FR2917623A1 - Use of Lactobacillus bacteria/culture supernatant of the bacteria cultivated on medium containing lactose/substance, for preparing drug/food supplement to prevent/delay colonization of respiratory system of a patient by pathogenic bacteria - Google Patents

Abstract

Use of Lactobacillusbacteria under living form or culture supernatant of Lactobacillus bacteriacultivated on a medium containing lactose or a substance extracted from the supernatant culture, for the preparation of a drug or food supplement to prevent or delay the colonization of respiratory system of a patient by pathogenic bacteria, which colonize the respiratory by retrograde way after residing in the gastrointestinal tract, preferably in the stomach, is claimed. ACTIVITY : Antiinflammatory; Respiratory-Gen; Antidiarrheic; Uropathic; Gastrointestinal-Gen; Dermatological; Antimicrobial. MECHANISM OF ACTION : None given.

Description

2917623 Use of a probiotic for the prevention of infections of

respiratory tracts

  Infections acquired in hospitals are considered to be an important factor in the future of patients admitted to an Intensive Care Unit (ICU). In critically ill patients, colonization of the gastrointestinal tract, and in particular of the stomach, is considered a risk factor for pneumopathies acquired under mechanical ventilation (1, 11, 38). Indeed, the gastrointestinal tract is a reservoir for many nosocomial pathogens (34), and selective digestive decontamination (DDS) is generally used as a prophylaxis strategy for infections. The DDS includes the administration of antibiotics locally, non-absorbable, in the oropharynx and enteral, in combination or not with systemic antibiotic therapy (intravenously). This approach aims at eradicating the colonization of the oropharynx, the stomach and the intestine by potentially pathogenic aerobic microorganisms, while not disturbing or minimizing the endogenous anaerobic flora. However, the selective pressure exerted by the antibiotic DDS can cause significant side effects, such as the overgrowth of microorganisms of the endogenous microflora or the resistance of pathogenic microorganisms of exogenous origin to the antibiotics administered (8, 22, 23, 28). . In addition, anti-ulcer prophylaxis, widely used in ICU patients, increases the pH of the stomach and thereby facilitates bacterial overgrowth. As a result, the stomach is the main source of nosocomial pneumopathies retrograde (7, 12, 19).

  Probiotics are defined as living or viable microorganisms that produce a benefit to health and especially in the treatment and prevention of certain pathologies (17). This is usually bacteria or yeasts. Most bacterial probiotics belong to the genera Lactobacillus and Bifidobacterium; they must be non-pathogenic, non-toxic and able to survive in the gastrointestinal tract. The applications of probiotics have been mainly limited to the treatment of intestinal disorders such as diarrhea and inflammatory diseases (13, 16, 24, 27, 33, 36). Although the mechanism of action remains to be explored, the beneficial effect of probiotics clinically has also been demonstrated in the prevention of vaginitis, urinary tract infections and gastritis caused by Helicobacter pylori (21, 31 , 32, 41). The use of probiotics to treat atopic dermatitis, more particularly in children, has also been described, in particular in WO2007023226. Other results suggest that probiotics may also be an alternative strategy for selective digestive decontamination (DDS) in the future. A decrease in the rate of postoperative infections was observed in patients receiving orally L. plantarum in combination with enteral fiber nutrition (24). Similar effects were obtained in studies involving patients undergoing major abdominal surgery or with acute pancreatitis (13, 16). Lactobacillus rhamnosus strain Lcr35 has been shown to adhere to intestinal cells and to transiently colonize the intestinal tract (10, 14). In vitro tests have demonstrated the inhibitory effect of Lcr35 on the growth of Gram-positive shells and gram-positive and negative bacilli, including Pseudomonas aeruginosa (14). It has also been shown that the culture supernatant of Lactobacillus rhamnosus bacteria cultured on a lactose-containing culture medium, acts as a growth inhibitor of pathogenic bacteria in vitro (14). However no use of a bacterial probiotic has been described against respiratory pathologies such as nosocomial pneumopathies related to the colonization of the respiratory tract by pathogenic bacteria.

  The present invention relates to the use of (i) probiotic bacteria in living form, or (ii) culture supernatant of probiotic bacteria cultured on a medium comprising lactose, or a substance extracted from said culture supernatant, for the manufacture a drug or dietary supplement intended to prevent or delay the colonization of the respiratory system of a patient by pathogenic bacteria. More particularly, the drug or dietary supplement according to the invention aims to prevent or delay the colonization of the respiratory system of a patient by pathogenic bacteria that colonize the respiratory tract retrograde after having stayed in the gastrointestinal tract, and more especially in the stomach. In a preferred embodiment, the drug or dietary supplement according to the invention also prevents or delays the colonization of the gastrointestinal tract, including the stomach, by pathogenic bacteria.

  By drug or dietary supplement is meant any form acceptable pharmacologically, especially in the form of dietary food of oral supplementation, hygiene product, foodstuff or dietetic for special medical purposes (or special), or medical device, or any other suitable form well known to those skilled in the art. The drug or dietary supplement is preferably in a dosage form suitable for oral administration, for example in the form of oral suspension or capsule. It can also be in the form of powder or granule to add to food or drink, or to suck. Preventing or delaying the colonization of the respiratory tract by pathogenic bacteria includes in particular the increase in the duration of non-contamination (NAE) of a pathology of the respiratory tract caused by said pathogenic bacteria or the increase in the median duration of stay in a hospital or intubation before acquisition of this pathology, compared to the NAE or the median time before acquisition observed in the absence of treatment with the drug or food supplement according to the invention. The increase in the NAE or the median length of stay in hospital or intubation before acquisition is preferably at least 10 days, preferably at least 20 or 30 days. By gastrointestinal tract (or digestive tract or digestive system) is meant all the elements involved in the digestion, namely the mouth, the esophagus, the stomach, the small intestine, the colon and the rectum. Respiratory (or airway) means all the pathways involved in breathing, namely the nose, mouth, pharynx, larynx, trachea, lungs and bronchi. By retrograde colonization, it is meant that the pathogenic bacteria initially stay in the gastrointestinal tract, and more particularly in the stomach, before colonizing the airways, including the bronchi and lungs. The length of stay in the stomach before contamination of the respiratory tract varies in particular depending on the pathogen, the state of health of the patient and the treatments that are administered. This duration may for example be between 2 and 30 days, between 5 and 20 days or between 10 and 20 days. In one study, it was found that in more than 50% of cases, germs are isolated from the stomach up to 16 days before being isolated at the level of the trachea (19). To stay means the fact of being able to detect pathogenic bacteria by conventional techniques implemented by those skilled in the art on the date of the invention.

  The probiotic bacteria used for the implementation of the present invention are preferably bacteria of the genus Lactobacillus, for example bacteria of the species L. rhamnosus (also called Lactobacillus casei subsp. Rhamnosus), L. casei, L. reuteri , L. gasseri, L. johnsonii or L. acidophilus. Preferentially, these are bacteria of the species L. rhamnosus. Examples of strains of L. rhamnosus bacteria are strains Lcr35, GG (deposited with ATCC as ATCC number 53103) and GR-1.

  According to a preferred embodiment of the invention, the probiotic bacteria are bacteria of the strain Lcr35, derived from the species Lactobacillus rhamnosus, the latter being deposited with the ATCC under the number ATCC 7469. The particular strain Lactobacillus rhamnosus Lcr35 may in particular be obtained according to the protocol defined in Example 1 Preferably the probiotic is a bacterium capable of fermenting lactose.

  In choosing a probiotic to obtain a product according to the invention, it is essential to look for a bacterium which has no pathogenic effect and which is preferably known to have good properties as regards its survival rate in the digestive tract. Another parameter to take into account is its resistance to technological processes. Inasmuch as it has the various advantages mentioned above, the specific strain Lactobacillus rhamnosus Lcr35 is particularly preferred in the context of the present invention. Moreover, the fermentative characters of this strain are particularly advantageous.

  It may also be advantageous for the probiotic in question to have resistance to most conventional antibiotics. The Lcr35 strain in particular is resistant to streptomycin, tetracycline, erythromycin, nalidixic acid, pipemidic acid, fusidic acid, colistin, polymyxin, sulfamethoxazole and trimethoprim.

  Conversely, there are situations where the probiotic is preferably sensitive to the majority of known antibiotics. Advantageously, the drug or dietary supplement also comprises a prebiotic, that is to say a substance that can not be digested by the patient's body and that has beneficial effects on the person who consumes them by stimulating growth or growth. activity of certain non-pathogenic bacteria naturally present in the body, and more particularly in the gastrointestinal tract, or facilitating the survival of a probiotic administered simultaneously.

  Preferably the prebiotic is lactose and / or potato starch. The drug or food supplement used in the context of the present invention preferably comprises probiotic bacteria in living form. By living it is to be understood that the integrity of the cell is maintained and that cellular processes occur or can occur if the bacterium is cultured in a suitable medium and conditions. Any live bacteria can be re-seeded on a suitable culture medium and grow under suitable conditions. The present invention therefore includes the use of probiotic bacteria in freeze-dried or frozen form. The drug or food supplement according to the invention may be in any suitable form. Suitable forms are well known to those skilled in the art. In particular, they must ensure the survival of the probiotic. It is very important to consider three essential factors which are the atmosphere, the humidity and the temperature. The various means of preserving probiotics are well known to those skilled in the art. Preferably, the drug or food supplement is in lyophilized form. Indeed, this form ensures not only excellent preservation, but it also preserves any living bacteria that are part of the drug or food supplement according to the invention. It is important to determine the enumeration of the probiotic when obtaining a freeze-dried drug or food supplement according to the invention. The probiotic bacterium contained in the drug or food supplement may be the only lyophilized element, but preferably the bacterium is lyophilized with a culture medium, for example a medium based on lactose. The drug or food supplement may of course include additional components, in addition to the bacterium and the medium based for example on lactose. Such constituents are either added before lyophilization of the other components, or after (for example, excipients for the galenic shaping of the product). In a drug or dietary supplement according to the present invention, the number of bacteria or seeds per gram of product is preferably greater than 1 x 108, for example between 1 x 108 and 1 x 1010, more particularly between 1 x 108 and 1 x 109. Before any freeze-drying of the drug or food supplement according to the invention, it is generally in liquid form. In such a situation, the drug or dietary supplement preferably comprises a concentration in the medium equal to or greater than 1 x 108 CFU (colony forming units or seeds) per mL. To prevent or delay the colonization of a patient's respiratory system by pathogenic bacteria, doses each preferably of between about 1 x 10 8 and 1 x 10 10 CFU of probiotic bacteria are administered to the patient. 6 2917623 Several doses, preferably between one and four, may be administered per day. The doses can be adjusted, in particular according to the corpulence of the patient to be treated and its possible reaction to treatment. In a preferred embodiment, two daily doses of one gram each containing at least 1 x 108 probiotic bacteria per gram are administered with at least 8 hours between each set.

  Advantageously, the drug or dietary supplement according to the invention comprises a probiotic in living form and one or more prebiotics that can be degraded by the probiotic. The use of one or more prebiotics potentially increases the life of the probiotic after administration to the patient. Indeed, because the prebiotic corresponds to a culture medium adapted to the probiotic, when the drug or food supplement according to the invention is administered (and especially when it is in lyophilized form and it is resuspended before ingestion), the probiotic is immediately in an environment compatible with its survival. This combination of prebiotic (s) and probiotic is a symbiotic. In a preferred embodiment, the probiotic is a bacterium capable of fermenting lactose, for example a bacterium of the Lcr35 strain, and the one or one of the prebiotics is lactose or potato starch. It is particularly advantageous that, prior to its incorporation into the drug or food supplement according to the invention, the probiotic is cultured on a medium containing the prebiotic (s), preferably a medium containing lactose and / or apple starch. Earth. Indeed, under these conditions, the medium also contains metabolites resulting from this culture. Some of these metabolites may have advantageous antimicrobial properties. The combination of these compounds (probiotic, prebiotics and metabolites) contributes to the beneficial effect on respiratory infections. More preferably, in order to obtain the drug or dietary supplement, the medium in which the probiotic is found remains unchanged. This avoids too many centrifugation steps; indeed, during centrifugation, the cell walls of microorganisms can be damaged, which affects their survival. Thus, by avoiding changing the probiotic medium and consequently reducing the number of centrifugations, a better probiotic survival rate is ensured. According to a preferred embodiment of the invention, the probiotic bacterium is cultured on a lactose-containing culture medium, and the set consisting of the probiotic and this medium is then used for the preparation of the drug or food supplement according to the invention. 'invention.

  Advantageously, the bacterium is cultured for at least 4 hours, at least 12 hours, preferably at least 24, 48 or 72 hours, or more, on a culture medium based in particular on lactose which is then optionally supplemented to form the medicament or food supplement according to the invention. Alternatively, the drug or dietary supplement used in the context of the present invention does not include the probiotic but the culture supernatant of probiotic bacteria grown on a medium comprising lactose, and this supernatant has the ability to prevent or delay colonization, and particularly the retrograde colonization of the respiratory system of a patient by pathogenic bacteria.

  Advantageously, the probiotic bacterium is cultured for at least 4 hours, at least 12 hours, preferably at least 24, 48 or 72 hours, or more, on the lactose-based culture medium before the supernatant is collected and then used. for the preparation of the drug or food supplement according to the invention. Alternatively, the drug or food supplement used in the context of the present invention does not include the probiotic but an active substance produced by the probiotic bacteria, extracted from the culture supernatant probiotic bacteria grown on a medium comprising lactose, and having the ability to prevent or delay colonization, particularly retrograde colonization, of a patient's respiratory system by pathogenic bacteria.

  Advantageously, the probiotic bacterium is cultured for at least 4 hours, at least 12 hours, preferably at least 24, 48 or 72 hours or more, on the lactose-based culture medium before said active substance is extracted from the supernatant. , then used for the preparation of the drug or food supplement according to the invention.

  Whether the medicament or dietary supplement used for the implementation of the present invention comprises the probiotic, the probiotic culture supernatant or a substance extracted from this culture supernatant, the probiotic is advantageously cultivated in a first time on a medium based on lactose containing at least 5% by weight of lactose, preferably at least 20%.

  Said medium is preferably sterile before inoculation with the probiotic.

  Preferably, such a medium is a culture medium (fermentation) thus containing other substances. The medium is preferably a medium consisting essentially of milk, for example skimmed milk. The lactose-based medium advantageously also contains potato starch. It may also contain maltose dextrin. Any other pharmacologically acceptable compound or excipient may also be included in the composition of a lactose-based medium as defined. According to a preferred embodiment of the present invention, the lactose-based medium corresponds to a culture medium compatible with the fermentation of lactose by the probiotic bacterium included in the product according to the invention. Preferably, the probiotic used is not seeded or cultured on SRM.

  The pathogenic bacteria may include but not limited to bacteria of the family Pseudomonadaceae, Moraxellaceae, Streptococcaceae, Pasteurellaceae, Staphylococcaceae, Enterobacteriaceae, Enterococcaceae or Xanthomonadaceae. It is more particularly bacteria recognized as being the cause of pneumopathies, including pneumopathies acquired in a hospital environment, such as for example Pseudomonas aeruginosa, Acinetobacter baumannii, Acinetobacter calcoaceticus, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenza, Stenotophomonas maltophilia , Staphylococcus aureus, Enterobacter, Enterococcus faecium or Xanthomonas maltophilia. More particularly, it is Pseudomonas aeruginosa bacteria. The pathogenic bacteria may also be bacteria that are resistant to one or more antibiotics, including but not limited to resistance to an antibiotic belonging to the beta-lactam family (including Penicillins, Carbapenems and Cephalosporins), Aminosides, Phenicols, Tetracyclines , Sulfamides-Trimethoprim or Quinolones. More particularly, it may be a resistance to Penicillin G, Methycillin, Oxacillin, Ampicillin, Amoxicillin, Ertapenem, Ceftizoxime, Ceftriazone, Ceftazidime, Streptomycin, Gentamicin , Kanamycin, Chloramphenicol, Tetracycline, Trimethoprim or Rifampicin. It may more particularly be resistant Pseudomonas aeruginosa bacteria, including Ceftazidime resistant Pseudomonas aeruginosa bacteria.

  Pneumopathy means an infection of the lung tissue most often caused by a lung infection caused by bacteria, viruses, atypical germs, fungus or other parasites. The present invention more particularly covers the case where the pathogenic bacteria are at the origin of a nosocomial infection. Nosocomial infection refers to an infection contracted in a health facility. An infection is considered as such when she was absent at the time of admission. When the infectious state of the patient on admission is unknown, the infection is classically considered nosocomial if it appears after 48 hours of hospitalization. Infectious nosocomial pneumonia (PN) is defined as a pulmonary infection acquired after at least 48 hours of hospitalization. The term "mechanically ventilated nosocomial pneumonitis" refers to infections acquired after at least 48 hours of invasive mechanical ventilation. The mortality rate of patients with nosocomial pneumonitis is approximately 50%. Mechanical ventilation refers to providing or assisting the spontaneous breathing of a patient with a device (respirator or ventilator). Mechanical ventilation is generally practiced in the context of critical care (emergencies, intensive care) and anesthesia. The patient-ventilator interface is usually performed via an endotracheal tube, a waterproof mask, a tracheostomy tube or a laryngeal mask.

  The drug or dietary supplement according to the invention can be administered by various routes. According to a preferred embodiment, it is administered orally or nasally. According to one particular embodiment of the invention, the drug or dietary supplement is administered via a nasogastric tube. The treated patient receiving the drug or dietary supplement as described is preferably a human, preferably an adult. Adult means any person who is at least 18 years old.

  Alternatively, the drug or dietary supplement according to the invention may be used for veterinary purposes, the patient in question may in this particular case be a mammal other than man, for example a monkey, a dog, a cat or a rabbit. The drug or dietary supplement according to the invention can be administered to hospitalized patients or not. According to a preferred embodiment, the composition is administered to hospitalized patients, for example in an anesthesia, resuscitation or surgery department. These include patients undergoing respiratory assistance.

  The drug or food supplement is for example intended to be administered to the patient for a period of one to six weeks, or two to four weeks. The drug or dietary supplement according to the invention can be administered to patients prior to their hospitalization, for example for one to three weeks, and / or during the period of their hospitalization or placement under respiratory assistance, and / or after discharge from the hospital, for example for one to three weeks. Preferably, the drug or dietary supplement is administered to the patient for the duration of his hospital stay. Preferably, the drug or dietary supplement as described is intended to be administered to hospital patients for a period of at least 2 days, at least 4 days, at least 7 days or at least 2 weeks . More preferably, in the case of patients placed under mechanical ventilation, the drug or food supplement is administered to the patient during the entire period during which the patient is placed under mechanical ventilation.

  The stomach pH of hospitalized patients, for example in an intensive care unit, is generally higher than normal, for example greater than about 4, because of the administration of prophylactic drugs intended in particular to minimize the risk of occurrence of a disease. ulcer. It is believed that this rise in pH can facilitate the survival or proliferation of pathogenic bacteria but also the probiotic in the stomach. In a particular embodiment of the invention, the medicament or dietary supplement as described is administered to patients whose stomach pH is greater than 4.

  The drug or dietary supplement may be administered in combination with another drug, such as an antibiotic or an antiulcer. According to another preferred embodiment, it is a gastric anti-ulcer. According to one particular embodiment, it is an inhibitor of the proton pump. According to a preferred embodiment, the drug or dietary supplement is intended to be administered in combination, prevention and / or following a preventive antibiotic treatment.

  A study was conducted to determine whether oral administration of a drug according to the invention, containing the probiotic L. rhamnosus strain 35 (Lcr 35), could prevent colonization of the stomach and respiratory tract by the pathogenic P. aeruginosa, and thus impact the development of an infectious process. Lcr35 was administered as a powder orally or via a nasogastric tube, which differs from previous studies aimed at modifying the intestinal flora. Previous in vivo studies in human volunteers have shown that L. rhamnosus colonizes the intestinal tract when 1 x 108 CFU / day is administered (10), it was chosen to use a minimum dose of 1 x 108 CFU / 12h. The main result of this study is to have shown that the administration of probiotic organisms to reduce the risk of P. aeruginosa infection represents an alternative to DDS. In fact, bronchial, nasal and digestive colonizations occurred more frequently in patients receiving placebo (group A) than in patients receiving Lcr35 (group B). Previous studies had shown that P.

  aeruginosa, and in particular multidrug-resistant P. aeruginosa strains, are mainly isolated after a long hospital stay, including a period of more than 7 days of respiratory assistance (2, 6, 29, 39, 40). The results of this study showed that the average time to P. aeruginosa was higher in Group B patients than in Group A patients, particularly for bronchial samples. Finally, a statistically significant difference was observed in the delay in occurrence of mechanically ventilated pneumonia (VAP) between the two groups (with a lower number of cases for patients in group B.

  This study confirms that the oral administration of probiotics represents an alternative to prevent the colonization of the respiratory tract by this pathogen and can be a means of limiting this contamination until the cessation of the use of respiratory assistance. Overall, the results indicate that the administration of Lcr35 to prevent infectious complications related to selective decontamination of the enteral gastrointestinal tract represents a partial but interesting alternative strategy.

  On the other hand, although probiotics have been widely used in the food industry for many years and have generally shown excellent safety results (20), the main concern associated with their use risk of sepsis. In this study, which did not include immunodepressed patients, no cases of sepsis were observed among patients in group B receiving 2 daily doses of the Lactobacillus Lcr35 strain.

  Experimental part Example 1: Obtaining the Lyophilized strain Lactobacillus rhamnosus Lcr35 and packaging.

  Reactivation The commercially available microorganisms (Bacilor from the Lyocentre laboratory) or from the Lyocentre laboratories' souchothèque, are put back into activity by sowing with 100 to 200 mg of the freeze-dried product 10mL of skimmed enriched milk medium (glucose 15g / L, extract yeast 5g / L). This tube is placed in an oven at 37 C for 72 hours. 1 ml of the culture of this first tube is introduced into a second tube containing 10 ml of sterile enriched milk medium, this tube is placed in an oven at 37 ° C. for 72 hours. This operation is repeated several times so as to obtain the largest population possible, greater than 1 x 109 per ml.

  Preparation of the stock 1 ml of this reactivated strain is introduced into a flask containing 180 ml of sterilized enriched skimmed milk and then cultured at 37 ° C. for 72 hours, this stock is used to seed the flasks.

  Culture medium In order to be able to dispose of a lyophilized culture of Lactobacillus rhamnosus (also called Lactobacillus casei var.rhamnosus) of adapted title, this one is elaborated simultaneously according to 2 different methods of culture: on simplified medium on enriched medium a) constituents media: 1. Simplified medium: 100g skimmed milk powder Water, qs 1,000 L 2. Fortified medium: 16,650g skimmed milk powder Glucose 4,450g Manganese sulphate 0,028g Sodium citrate 0,560g Monopotassium phosphate 1,780g Dipotassium phosphate 3,560g25 13 Autolysate of beer yeast 1,670g Water, qs 1,000 L b) Preparation and sterilization of media: 100g 1. Simplified medium: formula: skimmed milk powder Water, qs 1,000 L Method: In a container, put 200 liters of water and add in small portions with slow stirring the 28 kg of skimmed milk powder. After complete dispersion, adjust to 280 L with water. This milk is divided into properly capped containers and sterilized by autoclaving for 30 minutes at 115 ° C. 2. Enriched medium: Formula for a 2000-liter container Method: In a container, put 1000L of water, then with rapid stirring, add the milk; after complete dispersion add glucose, manganese sulphate, sodium citrate, mono and dipotassium phosphates, and finally the autolysate of beer yeast. After complete dispersion, adjust to 1800 liters with water. Then, still stirring, sterilize this medium at 115 ° C. for 30 minutes and then cool to 37 ° C.

  Seeding and culture a) Simplified medium: Introduce 1 to 2 ml of preculture from the stock base into a vertical laminar airflow hood. powdered skimmed milk 30.00kg Glucose 8.00kg Manganese sulphate 0.05kg Sodium citrate 1.00kg Potassium dihydrogen phosphate 3.20kg Dipotassium phosphate 6.40kg Autolysate of brewer's yeast 3.00kg Water, qsp 1800.00 L Then these containers are placed for 72 hours in bacteriological incubators maintained at 37 C, plus or minus 2 C.

  b) enriched medium: Introduce, by 1800 liter container, 2.4 liters of preculture from the stock base. Maintain, thanks to a suitable thermostatic device, the temperature at 37 ° C. for 48 hours with slow stirring.

  Preparation of the culture for freeze-drying: a) simplified medium: constituents: - culture of Lactobacillus rhamnosus 136.8 L - potato starch 35.0 kg - lactose 15.0 kg - suspension of calcium hydroxide q.s. for neutralization pH = 7.0 Method of preparation: Under a laminar air flow hood, pour the resulting culture into a 200 liter vat, shake vigorously to break the curd and add with rapid stirring the potato starch and lactose. When their dispersion is complete, measure the pH which must be close to 3. Then, with calcium hydroxide, neutralize this mixture up to pH = 7.0 t 0.1. b) enriched medium constituents: 25 - suspension of calcium hydroxide q.s. for neutralization pH = 7.0 Preparation method: Lactobacillus culture. rhamnosus is centrifuged continuously in a centrifuge to totally eliminate the supernatant. The pellet is recovered in a laminar airflow hood, it is mixed with rapid stirring with the freeze-drying excipients. Then, using calcium hydroxide, neutralize this mixture to pH = 7.0 0.1. Lyophilization - culture of Lactobacillus rhamnosus 1800.0 L - potato starch 6.6 kg - lactose 3.3 kg - maltose dextrin 6.0 kg 2917623 The culture preparations of Lactobacillus rhamnosus for lyophilization are aseptically distributed in stainless steel trays. Freeze rapidly at -40 ° C., then sublimate at -22 ° C. When the product has exceeded the threshold of 0 ° C., bake at + 37 ° C. According to the preparations used, the quantities obtained of freeze-dried dry product. are about the following: Lyophilized culture of Lactobacillus. rhamnosus Simplified medium 57 kg Enriched medium 17 kg Crushing, mixing, conditioning a) grinding 10 In a room with a dehumidified atmosphere (RH less than 50% and filtered (filtration 99.999)), the lyophilized culture of Lactobacillus rhamnosus is recovered, crushed and sieved. on an oscillating type calibrator equipped with a grid of 60 MESH, a sample is taken for enumeration of lactobacilli. The product is stored in hermetically sealed containers. After titration of live Lactobacillus rhamnosus, the products are homogenized to obtain a desired title raw material.

  b) Mixing In order to obtain batches of freeze-dried Lactobacillus rhamnosus culture from 200 to 225 kg, the titre of which is between 1 x 109 and 1 x 10 10 germs per gram, mixing is carried out in a mild mixer according to the following method : -QS lyophilized culture of Lactobacillus rhamnosus in a simplified medium. -Q.S. lyophilized culture of Lactobacillus rhamnosus on enriched medium, such that the weight of the final mixture is between 200 and 225 kg of a powder whose title is between 1 x 109 and 1 x 1010 germs per gram.

  c) conditioning After mixing, samples are taken for testing. The rest of the mixture is conditioned according to the demand in hermetically sealed containers. Then this powder is formulated galenically.

  Example 2: Prevention of colonization of the respiratory tract by Pseudomonas aeru_ginosa

  Colonization of the gastrointestinal tract by nosocomial pathogens is a risk factor for intensive care unit (ICU) patients and can frequently lead to ventilator-acquired pneumopathy. In addition, selective digestive decontamination using antibiotics can promote the emergence of microbial resistance. Therefore, the identification of alternative methods to reduce the concentration of opportunistic or exogenous pathogens in the gastrointestinal tract and upper respiratory tract without increasing the selective pressure related to antibiotics is of great interest. Preventing the colonization of patients' stomachs with pathogens susceptible to antibiotic resistance, using oral probiotics, is therefore an interesting alternative. By modifying the composition of the endogenous flora, probiotics and more particularly lactic acid bacteria are well adapted to acidic environments, known to prevent the growth of non-acidophilic bacteria, the barrier function of the intestine could be strengthened and contribute to the prevention of nosocomial infections associated with the contamination of the intestine. To test this hypothesis, a prospective clinical study was conducted on 211 ICU patients to study the prevention of digestive carriage of a pathogen causing nosocomial infections, P. aeruginosa, by means of administration. of a known probiotic, Lactobacillus rhamnosus Lcr35 (10, 14). The patients were randomized into two groups, 104 of them belonging to the probiotic group (group B) received two daily doses of at least 1 x 108 CFU of Lcr35, the remaining 107 belonging to the placebo group (group A) received two doses. daily culture medium without the prebiotic. The search for Pseudomonas aeruginosa was performed on swab samples of the nose and anus, aspiration of gastric fluid and bronchial samples. Bacteriological analyzes of samples taken to monitor patients' infection status were also monitored. Regardless of the type of specimen, P. aeruginosa was isolated from fewer Group B than Group A patients, with a significant difference in mean acquisition time of 50 vs. 11 days (p = 0.001), and average duration of non-contamination of 48 to 15 days (p = 0.01) of isolated P. aeruginosa in bronchial samples. In addition, the occurrence of pneumopathies acquired under mechanical ventilation was less frequent in patients in group B (2.9%) than in group A (7.5%).

  MATERIALS AND METHODS A - Subjects Patients 18 years of age and older with a length of stay in the ICU of at least 48 hours were included in the study. Patients with any of the following characteristics were excluded: less than 18 years of age, immunodeficient, neutropenia less than 500 / mm3, with gastrointestinal digestive syndrome, contraindication to fluid administration enterally, with Ceftazidime-resistant P. aeruginosa colonization detected by admission sampling or by respiratory sampling in the first 4 days of hospitalization in intensive care unit.

  B - Scope of the study This randomized, double-blind, prospective study was conducted in a 17-bed ICU at the Clermont-Ferrand University Hospital Center (France) between March 2003 and October 2004. The study was conducted in accordance with with the Helsinki Declaration and was approved ethically by the Advisory Committee for the Protection of Persons in Biomedical Research in Auvergne (CCPRB, AU 479). Before being included in the study, patients or their closest relatives gave informed consent. The culture medium without the probiotic (placebo) or the probiotic in its available pharmaceutical form (freeze-dried L. rhamnosus containing at least 1 x 108 CFU / g) was administered twice daily to the patients of groups A and B respectively by the intermediate of a double-lumen nasogastric tube (Maxter-catheters, Marseille, France) or orally after removal of the catheter, from the third day after admission to the end of stay in the department.

  C - Clinical Data The following clinical data were collected: age, sex, admission severity score (SAPS) (25), comorbidity, and previous antibiotic treatments. Throughout the study, antibiotic administration and bacteriological data were recorded. Mechanically ventilated pneumonitis (VAP) was defined by at least one sample obtained by protected distal sampling (PDP) or protected telescopic brush (PWB) containing more than 103 CFU / mI, or an enterotracheal aspirate containing more than of 105 CFU / ml, by the presence of a new parenchymal infiltrate on a chest x-ray, and by one of the following signs: purulent tracheal secretion, fever (temperature> 38.5 C), presence of pathogenic bacteria in a blood culture outside of another source of infection or bronchoalveolar lavage (BAL) with more than 5% of cells with intracellular bacteria.

  D - Microbiological samples Nose, anus and, as long as the gastric tube was in place, gastric samples were taken at the admission, once a week and at the exit. In patients receiving enteral nutrition, gastric samples were taken before the bottles of nutrient were changed. When no gastric residue was obtained, 10 ml of physiological saline was injected into the probe and aspirated.

  The gastric samples were seeded on Drigalski agar in the presence or absence of Ceftazidime (4 mg / l) and on Mueller-Hinton agar, and then after 10 serial dilutions on MRS agar (Oxoid, Basinstoke, UK) to perform a quantitative analysis of the rate. of L. rhamnosus Lcr35. The nose and anus samples were immersed in 1 ml of physiological saline solution and seeded on Drigalski agar containing 4 mg / l of Ceftazidime. The colonies that appeared were identified using an ID32GN gallery (BioMérieux, Marcy l'Etoile, France). The results of analyzes of samples taken by the hospital laboratory for routine microbiological diagnoses due to the infection status of the patients were also included in the study.

  E - Statistical analysis The statistical analyzes were carried out with the SPSS 11.0 program (SPSS, Paris, France). The Chi-square test or the Fisher exact test were used to compare the qualitative variables, and the Student's t-test or the non-parametric Mann-Whitney test were used for the quantitative variables. For each patient, the geometric mean bacterial concentration was calculated for each of the sample types. The median obtained for the different patients was included in the results. The threshold of statistical significance was set at 0.05. Rates of non-colonized P. aeruginosa patients were estimated and both groups were compared using the Kaplan Meier survival curve method and the Logrank test. The mean time of non-infection (NAE), as well as the length of stay without acquisition of Pseudomonas aeruginosa, were calculated after exclusion of patients in which the bacteria considered were isolated during the first 4 days of stay in the unit.

  RESULTS The results are detailed in Table 1 below. Sampling Nasal swab Gastric liquid Respiratory tract Group2 ABABAB P. aeruginosa Presence during 2 2 0 2 1 1 admission Acquisition at 7 3 6 3 13 5 during stay Median duration 17 (8-46) 37 (31-69) 30 (6-53) 16 (6-18) 11 (5-40) 50 (11-75) before acquisition (extremes) 3 NAE mean (IC) 4 75 (65-84) 83 (74-91) 73 (61) -85) 87 (82-92) 15 (9-21) 48 (26-70)% NA at day 21 92.8 100 97.6 94.6 85.4 98.5% NA at day 42 85.3 90.1 84.8 94.6 71.6 92.9 P. aeruginosa CAZK Presence at 0 1 0 0 0 0 admission Acquisition at 5 2 3 1 5 0 course of stay Median duration 17 (8-33) 50 (31-69) 10 (6-33) 18 20 (5-40) NA before acquisition Mean NAE 79 (72-87) 85 (77-93) 83 (78-89) 89 (86-92) 78 (69-87) NA oc) '% NA at day 21 94.3 100 97.6 97.9 93.6 100% NA at 42nd day 86.8 95.8 93.2 97.9 82.3 100 1 0 1. Sample obtained by protected distal sampling (PDP) or by protected telescopic brush (BTP) with more than 10 'CFU / ml or endotracheal aspirate containing t more than 105 CFU / ml 2. Group A (n = 107), placebo; group B (n = 104), patients receiving Lcr35 orally twice daily 3. Mean length of hospital stay before bacterial acquisition calculated using Kaplan Meier and Logrank methods 4. NAE = mean time of non-infection; CI = Confidence interval Table 1 - Frequency of patients infected with P. aeruginosa at admission and during stay, median duration before acquisition and mean duration of non-infection (AIN) A Inclusion of patients A total of 211 patients were including 104 received the probiotic strain (group B). Most patients were hospitalized following surgery (29.4%), trauma (24.2%) or respiratory distress (11.4%). There was no significant difference between patients in groups A and B in terms of age (mean age 51, SD 15.2 vs 57.2 SD 17.1) (SD = standard deviation), sex (sex ratio male / female, 0.30 vs 0.55), the admission severity score (mean SAPS, 44 SD 15.2 vs 44.6 SD 15.9), the median duration of hospitalization (14 vs. 14 days), median duration of gastric intubation (11 vs 12 days), tracheal (9 vs 12 days), mechanical ventilation (13.5 vs. 14 days), or use of catheters (14 vs 14.5 days).

  There was no significant difference between the antibiotics administered before and during the study. None of the patients received piperacillin or first-generation cephalosporins before or during their stay; the only difference is fluconazole, administered to 27 patients in group A and 45 patients in group B, respectively (p = 0.006). Omeprazole doses of 20mg / day were administered at 90 (84.7%) and 93 (89.4%) patients respectively in groups A and B as prophylactic treatment for stress ulcer.

  B - Detection of Lcr35 in gastric aspirates Lactobacillus rhamnosus was detected in the gastric fluid of 53 patients in group B.

  It was detected in 49.6% (SD 24.9) of hospital stays and the median length of stay before detection was 13 days (CI 9-17). The median bacterial concentration was 103 / ml (range 102-106 / ml). No patients contracted Lactobacillus infection during the study. Lcr35 did not colonize the stomach of all patients in group B since it was only detected in the stomach of 51% of patients tested. Unknown individual factors, such as the composition of the endogenous flora, may explain why some patients were not colonized because no statistical link was observed between Lcr35 colonization and antibiotic treatment.

  C - Detection of P. aeruginosa In each group, P. aeruginosa strains were isolated during the first 3 days following admission, using nasal swabs; only one patient in group B had P. aeruginosa colonization resistant to Ceftazidime. During the duration of the study and among the other patients (105 and 104 respectively for groups A and B), P. aeruginosa was detected in the nose swabs of 7 and 3 patients. Only 5 (4.7%) and 2 (1.9%) of these samples from Groups A and B, respectively, exhibited resistance to Ceftazidime. The median time to P. aeruginosa in nose swabs was 17 days (range 8-46) in group A and 37 days (range 31-69) in group B, and mean NAE were 75 days (IC 65-84) in group A and 83 days (IC 74-91) in group B. In groups A and B respectively, 92.8% and 100% of patients at day 21, and 85.3% and 90.1% at day 42 had not acquired P. aeruginosa. In addition, the difference between patients in the two groups with Ceftazidime-resistant samples was not significant.

  Strains of P. aeruginosa were isolated in the gastric fluid of only two patients in group B during the first three days of stay; none of these samples exhibiting resistance to Ceftazidime. During the study, 6 and 3 patients among the 107 (group A) and 102 (group B) patients respectively, acquired P. aeruginosa at the gastric level. Only 3 (2.8%) and 1 (1.0%) patients in Groups A and B, respectively, acquired ceftazidime-resistant strains. Three patients in group B concurrently acquired Lcr35 and P. aeruginosa in the gastric fluid. The median time to P. aeruginosa at the gastric level was 30 days (range 6-53) in group A and 16 days (range 6-18) in group B, and the NAE was 73 days ( IC 61-85) for group A and 87 days (IC 82-92) for group B. In the same way as for nasal swab specimens, the difference observed between the two groups of patients with strains resistant to Ceftazidime was not significant. Analysis of airway samples showed P. aeruginosa strains in one patient in each group at the time of admission; none of these samples showed resistance to Ceftazidime. During the study, 13 positive samples, 5 of which were resistant to ceftazidime, were detected in group A compared to only 5, all of which were sensitive to Ceftazidime in group B. A significant difference was observed with respect to the median time delays. respiratory tract acquisition of P. aeruginosa which was 11 days (range 5-40) in group A and 50 days (range 11-75) in group B (p = 0.01), and NAE were 15 days (CI 9-21) for group A and 48 days (CI 26-70) for group B (p = 0.01, Logrank test). In groups A and B respectively, 85.4% and 98.5% of patients at day 21; and 71.6% and 92.9% on the 42nd day, had not acquired P. aeruginosa. This microorganism was also responsible for ventilator-acquired pneumonia (VAP) in 8 patients in group A and 3 patients in group B. The median time to acquire a PAVM was 15 days (range 5-40), respectively. for group A and 65 days (range 11-75) for group B. The presence of P. aeruginosa was also sought in anal samples. None were positive at admission, but over the course of the study, anal swabs from one patient in each group were positive. Both strains were resistant to Ceftazidime. P. aeruginosa was detected in other samples in three other patients during their stay, a Ceftazidime isolate in group A and two isolates sensitive to ceftazidime in group B.

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Claims (28)

claims   1. Use of (i) Lactobacillus bacteria in live form, or (ii) culture supernatant of Lactobacillus bacteria grown on a medium comprising lactose, or a substance extracted from said culture supernatant, for the manufacture of a medicament or dietary supplement intended to prevent or delay the colonization of the respiratory system of a patient by pathogenic bacteria that colonize the respiratory tract retrograde after having stayed in the gastrointestinal tract, and more particularly in the stomach.   2. Use according to claim 1, wherein said drug or food supplement also prevents or delays the colonization of the gastrointestinal tract, including the stomach, by said pathogenic bacteria.   3. Use according to claim 1 or 2, wherein said pathogenic bacteria belong to the family of Pseudomonadaceae, Moraxellaceae, Streptococcaceae, Pasteurellaceae, Enterococcaceae or Xanthomonadaceae.   4. Use according to claim 3, wherein the pathogenic bacteria are bacteria of the species Pseudomonas aeruginosa.   5. Use according to any one of claims 1 to 4, wherein the Lactobacillus bacteria are Lactobacillus rhamnosus bacteria or Lactobacillus casei, for example Lactobacillus rhamnosus of the Icr 35 strain.   6. Use according to any one of claims 1 to 5, wherein said drug or dietary supplement comprises live Lactobacillus bacteria as well as a prebiotic.   The use according to claim 6, wherein said prebiotic is lactose and / or potato starch.   The use of claim 6 or 7, wherein said drug or dietary supplement also comprises metabolites produced by said Lactobacillus bacteria.   9. Use according to any one of claims 1 to 8, wherein the patient is a human.   The use of claim 9 wherein the patient is 18 years of age or older.   11. Use according to claim 9, wherein the patient is hospitalized, more particularly where the patient is hospitalized in an anesthesia, resuscitation or surgery department.   12. Use according to claim 11, wherein said patient is hospitalized for a period of at least 2 days.   13. Use according to claim 11 or 12, wherein the patient is placed under mechanical ventilation.   14. Use according to claim 13, wherein said medicament or food supplement is intended to be administered to said patient for the entire period during which it is placed under mechanical ventilation.   15. Use according to any one of claims 11 to 14, wherein said pathogenic bacteria are causative agents of a nosocomial infection.   16. Use according to claim 15, wherein said nosocomial infection is a pneumopathy, more particularly a pneumopathy acquired under mechanical ventilation.   17. Use according to any one of claims 1 to 16, wherein the pathogenic bacteria are resistant to antibiotics.   18. Use according to any one of claims 1 to 17, wherein said drug or dietary supplement is to be administered in freeze-dried form.   19. Use according to any one of claims 1 to 18, wherein said medicament or dietary supplement is intended to be administered orally.   20. Use according to any one of claims 1 to 18, wherein said drug or food supplement is intended to be administered nasally, more particularly via a nasogastric tube.   21. Use according to any one of claims 1 to 20, wherein said drug or food supplement comprises a number of Lactobacillus bacteria between 1 x 108 and 1 x 1010 colony forming units, preferably between about 1 x 108 and 1 x 109 units. forming colonies per ml or per gram.   22. Use according to any one of claims 1 to 21, wherein said drug or dietary supplement is intended to be administered in combination with another drug.   The use of claim 22, wherein said drug or dietary supplement is for administration in combination with an antiulcer.   24. Use according to claim 23, wherein said anti-ulcer is a proton pump inhibitor.   25. Use according to any one of claims 1 to 24, wherein said medicament or dietary supplement is intended to be administered in combination, in prevention or as a result of preventive antibiotic treatment.   26. Use according to any one of claims 1 to 25, wherein the stomach pH of the patient is greater than 4.   27. Use according to any one of claims 1 to 26, wherein said drug or dietary supplement is intended to be administered at two doses of one gram per day, each dose comprising a number of bacteria equal to or greater than 1 x 108 germs per gram.   28. Use according to any one of claims 1 to 27, wherein said Lactobacillus bacteria have previously been cultured on a lactose-based medium for at least 24, 48 or 72 hours.