Nutritional influences on gut microbiology

Nutrition, intestinal health, and the well-being of animals are intricately related. The gastrointestinal tract is a direct path by which pathogens can enter an animal, and if the protective mechanisms of these organs fail, those pathogens can colonize and/or gain entry into host cells and tissues. Mechanisms by which the gut protects against pathogen invasion include secretion of IgA, maintenance of a viable commensal microflora, maintenance of inhibitory physiological conditions, production of a mucin barrier, and peristalsis. Nutrition can affect all of the above mechanisms, including the microflora, while in turn, activities of the microflora can directly affect the secretion of antibodies (Majamaa et al., 1995), the gut environment (Tazume et al., 1990; Campbell et al., 1997), intestinal mucins (Meslin et al., 1993; Pestova et al., 2000) and even peristalsis (Rolfe, 1984).

While we might assume that enteric diseases result directly following the incidental ingestion of a virulent organism, it is often the case where a preexisting organism suddenly blooms to high numbers resulting in a disease state. During that process, a complex microflora composed of many species is transformed into a flora of few species, with the pathogen comprising the most significant component (McAllister et al., 1979). As an example, colibacillosis in pigs is typically caused by a few distinct serogroups of E. coli. As these pathogens colonize the gut they produce a variety of enterotoxins that can lead to high morbidity and/or mortalities. Yet, these E. coli serogroups are also found in apparently healthy swine (Echeverria et al., 1984; Gannon et al., 1988). It is also not uncommon to isolate other enteric pathogens, including various serovars of Salmonella enterica in healthy animals (Stege et al., 2000), where again, these organisms may remain in low numbers, with little significance to the animal, until some unpredictable event causes rapid increases in numbers resulting in illness. It is probable then, that specific conditions cause a decreased resistance of the gut and/or increased virulence of the pathogen, or other events occur that provide an opportunity for the rapid colonization by opportunistic organisms.

The small intestine is of particular importance with regard to enteric function and disease. It is the major organ of nutrient absorption, as well as the site of colonization by a wide variety of enteric pathogens. Thus, while direct effects of enteric diseases often result from pathogen toxin production, disruption of mucosal enterocytes, electrolyte imbalance and hemorrhaging, an indirect effect of decreased nutrient absorption nearly always also results, thus further compromising the health of the animal. It is likely that nutrition is among those factors affecting pathogen colonization. It has long been assumed that the change of diet following weaning may be among the key factors that predispose young animals to enteric pathogens. Type of diet and degree of intake have been reported to play a role in the promotion of edema disease, a severe form of colibacillosis in weaned pigs (Smith and Halls, 1968; Bertshinger et al., 1978). Additionally, because foodstuffs directly affect the composition of the intestinal chyme, a primary bacterial niche within the gut, it is likely that diet affects a variety of other enteric organisms as well.


pH of the gastrointestinal tract

While pH of the gastric and intestinal chyme directly affects activity of various digestive enzymes and rates of digestion of foodstuffs, it likely also affects the species composition of the enteric microflora and the prevalence of potential pathogens. The low pH of the gastric juices has been widely reported as providing a barrier to pathogens (Dinsmore et al., 1997; Carrion and Egan, 1990). However, pH is also of significant importance in other regions of the gastrointestinal (GI) tract. Beyond affecting activity of digestive enzymes in the mid and lower gut, pH also appears to affect the ability of enteric pathogens to colonize. Earlier work by our group noted a correlation between above normal ileal pH (>7.0), high concentrations of E. coli, including pathogenic K88 serogroups, and low concentrations of lactobacilli postweaning (Mathew et al., 1993) (Figures 1 and 2). Additionally, growth of opportunistic organisms, including pathogenic E. coli and salmonella, is known to be favored by near neutral pH, whereas lower pH values are more conducive to growth of resident bacteria, including lactobacilli (Hampson et al., 1985; Drasar and Barrow, 1985).

Diet can affect pH of the GI tract through innate buffering capacity of foodstuffs (Roth and Kirchgessner, 1989), through the addition of buffering agents (Kellaway et al., 1976) and acidic additives (White et al., 1969) and through promotion of short chain fatty acid production by the microflora (Andrieux et al., 1989; Campbell et al., 1997). Effects of buffering or acidic agents are generally insignificant beyond the stomach and/or anterior small intestine, rarely affecting the pH downstream of those sites. On the other hand, feed components that promote VFA production can affect pH in more distal regions of the GI tract, including the cecum and large intestine (Campbell et al., 1997; Roberfroid, 1993). A variety of oligosaccharides have been shown to affect pH of the lower GI tract. For example, Campbell et al. (1997) noted that when fructooligosaccharides, oligofructose or xylooligosaccharides were included in the diet, pH of the cecal contents were lowered, while short chain fatty acid concentrations were increased over control diets and diets containing microcrystalline cellulose.




Figure 1.Ileal pH of pigs versus days postweaning, depicting an increase soon after weaning. Data are Least Squares means from 12 cannulated pigs weaned at 21 days of age. Bars not sharing like superscripts differ (P < 0.05). Summarized from Mathew et al., 1996a.




Figure 2. Ileal E. coli and lactobacilli concentrations from pigs versus days postweaning. Data are Least Squares means of ileal bacterial concentrations expressed as Log10 colony forming units (CFU) from 12 cannulated pigs weaned at 21 days of age. Time (day) effect: P < .01. Bars within bacterial group not sharing like superscripts differ (P < 0.05). Summarized from Mathew et al., 1996a.


Reduced feed intake may also affect pH of the gut. In an earlier report (Mathew et al., 1996a), we hypothesized that temporary reduced intake following weaning may be partly responsible for increased pH of ileal contents in pigs, which in turn promoted greater E. coli concentrations and lower lactobacilli concentrations. We also noted a sharp drop in ileal VFA concentrations at that time. Earlier, Illman et al. (1986) had demonstrated that food restriction in rats resulted in increased pH of cecal contents, which was correlated with lower VFA concentrations.


Role of the volatile fatty acids

Because short-chain fatty acids have not been recognized as an important energy source in nonruminants, far less work has been conducted to characterize their occurrence in those animals compared to ruminants. Research suggests, however, that these compounds play several important roles in resisting pathogens. A number of investigations suggest that VFA and other short-chain fatty acids increase resistance to opportunistic organisms, including pathogenic E. coli, in the intestinal tract. Hentges (1970) suggested that acetic and propionic acid produced by Bacteriodes fragilis inhibited Shigella flexneri growth in vitro. Other in vitro work indicated that high concentrations of VFA in an anaerobic digester for cattle feces inhibited Salmonella typhi (Kunte et al., 1998). Using intestinally cannulated pigs, we observed that ileal VFA concentrations decreased significantly during the period of postweaning lag in pigs (Figure 3), and this decrease corresponded to an increase in ileal E. coli and a concomitant decrease in lactobacilli concentrations (Mathew et al., 1996b).

Beyond direct effects of VFAs on pathogens, these compounds also contribute to the overall health of the intestinal mass by providing a ready source of energy and metabolites. It has been noted that the energy needs of the intestinal mass are disproportionately high compared to other tissues. Britton and Krehbiel (1993) reported that the gut mass accounts for up to 25% of total oxygen consumption while comprising only about 6% of total body mass. They further indicated that VFAs provide a large proportion of the metabolic needs for those organs. Waechtershaeuser and Stein (2000) noted that short chain fatty acids, primarily butyrate, help maintain the mucosal barrier of the gut, and that a lack of these acids may be a cause of ulcerative colitis and other inflammatory conditions. Of further interest, Malbert et al. (1994) suggested that ileal VFA concentrations may affect gastric emptying patterns and ileal contractions in the pig. In that work, infusion of VFA into the ileum decreased gastric contractions while increasing ileal contractions. It is possible that the decrease in ileal VFA concentrations that we observed postweaning in pigs (Mathew et al., 1996b) may thus lead to a slowing of food material in that region of the gut, possibly causing a stagnation of the chyme and thus further contributing to the increased colonization of pathogens during the postweaning lag. As discussed earlier, decreased intake has been associated with undesirable intestinal traits such as abnormally high pH (Illman et al., 1986). As such, determining the response of short-chain fatty acid production to varying diet and management regimes should add to our knowledge of the complex interactions associated with enteric health.




Figure 3.Ileal concentrations of primary VFA from pigs versus days postweaning. Data are Least Squares means of concentrations (mmole/L) from ileal chyme of a total of 36 cannulated pigs weaned at 21 days of age. Time (day) effect; P < 0.0001. Summarized from 3 studies. (Mathew et al., unpublished data).


VFA concentrations in the lower GI tract of nonruminants can rival those in the bovine rumen. In an earlier study comparing carbohydrate sources in weaned pig diets, total VFA concentrations in the cecum and colon ranged from 128 to 178 mmole/L, with acetate, propionate and butyrate comprising approximately 55%, 25% and 15% of the total, respectively, and valerate, isovalerate and isobutyrate making up the difference (Sutton et al., 1991). A significant concentration of VFA can also be found in the ileum (Mathew et al., 1996b), with lesser but measurable amounts being found in the mid and anterior small intestine (Tortuero et al., 1994). A number of studies have determined that diet can affect VFA concentrations of the GI tract (Andrieux et al., 1989; Tortuero et al., 1994; Ehle et al., 1982), with fiber and/or indigestible carbohydrates being the most effective in promoting increases of these fermentation by-products. In view of the importance of

VFAs as described above, it is reasonable to assume that such dietary components can affect enteric health. It remains problematic however, that significant fiber inclusion in diets of growing, nonruminant animals negatively affects gain and feed efficiency (Drewry, 1981). On the other hand, during the early postweaning phase and other times of stress, control of enteric disease may become a priority, thus possibly justifying inclusion of moderate quantities of VFA-promoting compounds, including fiber in starter diets. Certainly, further research in this area is warranted.


Role of the intestinal mucins

Mucins are a large class of secretory glycoproteins produced by the epithelia of the intestine and other mucosal surfaces. It is widely accepted that mucins protect the gut mucosa from the abrasive action of feedstuffs and from bacterial colonization (Neutra and Forstner, 1987). Importantly, recent work by some investigators has suggested that components of gut mucins specifically bind pathogen adhesins, thus reducing the risk of adhesion to similar receptors on host cells (Depicted in Figure 4). Schroten et al. (1992) reported that mucins comprised the major inhibitory component against Sfimbriated Escherichia coli. Dean-Nystrom and Samuel (1994) found that E. coli possessing 987P adhesins bound to enterocytes from both neonatal and weaned pigs; however, glycolipids found in the mucus of older, postweaned pigs also bound the 987P adhesin. These authors hypothesized that the binding of adhesins to the mucus of older pigs decreased the exposure of the adhesins to the brush border and may partly explain the absence of 987P-mediated infection in older pigs. Furthermore, Blomberg et al. (1993a) found similar binding of K88 adhesins to ileal mucus from pigs; thus suggesting a similar protective mechanism against postweaning colonization by that enterotoxigenic strain. The above studies indicate the fundamental role intestinal mucus plays in protection against adhesive enteric pathogens.

Mucins and other glycoproteins are widely known to be fermentable by the microflora and are degraded in the colonized gut, thereby providing a usable substrate for various microbial species. The impact of an active flora on mucins can be seen in an earlier report by Gustafsson and Carlstedt- Duke (1984), where it was observed that mucins are relatively stable in the gnotobiotic animal but are degraded at a predictable rate upon colonization of the intestine by the normal flora. Enteric species may select specific carbohydrate components of the glycoproteins, including galactosyl units, for use as nutrient substrates (Roy et al., 1991). Thus, a complex balance exists between conditions in the gut, intestinal mucins and the microflora; and this balance may be altered as a consequence of health, diet, weaning and other factors.




Figure 4.Depiction of intestinal mucosa showing brush border glycoproteins and protection by mucin layer that contains similar glycoproteins. Mucin glycoproteins act to intercept adhesive E. coli helping to prevent attachment to the underlying enterocyte layer.


Using pigs cannulated at the ileum, we observed significant changes in intestinal mucins following weaning, and some of these changes were alleviated by inclusion of galactose in the diet (Pestova et al., 2000) (Figure 5). In agreement with the work of Enss et al. (1992) a significant increase in the acid subclasses of mucin was observed following weaning. Additionally, O-linked polymerization patterns were changed, and increased degradation of ileal mucins was observed postweaning. We hypothesized that lack of galactose in postweaning starch-based diets, which provide primarily glucose, cause increased scavenging of galactosyl units by some groups of the microflora, thus increasing the rate of degradation of these compounds. In contrast, nursing pigs digesting milk are provided with galactose and glucose in approximately equal concentrations through the cleaving of lactose, the primary sugar dimer in milk. Galactose was shown to be a preferred substrate of some enteric microflora in a study by Roy et al. (1991). In that study, Bifidobacterium infantis grew slowest when provided only glucose as a carbohydrate source; whereas that species grew more rapidly when provided galactose or galactose-containing sugars. It is possible then, that such species may utilize mucins more rapidly when free galactose is no longer available in the intestinal chyme. In vitro work has shown that Bifidobacteria utilize mucins as a substrate. Poch and Bezkorovainy (1988) observed that pig gastric mucin was an effective growth-enhancing supplement for some species of Bifidobacteria when grown on a trypticase-peptone-yeast medium with no additional carbon source. Thus, galactosyl units on the mucins likely provide a primary carbon and energy source for such organisms. This may explain the moderating effect of galactose inclusion on degradation of mucins that we observed postweaning. In view of the importance of mucins in protecting the gut epithelial layer, increased degradation of these glycoproteins postweaning would have major implications with regard to pathogen colonization and intestinal health.




Figure 5. Amount of soluble mucin in ileal digesta of weaned pigs fed a control diet versus a diet containing galactose. Data are Least Squares means + SE of stain intensity via area under the curve by line profile using image analysis (Image Pro Plus, Silver Spring MD, USA). Ileal digesta were collected from cannulated pigs 7 days postweaning. Pigs were fed either a control diet (n = 4) or a diet containing 13% galactose (n = 6).


Protective function of the microflora

Much evidence has been established to indicate that a stable resident microflora provides resistance against enteric pathogens. Substantial evidence for such protection can be derived from earlier works such as by Bohnoff et al. (1954) who noted that mice that were orally treated with streptomycin were much more susceptible to invasion by Salmonella enteriditis. They speculated that antibiotic administration caused a loss of resident microflora, which increased the opportunity for invasion by that pathogen. Similarly, Freter (1955) noted that administration of antibiotics to guinea pigs rendered them more susceptible to infection by Vibrio cholerae.

More recently, Blomberg et al. (1993b) reported inhibition of K88 adherence by a proteinaceous component in spent culture fluid from porcine derived Lactobacillus spp. In that investigation, an in vitro assay was conducted using tritium-labeled K88ab E. coli to study adhesion to ileal mucus from 35-day-old pigs. They found that adherence of K88ac E. coli was reduced 36 to 48% when, prior to the adhesion assay, E. coli cells were resuspended in BHI spent culture from L. murinus C39 and L. crispatus 152, respectively. They suggested that lactobacillus components bound to the K88 receptor thereby preventing colonization of the intestinal brush border (Depicted in Figure 6).




Figure 6.Depiction of intestinal mucosal structure showing enterocyte brush border glycoproteins with E. coli adhesins binding to sugar units (right), and blocking of adhesin targets by undefined factors from lactobacilli.


Considerable work continues in an attempt to define competitive exclusion cultures that inhibit pathogen carriage by food animals. Hollister et al. (1999) was successful in reducing salmonella colonization in chicks by use of a live cecal culture from salmonella-free poultry. Similarly, Fedorka-Cray et al. (1999) showed similar promise for such cultures in young swine. Additionally, some work suggests that the resident microflora may stimulate production of immune factors, including IgA and inflammatory response effectors (Majamaa et al., 1995; Perdigon et al., 1991). Perdigon et al. (1991) noted that specific lactobacilli subgroups, when fed to mice, resulted in enhanced protection against Salmonella typhimurium and E. coli by way of increased IgA production (Maassen et al., 1998). Additionally Maassen et al. (1998) noted increased cytokine production in mice fed lactobacilli, and observed that host response was highly dependent upon lactobacillus strain.

In total, these works indicate direct effects of the microflora on pathogen colonization. However, as was discussed earlier, it should be noted that other activities of the resident microflora, including production of VFA and maintenance of appropriate pH, are also important factors in providing resistance to enteric pathogens. As more information comes forth regarding appropriate bacterial groups, species, or subtypes most suitable for competitive exclusion and/or probiotic strategies, more specific dietary regimens will need to be formulated to promote or maintain those organisms in the GI tract.

Diet has been shown to affect concentrations of resident bacteria and specific makeup of the flora. It has been known for some time that the microflora of human infants fed breast milk differs considerably from those receiving synthetic formulas (Yoshioka et al., 1983; Rubaltelli et al., 1998); and the microflora of humans consuming primarily vegetarian diets differs considerably from those consuming a wider variety of foods (Norin et al., 1998). However, given the current restraints on livestock diets, it may be more difficult to effect such large-scale changes through dietary manipulation in production animals. Among these constraints is the need to maximize energy content to support rapid, efficient growth of animals bound for market. Thus, there will remain a need to incorporate a large amount of cereal grains into the rations, thereby making starch the most abundant carbohydrate by far in the intestinal chyme. Additionally, the amount of non-digestible components aimed at feeding the microflora must be added to the exclusion of energy and protein components for the animal, thus prebiotic additives are invariably included as a small percentage (typically less than 1%) of the diet.

Still, type of diet has been shown to alter intestinal microflora in a number of animal species. Bedbury and Duke (1983) reported that the cecal microflora in turkeys was altered by fiber inclusion in the diet, noting that E. coli concentrations were higher in birds fed low fiber diets. Canzi et al. (1994) observed that guar gum increased Bacteroidaceae and bifidobacteria in the cecum of rats, and pectin also increased cecal Bacteroidaceae concentrations. Noack et al. (1998) produced similar effects on bifidobacteria through guar gum inclusion in the diet. Also using rats, Ryhanen (1996) noted that diet affected the ability of probiotics to alter intestinal microflora, and that the changes in intestinal flora induced by diet were more pronounced than changes due to probiotic inclusion. Such studies thus provide the basis for use of prebiotics to enhance growth of or maintain more desirable groups of enteric bacteria.


The next step

If we are to move toward development of more effective non-antibiotic alternatives for maintenance of health and productivity of animals, we must fully define the interactions of the gut flora, dietary components and diseasecausing agents. We will also need to better characterize the key species and subgroups of protective bacteria, understand their nutritional and environmental requirements, and find a way to provide those factors through dietary formulations. More advanced techniques for identification and differentiation of enteric species and strains will also need to be incorporated into our efforts. It is clear that the associations of commensal bacteria and their hosts are highly specific and often change during the life cycle and health status of the animal. Additionally, the associations have been shown to be dependent upon the specific strains of microorganisms within a given species. To better define these symbiotic interactions, DNA-based techniques such as macro restriction profiling, 16S rDNA characterizations, PCR-based fingerprinting, and other more definitive methods to help characterize enteric bacterial communities will be required. Such techniques will also aid in characterizing important species that are difficult to culture and thus are often missed through use of traditional microbial techniques. We will also need to better define the physiology and metabolic pathways of those key species under varying conditions so that more effective prebiotic strategies can be formulated. As we advance our knowledge in these areas, we will improve the efficacy of dietary strategies for enhancement of animal and human health.

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