Intestinal microbiome of poultry and its interaction with host and diet

Abstract

The gastrointestinal (GI) tract of poultry is densely populated with microorganisms which closely and intensively interact with the host and ingested feed. The gut microbiome benefits the host by providing nutrients from otherwise poorly utilized dietary substrates and modulating the development and function of the digestive and immune system. In return, the host provides a permissive habitat and nutrients for bacterial colonization and growth. Gut microbiome can be affected by diet, and different dietary interventions are used by poultry producers to enhance bird growth and reduce risk of enteric infection by pathogens. There also exist extensive interactions among members of the gut microbiome. A comprehensive understanding of these interactions will help develop new dietary or managerial interventions that can enhance bird growth, maximize host feed utilization, and protect birds from enteric diseases caused by pathogenic bacteria.

Introduction

The gastrointestinal (GI) tract of poultry comes into contact with exogenous microorganisms immediately after hatch and thereafter it becomes a warm shelter for a complex microbiome consisting primarily anaerobic bacteria. As host grows, this microbiome becomes very diverse until it reaches a relatively stable yet dynamic state. Compared with other food animals that are mammalians, poultry (chicken, turkey, and duck) has a shorter GI tract and faster digesta transit. This anatomic feature selects a very different intestinal microbiome in poultry than in other food animals. There exist extensive interactions of this intestinal microbiome with poultry host and diet, and also interactions among individual gut microbes (Fig. 1), which have profound effects on poultry nutrition and health, and are therefore of great importance to poultry production. The objective of this review is to give an overview of the current state of knowledge of the microbe-host, microbe-diet, and microbe-microbe interactions in poultry (primarily chicken) gut.

Figure 1. Conceptual model of the interactions among gut microbiome, avian host, diet, and litter microbiome

Intestinal Microbiome of Poultry

The GI tract of poultry (e.g., chicken, turkey, and duck) consists of esophagus, crop, proventriculus, gizzard, small intestines (duodenum, jejunum, and ileum), cecum, colon, and cloaca. Relative to body length, the poultry GI tract is much shorter than that of mammalian animals. As such, the digesta passes through the entire GI tract faster in poultry than in mammalians. Although diet and feeding can have an effect on passage rate, the average whole tract transit time is less than 3.5 h.¹ Such short retention time selects bacteria that can adhere to the mucosal layer and/or grow fast. However, the ceca, which are two blind pouches that have rather slow passage rate, are ideal habitats for a diverse microbiome that has considerable effect on host nutrition and health. The cecal microbiome is indeed the most studied intestinal microbiome of poultry.

The cecum of both chickens and turkeys harbors a complex microbiome, which is almost exclusively composed of bacteria.² Early cultivation-based studies revealed low abundances of lactobacilli (>10⁴/g colony forming units, CFUs) and clostridia (10²–10⁴/g CFUs) in the small intestines and high abundance (10¹⁰‒10¹¹/g microscope counts) of anaerobic bacteria in the cecum of chickens.^3,4 Identified bacteria included anaerobic Gram-negative cocci, facultative anaerobic cocci, and streptococci. Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium were the major genera that were recovered from cecum by cultivation. Between 20–60% of the total cecal bacteria could be cultivated depending on the media used.^3,4 Temporal changes were also observed as chicken aged.³ The first cultivation-based study on the intestinal microbiome of domesticated turkeys was reported in 1983.⁵ Most (77%) of the microbes were Gram-positive rods, followed by Gram-negative rods (14%), and Gram-positive cocci (9%). Bacteria of Eubacterium, Lactobacillus, Peptostreptococcus, Escherichia coli, Propionibacterium, and Bacteroides were isolated as predominant microorganisms. Although only revealing a limited number and diversity of bacteria, these early studies laid the foundation for microbiological studies of the intestinal microbiome in poultry.

Sequencing of 16S rRNA genes by first the Sanger sequencing technology and recently next-generation sequencing (NGS) technologies make it possible to comprehensively characterize the intestinal microbiome of poultry, and the sequence information has greatly expanded our knowledge on the bacterial diversity present in the intestinal tract, particularly the cecum, of chickens and turkeys.² Through phylogenetic and statistical analysis of 16S rRNA gene sequences recovered from intestinal microbiome of both chickens and turkeys, a global bacterial census was created for poultry intestinal microbiome.² Although this census is not complete, it serves as a phylogenetic framework for the bacterial diversity in the intestinal microbiome of both chickens and turkeys. In total, 13 phyla of bacteria were found, but Firmicutes, Bacteroidetes, and Proteobacteria accounted for most (> 90%) of the intestinal bacteria of chickens and turkeys. More than 900 species-equivalent operational taxonomic units (OTUs, defined at 0.03 phylogenetic distance) were found in chicken, and these OTUs represent 117 established genera of bacteria. For turkey, the census contained nearly 500 OTUs of bacteria within 69 existing genera. The most predominant genera found in both chicken and turkey were Clostridium, Ruminococcus, Lactobacillus, and Bacteroides, but with different distribution between the two bird species. Chickens and turkeys have distinct intestinal microbiomes, sharing only 16% similarity at species-equivalent level. Genetic and other factors (e.g., diet, digesta passage rate, and rearing environment) may be attributable to the difference in intestinal microbiome composition between chicken and turkey.

Interactions between Gut Microbiome and Host

Extensive interactions occur between poultry host and its gut microbiome (Fig. 1). These interactions are manifested particularly through exchange of nutrients, modulation of host gut morphology, physiology, and immunity.

Nutritional interactions

Most readily digestible dietary carbohydrates are digested and absorbed by the host in the proximal gut, leaving indigestible carbohydrates and residual digestible carbohydrates to bacteria residing the distal gut.⁶ Many intestinal bacteria can hydrolyze indigestible dietary polysaccharides, oligosaccharides, and disaccharides to their compositional sugars, which can then be fermented by intestinal bacteria, yielding short chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate. The SCFAs can be utilized by the host as energy and carbon source.^6-9 Such fermentation can be observed in most part of the avian gut (from crop to cecum) but primarily takes place in the cecum, which is densely populated with bacteria.¹⁰ The above fermentation increases as young birds grow. Cecal acetate, propionate, and butyrate are undetectable in 1-d-old broilers. As the cecal microbiome becomes established, these SCFAs reach high concentrations in 15-d-old broilers and remain stable afterwards.⁷ In the cecum, SCFAs are absorbed across the epithelium by passive diffusion and enter a variety of metabolic pathways.⁶ A previous study has provided evidence that SCFAs, especially butyrate, can serve as an important energy source for intestinal epithelial cells.¹¹ In addition, it is reported that SCFAs can regulate intestinal blood flow, stimulate enterocyte growth and proliferation, regulate mucin production, and affect intestinal immune responses.^6,9,12

Gut bacteria also contribute to host nitrogen metabolism. In birds, the intestinal and urogenital tracts meet at the cloaca where urine mixes with feces. Some urine may travel to the ceca due to the retrograde peristalsis in the rectum.¹³ Cecal bacteria can then catabolize uric acid to ammonia, which can be absorbed by the host and used to synthesize a few amino acids such as glutamine.¹⁴ Some of the dietary nitrogen is incorporated into bacterial cellular proteins. Therefore, gut bacteria themselves can be a source of amino acids.¹⁵ However, the majority of these bacterial proteins are lost to the host with the excretion of feces because most of the intestinal bacteria in birds reside in the cecum which does not have the ability to digest and absorb protein. Utilization of bacterial proteins is possible when chickens are housed on hard floors, where coprophagy (ingestion of feces) can occur and bacterial proteins can be digested and absorbed in proximal gut.^8,14

A recent study demonstrated in vitro that the chicken intestinal microbiome required simple sugars and peptides for balanced growth whereas human intestinal microbiome preferred polysaccharides and proteins.¹⁶ Chicken microbiome also produced greater concentrations of SCFAs than human microbiome. Given the shorter digestive tract and faster digesta transit in poultry than in mammalian animals, more sugars and peptides may be available in the intestines of poultry than in the colon of human, which in turn selected an intestinal microbiome adapted to simple sugars and peptides.

Gut microbiome of poultry may also serve as a vitamin (especially B vitamins) supplier to its host.^6,17 Similar as bacterial protein, most of the vitamins synthesized by gut bacteria are excreted with feces because they cannot be absorbed in the cecum.⁶ However, coprophagic birds may benefit from bacterial vitamin synthesis. This is evidenced by a greater vitamin requirement by chickens housed in wire cages, where coprophagy is prevented, than by chickens raised on hard floors.¹⁴

In a reciprocal manner, birds can also provide some nutrients to intestinal bacteria. For instance, mucins produced by goblet cells of the gut are important sources of carbon, nitrogen, and energy for some commensal and pathogenic bacteria.^6,18 Few reports are available on mucin-utilizing bacteria of poultry origin, but studies on other animal species showed that a variety of bacteria can degrade mucins, including some species of Bifidobacterium,^19,20 Bacteroides,⁶ and Akkermansia muciniphila.²¹ These bacteria are able to attach to the mucus layer and secrete specific enzymes for mucin degradation.¹⁸ Although mucin degradation by these bacteria has not been demonstrated in poultry yet, members of these species have been found in the gut of poultry, and it is reasonable to assume that some of the intestinal bacteria can and do degrade mucins in birds. The mucus layer of GI tract serves as a protective barrier for attached bacteria, and the constantly replenished mucin is an excellent source of nutrient for some gut bacteria. The ability to attach to and utilize mucin enables mucin-utilizing bacteria to outcompete other species on the surface of the mucus layer. As a result, these bacteria play an important role in enteric disease and health.

Despite the fact that birds and its intestinal inhabitants both benefit from the host-microbe nutrient exchange, some of the intestinal bacteria are sometimes found to compete with the host for nutrients. Gut microbiome has evolved with the host toward a symbiotic relationship, and in healthy birds direct competition for nutrients is limited, as most digestible nutrients are absorbed by the host in the small intestine, where bacterial density is low and bacterial utilization of nutrient is suppressed due to the low pH and short retention time.¹⁰ However, when bacteria overgrow in the small intestine under certain circumstances, nutrients are captured and utilized by bacteria before normal absorption by host can take place.²² In humans and mice, some intestinal bacteria can deconjugate bile acids thereby suppressing lipid digestion by the host.^23,24 Clostridium perfringens, streptococci, and some of the bifidobacteria and lactobacilli isolated from chickens are able to deconjugate bile acids, but it remains to be determined to what extent bacterial deconjugation of bile acids decreases lipid digestion in chicken.

In modern broiler production industries, feed represents the major portion of production cost. Efficiency in converting feed into body mass is thus of critical concern for broiler producers. Because gut microbiome plays such an important role in feed digestion and absorption, attentions have been drawn to the associations between gut microbiome and host feed utilization efficiency. By using microbial profiling on broiler chickens across various feeding trails, Torok et al.²⁵ were able to identify groups of bacteria that are potentially associated with broiler growth performance. Recently, more comprehensive analyses using a NGS technology also revealed certain bacteria that might be associated with growth performance of broiler chickens.^26,27 Future studies are needed to determine if these bacteria are the cause or consequence of variations in feed utilization efficiency.

Microbiome affects intestinal morphology and physiology

The early post-hatch period is a critical stage for poultry growth and health as the new hatchling switches its nutrient source from the yolk to carbohydrate- and protein-based diet.^28,29 In order to accommodate the rapid transition of nutrient source, the digestive organs of newly hatched poults undergo both anatomical and physiological changes and are the most rapidly developing organs during the early post-hatch period.³⁰ The rapidly developed intestinal tract provides an ideal niche for microbial colonization. In the meantime, gut microbiome also plays an important role in intestinal development. Previous studies using germ-free (GF) chickens indicated that, comparing with conventional birds, the small intestine and cecum of GF birds had a reduced weight and a thinner wall.^31,32 It has been suggested that SCFAs increases enterocyte growth and proliferation, which may partially explain the stimulating effect on intestinal growth by gut microbiome.^33-35 This premise was supported by the study of Muramatsu et al.³⁶ who reported that feeding fermentable carbohydrates, which can stimulate microbial fermentation and consequently SCFAs production, increased the gut weight in chicken.

Gut microbiome can also affect intestinal morphology of poultry. Intestinal villi are shorter and the crypts are shallower in GF birds or birds colonized with a low load of bacteria than in conventionally-raised birds.^32,37 Dietary supplementation of three different probiotic species (Lactobacillus acidophilus, Bacillus subtilis, and Saccharomyces cerevisiae) also increased villus height in duodenum and villus height:crypt depth ratio in ileum of broilers.³⁸ Similarly, it has been reported that dietary inclusion of prebiotics (e.g., fructooligosaccharide and mannanoligosaccharide) or fermented feed (e.g., fermented cottonseed, soybean, and rapeseed meal) also result in increased villus height and villus height:crypt depth ratio in the small intestine of chicken.^39-43 Such morphology alterations are not likely a direct effect of these dietary supplements, but an indirect effect through the manipulation of gut microbiome structure.⁴⁰ Intestinal morphology change can also be an outcome of infections caused by enteric pathogens. For instance, chickens with Eimeria spp/C. perfringens-induced necrotic enteritis had significantly reduced villus heights and villus height:crypt depth ratio in comparison to unchallenged controls or challenged chickens fed zinc bacitracin/monensin.⁴⁴ Fasina et al.⁴⁵ also demonstrated that mock-challenged chicks had significantly greater villus height, villus area, crypt depth, and villus height:crypt depth ratio than chicks challenged with Salmonella Typhimurium.

The activity of intestinal digestive enzymes can be affected by gut microbiome as well. Compared with GF chickens, conventional birds had greater activity of intestinal alkaline phosphatase.⁴⁶ Diets that can induce changes in gut microbiome structure may also influence intestinal digestive enzyme activity. For instance, the activities of amylase and protease are elevated in broilers fed diets containing fermented cottonseed meal or fructooligosaccharides.^40,43 Feeding broilers with fermented soybean meal instead of unfermented soybean meal increased the activities of protease, trypsin and lipase.⁴¹ It was concluded that these diets stimulate certain bacteria (e.g., Bifidobacterium and Lactobacillus) that can increase digestive enzyme activity, while suppressing some bacteria (e.g., Escherichia coli) that can either impair digestive enzyme secretion by damaging the villus and microvillus of mucosa or secrete proteolytic enzyme to degrade digestive enzymes.⁴⁰

Microbiome and immunity

Colonization with microorganisms in the poultry gut occurs immediately after hatch and microbial succession follows until eventual establishment of a complex and dynamic microbiome.⁴⁷ Digestive tract is the most important reservoir of microorganisms and extensive interaction between these non-self cells and host immune system takes place in the GI tract.

The inner surface of avian gut is coated with a gel-like mucus layer which is formed from mucin glycoprotein secreted by the goblet cells.⁴⁸ This layer of mucin consists of an outer loose layer in which microorganisms can colonize and an inner compact layer which repels most bacteria.⁴⁹ As a component of the intestinal mucosal innate immune system, the mucus layer prevents gut microorganisms from penetrating into the intestinal epithelium and serves as the first line of defense against infection.⁴⁷ A good example is the different pathogenicity of Campylobacter jejuni in chicken and human. In vitro studies have shown that C. jejuni is able to adhere and invade both chicken and human intestinal epithelial cells.^50,51 However, C. jejuni does not cause disease in chicken even though the chicken gut is heavily populated with this bacterium, whereas ingestion of C. jejuni-contaminated food may lead to severe gastroenteritis in human.⁵² It has been shown that the chicken intestinal mucus is able to attenuate C. jejuni virulence by inhibiting its ability to adhere and invade intestinal epithelial cells,^51,53 whereas the human mucus-adherent E12 cell line was found to enhance C. jejuni adhesion and invasion.⁵⁴ Thus, it has been suggested that the difference in intestinal mucus layer between chicken and human may contribute to the distinct pathogenesis of C. jejuni seen in these two hosts.⁵⁵ Another study reported that sialylated mucin is more abundant in conventional chicks while sulfated mucin is more predominant in birds with a low bacterial load.³⁷ Such change in mucin composition can be observed as early as 4 d post-hatch and indicates a potential role gut microbiome plays in regulating the establishment of mucus layer.³⁷ By using a chicken necrotic enteritis model (coccidial infection followed by C. perfringens inoculation), Collier et al.⁵⁶ also showed that infection with Eimeria acervulina and E. maxima enhanced host mucogenesis, which benefits the growth of mucolytic bacteria C. perfringens. Interestingly, as the severity of necrotic enteritis becomes greater, the expression of mucin gene (e.g., MUC2) decreases.⁴⁸ Such decline in mucogenesis is probably due to the severe necrosis of the intestinal mucosa which results in extensive shedding of goblet cells.

Another important component of the innate immune system that functions in the avian gut is the antimicrobial peptides present on the intestinal epithelial surface.⁴⁷ In poultry, the most important and well-studied antimicrobial peptides are β-defensins. They are small cationic peptides produced by avian macrophages, heterophils, and epithelial cells, and they can kill various intestinal pathogens by disrupting cell membrane permeability, which leads to cell lysis.^57,58 Brisbin et al.⁴⁷ indicated that Salmonella infection increased the expression of β-defensin genes in chicken, whereas administration of probiotics prior to Salmonella inoculation resulted in a decline in the gene expression of β-defensins. However, in a study conducted by Derache et al.,⁵⁸ in vitro infection with live Salmonella Enteritidis did not increase the expression of β-defensin genes in avian epithelial cell cultures. A possible explanation for this discrepancy is that the increase in β-defensin gene expression after in vivo Salmonella challenge was due to the recruitment of heterophils to the gut in response to Salmonella infection. Interestingly, in their study, Derache et al.⁵⁸ found that avian epithelial cells responded differently to live and heat-inactivated Salmonella Enteritidis: the expression of β-defensin gene AvBD2 in epithelial cells was increased after incubation with heat-inactivated Salmonella Enteritidis. Such finding indicates that live Salmonella Enteritidis may be able to block the induction of β-defensin gene expression in epithelial cells by a yet unknown mechanism and use this mechanism as a strategy to prevent itself from being eliminated by host immunity. Such a strategy may subsequently facilitate Salmonella Enteritidis to adhere to and invade the intestinal epithelium.

The cellular component of the avian innate system, such as macrophages and heterophils, also protects host from enteric infection. These cells can be found in peripheral circulation and the lamina propria. When intestinal microorganisms breach the intestinal epithelial barrier these immune cells are recruited to the site of infection, where they kill the invaders using a variety of strategies, such as phagocytosis and oxidative burst.⁴⁷ The post-hatch colonization of avian gut by commensal microorganisms typically leads to a mild inflammation, which in turn results in macrophage and heterophil infiltration into the lamina propria.⁵⁹ In addition, increased influx of macrophages and heterophils to the lamina propria and villus epithelium can be observed in chickens infected with enteric pathogens such as Salmonella Typhimurium and Salmonella Enteritidis.^45,60 Although leukocyte infiltration is a defense mechanism against microbial infection during acute inflammatory response, it is worth noting that some pathogens are able to take advantage of this defense mechanism and use it to facilitate its pathogenicity. For instance, Salmonella is known as an intracellular pathogen which is able to survive and replicate in some host cells such as macrophages.^61,62 The influx of macrophages to lamina propria and villus epithelium may therefore help spreading the pathogen to other organs and causing systemic infection.

The interaction between gut microbiome and host innate immune system can leads to subsequent adaptive immune response. B cells and T cells, which elicit antibody-mediated and cell-mediated immune responses, respectively, are the two primary types of lymphocytes that are of fundamental importance in the adaptive immune system. In avian gut, B cells and T cells can be found in organized lymphoid tissues (e.g., cecal tonsils, Peyer’s patches, and the bursa of Fabricius) and in more dispersed areas such as lamina propria and epithelium.^47,63 It has been shown that manipulation of gut microbiome through administration of probiotics can influence antibody-mediated immune response. Birds receiving probiotics containing L. acidophilus, Bifidobacterium bifidum, and Streptococcus faecalis showed enhanced systemic antibody response to sheep red blood cells.⁶⁴ In addition, intestinal IgG reactive to tetanus toxoid, and serum IgG and IgM reactive to tetanus toxoid and C. perfringens α-toxin were also increased in chickens fed the same probiotic product.⁶⁵ Other studies suggest that various strains of lactobacilli have a stimulating effect on antibody-mediated response in chicken and such effect is dependent on the strain of Lactobacillus used and the type (layer- or meat-type) and age of the chicken.^66,67 However, it remains to be elucidated how probiotics enhance antibody-mediated immune response. It is speculated that probiotics can stimulate the production of Th2 cytokines (e.g., IL-4 and IL-10), which may subsequently enhance the immune response mediated by antibody.⁶⁴

Besides antibody-mediated response, cell-mediated immune response is also found to be affected by gut microbiome. By using germ-free, conventional, and gnotobiotic chickens, Mwangi et al.⁶⁸ demonstrated that enteric microbiome complexity had a dramatic influence on the gut T cell repertoire. Brisbin et al.⁶⁹ reported that various Lactobacillus species had the capacity to induce differential cytokine expression in T cells of chicken cecal tonsils which could contribute to intestinal homeostasis. In addition, it has been shown that after being challenged with Salmonella Typhimurium, broiler chickens treated with probiotics containing L. acidophilus, Bifidobacterium bifidum, and Streptococcus faecalis had a significant decrease in gene expression of IL-12 and IFN-γ, which are important cytokines in cell-mediated response against intracellular pathogens, in cecal tonsil.⁷⁰ It should be noted that besides pathogens and probiotic strains, commensal bacteria, at least some of them, may also affect the immune response. Future studies are needed to determine the types of such commensal bacteria and their importance to immune response in poultry.

Interactions between Gut Microbiome and Diet

Dietary components affect gut microbiome

Diet has the greatest potential impact on the intestinal microbiome in poultry as dietary components that escape host digestion and absorption serve as the substrates for the growth of intestinal bacteria (Fig. 1). One of the most important impacts stems from the use of wheat-, barley-, or rye-based diets. These diets contain high levels of indigestible, water-soluble, non-starch polysaccharides, favor the proliferation of C. perfringens and predispose young chicks to necrotic enteritis, whereas diets poor in non-starch polysaccharides, such as corn-based diets, do not.^71,72 It has been suggested that high level of non-starch polysaccharides leads to increased digesta viscosity, decreased digesta passage rate, and a decline in nutrient digestibility, which in turn favors the growth of C. perfringens.^73,74 When compared with corn-based diet, wheat-based diets also affect a number of other bacteria.^75,76 Even a small variation in dietary cereal grain composition can potentially affect the intestinal bacteria at strain level as demonstrated by Hammons et al.⁷⁷ who showed that a standard corn-soybean ration favored Lactobacillus agilis type R5, whereas a ration high in wheat middlings favored L. agilis type R1. The source and level of dietary protein may also affect gut microbiome. It has been demonstrated that unlike soybean meal, which is widely used as a source of protein in poultry industry, fermented cottonseed meal as a protein source increases the population of lactobacilli and decreases the number of coliforms in cecum of broiler chickens.⁴³ Diets with high percentages of animal protein (e.g., fishmeal) favors the growth of C. perfringens in the hind-gut of chicken and is considered as one of the predisposing factors of necrotic enteritis.⁷⁸ In addition, it has been reported that C. perfringens was more abundant in the ileum of broiler chickens fed diet with animal fat (a mixture of lard and tallow) than chickens fed diet with soy oil, indicating that gut microbiome can also be influenced by dietary fat source.⁷⁹

Various feed additives in poultry diet can influence gut microbiome and some of them are used to modulate intestinal microbiome to reduce enteric pathogens. Dietary enzymes, such as xylanase and β-glucanase, increase intestinal lactic acid bacteria and decrease the population of adverse and pathogenic bacteria such as E. coli.⁸⁰ Dietary supplementation with xylanase and β-glucanase can also offer chickens some protection against necrotic enteritis as the enzymes break down the non-starch polysaccharides in the diet and reduce the digesta viscosity.^81-83 Dietary inclusion of some plant-derived essential oils has also been used to protect chickens from enteric disease. For instance, plant-derived trans-cinnamaldehyde and eugenol were shown to be effective at reducing Salmonella Enteritidis colonization in 20-d-old broiler chickens.⁸⁴ In addition, it has been demonstrated that a blend of essential oils containing thymol, carvacrol, eugenol, curcumin, and piperin reduced the colonization and proliferation of C. perfringens in the gut of broiler chickens.⁸⁵

Antibiotic growth promoters

Another class of feed additives that has drastic effect on intestinal microbiome is antibiotic growth promoters (AGPs) (Fig. 1). AGPs are a group of dietary antibiotics used at sub-therapeutic levels to improve feed efficiency, increase animal growth, and maintain animal health.⁸⁶ Dietary inclusion of AGPs has been practiced in food animal industry for more than 50 y.^86,87 Although the precise mode of action of AGPs still remains to be elucidated, it is widely accepted that the growth-promoting effect of AGPs is primarily brought about through modulation of intestinal microbiome.⁸⁸ Adverse and pathogenic bacteria in the GI tract of chicken, such as E. coli, Salmonella ssp., and C. perfringens, compete with the host for nutrient and may also damage the intestinal epithelium, which adversely affect the digestion and absorption function of the host.⁸⁹ Inclusion of AGPs in poultry diet can inhibit the growth of enteric pathogens, reduce the incidence of disease and promote growth of the birds. However, due to the growing concern over widespread antibiotic resistance, there is a trend toward abolishing the use of AGPs. Most AGPs are banned in the European Union, and the United States has started to reduce the use of AGPs, with a possible ban on AGPs in the not-so-distant future.⁹⁰ A negative outcome of banning AGPs is potential increase in incidence of disease in chicken. For instance, after the AGPs ban, C. perfringens-induced necrotic enteritis has become one of the most noticeable, emerging diseases of broiler chickens in Europe.⁹¹ Therefore, non-antibiotic alternatives which can control disease and promote growth of chicken are of great interest.

Prebiotics

Prebiotics are indigestible food ingredients which benefits the host animal by serving as a substrate for one or several beneficial bacteria present in the intestine (Fig. 1), granting these beneficial bacteria proliferative advantages over other bacteria.^9,90,92 Most prebiotics are polysaccharides such as galactooligosaccharides (GOS) and fructooligosaccharides (FOS). It has been reported that dietary inclusion of GOS favored the growth of bifidobacteria in the GI tract of broiler chickens.⁹³ Inclusion of FOS in an alfalfa molting diet significantly decreased cecal Salmonella Enteritidis counts in laying hens.⁹⁴ Dietary supplementation with FOS also decreased C. perfringens and E. coli, and increased Lactobacillus diversity in chicken gut.⁹⁵ Mannanoligosaccharides (MOS) is another prebiotic used in poultry industry. In addition to stimulating beneficial bacteria, MOS can also block pathogen binding to mannan receptors on the mucosal surface, thus hampering the attachment to and colonization of intestinal epithelia by certain pathogenic bacteria, particularly Salmonella Typhimurium.⁹⁶

Interactions among Avian Gut Microbes

As in other microbiome, different members of the GI microbiome can have different interactions, such as competition, cooperation, and antagonism (Fig. 1). The interactions among different bacteria that are important to poultry production are overviewed.

Competition for nutrient and attachment site

Although the avian GI tract is an ideal habitat for microorganisms, it does not support unrestricted microbial growth or proliferation due to the limited availability of nutrient and space therein. Therefore, competition for these resources (i.e., nutrient and attachment site) among microorganisms is a common phenomenon in intestinal ecosystem.⁹⁷ A good example is the competition for zinc among GI microbes. Zinc is an essential trace element required by both eukaryotic and prokaryotic cells and is involved in various cellular functions, such as enzymatic reactions and gene expression.^98,99 Under low-zinc conditions, C. jejuni uses the high-affinity ZnuABC transporter to bring zinc into cell.¹⁰⁰ In a recent study, Gielda and DiRita showed that both a wild-type C. jejuni strain and a znuABC^- mutant strain of C. jejuni were able to colonize limited-mirobiota chicks at similar efficiencies, but only the wild-type C. jejuni strain was able to colonize conventional chicks.⁹⁹ In the same study, it was also shown that the zinc level in cecal content was significantly lower in the conventional chicks than in the limited-microbiota chicks, suggesting that under low zinc conditions, C. jejuni lacking the high-affinity zinc uptake system was outcompeted by other bacteria present in the GI tract. The ZnuABC transporter system is found not only in C. jejuni but also in some pathogenic bacteria (e.g., Salmonella Typhimurium and E. coli), making it a potential target for development of broad-spectrum antimicrobials.^99,101,102

In order to cause infections in birds, enteric pathogens need to first attach to and breach the intestinal epithelial barrier.¹⁰³ In healthy birds, the commensal bacterial communities in the GI tract colonize intestinal mucosa and form a layer of bacteria covering the mucosal surface. By occupying a diverse array of adhering niches along the GI tract, this layer of dense and complex microbial communities can effectively block the attachment and subsequent colonization by most invading enteric pathogens.^103,104 This phenomenon is called “competitive exclusion.”³² The GI tract of newly hatched chick is sterile, but is immediately colonized by microorganisms present in the surrounding environment.⁴⁷ In the wild, the GI tract of the new hatchling is rapidly colonized by members of the gut microbiome from its mother’s feces and is therefore protected from pathogen invasion.¹⁰⁵ However, in commercial poultry production the chicks are hatched in incubators and have no contact with hens. The surrounding environment is therefore relatively clean and usually has a microbial community distinct from the microbiome in a healthy adult chicken’s gut, which may lead to a delay in normal colonization and succession of intestinal microbiome.^92,105 Enteric pathogens in the environment may thus have a greater opportunity to attach to and breach intestinal mucosal layer and cause infection in new hatchlings as a result of the absence of a normal gut microbiome. This may partially explain why newly hatched chicks are particularly vulnerable to enteric infections such as necrotic enteritis.^92,103 In order to protect newly hatched chicks from enteric disease, competitive exclusion cultures have been used by poultry producers to help newly hatched chicks to rapidly establish a healthy gut microbiome. Competitive exclusion cultures are suspensions of intestinal contents obtained from healthy adult birds.¹⁰⁶ By oral administration to newly hatched poults, competitive exclusion cultures have been shown to be effective in protecting new hatchings from being infected by some pathogens such as Salmonella and C. perfringens.^107-109

Production of bacteriostatic and bactericidal substances

Another widely used strategy for some bacteria to gain competitive advantages is to produce bacteriostatic or bactericidal substances hostile to competitors. Previous studies have shown that lactic acid and other SCFAs produced by various commensal bacteria are inhibitory to certain pathogens. For instance, in vitro studies have shown that lactic acid bacteria ferment carbohydrates present in chickens’ feed and produce lactic acid, which lowers the pH in the surrounding environment and inhibits the growth of certain pathogens such as E. coli, Salmonella Typhimurium, and C. perfringens.^110,111 An in vivo study also demonstrated a negative correlation between concentrations of SCFAs (in particular acetate, propionate, and butyrate) and abundance of the family Enterobacteriaceae in broilers’ ceca.⁷ Such a negative correlation was further substantiated by an in vitro study conducted by the same researchers. It was proposed that, in addition to lowering extracellular pH, SCFAs in undissociated form can diffuse freely across the bacterial cell membrane into the cell, where they dissociate, lowering the intracellular pH that inhibits some essential enzymes or metabolism.^7,112,113

Certain bacteria can also produce bacteriocins to selectively inhibit the growth of other bacteria. Bacteriocins are a group of antimicrobial peptides produced by bacteria and archaea.¹¹⁴ Various strains of Lactobacillus salivarius isolated from chicken GI tract can produce bacteriocins which are inhibitory to some Gram-negative and Gram-positive bacteria such as Salmonella Enteritidis and C. jejuni.^115-117 Bacteriocins produced by strains of Enterococcus faecium, Pediococcus pentosaceus, and Bacillus subtilis isolated from broiler chicken are able to inhibit C. perfringens and Listeria monocytogenes.^118,119 In addition, it has been shown that several strains of E. faecium produce bacteriocins against the oocysts of poultry Eimeria spp.¹²⁰ The inhibitory effect on various adverse bacteria and pathogens makes bacteriocin production a frequently considered trait in selection of probiotics. Nevertheless, it is worth noting that a variety of pathogenic bacteria (e.g., Staphylococcus aureus) also produce bacteriocin effective against competing bacteria.¹²¹

Horizontal gene transfer

Horizontal gene transfer is “the non-genealogical transmission of genetic material from one organism to another.”¹²² It is mediated by processes such as conjugation, transformation, and transduction and is an effective mechanism which contributes to bacterial diversification and facilitates bacterial adaptation to new environments.^123,124 In modern poultry industry, litter, which contains bacteria excreted from chickens or turkey, is often used for multiple growth cycles. Once developed in the GI tract, antibiotic resistant bacteria can accumulate in the litter and recycle between the litter and GI tract over multiple growth cycles (Fig. 1). Such a practice can greatly increase the incidence of horizontal transfer of resistance genes and may contribute to the wide spread of antimicrobial resistance among adverse and pathogenic bacteria and is thus of particular interest.^125-127 In addition, virulence genes can also be exchanged among poultry enteric pathogens, increasing the recipient’s pathogenicity.¹²⁸ The predominant commensal intestinal microorganisms usually possess certain traits which enable them to outcompete other bacteria (especially adverse and pathogenic bacteria) and survive in the GI tract. These traits, however, may be acquired by pathogens via horizontal gene transfer, making those pathogens more competitive. On the other hand, commensal bacteria may also become pathogenic to the poultry host by obtaining virulence factors from pathogens.¹²⁹ Therefore, caution should be taken when using direct-fed microbials such as probiotics.

Probiotics

Probiotics are live microbial feed supplement used by livestock and poultry producers to protect animals from enteric pathogen infection and improve animal health.^92,130 The mode of action of probiotics can vary depending on the traits of the specific probiotic strains/species used, but most probiotics benefit the host through the following mechanisms: (1) inhibition of colonization by and proliferation of pathogenic bacteria through competition for nutrient and attachment site,^103,104 (2) production of bacteriostatic and bactericidal substances against pathogens,^7,111 (3) neutralizing enterotoxins,¹³¹ (4) enhancing gut barrier function,¹³² and (5) enhancing host immunity.^132,133 The effect of different probiotics on chicken gut microbiome has been extensively investigated. Several lactobacilli strains have been shown to decrease the population of Salmonella, Campylobacter and some other non-beneficial bacterial groups in chicken gut.^134,135 Molnár et al.¹³⁶ reported that dietary supplementation of Bacillus subtilis significantly decreased E. coli population in the ileum of chicken. Another study demonstrated that spores of Bacillus licheniformis could prevent C. perfringens-induced necrotic enteritis in broiler chickens.¹³¹ Some strains of Clostridium butyricum are also potential probiotics that can be used in poultry production. This was demonstrated by Yang et al.¹³² who showed that C. butyricum HJCB998 significantly decreased cecal Salmonella and C. perfringens population while increasing Lactobacillus and Bifidobacterium populations in the cecum. The protective effect of multispecies probiotics has also been investigated. A multispecies probiotics containing Enterococcus faecium, Bifidobacterium animalis, Pediococcus acidilactici, L. salivarius, and Lactobacillus reuteri isolated from chicken gut decreased cecal coliform population.¹³⁷ Ghareeb et al. also demonstrated that multispecies probiotics containing E. faecium, P. acidilactici, L. salivarius, and L. reuteri significantly reduced cecal colonization by C. jejuni, indicating that probiotic products can also be used to improve food safety by reducing the population of human pathogens, such as C. jejuni, in chicken.¹³⁸

Poultry litter microorganisms influence gut microbiome

During their growth cycle, chickens continuously take up microorganisms from the surrounding environment. Poultry litter, the bedding material used in chicken houses, is usually mixed with chicken excreta and thus harbors a complex microbial community (mostly intestinal bacteria), and is thus of a potential impact on chicken gut microbiome (Fig. 1). Reusing litter for several growth cycles before a thorough clean-out is a management practice commonly used by poultry producers to reduce production cost and to help alleviate the challenges faced in litter disposal.¹³⁹ Reuse of poultry litter influences the microbial community resident in the litter, which may in turn affect chicken gut microbiome. In a recent study, Cressman et al.¹⁴⁰ demonstrated that more environmental bacteria were found in fresh litter, while more bacteria of intestinal origin resided in reused litter. It was also found in the same study that the ileal mucosal microbiome of chickens reared on fresh litter was dominated by Lactobacillus spp, whereas a group of unclassified Clostridiales were the dominating bacteria in chickens reared on reused litter. It was also reported that microorganisms in reused poultry litter can function as competitive exclusion culture and delay ileal mucosal colonization by C. perfringens during early post-hatch period.¹⁴¹ On the other hand, reused litter may also harbor disease-causing microorganisms from the previous flock and thus serves as a source of pathogens to the subsequent flock.¹⁴²

Conclusions and Future Perspectives

Gut microbiome is now recognized as an essential component of the intestinal ecosystem and is referred to as a forgotten organ, which contributes to the wellbeing of animal host in a range of aspects, especially nutrition and disease resistance.¹⁴³ Thanks to the new technologies such as NGS, it is now possible to gain a comprehensive knowledge of not only the compositional but also the metabolic characteristics of the gut microbiome, which allows researchers to better understand the interactions among gut microbiome, diet, and host. Manipulations of gut microbiome through dietary and managerial interventions have been used by poultry producers to enhance bird growth and reduce the incidence of disease. Undeniably, however, AGPs are still the most effective and cost-efficient strategy to do that job. Further studies on poultry gut microbiome and its interaction with host and diet can potentially provide the knowledge base needed to develop alternative strategies that can completely replace AGPs in modern poultry production.

Glossary

Abbreviations:

GI

gastrointestinal

CFUs

colony forming units

NGS

next-generation sequencing

SCFAs

short chain fatty acids

GF

germ-free

AGPs

antibiotic growth promoters

GOS

galactooligosaccharides

FOS

fructooligosaccharides

MOS

mannanoligosaccharides

10.4161/gmic.26945

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.