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• • G—PHYSICS
  • G01—MEASURING; TESTING
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  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/06—Gastro-intestinal diseases

Definitions

• the present invention relates to the field of medical diagnostics, pharmacy, biotechnology.
• Optimal breastfeeding actually remains a highly effective public health strategy for infant survival, especially to reduce mortality from gastroenteritis and pneumonia in developing countries(Bhutta et al., 2013).
• Breastfeeding studies showed the protective role of breast milk against many chronic and immune conditions, in particular, type 1 diabetes, necrotizing enterocolitis, asthma and leukemia.(Bartick and Reinhold, 2010).
• the development of the microbiota in the newbom's digestive system is a gradual and dynamic process, which is determined by several factors, such as mode of birth, prematurity, diet, related diseases and antibiotic therapy, as well as the impact of the environment, which requires personalized approach (Wall et al. 2009).
• the colonization process is strongly influenced by diet (breast milk and / or formula.
• diet milk and / or formula.
• bifidobacteria and coliforms are predominant, followed by Lactobacillus spp. and Bacteroides and varies widely between individuals. (Wall et al., 2009).
• oligosaccharides are the most common and include significantly more than 100 different bioactive carbohydrates, composed of 3 to 32 monosaccharides (Newburg et al., 2005). As a class, oligosaccharides are 6-12 g / L from breast milk and are largely synthesized from lactose. (Bode, 2012). Human colostrum contains approximately 22-24 g / L of milk oligosaccharides.
• oligosaccharides are the third most important component after lactose and lipids and their amount is much higher than the amount of total protein. (Newburg et al., 1995).
• the structure of more than 100 human oligosaccharides has been characterized up to date (Urashima et al., 2011a,b; Kobata, 2010). Characterization of the physiology of bifidobacteria is largely focused on their ability to metabolize various dietary oligosaccharides.
• This genus of bacteria has a set of transmembrane permeases that ensure the metabolism of various carbohydrate polymers, such as dietary fiber, including human milk oligosaccharides, which pass undigested to the distal parts of the digestive tract. (Moro et al., 2006; Macfarlane and Cummings, 1999). Only a few species of bifidobacteria are capable of metabolizing and using human milk oligosaccharides as a carbon source.
• Such species are typically isolated mainly from the microflora of the gastrointestinal tract of newborns.lt has been found that bifidobacteria associated with the newborn are able to assimilate mainly lacto-N-tetraoses (LNT; Gal ⁇ 1-3GlcNAc ⁇ 1-3 Gal ⁇ 1-4Glc) or lacto-N-neotetraoses (LNnT; Gal ⁇ 1- 4GlcNAc ⁇ 1-3 Gal ⁇ 1-4Glc (LoCascio et al., 2007).
• LNT lacto-N-tetraoses
• LNnT lacto-N-neotetraoses
• Gal ⁇ 1- 4GlcNAc ⁇ 1-3 Gal ⁇ 1-4Glc lacto-N-neotetraoses
• the spices B. bifidum secretes extracellular 1,2- ⁇ -1-fucosidase (EC 3.2.1.63), which cleaves terminal 1,2- ⁇ -fucosyl bonds that protect galactose residues in lacto-N-biosis, thus allowing continuation of catabolic processes until their full assimilation (Katayama et al., 2004; Nagae et al., 2007).
• lacto-N-biosidase (EC3.2.1.140) releases lacto-N-biosis from lacto- N-tetraose and other milk oligosaccharides that lack fucosylation or sialylation.(Wada et al., 2008).
• the lacto-N-biose is transported across the cell membrane by a special ABC transporter that integrates the lacto-N-biose binding subunit.
• a special ABC transporter that integrates the lacto-N-biose binding subunit.
• B. longurn uses endo-ot-N -acetylgalactosaminidase (EC 3.2.1.97) to extract galacto-N-biosis from O-linked mucin glycans(Fujita et al., 2005).
• endo-N-acetylgalactosaminidase and fucosidase activity allows B.
• Metabolites are a consequence of the physiological state of the human body from one side, and from the other of the physiological activity of the accompanying microflora in the intestinal tract. This makes them an ideal way to track changes caused by illness or treatment.Assessing the amount and diversity of metabolites is a particularly important point in understanding the many interactions between genetic factors, the environment, and the microbiota. At key points in metabolic processes they inter sect, where the low molecular weight metabolites mediate these relationships.
• biomarkers for various diseases. Although this may be a favorable area for biomarkers, there are many examples of different metabolites being associated with the same disease, which reduces their reliability as biomarkers.
• pH values of faecal samples Another indicator whose potential is being investigated for a biomarker is the pH values of faecal samples. There is evidence that they directly correlate with the bacterial species that colonize the baby's intestines. For example, a direct relationship has been found between lower pH values of faecal samples and significantly reduced amounts of potentially pathogenic bacterial populations (ie Clostridiaceae, Enterobacteriaceae, Peptosteptoccocaceae and Veillonellaceae). Another example is the found correlation between the abundance of specific species of Enterobacteriaceae, which cause inflammation of the intestines and the presence of colic in infants.
• Asthma is the most common chronic disease of childhood. Canadian scientists have found a correlation between changes in the intestinal microbiome in infants up to one year (dysbiosis), which affect the development of asthma. The effects of microbial intestinal dysbiosis on atopic wheezing in a population living in developing countries with a low economic standard have been studied. Microbial dysbiosis at 3 months of age is associated with the later development of atopic wheezing. For example, dysbiosis has been observed in Ecuadorian infants involving various bacterial taxa, including several microscopic fungal taxa. Fecal short-chain fatty acid levels in three-month-old infants with atopic wheezing show clear trends of increased acetate concentration and decreased caproic acid concentration.
• the objective of the invention is to provide a method based on in vitro quantitative assessment, which provides the ability to quickly predict dysbiosis in infants up to 1 year age, which can be used to create a model (algorithm) for accurate diagnosis of intestinal microbiota in newborns from one month to one year age and the method helps to quickly establish the type of dysbiosis and the degree of disorder.
• the method is based on an interdisciplinary approach in analyzing the quantitative and qualitative composition of the microbiota, the type and quantity of key enzymes and metabolites secreted by the microbiome and data analysis through an information system.
• milk proteins such as caseins and lactoferrin
• milk proteins have been found to have the ability to inhibit the attachment of cariogenic streptococci to hydroxyapatite and to promote the attachment of commensal bacteria in vitro (Johansson and Lif Holgerson, 2011).
• oral colonization by bacteria that cause dental caries can occur after emergence of the first milk teeth in the newborn, as later, however, it has been shown that the cariogenic Streptococcus mutans may be present in the oral cavity of the baby before the appearance of hard tissues there (Law et al., 2007).
• the intestinal microbiome is characterized by: (1) no disturbances in the composition and amounts of taxa when changing diet or taking antibiotics; (2) high susceptibility to invasion by external taxa; and (3) poor use of the available resources (including breast milk oligosaccharides).
• the healthy/balanced state of the microbiota is characterized as a highly functioning ecosystem when the intestinal microbiome is: (1) stable over time in the composition and amount of taxa; (2) resistant to invasion by allochthonous bacteria; and (3) demonstrate high conversion of breast milk oligosaccharides into end products and biomass beneficial to infants.
• This model is agnostic by method and index of choice and provides a quantitative indicator and objective approach to assessing the microbiome, but it is limited mainly to data on the composition and quantity of microorganisms, and is not based on correlations between composition and quantity of microorganisms, enzyme activity responsible for the absorption of oligosaccharides from breast milk and the amount of metabolites secreted by the microbiota.
• the present patent application refers to the development of a specific method for in vitro quantification of fast prediction of dysbiosis in infants up to 1 year age at which it achieves the ability to control the human microbiome, as in the method, according to the description takes into account on the one hand common indicators such as age and gender, and on the other hand the specific indicators of human health.
• the proposed method is based on the processing of data from analyzes of the diversity and ratio of microorganisms in the intestinal tract and other body fluids (saliva, breast milk, feces), amount of metabolites used as biomarkers and reporting the level of enzyme activity of enzymes used as biomarkers, allowing the individual characteristics of people to be taken into account.
• the method covered by this patent application is based on an integrated approach for in vitro analysis of the intestinal microbiota balance in children from one to twelve months age and a prognosis for its recovery in established dysbiosis by calculating correlation coefficients between key indicators divided into three main groups:
• the in vitro quantitative assessment method for the rapid prediction of dysbiosis in infants up to one year of age and for multifactorial diseases requires the following course of analysis: 1. Protocol for quantitative analysis of the human microbiota by real-time qPCR. The presented protocol can be used to identify abnormalities for dysbiosis analysis in newborns from one to twelve months and to be included in an algorithm for identification of condition, abnormalities and rules for restoring the balance of the microbiota. It will be possible to quantify different groups of microorganisms from the beneficial and pathogenic microflora which to be used for calculation of the ratios between individual species (Table 1).
• Essential for determining the degree of dysbiosis represent ratios bifidobacteria/bacteroids; lactobacilli/bacteroids; (bifidobacteria + lactobacilli)/bacteroids; (bifidobacteria + lactobacilli/yeast; (bifidobacteria + lactobacilli)/E. coli.
• Another important indicator is the ratio between bifidobacteria and the main enzymes responsible for the utization of the main components of breast milk (lactose, lipids, proteins, oligosaccharides).
• the enzyme ⁇ -fucosidase provides direct information on the adhesion potential of the beneficial microflora (bifidobacteria and lactobacilli) on the epithelial cells of the intestinal mucosa, while the other enzymes provide information on the rate of reproduction of beneficial microorganisms and their place in this specific ecological niche.
• the data on enzyme activity correlate with the data on the amount of metabolites found in the respective samples.
• the range of ratios of the values of the enzyme activities is described in Table 6.
• the ratios between the enzymatic activities of the enzymes of the second point (the second stage of the analysis) and the amount of short-chain fatty acids can be calculated.
• the range of ratios of the values of the enzyme activities is described in Table 6.
• Example 1 In vitro study of 12 indicators by assessing the balance of the microbiota in infants aged from one to twelve months:
• the presented protocol can be used to identify deviations for analysis of dysbiosis in newborns aged from one to twelve months and to be included in an algorithm for identifying the condition, deviations and rules for restoring the balance of the microbiota.
• the primer pairs and fluorescently labeled probes for real-time qPCR detection of bacterial species and communities in samples from different target groups are presented in Table 2.
• Bacterial strains used to control the specificity of the primers and labeled probes, as well as for quantitative assessment of individual bacterial species and communities are presented in Table 3.
• the strains were purchased from the Bulgarian National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ).
• the strains were grown aerobically or anaerobically in selective growth media as recommended by DSMZ and NBIMCC.
• For each culture are made a series of dilutions, which were plated onto appropriate agar medium to determine the total number of bacteria - colony-forming units (CFU). Additionally, 1.0 ml samples were taken from the dilutions and centrifuged under the following conditions: 12000rpm / 3min.
• the bacterial cell pellets were stored at -80 ° C until use for analysis.
• Bacterial genomic DNA from control strains and isolates from target groups was extracted using the Blood and Tissue kit DNeasy (Qiagen, Hilden, Germany).
• Microbial DNA from faecal samples and breast milk was isolated using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). Initially, 200 mg of faecal sample / breast milk was diluted in 1.8 ml of sterile PBS buffer. To diluted faecal sample (200 ⁇ l), 20 ⁇ l of lysozyme (150 mg/ml) and 130 ⁇ l of lysis buffer (2.0 mM Na 2 EDTA; 20 mM Tris-HCl; 1.2% Triton X-100) were added. The suspension was incubated at 37°C for 30 minutes, and DNA was extracted according to the manufacturer's instructions.
• the amount and purity of bacterial genomic DNA isolated from the standard strains and from the tested samples are determined spectrophotometrically.
• the absorption of suitably diluted DNA samples at wavelengths of 260 nm and 280 nm is determined.
• the measurements are performed on a Shimadzu UV-2600 spectrophotometer at a light path of 8.5 mm, after resetting the apparatus against the cuvette with elution buffer.
• the homogeneity of DNA was determined by by electrophoresis in 1% (w/v) agarose gel and ethidium bromide staining.
• the samples with DNA were stored at -20 °C.
• the real-time qPCR reactions for detection and quantification of bacteria were performed using SYBR Green PCR Master Mix (Applied Biosystems). The reactions were carried out in 96-well plates with a final volume of 25 ⁇ L.
• the Real-time qPCR was performed using a Real-Time PCR CFX96 Touch (BIO-RAD) with software version 3.0.1224 under conditions for each of the target bacterial groups listed in Table 4.
• TaqMan Universal PCR Master Mix (Applied Biosystems) was used in real-time qPCR reactions to detect and quantify bacteria with fluorescently labeled probes.
• the reactions were carried out in 96-well plates with a final volume of 25 ⁇ L, under conditions for each of the target bacterial groups listed in Table 5.
• agarose gel electrophoresis of the reaction products was performed. Separation of the DNA fragments was performed in a 2% agarose gel at 35V and 25 mA current. The separated fragments were visualized fluorescently - by treating the gel with ethidium bromide and illuminating with UV light. The approximate size of the DNA fragments is determined by comparison with a standard containing small DNA fragments of known size - SmartLadder SF (Eurogentec, Belgium).
• the enzymatic reaction was performed under the following conditions: 100 mM sodium acetate buffer, pH 6.0, containing 2 mM o-nitrophenyl-ß-D-galactopyranoside (ONP-Gal) solution and suitably diluted cells or supernatant after cell disintegration at 37 °C.
• the reaction was stopped with 1 M sodium carbonate.
• One unit of enzyme activity catalyzes the production of 1.0 ⁇ mol o-nitrophenol per minute at pH 6.0 and 37 °C.
• the o-nitrophenol content was determined spectrophotometrically at 410 nm wavelength on a Beckman Coulter DU 800 spectrophotometer.
• the enzyme reaction was performed under the following conditions: 400 mM citrate buffer, pH 4.0, containing 9.9 mM solution of p-nitrophenyl- ⁇ -D-galactopyranoside (PNP-Gal) (prepared in distilled water by dissolving p-nitrophenyl- ⁇ -D-galactopyranoside, Sigma cat. No N-0877) and suitably diluted cells or supernatant after cell disintegration at 37 °C.
• PNP-Gal p-nitrophenyl- ⁇ -D-galactopyranoside
• the reaction was stopped with 200 mM borate buffer, pH 9.8.
• One unit of activity converts 1.0 ⁇ mole of p-nitrophenyl- ⁇ -D-galactopyranoside to o-nitrophenol and D-galactose in one minute at pH 4.0 and 37 °C.
• the p-nitrophenol content was determined spectrophotometrically at 405 nm wavelength on a Beckman Coulter DU 800 spectrophotometer.
• the enzyme reaction was performed under the following conditions: 100 mM citrate buffer, pH 6.5, containing 10 mM solution of p-nitrophenyl- ⁇ -L-fucopyranoside (PNP-FUC) (prepared by diluting p-nitrophenyl ⁇ -L-fucopyranoside, Sigma cat. No N3628) with distilled water and suitably diluted cells at 37 °C.
• PNP-FUC p-nitrophenyl- ⁇ -L-fucopyranoside
• the reaction was stopped with 200 mM borate buffer, pH 9.8.
• One unit of activity converts 1.0 ⁇ mole of p-nitrophenyl- ⁇ -L-fucopyranoside to p-nitrophenol and L-fucopyranoside in one minute at pH 6.5 and 37 °C.
• the p-nitrophenol content was determined spectrophotometrically at 405 nm wavelength on a Beckman Coulter DU 800 spectrophotometer.
• the enzymatic reaction was performed under the following conditions: 100 mM acetate buffer, pH 6.0, containing 1.25 mM p-nitrophenyl- ⁇ -D-glucopyranoside solution (prepared by diluting p-nitrophenyl- ⁇ -D-glucopyranoside, Sigma cat. No N3628 with distilled water and suitably diluted cells at 37 °C.
• the reaction was stopped with 0.5 M sodium carbonate.
• One unit of activity converted 1.0 ⁇ mole of p-nitrophenyl ⁇ -D-glucopyranoside to p-nitrophenol and D-glucopyranoside per minute at pH 6.0 and 37 °C.
• the p-nitrophenol content was determined spectrophotometrically at 405 nm wavelength on a Beckman Coulter DU800 spectrophotometer.
• the enzyme reaction was performed under conditions: 0.1 M acetate buffer, pH 5.0, containing 20 mM solution of p-nitrophenyl-ß-D-glucopyranoside (ONP-Glu) and suitably diluted cells at 37 °C.
• the reaction was stopped with 0.2 M sodium carbonate.
• One unit of activity converts 1.0 ⁇ mole of p-nitrophenyl- ⁇ -D-glucopyranoside to p-nitrophenol and D- glucopyranoside in one minute at pH 5.0 and 37 °C.
• the p-nitrophenol content was determined spectrophotometrically at 405 nm wavelength on a Beckman Coulter DU 800 spectrophotometer.
• protease activity The protease activity of the cells and the supernatant was examined in the presence of casein (Fluka) as a substrate.
• One unit (U) of protease activity is defined as the amount of enzyme that hydrolyzes casein to 1 ⁇ mol tyrosine in 1 minute at pH 7.5 and 37 °C.
• the enzyme reaction was performed under the following conditions: in 50 mM phosphate buffer, pH 7.5, dissolved 0.65% (w/v) casein and 1.0 ml of appropriately diluted sample (cells or supernatant). The reaction proceeds for 10 minutes at 37 °C in a water bath. The reaction was stopped by the addition of 110 mM trichloroacetic acid (TCA) (Merck) at 37 °C. The samples were incubated with TCA for 30 minutes at the same temperature. To remove undigested casein, the samples were centrifuged at 9000 rpm/15 min, 4 °C.
• TCA trichloroacetic acid
• the supernatant was analyzed for the amount of tyrosine released from casein degradation in the presence of 500 mM Na 2 CO 3 and 0.5 M Folin reagent solution (Merck) at 37 °C for 30 minutes.
• the absorbance at ⁇ 660 nm (A 660 nm ) was measured on a Beckman Coulter DU 800 spectrophotometer against a control containing distilled water instead of a sample. From the measured values at A 660 nm , the concentration of tyrosine ( ⁇ mol) is determined by standard curve.
• L-tyrosine Sigma- Aldrich is used as a standard to construct the standard curve.
• the enzymatic reaction was performed under the following conditions: 0.1 M acetate buffer, pH 7.6, containing 0.8 mM p-nitrophenyl palmitate solution and suitably diluted cells at 37 °C.
• the reaction was stopped with 0.2 M lead acetate (II).
• One unit of activity converts 1.0 ⁇ mole from p-nitrophenyl palmitate to p-nitrophenol and palmitate in one minute at pH 7.6 and 37 °C.
• the p-nitrophenol content was determined spectrophotometrically at 405 nm wavelength on a Beckman Coulter DU 800 spectrophotometer.
• Table 6 Range of values of the measured indicators in healthy children aged from one to twelve months.
• Example 2 In vitro study of 6 indicators by assessing the balance of the microbiota in infants aged one to twelve months:
• a protocol for the quantitative analysis of the human microbiota by real-time qPCR The presented protocol can be used to identify deviations for analysis of dysbiosis in newborns aged from one to twelve months and to be included in an algorithm for identifying the condition, abnormalities deviations and rules for restoring the balance of the microbiota.
• the primer pairs and fluorescently labeled probes for real-time qPCR detection of bacterial species and communities in samples from different target groups are presented in Table 8.
• Bacterial strains used to control the specificity of the primers and labeled probes, as well as for quantitative assessment of individual bacterial species and communities are presented in Table 9.
• the strains were purchased from the Bulgarian National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ).
• the strains were grown aerobically or anaerobically in selective growth media as recommended by DSMZ and NBIMCC. For each culture are made a series of dilutions, which were plated onto appropriate agar medium to determine the total number of bacteria - colony-forming units (CFU).
• CFU colony-forming units
• Bacterial genomic DNA from control strains and isolates from target groups was extracted using the Blood and Tissue kit DNeasy (Qiagen, Hilden, Germany).
• Microbial DNA from faecal samples and breast milk was isolated using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). Initially, 200 mg of faecal sample / breast milk was diluted in 1.8 ml of sterile PBS buffer. To diluted faecal sample (200 ⁇ l), 20 ⁇ l of lysozyme (150 mg/ml) and 130 ⁇ l of lysis buffer (2.0 mM Na 2 EDTA; 20 mM Tris-HCl; 1.2% Triton X-100) were added. The suspension was incubated at 37°C for 30 minutes, and DNA was extracted according to the manufacturer's instructions.
• the amount and purity of bacterial genomic DNA isolated from the standard strains and from the tested samples are determined spectrophotometrically.
• the absorption of suitably diluted DNA samples at wavelengths of 260 nm and 280 nm is determined.
• the measurements are performed on a Shimadzu UV-2600 spectrophotometer at a light path of 8.5 mm, after resetting the apparatus against the cuvette with elution buffer.
• the homogeneity of DNA was determined by by electrophoresis in 1% (w/v) agarose gel and ethidium bromide staining.
• the samples with DNA were stored at -20 °C.
• Real-time qPCR The real-time qPCR reactions for detection and quantification of bacteria were performed using SYBR Green PCR Master Mix (Applied Biosystems). The reactions were carried out in 96-well plates with a final volume of 25 ⁇ L. The Real-time qPCR was performed using a Real-Time PCR CFX96 Touch (BIO-RAD) with software version 3.0.1224 under conditions for each of the target bacterial groups listed in Table 10.
• TaqMan Universal PCR Master Mix (Applied Biosystems) was used in real-time qPCR reactions to detect and quantify bacteria with fluorescently labeled probes.
• the reactions were carried out in 96-well plates with a final volume of 25 ⁇ L, under conditions for each of the target bacterial groups listed in Table 11.
• Table 11. Primers, fluorescently labeled probes and reaction conditions for real-time qPCR quantification of bacteria in fecal samples.
• agarose gel electrophoresis of the reaction products was performed. Separation of the DNA fragments was performed in a 2% agarose gel at 35V and 25 mA current. The separated fragments were visualized fluorescently - by treating the gel with ethidium bromide and illuminating with UV light. The approximate size of the DNA fragments is determined by comparison with a standard containing small DNA fragments of known size - SmartLadder SF (Eurogentec, Belgium).
• the enzymatic reaction was performed under the following conditions: 100 mM sodium acetate buffer, pH 6.0, containing 2 mM o-nitrophenyl-ß-D-galactopyranoside (ONP-Gal) solution and suitably diluted cells or supernatant after cell disintegration at 37 °C.
• the reaction was stopped with 1 M sodium carbonate.
• One unit of enzyme activity catalyzes the production of 1.0 ⁇ mol o-nitrophenol per minute at pH 6.0 and 37 °C.
• the o-nitrophenol content was determined spectrophotometrically at 410 nm wavelength on a Beckman Coulter DU 800 spectrophotometer.
• ⁇ -L-fucosidase activity (EC 3.2.1.51) The enzyme reaction was performed under the following conditions: 100 mM citrate buffer, pH 6.5, containing 10 mM solution of p-nitrophenyl- ⁇ -L-fucopyranoside (PNP-FUC) (prepared by diluting p-nitrophenyl- ⁇ -L-fucopyranoside, Sigma cat. No N3628) with distilled water and suitably diluted cells at 37 °C. The reaction was stopped with 200 mM borate buffer, pH 9.8.
• PNP-FUC p-nitrophenyl- ⁇ -L-fucopyranoside
• One unit of activity converts 1.0 ⁇ mol of p-nitrophenyl- ⁇ -L-fucopyranoside to p-nitrophenol and L-fucopyranoside in one minute at pH 6.5 and 37 °C.
• the p-nitrophenol content was determined spectrophotometrically at 405 nm wavelength on a Beckman Coulter DU 800 spectrophotometer.
• Table 12 Range of values of the measured indicators in healthy children aged from one to twelve months.

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