Chapter 7.

INTESTINAL MICROBIOLOGY

E.M. JENKINS

 

7.1 INTRODUCTION

In the foregoing lecture we will discuss the relationship of microorganisms with each other and their interaction with the host subject. These reactions involve microbes found in the small and large intestine, colon, cecum and rumen. The reactions taking place here involve a number of symbiotic, synergistic and commensalistic mechanisms. Many of these reactions entail synthesis and/or energy production. Relationships between protozoa and bacteria of the alimentary tract are discussed. In these models, bacterias are extremely important. They are unique because of the diversity of substrate that they can use, their resistance to anaerobiosis, their rapid growth at body temperature, and serving as nutritional models.

The intestinal bacteria control the synthesis of bacterial products essential to the survival and their inter-relationship with the host. Some of the products that they synthesize include hemolysin, colicin, substrates that confer resistance to antibiotics and chemotherapeutic agents fimbriae and enterotoxin.

Of much interest is the way in which ruminants use the rumen for the digestion of terrestrial plants since they lack the enzymes necessary to digest cellulose. Mammals that subsist primarily on grasses and leaf plants can break it down by making use of microorganisms as digestive agents.

 

Key Words:

metabolism,

anaerobic methanogenic bacteria,

competition nutritive model,

continuous nutritive model,

faunated,

plasmids,

hemolysin,

colicin,

enteropathogenicity,

enterotoxin,

symbiosis,

synergistic,

commensalism,

synergism.

 

7.2 Interactions among Intestinal microorganisms

Microbe-microbe interactions, in addition to the interactions of microbes within their animal hosts are major areas for research in which much more qualitative, quantitative and kinetic biochemical and microbiological information must be obtained before we can hope to adequately understand the microbial ecosystem(s) of the gastrointestinal tract. When characterizing bacteria from the rumen, cecum or colon using adequate bacteriological techniques, one is very soon impressed by the great variety of quite different kinds of bacteria, each present in extremely large numbers and apparently exceptionally well adapted to life together. The study of these individual species has brought to light a number of interesting symbiotic, synergistic, or commensalistic interactions involving promotion of growth, and therefore of metabolism, by these organisms.

There are a number of interactions involving cross-feeding of growth factors such as B vitamins and amino acids among bacteria. Some interesting examples of this general type of phenomenon among gastrointestinal bacteria include the requirement of vitamin K, bio-synthesized by many species such as Bacteroides fragilis and Veillonella alkalescence and by certain Bacteroides melaninogenicus strains. The production of branched-chain volatile acids such as 2-methylbutyrate by certain bacteria such as Bacteroides ruminicola is essential for the growth of many other bacteria which may require this, or similar acids. The latter include many and diverse species of anaerobic bacteria, e.g. Ruminococci, Bacteroides succinogenes, methanogenic bacteria, Treponema (Borrelia) spp. and others. The requirement for heme or related tetrapyrroles produced by other bacteria by most strains of Bacteroides ruminicola is well documented, and several other Bacteroides spp. are known to require heme for growth.

7.3 Energy Sources for Growth

Relatively well documented interactions involving energy sources for growth are numerous and only a few will be mentioned. Lactate fermenting bacteria such as Megasphaera elsdenii and Veillonella alkalescence probably subsist mainly on lactate produced by other bacteria in the gastrointestinal tract. The rumen treponemes may grow at the expense of the hydrolytic products of cellulytic species. of great importance to the understanding of many anaerobic ecosystems(s) including those of the organic, sediments of natural waters, anaerobic digesters of sewage disposal as well as the rumen, and probably the cecum and colon of many animals, is the interaction in which hydrogen produced by hydrogenase-containing alcohol, lactic acid, or carbohydrate-fermenting bacteria is required and utilized for growth of methanogenic bacteria.

There are indications that this type of interaction not only allows growth of the methanogenic bacteria and benefits the fermentative bacteria in that they obtain more energy for growth but also dramatically affects the balance of fermentation products produced in the habitat. Pure culture studies of rumen bacteria showed that many species which produce relatively large amounts of ethanol or lactate in fermentation of carbohydrates also produce hydrogen gas. However, several workers showed that in mixed culture in vivo fermentation, no ethanol and little lactate was usually produced by these species.

An explanation for these results became evident through studies on the anaerobic bacteria previously known as Methanobacillus omelianskii, isolated from bay mud. It was shown that Methanobacillus melianski grown on ethanol was actually a synergistic association of a methanogenic bacterium (S organism) which ferments ethanol with formation of acetate and hydrogen, but can not grow unless another system such as above methanogenic bacterium allows maintenance of a low partial pressure hydrogen. 

7.4 Relationships Between Protozoa and Bacteria of the Alimentary Tract

The first type of relationship to the intestinal microbes may be called the

1. competition model,

and the second the

2. continuous fermentation model. 

7.5 The Competition Model

In the competition model there are three methods for competing with the microbes. The first is their inhibition by stomach acidity. The second is secretion of large quantities of enzymes for rapid digestion, together with a large area for rapid digestion, together with a large area for rapid absorption. The third, a corollary of the second, is rapid passage of digest through the small intestine. The rate of passage is so fast that, unless they have some means of attachment, microorganisms are ordinarily unable to maintain themselves in high numbers in the small intestine of animals in a normal state of nutrition.

7.6 The Continuous Fermentation model

In the continuous fermentation model, for which the food is ordinarily plant fibrous material, the storage organ is modified to separate the storage from the acid region, and the storage portion is increased in size to permit longer fermentation. The host absorbs and oxidizes the fermentation acids as a source ATP, and it digests the microorganisms as a source of protein. This type of nutrition is found not only in ungulate ruminants, but also in many other animals. Still another nutritional model, the cecal model, is important and of wide occurrence. It combines the other two. The host first digests the food and absorbs the products, then the microorganisms ferment it in a much enlarged cecum or colon. The acid fermentation products are absorbed and used for ATP production. The bodies of the microorganisms ferment it in a much enlarged cecum or colon. The acid fermentation products are absorbed and used for ATP production. The bodies of the microorganisms produced may become available to the host through coprophage. Feces are consumed by many rodents, lagomorphs termites and other animals. The cecal model is found also in elephants, horses and related forms without appreciable coprophagy.

In all these nutritional models, bacteria is extremely important. The diversity of substrates that they can use, their resistance to anaerobiosis and their rapid growth at body temperatures make them denizeils of all intestinal habitats.

Formation of ATP under anaerobic conditions requires more substrate than is needed for aerobic genesis of ATP. Most anaerobic protozoa and some anaerobic bacteria deposit relatively large quantities of reserve carbohydrate, starch or glycogen in their cytoplasm, using it when external substrate is not available. Most protozoa cannot compete with bacteria in absorbing dissolved carbohydrates, but are able to ingest insoluble carbohydrate such as starch and cellulose, digesting them internally, safe from bacterial attack. Evidence that the rumen protozoa ingest bacteria has been obtained by microscopic observation and by using radioactively labeled cells of Escherichia coli. The number of bacteria in a culture including protozoa is less than in a culture without; and their concentration in the rumen of the normal faunated animal is less than in an animal from which the protozoa has been removed.

Part of the reduction in bacterial numbers may be due to competition for food but much of it is due to ingestion and digestion of the bacteria by the protozoa. It has been found that growth of lambs is favored by the presence of the protozoa, defaunated lambs showing less growth than do faunated lambs on the same feed. This is difficult to understand. In the process of consumption of bacteria by the protozoa and their conversion into protozoan protoplasm a decrement of nutritional material would be expected. The results suggest a qualitative superiority of the protozoa over the bacteria as food for the host.

This review of contributions by many workers to our knowledge of specific relationships between bacteria and protozoa of the alimentary tract indicates that in many cases the interrelationship is so strong as to lead to symbiosis. Many interrelationships do not lead to such close associations, yet may be important in the overall balance in a microbial ecosystem.

The effect of the normal microbial ecosystem of the alimentary tract is favorable to the host. Potentially pathogenic organism(s) , may be present but the ecological balance is such that they remain in a harmless niche in which the host is not injured. But the disturbance of the ecosystem brings a change balance in which the potential pathogen is favored and moves into a niche including harmful effects on the host.

The concept that microbial diseases should be viewed as the result of changes in ecosystems is nowhere more applicable than in the alimentary tract. Changes in ecosystems brought about by antibiotics, overfeeding, and other stresses can change the normal microbiota from normal at harmony with the host to one not functioning effectively and even causing damage. In man some of these changes involve protozoa-bacteria interrelationship. In animals with a large protozoan population they may be even more important.

 7.7 Plasmids in Intestinal Bacteria

Plasmids in intestinal bacteria control the synthesis of a wide variety of bacterial products many of which are significant in the survival of the bacteria and in their interrelationship in their host and with other microorganisms. These products include homolysin, colicins, substances that confer resistance to antibiotics and chemotherapeutic agents, fimbriae, and enterotoxins. This presentation is confined to the plasmids in Enterobacteriaceae and is concerned primarily with the significance of these plasmids in the ecology of the intestinal tract and in the health of the animal.

7.8 Hemolysin

Plasmid control of alpha hemolysin production in E. coli was first reported in 1968. In that study, transmissibility was demonstrated for 13 of 53 E. coli strains enteropathogenic for pigs. The rate of transfer of homiletic activity was shown to be high, 10 to 20% after 2hr incubation of mixed cultures. The hemolysin plasmid (Hly) could be transferred by conjugation, not only to other E. coli strains but also to Shigella and Salmonella. In E. coli and Salmonella, the Hly plasmid tended to be stable, whereas in Shigella it is usually unstable.

7.9 Colicins

A colicin is capable of killing strains of E. coli but is ineffective against the producer strain. It is clearly a potential asset to an E. coli to determine whether colicin production enhances the colonizing ability of E. coli strains in the intestinal tract. In one study, 42% of the E. coli isolated from the feces of young pigs were shown to be colicinogenic, but could not be concluded that colicin production contributed to the establishment of these organisms in the pigs intestine.

Colicin has been shown to be of benefit to the producer organism in the urinary tract, and it is probable that its ineffectiveness in the intestine is due to inactivation by proteolytic enzymes. However, colicin should not be discounted as an insignificant factor in the ecology of the intestinal tract. In the extra-intestinal environment, colicin may play a role in the survival of E. coli strains and thereby increase the chances of colonization of the intestine by producer strains.

 7.10 Drug Resistance Plasmids

Drug resistance plasmids have been the subject of extensive investigation because of their obvious importance. It is well documented that under the influence of the selective pressure applied by the use of antibiotics the presence of the plasmids favors survival of organisms possessing them and leads to an increasing proportion of intestinal organisms resistant to drugs. The ability of these plasmids to "pick up" genes or other plasmids could lead to more profound effects of the selective pressure of antibiotics in these genes or plasmids contribute to the intra-intestinal survival and pathogenicity of the R+ bacteria.

7.11 K88 Plasmid

The K88 plasmid codes for the K88 fimbrial antigen which is found almost exclusively on E. coli strains enteropathogenic for swine. This plasmid does not appear to have its own transfer factor, but this is not a serious handicap in a population of cells that frequently possesses more than one transfer factor.

It is of interest that the K88 fimbriae does not develops when the organisms grow at room temperature but are produced when the E. coli grow at body temperature. Furthermore, as these surfaces, it has long been postulated that they are a dominant factor in the ability of certain E. coli to colonize the upper small intestine of swine.

Only recently however has it been shown that the K88 fimbriae play an important role in the colonizing ability and hence the enteropathogenicity of E. coli strains in pigs.

It has also been suggested that there must be other surface structures or properties of enteropathogenic E. coli that play a role analogous to that demonstrated for the K88 fimbriae. Such a structure is demonstrable for E. coli with various hosts is a remarkable phenomenon. One component of host specificity probably lies in the specificity of association of E. coli with intestinal epithelium. Thus the K88 fimbriae seem to contribute to the specificity of certain E. coli strains for the pig intestine. Other structures, no doubt, confer specificity for the intestine of other host species.

Other types of plasmid are K99 primarily found in cattle and K987 in swine. Pili or fimbrial are minute, proteinaceous, filamentous appendages that are produced by bacteria. Pili of enterotoxigenic Escherichia coli (ETEC) facilitate intestinal colonization by ETEC. Suckling newborn pigs, lambs, and calves whose dams have been vaccinated parenterally with such pili (K88, K99 or, K987P antigen types) are protected against fatal diarrheal disease caused by ETEC bearing pili homologous to those in the vaccines. The K99 antigen is a logical basis for the development of practical vaccines against ETEC infections in calves because most ETEC from calves produce K99. Furthermore most fatal ETEC infections in calves apparently occur in the first day or 2 after birth. Thus, the highest prevalence of the disease occurs at the age when the colostral antibody is repeatedly introduced into the intestine of calves suckling vaccinated dams. However, some cases of ETEC infection in calves do occur during the postcolostral period. The immunoglobulin (Ig) content of bovine milk is high during the colostral period. This Ig is principally serum-derived IgG, and it decreases precipitously in the postcolostral period. Milk antibody stimulated by parenteral vaccination of pregnant cows with K99 follows this typical pattern of decrease in the immediate postcolostral period. Thus, existing procedures for passive immunolization via K99 antibody in mild may be comparatively ineffective against ETEC infections that occur during the postcolostral period.

Oral vaccination with some antigens stimulates lymphoblasts in aggregated lymphatic follicles (Payer's Patches) to proliferate to circulate via lymphatics and blood, and to differentiate to plasma cells producing IgA or IgM specific for the stimulating antigen.

Oral vaccination of the cows with live E. coli stimulates the production of serum and colostral antibodies against the cell wall antigens of the vaccine strains of E. coli. Oral vaccination of pregnant swine (gilts) with live K99+ E. coli results in higher titers of K99 antibody in serum and colostrum.

7.10 Enterotoxin Plasmid

The term enterotoxin as applied to Gram-negative rods; refers to a toxic substance that caused fluid accumulation in the ligated intestine of a suitable test animal. A heat stable enterotoxin (ST) was first shown to be produced by porcine enteropathogenic E. coli. Shortly afterwards, it was demonstrated that ST was produced in response to an extrachromosomal genetic element. Subsequently, a heat-liable enterotoxin (LT) was demonstrated and also shown to be under plasmid control. Recently an E. coli strain implicated in infantile diarrhea and another strain incriminated in diarrhea in human adults have both been shown to possess a plasmid that codes for enterotoxin production.

In terms of production of ST and LT, it appears that there are two classes of enterotoxin plasmids. One codes for ST only, the other for ST and LT. In terms of transmissibility of the plasmid, two groups have been recognized; one in which the transfer factor and the genes for enterotoxin are not closely associated and one in which they are closely associated (unpublished observation).

The rate of transfer of the enterotoxin plasmid in in vitro studies is extremely high. There is no evidence that long-term selective advantage is gained by organisms possessing the Ent plasmid, and this is probably one reason that the rate of emergence of new serotypes of enteropathogenic E. coli is low. Another reason is that transfer of the Ent plasmid to an E. coli strain does not create a new enteropathogen unless that strain has sufficient capacity for colonization of the upper small intestine. 

Quantitative factors probably play a role as well in the E. coli-intestine disease relationship, as far as enterotoxin is concerned. Some strains and serotypes of E. coli appear to produce enterotoxin or to production of enterotoxin of greater potency. At a given degree of colonization of the intestine, therefore, one strain might cause diarrhea whereas another might not. This degree of enterotoxicity of an E. coli strain appears to be a direct effect of the Ent plasmid because it can be shown to be unchanged regardless of the E. coli strain to which the plasmid has been transferred.

 7.11 Plasmid Interrelationship

To date, no significant functional relationship have been shown between these different plasmids in intestinal bacteria. However, it is quite conceivable that a plasmid that codes for a virulence determinant, such as the Ent plasmid, could become linked with an R factor. Such a linkage combined with the continued use of antibiotics, could provide the selection required to markedly increase of enteropathogenic types of E. coli.

Much work needs to be done on the relationship between enterotoxins and enterotoxin plasmids from E. coli which cause diarrhea in animals and those which cause diarrhea in man. E. coli strains from adult human diarrhea cases produce enterotoxins that behave much like the enterotoxins from the strains isolated from pigs, and it is important to determine whether the enterotoxins from E. coli of animal origin can effect human intestines. That the antigenic relatedness between LT from human E. coli strains and LT from porcine E. coli strains appears to be low is encouraging.

 7.12 The Rumen Microorganisms

The biochemical reactions occurring in the rumen are complex and involve a wide variety of microorganisms. Properties of some of the important rumen bacteria are outlined in the table that follows. All of these organisms are obligate anaerobes and must be cultured under strictly anaerobic conditions. A number of rumen bacteria require as growth factors certain branched-chain acids (e.g. isovaleric and isobutyric) that are present in the rumen fluid.

As seen in the table, different rumen bacteria carry out specific functions. A variety of types hydrolyze cellulose to sugars and then ferment these to acids. If the animal is fed a diet high in starch (grain, for instance) then the starch-digesting bacteria are common, although on a low starch diet these organisms are usually in minority. If the animal is fed on legume pasture, which is high in pectin, then the pectin-digesting bacterium Lachnospira multiparous is a common member of the rumen flora. Some of the fermentation products are themselves used by other rumen bacteria. Thus, succinate is converted to propionate and CO2, and lactate is fermented to acetic and other acids by Selenomonas and Peptostreptococcus. A number of rumen bacteria produce ethanol as fermentation product when grown in pure culture, yet ethanol rarely accumulates in the rumen because it is fermented to H2 and CO2. Also, a variety of bacteria produce H2 during growth in pure culture, yet H2 never occur in the production of methane. Another source of H2 and CO2 for the methane-producing bacterium is formate.

In addition to the bacteria, the rumen has a characteristic protozoal fauna, composed almost exclusively of ciliates. Although these are not essential for rumen fermentation, the protozoa definitely contribute to the process. They are able to ferment sugars, starch, and cellulose with the production of the same organic acids that are formed by the bacteria. Protozoal population densities are usually larger in animals on a good ration than in those fed a poor diet, and there are some reasons to believe that protozoal counts could be used as indicators of the well-being of the animal. It has been well established that the protozoa ingest rumen bacteria; animals without protozoa usually have higher bacterial populations, which suggest that the protozoa may control bacterial density to at least some extent.

7.11.1 Rumen Fermentation

The bulk of the organic matter in the terrestrial plants is present in the insoluble polysaccharides, of which cellulose is the most important. Mammals, and indeed most animals, lack the enzymes necessary to digest cellulose, but mammals that subsist primarily on grasses and leafy plants can break it down by making use of microorganisms as digestive agents. Unique features of the rumen as a site of cellulose digestion are its relatively large size (100 liters in a cow, 6 liters in a sheep) and its position in the alimentary tract as the organ where ingested food goes first. The high constant temperature (390C) and the rumen are also important factors. the rumen operates in a more or less continuous fashion, and in some ways can be considered analogous to a chemostat.

The relationship of the rumen to other parts of the digestive system is shown below. Food enters the rumen mixed with saliva and is churned in a rotary motion during which the microbial fermentation occurs. The food mass then passes gradually into the reticulum, where it is formed into small portions called cuds, which are regurgitated into the mouth where they are chewed again. the now finely divided solids, well mixed with saliva, are swallowed again, but this time the material passes down a different route, ending in the abomasum, an organ more like a true stomach. Here true digestion begins and continues into small and large intestine.

Food entering the rumen is mixed with the resident microbial populations and remains there on the average about nine hours. During this time, cellulolytic bacteria and protozoa hydrolyze cellulose to the disaccharide cellobiose and the monosaccharide glucose. These sugars then undergo a microbial fermentation with the production of organic acids, primarily acetic, propionic, and butyric, and the gases carbon dioxide and methane. The organic acids pass through the rumen wall into the bloodstream and are oxidized by the animal as its main source of energy. In addition to their digestive functions, the rumen microorganisms synthesize amino acids and vitamins that are the main source for the animal of these essential nutrients. The rumen contents after fermentation consist of enormous number of microbial cells plus partially digested plant materials, which pass through the gastrointestinal tract of the animal, where they undergo digestive processes similar to those of other animals. Microbial cells formed in the rumen are able to grow on urea as a sole nitrogen source, it is often supplied in cattle feed in order to promote microbial protein synthesis. The bulk of this protein will end up in the animal itself. A ruminant is thus nutritionally superior to a non-ruminant when subsisting on foods that are deficient in protein, such as grasses.

 

Figure 1. Polygastric Alimentary Tract

 

 

 

 

Figure 2: Bichemical aspects of polygastric alimentary tract

 

 

 

 

 

 

TABLE 1

 

CHARACTERISTICS OF SOME RUMENAL BACTERIA

__________________________________________________________

GRAMORGANISM STAIN SHAPE MOTILITY FERMENTATION PRODUCTS

__________________________________________________________

 

Cellulose decomposers:

Bacteroides succinogens Neg. Rod - Succinate, acetate, formate

Butyvibrio fibrisolvens Neg. Curved rod + Acetate, formate, lactate

butyrate, H2, CO2

Ruminococcus albus Pos. Coccus - Acetate, formate, H, CO

Clostridium lochheadii Pos. Rod (spore) - Acetate, formate, butyrate

H, CO

 Starch decomposers:

Bacteroides amylophiilus Neg. Rod - Formate, acetate, succinate

Bacteroides ruminicola Neg. Rod - Formate, acetate, succinate

Selenomonas ruminantium Neg. Rod - Acetate, propionate, lactate

Succimonas amylolytica Neg. Oval - Acetate,propionate, succinate

Streptococcus bovis Pos. Coccus - Lactate

 

Lactate decomposers:

Selenomonas lactilytica Neg. Curved rod + Acetate, succinate

Peptostreptococcus elsdenii Pos. Coccus - Acetate, propionate, butyrate

Valerate, H, CO

 Pectin decomposres:

Lachnospira multiparous Pos. Curved rod + Acetate, formate, lactate

H, CO

 Methane producers:

Methanobacterium ruminantium Pos. Rod - CH (from H+CO)

 

 

 References:

Microbiology-Davis, Dulbaco, Eisen, Ginsberg, and Wood. 4th edition, 1990.