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CFS and FMS Research
Gastrointestinal
Microbiology and Biochemistry
Faecal Microbiology and Biochemistry
Analysing the Faecal Microbiology
Introduction
Due to the vast diversity in faecal microbiota, the CFS research team has decided to focus on assessing a number of selected key micro-organisms. This approach has provided researchers with a good indication of gastrointestinal tract homeostasis. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
Quantitative data on selected microbial flora is used to indicate if there is an imbalance in the intestinal ecosystem.
The following groups of organisms have been found to be important:
1. Anaerobic bacteria (bacteria that doesn’t require oxygen.
2. Aerobic bacteria (bacteria that requires oxygen to survive.
3. Fungi
4. Parasites
Important Gastrointestinal Micro-organisms (part a)
1. Anaerobic bacteria
2. Lactobacillus species
This genus consists of gram-positive rods and represents the first colonisers in formula-fed infants. These organisms are gradually replaced by typically gram negative bacteria by adulthood.
Bifidobacterium species
Members of this genus are gram positive. They are the first bacteria to colonise in breast-fed infants since the breast milk contains an important growth factor (a disaccharide amino sugar).
Clostridium species
Riboflavin production is a product of fermentation by certain species of Clostridium bacteria. Clostridium difficile is a normal inhabitant of the intestinal flora and may cause disease if other organisms are destroyed by factors such as the use of antibiotics.
Several species in this genus may cause serious disease. These include:
1. Clostridium tetani (tetanus)
2. Clostridium perfringens (gas gangrene and food poisoning)
3. Clostridium botulinum (botulism)
Clostridia and other anaerobes are particularly important in the production of fatty acids. Fatty acids are the energy source for the colonic mucosa cells. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
Bacteroides species
These organisms are usually beneficial to humans and usually produce a range of organic acids as fermentation end products. This genus comprises about 30% of the bacteria isolated from human faeces. Some types, such as Bacteroide fragilis, can be harmful.
Important Gastrointestinal Micro-organisms (part b)
1. Aerobic bacteria
2. Escherichia coli
Escherichia coli (E coli) are rod shaped bacteria representing members of the family Enterobacteriaceae. These are gram negative facultative anaerobes.
(Note: The word ‘facultative’ refers to the way in which some bacteria have the ability to live under more than one specific type of environmental conditions.)
These enteric bacteria can produce large amounts of gas during sugar fermentation. Escherichia coli is a major facultative anaerobe inhabitant of the colon in humans. Some types can cause gastroenteritis.
Enterobacter/Klebsiella species
These bacteria are similar to Escherichia coli. This group also represents members of the family Enterobacteriaceae.
These are:
1. Facultative anaerobes
2. Common inhabitants of the human colon
3. Rarely responsible for causing enteritis
3. Fungi
Candida albicans
Candida albicans is a normal member of the gastrointestinal tract, and in normal individuals does not produce disease. This is because its growth is suppressed by other microbiota.
If the other types of microflora are disturbed by antibiotics or stress, Candida can rapidly multiply. Candidiasis is then likely to develop. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
4. Parasites
A number of parasites are commonly detected in stool samples. These may be responsible for causing serious gastrointestinal diseases.
These organisms include:
1. Entamoeba histolytica
2. Giardia lamblia
References:
1.Dubos R, Schaedler RW, Costello R, Hoet P. Indigenous, normal, and autochthonous flora of the gastrointestinal tract. J Exp Med, 1966; 122:66
2.Gorbach SL, Nahas L, Lerner PI, WeinsteinL. Studies of the intestinal microflora. I. Efects of dit, age and periodic sampling on numbers of faecal microorganisms in man. Gastroenterology, 1967; 53:845.
3.Croucher SC, Houston AP, Bayliss CE, Turner RJ. Bacterial populations associated with different regions of the human colon wall. Appl Environ Microbiol, 1983l 45:1025.
4.Holdeman LV, Good IJ, Moore WEC. Human faecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl Environ Microbiol, 1976; 31:359.
5.Sears HJ, Brownlee I, Uchiyama JK. Persistence of individual strains of Escherichia coli in the intestinal tract of man. J Bacteriol, 1950; 59:293.
6.Sears HJ, Brownlee I. Further observations on the persistence of individual strains of Escherichia coli in the intestinal tract of man. J Bacteriol, 1952; 63:47.
7.Goldberg MJ, Smith JW, Nichols RL.Comparison of the faecal microflora of Seventh-Day Adventists with individuals consuming a general diet. Ann Surg, 1977; 186:97.
8.Moore WEC, Holdeman LV. Human faecal flora: the normal flora of 20 Japanese-Hawaiians. Appl Microbiol, 1974; 27:961
9.Moore WEC, Cato EP, Holdeman LV. Some current concepts in intestinal bacteriology. Am J Clin Nutr, suppl., 1978; 31:S33.
10.Deitch EA,Winterton J, Berg R. Effect of starvation, malnutrition, and trauma on the gastrointestinal tract flora and bacterial translocation. Arch Surg, 1987; 122:1019.
11.Berg RD. Promotion of the translocation of enteric bacteria from the gastrointestinal tracts of mice by oral treatment with penicillin, clindamycin, or metronidazole. Infect Immun, 1981; 33:854.
12.Prescott, LM, Harley, JP, Klein, DA, Microbiology, Third edition, WCBrown, Melbourne, 1996.
Newcastle (CPRU) research has found that definite alterations in faecal lipid composition occur in patients with chronic fatigue and chronic pain disorders.
Changes in lipid composition may reflect:
1. Disturbances in the bile cycle metabolism
2. Malabsorption
3. Gastrointestinal flora imbalance
4. Tissue damage
Lipids
What are lipids?
Lipids are a group of organic compounds that make up the fats and other esters.They are relatively insoluble in water and readily soluble in chemicals such as alcohol, chloroform, hexane and ether. Lipids vary greatly in structure and include triacylglycerides, phospholipids, steroids, fatty acids and many other molecules.
Lipids serve as membrane components, storage molecules for carbon and energy, precursors for other cellular reactions and protective barriers against water loss.
Analysis of lipids
The analysis of the complex lipids is a difficult task due the range in sizes and molecular properties. During the sample preparation procedure, the lipids are broken down to their composite building blocks (by saponification). © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
These saponified products are then measured and identified by techniques such as gas chromatography mass spectrometry.
Faecal Lipids Components (part a)
Lipids found in faecal samples may be derived from several origins.
Bile:
Most of the bile acids secreted into the upper small intestine are absorbed into the lower intestine and returned to the liver. Enterohepatic circulation handles about 20-30g bile acids per day.
The most abundant bile acids are cholic and chenodeoxycholic acids. These are produced from cholesterol via cytochrome P-450 activity. These acids are usually conjugated to glycine or taurine to form bile salts. These bile salts have a detergent action which allows the digestion of lipids and their absorption through the intestinal mucosa. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
Faecal lipids Components (part b)
Dietary components:
Not all ingested fats are absorbed. Many plant sterols, such as sitosterol, are not normally well absorbed and pass through the digestive tract. Some dietary cholesterol and fatty acids will not be absorbed and will be excreted in the faeces.
Microbiota:
The bacteria in the gastrointestinal tract contain membranes and fatty acid components to sustain their cell integrity. They can release certain lipid products into the gastrointestinal environment.
Bacteria can be classified on the basis of their fatty acid composition. Future research will attempt to link characteristic fatty acids with alterations in bacterial composition of the colonic microbiota.
The lipid test may prove to be very useful in assessing microbial homeostasis without extensive microbiological testing. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
Bacterial formation of coprostanol:
The anaerobic bacteria in the gastrointestinal tract are very good at converting cholesterol (and sitosterol) to another sterol product called coprostanol. Coprostanol is not absorbed by humans and is normally the major sterol product excreted in the faeces. Low levels of coprostanol relative to cholesterol would, therefore, indicate a disturbance in the microbial homeostasis.
A practical example:
The analysis of a faecal sample from a healthy subject was compared with that from a long suffering CFS patient. Significant differences were found in the fatty acid composition and in the levels of sterols. Closer inspection of the sterol section of the analysis indicated a total absence of coprostanol production in the CFS patient. This is probably an extreme example but it clearly demonstrates the capacity of the lipid test to detect a disturbance to the normal microbial activities in the gastrointestinal tract.
Background Microbiology of the Gastrointestinal Tract
The intestinal microbial flora can be divided into three categories.
1. Autochthonous:
These are indigenous organisms that are present at a constantly high level in the host, regardless of differing environments.
2. Normal:
Normal organisms become established in all the individuals in a given community. They are not necessarily present in others when the environment is different.
3. Indigenous:
These include all organisms that are able to colonise in the intestine of healthy individuals.
Stomach
The acidic environment of the stomach may support the following microbial flora:
1. Streptococcus bacteria
2. Staphylococcus bacteria
3. Lactobacillus bacteria
4. Candida
5. Oral anaerobes
The density of microbial content of the stomach has been estimated to be over 100 000 organisms per gram wet weight. This flora does not appear to be resident and originate from food, nasopharyngeal and salivary secretions. Micro-organisms may survive if they are protected and pass rapidly through the stomach (eg Salmonella) or if they are acid resistant (eg mycobacteria).
Small Intestine
With the exception of the distal ileum, the small intestine usually accommodates transient microbial populations. When micro-organisms develop in the small intestine they are secondary to underlying diseases. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
Duodenum:
This region contains few micro-organisms due to the combined actions of -
1. stomach acid secretions
2. inhibitory action of bile and pancreatic secretions
Gram positive cocci (eg oral ‘viridian’ streptococcus, staphylococcus) and rods (eg lactobacilli) compose most of the microbiota.
Jejunum:
Enterococcus faecalis, lactobacilli, diptheroids and Candida albicans may be present.
Ileum:
The environment is more alkaline and, as a result, anaerobic gram-negative bacteria and members of the family Enterobacteriaceae become established. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
Large intestine
The colon has the largest microbial population of the body. Over 300 species have been isolated from human faeces. The microbiota consist primarily of:
1. anaerobic and facultative anaerobic gram-negative non-sporing bacteria
2. gram-positive, spore-forming, and non-spore-forming rods.
The four most numerous microbial species identified in the intestine of humans are:
1. Bacteroides
2. Eubacterium
3. Peptostreptococcus
4. Fusobacterium
Escherichia coli, the most abundant facultative anaerobe, represents less than 0.1% of the total intestinal microbial population.
Non-bacterial micro-organisms, such as Candida albicans and certain protozoa may also be detected.
References:
1.Sawamura-A; Kusunose-E; Satouchi-K; Kusunose-M. Catalytic properties of rabbit kidney fatty acid omega-hydroxylase cytochrome P-450ka2 (CYP4A7).Biochim-Biophys-Acta. 1993; 1168(1): 30-6
2.Dixon RM, Borden EC, Keim NL, et al. Decreases in serum high-density-lipoprotein cholesterol and total cholesterol resulting from naturally produced and recombinant DNA-derived leukocyte interferons. Metabolism 1984; 33:400-4.
Various physiological processes occur in an adult resulting in the potential excretion of vast numbers of micro-organisms per day. These processes include:
1. peristalsis and segmentation,
2. desquamation of the surface epithelial cells to which micro-organisms are attached, and
3. the continuous flow of mucus with its associated microbes.
Research has shown that the microbial flora of an individual is very consistent.
A single sample is a very good representation of the flora of that subject (ref. 5 and 6).
Changes in diet have been found to produce only slight changes in flora (ref. 7 and 8).
The individuality and stability of the faecal flora suggest that the host environment is the major determining factor in the composition of the flora. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
Under normal conditions, the resident microbial community is self-regulating.
If the intestinal environment is disturbed, the normal flora may be dramatically altered.
Disruptive factors include:
1. stress (ref. 9)
2. worry or anger (ref. 4)
3. altitude changes
4. starvation (ref. 10)
5. parasitic organisms
6. diarrhoea7. antibiotics (ref. 11)
The actual proportions of the individual populations within the indigenous microflora depend largely on a person’s diet.
References:
4. Holdeman LV, Good IJ, Moore WEC. Human faecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl Environ Microbiol, 1976; 31:359.
5. Sears HJ, Brownlee I, Uchiyama JK. Persistence of individual strains of Escherichia coli in the intestinal tract of man. J Bacteriol, 1950; 59:293.
6. Sears HJ, Brownlee I. Further observations on the persistence of individual strains of Escherichia coli in the intestinal tract of man. J Bacteriol, 1952; 63:47.
7. Goldberg MJ, Smith JW, Nichols RL. Comparison of the faecal microflora of Seventh-Day Adventists with individuals consuming a general diet. Ann Surg, 1977; 186:97.
8. Moore WEC, Holdeman LV. Human faecal flora: the normal flora of 20 Japanese-Hawaiians. Appl Microbiol, 1974; 27:961
9. Moore WEC, Cato EP, Holdeman LV. Some current concepts in intestinal bacteriology. Am J Clin Nutr, suppl., 1978; 31:S33.
10. Deitch EA,Winterton J, Berg R. Effect of starvation, malnutrition, and trauma on the gastrointestinal tract flora and bacterial translocation. Arch Surg, 1987; 122:1019.
11. Berg RD. Promotion of the translocation of enteric bacteria from the gastrointestinal tracts of mice by oral treatment with penicillin, clindamycin, or metronidazole. Infect Immun, 1981; 33:854.
New Roles for Colonic Bacteria
Newcastle research has indicated that several distinct alterations in the distribution of bacterial flora occur in patients with chronic fatigue and chronic pain related disorders. These changes in the microbial composition of the faeces may reflect disturbances to :
1. digestion
2. bile cycle metabolism
3. malabsorption
4. changes in bowel ecology
If the homeostasis of the colon microbiota is disturbed by toxic chemicals or antimicrobial agents, then many resilient organisms can proliferate to cause illness. These resilient organisms may also produce toxins or antibiotics that inhibit the growth of other species.
An important function of the gastrointestinal flora is, therefore, to prevent colonisation by harmful or pathogenic organisms. Newcastle CFS researchers have started investigating selected colonic bacterial species to determine what types of organic acids, amino acids and sugars are released during their growth. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
The first organisms tested were Escherichia coli, Proteus mirabilis and Enterobacter cloacae (clinical isolates). These organisms have been found to be capable of releasing substantial quantities of amino and organic acids. Many of these products are those that have been identified as being significantly deficient or in excess in CFS patients.
One organism, Enterobacter cloacae, produced substantial quantities of 4-amino-butyric acid, otherwise known as the inhibitory nerve transmitter GABA. Proteus mirabilis was characterised by producing large quantities of lysine and glutamic acid, whereas E coli produced large quantities of alanine and valine. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
All three species examined produced phosphoserine (CFSUM2) which is deficient in most CFS patients
These results suggest that certain bacterial species in the gastrointestinal tract can contribute to the nutrition of the patient by providing:
1. non-essential amino acids
2. essential amino acids
3. other growth factors
Considering the microbial mass within the colon, this could represent a very significant contribution to the nutrition of the patient.
Bioscreen's Gut Restoration Protocols
Unfortunately, there hasn't been a great deal of clinical trial data to confirm the outcomes of Bioscreen’s treatment recommendations. However, some strategies can be extrapolated from Newcastle’s research findings. © Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
Patient management
The work of Bioscreen’s CFS researchers has resulted in treatment protocols that aim to restore the bowel flora ecology and its associated functions. Depending on test results, the following supplements may need to be considered:
1. digestive enzymes
2. FOS (fructo-ologo-saccharide)
3. lactosucrose
4. enteric coated lactobacillus and bifidobacterium
5. specific amino acids
6. B group vitamins
© Bioscreen, CPRU, S Ashton and F Bartosy 1999 - 2001
If the relative abundance of serine is less than 6%, serine supplementation should also be considered. Note: The specific level of serine in the urine is obtained from the urine test result.
A management protocol should be used where there is evidence of disturbance to the gastrointestinal microflora. A gut restoration protocol can be used as a basis to develop a personalised management plan for the patient. This should be considered if:
1. the bacteroides % is greater than 95% and/or
2. the E. coli % is less than 50%.
If the bacteriodes are less than 20% please contact the laboratory for discussion.
Note:
For more information, refer to: “Interpretation notes for use by the consulting medical or health practitioner”. These notes are normally supplied with each completed faecal analysis report.
© CPRU and F Bartosy 1999, 2000
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