Study of human microecology by mass spectrometry of microbial markers

G.A. Osipov1 and N.V. Verkhovtseva2

1 Academician Yu. Isakov Research Group, Bakulev Scientific Center for Cardiovascular Surgery, Rublevskoe shosse 135, 121552 Moscow, Russia; -Lomonosov Moscow State University, Leninskie gory 1, 119991 Moscow, Russia; osipovga@mail.ru

Abstract

This review shows that mass spectrometry of microbial markers (MSMM) permits simultaneous in situ determination of more than one hundred microbial fatty acids in clinical, biotechnological or environmental samples, without precultivation and use of biochemical test materials and primers. Unprecedented information about the quantity of anaerobes and uncultivated aerobes, as well as actinobacteria, yeasts, viruses and microscopic fungi in one sample has provided a full understanding of microbial etiology in clinical conditions of patients. The study of intestine dysbiosis has confirmed the hypothesis about the nosological specificity of changes in the intestinal microbiota. It has been proven that infectious processes are polymicrobial. Measurements have shown that anaerobes dominate in number and functional activities in inflammation. The division of microbes into pathogenic and non- pathogenic is artificial. All microbes living in a human body simultaneously stay in both forms. Lactobacilli and bifidobacteria appear as agents of septic conditions and endocarditis. MSMM data confirm that anaerobes of Clostridium, Eubacterium, Propionibacterium, as well as actinobacteria of Streptomyces, Nocardia, Rhodococcus are mixed infection dominants. The data testify translocation of these microbes in inflammation loci from the intestine. Quantitative comparison of concentration of markers in the inflamed organ and blood proves reproduction of microorganisms in this locus. The current hypothesis is confirmed that the goal of translocation is not only infection, but also a biofilm formation similar to intestines, which stimulate local immunity, protection from local pathogens and restoration of the damaged tissues. Quantification using GC-MS revealed that the influence of antibiotics on the normal intestines microbiota are not as dramatic as believed. Growth-promoting effects are die most important benefits of probiotic applications. The probiotic essence is not die microbial biomass itself, but growth factors, alarm molecules, and other factors of intestinal microbes. There are new possibilities in improving probiotics by using microbial ‘consortia’, modelling real gut microbiota.

Keywords: microbiota, intestine wall, fatty acids, mass spectrometry, translocation

 

 

  1. Introduction

Cellular fatty acid (CFA) Composition of micro-organisms has been used extensively for bacterial identification and taxonomic purposes. Variability in number of carbon atoms, the number and position of double-bonds, cis or trans isomers and substitution groups are stable parameters, provided that highly standardised analysis and culture conditions are observed (Welch, 1991).

Environmental and human microbiota closely interact due to the fact that man himself is part of the environment like other living beings and plants. Humans are participants in the global nutrition and metabolic chain. This biological concept helps us to understand the clinical behaviour of microbial communities. Microbes have been living with humans for more than one million years. Their community in the mucous layers of the gut is present in the form of a pseudo-tissue (biofilm) and is the vital multicellular organ of a person. The microecological status of a person, or more precisely, the maintenance of its homeostasis, is a necessary condition for die stable functioning of all organs and systems, from resistance to diseases and longevity and maintaining a high quality of life. In their intestine humans possess an ‘extended genome’ of millions of microbial genes — the microbiome. Because this complex symbiosis influences host metabolism, physiology and gene expression, it has been proposed that humans are complex biologic ‘superorganisms’ (Kinross et al., 2008). It is known that microbes in a human body produce more than 60% of the substances necessary for the functioning of internal organs and skin. The intestinal microbiota is a source for body maintenance and intestinal immunity, and regulation of the microbial structure for health maintenance is a promising therapeutic strategy. One of the outcomes of stress on a human body is metabolic infringement as a consequence of changes in intestinal microbiota. There is a disproportion in the production of biologically active substances by microorganisms. Organs function abnormally due to a deficiency of vitamins, enzymes, cross-talking molecules and other hormone-like substances. The metabolism and reproduction of cells and systems, e.g. immune, nervous, endocrine, is broken. Consequently, one of the first stages in rehabilitation should be the control and restoration of their microecology.

Why have we not used die potential of health, quality of life management and longevity of the person through its own microbiota regulation? This is because there is no simple method to define and control the human microecological status and, in particular, intestinal wall microbiota. Genomic, proteomic and metabolomic technologies are expensive and time-consuming and mostly used as scientific tools. Therefore the development of molecular non-culturable methods was required to be able to study the molecular ecology. This has led to the recognition of tire importance of endogenous microbiota in various diseases and disorders. Even non-gut related disorders, such as obesity, atopic eczema and autism, have been correlated with the composition and activity of the microbiota. In this context new ‘omic’ technologies will be very helpful in better characterising and understanding the effects of probiotics on the balance of the gastrointestinal microbiota (Chaucheyras-Durand and Durand, 2010).

Currently the control of human microbiota is a problem in practical health care. This is due to the fact that classical bacteriological methods are difficult to use in practical applications. Ar. alternative non-culture method of human microbiota evaluation is gas chromatography-mass spectrometry (GC-MS) analysis of long chain cellular fatty acids, fatty aldehydes and sterin.es which form the phospholipids components of cellular membranes. The capacity of GC-MS detection of microorganisms by their markers, including fatty acids, in practical medicine has not been widely studied. Attempts to detect bacteria from blood muramic (Fox et al., 1996) and p-hydroxvmyristic (Maitza et al., 1978) acids have been reported. The control of meningococci from the presence of β-hydroxylauric acid in blood (Brandtzaeg et al., 1992) and gonococci from the concomitant presence of p-hydroxylauric and [3-hydroxypalmitic acids (Slid and Feingold, 1979) has been proposed. Other potential uses of GC-MS for diagnosis in clinical microbiology research have been discussed by Larsson (1994). Recently 3-hydroxy fatty acids in tissues (Szponar et al., 2003) and saliva (Ferrando et al., 2005) were investigated as diagnostic markers of endotoxin intraperitoneal infection and chronic periodontitis.

The purpose of this review is to outline another approach, which differs from the literature cited above by the inclusion in GC-MS screening of all (⁓170) known microbial markers in one run. Most of our cited publications are printed in Russian, however some features of methods and their clinical applications are available in English at www. rusmedserv.com/microbdiag/eng.

  1. Mass-spectrometry and microbial markers approach

We have previously described examples of microbial detection in infectious processes by GC-MS (Osipov and Dyomina, 1996). Homeostasis of small molecules of microbial origin has been found in human blood that is impaired in inflammations (Beloborodova and Osipov, 2000). The composition of the human microbial community is known, thus knowledge of the qualitative and quantitative structure of the microbiota of an individual will give a representation of the current condition of immunity. It, in turn, allows the risk of development of an infectious disease to be predicted. The method yields live data about the human microbiota without separating and cultivating microorganisms on media, and reduces test time from 2-5 days to 1.5-3 hours. Fast test results allow a timely recommendation for correcting the immune status of the patient.

GC-MS can provide unique information about the composition of specific monomeric chemical compounds of microbial cells. These compounds (markers) can be detected on the background of other chemical compounds of the biological fluids. The advantages of this analytical procedure consist in the direct extraction of CFA from samples by a chemical procedure. The specimens are separated by capillary column chromatography and detected by means of multi-ion detection mass spectrometry. The GC-MS method not only permits the detection of marker compounds (CFAs, aldehydes, alcohols and sterines) in clinical material, but can also determine and calculate (reconstruct) the composition of the microbial community, encoded in the markers of the sample (Osipov et al., 2009). Materials commonly used for clinical investigation are blood, saliva, urine, body fluids, synovial or ascetic liquid, mucosa, phlegm, throat, nose and ear swabs, and secretion of genitals, tissue sampling, and skin smears, depending on the aim of the investigation.

A GC-MS system costs as much as any microbial identification system, and consists of a mass spectrometry unit, a chromatography unit and a computer. Sample preparation is purely chemical and does not assume cultivation of microorganisms. This makes it possible to quantitatively define any microbes, without depending on their ability to grow in artificial conditions. The chromatograms obtained by the selective ion method (Figure 1) can confidently detect microbial components in the presence of the predominant human waste components. The paks of target substances are mostly superposition- free, and distant from the substrate peaks, making automated analysis possible. The list of fatty acids, aldehydes and sterols detected in specimens is given elsewhere (Osipov et al., 2001, 2003,2009) indicating the most likely microorganisms in whose cells they were usually found (Table 1). For instance, il6 (iso-hexadecanoic acid) is a marker of Streptomyces spp., 18:lw7 refers to Lactobacillus spp. and hl4 (3-hydroxy-tetradecanoic acid) is a well-known feature of Enterobacteriaceae, Haemophilus spp., Fusobacterium spp. and some other gram-negative microbes.

The method allows multi-ion detection for up to 30 ions in five groups during a 30-minute GC run. Such a run

includes the complete variety of microbes, which is why primers, standards, and every day calibration are not needed. The peak area of the microbial marker on the selective chromatogram is proportional to the biomass of the specified microbe.

  1. Human microecology

The human microbiota is mostly concentrated in the intestine. Resident bacteria are mostly localised (aside from those at the epithelial cells) within die mucus. One of the major possibilities of mass spectrometry of microbial markers (MSMM) is that is can be used to reconstruct the composition of the intestinal microbiota (Figure 2).

Human faeces have the same qualitative composition, but differ in quantity of single genera and species in it. There are more bacteroides, clostridia and eubacteria and less bifidobacteria and actinobacteria (Figure 3) in the faecal microbiota.

Quantitative measurement of the intestinal microbiota allows for a description of part of tire digestive food chain — from chyme to faeces (Figure 4). Chyme, food digestive products which pass from stomach to the small intestine (e.g. polymers, like cellulose, proteins, and fats) are first aerobically transformed by actinobacteria into sugars, carbon dioxide and water. This, in turn, serves as substrate for facultative and strict anaerobes — lactobacilli, eubacteria and dostridia. These bacteria produce volatile fatty acids, which may in turn be utilised by other members of intestines biofilm at the time of their formation. Eubacterium and Clostridium spp. produce hydrogen and formic acid which serve as feedback regulators for the methanogenic reactions. The human intestine (as well as the cattle rumen) can be seen as a methanogenic reactor (Davey and O’Toole, 2000), analogous to anaerobic treatment of industrial wastewaters (Lettinga, 1995; Wheatley, 1990).

A final hypothesis to explain the observed cluster morphology is that, analogous to what is found for aerobic biofilms, anaerobes also produce cell-to-cell signalling molecules. Therefore it is possible that the cluster (biofilm) architecture of the aggregates represents an optimal arrangement for the in- and outflux of substrate and gas products, respectively. We measured the composition of methane granules in the microbial community of Lettinga’s granules (Lettinga, 1995) and found that they included complex microbial consortia. These consisted of members of the genera Clostridium, Bacteroides, Butyrivibrio, Succinivibrio, Flavobacterium, Lactobacillus, Eubacterium, Methanothrix, Methanobacterium, Cytophaga. Acetobacterium, Pseudomonas, Streptococcus, Lachnospira, Propionibacterium, Cellulomonas, the phylum Actiriobacteria and microscopic fungi. The theory of methane digestion could also be applied to methanogenesis in the human intestine, and trophic and metabolic relations among the taxonomic groups in it could be proposed (Figure 4).

  1. Microbial ecology and health

The concentration of microbial markers in blood of healthy people is on average constant and correlates with the composition of small intestine’s microbiota ( Osipov etal., 2003). This allows for a new, non-invasive approach to monitoring human microecology by measuring microbial markers in blood. Only 40 µl of finger blood and 3 hours is needed for reconstruction of the human microbiome dominants.

Parallel measurements of the blood concentrations of microbial markers in the same patients whose small intestinal biopsy specimens were studied have shown that the tendency for their concentration to change coincides. Figure 5 compares the measurements of bacterial markers in the lejunal biopsy specimens and in the blood of a patient with irritable bowel syndrome. A biopsy specimen from the intestinal mucosa (4 mg) and a blood sample (50 mg) from the finger were taken for analysis. The results show that the composition of microorganisms in the blood is close to the

composition in the jejunal mucosa, expressed as a percentage.

The fact that there is a correlation between microbial markers in blood and the small intestinal microbiota corresponds to the known ideas on the biochemistry of absorption of lipids, vitamins, and other food components, which occurs just in the small bowel. Breakdown and new production of dietary glycerides occur in its epithelium and they enter the blood (Schmidt and Thews, 1993). Naturally, the same probably occurs with the lipids of cells of the microorganisms that colonise the small intestine or as a result of their phagocytosis. With this, the food component is absent under our sampling conditions as patients have not eaten for at least 15 hours before taking an intestinal mucosal biopsy specimen and a blood sample.

The presence of microbial markers in the blood is theoretically due to the mechanism of an immune response to the appearance of a causative agent. Human phagocytes adsorb and digest the antigens of microorganisms, including the whole cells, and release lysis products into the stream of the lymphatic and blood systems. The lymph flow that constantly washes tissues, the intestinal wall, and the epithelium of the entry of infection may be conceived to take the antigen of pathogens of chronic or acute infection. Serum proteins pass antigenic components to phagocytes where the known mechanism for elimination of the antigen and damaged cells of the organism is triggered and antibodies are produced for the subsequent acts of an immune reaction. It is important for us that the components of microbial cells enter the blood and may be determined in it by a sensitive GC-MS analytical method in the mode of fragment ions. It is not to be supposed that active microbial cells are presence in the blood. Blood inoculation to nutrient media is known to generally yield a negative result; microbial growth does not occur except in cases of bacteremia. Besides phagocytosis, microbial cells are destroyed by die action of other mechanisms, including the intrinsic cell autolysis and enzymatic lysis by the protein complement of blood and other components of immune defence. Ln any case the monomeric constituents of biopolymers ultimately degrade. From die principles of die human physiology of exchange of biological fluids (Schmidt and Thews, 2003), the exchange of 70% of the plasma with interstitial space occurs within one minute. So die minor fragments of microbial biopolymers formed in this space enter the blood almost immediately. Considering that the bulk of phagocytes are outside the vascular bed, in the intercellular space and that they readily pass through the walls of vessels, it is felt that, no matter what the focus of inflammation, along with phagocytes and protein carriers, microbial markers constantly and rapidly enter the blood. It is the presence of microbial markers in die blood that reflects the composition of human microbial communities irrespective of the habitat of microorganisms or the focus of inflammation.

However, the blood concentration of microbial markers remains rather high in normalcy, i.e. in the absence of symptoms of inflammation or local infection, which has not yet been adequately explained. The quantitative estimates obtained in the present study on the concentration of microorganisms on the intestinal wall clearly suggest that the intestinal microbiota is their main source. The ratio of the blood amount of fatty acids to the total number of intestinal microbes may be estimated as follows. Our experiments were carried out using biopsy specimens with a characteristic size of 2 mm. Let us assume that the measured concentration of microbial cells applies just to such an intestinal mucosal layer, 2 mm in thickness. Then by taking into account that the intestinal mucosal surface is die order of 0.3 m2 (without considering folds and microvilli), die volume of the probed mucosa is 3xl03 cm2 x 0.2 cm =

6xl02cm3, i.e. the weight is 600 g, where it should be 1.2xl0n cells/g x 600 g = 7.2xIO14 cells, as shown by our estimates. This is entirely consistent with the estimate of the total number of microorganisms (1011 cells) in the human. By the sum of the concentrations of microbial markers in the blood of healthy persons, the respective number of microbial cells which produce the markers is equal to 2.911х10ьсе115/т1 or 1.4x 1013 cells as a whole, assuming the volume of blood is 5 litres; that is, a fiftieth or 2% of fatty acids of the cells of microorganisms colonising the intestine enters the blood.

Familial Mediterranean fever fFMF) is a good example of microbial etiology investigations in diseases. There is no single fever agent, but specific changes in microbiota were revealed by the MSMM method. The highest concentrations of bacterial markers were observed in the blood of FMF patients in the remission period, whereas in the attack period the corresponding values were reduced (Figure 6). Notably, the results obtained for FMF patients cardinally differed from other investigated patient groups with non- genetic gastrointestinal disorders (Ktsoian et al., 2002; Ktsoyan et al., 2008).

Measurement of microbial markers was followed by carbonic and phenyl carbonic acid investigation, which completed the metabolome of FMF (Figure 7).

Measurement of carbonic and phenyl carbonic acid in the blood of FMF patients along with MSMM yielded hypothesis on the microbial etiology of FMF. A background of animal protein deficiency and a surplus of vegetative carbohydrates, citrus flavonoids and seafood lipids due to local tradition, causes accumulation of aromatic amino acids (tyrosine, phenylalanine). This type of diet predisposes abnormal growing Eubacterium spp., which could start the inflammatory process in FMF. The accumulation of phenolic compounds stops at a stage of anaerobiosis — on catechol. This is confirmed by the positive effect of treatment with metronidazole and oxygenation. Benzoic acid along with Eubacterium antigen is proposed as the triggering substance in periodic fever.

As the above example shows, we have developed a new technology to discover and monitor host-microbes crosstalking by GC-MS — micro-metabolomics — precise detection of microbial markers (more than 200 structural fatty acids, hydroxy fatty acids and aldehydes) in body Quids and tissues accomplished with quantification of human and microbe metabolic carbonic and phenylcarbonic acids (over 100 substances). Micro-metabolomic molecular technology can be used for quantitative monitoring microbial homeostasis and its deviation from the norm. ‘ There exist as many variants of human dysbiosis, as nosological variety of conditions’ (Shenderov, 1998).

Clinical application of micro-metabolomics will help to confirm this and can unravel many secrets of human health. For example, specific nosological changes in intestinal microbiota’s composition with organs and skin diseases. The preliminary data obtained (Beloborodova and Osipov, 2000) support the hypothesis on the constant presence microbial markers in the blood of healthy and ill individuals and allow for a formulation based on the concept of homeostasis of small molecules originating from microbes. Because of real right-side tailing of distribution, we apply the Kolmogorov- Smirnov test since it is more convenient than the Mann- Whitnev test, which should preferably be applied to normal, Gaussian frequency distribution.

According to this test microbial markers have shown valuable differences at P<0.05 between the patient and control groups. Extreme values exceeding +1.5 quartile distance are rarely found in donors, but are common in patients. Such values probably indicate inflammation, caused by corresponding microbes. As far as the composition of microorganisms markers in the blood reflect the intestine microbiota as described we use this approach in investigations. For atopic eczema we regularly found a deficiency of bifidobacteria in intestines and overgrowth of Eubacterium spp, Propionibacterium freudenreichii, Nocardia and other microorganisms (Figures).

For patients with acne we regularly found deficiency of lactobacilli and overgrowth of clostridia Clostridium ramosum group, bifidobacteria, herpes-virus and other microorganisms in the intestines (Polesko et al., 2007). Seborrhoea patients differ from healthy humans in deficiency of lactobacilli and propionibacteria in the intestine s wall, along with increased growth of the clostridia C. ramosum group and Eubacterium spp.

What is the advantage of micro-metabolomics in the study of infections? Unlike the previous examples, we have made quantitative measurements of mixed infection, and have shown a rating number of agents. This data also confirms the predominance of the endogenous way — translocation. In the majority of infectious processes, i.e. cystic fibrosis, post operational surgical infections, respiratory and abdominal infections, testes, uterus inflammation, mastitis, synovitis, peritonitis, pyelonephritis, pancreatitis, etc., anaerobes Propionibacterium, Eubacterium. Clostridium and Bifidobacterium dominate, and in the case of bacteraemia and septic conditions Lactobacillus takes part as well. Secondary infections include actinobacteria and cocci. Pseudomonas and other non-fermenting bacteria, like members of Enterobacteriaceae, as well as Staphylococcus are irregular and minor participants in infectious processes.

Recently, Tunney et al. (2008) showed that the lungs of cystic fibrosis (CF) patients were not only colonised by common bacteria, like Pseudomonas aeruginosa, but also by a range of potentially pathogenic anaerobic species. The involvement of 47 microbial taxa, including some fungi and viruses, in the process was confirmed by MSMM. Before antibiotic treatment anaerobic bacteria, including Eubacterium, Propionibacterium, Clostridium, Actinomyces and Eggerthella were found in high numbers (Зх 109 cells/ml, 1.4×10*’ cells/ml, 4×10s cells/ml, lx 10s cells/ml, lx 10- cells/ ml, respectively). In comparison, Staphylococcus aureus and P. aeruginosa were found at cell densities of 4.8x 10 cells/ml each. The concentration of other anaerobes — Bacteroides, Porphyromonas and Prevotella — mentioned as potentially significant in previous works (Rogers et al., 2004), were not clinically i mportant in our patients. Our results indicate that anaerobes are present in large numbers in the airway and could play a clinical role in CF patients, especially during an acute exacerbation of pulmonary infection, where they may contribute to the inflammatory process (Semykin et al.. 2008).

  1. Translocation phenomena and beneficial microbes

Translocation phenomena are general in character. We observed it in CF (Semykin et al., 2008), but also in meningitis (Osipov et al., 2007), abdominal infection (Fedosova and Lyadov, 2010), peritonitis (Boiko et al.. 2009). synovitis (Prochorova et al., 2009) and inflammations of internal genitals of women (Osipov et al., 2007). Long straight chain as well as branched, unsaturated, cyclopropane and hydroxy fatty acids, which are specific to microorganisms but not synthesised by the host’s cells, were found in patient’s blood taken before appendectomy and

in post-operative pus. Enterococcus, Mycobacteria, Streptornyces, Enterobacteriaceae, Bacteroides fragilis, Eubacterium, Helicobacter pylori, Klebsiella spp., and Clostridium perfrtngens among other microorganisms were detected by their specific markers. Presence of Staphylococcus haemolyticus and diphteroids was derived from the composition of the fatty acid solution. Only Escherichia coli, Peptostreptococcus spp., Bacteroides spp., Candida albicans, P. aeruginosa, Eusobacterium spp., anaerobic gram-positive rods and the Enterobacteriaceae spp. were discovered by culture method in post-operative pus or puncture. Blood culture was negative. The composition of the microbial markers in blood reflected the content in the post-operative pus of the same patient. The possibility of predicting the causative agents by detection of microbial markers in blood is proposed as a non-invasive method for abdominal infections such as abscess, infiltrate, peritonitis, etc.

Markers of microorganisms are also found in healthy organs. Sometimes the microbial community of the inflamed organ resembled the intestinal one. It appears that the inflamed organ translocates not only separate pathogens, but all intestinal microbiota — bifidobacteria and lactobacteria among others. Is the phenomenon of translocation of microorganisms not only an endogenous infection, but also an endogenous antimicrobial therapy? It has been hypothesised that this type of translocation is part of the mechanism of endogenous antimicrobial therapy and local cure, analogous to the intestines where these bacteria interfere with contamination by transient pathogens. Indeed, experiments by Van den Broek (1992) with gnotobiont and conventional rats allows such an assumption. Thus the role of pathogens can be attributed to those microorganisms which show disproportional growth in the intestines.

Furthermore, the protective mechanisms of commensal biota are increasingly being recognised, suggesting that perioperative modulation of the gut microbiome with pre-, pro- and synbiotics may beneficially influence surgical outcomes (Kinros etal., 2009).

We have made a quantitative measurement by MSMM in synovial fluid of an inflamed knee or elbow joint and

compared it with the blood of the same patients (Prochorova et al., 2009). It appeared that the relative concentration in the inflamed tissue as compared with the same patients blood values, in other words die total bodies’ microecology, showed a hundred fold increase in the number of streptococci, and a ten-fold increase in clostridia and nocardia. Several times above the norm were the concentration of markers of the genera Moraxella/Acinetobacter, Fusobacterium/ Haemophilus and C. perfringens.

In some patients with gonarthritis an increase of ten or more times the concentration was found of markers for P aeruginosa, Bacillus cereus, Eubacterium lentum, Campylobacter mucosalis, El. pylori, Prevotella and Candida species (Figure 9). These species are the main infectious agents of the joint (calculated quantity 10*’-103 cells/ ml). Minor groups (with quantities of 10 ‘-10(’ cells/’ml) include bacteria which are usually revealed by cultivation — Acinetohacter, Alcaligenes, Staphylococcus and others.

We found that translocations in synovitis are polymicrobial, as is spontaneous bacterial peritonitis in liver cirrhosis (Vinnitskaya et al., 2008). Is it therefore possible to expect the occurrence of any intestinal microbe in the i nflammation zone, or even all of them at once? This is a question of sensitivity and method capacity. The applied method allows for simultaneous scanning of 170 microbial markers of different taxonomic levels and groups. The level of the mixed infection and its composition reflects the specificity of the synovitis microbial etiology of each patient and provides additional information for antibiotic treatment and other medicinal actions for the restoration of the patient’s microecological status. As it seems, we have the same simple tool which will help in understanding individual human gut microbial activities as a necessary part of future personalised health care (Kinross etal., 2008; Nicholson and Holmes. 2005; Nicholson et al., 2006).

Proof of such translocations can be found in the literature. The concept of translocation of all potential pathogens from intestines to lymph nodes, liver, spleen and other organs is discussed as far as 1902 by Askoli. It has now become clear that not only gram-negative micrixirganisms, but also grampositive bacteria and fungi can pass the intestinal barrier (MacFie, 2004). It has been experimentally confirmed in one of the latest analysis of the joint microbiota by comparison with the data from culture and genetic methods. For 475 bacterial isolates (including 176 from a knee joint), the biochemical identification coincided with the 16S rRNA homology data. Thus it was shown that joint colonisation is polymicrobial (Fenollar et al., 2006) and that the stimulus for a translocation could be the intestinal microbiota’s overgrowth or stress.

Antibiotics, probiotics and microecology improvement

Quantifications using GC-MS revealed that the influence of antibiotics on the normal intestine’s microbiota is not so dramatic as believed. Antibiotics should be considered in the second phase at dysbiosis, e.g. for irritable bowel syndrome, and antibiotic-associated diarrhoea. The rats’ microbiota was shown to be resistant to a three-week course of daily 500 mg ampicillin (Dyachenko et al., 2006). Antibiotics can be applied safely to correct dysbiosis within the doses specified in the prescription.

Growth-promoting effects for farm animals are the most important expected benefits of probiotic applications. However, many probiotics fail to colonise the gastrointestinal tract (GIT) even transiently, as the GIT has many defences that inhibit colonisation.

The human GIT harbours dense and complex microbial communities, composed of bacteria, protozoa, fungi, archaea, and viruses. Considerable research has been devoted to characterise digestive ecosystems in terms of composition and functional diversity. This has led to a better understanding of die major contribution of the gut microbiota to human nutrition and health. The GIT consists of a large surface area with high levels of nutrients. Chyme and mucin are die principal components of the nutrient and media flow. Chyme moves along the intestines and mucin moves in a normal direction, t.e. perpendicular, providing optimum conditions for the intestinal microbial biofilm. Ln die GIT biofilm is formed in two locations: first, attached to food particles in the lumen and secondly, attached to the mucosa and within the mucous layer itself (Macfarlane and Macfarlane, 2006, Macfarlane et al., 2000). Intestinal biofilm looks like stocks which cover gut from top to bottom, and could be interpreted as another human organ due to its genetic and nutritional integrity and quorum sensing. Microbial communities associated closely with the mucosa are important because of their ability to interact with the host tissues, dius influencing the healdi of the individual (Macfarlane, 2008). Unfortunately all research of the human microbiota so far has been made in faeces. Faeces are not identical to the biofilm as they have no contact with most parts of the GIT wall and cannot take part in the upper GIT digestion. This is a common misunderstanding: substitution of intestinal microbiota with fecal microbiota, where they really are two different communities.

By measuring microbial markers in blood, we have the opportunity to directly monitor the effect of probiotics on real GIT microbiota, which are in close contact with the intestinal wall. For example, we followed the treatment of 30 patients with antibiotic-associated diarrhea (AAD). Binary probiotic Bifiform (Bifidobacterium longtnn 10 cfu and Enterococcus faecium 10 cfu) were administered daily for three weeks in six enterosoluble capsules. All patients showed up to five times the amount of deficiency in total intestinal colonisation and not only an overgrowth of Clostridium difficile, but also an increase in C. ramosum and C. perfringens group. This clinical case was interesting with regard to probiotic genera in the patients’ intestines. As it appeared, AAD patients had different numbers of target microbes. Five of them showed an initial surplus of Bifidobacterium spp. and an excess of eight times of Enterococcus spp. The question is: is it necessary to apply those microbes when they are already abundant in the intestines? As it turned out, it has no value. Positive dynamics in a treatment started to appear from the 2nd-3rd day of bifiform administration, and the normal intestine’s colonisation was restored more slowly.

Quantitative analysis of a wide range of microorganisms in faeces and biopsies from small intestines by MSMM in irritable bowel syndrome (IBS) revealed a complex picture of microbial rearrangements as a result of enterol application, a probiotic preparation of Saccharomyces boulardii (Parfenov et al., 2006). Before treatment, an increase in the number of separate representatives of the microbial community’ was found in all IBS patients.

After enterol treatment changes were found in the small intestine for all genera and species (Lactobacillus, Bifidobacterium, Clostridium, Eubacterium, Streptomyces, Rhodococcus, Streptococcus, Candida, etc.) directed towards restoration of the normal microbiota. However, there was no restoration of the colonisation level of healthy people, despite the positive clinical effect found. Thus, no specific influence of enterol on certain microorganisms or groups was detected. Comparison of microbial counts in faeces before and after treatment do not show enterol effectiveness. For all IBS patients it was found that the total number of bacteria in faeces was many times lower in comparison with the control group. Though enterol undoubtedly changed die number of some microorganisms towards the normal situation, the sum of microbes poorly increased, or even decreased due to the large reduction of C. perfringens and Lactobacillus in proportion to the growth of organisms of odiers taxons. It was not possible to find a stable tendency in the change in faecal microbiota after treatment, though the general decrease in the number of C. perfringens, Lactobacillus, some Eubacterium, Enterococcus and Propionibacteriurn spp. was more often observed using the MSMM approach. Measurement of the concentration of microbial markers in blood and reconstruction of the microecological status of the host by the MSMM algorithm has shown that this data quantitatively reflects the total changes in small intestinal and faecal microbiota. In conclusion, it is possible to non- invasively monitor the intestinal colonisation, based on the measurements of microbial markers in blood and feces.

  1. Using microbial consortia, modelling real gut microbiota

The mechanism of probiotic activity still remains unknown. However, we have identified some new clues from MSMM investigation. Firsdy, it confirms that a probiotic strain does not only stimulate the same genera or species to which it belongs. Secondly, the microbial strain does not add to the host microbiota in order to supplement the deficiency. The number of administered cells (108-1010) is too few, compared with the intestinal content (about 1014 microbes). Thirdly, adding probiotics stimulates restoration of the host microbial community, but it appears to be non-specific and depends on the patient’s condition. The fourth observation is that faeces is not the appropriate medium in which to measure probiotic efficiency.

In fact, many probiotics fail to colonise the intestine even transiently, as the GIT has many defences that inhibit colonisation. Consequently, the probiotic essence is not the microbial biomass itself. Growth factors, crosstalking molecules and other unknown factors promote the intestinal microbes. Investigators must apply their efforts towards the discovery of these factors. In the meantime it is necessary to develop advanced probiotics using new- theoretical and technological possibilities by microbial consortia, modelling real gut microbiota.

One can formulate probiotics of different types according to dysbiosis specificity. Binary, triple or multiple composition including Eubacterium spp. and actinobacteria may be developed as well as consortia modelling intestinal microbiota. One consortium-type product is a hypothetic complex probiotic, simulating a preferable real ratio and set of intestinal microbiota components. Ultimately one could preserve the faeces of a young man in good health (creating a bank of personal microbiota) with the purpose of later doping or spiking it in the difficult periods of his life.

Another variant of a spontaneously formed a consortium of microorganisms could be on the basis of natural inoculums (faeces or intestinal mucin) and supported by constant chemostat culture. Active sludge in waste water treatment is analogous to this technology. Such a probiotic should be effective, as it contains native strains of intestinal bacteria in the balanced composition, has natural trophic, energy, genetic and other communications appropriate to a biofilm of certain functional assignment. It has the chance to be included in the mucin in addition to the host’s own microbial aggregations and intestinal fermentation process as an artificial biofilm.

A third variant is a probiotic granular biofilm. Every granule is a completed, functionally independent community, which carries out the full exchange with the nutritious chyme. This technology is used in anaerobic processing, which can be industrial, or municipal for both agricultural waste and biogas production. At the heart of this technology are the methanogenic microorganisms including methane- producing archaebacteria, cellulolytic, peptolytic, or both bacteria and fungi.

We discover such spontaneously formed consortia of microorganisms in some liquid forms of probiotics fermented in milk media. As it happened, the real composition of liquid symbiotic (K)1» cells/g both lacto and bifido strains in ferment media as declared in product annotation) measured by mass spectrometry microbial markers approaches the consistency of a mixture of gut microbes. Real MSMM count Lactobacillus 8.7xl09 and Bifidobacterium 1.9xlO10 are close to that in annotation (Figure 10). Yet, environmental isolates invaded the process in spite of protective measures.

How can the broken balance of a microbial biofilm be restored? Antibiotics are recommended for bacterial overgrowth. Very effective is the use of co-trimoxazole or metronidazole when the number of Clostridium, Eubacterium or any other anaerobe exceeds the normal value, or amicacin when actinobacteria vegetate intensively. Concentrated IO10 1/ml liquid probiotics bifido, lacto or combined are useful for growth stimulation. Correction of die communication between microbiota and epithelium could be achieved with medications such as immune modulators, bismuth preparations, metronidazole, flavonoids and vitamins. Metronidazole stimulates the growth of beneficial microbes m the intestines, as found by’ our treatment of seborrhoea patients. Efficient treatment and correction of microbiota can be carried out by means of stage-by-stage control using MSMM technology.

Physicians must bear in mind that this new knowledge on advanced microecology of the patient and infection demands reconsideration of tactics of antibiotic treatment and other medical actions. Doctors must keep the microecology of his patients in balance. Diseases of this century such as FMF, AIDS, SARS, cystic fibrosis, and septic condition, are all associated witli severe disturbances in the patients microecology. People do not die of viral infections or genetic disturbances, but from multiple organ failure caused by their own microbes.

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Received: 29 March 2010 / Accepted: 11 October 2010

© 2011 Wageningen Academic Publishers. Beneficial Microbes. March 2011; 2(1): 63-78

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