Methods of treating <i>Pseudomonas aeruginosa </i>respiratory infections
11364271 · 2022-06-21
Assignee
- Inserm (Paris, FR)
- Etablissement Français Du Sang (EFS) (La Plaine Saint Denis, FR)
- UNIVERSITÉ DE BRETAGNE OCCIDENTALE (Brest, FR)
- Centre Hospitalier Regional et Universitaire de Brest (Brest, FR)
Inventors
- Rozenn Le Berre (Brest, FR)
- Marie-Sarah Fangous (Quimper, FR)
- Gwenaelle Le Blay (Plouzané, FR)
- Geneviève HERY-ARNAUD (BREST, FR)
- Rosyne Lagrafeuille (Brest, FR)
Cpc classification
International classification
A01N63/00
HUMAN NECESSITIES
Abstract
Pseudomonas aeruginosa (PA) leads to chronic respiratory infections especially in patients with cystic fibrosis patients and chronic obstructive pulmonary disease (COPD), characterized by a high morbidity. After screening Lactobacilli coming from CF expectoration, on their capacity to inhibit two Pseudomonas aeruginosa (PA) virulence factors (elastase, pyocyanin), the inventors evaluated the effect of intranasal administration of Lactobacilli on PA murine pneumonia. The primary outcome was the bacterial lung load 24 hours after PA induced pneumonia. To understand the role of Lactobacillus, the chemokines, the pro and anti-inflammatory BAL rates were also measured. The administration of Lactobacilli cocktail 18 h prior the PA lung infection decreases significantly the lung bacterial load at 24 h post-infection. Although the mechanisms need to be deeply explored, an immunomodulation effect may be involved, notably through the recruitment of neutrophils. Thus the present relates to a method of treating a Pseudomonas aeruginosa respiratory tract infection in a patient in need thereof comprising administering to the patient's respiratory tract a therapeutically effective amount of at least one Lactobacillus strain.
Claims
1. A method of treating a Pseudomonas aeruginosa respiratory tract infection in a patient in need thereof comprising administering to the patient's respiratory tract a therapeutically effective amount of at least one Lactobacillus strain, wherein the administration is performed by instillation or inhalation.
2. The method of claim 1 wherein the patient suffers from a chronic pulmonary disease selected from the group consisting of chronic obstructive pulmonary disease (COPD), ventilated acquired pneumonia, chronic bronchitis, recurrent bronchitis, acute bronchitis, rhinosinusitis, mild pulmonary disease, hereditary emphysema, and cystic fibrosis.
3. The method of claim 1 wherein the Lactobacillus strain is selected from the group consisting of Lactobacillus acetotolerans, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus arizonensis, Lactobacillus aviarius, Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus coelohominis, Lactobacillus collinoides, Lactobacillus coryniformis subsp. coryniformis, Lactobacillus coryniformis subsp. torquens, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus cypricasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp delbrueckii, Lactobacillus delbrueckii subsp. lactis, Lactobacillus durianus, Lactobacillus equi, Lactobacillus farciminis, Lactobacillus ferintoshensis, Lactobacillus fermentum, Lactobacillus formicalis, Lactobacillus fructivorans, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus hamsteri, Lactobacillus helveticus, Lactobacillus helveticus subsp. jugurti, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus intestinalis, Lactobacillus japonicus, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kefiri, Lactobacillus kimchii, Lactobacillus kunkeei, Lactobacillus leichmannii, Lactobacillus letivazi, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus mindensis, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus oris, Lactobacillus panis, Lactobacillus pantheri, Lactobacillus parabuchneri, Lactobacillus paracasei subsp. paracasei, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus paracasei subsp. tolerans, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus psittaci, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus salivarius subsp. salicinius, Lactobacillus salivarius subsp. salivarius, Lactobacillus sanfranciscensis, Lactobacillus sharpeae, Lactobacillus suebicus, Lactobacillus thermophilus, Lactobacillus thermotolerans, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vitulinus, Lactobacillus vermiforme, Lactobacillus zeae.
4. The method of claim 1 wherein at least 2, 3, 4 or 5 Lactobacillus strains are administered to the patient.
5. The method of claim 1 wherein Lactobacillus salivarius and Lactobacillus brevis are administered to the patient.
6. The method of claim 1 wherein the Lactobacillus strain is a probiotic strain.
7. The method of claim 1 wherein the probiotic Lactobacillus strain is a viable probiotic Lactobacillus strain.
Description
FIGURES
(1)
(2)
(3)
(4)
EXAMPLE 1
(5) Methods
(6) Ethics
(7) This study is approved by our local ethics committee and the ethics committee for animal experiments (DAP 2017040717237994).
(8) Bacterial Strains
(9) Pseudomonas aeruginosa Strain:
(10) P. aeruginosa PAO1 was chosen as reference strain for all the experiments [1]. It was frozen at −80° C. before subculture on Mueller Hilton agar plate before the experiments.
(11) Lactobacillus Strains:
(12) One hundred and thirty-seven Lactobacillus isolates were previously isolated from CF patient's respiratory samples (Fangous et al, Research in Microbiology). Fifty Lactobacillus isolates were selected from PA colonised (n=30) or not colonised patients (n=20), by respecting the species prevalence observed within each group of patients. These isolates were screened in vitro for their ability to decrease the synthesis of 2 PA virulence factors, the pyocyanin and the elastase.
(13) All isolates were frozen at −80° C. before subculture on 5% sheep-blood agar (bioMérieux, Marcy l'Etoile, France) in 5% CO.sub.2 at 37° C. for 2 days before the experiments.
(14) Inhibition Tests of Lactobacillus Strains on PAO1 Virulence Factors
(15) Elastase
(16) For the elastase assay, PAO1 and Lactobacillus isolates were cultivated overnight separately at 37° C. in Brain Heart Infusion broth (BHI). The inhibition of the elastolytic activity of Pseudomonas aeruginosa PAO1 by Lactobacilus isolates was investigated by colorimetric assay, using Elastin Congo Red (Sigma), as adapted by Alexandre and collaborators [2]. Succinctly, overnight culture of PAO1 in BHI broth was washed twice with isotonic saline solution and adjusted to 5×10.sup.7 CFU/ml in broth media. Overnight culture of Lactobacillus in BHI broth was neutralised with NaOH 0.1M and adjusted to 5×10.sup.7 CFU/ml in broth media. A vol/vol co-culture was made and incubated 20 hours under aerobic conditions at 37° C. After centrifugation (20′ at 3500 g), 50 μL of the supernatant was mixed with 1 ml of Elastin Congo Red solution (20 mg/ml in a 10 mM sodium phosphate buffer) and incubated for 20 hours more under agitation. Finally, the soluble fraction released in the supernatant by elastase was measured at 495 nm after centrifugation (20′ at 3500 g) with a spectrophotometer.
(17) The results were normalized to the OD.sub.595 of the co-culture and expressed as a ratio of the absorbance observed in presence of the Lactobacillus isolate to the absorbance observed with a monoculture of PAO1.
(18) The experiments were conducted twice to three times for each isolate of Lactobacillus.
(19) Pyocyanin
(20) For pyocyanin production, P. aeruginosa PAO1 was grown overnight in Bacto-Peptone (BP) broth (20 mg/L BP, MgCl.sub.2 1.4 g/L, K.sub.2SO.sub.4 10 g/L). Lactobacillus was grown overnight on MRS broth. A vol/vol co-culture was made as previously described for the elastase experiments, and incubate under aerobic conditions at 37° C. The inhibition of the pyocyanin synthesis was investigate by colorimetric assay after extraction in an acid solution as previously described [3].
(21) The results were normalized to the OD.sub.595 of the co-culture and expressed as a ratio of the absorbance observed in presence of the Lactobacillus isolate to the absorbance observed with a monoculture of PAO1.
(22) The experiments were conducted twice to three times for each isolate of Lactobacillus.
(23) Murine Model of Pneumonia:
(24) Preparation of the Bacterial Strains
(25) Lactobacillus were grown overnight on MRS broth under aerobic conditions at 37° C. The 3 strains of Lactobacillus with the better inhibitive abilities against P. aeruginosa PAO1 were equally mixed in a cocktail named “Strong Lactobacilli; SL”.
(26) The 3 strains with the weakest abilities were mixed and named “Weak Lactobacilli; WL”.
(27) P. aeruginosa PAO1 was grown overnight on Luria Bertani (LB) broth under aerobic conditions at 37° C.
(28) Each culture was washed twice with isotonic saline solution and adjust to 10.sup.8 CFU/ml for the P. aeruginosa PAO1 suspension, or to 10.sup.6 CFU/ml for the SL and WL suspensions, based to the OD.sub.595 nm and controlled by serial dilution and plating on Mueller Hinton agar plates in triplicates.
(29) Animals
(30) C57BL/6 mice, aged 6-8 weeks old, were purchased from Janvier Labs (Le Genest Saint Isle, France) and maintained at the University of Brest, France. Mice received water and food ad libitum, and were monitored every eight hours until being sacrificed.
(31) Seventy-one mice were divided in 5 groups: Control, n=10; Control SL, n=16; Control PAO1, n=16; SL+PAO1, n=16; WL+PAO1, n=13.
(32) Infection Model of Acute Pneumonia
(33) Bacteria were administrated by intranasal instillation of 20 μL of the bacterial suspension (10 μL per nostril), under a short intraperitoneal anaesthesia with ketamine/xylazine (100/10 mg/kg) allowing maintenance of spontaneous breathing.
(34) Lactobacillus suspension (SL or WL) was administrated 18 hours prior the infection with P. aeruginosa PAO1.
(35) Control groups received 20 μL of isotonic saline suspension instead of Lactobacillus suspension and/or P. aeruginosa suspension.
(36) Sampling Procedure
(37) Six or 24 hours post infection with PAO1, mice were anesthetized with intraperitoneal injection of ketamine/xylazine (100/10 mg/kg) and euthanasied by intracardiac exsanguination.
(38) Blood, bronchoalveolar lavage (BAL), lung and spleen tissues were harvested from animals under aseptic conditions.
(39) BAL was performed after euthanasia by cannulation of the trachea and injection and aspiration of 500 μl of isotonic saline solution three times.
(40) Bacterial Loads
(41) The lungs were removed and homogenized with 4 ml of isotonic saline solution with Ultra-Turrax. Bacterial loads of PAO1 and Lactobacillus cocktails were determined by plating serial dilutions of total lung homogenate on Cetrimide and MRS agar plate. Each dilution was plated in duplicate. Plates were incubated 24 to 48 h at 37° C. under aerobic conditions. Colonies were confirmed using MALDI-TOF mass spectrometry (Microflex LT, Bruker Daltonics, Bremen, Germany). Identifications were obtained when scores were strictly superior to 2.
(42) White Blood Cells (WBC) Count
(43) The total white blood cells count was enumerated by manual counting method with a hemocytometer by light microscopy.
(44) Macrophages, neutrophils and lymphocytes were differentiated after centrifugation, cytospins preparation and May-Grünwald-Giemsa staining.
(45) Cytokine Measurement
(46) The concentrations of the cytokines were performed on BAL supernatant, after centrifugation at 4° C., and freezing at −80° C.
(47) The cytokines studied were IL-1b, IL-12, IL-17-a, IL-22, IL-23, IFN-g and the 2 chimiokines CXCL-1 ad CXCL-2. Dosages were performed with single Elisa kits.
(48) Statistics
(49) Results are presented as mean and standard error of the mean. Comparisons between the groups were analysed by the Mann-Whitney test. Results were considered statistically significant for p<0.05. All statistical tests were performed using the R software.
(50) Results
(51) Screening In Vitro of Lactobacillus Strains
(52) The 50 selected strains were distributed as figured on the Table 1. Eleven species were represented. Considering the anti-elastolytic activity, 25 (83%) strains from PA colonized patients and 15 (75%) strains from PA non colonized patients exhibited an anti-PA activity with respectively average of 60.6±0.15% and 64.7±0.15% of activity.
(53) Considering the inhibition of the pyocyanin synthesis, 6 (20%) strains from PA colonized patients and 6 (30%) strains from PA non colonized patients exhibited an anti-PA activity with respectively average of 83.6±0.15% and 80.15±0.17% of activity.
(54) To constitute 2 blends of Lactobacillus to administrated to our mice model of PA pneumonia, 3 strains with the better anti-PA activities and 3 strains with the weakest anti-PA activities were selected.
(55) The “Strong Lactobacilli” (ST) blend was constituted with 2 L. fermentum strains and 1 L. rhamnosus strains. The three strains were from PA colonized patients.
(56) The “Weak Lactobacilli” (WK) blend was constituted with 1 L. paracasei, 1 L. salivarius and 1 L. brevis strains. The last two strains were from PA colonized patients.
(57) The anti-PA activity of these 6 strains are shown on the Table 2.
(58) Administration of Lactobacilli Decreases the Lung PA Load
(59) Significant decreases of 2 and 4 log of PAO1 were observed 24 h after PAO1 instillation in SL+PAO1 (4.6.10.sup.1 CFU/g) and WL+PA01 (<1 CFU/g) groups compared to PAO1 group (6.8.10.sup.4 CFU/g) (
(60) BAL Cytological Analysis
(61) An increase of the WBC count and PNN were observed following the administration of the SL cocktail in the Control SL group, but significantly less important than in the Control PAO1 group (
(62) BAL Cytokine Analysis
(63) Following the decrease of the recruitment of PNN due to prophylactic administration of Lactobacilli cocktail, the immunological response was studied through cytokines and chemokines dosages 6 h (T6) and 24 h (T24) post PAO1 administration.
(64) No production of CXCL1 and CXCL2 was observed in the Control and Control SL at T6 and T26 whereas an important increase was observed at Thin the Control PAO1 group. The chemokines CXCL1 and CXCL2 BAL's levels were decreased in SL+PAO1 (DNS) and WL+PA01 groups (DS) at 6 h post-infection compared to the Control PAO1 group.
(65) No difference of production of IL-1B was observed between the Control and Control SL groups at T6 and T24. However, an increase was observed at T6 in the Control PAO1 group (mean=115 pg/ml). The IL-1B BAL's level were decreased in SL+PAO1 (mean=67.5 pg/ml) and WL+PA01 groups (mean=33.6 pg/ml; p=0.01) at 6 h post-infection compared to the Control PAO1 group. A significant decrease was also observed at T24 in the SL+PAO1 (mean=9.0 pg/ml; p=0.01) compared to Control PAO1 (mean=22.2 pg/ml).
(66) No difference of production of IL-12 was observed between the Control, Control SL and Control PAO1 groups at T6. However, a slight increase was observed at T24 in the Control SL compared to the Control group, and a significant increase was observed in the PAO1 group (mean=105.5 pg/ml). No clear difference was observed in the SL+PAO1 and WL+PAO1 groups compared to Control PAO1 at T6 and T24.
(67) No production of IFN-G was observed in the Control, Control SL and Control PAO1 at T6. However, an increase was observed in the Control PAO1 group at T24 (mean=77.7 pg/ml) compared to the 2 others. Following the administration of Lactobacilli cocktail, a decrease of IFN-G was observed at T24 in the SL+PAO1 (mean<15 pg/ml) and WL+PAO1 (mean<15 pg/ml) groups.
(68) Finally, the immunomodulation mediated via the TH17 axis was explored through IL-17A and IL-23A dosages. No production of IL-17A was observed in the Control, Control SL and Control PAO1 at T6. However, an increase was observed in the Control PAO1 group at T24 (mean=96.4 pg/ml) compared to the 2 others. Following the administration of Lactobacilli cocktail, a decrease of IL-17A was observed at T24 in the SL+PAO1 (mean<5 pg/ml) and WL+PAO1 (mean=3.95 pg/ml) groups. A slight production of IL-23 was observed whatever the group at T6 and T24, with no significant difference following the prophylactic administration of Lactobacilli cocktails.
EXAMPLE 2
(69) Methods
(70) Ethics
(71) This study has been approved by the ethics committee for animal experiments (DAP2017040717237994 and DAP2017110311134961).
(72) Preparation of the Bacterial Strains
(73) Lactobacilli were grown overnight on MRS broth under aerobic conditions at 37° C. Three strains without inhibitory activity were mixed as a control in a blend named “L.psb”. PAO1 was grown overnight in Luria-Bertani broth (Sigma) under aerobic conditions at 37° C. Each culture was washed twice with isotonic saline solution (SS) and adjusted to 108 CFU.ml-1 for the PAO1 suspension, or to 107 CFU.ml-1 for the L.psb suspensions, based on the OD595 nm and controlled by serial dilution and plating on MH in triplicates.
(74) Animals
(75) C57BL/6 mice, aged 6-8 weeks old, were purchased from Janvier Labs (Le Genest Saint Isle, France) and maintained at the University of Brest, France. Mice received water and food ad libitum, and were monitored every eight hours until being sacrificed.
(76) 9 groups of C57BL/6 mice: Control PAO1, Control Lpsb (L. paracasei, L. salivarius and L. brevis), Control Lp (L. paracasei), Control Ls (L. salivarius), Control Lb (L. brevis), Lpsb+PAO1, Ls+PAO1 and Lb+PAO1.
(77) Infection Model of Acute Pneumonia
(78) Bacteria were administrated by intranasal instillation of 20 μL of the bacterial suspension (10 μL per nostril), under a short intraperitoneal anaesthesia with ketamine/xylazine (100/10 mg/kg) allowing maintenance of spontaneous breathing.
(79) Lactobacillus suspension was administrated 18 hours prior the infection with P. aeruginosa PAO1.
(80) Control groups PAO1 received isotonic saline suspension instead of Lactobacillus suspension. Control L.psb groups received isotonic saline suspension instead of PAO1.
(81) Survival
(82) Mice were monitored during 7 days after infection with PAO1. Fur aspect, activity, behaviour, posture, eye lids, respiration, chest sounds, and body weight were followed frequently during the whole experiment, and scored from 1 to 4 according to the M-CASS scoring system [17]. When mice reached a score of 11 during day, buprenorphine was administered subcutaneously (0.05 mg/kg/12 h) for analgesia. Mice were sacrificed when they reached a score of 4 in the 8 parameters during the day or in one parameter at night to prevent overnight death.
(83) Sampling Procedure
(84) Six (T6) or 24 hours (T24) post infection with PAO1, mice were anesthetized with intraperitoneal injection of ketamine/xylazine (100/10 mg/kg) and sacrificed by intracardiac exsanguination. Blood, BAL, lung and spleen tissues were harvested from animals under aseptic conditions. BAL was performed after euthanasia by cannulation of the trachea and injection and aspiration of 500 μl of SS three times.
(85) Bacterial Cell Count in Lung Homogenates
(86) Mice were sacrificed at T24 and lungs removed and homogenized with SS with Ultra-Turrax. Bacterial loads of PAO1 and Lactobacillus blends were determined by plating serial dilutions of total lung homogenate on Cetrimide (bioMérieux) and MRS agar plates. Each dilution was plated in duplicate. Plates were incubated 24 to 48 h at 37° C. under aerobic conditions. Colonies identification was confirmed using MALDITOF mass spectrometry (Microflex LT, Bruker Daltonics, Bremen, Germany).
(87) White Blood Cells Count
(88) The total white blood cells (WBC) count on BAL was enumerated by manual counting method with a hemocytometer (Kova slide) by light microscopy. Alveolar macrophages (AM), polymorphonuclear (PMN) and lymphocytes were differentiated after centrifugation, cytospins preparation and May-Grünwald-Giemsa staining.
(89) Cytokine Measurement on BAL
(90) The cytokines studied were IL-1β, IL-6, IL-1β, TNF-α, and the 2 chemokines CXCL-1 and CXCL-2. IL-1β, IL-6 and IL-10 (eBiosciences), TNF-α and the chemokines CXCL1 and CXCL2 (R&D System, Abingdon, UK) were determined in the BAL by enzyme-linked immunosorbent assay (ELISA), using commercial kits according to the manufacturer's recommendations. The lower levels of detection were 7 pg/ml for CXCL1 and CXCL2, 4 pg/ml for IL-1β and IL-6, 8 pg/ml for IL-10 and TNF-α.
(91) Results
(92) In Vitro Screening of Lactobacilli Isolated from CF Respiratory Samples
(93) Forty strains (80%) exhibited anti-elastolytic activity (mean activity=−37.4%±0.15), and 12 (24%) exhibited anti-pyocyanin activity (mean activity=−18.13%±0.15). To constitute 2 blends of Lactobacilli to administrate to the mice model of PA pneumonia, 3 strains with the highest anti-PA activities (L.rff) and 3 strains with no anti-PA activities (L.psb) were selected (Table 2).
“L.rff” was constituted with 2 L. fermentum strains and 1 L. rhamnosus strain. The three strains were isolated in PA colonised patients.
“L.psb” was constituted with 1 L. paracasei, 1 L. salivarius and 1 L. brevis strains. The last two strains were isolated in PA colonised patients.
(94) Nasal Priming with Lactobacilli Enhances the Survival Rate
(95) C57BL/6 mice were inoculated intranasally with each blend of Lactobacilli 18 hours prior to PAO1 administration. All Control PAO1 mice died but two (12% survival). Mice receiving L.psb were fully protected (100% survival) (p<0.001). None of the Control L.psb mice died nor exhibited any clinical signs of distress (
(96) Administration of Lactobacilli Decreases the Lung PA Load
(97) Significant decrease of PAO1 lung load observed 24 h postinfection in presence of Lpsb cocktail (9.49×102 CFU/g, p=0.010) and Ls strain (7.81×102 CFU/g p=0.011) whereas a moderate impact of Lb strain (1.16×103 CFU/g p=0.056) is observed compared to the control PAO1 group (9.34×103 CFU/g) (
(98) White Blood Cells Count and Cytokines Analysis in BAL
(99) To elucidate the mechanism of the PA lung load reduction, we investigated the WBC recruitment and cytokine synthesis in the BAL (
DISCUSSION
(100) Intranasal administration of Lactobacilli improves lung P. aeruginosa clearance at 24 hours post infection, associated with the decrease of chemokines and proinflammatory cytokines, and a lower neutrophils recruitment.
(101) Used as a prophylactic treatment, Lactobacilli reduce the inflammatory response triggered by PA, which is deleterious on the PA control group. No difference in the PA lung load, white blood cells count or cytokines production was observed between the 2 blends of Lactobacilli administered. This might suggest that there is no correlation between the anti-PA activities screened in vitro on Lactobacillus strains and their abilities to fight against the infection in vivo. The two virulence factors studied for the screening, the elastase and pyocyanin, were chosen because their pathogenicity was confirmed in vivo on murine model of pneumonia (Le Berre et Lau, 2004). Thus, the elastolytic activity was positively correlated to acute lung injury, and PA pyocyanin deficient isogenic mutants induce less tissue damages than the wild strains. As no difference was observed in our study whatever the anti-PA activities of the Lactobacillus strains used, both virulence factors which are quorum sensing dependent probably not interfere directly with the innate immune response in our murine model of PA pneumonia, and nor is the quorum sensing system.
(102) Considering the different mechanisms of action of the probiotics, direct antimicrobial activity, reinforcement of the epithelium barrier function and immunomodulation (Alexandre, 2014), other hypothesis than the inhibition of the PA virulence factors must be formulated to understand how Lactobacilli may act against PA infection. Several publications studying the effect of Lactobacillus administration by oral gavage suggest that the probiotic protective abilities of Lactobacillus are based on the immunomodulation mediated by the gut-lung axis. However, in our study, Lactobacilli were administrated intranasally to liberate from this axis and observed their action on the respiratory tract when directly administered in situ. Nevertheless, similarities probably exist, as the microbiota modification occurring in the gut and leading to the immunomodulation which certainly occurs in the lung too. Lactobacilli could sufficiently stimulate the respiratory mucosal immune system to protect from bacterial infection.
(103) In our study, the PA clearance lead by the Lactobacilli may be based on the modulation of the bactericidal activity and phagocytosis activity of the alveolar macrophage and neutrophils, which recruitment is decreased and associated with a lightest production of chemokines when Lactobacilli were administrated compared to the PAO1 group.
CONCLUSION
(104) The administration of Lactobacilli cocktail 18 h prior the PA lung infection decreases significantly the lung bacterial load at 24 h post-infection. Although the mechanisms need to be deeply explored, an immunomodulation effect may be involved, notably through the recruitment of PNN.
(105) Tables:
(106) TABLE-US-00001 TABLE 1 Lactobacillus strains selected for the in vitro screening Patients status of PA colonization Species Number of strains Colonized L. rhamnosus 9 L. fermentum 6 L. paracasei 5 L. gasseri 3 L. salivarius 1 L. crispatus 1 L. johnsonii 1 L. brevis 1 L. parabuchneri 1 L. casei 1 L. plantarum 1 Non colonized L. rhamnosus 5 L. fermentum 4 L. paracasei 4 L. salivarius 2 L. parabuchneri 1 L. plantarum 1 L. gasseri 2 L. johnsonii 1
(107) TABLE-US-00002 TABLE 2 anti-PA activities of the 6 Lactobacillus strains selected to be administrated to the mice (expressed as a ratio of the absorbance observed in presence of the Lactobacillus isolate to the absorbance observed with a monoculture of PAO1). Anti- Anti- elastolytic pyocyanin activity synthesis Blend of Lactobacillus Strains of Lactobacillus (%) (%) “Strong Lactobacilli” L. rhamnosus 2C 61 71 L. fermentum 9C 50 94 L. fermentum 10C 69 76 “Weak Lactobacilli” L. paracasei 9N 114 116 L. salivarius 20C 187 150 L. brevis 24C 141 174
REFERENCES
(108) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.