10^{^-1}dilution in sterile distilled water. To this, 3 drops of 1% phenolphthalein was added, and was titrated with 0.1 N NaOH solution until a light pink colour was observed (15). The volume of 0.1 N NaOH required to achieve neutralization was used to calculate the percentage yield of lactic acid using by the following calculation (16, 17):

The actual yield is the amount of lactic acid formed as a result of bacterial fermentation during overnight incubation, and theoretical yield is the maximum amount of lactic acid presumed to be produced by homo-fermentative LAB strains from available amount of fermentable sugar present in the MRS broth (i.e., 0.02 gm/ml).

Test for acid tolerance

Overnight incubated MRS broth of potential Lactobacillus spp. was centrifuged at 1,000 rpm for 10 minutes. Resulting bacterial pellet was re-suspended in 2ml of sterile saline. One ml of this bacterial suspension was added to 9 ml of sterile artificial gastric juice media (0.2% NaCl, 0.35% pepsin, adjusted to pH 3.0). Another one ml of bacterial suspension was added to 9 ml of sterile MRS broth (adjusted to pH 7.0). The tubes were incubated at 37 °C for 3 hours and O.D₆₀₀ was measured for both the tubes after incubation (18). For calculation, bacterial density at O.D₆₀₀ was empirically equated to the bacterial count in colony forming unit (CFU)/ml as per the growth curve calculation of Lactobacillus plantarum as described by Trabelsi et al.(19). Acid tolerance ability of bacterial isolates was estimated using the following formula (18):

Test for bile salt tolerance:

A set of MRS broth with 0.2% sodium acetate, each supplemented with cattle bile salt (concentrations of 0.0%, 0.3%, 0.5%, and 1%) were prepared. Overnight activated MRS bacterial suspension (20 μl) was inoculated in the broth (180 μl) and incubated at 37 °C for 24 hours. After incubation, O.D₆₀₀ was measured using micro-plate absorbance reader (BioTek, USA). Bacterial absorbance density at O.D₆₀₀ was empirically equated to the bacterial count in CFU/ml as per the growth curve calculation of Lactobacillus plantarum as described by Trabelsi et al.(19).The bile tolerance of the test isolates was calculated using the following formula (20):

Bacterial species identification by 16S rDNA sequencing

Bacterial DNA Extraction

Phenotypically identified presumptive Lactobacillus spp. yielding optimal in vitro probiotics characteristics were subjected to bacterial DNA extraction as per kit instructions (Zymo BIOMICS DNA Miniprep kit, USA). The 16S rDNA PCR was performed in a 10 µL reaction containing 1X KAPA HiFi-Hotstart Ready mix (KAPA, USA), 0.2 µM of each forward (5’CCTACGGGNGGCWGCAG3’) and reverse primers (5’GACTACHVGGGTATCTAATCC3’)(21), and1 µL of template DNA. PCR was conducted at 95 °C/15 minutes for initial denaturation, followed by 40 cycles of each 98 °C/30 s, 65 °C/30 s and 72 °C/30 s, with final extension at 72 °C/5 minutes. The amplicons (550 bp) were excised from gel and purified using 1X Agencourt AMPure beads (Beckman Coulter, USA) and sequenced using both the forward and reverse primers on ABI-310 Genetic Analyzer (Applied Biosystems, USA). An M13 sequence was used as a positive control.

After initial quality assessment, the resulting DNA sequences were referenced in the NCBI database using BLAST (Basic Local Alignment Search Tool) (22). For taxonomic assignment, phylogenetic analysis was performed by comparing sample sequences with a set of Lactobacillus16S rDNA sequences extracted from the NCBI GenBank. The best-fit nucleotide substitution model, K2+G, was selected based on the Bayesian Information Criterion (BIC) score in MEGA X v10.2.5 (23). Maximum likelihood (ML) phylogeny was inferred using the selected model in MEGA X, with 1000 bootstrap replications. Phylogenetic tree annotation and visualization was performed using FigTree v1.4.4 (24).

Results

A total of 41 cloacal samples were collected from various indigenous breeds of free-ranging backyard chicken from rural outskirt hills of the Kathmandu valley. A total of 90 potential LAB isolates were selected based on CaCO3 hydrolysis in MRS agar.

Of 90 potential LAB isolates, 52 isolates (57%) showed anti-bacterial activity against at least one bacterial pathogen with zone of inhibition ranging from 7 to 16 mm (Supplemental table 2). In general, higher proportion of LAB isolates inhibited Gram-negative enteric bacilli (coliforms) compared to Gram-positive cocci (S. aureus). Majority (69%, 36/52) of LAB isolates inhibited Salmonella spp., followed by inhibition against C. freundi (53%, 28/52), Shigella spp. (44%, 23/52), K. pneumonia (30%, 16/52), E. coli (26%, 14/52) and S. aureus (13%, 7/52). Nine of these LAB isolates (17%) showed inhibition against all tested Enterobacterales bacteria, of which 5 isolates also inhibited S. aureus.

Of 52 LAB isolates showing varying anti-bacterial activity, 46 fulfilled basic phenotypic criteria of Lactobacillus spp.-Gram positive rods giving negative reactions for each sulphur, indole, catalase, and oxidase tests. The selected 46 potential Lactobacillus spp. isolates were further subjected to in vitro screening tests for probiotics properties. Of 46 isolates, 37 produced acid of varying yields (38%-81%). Among these 37 isolates, 27 isolates showed survival at pH 3.0 with varying survival rates (1.2%-62.1%). Tolerance to bile salt varied with the bile concentrations. 17 isolates had 0.3% and 0.5% bile salts tolerance, while14 isolates survived in the presence of 1.0% bile salt. The resulting profile of 52 LAB isolates on in vitro screening tests for probiotics is given Supplemental table 2.

Of 31phenotypically identified Lactobacillus spp. exhibiting optimal in vitro probiotics properties, 3 isolates (26B, 28B, and 30B) showing anti-bacterial activity against the widest range of tested bacterial pathogens, and one (C4/36(4)) showing high bile tolerance (1.0%) were randomly selected for 16S rDNA sequencing. Based on phylogenetic analyses of 16S rDNA sequences of the isolates, 26B (GenBank accession number ON955508) , 28B (GenBank accession number ON955509) , and 30B (GenBank accession number ON955510) isolates clustered into the clade of Lactobacillus plantarum with average p-distance of 0.0061-inferring a genetic similarity of 99.38% with L. plantarum. The isolate C4/36(4) (GenBank accession number ON955511), on the other hand, grouped into the cluster of Lactobacillus. reuteri, L. antri, and L. panis with average p-distance of 0.0014 and a genetic similarity of 99.85% with the given cluster(Figure 1).

Figure 1. Maximum likelihood tree based on 16S rDNA gene sequences of different Lactobacillus spp. The isolates of the study are indicated in red font. Lactococcus plantarum was chosen as an out-group. The scale bar (0.03) shows the nucleotide substitution rate per site. Bootstrap probabilities as determined for 1000 replicates are shown at nodes. The isolate C4/36(4) has been labeled a C4 in the given phylogenetic tree.

Discussion

In our study, we have isolated and identified potential LAB from 41 non-duplicate cloacal samples collected from a group of diverse backyard chicken breeds reared at a rural outskirt of the Kathmandu valley (Nepal). Of 90 potential LAB isolates, 31 isolates were phenotypically identified to be Lactobacillus spp. fulfilling basic in vitro probiotics characteristics. Of these, at least 4 were genetically confirmed to be Lactobacillus spp. Further in vivo experiments need to be conducted to assess gut adaptation and probiotics performance to develop these isolates as poultry probiotics feed supplement. Industrial production parameters, such as ability to propagate in cheaper growth media, and maintain viability throughout production steps till storage and final application also need to be assessed (10).

With an increasing global threat of antimicrobial resistance (AMR), there is an urgent need to explore alternative solutions to antibiotic use. Routine use of sub-optimal dosages of antimicrobials in poultry sector with an aim to enhance their growth and performance has been one of the drivers of accelerated AMR (25). The discontinuation of anti-microbial use in poultry, however, as a result of stringent regulation in high income countries, has resulted in poor poultry production (25). This underscored the importance of prophylactics like antimicrobials for desired poultry yields. There are numerous studies indicating the use of probiotics as alternative solution replacing antimicrobials in poultry industry with similar benefits but without risk of AMR emergence (25). Additionally, probiotics are also known to inhibit aflatoxigenic molds and degrade aflatoxins (25).

There are various native breeds of chicken found in Nepal, generally known as “local breeds”, but some with specific names like Shakini, Giriraj, Ghanti khuile, Kadaknath and Pwankh Ulte (Dumse) (26). Based on various factors, gut microbiota of free-ranging indigenous chicken breeds is different from that of commercial breeds (27). Free-ranging chicken forage on a variety of natural food sources, such as insects and green foliage, which may enrich their microbiome diversity (28). As a result, compared to commercial breeds, the backyard breeds have higher immunity, and therefore, can be more resilient against various infectious diseases (28).

We have isolated potential probiotics from indigenous chicken breeds. Unlike probiotics that are often isolated from abiotic sources, such as fermented food, the probiotics isolated in our study are host-specific to chicken with potential enhanced intestinal mucosa colonization abilities for greater natural integration into host gut microbiota (29). However, further in vivo investigation is still needed to fully assess this probiotics feature.

Bacterial infections are one of the major reasons of morbidity and mortality in poultry, especially in commercial broiler and layer breeds-leading to production loss (30). Colibacillosis and salpingitis caused by pathogenic strains of E. coli, mycoplasmosis caused by Mycoplasma spp., fowl cholera by Pasteurella multocida, necrotic enteritis by Clostridium perfringens, ulcerative enteritis by Clostridium colinum, salmonellosis, fowl typhoid and paratyphoid by diverse serovars of Salmonella spp., omphalitis by coliforms, pododermatitis and staphylococcosis by Staphylococcus aureus, and shigellosis by Shigella spp. are some of the common diseases caused by bacterial infections in poultry (30, 31).

Probiotics are known to protect host from infections through various mechanisms, including inhibiting colonization by pathogens and producing antibacterial metabolites such as acetic acid, lactic acid, alcohols, and bacteriocins (32). Effective inhibition property against known pathogenic bacteria is one of the important selection criteria for good probiotics candidate. In our study, inhibition of coliform bacteria by LAB isolates was more common compared to inhibition against Gram-positive cocci, where 67% (31/46) phenotypic LAB isolates inhibited Salmonella spp., while 19% (9/46) isolates inhibited all tested enteric bacilli, in contrast to 15% (7/46) isolates that inhibited S. aureus with or without inhibiting enteric bacilli. Taheri et. al also reported higher inhibition of enteric bacilli, where 18% (62/332) of selected LAB strains isolated from gut of broiler chicken inhibited Salmonella enteritidis, Salmonella typhimurium, and E. coli O78:K80 (33). Kizerwette-Swida et al. reported higher inhibition activity against Gram-positive pathogens including C. perfringes and S. aureus than coliforms including E.coli and Salmonella (34). In our study, 5 of 46 LAB isolates inhibited all tested Gram-positive cocci and enteric bacilli, 3 (26B, 28B, and 30B) of which were genetically identified to be L. plantarum. Probiotics cultures of L. plantarum has been reported to have efficient antibacterial activity against broad spectrum of bacteria including clinical isolates S. aureus and E. coli via direct cell competitive exclusion as well as production of acids or bacteriocin-like inhibitors (35, 36).

The ability to survive in acidic gut environment, as they pass through stomach and intestine and colonize in the host gut, is an essential property of probiotics (29). In our study, 27 of 46 isolates fulfilling phenotypic characteristics of LAB isolates showed survival tolerance in simulated gastric juice medium of pH 3.0, with the survival ratio ranging from 1.2% to 62.1%. This infers that the isolates would survive harsh acidic condition of the host gut upon introduction. Feng et al. reported that of 52 potential probiotics strains isolated from gut of piglets, 100% of isolates showed survival at pH 3.0, though 8 isolates failed to survive at an acidic pH of 1.0 (37). In another study aiming to isolate potential probiotics from poultry fecal samples, of 42 potential isolates, 16% (n=7) of selected isolates tolerated acidic pH of 2.5 and 4.0 compared to normal pH (38). Further, lactic acid produced by LAB isolates provides various health benefits including immune-modulatory functions and prevention of diarrheal diseases (1). In our study, 37 of 46 potential probiotics produced lactic acid with the yield ranging from 38 to 81%.

Bile salts are one of essential constituents of mammalian gut that help solubilize dietary fats. Hydrophobicity and detergent properties of bile salts exert strong antimicrobial effects. Thus, tolerance to bile is considered as one of important qualities of probiotics. Bile concentration varies along duodenum, jejunum and cecum of chicken gut, which were estimated to be 0.17, 0.7, and 0.0085 % respectively (39). In our study, 18 of phenotypic lactobacilli isolates fulfilling probiotics characteristics of acid production and acid tolerance showed 24 hour in vitro survival in the presence of 0.3% and 0.5% bile salts. Shin et al. reported that all 3 sequenced potential probiotics isolates obtained from gut of broiler chicken tolerated the presence of 0.3% bile salts (40). Oh et al. reported 7 of the potential probiotics isolates tolerated 0.3% bile salts for 24 hours (41).

Conclusion

With increasing global crisis of AMR, substitution of antibiotic growth promoters with effective probiotics in poultry and other veterinary fields seems to be a promising alternative solution. Our study isolated, characterized and identified 4 Lactobacillus spp. demonstrating optimal in vitro probiotics properties. With further evaluation these identified Lactobacillus spp. candidates can be developed as effective probiotics which can be used as poultry feed supplement replacing regularly used antimicrobials.

Data Availability Statement

The data supporting the findings of this study are available as supplementary materials.

Funding

This research project was funded by BIOVAC Nepal and the Center for Molecular Dynamics Nepal (CMDN) with partial thesis support from the SANN International College, Purbanchal University.

Competing interests

No potential competing interest was reported by the authors.

Acknowledgment

We acknowledge the field and laboratory team of the Center for Molecular Dynamics Nepal (CMDN) for providing logistical, operational and supervisory support throughout the study.We thank the SANN International College for their support. And our gratitude to the members of local community where we collected our samples.

Supplemental table 1: Demographic details of chicken cloacal samples shown by identification number, sampling locations and the chicken breed.

Supplemental table 1: Demographic details of chicken cloacal samples shown by identification number, 427 sampling locations and the chicken breed.

Supplemental table 2: Laboratory results of 52 LAB isolates on antibacterial inhibition, biochemical test, and in vitro test for potential probiotics strains

This article was originally published in bioRxiv 2022.07.12.499701; doi: https://doi.org/10.1101/2022.07.12.499701. Reproduced here with permission from the author.

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