Compatibility between the plant growth-promoting rhizobacteria Azospirillum and Pseudomonas on roots

Plant Growth-Promoting Rhizobacteria (PGPR) can form an associative symbiosis with plants, which results in stimulation of plant growth. PGPR harbour different phytobeneficial mechanisms (non-symbiotic nitrogen fixation, phytohormone synthesis, etc.). Various PGPR can interact with the same host plant, and it is possible that their phytobeneficial effects will be influenced by the interactions between these PGPR. The objective of this doctoral work was to characterize PGPR compatibility in the rhizosphere of the same host plant, in the case of model bacteria belonging to the genera Azospirillum and Pseudomonas. Because certain phytobeneficial Pseudomonas produce antimicrobial metabolites, such as 2,4-diacetylphloroglucinol (DAPG), we have first examined if DAPG production capacity could be involved in Azospirillum inhibition. In vivo experiments, performed with P. fluorescens F113 and a DAPG-negative mutant in gnotobiotic systems, showed that root colonization and phytostimulation activity of certain Azospirillum PGPR was indeed affected in the presence of DAPG-producing Pseudomonas. In order to evaluate Azospirillum root colonization in non-sterile soil, real-time quantitative PCR tools were developed and validated for three prominent Azospirillum strains (A. lipoferum CRT1, A. brasilense UAP-154 and CFN-535). The use of these real-time PCR tools enabled the comparison of the three Azospirillum strains, each co-inoculated with the DAPG-producing strain P. fluorescens F113, in the rhizosphere of maize grown in non-sterile soil. Root colonization levels differed according to the Azospirillum strain, and the combination of phytobeneficial microorganisms led to enhanced maize growth in comparison with non-inoculated plants. These results suggest that PGPR belonging to the genera Pseudomonas and Azospirillum may be compatible in the rhizosphere of a same plant, even if the former have the potential to inhibit some of the latter by producing antimicrobial secondary metabolitesLes bactéries rhizosphériques qualifiées de PGPR (Plant Growth-Promoting Rhizobacteria) forment des symbioses associatives avec les plantes, stimulant la croissance de ces dernières. Les PGPR présentent différents mécanismes phytobénéfiques (production de phytohormones, fixation non symbiotique de l'azote, etc.). Plusieurs PGPR sont susceptibles d'interagir avec la même plante hôte, et il est possible que leurs effets phytobénéfiques soient influencés par les interactions qu'elles auront les unes avec les autres. L'objectif de cette thèse était de caractériser la compatibilité des PGPR dans la rhizosphère d'une même plante hôte, dans le cas de modèles bactériens appartenant aux genres Azospirillum et Pseudomonas. Certains Pseudomonas phytobénéfiques produisant des métabolites antimicrobiens, comme le 2,4-diacétylphloroglucinol (DAPG), nous avons tout d'abord examiné si la capacité à produire du DAPG pouvait inhiber Azospirillum. Les expériences de confrontation réalisées in vivo avec P. fluorescens F113 et un mutant DAPG-négatif, en système gnotobiotique, ont montré que la colonisation racinaire et l'activité phytostimulatrice de certaines PGPR Azospirillum pouvaient effectivement être diminuées en présence de Pseudomonas producteurs de DAPG. Pour évaluer la colonisation racinaire par Azospirillum en sol non stérile, des outils de PCR quantitative en temps réel ont été développés et validés pour trois souches de premier plan (A. lipoferum CRT1, A. brasilense UAP-154 et CFN-535). L'utilisation de ces outils a permis la comparaison de ces trois souches d'Azospirillum, chacune co-inoculée avec la souche P. fluorescens F113 productrice de DAPG, sur du maïs cultivé en sol non stérile. Les niveaux de colonisation racinaire différaient selon la souche d'Azospirillum, et la combinaison de microorganismes phytobénéfiques conduisait à une meilleure croissance du maïs par comparaison avec des plantes non inoculées. Les résultats suggèrent que des PGPR des genres Pseudomonas et Azospirillum peuvent être compatibles dans la rhizosphère d'une même plante, même si les premiers ont le potentiel d'inhiber certains des seconds par la production de métabolites secondaires antimicrobien

Compatibilité des bactéries phytobénéfiques Azospirillum et Pseudomonas dans la rhizosphère

Plant Growth-Promoting Rhizobacteria (PGPR) can form an associative symbiosis with plants, which results in stimulation of plant growth. PGPR harbour different phytobeneficial mechanisms (non-symbiotic nitrogen fixation, phytohormone synthesis, etc.). Various PGPR can interact with the same host plant, and it is possible that their phytobeneficial effects will be influenced by the interactions between these PGPR. The objective of this doctoral work was to characterize PGPR compatibility in the rhizosphere of the same host plant, in the case of model bacteria belonging to the genera Azospirillum and Pseudomonas. Because certain phytobeneficial Pseudomonas produce antimicrobial metabolites, such as 2,4-diacetylphloroglucinol (DAPG), we have first examined if DAPG production capacity could be involved in Azospirillum inhibition. In vivo experiments, performed with P. fluorescens F113 and a DAPG-negative mutant in gnotobiotic systems, showed that root colonization and phytostimulation activity of certain Azospirillum PGPR was indeed affected in the presence of DAPG-producing Pseudomonas. In order to evaluate Azospirillum root colonization in non-sterile soil, real-time quantitative PCR tools were developed and validated for three prominent Azospirillum strains (A. lipoferum CRT1, A. brasilense UAP-154 and CFN-535). The use of these real-time PCR tools enabled the comparison of the three Azospirillum strains, each co-inoculated with the DAPG-producing strain P. fluorescens F113, in the rhizosphere of maize grown in non-sterile soil. Root colonization levels differed according to the Azospirillum strain, and the combination of phytobeneficial microorganisms led to enhanced maize growth in comparison with non-inoculated plants. These results suggest that PGPR belonging to the genera Pseudomonas and Azospirillum may be compatible in the rhizosphere of a same plant, even if the former have the potential to inhibit some of the latter by producing antimicrobial secondary metabolitesLes bactéries rhizosphériques qualifiées de PGPR (Plant Growth-Promoting Rhizobacteria) forment des symbioses associatives avec les plantes, stimulant la croissance de ces dernières. Les PGPR présentent différents mécanismes phytobénéfiques (production de phytohormones, fixation non symbiotique de l’azote, etc.). Plusieurs PGPR sont susceptibles d’interagir avec la même plante hôte, et il est possible que leurs effets phytobénéfiques soient influencés par les interactions qu’elles auront les unes avec les autres. L’objectif de cette thèse était de caractériser la compatibilité des PGPR dans la rhizosphère d’une même plante hôte, dans le cas de modèles bactériens appartenant aux genres Azospirillum et Pseudomonas. Certains Pseudomonas phytobénéfiques produisant des métabolites antimicrobiens, comme le 2,4-diacétylphloroglucinol (DAPG), nous avons tout d’abord examiné si la capacité à produire du DAPG pouvait inhiber Azospirillum. Les expériences de confrontation réalisées in vivo avec P. fluorescens F113 et un mutant DAPG-négatif, en système gnotobiotique, ont montré que la colonisation racinaire et l’activité phytostimulatrice de certaines PGPR Azospirillum pouvaient effectivement être diminuées en présence de Pseudomonas producteurs de DAPG. Pour évaluer la colonisation racinaire par Azospirillum en sol non stérile, des outils de PCR quantitative en temps réel ont été développés et validés pour trois souches de premier plan (A. lipoferum CRT1, A. brasilense UAP-154 et CFN-535). L’utilisation de ces outils a permis la comparaison de ces trois souches d’Azospirillum, chacune co-inoculée avec la souche P. fluorescens F113 productrice de DAPG, sur du maïs cultivé en sol non stérile. Les niveaux de colonisation racinaire différaient selon la souche d’Azospirillum, et la combinaison de microorganismes phytobénéfiques conduisait à une meilleure croissance du maïs par comparaison avec des plantes non inoculées. Les résultats suggèrent que des PGPR des genres Pseudomonas et Azospirillum peuvent être compatibles dans la rhizosphère d’une même plante, même si les premiers ont le potentiel d’inhiber certains des seconds par la production de métabolites secondaires antimicrobien

Etude de l'antibiorésistance émergente à l'hôpital de Montpellier par une approche d'épidémiologie basée sur les eaux usées

International audience1. Introduction et objectif La surveillance de l’antibiorésistance permet une prévention argumentée et permet d’être réactif pour contrôler les crises. Le suivi individualisé des patients porteurs de bactéries hautement résistantes émergentes (BHRe) et de leurs contacts est recommandé mais les données épidémiologiques qui en découlent sont parcellaires. De nouveaux outils pourraient alléger le suivi et rendre la surveillance plus exhaustive. L’épidémiologie basée sur les eaux usées s’est montrée efficace lors de la pandémie COVID-19. Nous proposons une surveillance sur 18 semaines par quantification de 3 gènes associés aux BHRe (blaOXA-48, blaNDM, vanA) et blaCTX-M en tant que « standard d’endémicité » dans les eaux usées de 3 bâtiments hospitaliers accueillant des patients avec différents risques de portage de BHRe.2. Matériel et MéthodesLes eaux usées sont collectées par préleveur automatique (IAGE®) à la sortie de 3 bâtiments, HOM, DIG et LN. HOM et DIG accueillent un taux élevé de patients BHRe. LN (400 lits), accueillant des patients sans risque ajouté de portage BHRe, est pris pour déterminer un signal de base. Les gènes blaCTX-M, blaOXA-48, blaNDM et vanA sont quantifiés par qPCR et ddPCR par rapport au gène d’ARNr 16S. Enterobactéries productrices de carbapénémase (EPC) et entérocoques résistants à la vancomycine (ERV) sont dénombrés par culture sur milieux sélectifs et identifiés par MALDI-TOF-SM.3. RésultatsLe signal varie selon les gènes, les bâtiments et dans le temps, confirmant l’intérêt potentiel de cette approche comme outil de surveillance. La quantification des gènes dans le bâtiment LN donne un signal de base régulier dans le temps et significativement différent des signaux HOM et DIG, ce qui permet d’envisager la détermination de seuils cible/alerte. Le signal blaOXA-48 est souvent comparable ou supérieur au signal blaCTX-M y compris dans le bâtiment LN, suggérant la possible sous-évaluation de la circulation des EPC OXA-48. Les EPC du genre Citrobacter ont une forte prévalence. vanA et blaNDM présentent une dynamique épidémique / sporadique comme attendu. La corrélation entre le signal et la présence d’un patient porteur connu est faible dû au faible nombre de patients porteurs connus sur la période. 4. ConclusionNous montrons la faisabilité de l’épidémiologie de l’antibiorésistance basée sur les eaux usées à l’hôpital. La relation nombre de patients porteurs / copies de gènes de résistance dans les eaux usées doit être étudiée. Elle est certainement moins directe que celle observée pour le SARS-CoV-2 du fait des transferts de gènes entre bactéries

Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings

International audienceAzospirillum lipoferum CRT1 is a promising phytostimulatory PGPR for maize, whose effect on the plant is cell density-dependent. A nested PCR method is available for detection of the strain but does not allow quantification. The objective was to develop a real-time PCR method for quantification of A. lipoferum CRT1 in the rhizosphere of maize seedlings. Primers were designed based on a strain-specific RFLP marker, and their specificity was verified under qualitative and quantitative PCR conditions based on successful CRT1 amplification and absence of cross-reaction with genomic DNA from various rhizosphere strains. Real-time PCR conditions were then optimized using DNA from inoculated or non-inoculated maize rhizosphere samples. The detection limit was 60 fg DNA (corresponding to 19 cells) with pure cultures and 4 × 104 CFU equivalents g−1 lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 104 CFU equivalents g−1. Assessment of CRT1 rhizosphere levels in a field trial was in accordance with estimates from semi-quantitative PCR targeting another locus. This real-time PCR method, which is now available for direct rhizosphere monitoring of A. lipoferum CRT1 in greenhouse and field experiments, could be used as a reference for developing quantification tools for other Azospirillum inoculants

Wastewater-based Epidemiology (WBE): toward a relevant tool to monitor hospital antimicrobial resistance

International audienceResistance to antibiotics can lead to therapeutic failures of infectious diseases but also can slow down advances in intensive medicine or surgery. Among extremely resistant bacteria (XDR), carbapenemase-producing enterobacteria (CPE) are of particular concern because of their capacities to hydrolyze most of the β-lactam antibiotics including the last resort antibiotic carbapenems [1]. In France, blaOXA-48 and blaNDM are the most widespread carbapenemases. Another kind of XDR of concern are vancomycin-resistant Enterococci (VRE) associated with vanA gene [2]. Based on current knowledge, these 3 antimicrobial resistance genes (ARG) are emerging in France and spread through an epidemic mode. However, data on XDR carriage is patchy in general population because it is monitored only in high risk-hospitalized patients. New tools are needed to make surveillance of emerging resistances more comprehensive.During SARS-CoV-2 pandemic, efficiency of wastewater-based epidemiology (WBE) has been showed [3]. Because CPE and VRE are gastrointestinal bacteria, we propose WBE to improve the surveillance in human population. To achieve this goal, we developed a strategy to monitor blaOXA-48, blaNDM and vanA genes in wastewater from three hospital wards. Two wards were at high-risk for XDR carriage in patients. The last one was considered at low-risk and was used as a standard. blaCTX-M gene was also monitored as “endemic standard” because its endemic character both inside and outside the hospital. Genes’ quantifications were performed by qPCR and dPCR from hospital wastewater samples. CPE and VRE were cultured on selective media, enumerated and identified by MALDI-TOF-MS. Molecular signals were observed both for endemic (blaCTX-M) and emerging ARG (blaOXA-48, blaNDM and vanA) with space-time variations. It was supported by significant positive correlations between results obtained by qPCR and dPCR. For the low-risk ward, a stable baseline signal was observed, with quantities of genes significantly different from those of the 2 high-risk wards. This suggests the possibility to determine a basal state of ARG quantifications in hospital wastewater to set up alert thresholds. blaNDM and vanA had epidemic dynamics as expected. Furthermore, blaOXA-48 signal was either similar or superior to blaCTX-M signal in all the wards, suggesting an undervaluation of OXA-48 diffusion in France, or a possible amplification of the signal in the hydric bacterial community of wastewater pipes. Finally, there was no taxonomic overlap between resistant bacterial communities from patients and from wastewater. These results could be in favor of ARG exchanges between patients’ and waterborne bacteria.This study highlights the feasibility of AMR monitoring with WBE in a hospital. To validate this approach, further studies had to be performed considering ARG transfers and persistence

Is environmental epidemiology suitable for monitoring emerging antibiotic resistance in human ? A pilot study in wastewater of hospital buildings

International audienceAntibiotic resistance is a major global threat, especially to human health. Bacteria display more and more resistance genes and Multi-Dug Resistant (MDR) bacteria are now endemic worldwide, for instance enterobacteria producing extended spectrum betalactamase (EBLSE) such as CTX-M betalactamase. Extremely resistant bacteria (XDR) are emerging and their epidemiology should be monitored thoroughly. Among XDR bacteria, carbapenemase-producing enterobacteria (CPE) are of particular concern [1,2] because they produce specific enzymes that hydrolyze carbapenems (last ressort antibiotics in severe infections [2,3]) [4] but also most molecules in the β-lactam antibiotic class. In France, the most widespread carbapenemases are firstly oxacillinases of type OXA-48, then NDM metallo-β-lactamases [5]. A second type of emerging XDR bacteria are vancomycin-resistant enterococci (VRE) carrying vanA resistance gene. Surveillance of antibiotic resistance is a key to prevention mainly for emerging XDR [6]. Monitoring of pathogens in wastewater has been effective during the SARS-CoV-2 pandemic. In this study, we developed a strategy to monitor XDR genes blaOXA-48, blaNDM et vanA in wastewater from three hospital buildings occupied by patients with various risk for XDR carriage. We also compared the rate of emerging XDR genes to the rate of blaCTX-M chosen as marker of endemic MDR

Biocontrol and biofertilizer activities of the Streptomyces anulatus S37: an endophytic actinomycete with biocontrol and plant-growth promoting activities

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Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap

This work describes the formation of transformation products (TPs) by the enzymatic degradation at laboratory scale of two highly consumed antibiotics: tetracycline (Tc) and erythromycin (ERY). The analysis of the samples was carried out by a fast and simple method based on the novel configuration of the on-line turbulent flow system coupled to a hybrid linear ion trap – high resolution mass spectrometer. The method was optimized and validated for the complete analysis of ERY, Tc and their transformation products within 10 min without any other sample manipulation. Furthermore, the applicability of the on-line procedure was evaluated for 25 additional antibiotics, covering a wide range of chemical classes in different environmental waters with satisfactory quality parameters. Degradation rates obtained for Tc by laccase enzyme and ERY by EreB esterase enzyme without the presence of mediators were ∼78% and ∼50%, respectively. Concerning the identification of TPs, three suspected compounds for Tc and five of ERY have been proposed. In the case of Tc, the tentative molecular formulas with errors mass within 2 ppm have been based on the hypothesis of dehydroxylation, (bi)demethylation and oxidation of the rings A and C as major reactions. In contrast, the major TP detected for ERY has been identified as the “dehydration ERY-A”, with the same molecular formula of its parent compound. In addition, the evaluation of the antibiotic activity of the samples along the enzymatic treatments showed a decrease around 100% in both case