Enterococcus isolated from poultry intestine for potential probiotic use

Background and Aim: To develop species-specific probiotics for poultry, it is ideal to obtain these probiotic microorganisms directly from the intestinal tract of broiler and egg-laying chicks in production environments to ensure adaptation to actual conditions. This study aimed to isolate lactic acid bacteria (LAB) from the intestinal tract of broiler and egg-laying chicks to determine their probiotic potential. Materials and Methods: Twenty-five Ross-308 broilers and 25 Isa Brown egg-laying chicks were raised until days 42 and 120, respectively; they were housed in an individual poultry building. Lactic acid bacteria were isolated and identified from the small intestine mucus of broiler and layer chicks and then evaluated based on resistance to acidic pH levels, bile salt concentration, and antagonistic activity against wild strains of Escherichia coli and Salmonella spp. selected strains with probiotic potential were identified by polymerase chain reaction and confirmed by rDNA sequencing. Results: One hundred and fifty Gram-positive isolates were obtained; 28% (42) were catalase and oxidase negative and biochemical identification was made by crystal system: 76.2% (32) Enterococcus spp., 16.6% (7) Lactococcus spp., and 7.2% (3) Streptococcus spp.; and evaluated for hemolysin production; tolerance to low pH and bile salts, and antagonistic potential were carried out. Molecular characterization yielded 56% (24) Enterococcus faecium, and 44% (18) Enterococcus faecalis. About 81% (34) of strains were without vancomycin resistance genes criterion. Conclusion: This study isolated and characterized 36 strains of LAB with probiotic qualities, from the intestines of broiler and egg-laying chicks, selecting E. faecium, Enterococcus avium, and Enterococcus casseliflavus, Lactococcus garviae as promising strains for further in vitro and in vivo research.


Introduction
Probiotics are toxin-free producer microorganisms, which, if supplied appropriately, balance and maintain gastrointestinal health.In humans, there is evidence of the probiotics' ability to restore intestinal ecology, reducing the burden of pathogenic microorganisms [1].Probiotic microorganisms include lactic acid bacteria (LAB), widely known as lactic acid producers from carbohydrate metabolism LAB pathways.These Gram-positive microorganisms are facultative anaerobic fermenters, catalase and oxidase-negative [2].Lactobacillus, Bifidobacterium, Enterococcus, and Bacillus are some of the most frequently used bacterial species as probiotic microorganisms.However, fungi species such as Aspergillus oryzae and yeast such as Candida pintolopesii, Saccharomyces boulardii, and Saccharomyces cerevisiae are also employed as probiotics [3].
Some selection criteria for bacteria probiotics are: (a) To be present in the intestinal flora of the animal species of interest, since in this way, they will adapt more efficiently to the gastrointestinal environment in each specie conditions; (b) tolerate low pH conditions, since they must pass through the stomach; (c) tolerate the bile salts action as in the first portion of the small intestine (duodenum) [4]; and (d), recently the International Scientific Association of Probiotics and Prebiotic also established that there must not exist vancomycin resistance genes (VRGs) [5].Compliance with these criteria and the functionality and safety of probiotic microorganisms used in the industry are regulated and guaranteed by the Food and Agriculture Organization and the World Health Organization [6].Lactic acid bacteria antagonize pathogenic bacteria by competing on adhesion sites and nutrients as well as producing bacteriocins (proteins and peptides with bactericidal properties) and volatile fatty acids [7].Benefits in gastrointestinal functioning and strengthening of native intestinal flora Available at www.veterinaryworld.org/Vol.16/August-2023/5.pdf in animals supplemented with LAB have been demonstrated in humans [8], fishes [9], pigs [10], goats [11], cattle [12], and birds [13].Furthermore, some effects on the immune system have been described, such as the modulation of gut-associated lymphoid tissue response by stimulating immunoglobulin A and interferon-gamma production [14,15], and the activation of CD4 and CD8 T lymphocytes [16].Poultry production operates under stressful conditions and can promote infectious disease spread due to the high population density, thus affecting health and productivity [17].To reduce or treat these situations, producers use prophylactic antibiotics; however, the short production periods in poultry are sometimes not enough for antibiotics to be entirely metabolized and then some traces can be found in meat and eggs [18,19], facilitating the occurrence of bacterial resistance to antibiotics.Therefore, it is necessary to develop more efficient, effective, and innocuous therapeutic and/or nutritional alternatives to reduce the use of antibiotics in animals for human consumption.In some instances, probiotic microorganisms have been derived from native chickens under field conditions, providing the opportunity to incorporate biodiversity into the microorganism collections used for commercial purposes [20], including Lactobacillus spp.genus [21,22].Following these examples, we raised broiler and egg-laying chicks in field conditions, taking care of not using growth promoters (currently used in commercial poultry food in Colombia), so we can recover and select intestinal microorganisms with probiotic potential.
This study aimed to isolate LAB from the intestinal tract of broiler and egg-laying chicks raised under field conditions and fed without growth promoter to determine their probiotic potential (tolerance to low pH and bile salts, and antagonistic performance against strains of Escherichia coli and Salmonella spp.), as well as to assess the presence of antibiotic resistance genes.
Twenty-five Ross-308 broiler chicks and 25 Isa Brown egg-laying chicks were bred until days 42 and 120, respectively; they were housed in poultry cages under field conditions in a rural location of the city of Bucaramanga, province of Santander (Colombia).The birds were fed and watered ad libitum, and lighting and heating were provided according to breed requirements (Ross-308 and Isa Brown Manual).All animals were fed with antibiotic-free food whose formulation was based on the nutritional requirements established by the Spanish Federation for the Development of Animal Nutrition.

Isolation and identification of LAB
Birds were sacrificed by cervical deviation at weeks 1, 2, 3, 4, and 5 for broilers and 2, 6, 17, and 35 for egg-laying chicks, and the intestines were removed from the body of each bird according to the necropsy method described by Martínez-Acevedo [23].Deep mucous membrane scraping was performed in the duodenum, jejunum, and ileum; subsequently serial dilutions were made up to 10-10 and last five dilutions of each intestinal segment were seeded in Man Rogosa Sharpe (MRS) agar (Merck, Darmstadt, Germany), to obtain axenic cultures.The bacteria obtained were selected according to their morphological characteristics.Gram staining, catalase test, and oxidase test were performed and LAB were identified using the semiautomated system Crystal for Grampositive (BBL TM Crystal TM Gram Positive ID Kit (BD, New Jersey, USA).

Isolates hemolytic activity
Twenty-five milliliters of blood agar was served in Petri dishes, three wells were made using a sterile element, and each well was inoculated with 50 μL of axenic culture of the strains previously isolated by Miller et al. [24] in Brain Heart Infusion broth and incubated for 48 h at 37°C with AnaeroGen® (Oxoid, UK), 13% CO 2 .Afterward, the halos produced were read according to the type of hemolysis (Alpha, Beta, and Gamma) [25].

Resistance of isolates to low pH and bile tolerance
Isolates were inoculated in test tubes with MRS broth, whose pH was modified with 1N HCl at the following values: pH2, pH3, pH4, and pH5.They were incubated at 41°C for 4 h until a turbidity of 0.5 was obtained in Farlanthe scale [26]; three inocula of 50 μL were made on MRS agar and incubated for 72 h with AnaeroGen system 13% CO 2 [27].The percentage of resistance was calculated using the Kociubinski formula [28], by comparing colony-forming units per microliter of the control (no treat culture) (CCFU/μL), and the treated culture (pHCFU/μL) tested.

Resistance of isolates to bile salt
Isolates were inoculated in test tubes with MRS broth modified with chicken bile at: 0.1%, 0.15%, 0.2%, and 0.3% concentrations; incubated at 41°C for 4 h, then three inocula of 50 μL were made on MRS agar (Merck) and incubated for 72 h with the AnaeroGensystem (Hampshire, UK), at a concentration of 13% of CO 2 [27].The percentage of survival was calculated by modifying the equation of [28].

Antagonistic activity
Four strains of E. coli and two of Salmonella spp.were seeded on soybean trypticase agar (STA) plates, five wells were made in each plate, then 50 μL of cultures of each isolated strain were inoculated in each well, incubated at 41°C by 72 h with the AnaeroGen system 13% CO 2 [27]; inhibition halos were measured [29].

DNA extraction
DNA extraction was carried out from pure cells suspended in sterile water with the MOBIO Ultraclean Microbial DNA Isolation commercial kit (Irvine, CA, USA) was used, following the manufacturer's instructions.At the end of the procedure, the concentration and purity of DNA obtained by spectrophotometry were verified in Nanodrop equipment and its subsequent visualization was carried out in 1% agarose gel.

Polymerase chain reaction (PCR) and bioinformatics analysis
Fragments amplification was carried out with 16S universal initiators (1492R-27F).The PCR assays were performed in 25 μL of reaction mixture containing 21 μL of Master mix, 1 μL of reverse primer, 1 μL of reverse primer, and 2 μL of genomic DNA, under the following conditions: A denaturation initial step at 94°C for 3 min, followed by 30 denaturation cycles at 94°C for 45 s, annealing at 63°C for 40 s and extension at 72°C for 90 s, and finally a 72°C extension stage for 10 min.The amplified PCR products (2 μL) were visualized in 1.5% agarose gel, and documented by a light transilluminator.Polymerase chain reaction products were sequenced by Corpogen (Bogotá) in alliance with Macrogen (Korea).The sequences were subjected to a Blast in the National Center for Biotechnology Information database and subsequently analyzed.

Statistical analysis
Continuous variables are expressed as means (standard deviations) and categorical variables as percentages.The percentage resistance to pH levels (2, 3, 4, and 5) and bile concentrations (0.1, 0.15, 0.2, and 0.3) are expressed as mean and standard deviation, and the antagonistic behavior of the isolated strains to challenging strains of E. coli and Salmonella is expressed as the percentage of antagonistic isolates by strain.
Cluster analysis using Gower distance for dissimilarity matrix was accomplished for the isolated strains for the resistance percentage to pH and bile salts concentration, and for the antagonistic behavior to challenging strains of E. coli and Salmonella.We employed the divisive clustering method, starting from all isolated strains and deriving clusters based on similar pH and bile salt resistance characteristics for one side and antagonistic behavior to challenging strains for the other side.Dendrograms were elaborated based on clusters obtained using the divisive clustering method.Then, we describe the average resistance percentage by cluster to the pH and bile salt levels, presenting the isolated strains in every cluster.In addition, we show the isolated strains' average percentage of antagonistic behavior to challenge strains by cluster and the isolated strains' names by cluster.The R software version 4.1 was used for the computations [31].

Hemolytic activity
A hemolysin production test was performed on 42 strains, of which 6 (14.3%) strains presented alpha and/or beta hemolysis.Only bacteria of the genus Enterococcus spp.showed some type of hemolysis; all hemolytic isolates were dismissed.

Low pH and bile salt resistance
Table-1 shows the mean and standard deviation of the dilution and the resistance percentage to the pH levels and to the bile salt concentration levels.Most of the strains were isolated from broiler chicks (32) versus laying chicks (4).From broiler chicks, we isolated eight strains from duodenum, 12 strains from jejunum, and 12 strains from Ileum, while from laying chicks, we isolated two strains from duodenum, and one strain in jejunum and ileum.The resistance percentage to pH 2 ranged from 20% for L. rafinolactis in broiler chick's ileum, to 70% for S. vestibularis in duodenum.The resistance percentage to pH 3 ranged from 39.7% for E. faecalis strains in broiler chick's ileum to 85% for L. lactis lactis in broiler chicks' jejunum.The resistance percentage to pH 4 and pH 5 was above 50% in all isolated strains for both pH levels.The average resistance percentage to four bile concentrations is shown in Table-1.The resistance percentage to a bilis concentration of 0.3 ranged from 25% for L. rafinolactis in duodenum and L. lactis cremoris and S. vestibularis in jejunum to 90% for E. faecalis in duodeum, while for a 0.2 bile concentration, it ranged from 35% for L. rafinolactis in duodenum to 70% L. rafinolactis in ileum.
The dendrogram obtained from the clustering method for the combined clustering effects of pH levels and bile salt levels is shown in Figure -1.Based on the within clusters sums of squares, we select seven clusters shown in Figure -1.
Table-2 displays the average percentage resistance to four pH levels and four bile salt concentrations by cluster.We observed that cluster 3 shows the highest average resistance to pH 2 (41%) and the highest average resistance to bile salts concentration of 0.3 (87%).Table-3 presents the isolated strains by cluster and from that we found that cluster 3 contains E. casseliflavus and E. avium from Broiler chick's Jejunum, and two strains of E. avium from broiler chick's Ileum.

Antagonistic activity
The antagonistic activity was performed against (4) E. coli and (2) Salmonella spp.wild-type strains.Table-4 shows the antagonistic characteristics of isolated strains to challenging strains of E. coli and Salmonella spp.E. durans in laying chicks' ileum and E. avium in broiler chicks' jejunum showed antagonistic behavior to five challenging strains, L. garviae in broiler chicks' duodenum, and E. avium in broiler chicks' jejunum to four challenging strains.In contrast, E. faecium strains in broiler chicks' jejunum showed antagonistic behavior to all challenging strains.Figure -2 shows the dendrogram for the clustering method, for the antagonistic characteristics of the isolated strains to challenging strains of E. coli and Salmonella spp.We again established seven clusters based on the within cluster sum of squares.Table -5 shows the average percentage of isolated strains antagonist to the challenging strains.
From Table-5, we observed that cluster 6 contains 10 isolated strains having antagonistic behavior in 70% to E. coli 32, 50% to E. coli 40, 100% to E. coli 46, 100% to Salmonella 1, and 100% to Salmonella 2, making those strains in this cluster to be strong candidates to be considered as probiotic strains, where isolated strains in cluster 6 are comprised by E. faecium, L. garviae in Broiler chicks Duodenum, E. faecium (two strains), E. casseliflavus, E. avium in broiler chicks jejunum, E. faecalis (two strains), E. faecium in broiler chicks ileum, and E. durans in laying chicks  ileum (Table -6).Finally, we would like to add that from the most resistant cluster 3 to pH and bile salts levels, we found E. casseliflavus in broiler chicks' duodenum and E. avium in broiler chicks jejunum, which are also included in the clustering results for cluster 6 in the antagonistic behavior to the challenger strains of E. coli and Salmonella spp.

Vancomycin resistance
Duplex PCR was performed to identify VRGs.No VRG was found in the 25 strains (Figure -3).

Discussion
This study shows the selection process of field strains candidates to be probiotic bacterial strains  [32], and it has been evaluated in piglets for managing weaning diarrhea [33], and the immunomodulating activity in the nematode model [34].
Chicken meat-associated isolates of Enterococcus are often multidrug-resistant, closely related to pathogenic strains, and harbor perturbing virulence factors, representing enterococci evolving into drug-resistant human pathogens [35].We found that the strains in this study were negative for harboring VRGs.Although the probiotic potential has been explored in Enterococci, we also found studies showing no effect on the productive responses but influencing positively  over the feed and chick cost [36].Enterococcus faecium are probiotic candidates which can be applied in commercial scenarios to improve poultry performance and control of pathogens, hence decreasing further transmission to humans [37].Enterococcus faecium, E. faecalis, and E. durans have been employed as probiotics to prevent subclinical necrotic enteritis (SNE) [38,39].Comprehensive evaluation showed that E. faecium could be a candidate as a probiotic strain used in humans or animals, with the potential to restrain the effect of Salmonella strains [40][41][42].We also found evidence in the literature that E. faecium strains isolated from chicken are alternatives to antibiotics to reduce feed conversion rate in broiler chicken, possibly mediated by increasing the villus height of the intestinal epithelium, so the mechanisms involve keeping healthy the intestinal architecture of the epithelium [43].In this study, we provide the microorganisms population biodiversity from the small intestine parts (duodenum, jejunum, and ileum) from broiler and egg-laying chicks raised in field conditions without the use of growth promoters from a rural location in a Colombian town, isolating multi-genus populations such as E. faecalis, E. faecium [44], E. avium, E. casseliflavus, and L. garviae, similar to the findings of Neveling et al. [44].Finally, we conclude that we have found a set of potential LAB strains to evaluate in vivo the probiotic effect on the commercial and productive features in broiler and egg-laying chicks.The study's findings demonstrate the value of the research in this field and that future research must be accomplished to develop and advance alternatives to prophylactic probiotics in poultry production.

Conclusion
In this study, we isolated and characterized probiotic potential LAB strains from poultry.Isolates were identified as E. faecium, E. avium, E. casseliflavus, and L. garviae, some promising microorganism strains for further research and use as probiotics in animal production.However, the actual relevance of our findings lies in the possibility of finding microorganisms with the potential to reduce the administration of antibiotics in animal industries, such as the poultry industry, where antibiotics are widely accepted and improve animal and human health, according to the One Health concept.

Figure- 2 :
Figure-2: Dendrogram of the isolated strains clusters selected by antagonistic behavior to challenging Escherichia coli and Salmonella strains.B=Broiler chick, L=Egg laying chick, D=Duodenum, J=Jejunum, I=Ileum.
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Table - 2
: Average of the percentage resistance to four pH levels [mean + -(SD)] and four bile concentration [mean + -(SD)] by cluster.

Table - 3
: Isolated strains selected from the percentage resistance to four pH levels [mean + -(SD)] and four bile concentrations [mean + -(SD)] by cluster.

Table - 4
: Percentage of isolated strains showing antagonistic behavior to the challenging strains of Escherichia coli and Salmonella by strain, region of the minor intestine, and chick type (broiler or laying).

Table - 5
: Mean percentage of antagonistic behavior by challenging strains of Escherichia coli and Salmonella by cluster.