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2005 Poultry Science Association, Inc. Comparative Gut Microflora, Metabolic Challenges, and Potential Opportunities J. Apajalahti1 Alimetrics Ltd, Höyläämötie 14, FIN-00380 Helsinki, Finland Primary Audience: Researchers, Nutritionists, Veterinarians SUMMARY Bacteria colonize practically every habitat in the nature. Bacterial community of the gastrointestinal tract (GIT) is one of the major metabolic tissues of animals. Still, its species composition is not known, because methods that have been previously used for bacterial analysis do not capture the species present. The bacterial community within the GIT not only has intracommunity interactions, but it also interacts with the host tissues. For the host, it is essential to tolerate commensal bacteria and to recognize and fight intestinal pathogens. In addition to recognized pathogens, there are many functional bacterial groups, which produce metabolites considered harmful for the host. Such functional bacterial groups include protein-fermenting bacteria that produce toxic end products in their amino acid metabolism, which is why a highprotein diet is often considered unhealthy. Carbohydrates are preferred substrates for gastrointestinal bacteria, but in the distal colon, they may become depleted, and putrefaction becomes the dominating type of fermentation. This largely accounts for the health effects of slowly fermenting dietary fibers. Microbial communities in the GIT of different animals have developed hand-in-hand with their digestion strategy. In monogastric animals, bacterial fermentation has concentrated in the lower GIT, whereas in ruminants bacteria are responsible for the initial digestion of the diet. Rumenlike sites with intense bacterial fermentation are found in most animals, but no other group of animals totally depends on bacteria. The response of rumen bacteria to feed components can be evaluated by an in vitro rumen simulation, which measures relevant fermentation parameters, such as the yield of bacterial biomass (protein) and volatile fatty acids (energy). In monogastric animals, it is essential that the host has measures to prevent bacterial competition for substrates in regions in which absorption takes place; the use of prophylactic antibiotics for production animals has aided this. Availability of good methods for monitoring the total bacterial communities and their metabolism is a prerequisite for informative studies in the future. Key words: intestinal bacteria, chicken, rumen, human, putrefaction, bacterial metabolite, antimicrobial growth promoter 2005 J. Appl. Poult. Res. 14:444–453 1 To whom correspondence should be addressed: [email protected]. APAJALAHTI: INFORMAL NUTRITION SYMPOSIUM DEVELOPMENT OF GASTROINTESTINAL MICROBIAL COMMUNITIES Bacteria are spread everywhere in nature and readily colonize a new habitat with available nutrients and energy for growth and maintenance. Bacterial cells are carried by water, soil, dust, aerosols, or simply by air. Indeed, there are few bacteria-free zones in our environment. Consequently, newborn animals are exposed to bacteria from the very moment of birth or hatching. The gastrointestinal tract (GIT) of mammals receive the first inoculum of bacteria from feces and breast milk of the mother [1, 2], whereas newly hatched chicks get bacteria from the surface of the eggshells [3, 4]. Very little information is available on the bacterial colonization of the fish gut, but it seems logical that the original inoculum for the fry comes from the surrounding water. In chickens the GIT becomes rapidly colonized by bacteria, with the maximum bacterial densities being reached within the first 5 d after hatching [5]. During the following weeks, the composition of microflora changes markedly [6, 7]. The ingested bacteria from habitats other than GIT mainly just pass through the intestine unable to colonize any compartment of the tract. However, some bacteria thrive under the gut conditions and become members of the resident microbial community. In such communities, bacteria do not live independently of the other species but interact and depend on each other and their host in many ways. Bacterial communities are metabolically versatile mixtures of different bacteria whose relative abundance is regulated by environmental factors, such as substrate flows, antibacterial compounds, and the structure and function of the host epithelium. As entities, bacterial communities are energetically more efficient and metabolically more flexible than representatives of any single bacterial species present or the host itself, because natural selection inevitably forces out bacteria, which do not have a unique role (ecological niche) in the community. In addition, any strain becomes replaced if a more efficient one appears. Following this general mechanism of bacterial community evolution, site- and diet-specific bacterial 445 communities evolve in the GIT compartments of different animal species. The GIT of an adult homeothermic animal maintains bacteria in quantities exceeding the total number of host cells and the metabolic activity of any host tissue. Yet, the composition and metabolic function of this bacterial tissue is poorly known today, because most methods used for bacterial analysis have been based on dilution and cultivation; these methods separate the interdependent bacterial species from each other and the host. Only a fraction of gastrointestinal bacteria grows independently as pure cultures with the practices applied in most laboratories today [8, 9, 10]. Intestinal lumen should be considered as a part of the external world in which the animal is at its closest vicinity to the bacteria of the surrounding environment but still clearly separated by the intestinal epithelium. The bacterial community residing within the GIT not only has intracommunity interactions, but it also interacts with the intestinal tissue of the host. A difficult task of the defense mechanism of the intestinal tissues is to allow close contact with the friendly commensal bacterial community while recognizing and fighting pathogenic bacteria. Indeed, well-directed immunological tolerance and defense mechanisms are important determinants of intestinal health [11, 12]. While natural selection drives the development of the bacterial community to the best possible fit for its habitat, a similar selection drives the evolution of host animals to accommodate a beneficial bacterial community. Failure to tolerate commensal bacteria may be as detrimental as the failure to recognize and successfully fight intestinal pathogens. Indeed, one of the challenges of today’s animal breeding is to select for animals, which show both good performance and high resistance against incidental intestinal pathogens. DISTRIBUTION OF MICROBES IS DETERMINED BY THE DIGESTIVE SYSTEM OF THE HOST Microbial communities in the GIT of different animal species have developed hand-in-hand with their digestion strategy, because the structure and function of the digestive machinery determines the sites of intestine in which both 446 JAPR: Symposium FIGURE 1. Relative intensity of bacterial fermentation in compartments of the GIT of various animals. Intensity of bacterial metabolism in the intestinal compartments is indicated by the fill intensity in respective textboxes. physicochemical and nutritional requirements for bacterial growth are fulfilled. Still, there are no sites in the GIT in which bacterial growth would be entirely missing. The overall bacterial activity may be modest in compartments, such as an acidic stomach, but specialized bacteria thrive under difficult conditions and benefit from the reduced competition by other bacteria. The nutritional diversity and digestion potential of bacterial communities clearly exceed that of any host animals. Consequently, many components in the diet cannot be attacked by the host digestive enzymes but can be utilized by intestinal bacteria. Coevolution of animal species and their intestinal microflora has led to a situation in which hot spots of bacterial fermentation have been restricted to defined parts of the GIT. In monogastric animals, bacterial fermentation has concentrated in the cecum and colon (Figure 1). These parts of the GIT receive dietary compounds that escape host digestion and absorption; therefore, bacteria do not compete with the host when they ferment the entering substrates. However, in the upper GIT competition occurs for all the simple sugars and amino acids utilized by the host, which are also available for the bacteria. In poults, equines, and rodents, the site for intense bacterial fermentation is a well-developed appendix or cecum. In other monogastric animals, such as humans and swine, the cecal appendix is diminished, and the bacterial fermentation mainly occurs in the colon (Figure 1). In ruminants, bacteria are responsible for the primary digestion of the diet. This strategy saves energy, since the costs for feed digestion and fighting against bacterial overgrowth are clearly reduced. The drawback is that the metabolism of the host depends on the end products of bacterial metabolism, such as short-chain fatty acids, which have only half of the energy content of the carbohydrates. However, since the metabolic potential and nutritional diversity of the rumen bacterial community is far beyond that of the host, ruminants can benefit from dietary components, such as cellulose and hemicellulose, which are not available for monogastric animals. Provided that feed intake is adequate, the microbial community of an adult cow with more than 1016 fully adapted bacteria is a high-capacity fermentor capable of providing enough energy for the host. All the homeothermic animals mentioned above have 1 compartment in the intestinal tract with high fermentation intensity (Figure 1). Bacterial density in the cecum, colon, and rumen exceeds 1011/g of digesta, the habitats are anoxic, and their redox potential is in the range of −200 to −400 mV [13, 14, 15, 16]. Typically, such bacterial communities are methanogenic or sulfidogenic, and the major end products of fermentation are highly reduced volatile fatty acids, such as acetic, propionic, and butyric acids and gases, such as CO2 and CH4. Not all animals have such bacterial concentrates in their intestine. The GIT of Atlantic salmon has a stomach, APAJALAHTI: INFORMAL NUTRITION SYMPOSIUM proximal intestine with a number of pyloric ceca, and distal intestine. All these compartments are characterized by flat bacterial densities lower than those in the ileum of the homeothermic animals and modest bacterial fermentation, producing lactic and acetic acids [17] (Figure 1). Such low bacterial density and the lack of bacterial gradients are likely to reflect the low body temperature and highly digestible, carbohydratepoor diet of these carnivorous species. PROTEIN FERMENTATION IN THE GUT PRODUCES HARMFUL METABOLITES In the GIT, we can name obvious pathogenic bacteria, which invade host tissues and produce potent toxins. Well-known examples are some species of the genera Salmonella, Campylobacter, and Clostridium, which lead to acute diarrhea, intestinal inflammation, and sometimes lethal necrosis of the intestinal tissue [18, 19, 20]. In addition to such indisputably recognized pathogens, there are many functional bacterial groups, which under certain conditions release metabolic products considered harmful for the well-being of the host. Such functional bacterial groups include putrefactive, protein-fermenting bacteria, which produce toxic end products in their amino acid metabolism. The metabolism of all amino acids produces, in addition to shortchain fatty acids, ammonia, and amines, which tend to increase the pH of the intestinal contents (Figure 2). Saccharolytic fermentation (fermentation of carbohydrates) produces mainly organic acids, which tend to reduce luminal pH. The general assumption is that a low intestinal pH is good for intestinal health, because the growth of many known pathogens is inhibited under such conditions [21, 22, 23, 24, 25]. Indeed, the relative intensity of the fermentation types determines the effect of the bacteriological activity on the acidity of the intestinal contents (Figure 2). Products of putrefaction have several undesired effects on the host health, which is why high-protein diets are often considered unhealthy for humans. Firstly, amino acid assimilation always produces ammonia, which may lead to neoplastic growth of colon epithelium and harmful accumulation of ammonia in body fluids 447 [26]. For risk groups, this can be especially hazardous. Aromatic amino acids, such as tyrosine, phenylalanine, and tryptophan are converted to phenols and indoles, which have been shown to express hypertensiveness, schizophrenia, and migraine, and phenols are, in addition, cocarcinogens [27, 28, 29]. Generally, bacteria favor carbohydrates, if those are available. In practice, this means that saccharolytic fermentation is characteristic of proximal colon, which is rich in carbohydrates. Carbohydrates in the distal colon become depleted; thus, putrefaction becomes the dominating type of fermentation. This largely accounts for the health effects of prebiotics and dietary fibers; as slowly digestible structures, they provide carbohydrates also to distal colon, thus suppressing putrefaction [30]. An obvious alternative for the inclusion of nonstarch polysaccharides in the diet is to limit the intake of poorly digestible protein. If dietary protein became readily digested in the upper GIT by the digestive system, there would be less substrate available for the colonic putrefaction. Predatory animals have their meals raw, which keeps the meat-derived proteins native and far more susceptible to digestive enzymes. Little is known about the extent to which the end products of putrefaction affect the performance of production animals. It seems likely, however, that any protein escaping digestion in the small intestine of chickens and pigs is a potential risk for the health of the animal. MICROBIAL GROWTH IN THE SMALL INTESTINE IS COSTLY FOR THE PRODUCTION ANIMALS Taking into account that small intestinal digesta is very rich in nutrients, it is striking how efficiently animals can keep bacterial numbers low in this critical part of the digestive tract. Monogastric animals have several mechanisms that restrict bacterial growth in the proximal GIT, including chemical inhibition (e.g., acid and bile), highly competitive rate of nutrient absorption (large absorptive surface and active transport), high passage rate of digesta (washout of free bacteria), continuous sloughing of the epithelial cells and mucus (washout of adhered bacteria), and the immunological defense mech- 448 JAPR: Symposium FIGURE 2. Schematic presentation of characteristic pathways and potential health effects of saccharolytic and putrefactive fermentations by intestinal bacteria. anisms (IgA production). Failure to prevent bacterial growth in the proximal small intestine would, no doubt, lead to starvation of the host. Physiological functions, such as those mentioned above, prevent the wild competition for substrates, and bacteria are restricted to densely populated sites, such as rumen, cecum, or colon. In these compartments, bacteria benefit the host by converting unavailable dietary compounds to end products of fermentation, which can then be utilized by the host. However, in spite of all the measures to limit bacterial growth to described compartments, bacteria do grow throughout the GIT. Even though the density of actively metabolizing bacteria in the low-density regions rarely exceeds 1% of those in the most active sites, bacteria can still capture a significant proportion of simple substrates that could be readily absorbed and utilized by the host. In broiler chickens, as much as 20% of the total absorptive surface in the small intestine can be bacterial. If we assume that the respected absorp- tive surfaces would be evenly distributed in the small intestine and the rate of uptake through bacterial membranes would be comparable to that of the epithelial membranes of the host, bacteria could capture up to 20% of the nutrients. The true figure is most likely somewhat lower because of an increasing gradient of bacterial density and a decreasing concentration of simple substrates towards the distal end of the small intestine. Animal production has been commonly using prophylactic antibiotics to support the animals’ inherited measures to fight bacteria in the intestinal regions that are essential for the nutrient extraction and absorption. The total bacterial numbers in the ileum of broiler chickens are reduced by 90% with Avoparcin [13]. Possibly based on such bacterial growth reduction, growth-promoting antibiotics improve weight gain and feed conversion efficiency in pigs and broiler chickens [31, 32]. Recently, the use of some prophylactic antibiotics has been banned APAJALAHTI: INFORMAL NUTRITION SYMPOSIUM 449 FIGURE 3. Effect of antibacterial agents on the growth of broiler chickens. Birds were grown on a wheat-based diet amended with monensin, bacitracin methylene disalicylate (BMD), and phenoxy methyl penicillin as indicated in the figure. Bars in columns indicate SE between replicate animals [33]. in many countries in an attempt to limit the spread of antibiotic-resistant organisms. The trend will likely expand to limitation of the remaining antimicrobial growth promoters and perhaps even the coccidiostats. The consequence of the present development varies and will continue to vary from country to country. In countries in which the feed production and farm management practices follow high standards, the situation is relatively well under control. However, in many countries, abandoning the prophylactic antibiotics has led to serious problems in animal health and performance. Figure 3 summarizes the effect of a coccidiostat (monensin) alone and in combinations with a growth promoter bacitracin methylene disalicylate (BMD) or a therapeutic antibiotic (penicillin) on the growth of broiler chickens [33]. At the age of 11 d, none of the antimicrobials had an effect on the growth of the animals, whereas 1 wk later all the treatment groups had gained significantly more weight than the control group. It was not until after 4 wk from hatching that the antibiotics combined with monensin started to affect the growth of the chicken (Figure 3). Even though some antimicrobial compounds are called and registered as coccidiostats instead of growth pro- moters, their effect on the growth of total microflora may not differ significantly. The presence of antibiotics in feed has been one of the major selective factors affecting the composition of microflora in the GIT of animals. For decades, microbe selection has favored those tolerating antibiotics in comparison to those sensitive to antibiotics. This antibiotic-tolerant microflora has become the normal intestinal microflora as we know it, and indeed, most of our knowledge and experience on animal management leans against this background. Veterinarians have been able to recognize the microbemediated disorders developing from this normal microflora base. Likewise, most feed companies, animal breeders, and so on have developed their products for conditions using growth-promoting antibiotics. Removing antibiotics from feeds has changed the rules of competition (selection pressure) in the intestinal microbial community from favoring antibiotic-resistant microbes to the advantage of microbes growing efficiently on feed residues in the absence of antibiotics. Since most microbes in the GIT of animals have been and are still unknown, the new selection criteria may lead to the outgrowth and establishment of unforeseeable bacterial species. JAPR: Symposium 450 FIGURE 4. Flow chart illustrating the role of rumen fermentation in ruminant nutrition. Following the recent ban of growth promoters, feed and feed ingredient companies are actively seeking for alternative strategies to control unwanted growth of small-intestinal microflora. As the tools for microbial community analysis have become more accurate and independent of bacterial culturability we have learned that bacteria in the GIT can be modulated by numerous dietary vehicles, such as grain, feed types, and feed enzymes [13, 34, 35]. RUMEN BACTERIA BUILD PROTEIN AND ENERGY FOR THE HOST Gastrointestinal bacteria have a central role in the life of ruminants, not only on the health of individual animals but also on the evolution of a whole animal species. In a nutrient-poor environment, animals with emerging characteristics of ruminants would benefit from diversi- fied selection of utilizable feed and retrieval of energy from cellulose and hemicellulose. Rumen-like sites with intense bacterial fermentation are found in most animal species, but no other group of animals depends on bacterial fermentation to the same extent as the ruminants, which have been estimated to derive 70% of their energy from the products of rumen fermentation, mainly acetic, propionic, and butyric acids [36]. Not all acids have the same value for the host. Different fatty acids are preferred energy sources for different tissues; therefore, their relative value for the host varies, depending on the physiological status of the animal. Monogastric animals, such as swine and human, may significantly gain from bacterial fermentation; they have been estimated to achieve 10 to 20% of their energy requirements by absorbing shortchain fatty acids produced by bacteria in the hindgut [36, 37]. APAJALAHTI: INFORMAL NUTRITION SYMPOSIUM 451 FIGURE 5. Yield of volatile fatty acids and microbial biomass in rumen fermentation simulation. In vitro fermentation was carried out for 12 h, and then fermentation products were quantified. Volatile fatty acids were analyzed by gas chromatography and bacteria by flow cytometry as described previously [17, 38]. Bars in columns indicate SE between the replicate fermentation vessels [33]. The bulk of dietary nitrogen is assimilated by the actively growing rumen microflora. Subsequently, the majority of the protein assimilated by the animal comes from the digestion of bacterial biomass in abomasum, the true stomach of the ruminants. Optimization of the diet for ruminants is challenging due to the involvement of the complicated rumen parameters. How to optimize dietary protein and energy when they cannot be readily analyzed from the components of the ration? True protein and energy available for the host are not in the diet but are being produced by rumen bacteria from the components of the diet (Figure 4). Feed materials used for ruminant rations are many, and their quality may vary. Quality of forages depends on plant varieties, fertilization, and seasonal conditions, such as light, temperature, and humidity. Quality of fermented silages depends, in addition to the factors listed above, on the bacteria present and the fermentation conditions. Also, farmers use many types of byproducts, the specifications of which are difficult to control. One way to evaluate the response of rumen bacteria to feed components is to use an in vitro rumen simulation and to measure relevant fermentation parameters, such as the yield of bacterial biomass (protein) and volatile fatty acids (energy). As an example, we tested fermentation characteristics of different fractions of corn silage, using such an approach. Significant differences were observed in the yield of bacterial biomass, volatile fatty acids, and gas production of different fractions (Figure 5). The highest yields of both microbial protein and volatile fatty acids were found with the grain fraction; rumen fermentation produced 800 g of acids and bacteria from 1 kg (DM) of ensiled grain [33]. The other fractions and the mixed silage produced about 500 g of the fermentation products. It is noteworthy that not only the total yield, but also the energy-to-protein ratio of different fractions of corn silage varied significantly (Figure 5). The approximate ratio of microbial biomass to volatile fatty acids was 1:4 in mixed corn silage but close to 1:2 in the leaf fraction of the corn silage. How accurately can protein and energy yields of different feed raw materials be estimated from their chemical composition? Is it possible that the production efficiency of dairy and beef cattle could be significantly improved if JAPR: Symposium 452 we knew how to optimize the relative production kinetics of the true protein and energy? FUTURE POTENTIAL OF KNOWLEDGE-BASED MICROBIAL MANAGEMENT The structure and function of gastrointestinal bacteria affect our daily life and society. Gut bacteria may become recognized because of the intestinal disorders caused by enteric pathogens or the high price of farm products affected by the efficiency of rumen fermentation. Obviously, we should regulate the growth of beneficial and harmful bacteria and thus improve the quality of life. However, it is only within the last decade that scientists have shown that there is an undiscovered microbial world in the GIT of animals. This discovery has emerged with the new tools for microbial community analysis. Earlier methods were based on the cultivation of bacteria under artificial laboratory conditions, whereas the new analytical tools are based on direct extraction of bacterial DNA from intestinal samples and subsequent sequencing of the taxonomically relevant genes. Such approaches have re- vealed that we used to know only a fraction of the total bacteria. Now we acknowledge the true diversity of bacteria, but for most newly discovered species, we only know a partial DNA sequence. In the present situation, microbial management based on true understanding is challenging. Modulation of the intestinal bacterial community to a beneficial direction by feeding of live bacteria (probiotics) or specialty carbohydrates for the beneficial bacteria (prebiotics) are currently under active research and development. Since the growth requirements of bacteria differ, it should be possible to shift the microbial community from harmful to nonharmful direction by changing the gut dynamics through dietary modulations. Probiotic bacteria are meant to improve the health of the GIT, but these are likely to be effective only if the requirements for their growth are fulfilled or if their physiological effect is independent of their viability. If viability is a requirement, a synbiotic product that contains both a probiotic strain and a prebiotic substance, favoring the growth of that particular probiotic, might be a good solution. CONCLUSIONS AND APPLICATIONS 1. Modern tools to detect each member of the microbial communities have brought up new questions about the role of microflora. What are all the bacterial species doing? Are they good or bad for the health of the host? 2. Thorough epidemiological studies together with well-designed animal trials, measuring parameters that reflect animal performance and health should be carried out to improve our understanding of gastrointestinal interactions and the role of individual bacteria in the gastrointestinal microbial community. 3. When relevant indicator bacteria and their metabolites are known, it is possible to design animal trials and laboratory simulations, which can be effectively used for product evaluation and screening of novel microbial modulators. REFERENCES AND NOTES 1. Martin, R., S. Langa, C. Reviriego, E. Jiminez, M. L. Marin, J. Xaus, L. Fernandez, and J. M. Rodriguez. 2003. Human milk is a source of lactic acid bacteria for the infant gut. J. Pediatr. 143:754–758. 2. Tannock, G. 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