New anti-Pseudomonas aeruginosa therapy for cystic fibrosis patients : the use of Lactobacillus spp.

L’augmentation préoccupante de la résistance aux antibiotiques de Pseudomonas aeruginosa (PA) nécessite la recherche de thérapies alternatives telles que les Lactobacillus. Dans cette thèse, différents travaux ont été réalisés :1) Sur un modèle murin de pneumonie aigüe à PA, nous avons démontré l’effet bénéfique de l’administration intratrachéale d’un mélange de 3 souches de Lactobacillus provenant de lait ou de la cavité orale de patients sains.2) Nous avons ensuite prospectivement étudié la population en Lactobacillus des expectorations de patients atteints de mucoviscidose (CF). La prévalence moyenne de portage était de 61%.3) Parmi ces Lactobacillus issus de l’écosystème respiratoire des patients CF, nous avons constitué deux mélanges de 3 souches de Lactobacillus, sélectionnées pour leur activité anti-élastolytique et antipyocyanine in vitro. L’administration intranasale de ces mélanges à des souris C57Bl/6, 18h avant leur infection par PAO1, améliore significativement la survie à 7 jours des souris, ainsi que la clairance pulmonaire en PAO1 à 24h post-infection. Une diminution significative du recrutement pulmonaire des neutrophiles et des cytokines proinflammatoires était observée, associée à une augmentation de celle en IL-10.Ainsi, cette thèse démontre les effets bénéfiques de l’administration prophylactique des Lactobacillus par voie respiratoire sur la pneumonie aigue à PA. Ce résultat serait lié à l’immunomodulation de ces bactéries.Enfin, les propriétés contre l’élastase et la pyocyanine de ces souches in vitro ne présagent pas de leur activité in vivo, avec le mélange L. paracasei 9N, L. brevis 24C et L. salivarius 20C présentant seulement in vivo la meilleure activité (Brevet BIO17555).The alarming increase in antibiotic resistance of Pseudomonas aeruginosa (PA) requires studying alternative therapies, such as Lactobacillus. In this thesis, several studies were carried out:1) In a murine model of acute PA pneumonia, we have demonstrated the beneficial effect of intratracheal administration of a mixture of three Lactobacillus strains from milk or the oral cavity of healthy patients.2) We then prospectively studied the Lactobacillus population in sputum of patients with cystic fibrosis (CF). The average prevalence of carry was 61%.3) Lactobacillus from the respiratory ecosystem of CF patients were used to establish two mixtures of 3 Lactobacillus strains. Strains were selected for their antielastolytic and anti-pyocyanin effects in vitro.Intranasal administration of these mixtures to C57Bl / 6 mice, 18h prior to PAO1 infection significantly improves 7-day survival and pulmonary clearance of PAO1 24h postinfection.A significant decrease in lung neutrophil recruitment and pro-inflammatory cytokines was observed, while the production of IL-10 increased.This thesis demonstrates the beneficial effects of prophylactic respiratory administration of Lactobacillus on acute PA pneumonia. Anti-PA properties in vitro are not an indication of the in vivo activity. The mixture containing L. paracasei 9N, L. brevis 24C, and L. salivarius 20C show the best anti-PA activity (Patent BIO17555). This result is likely related to an immunomodulating effect of these bacteria

Nouvelle thérapeutique anti-Pseudomonas aeruginosa dans la mucoviscidose : les Lactobacillus spp.

The alarming increase in antibiotic resistance of Pseudomonas aeruginosa (PA) requires studying alternative therapies, such as Lactobacillus. In this thesis, several studies were carried out:1) In a murine model of acute PA pneumonia, we have demonstrated the beneficial effect of intratracheal administration of a mixture of three Lactobacillus strains from milk or the oral cavity of healthy patients.2) We then prospectively studied the Lactobacillus population in sputum of patients with cystic fibrosis (CF). The average prevalence of carry was 61%.3) Lactobacillus from the respiratory ecosystem of CF patients were used to establish two mixtures of 3 Lactobacillus strains. Strains were selected for their antielastolytic and anti-pyocyanin effects in vitro.Intranasal administration of these mixtures to C57Bl / 6 mice, 18h prior to PAO1 infection significantly improves 7-day survival and pulmonary clearance of PAO1 24h postinfection.A significant decrease in lung neutrophil recruitment and pro-inflammatory cytokines was observed, while the production of IL-10 increased.This thesis demonstrates the beneficial effects of prophylactic respiratory administration of Lactobacillus on acute PA pneumonia. Anti-PA properties in vitro are not an indication of the in vivo activity. The mixture containing L. paracasei 9N, L. brevis 24C, and L. salivarius 20C show the best anti-PA activity (Patent BIO17555). This result is likely related to an immunomodulating effect of these bacteria.L’augmentation préoccupante de la résistance aux antibiotiques de Pseudomonas aeruginosa (PA) nécessite la recherche de thérapies alternatives telles que les Lactobacillus. Dans cette thèse, différents travaux ont été réalisés :1) Sur un modèle murin de pneumonie aigüe à PA, nous avons démontré l’effet bénéfique de l’administration intratrachéale d’un mélange de 3 souches de Lactobacillus provenant de lait ou de la cavité orale de patients sains.2) Nous avons ensuite prospectivement étudié la population en Lactobacillus des expectorations de patients atteints de mucoviscidose (CF). La prévalence moyenne de portage était de 61%.3) Parmi ces Lactobacillus issus de l’écosystème respiratoire des patients CF, nous avons constitué deux mélanges de 3 souches de Lactobacillus, sélectionnées pour leur activité anti-élastolytique et antipyocyanine in vitro. L’administration intranasale de ces mélanges à des souris C57Bl/6, 18h avant leur infection par PAO1, améliore significativement la survie à 7 jours des souris, ainsi que la clairance pulmonaire en PAO1 à 24h post-infection. Une diminution significative du recrutement pulmonaire des neutrophiles et des cytokines proinflammatoires était observée, associée à une augmentation de celle en IL-10.Ainsi, cette thèse démontre les effets bénéfiques de l’administration prophylactique des Lactobacillus par voie respiratoire sur la pneumonie aigue à PA. Ce résultat serait lié à l’immunomodulation de ces bactéries.Enfin, les propriétés contre l’élastase et la pyocyanine de ces souches in vitro ne présagent pas de leur activité in vivo, avec le mélange L. paracasei 9N, L. brevis 24C et L. salivarius 20C présentant seulement in vivo la meilleure activité (Brevet BIO17555)

La spectrométrie de masse Maldi-Tof appliquée à l'identification des espèces du complexe mycobacterium abscessus

Mycobacterium abscessus est un pathogène émergent chez le patient mucoviscidose. Trois espèces non différenciables en routine diagnostique en constituent le complexe: M. abscessus, M. Massiliense et M. bolletii. Des différences substantielles dans leur profil clinique et de sensibilité aux antibiotiques justifient leur typage à l'espèce. L'implantation croissante de la spectrométrie de masse (SM) dans les laboratoires offre une nouvelle perspective pour leur identification. Les performances de la SM MALDI-TOF dans l'identification des espèces du complexe ont été évaluées sur un souchier de 36 isolats cliniques et 3 souches de référence. L'analyse des spectres de masse de 9 isolats et des souches de référence a permis la caractérisation de 5 pics discriminants, ensuite validés sur le reste du souchier. Tous les isolats de M. abscessus et M. massiliense ont été correctement identifiés. Une erreur d'identification a été observée avec un isolat de M. bolletii confondu avec M. abscessus.Mycobacterium abscessus is an emerging pathogen in cystic fibrosis patients. M. abscessus complex is composed of 3 species: M. abscessus, M. massiliense et M. bolletii, which are not distinguishable in routine by usual techniques. There are differences considering their clinical profiles and antibiotics susceptibilities, which require correct typing to the specie level. The increasingly use of mass spectrometry (MS) in laboratories is a new opportunity for there identification. In this study, the ability to discriminate MABC at the specie level using MALDI-TOF MS was evaluated on 36 clinical isolates and 3 reference strains. Mass spectra obtained for 9 isolates and 3 reference strains were analyzed and highlighted 5 discriminating peaks, which were confirmed by analyzing the other isolates. Every isolates of M. abscessus and M. massiliense were correctly typed. One misidentification was observed with a M. bolletii isolate, mistaken with M. abscessus.LYON1-BU Santé (693882101) / SudocRENNES1-BU Santé (352382103) / SudocSudocFranceF

The hook effect in calcitonin immunoradiometric assay: a case report.

International audienceUNLABELLED: The hook effect, which has long been detected and documented for immunoradiometric assays (IRMA) such as those measuring prolactin or thyroglobulin, occurs when the serum antigen level is extremely high, thus inducing a bias in the methodology of measurement. RESULTS: We report the case of an 80-year-old man with confirmed medullary thyroid carcinoma (MTC). In the case reported here, the clinical status of the patient contrasts with his tumor antigen, serum calcitonin (CT), concentrations. The measured increased CT concentrations revealed the presence of a hook effect. This phenomenon occurs due to an excess of antigen during the one-step IRMA where the signal antibodies, bound to the non-captured antigens, are washed out during the measurement, inducing the loss of signal. Aiming to prevent the "hook effect", successive dilutions of the same sample of serum were done. CONCLUSIONS: Previous studies have shown when one-step IRMA reveals high concentrations of a tumor serum antigen (i.e. prolactin or thyroglobulin), a two-step IRMA or a systematic 1:10 dilution of the serum sample prevents the formation of the "hook effect". In our case report, the CT "hook effect" formation was prevented by performing serial dilutions of the serum sample

Priming with intranasal lactobacilli prevents Pseudomonas aeruginosa acute pneumonia in mice

Abstract Background Increasing resistance to antibiotics of Pseudomonas aeruginosa leads to therapeutic deadlock and alternative therapies are needed. We aimed to evaluate the effects of Lactobacillus clinical isolates in vivo, through intranasal administration on a murine model of Pseudomonas aeruginosa pneumonia. Results We screened in vitro 50 pulmonary clinical isolates of Lactobacillus for their ability to decrease the synthesis of two QS dependent-virulence factors (elastase and pyocyanin) produced by Pseudomonas aeruginosa strain PAO1. Two blends of three Lactobacillus isolates were then tested in vivo: one with highly effective anti-PAO1 virulence factors properties (blend named L.rff for L. rhamnosus, two L. fermentum strains), and the second with no properties (blend named L.psb, for L. paracasei, L. salivarius and L. brevis). Each blend was administered intranasally to mice 18 h prior to PAO1 pulmonary infection. Animal survival, bacterial loads, cytological analysis, and cytokines secretion in the lungs were evaluated at 6 or 24 h post infection with PAO1. Intranasal priming with both lactobacilli blends significantly improved 7-day mice survival from 12% for the control PAO1 group to 71 and 100% for the two groups receiving L.rff and L.psb respectively. No mortality was observed for both control groups receiving either L.rff or L.psb. Additionally, the PAO1 lung clearance was significantly enhanced at 24 h. A 2-log and 4-log reduction was observed in the L.rff + PAO1 and L.psb + PAO1 groups respectively, compared to the control PAO1 group. Significant reductions in neutrophil recruitment and proinflammatory cytokine and chemokine secretion were observed after lactobacilli administration compared to saline solution, whereas IL-10 production was increased. Conclusions These results demonstrate that intranasal priming with lactobacilli acts as a prophylaxis, and avoids fatal complications caused by Pseudomonas aeruginosa pneumonia in mice. These results were independent of in vitro anti-Pseudomonas aeruginosa activity on QS-dependent virulence factors. Further experiments are required to identify the immune mechanism before initiating clinical trials

The Emergence of Non-Tuberculous Mycobacterial Lymphadenitis in Children After the Discontinuation of Mandatory BCG Immunization in France

International audienceEighty-five children were diagnosed with culture-confirmed non-tuberculous mycobacterial cervical lymphadenitis within the MYCOMED surveillance network in 2004-2013. The mean incidence sharply increased from 0.57 to 3.7 per 100,000 children/year, following discontinuation of mandatory BCG immunization in 2007. Cases were due to Mycobacterium avium (62.3%), M. intracellulare (15.3%), and M. lentiflavum(12.9%). Outcome was favorable in all, with or without surgery or antimycobacterial treatment

<em>Porphyromonas</em>, a potential predictive biomarker of Pseudomonas aeruginosa pulmonary infection in cystic fibrosis

Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/bmjresp-2018-000374).International audienceIntroduction : Pseudomonas aeruginosa pulmonary infections are the primary cause of morbi-mortality in patients with cystic fibrosis (CF). In this cohort study, the objective was to identify candidate biomarkers of P. aeruginosa infection within the airway microbiota. Methods : A 3-year prospective multicentre study (PYOMUCO study) was conducted in Western France and included patients initially P. aeruginosa free for at least 1year. A 16S-targeted metagenomics approach was applied on iterative sputum samples of a first set of patients (n=33). The composition of airway microbiota was compared according to their P. aeruginosa status at the end of the follow-up (colonised vs non-colonised), and biomarkers associated with P. aeruginosa were screened. In a second step, the distribution of a candidate biomarker according to the two groups of patients was verified by qPCR on a second set of patients (n=52) coming from the same cohort and its load quantified throughout the follow-up. Results : Porphyromonas (mainly P. catoniae) was found to be an enriched phylotype in patients uninfected by P. aeruginosa (pDiscussion : Further studies on replication cohorts are needed to validate this potential predictive biomarker, which may be relevant for the follow-up in the early years of patients with CF. The identification of infection candidate biomarkers may offer new strategies for CF prevision medicine

High prevalence of OXA-23 carbapenemase-producing Proteus mirabilis among amoxicillin-clavulanate resistant isolates in France

International audienceIn this multicentric study performed in 12 French hospitals, we reported that 26.9% (14/52) of the amoxicillin/clavulanate-resistant Proteus mirabilis isolates produced the OXA-23 carbapenemase. We found that inhibition zone diameter less than 11 mm around amoxicillin/clavulanate disc was an accurate screening cut-off to detect these OXA-23 producers. We confirmed by whole genome sequencing that these OXA-23-producers all belonged to the same lineage that has been demonstrated to disseminate OXA-23 or OXA-58 in P. mirabilis

French national epidemiology of bacterial superinfections in ventilator-associated pneumonia in patients infected with COVID-19: the COVAP study

International audienceAbstract Background Description and comparison of bacterial characteristics of ventilator-associated pneumonia (VAP) between critically ill intensive care unit (ICU) patients with COVID-19-positive, COVID + ; and non-COVID-19, COVID-. Methods Retrospective, observational, multicenter study that focused on French patients during the first wave of the pandemic (March–April 2020). Results 935 patients with identification of at least one bacteriologically proven VAP were included (including 802 COVID +). Among Gram-positive bacteria, S. aureus accounted for more than two-thirds of the bacteria involved, followed by Streptococcaceae and enterococci without difference between clinical groups regarding antibiotic resistance. Among Gram-negative bacteria, Klebsiella spp. was the most frequently observed bacterial genus in both groups, with K. oxytoca overrepresented in the COVID- group (14.3% vs . 5.3%; p &lt; 0.05). Cotrimoxazole-resistant bacteria were over-observed in the COVID + group (18.5% vs . 6.1%; p &lt;0.05), and after stratification for K. pneumoniae (39.6% vs . 0%; p &lt;0.05). In contrast, overrepresentation of aminoglycoside-resistant strains was observed in the COVID- group (20% vs . 13.9%; p &lt; 0.01). Pseudomonas sp. was more frequently isolated from COVID + VAPs (23.9% vs . 16.7%; p &lt;0.01) but in COVID- showed more carbapenem resistance (11.1% vs . 0.8%; p &lt;0.05) and greater resistance to at least two aminoglycosides (11.8% vs . 1.4%; p &lt; 0.05) and to quinolones (53.6% vs . 7.0%; p &lt;0.05). These patients were more frequently infected with multidrug-resistant bacteria than COVID + (40.1% vs . 13.8%; p &lt; 0.01). Conclusions The present study demonstrated that the bacterial epidemiology and antibiotic resistance of VAP in COVID + is different from that of COVID- patients. These features call for further study to tailor antibiotic therapies in VAP patients