Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria

Definitions

• the invention relates to a method for selecting strains of Gram-positive lactic acid bacteria for combating infections of a pathogenic nature by strains of Gram-negative or Gram-positive bacteria, such as strains of enteropathogenic bacteria.
• the pathogenicity of strains of the Escherichia coli species is due to tissue adhesion to the wall of the duodenum.
• the adhesion is often stereospecific and can only take place if the tissue carries a very specific type of receptor.
• the interaction between a bacterial lectin and a tissue sugar is a typical example of a stereospecific interaction.
• These lectin molecules are frequently carried by filamentous appendages called fimbriae.
• Fimbriae are very fine protein filaments found mainly, and very commonly, in Gram negative bacteria. They can be distributed over the entire surface of the cell or be more localized. A fimbriae consists of repeated linear protein subunits. These subunits are often rich in non-polar amino acids, so that cells carrying fimbriae tend to have more hydrophobic surfaces than those of cells that lack them. In Gram-negative bacteria, many types of fimbriae ensure the adhesion of cells to each other or to cells on any surface. Each type of fimbriae has its lectin and only adheres to a specific receptor composed of glycoprotein and / or glycolipid epitopes.
• enteropathogenic bacteria do not express their attachment appendages, such as fimbriae, in the environment but only once ingested, the synthesis of these appendages being favored by the physicochemical conditions of the digestive tract.
• Escherichia coli K88 easily colonizes the duodenum, which does not contain a protective flora as complex as the other distal regions of the digestive tract. It is through chemotaxis that the pathogen approaches the lining of the duodenum and attaches to the first receptors present in the mucus. The pathogen then accesses, by its passage through the mucous gel, the epithelium of the underlying duodenal villi to which it attaches with avidity. It is the fixation of the pathogen which induces a cascade of intracellular mechanisms leading to the expression of virulence.
• infections with bacterial strains of serotype K88 are frequent in animals in pre-weaning and up to two weeks in post-weaning.
• infections are generally caused by bacterial strains of serotype 0138 , K8 or K85.
• bacterial infections are combated by the administration of antibiotics.
• Antibiotics are only effective at increasingly higher doses, which has the disadvantage of increasing the negative side effects resulting from too heavy antibiotic therapy (Bartlett JG, Clinical of Infectious Diseases, 1992, 15 (4), 573-581). This phenomenon is however inevitable insofar as pathogenic bacteria on repeated contact with antibiotics tend to develop antimicrobial resistance which is responsible for the ineffectiveness of the treatments.
• Other alternatives to antibiotic treatment have been proposed:
• a first solution consists in using natural prebiotic compounds added to the food which are capable of selectively amplifying the growth of certain harmless bacterial species of the intestinal flora so that it ensures an optimal protective function in a minimum of time;
• a second solution is based on the in vitro production of faecal flora or cultures of bacterial strains in order to administer to animals, via food or drinking water, harmless exogenous bacteria which would reinforce the constituent elements of the flora against unwanted pathogens.
• a third solution consists in using a much more limited number of bacterial strains isolated from a flora. The stages of selection of the most interesting bacteria as well as the knowledge of their fate in the digestive tract have often been overlooked.
• the invention aims to improve the fight against bacterial strains by providing a method for selecting strains of probiotic bacteria which are particularly effective under physiological conditions.
• the invention relates to a method for selecting strains of non-pathogenic Gram-positive lactic acid bacteria capable of fighting against infections by pathogenic bacteria for which the pathogenicity is mediated by tissue adhesion, consisting in choosing a strain exhibiting the set of the following properties: a - the ability to adhere to the tissues of an organ susceptible to infection; b - the ability of bacteria of this same strain to bind to each other via mutual adhesion sites; and c - the capacity to bind to the determinants of attachment of the pathogenic bacterial strain responsible for the infection.
• the strains of Gram-positive lactic acid bacteria are selected from the bacteria naturally present in the infected organ of the host.
• pathogenic enterobacteria whose pathogenicity results from tissue adhesion are Salmonella.
• fimbriae are essential to initiate colonization, in particular in the cecum of poultry, a preliminary step to infection.
• Helicobacter pylori which is responsible for chronic gastric inflammatory diseases and gastric and duodenal ulcerations.
• the adhesion of Helicobacter on gastric cells by means of adhesins on the surface of the pathogen attaching to a receptor on the gastric mucosa is essential to trigger the process of ulceration.
• Campylobacter jejuni it is the liposaccharides which play the function of adhesin allowing attachment to epithelial cells and mucus causing a severe diarrheal condition in humans.
• the method of the invention is more particularly advantageous for selecting bacteria capable of fighting against pathogenic bacteria expressing fimbriae with a view to stereospecific tissue adhesion.
• the triple adhesion characteristic which characterizes the Gram positive lactic acid bacteria selected according to the process of the invention ensures an improved antimicrobial activity.
• This activity can be specific or non-specific.
• the lactic acid bacteria selected also have the particularity of being able to bind selectively to the determinants of attachment of the pathogenic bacterial strain expressing the fimbriae.
• Selectivity can be demonstrated by demonstrating the absence of adhesion of the strain of lactic acid bacteria to bacteria not expressing fimbriae and resulting from the mutation of the pathogenic bacterial strain expressing fimbriae.
• the properties a), b) and c) of lactic acid bacteria can be demonstrated independently.
• the properties b) and c) are demonstrated simultaneously in an appropriate buffered physiological medium.
• the physiological medium which can be used for this must reflect the physiological conditions of the medium in which the lactic acid bacterium must ultimately exercise its activity.
• the composition of the physiological medium can be determined by a person skilled in the art in the light of his knowledge of the state of the art as a function of the portion of organ infected.
• the buffered physiological medium will contain essentially different electrolytes influencing the pH, bile secretions, pancreatic secretions, gastric secretions and extracts of mucus.
• the infected organ is a portion of the digestive tract.
• the physiological medium contains in particular an extract of the duodenal mucus and the enzymes of the digestive secretions and more particularly of bile salts, gastric secretions and pancreatic digestive secretions.
• chlorine ions will advantageously be introduced into the physiological medium concerned in the form of a salt and / or hydrochloric acid, the chloride ions being naturally secreted and present in the food bowl of the pyloric region.
• chloride ions When the chloride ions are added in the form of hydrochloric acid, and depending on the quantity of hydrochloric acid introduced, it may be necessary to neutralize the acidity of the medium by adding a base so as to restore the pH to physiological pH value, which is about 6 at the level of the lining of the middle duodenum.
• the salts which can be used for the incorporation of chloride ions are preferably the alkali or alkaline earth metal salts, sodium chloride being preferred.
• mineral bases such as NaHC0 3 , Na 2 C0 3 , KHCO 3 and K2CO 3 , better still NaHCO 3 and KHCO 3 are used.
• the physiological buffer contains:
• properties b) and c) are evaluated simultaneously in a buffered physiological medium reflecting the physiological conditions of the infected organ.
• the buffered physiological medium includes an extract of duodenal mucus, the enzymes of the digestive secretions and more particularly bile salts, gastric secretions and pancreatic digestive secretions as well as the electrolytes naturally prescribed in the duodenum and influencing pH.
• the invention further relates to the use of strains of Gram-positive lactic acid bacteria selected according to the method of the invention for the manufacture of a therapeutic composition intended for preventing or treating pathological disorders associated with an infection of the organ of host by pathogenic bacterial strains.
• the strain of Gram-positive lactic acid bacteria is naturally present in the infected organ of the host.
• the pathogenic bacterial strain is an enterotoxinogenic or verotoxinogenic strain and, for example, a strain of the species Escherichia coli.
• the host is preferably an animal such as a poultry, a pig, a cattle, a dog, a cat, a horse, a sheep, a goat or a fish, better still a pig.
• the lactobacillus strains tested are strains freshly isolated from the duodenum of piglets aged 6 to 16 weeks or sows aged 2 years. Only one strain was isolated per animal. The animals have been selected in different European farms. The strains were all isolated from an initial culture on MRS agar at 37 ° C in micro-aerobiosis and were then identified according to a sugar fermentation study on an API gallery.
• the main bacterial genera finally tested are Lactobacillus and
• Leuconostoc. MRS agar (from Man. Rogosa and Sharpe) is prepared by dissolving 70 g of the following mixture A in 1 liter of water:
• the final pH of the agar is 6.5 + 0.2 at 25 ° C.
• Cultures of these lactobacilli are prepared simply by removing bacteria from the surface of the MRS agar and suspending in Krebs-Heuselait buffer for reach a value of 10 9 bacteria / ml.
• the strain of pathogenic bacteria whose eradication is desired belongs to the species Escherichia coli. This strain was isolated from the digestive tract of a very sick animal. The severe diarrheal collibacillosis that had spread to an entire farm was caused by this strain ETEC LT +, 0149; K91, K88ac. - »The screening of lactobacilli is carried out by implementing the following three operating protocols.
• enterocytes The preparation of enterocytes was carried out from the washed intestinal parts.
• the duodenal mucosa (microvilli) was carefully scraped on the surface with a clean glass microscope slide and then resuspended in 2 ml of buffer.
• the enterocyte cells were dissociated from each other and the traces of glycerol as well as the damaged or ruptured cells were eliminated by a succession of washes and centrifugations at 3000 revolutions / minute. Between each washing step, a sample was observed in Gram staining until the cells appeared isolated and in perfect condition.
• the well prepared enterocyte solution can be stored for 4 to 5 days at 4 ° C without risk of contamination.
• the purple lactic bacteria are anarchically dispersed around the enterocytes (dews).
• the bacteria are partially or totally attached to the enterocytes.
• Tris-EDTA buffer which has a pH of 7.6 being as follows:
• the pepsin and the aqueous NaCl solution are brought into contact, and the pH is adjusted to 2 by addition of a 10M aqueous HCl solution. To this mixture are added in two stages the rest of the enzymes (mucus, pancreatin and bile extract). Intermediate, the pH of an intermediate solution is brought to 5 using a carbonate buffer 4 (30 ml of Na 2 CO 3 : 0.2M; 20 ml of NaHCO 3 : 0.2M; and 200 ml of 'water) in order to limit the risks of enzymatic denaturation. The pH of the final solution is then adjusted to 6 by using the same buffer 4 described above.
• the volume of the solution is then adjusted to 1 liter with the NaCl solution at 5 g / l in a volumetric flask and the pH is again checked to confirm a pH value 6.
• the strains of Escherichia coli were cultivated for 18 hours at 37 ° C on an aerobic BHI agar.
• BHI Brain heart Infusion Agar
• This agar has a pH of 7.4 + 0.2 at 25 ° C.
• Mixture B
• the colonies of cherscherichia coli and lactic acid bacteria were removed with a sterile platinum loop on the surface of the agar and disaggregated in a volume of 2 ml of NaCl solution at 5 g / l in order to constitute a homogeneous suspension. These suspensions were then diluted in the same buffer in order to reach an approximate absorbance value of 1.5 + 0.1 measured at a wavelength of 600 nm. A volume of 1 ml of lactic acid bacteria suspension was mixed with 2 ml of buffer 3 described above. The same dilution was carried out with the suspension ⁇ scherichia coli. The absorbances are thus adjusted to a value close to 0.5 (10 8 bacteria / ml) for a wavelength of 600 nm.
• the 3 ml of suspension of lactic acid bacteria are then added to the 3 ml of suspension of pathogenic bacteria in a single sterile and hermetically sealed tube. After several inversion of the tube over a period of 10 seconds, a 1 ml sample was taken in order to measure the absorbance (TOh time). Another sample was then carried out after 4 hours of incubation at 37 ° C with gentle shaking at 30 revolutions per minute (on a vertical rotational shaking table) for a last absorbance measurement (time T4h). Under the same incubation conditions, the controls necessary to measure the phenomenon of adhesion between bacteria of the same strain were carried out in the same manner except that the 3 ml of suspension of lactic acid bacteria were diluted in 3 ml of buffer 3 described above.
• concentrations of the various components of the enzyme buffer during the aggregation tests are reduced (due to the addition of the bacterial suspensions) and thus adjusted to desired values which are: 1.3 g / l of pepsin, 0.6 g / l of gastric mucus, 0.6 g / l of pancreatin and 0.3% of bile extract.
• O.D. Le (T4h) absorbance of the lactobacillus suspension measured after 4 hours
• O.D. Le (TOh) absorbance of the lactobacillus suspension measured at T0.
• FIG. 1 reports the results obtained concerning the self-aggregation and co-aggregation properties in the case of 21 strains of lactobacteria adhering to the porcine duodenal mucosa.
• the intensity of the co-aggregation phenomenon may be greater than that of the self-aggregation phenomenon, even if it already reaches a satisfactory level.
• This strain is cultured on BHI agar at 37 ° C., the BHI agar being as defined above.
• the self-aggregation and co-aggregation properties are demonstrated from the 10 strains of lactobacillus previously selected which have in the previous test ( Figure 1) co-aggregation values greater than or equal to 10.
• the bacteria selected for the preparation of medicinal preparations are represented by 3 strains of Lactobacillus fermentum, 2 strains of Lactobacillus acidophilus, 1 strain of Lactobacillus helveticus and 1 strain of Leuconostoc lactis.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• General Health & Medical Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Biochemistry (AREA)
• Biotechnology (AREA)
• Analytical Chemistry (AREA)
• Physics & Mathematics (AREA)
• General Engineering & Computer Science (AREA)
• Biophysics (AREA)
• Urology & Nephrology (AREA)
• Medicinal Chemistry (AREA)
• Mycology (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Epidemiology (AREA)
• Veterinary Medicine (AREA)
• Tropical Medicine & Parasitology (AREA)
• Virology (AREA)
• Pharmacology & Pharmacy (AREA)
• Cell Biology (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Food Science & Technology (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Toxicology (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26213050

Family Applications (1)

Country Status (9)

Family Cites Families (8)

• 2002
  • 2002-06-14 EP EP02755074A patent/EP1395672A1/de not_active Withdrawn
  • 2002-06-14 CA CA002450444A patent/CA2450444A1/fr not_active Abandoned
  • 2002-06-14 PL PL02364429A patent/PL364429A1/xx not_active Application Discontinuation
  • 2002-06-14 SK SK1533-2003A patent/SK15332003A3/sk unknown
  • 2002-06-14 RU RU2004100545/13A patent/RU2004100545A/ru not_active Application Discontinuation
  • 2002-06-14 HU HU0400199A patent/HUP0400199A3/hu unknown
  • 2002-06-14 WO PCT/FR2002/002058 patent/WO2002103034A1/fr not_active Ceased
  • 2002-06-14 CZ CZ20033384A patent/CZ20033384A3/cs unknown
  • 2002-06-14 US US10/480,472 patent/US20040241784A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events