A synergistic plant-based formulation as an alternative of antibiotics for poultry and its method of preparation

Abstract

The present invention relates to a synergistic plant-based formulation as an alternative of antibiotics for poultry and its method of preparation. The formulation is an alternative to the antibiotic used currently in poultry farming are inhibition of pathogenic bacterial species, better feed assimilation leading to impressive growth rate, no obvious tissue toxicity, harmonious relationship with probiotic bacteria, no adverse immune response, and absence of feed induced stress. It discourages the evolution of antibiotic-resistant bacterial species.

Description

[0001] P_W0100743

[0002] A SYNERGISTIC PLANT-BASED FORMULATION AS AN ALTERNATIVE OF ANTIBIOTICS FOR POULTRY AND ITS METHOD OF PREPARATION

[0003] FIELD OF INVENTION:

[0004] The present invention relates to the field of formulation for poultry and its method of preparation. The present invention in particular relates to a synergistic plantbased formulation as an alternative of antibiotics for poultry and its method of preparation.

[0005] BACKGROUND OF THE INVENTION:

[0006] Livestock and poultry products have provided the major sources of proteins in the human diet. The demand for animal protein has exponentially increased in recent times, which has put pressure on the animal production system. To keep a balance between demand and supply, industry has resorted to intensive animal production. Use of antibiotics has proven to be a lynchpin in enhancing the animal production. Antibiotics are not only used for disease treatment, but also as prophylaxis and growth promoters in the livestock sector. Two-thirds of the total global production of antibiotics is utilized for the livestock sector alone, which contributes to increased resistance against clinically relevant antibiotics. In the livestock sector, antibiotics are routinely used at subtherapeutic doses usually after mixing them in the animal feed. The antibiotic classes used in the livestock sector in the Indian subcontinent include penicillins, cephalosporins, tetracyclines, fluoroquinolones, sulphonamides, and aminoglycosides; representing nearly every class of antibiotics available for human use. This includes colistin, which is an antibiotic of last resort for Gram-negative pathogens. The zoonotic potential of foodborne pathogens and their ability to cause illness possess a serious health threat. India is one of the biggest markets and suppliers of animal products. Antibiotic consumption for the production of animals in India is consumption in New Delhi (India) revealed 40% samples positive for enrofloxacin, ciprofloxacin, doxycycline, oxytetracycline and chlortetracycline antibiotics. A recent report from Punjab (India) revealed extensive presence of extended-spectrum P-lactamase (ESBL) in Enterobacteriaceae isolated from the poultry. Due to thereports linking fluoroquinolone use in poultry with emergence of resistant pathogens, the use of fluoroquinolones as growth promoters was banned in USA and European Union. Recently, China and India have also banned colistin for use in the animal sector. Additionally, India has also banned streptomycin -tetracycline combination for use in agriculture sector.

[0007] To meet increasing demand for animal protein, poultry sector has resorted to intensive farming practices. Limitation of holding spaces, lack of proper hygiene, sanitation and skilled workers leads farmers to rely on antibiotic usage. Further, lack of strong regulations for antibiotic usage in animals and easy availability of antibiotics tempts farmers to use subtherapeutic dosage of antibiotics for growth enhancement of birds.

[0008] Reference may be made to the following:

[0009] Publication No. EP2170100 relates to an animal feed additive obtainable by: mixing water, an emulsion stabilizer and a physiologically acceptable carrier to form a gum solution; adding to the gum solution, separately or combined, an oil blend comprising: cinnamaldehyde and a compound selected from the group consisting of citral, eugenol, limonene, thymol, vanillin and combinations thereof; mixing the gum solution and the oil blend and homogenizing this mixture under pressure to form an emulsion; and spray drying and / or agglomerating the mixture to form a powder.

[0010] Publication No. CN115137017 relates to mixed feed additives, and provides a plant essential oil feed additive and application thereof. The plant essential oil feed additive is composed of essential oil and auxiliary materials, and by adjusting the different composition ratios of thymol, cinnamyl aldehyde and carvacrol of all the components of the essential oil, the multiple effects that growth of pathogenic bacteria is inhibited, meanwhile, growth of probiotics is not remarkably affected, and absorption of intestinal tracts to nutrient substances is enhanced are finally achieved; therefore, the effect of regulating the balance of flora in the intestinal tract is achieved.

[0011] Publication No. JP2022153806 relates to a chicken mite repellent effect which can be safe for domestic fowl and also safe for human bodies, has high chickenmite repellent effect and can be used instead of agents. There is provided a feed additive including, a plant extract and herb essence, and having a chicken mite repellent effect, in which the plant extract is at least two kinds selected from, an alfalfa extract, a Glycyrrhizae Radix extract, a rosemary extract, a Tanacetum vulgare extract, the herb essence is at least two kinds selected from, thyme essence, peppermint essence, Eucalyptus essence, coriander essence, vulgare essence, lavender essence, and Borrago officinalis essence.

[0012] Publication No. US 10688145 relates to the use of a poultry feed additive composition which is a flowable mixture of phytogenic compounds including at least an oil component which is microencapsulated essential thyme oil, and a saponin component which is the saponin contained in particulate dried quillaja bark powder. The mixture contains at least 0.5% saponin component (w / w), and the oil component in an effective ratio of at least 0.2:1 (w / w, oil component per saponin component), for improving the feed conversion efficiency in antibiotic-free poultry production.

[0013] Publication No. US2008032021 relates to a livestock and poultry feed additive composition includes at least one essential oil derived from at least one of an herb and a spice and containing thymol and carvacrol as its main ingredients, at least one organic acid derived from at least one of citric, fumaric, fulvic and humic acid and an organic pharmaceutically acceptable carrier. The total amount of the essential oil in the composition is present in an amount between five percent and forty percent (5% and 40%) by weight of the composition, the total amount of the organic acid in the composition is present in an amount between fifty percent and eighty-five percent (50% and 85%) of the composition by weight and the remainder of the composition consists of the organic pharmaceutically acceptable carrier.

[0014] Publication No. CN117730950 relates to feed additives, and a zinc sulfate -containing feed additive and a production process thereof. The feed additive contains an essential oil coating agent, a zinc sulfate slow-release agent, vitamin C, leucine and soybean hull powder and is prepared through the processes of mixing, pelletizing and particle finishing, the zinc sulfate slow-release agent can slowly release zinc ions at target parts of intestinal tracts, and the zinc ions can be slowly released at the target parts of the intestinal tracts; and after the vermiculiteis desorbed, alkaline substances such as ammonia gas in the intestinal tract can be adsorbed, the pH value in the intestinal tract can be reduced, and the reproduction of beneficial bacteria such as lactic acid bacteria is facilitated, so that the production performance of the broiler chicken is effectively improved, and the production efficiency of the broiler chicken is improved.

[0015] The increasing threat of antibiotic resistant bacteria across the globe demands for an effective alternative solution to sustain the efficiency of current livestock production. Several solutions like probiotics, prebiotics, organic acids, peptides and phytogenic substances have been proposed as alternatives to the conventional antibiotics. Due to their multifaceted characteristics like antioxidant, bactericidal, viricidal, fungicidal, antiparasitic, insecticidal, and immune-modulatory properties, some phytochemicals belonging to secondary metabolite category have been promoted as promising alternative for antibiotics. These metabololites which are represented by phenylpropanoids, terpenoids and their compounds have also shown promising results in feed assimilation in poultry birds due to enhanced secretion of bile and digestive enzymes. Overall, these metabolites have also been recognized as growth promoters for poultry; however, due to their weak antibacterial activity, a large amount is required to be given to birds which alters the organoleptic properties negatively and leads to reduced feed intake in birds. Amongst other promising GRAS status compounds, some vitamins such as vitamin C, E and K are essential dietary micronutrient in animals, possess antibacterial activity and also serve as a cofactor for biosynthetic enzymes. In some cases, vitamins such as vitamin C may possess anti-oxidant and pro-oxidant activities depending on the cellular milieu. Further, many metals including heavy metals and metalloids (such as chromium, copper, iron, iodine, manganese, molybdenum, selenium, vanadium and zinc are essential micronutrients for animals and exhibit weak antibacterial activity. These metals after carefully scrutiny can be tested in combination to assess their cumulative / synergistic antibacterial activity. Amongst above, copper serves as an essential element for all life forms. Non-ruminant species, particularly pig and poultry, can tolerate high levels of dietary copper due to adequate copper biliary excretion system.

[0016] In order to overcome above listed prior art, the present invention aims to provide a synergistic plant-based formulation as an alternative of antibiotics for poultryand its method of preparation. The formulation is a combination of GRAS status chemicals can effectively be used to mitigate foodbome pathogens.

[0017] OBJECTS OF THE INVENTION:

[0018] The principal to provide object of the present invention is a synergistic plantbased formulation as an alternative of antibiotics for poultry and its method of preparation.

[0019] Another object of the present invention is to provide a synergistic plant-based formulation which effectively mitigate bacterial pathogens, is non-toxic, accords equal growth-promoting ability as of antibiotics, and increases the production and immune parameters

[0020] Yet another object of the present invention is to provide an effective plant metabolite-based antimicrobial formulation.

[0021] SUMMARY OF THE INVENTION:

[0022] The present invention relates to a to a synergistic plant-based formulation as an alternative of antibiotics for poultry and its method of preparation. The formulation is an alternative to the antibiotic used currently in poultry farming are inhibition of pathogenic bacterial species, better feed assimilation leading to impressive growth rate, no obvious tissue toxicity, harmonious relationship with probiotic bacteria, no adverse immune response, and absence of feed induced stress. It discourages the evolution of antibiotic -resistant bacterial species.

[0023] BREIF DESCRIPTION OF THE INVENTION

[0024] It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments.

[0025] Fig.l shows A. schematic of different samples collected and respective sample collection site. B. heat map showing antibiotic resistance pattern of poultry isolates in different samples.Fig 2. shows interaction network showing antibacterial-antibacterial interactions against Salmonella enterica serovar Enteritidis. The grey line represents no interaction, while the black line represents synergistic interaction

[0026] Fig. 3. A. shows time kill kinetics of Salmonella enterica serovar Enteritidis in the presence of a different concentrations of formulation. Time-kill kinetics data shows that combination of these metabolites is bactericidal in nature. * represents complete killing of bacteria. The limit of detection corresponds to 30 CFU / mL. B. Time kill kinetics of Candida albicans in the presence of a different concentrations of formulation. Time -kill kinetics data in one combination example shows that combination of metabolites is bactericidal in nature. * represents complete killing of bacteria. The limit of detection corresponds to 30 CFU / mL.

[0027] C. Culture tubes showing frequency of resistance (FOR) against formulation. Formulation at IX MIC killed all bacteria and no turbidity can be seen.

[0028] D. Graph showing generation of resistant mutants over a period of time. Tetracycline was used as control antibiotic (a commonly used antibiotic in Indian poultry system). Each data point represents single day result. E. Bar graph showing potency of formulation against multiple antibiotic resistant poultry pathogens as well as probiotic strains. IX MIC of formulation represents as MIC against Salmonella enterica serovar Enteritidis.

[0029] Fig. 4. shows bar graph showing A. Normalized meat yield per bird, each dot represents four subgroups B. Feed conversion ratio, each dot represents four subgroups C. Concentration of IL10, D. TNFa, E. ILlb, F. IFNy, G. serum corticosterone, each dot represents independent birds (Unpaired student t-test with Mann- Whitney post-test, * = p-value <0.05).

[0030] Fig. 5. shows bar graph showing number of A. Enterobacteriaceae B.

[0031] Lactobacillus bacteria / gram of feces. Graph showing C. Alpha and D. Beta diversity in cecal microbiome. CONT represents control samples while TAMY represents formulation fed samples. E. Heatmap showing abundance of different genus in control or formulation fed cecal microbiome. CONT represents control samples formulation fed samples. Data from three independent samples are presented here.

[0032] Fig. 6. shows A. Images representing tissue histology of different vital organs like spleen, intestine, and liver in control or formulation fed group. Scale bar represents pm. B. Bar graph representing normalized fold change expression ofnutrient uptake genes under different feeding regimen. Data of bird (n=3) are plotted here. GAPDH gene was selected as a housekeeping gene. Error bar represents standard deviation (Unpaired student t-test with Mann- Whitney posttest, ** =p-value <0.01).

[0033] Figure 7. shows A. Heatmap representing the susceptibility of poultry pathogens isolated from different treatment groups. 60 different bacteria were analyzed from each group. The color code indicates the degree of resistance, ranging from white (susceptible) to the black (resistant). B. Bar graph showing number of resistant isolates out of 60 against a particular antibiotic.

[0034] DETAILED DESCRIPTION OF THE INVENTION:

[0035] The present invention provides a synergistic plant-based formulation as an alternative of antibiotics for poultry and its method of preparation. This is an antibiotic alternatives exclusive to farming sector. Such alternatives must effectively mitigate bacterial pathogens, be non-toxic, accords equal growthpromoting ability as of antibiotics, and does not affect production and immune parameters negatively. This is an effective plant metabolite-based antimicrobial combination and assessed the effectiveness of this combination on bacterial pathogens isolated from the poultry ecosystem.

[0036] In vitro optimized combination (of secondary plant metabolites with GRAS status chemicals) has been assessed on cohorts of broiler poultry birds. To evaluate the effect of the combination, the cecal microbiome of treated birds has been analysed with the in vitro optimized combinations and compared it with antibiotic fed birds. A detailed analysis of performance, growth rate, toxicity, cell-mediated and humoral immunity, fecal parameters, and physiological stress correlated with the cecal microbiome. The selected GRAS status molecules have potential to be promoted as effective alternatives of antibiotics for poultry birds without compromising on production parameters significantly.

[0037] Results

[0038] High level of resistance against clinically relevant antibiotics is present in poultry ecosystem

[0039] Figure 1. A. Schematic of different samples collected and respective sample collection site. B. Heat map showing antibiotic resistance pattern of poultryisolates in different samples. The color scale indicates the percentage of resistance, ranging from blue (susceptible) through yellow (intermediate) to the red (resistant) in each sample. Number of isolates in each sample are shown in bracket, 1. Packaged feed (n=2), 2. Drinking Water (n=4), 3. Cattle egret (n=12), 4. Feces (n=14), 5. House Fly (n=14), 6. Rice Bran (n=4), 7. Mustard Feed (n=3), 8. Commercial Feed (n=3), 9. Farm tap water (n=2), 10. Live poultry feed (n=3), 11. Fecal Camp (n=4), 12. Extruded Ground Nut (n=6), 13. Soya Meal (n=3), 14. Farm Soil (n=22), 15. Farm Sewage (n=33), 16. Egg Shell (n=8), 17. Sewage Camp (n=4). Fecal Camp and Sewage Camp isolates were isolated on Campylobacter selective media. Susceptibility criteria were decided based on the minimum zone of inhibition for the given antibiotics (available either at CLSI or EUCAST). Resistance profile of polymyxin B, colistin and vancomycin may not reflect the true cases. For hierarchical clustering of different samples Spearman ranked correlation (average linkage) was performed.

[0040] Understanding the prevalence of antibiotic -resistant bacteria belonging to different genera is particularly important because antimicrobial resistance (AMR) is not confined to one family only. The resistance pattern of all possible bacterial families prevalent in poultry was evaluated. 1745 isolates from 17 different samples has been collected within a large poultry farm rearing approximately 1,00,000 birds (Fig. 1.A). Out of the total 1745 isolates, 141 isolates, which showed a unique antibiogram (represented as a percentage heatmap Fig. l.B). A high level of resistance is observed against clinically relevant antibiotics such as cefotaxime / clavulanic acid (80.70%), ampicillin / sulbactam (34.80%), aztreonam (76.43%), imipenem (27.06%), cefixime (73.87%), amoxicillin (49.08%), ampicillin (65.82%), penicillin (79.79%), colistin (46.67%), polymyxin B (70.09%), vancomycin (53.88%), sulfafurazole (66.24%), rifampicin (71.46%), nitrofurantoin (64%), co-trimoxazole (35.78%), ciprofloxacin (26.22%), gatifloxacin (18.59%), nalidixic acid (81.51%), tobramycin (23.38%), kanamycin (57.87%), chloramphenicol (24.71%), azithromycin (76.78%) and amikacin (24.78%). Overall, the antibiotic resistance profile of the bacterial species isolated from poultry ecosystem displayed high level of resistance.

[0041] Screening of a combination of plant secondary metabolites as antibacterialsDue to their multiple targets in bacterial pathogens, plant secondary metabolites like phenylpropanoids, terpenoids and their compounds are promising potential alternatives of antibiotics agents. Several phenolics, terpenes and other related plant metabolites were selected to assess their efficacy against Salmonella enterica serovar Enteritidis (a model clinical isolate from poultry). The minimum inhibitory concentration (MIC) of selected plant metabolites was found to be ranging from 512 to >2048 mg / L (Table 1). Generally recognized as safe (GRAS) status chemicals are selected that exert antibacterial effect (Table 2). Since a negative organoleptic effect is exerted by strong odour of phenolics and some phytochemicals, a combinatorial approach is followed to decrease the concentration of individual components and other active constituents while increasing their antibacterial effectiveness. The combinatorial approach yielded synergistic combinations that shows a very high potency against Salmonella enterica serovar Enteritidis (Fig. 2). Specifically, the combinations comprise of 2-isopropyl-5-methylphenol (IPMP) in the ranges varying between 10 - 100 mg / L, 2-methyl-5-isopropylphenol at 25 - 250 mg / L, 3,7-dimethylocta-l,6-dien- 3-ol at 40 - 400 mg / L, (5R)-5-[(lS)-l,2-dihydroxyethyl]-3,4-dihydroxy-2,5- dihydrofuran-2-one -ascorbic acid at 150 - 1500 mg / L, and copper sulphate at 4 - 40 mg / L was used, which represents IX MIC.

[0042] Table 1: Table showing minimum inhibitory concentration of selected phytochemicals against Salmonella enterica serovar Enteritidis.

[0043]

[0044]

[0045]

[0046] Table 2: Table showing minimum inhibitory concentration of selected generally recognized as safe (GRAS) status chemicals against Salmonella enterica serovar Enteritidis.

[0047]

[0048]

[0049] Figure 2. shows interaction network showing antibacterial-antibacterial interactions against Salmonella enterica serovar Enteritidis. The grey line represents no interaction, while the black line represents synergistic interaction. To test the killing potential of newly discovered formulation combination, time- dependent kill-kinetics assay was performed. The in vitro time-kill kinetics of formulation against Salmonella enterica serovar Enteritidis showed a bactericidal pattern of killing at IX MIC. Within 6 hours, formulation combination reduced CFU counts by 2 and 5 Logio values at IX and 2X MIC respectively (Fig. 3.A). Since, Candida albicans is an opportunistic pathogen for poultry. It has similar killing potential of formulation against C. albicans also. More specifically, formulation was able to completely kill C. albicans within 8 hours and 1 hour,respectively at IX and 2X of MIC (Fig. 3.B) (IX MIC of formulation is same for Salmonella enterica serovar Enteritidis and C. albicans'). Frequency of Resistance (FOR) for formulation was >1O10, which highlights the effectiveness of combination in terms of pathogen inhibition and not letting pathogens to evolve resistance. Formulation effectively inhibited bacteria without detectable resistance even at IX of MIC (Fig. 3.C). Since, long term usage of any antibacterial agent give rise to generation of resistant mutant over the time specifically at when used at sub-therapeutic level. Hence, the effect of formulation is checked on generation of resistant mutant over a long period of time against Salmonella enterica serovar Enteritidis. Formulatipn prevented generation of any resistant mutant even after 28 days of repetitive passage, this is equivalent to approximately 2000 bacterial generations. However, Salmonella enterica serovar Enteritidis was able to mutate against tetracycline within 8 days of exposure (Fig. 3.D).

[0050] In any real world poultry settings multiple pathogenic bacteria poses threat to birds including eggs. Hence, the efficacy of formulation was tested against panel of pathogenic bacteria previously isolated from poultry settings. Formulation displayed potent activity against antibiotic resistant poultry pathogens like Campylobacter jejuni, Escherichia coli, Aerococcus urinaeequi, and Salmonella enterica, Bacillus cereus, Enterococcus faecium, Enterococcus gallinarum, Ochrobacterum intermedium, Enterococcus faecalis, Aeromonas veronii, Proteus mirabilis, Klebsiella oxytoca and Providentia stuartii (Fig. 3.E). Unfortunately, any strong antibacterial agent kills beneficial microflora like Lactobacillus spp. Hence, it was tested that the whether formulation kills probiotic bacteria or not. Surprisingly, Tamyaa did not show any inhibition of common lactobacillus probiotic strains like Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei upto 4X of the MIC value observed against poultry pathogen Salmonella enterica serovar Enteritidis (Fig. 3.E).

[0051] In vivo efficacy of formulation in broilers leads to better growth parameters Encouraging in vitro efficacy of formulation on bacterial pathogens prompted us to try this combination as a poultry feed additive. A single blind field trial was conducted at a commercial poultry farm located in Manglaur, Uttarakhand, India (29°47'27.38" N 77°52'42.10" E). The trial was conducted on two cohorts of 2000birds (divided into 4 groups each comprising 500), each belonging to the COBB 430Y. Birds were given standard feed (control cohort) or were supplemented with formulation. Both physiological and performance parameters were considered during the study. At the end of study (42ndday), supplementation of IX formulation resulted in 69.75 g higher weight gain per bird (p < 0.05) than the control group (Fig. 4. A). In terms of feed conversion ratio (FCR), the formulation group displayed a statistically significant reduction of 0.295 (p < 0.05) as compared to the control group (Fig. 4.B).

[0052] Since feed supplements can affect immunity as well as induce stress in broilers, we measured both immunological as well as stress indicators in the cohorts. Several immunological parameters such as IL10, TNFa, ILip, and fFNy were analyzed; while, cortisol level was measured to test stress level, formulation did not show any adverse effect on immunity (Fig. 4.C, 4.D, 4.E, 4.F). Furthermore, it did not show any elevation in cortisol level (Fig. 4.G).

[0053] Tamyaa positively affects broilers by reducing pathogenic bacterial burden:

[0054] Since majority of the pathogens of poultry belong to Enter obacteriaceae, the performance of formulation combinations was compared with control group for their effect on reducing the enterobacterial counts in feces, formulation decreased Enterobacteriaceae bacterial count by 2 logio CFU / g of feces (p < 0.01) (Fig.

[0055] 5.A). Interestingly, formulation did not affect the count of probiotic bacteria (Fig.

[0056] 5.B). To further probe organismal features of cecal microbiota in terms of how different feed additives drive the cecal microbiota change, we undertook 16S rRNA gene amplicon sequencing to assess the cecal microbiome changes. Alpha diversity was calculated using the Chao 1 index (Fig. 5.C). The results show that the cecal microbiota of both cohorts had overall similar diversity. The evaluation of beta diversity using NMDS suggested all formulation fed cohorts and control cohort are more distinctly related (Fig. 5.D). One of the most striking observation from 16s rRNA gene amplicon sequencing was validation of the previous observation regarding curtailing the growth of Escherichia while supporting the growth of Lactobacillus genera (Fig. 5.E).

[0057] Effect of Tamyaa supplementation on vital organs and nutrient uptakeThe spleen, intestine, and liver are vital organs of the body as these organs play a major role in digestion, metabolism, immunity and utilization of feed nutrients. External dietary supplemental can alter pathophysiology of these vital organs in poultry birds ( 7). Hence, we followed the effect of formulation on vital organ using histopathological examinations. The supplementation of formulation combinations did not exert any deleterious effect on vital organs (Fig.6.A).

[0058] Nutrient absorption is a complex physiological process and is directly linked to feed supplementation, we assessed the expression of four essential genes; Peptide Transporter 1 (PepTl), Excitatory Amino Acid Transporter 3 (EAAT3), Sodium-Glucose Linked Transporter 1 (SGLT1), and Fructose Transporter Type 5 (GLUT5) from samples belonging to different cohorts and compared them with a housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The supplemental addition of formulation led to significant enhancement in the expression of PepTl and GLUT5 by 2.6 and 1.6-folds whereas, there was no noticeable effect on the EAAT3 and SGLT1 gene expression (Fig.6.B).

[0059] Effect of long term feeding formulation on antibiotic resistance pattern in cecal bacteria (Enterobacteriaceae family)

[0060] The sub-therapeutic and prolonged use of supplemental antibiotics in feed leads to the generation of antibiotic resistance in livestock pathogens (4-6). In order to assess the effect of formulation supplementation on antibiotic resistance development, we evaluated the antibiotic resistance pattern of enterobacteria isolated from caecum. There is no significant difference in resistance against antibiotics representing different classes. (Fig.7.A, B, C, D).

[0061] Use of antibiotics either for disease prevention or growth enhancement in food animals is known to increase selection pressure on bacteria to evolve resistance against clinically relevant antibiotics (2-4). In a commercial poultry ecosystem, we found a high level of resistance against many clinically used antibiotics including colistin and carbapenems - the last-resort antibiotics. Due to rampant use of antibiotics in animal sector and its correlation with the emergence of antibiotic -resistant pathogens, there is a need for developing dedicated solutions to curtail the use of antibiotics meant for human use. In this direction, we present novel formulations of phytochemicals and GRAS status chemicals as a possiblealternative of antibiotics for the poultry sector. Supplemental addition of formulation as a poultry feed additive effectively controlled poultry pathogens, both in vitro and in vivo, reflecting its strong bactericidal activity. Since antibiotics under subtherapeutic dosage are given to animals as growth enhancers, in order for the farmers to adopt, any proposed alternative of antibiotics ideally should display comparable in vivo growth performance. The data suggested that supplementation of formulation, significantly improved the growth-related matrices (lower feed conversion ratio and higher body weight gain) of broilers. Further, enhanced lipase, trypsin and chymotrypsin production after supplemental feeding of the phyto-formulation may also contribute to the growth improvement in birds (20, 24). Accumulation in the animals' meat is one of the undesirable effects of feeding antibiotics to the meat animals. The final composition act in synergistic mode, which necessitates the addition of individual components in low enough concentrations that are safe for the poultry birds. This was obvious in very low copper accumulation in bird tissues. Further, the phyto -components are also reported to be quickly eliminated from the animal system and cause no tissue toxicity in poultry birds.

[0062] Formulation fed poultry birds showed growth enhancement to control fed cohort. This was also supported by enhanced expression of nutrient uptake genes PepTl and GLUT5 which is known to result in better nutrient assimilation. Few extant studies have reported immunomodulatory effects of 5-methyl-2-propan-2-ylphenol and 2-methyl-5-propan-2-ylphenol individually, which was attributed to increased expression of interleukins, TNF-a, INF-y, and secretory immunoglobulin A. Serum corticosterone is important stress parameter in broilersWe did not observe any change in serum corticosterone after supplemental addition of formulation, thus ruling-out any potential feed-induced stress.

[0063] A change in the intestinal microbiota of chickens can influence their immunity and health. Earlier reports have shown the effect of individual phytochemicals like essential oils on livestock species. Feeling of a monoterpene or phenolic like phyto compound in large amounts can alter microbiome of the host; for example -supplemental addition of essential oils in feed resulted in decreased beta diversity in goat. Similarly, supplemental addition of 5-methyl-2-propan-2-ylphenol and 2-methyl-5-propan-2-ylphenol resulted in reduced Shannon and Simpson diversityin chicken. Formulation supplementation, Chao 1 diversity was decreased as compared to control cohort (Fig. 5). It doesn’t show an adverse effect on commensal probiotic bacteria like Lactobacillus spp. in vitro.

[0064] Materials and methods

[0065] Chemicals and biological materials

[0066] All phytochemicals and GRAS status chemicals used in this study were purchased from Sigma Aldrich, USA. All culture media and antibiotic discs for disc diffusion tests were purchased from HIMEDIA, Mumbai, India. ELISA kits were purchased from Geno Technology Inc., USA.

[0067] Minimum Inhibitory Concentration (MIC) fractional inhibitory concentration (FIC) determination of plant secondary phytochemicals For identification of the best combination of phytochemicals, antimicrobial susceptibility testing was performed using the micro broth dilution method in Cation-adjusted Mueller Hinton broth (CAMHB) (HIMEDIA, Mumbai, India). MIC values were determined in triplicate in a flat clear bottom 96-well polystyrene plate (Genaxy Scientific, India). Growth was monitored by O.D. readings at 600nm (ODeoo) after 18 hours of incubation using Spectramax plus plate reader (Molecular Devices, USA). In order to unveil the interaction effects of each selected metabolite and chemical pairs, two-dimensional microdilution chequerboard assays were performed in 96-well plates. To evaluate the interaction of metabolites, the fractional inhibitory concentration (FIC) was calculated for each combination using the formula earlier described (32). Synergy was defined by an FICI value of <0.5 and additivity was defined as an FICI value of > 0.5 to 4, whereas antagonism was defined as an FICI value of > 4(32).

[0068] Time-kill kinetics

[0069] Time-kill experiments were performed in Cation-adjusted Mueller Hinton broth (CAMHB) (HIMEDIA, Mumbai, India) using Salmonella enterica serovar Enteritidis (standard poultry pathogen) or Candida albicans ATCC 10231. An overnight culture of Salmonella enterica serovar Enteritidis and C. albicans was diluted freshly 1: 100 in 5ml CAMHB and incubated at 37°C with aeration at 150 r.p.m. for 3 hours. Two different concentrations of the formulation were then added to the culture (Initial inoculum at the time of antibiotic addition was adjusted to 107CFU / mL) was incubated at 37°C with aeration at 150 r.p.m. Aliquots of culture were periodically sampled and tenfold serially diluted inphosphate buffer saline (IX PBS), plated on CAMH agar and incubated overnight at 37°C. The time-kill curve was plotted using the mean colony count (Logio CFU / mL) versus time over 24 hours. All experiments were performed in triplicate.

[0070] In vitro Frequency of Resistance (FOR)

[0071] The emergence of spontaneous resistance in Salmonella enterica serovar Enteritidis against formulation was determined in the CAMH broth medium (3 mL having IxlO9CFU / mL initial culture). Formulation was added at IX, 2X, and 4X MIC. After 48 h, samples were plated on CAMH agar plates. The plates were incubated for 48 h at 37 °C in static conditions in a humidity -controlled incubator. The frequency of resistance was determined by dividing the average number of colonies obtained after antibiotic treatment by the initial inoculum at the end of 48 h. All experiments were performed in triplicate.

[0072] Generation of antibiotic resistant temporal mutants

[0073] Single-step resistant mutants were generated using serial passaging under constant selection pressure according to the previously described method (33). Briefly, Salmonella enterica serovar Enteritidis (105CFU / mL) cells were treated with 2-fold serial dilution of formulation or tetracycline in CAMH broth for 28 days. Every day cells grown at sub-MIC concentrations were exposed to different concentrations of formulation or tetracycline (ranging from sub-MIC to 8x MIC) to isolate resistant mutants.

[0074] Sampling protocol- Sample collection from an industrial poultry farm

[0075] To evaluate the Samples were collected from 17 different sites. Bacterial microflora from different poultry samples such as bird excreta, bird feed, feed raw material, eggshell, soil, sewage, house flies, and drinking water were isolated. The fecal dropping of white egrets (common migratory birds) was also collected to check the potential spread of antibiotic -resistant bacteria by them. For the isolation of bacterial species, samples (n = 17) (1 g for solid samples, 1 ml for liquid samples) were resuspended in 9 ml 1 x phosphate buffer saline (PBS) and plated on nutrient agar plates. Out of total 1745 isolates, 141 different isolates (presumed different isolates based on the difference in colony morphology) were selected for further antibiotic susceptibility analysis using Kirby-Bauer disk diffusion assay as per CLSI and EUCAST guidelines (34)’(35). The antibiotics chosen for this study are clinically relevant and routinely used for human consumption for many diseases.In vitro efficacy of formulation on bacterial species prevalent in poultry To check the sensitivity of different isolates (MDR isolates) against formulation, the susceptibility assay was performed in CAMH broth. 13 different bacterial species were selected based on their high resistance against multiple classes of antibiotics. Five different probiotic bacterial species were also subjected to sensitivity analysis.

[0076] In vivo efficacy of formulation combination

[0077] A single blind field trial was conducted at a commercial poultry farm located in Manglaur, Uttarakhand, India (29°47'27.38" N 77°52'42.10" E). The trial was conducted on two cohorts of 2000 birds (divided into 4 subgroups each comprising 500), each belonging to the COBB 430Y. The control group was fed the base diet. The Tamyaa group was fed the base diet plus IX MIC of formulation (IX MIC premixed as obtained against Salmonella enterica serovar Enteritidis). The chickens were housed in independent sheds. Feed and water were provided for ad libitum consumption. Gross feed intake (FI) and body weight (BW) were recorded at the end of 42ndday. Feed conversion ratio (FCR = feed requirement per unit of body weight gain) were determined from these data obtained at the end of 42ndday.

[0078] Assessment of physiological parameters - immunity and stress after feeding the supplements

[0079] Fecal microbial enumeration - Eight birds (n = 6) from each group were randomly selected to count the colony-forming units (CFU) in caeca content using standard total plate counts method. The samples (cecal digesta, one gram) were decanted into sterile plastic containers and thoroughly mixed and vortexed with 9 ml sterile IX PBS. The supernatant was serially diluted ten-fold in IX PBS. Viable cell counts were determined using Eosin Methylene Blue Agar (HIMEDIA, Mumbai, India). For enumeration of probiotic bacteria, De Man-Rogosa-Sharpe agar was used and plates were incubated under anaerobic condition in Whitley A35 anaerobic workstation (Don Whitley Scientific Limited, UK).

[0080] Immunological and stress markers - Blood samples were collected from four randomly selected birds. The serum was collected and stored at -80°C until analysis. IL10, TNFa, ILlb, IFNy, and serum corticosterone level was measuredusing the chicken specific ELISA kits of respective biomarkers as per manufacturer’s instruction (Geno Technology Inc., USA).

[0081] Vital organs Histopathology

[0082] At the end of the experiment (on 42ndday) three randomly selected birds were euthanized. For histopathological examination, samples of intestine, liver, and spleen were immediately fixed with 10% neutral buffered formalin. Formalin- fixed tissues were processed, sectioned (5 pm), and stained with hematoxylin and eosin. Microscopy was performed using a brightfield microscope (Axioscope Al, Zeiss, Germany) equipped with a CMOS camera (Axiocam 305, Zeiss, Germany).

[0083] Real-time quantitative PCR

[0084] The effect of different feed additives on the expression of different nutrient uptake genes (PepTl, EAAT3, SGLT1, and GLUT5) was analyzed using quantitative real-time PCR. Total RNA was extracted from frozen intestinal samples and stored at -80°C (n = 3) using RNeasy™ Mini kit according to the manufacturer's protocol (QIAGEN, Germany). cDNA was synthesized from 500 ng of total RNA using the First Strand cDNA Synthesis Kit containing Oligo(dT)18 Primer (Thermo Scientific™, USA). Each qPCR reaction contained 5 pL of Fast SYBR™ Green Master Mix (Applied Biosystems, USA), 3 pL of cDNA (diluted 1:10), 0.25 pL of forward primer (5 pM), 0.25 pL of reverse primer (5 pM) and 1.5 pL of NFW and run in a QuantS tudio5™ Real-time PCR instrument (Applied Biosystems, USA). GAPDH was used as the reference gene. The list of primers and sequences are described in table 1. Fold change was calculated using the

[0085]

[0086] mers and their sequence used

[0087]

[0088] Metagenomic analysis of microbiome using nextgen sequencing of V3 - V4 hypervariable region of 16S rRNAEffect of supplemental formulation in controlling microbial burden and species richness in birds cecal sample was evaluated by metagenomic analysis of 16s rRNA gene. DNA samples for metagenomic analysis from cecal contents were extracted using the Power soil® DNA isolation kit (Qiagen, Germany). The sequencing was performed on Illumina platform (Illumina HiSeq 4000). The paired-end reads were assembled, filtered, trimmed, and aligned to the SILVA 16S database using Mothur software v.1.48.0. The paired-end reads were merged to make contigs leading to a total of 1070013 sequences. Next, the contigs were screened to remove sequences with ambiguous bases, and sequences shorter than 200 bp and longer than 500 bp according to the V3-V4 region which is around 464 bp in length. This removed 477451 sequences and the remaining 592562 were moved further for analysis. Out of these, duplicates were screened to find out the unique sequences. Out of 592562 screened sequences, 295151 were found to be unique. Next, the unique sequences were aligned to the reference (16S Silva V3V4 region). These aligned sequences were then filtered to remove any overhangs at the ends, this removed 13048 terminal gap characters and vertical gap characters and 293526 unique sequences. Next, to further de-noise these sequences, pre-clustering was performed allowing for up to 2 differences between sequences, this led to 162840 unique sequences. Chimera were removed next using the VSEARCH implemented in mothur, leading to 153021 unique sequences (a reduction of 6.03%). As a final quality control step, any fragments belonging to Archaea, chloroplasts, and mitochondria, and unknown were removed. This removed 229 sequences leading to 152929 unique and 583610 total sequences for final analysis. A distance matrix was created with a distance cut-off of 0.03 among sequences and this distance matrix was used to do the final clustering using the OptiClust method. The reads were clustered based into OTUs (Operational Taxonomic Unit) at 97% identity against the greengenes database “gg_13_8_99”. An OTU table containing the abundance of each OTU for each sample was generated and used for further analysis. Alpha and Beta diversities were calculated and plotted using Mothur package.

[0089] Antibiotic resistance evolution and statistical analysis of Enterobacteriaceae isolates from bird cecal samples

[0090] Evaluation of susceptibility of bacteria (Enterobacteriaceae family), i.e., E. coli (green metallic sheen on EMB agar) was carried out using the Kirby-Bauer diskdiffusion assay as per CLSI guidelines. 60 different presumptive colonies (from EMB agar plate) were selected for analysis. All 9 antimicrobial disks used ampicillin, amoxycillin-clavulanic acid, meropenem, tobramycin, amikacin, chloramphenicol, nitrofurantoin, colistin, and ciprofloxacin (HIMEDIA, Mumbai, India) were routinely tested for its efficacy on E. coli ATCC 25922 as a quality control standard.

[0091] Numerous modifications and adaptations of the system of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the true spirit and scope of this invention.

Claims

WE CLAIM:

1. A synergistic plant-based formulation comprising:(a) 2-isopropyl-5-methylphenol (IPMP) ranging from 10 - 100 mg / L, (b) 2-methyl-5-isopropylphenol ranging from 25 - 250 mg / L,(c) 3,7-dimethylocta-l,6-dien-3-ol ranging from 40 - 400 mg / L,(d) (5R)-5-[(lS)-l,2-dihydroxyethyl]-3,4-dihydroxy-2,5-dihydrofuran-2-one - ascorbic acid ranging from 150 - 1500 mg / L, and(e) copper sulphate ranging from 4 -40 mg / L2. The synergistic plant-based formulation as claimed in claim 1, wherein the frequency of Resistance (FOR) for formulation is >1010.

3. The synergistic plant-based formulation as claimed in claim 1, wherein the formulation for inhibiting C. albicans is having minimum inhibitory concentration (MIC) of 8 hours at IX concentration.

4. The synergistic plant-based formulation as claimed in claim 1, wherein the formulation for inhibiting C. albicans is having minimum inhibitory concentration (MIC) of 1 hour at 2X concentration.

5. The synergistic plant-based formulation as claimed in claim 1, for use as alternative of antibiotics for poultry.

6. The synergistic plant-based formulation as claimed in claim 1, wherein the formulation maintains commensal probiotic bacteria Lactobacillus spp.

7. A method of preparing synergistic plant-based formulation as claimed in claim 1, wherein the method comprising the steps of:(a) vigorous mixing of 2-isopropyl-5-methylphenol (IPMP) in the ranging from 100 - 1000 mg / L, 2-methyl-5-isopropylphenol at 250 - 2500 mg / L, 3,7-dimethylocta-l,6-dien-3-ol ranging from 400 - 4000 mg / L, (5R)-5-[(lS)-l,2-dihydroxyethyl]-3,4-dihydroxy-2,5-dihydrofuran-2-one -ascorbic acid ranging from 1500 - 15000 mg / L, and copper sulphateranging from 40 - 400 mg / L concentration in water to obtain forming emulsion;(b) mixing 1 part of the emulsion as obtained in step (a) with poultry feed in 9 parts of feed.

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