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Pseudomonas aeruginosa (PA) remains one of the leading causes of nosocomial acute pneumonia. The array of virulence factors expressed by PA and the intense immune response associated with PA pneumonia play a major role in the severity of these infections. New therapeutic approaches are needed to overcome the high resistance of PA to antibiotics and to reduce the direct damage to host tissues. Through its immunomodulatory and anti‐virulence effects, azithromycin (AZM) has demonstrated clinical benefits in patients with chronic PA respiratory infections. However, there is relatively little evidence in PA acute pneumonia. We investigated the effects of AZM, as an adjunctive therapy combined with ceftazidime (CAZ), in a murine model of PA acute pneumonia. We observed that the combined therapy (i) reduces the weight loss of mice 24 h post‐infection (hpi), (ii) decreases neutrophil influx into the lungs at 6 and 24 hpi, while this effect is absent in a LPS‐induced pneumonia or when PA is pretreated with antibiotics and mice do not receive any antibiotics, and that (iii) AZM, alone or with CAZ, modulates the expression of PA quorum sensing regulators and virulence factors (LasI, LasA, PqsE, PhzM, ExoS). Our findings support beneficial effects of AZM with CAZ on PA acute pneumonia by both bacterial virulence and immune response modulations. Further investigations are needed to clarify the exact underlying mechanisms responsible for the reduction of the neutrophils influx and to better discriminate between direct immunomodulatory properties of AZM, and indirect effects on neutrophilia resulting from bacterial virulence modulation. [ABSTRACT FROM AUTHOR]

Down syndrome (DS), the most frequent chromosomic aberration, results from the presence of an extra copy of chromosome 21. The identification of genes which overexpression contributes to intellectual disability (ID) in DS is important to understand the pathophysiological mechanisms involved and develop new pharmacological therapies. In particular, gene dosage of Dual specificity tyrosine phosphorylation Regulated Kinase 1A ( DYRK1A ) and of Cystathionine beta synthase ( CBS ) are crucial for cognitive function. As these two enzymes have lately been the main targets for therapeutic research on ID, we sought to decipher the genetic relationship between them. We also used a combination of genetic and drug screenings using a cellular model overexpressing CYS4 , the homolog of CBS in Saccharomyces cerevisiae , to get further insights into the molecular mechanisms involved in the regulation of CBS activity. We showed that overexpression of YAK1 , the homolog of DYRK1A in yeast, increased CYS4 activity whereas GSK3 β was identified as a genetic suppressor of CBS . In addition, analysis of the signaling pathways targeted by the drugs identified through the yeast-based pharmacological screening, and confirmed using human HepG2 cells, emphasized the importance of Akt/GSK3β and NF-κB pathways into the regulation of CBS activity and expression. Taken together, these data provide further understanding into the regulation of CBS and in particular into the genetic relationship between DYRK1A and CBS through the Akt/GSK3β and NF-κB pathways, which should help develop more effective therapies to reduce cognitive deficits in people with DS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Conan, Léon, Caroff, Rollet, Chaïr, Martin, Bihel, Mignen, Voisset and Friocourt.)

CBS encodes a pyridoxal 5'-phosphate-dependent enzyme that catalyses the condensation of homocysteine and serine to form cystathionine. Due to its implication in some cancers and in the cognitive pathophysiology of Down syndrome, the identification of pharmacological inhibitors of this enzyme is urgently required. However, thus far, attempts to identify such molecules have only led to the identification of compounds with low potency and limited selectivity. We consequently developed an original, yeast-based screening method that identified three FDA-approved drugs of the 8-hydroxyquinoline family: clioquinol, chloroxine and nitroxoline. These molecules reduce CBS enzymatic activity in different cellular models, proving that the molecular mechanisms involved in yeast phenotypic rescue are conserved in mammalian cells. A combination of genetic and chemical biology approaches also revealed the importance of copper and zinc intracellular levels in the regulation of CBS enzymatic activity-copper promoting CBS activity and zinc inhibiting its activity. Taken together, these results indicate that our effective screening approach identified three new potent CBS inhibitors and provides new findings for the regulation of CBS activity, which is crucial to develop new therapies for CBS-related human disorders.

Very little is known about chemical interactions between fungi and their mollusc host within marine environments. Here, we investigated the metabolome of a Penicillium restrictum MMS417 strain isolated from the blue mussel Mytilus edulis collected on the Loire estuary, France. Following the OSMAC approach with the use of 14 culture media, the effect of salinity and of a mussel-derived medium on the metabolic expression were analysed using HPLC-UV/DAD-HRMS/MS. An untargeted metabolomics study was performed using principal component analysis (PCA), orthogonal projection to latent structure discriminant analysis (O-PLSDA) and molecular networking (MN). It highlighted some compounds belonging to sterols, macrolides and pyran-2-ones, which were specifically induced in marine conditions. In particular, a high chemical diversity of pyran-2-ones was found to be related to the presence of mussel extract in the culture medium. Mass spectrometry (MS)- and UV-guided purification resulted in the isolation of five new natural fungal pyran-2-one derivatives-5,6-dihydro-6 S -hydroxymethyl-4-methoxy-2 H -pyran-2-one ( 1 ), (6S, 1' R , 2' S )-LL-P880 β ( 3 ), 5,6-dihydro-4-methoxy-6 S -(1' S , 2' S -dihydroxy pent-3'( E) -enyl)-2 H -pyran-2-one ( 4 ), 4-methoxy-6-(1' R , 2' S -dihydroxy pent-3'( E) -enyl)-2 H -pyran-2-one ( 6 ) and 4-methoxy-2 H -pyran-2-one ( 7 )-together with the known (6S, 1' S , 2' S )-LL-P880 β ( 2 ), (1' R , 2' S )-LL-P880 γ ( 5 ), 5,6-dihydro-4-methoxy-2 H -pyran-2-one ( 8 ), (6 S , 1' S , 2' R) -LL-P880 β ( 9 ), (6 S , 1' S )-pestalotin ( 10 ), 1' R -dehydropestalotin ( 11 ) and 6-pentyl-4-methoxy-2 H -pyran-2-one ( 12 ) from the mussel-derived culture medium extract. The structures of 1 - 12 were determined by 1D- and 2D-MMR experiments as well as high-resolution tandem MS, ECD and DP4 calculations. Some of these compounds were evaluated for their cytotoxic, antibacterial, antileishmanial and in-silico PTP1B inhibitory activities. These results illustrate the utility in using host-derived media for the discovery of new natural products.

This study investigated within-host heterogeneity of 66 Pseudomonas aeruginosa populations from pneumonia in 51 critically ill ventilated patients by examining 30 colonies per bronchoalveolar lavage (BAL). Differences in antibiotic susceptibility and quorum-sensing (QS) phenotypes were observed between the members of 14 (21.2%) and 10 (15.2%) populations, respectively. A significant association was found between QS deficiency and ceftazidime resistance. QS deficiency was associated with various lasR modifications, and was observed in 25 of 51 (49.0%) patients, including seven patients who received ≤48 h of ventilation. This study confirms the need to examine diverse colonies when analysing BAL cultures, particularly in β-lactam-exposed patients, to avoid missing ceftazidime- or imipenem-resistant isolates., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Azithromycin (AZM) is a 15-membered-ring macrolide that presents a broad-spectrum antimicrobial activity against Gram-positive bacteria and atypical microorganisms but suffers from a poor diffusion across the outer-membrane of Gram-negative bacilli, including Pseudomonas aeruginosa (PA). However, AZM has demonstrated clinical benefits in patients suffering from chronic PA respiratory infections, especially cystic fibrosis patients. Since the rise of multidrug-resistant PA has led to a growing need for new therapeutic options, this macrolide has been proposed as an adjunctive therapy. Clinical trials assessing AZM in PA acute pneumonia are scarce. However, a careful examination of the available literature provides good rationales for its use in that context. In fact, 14- and 15-membered-ring macrolides have demonstrated immunomodulatory and immunosuppressive effects that could be of major interest in the management of acute illness. Furthermore, growing evidence supports a downregulation of PA virulence dependent on direct interaction with the ribosomes, and based on the modulation of several key regulators from the Quorum Sensing network. First highlighted in vitro , these interesting properties of AZM have subsequently been confirmed in the animal models. In this review, we systematically analyzed the literature regarding AZM immunomodulatory and anti-PA effects. In vitro and in vivo studies, as well as clinical trials were reviewed, looking for rationales for AZM use in PA acute pneumonia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Leroy, Caillon, Caroff, Broquet, Corvec, Asehnoune, Roquilly and Crémet.)

Background: The risk benefit ratio has been clearly proven for a long time to be in favor of vaccination. However, the vaccine distrust is still increasing in the French population and vaccination coverage could be improved. There is a real need to increase confidence in vaccination. In this study, we interviewed heath care workers to collect their opinion concerning vaccination hesitancy amongst their patients., Methods: A qualitative study, using semi structured interviews, was performed. All the Pharmacists and doctors were interviewed by the same person using an interview guide prepared by a multidisciplinary team. Interviews were recorded and transcribed verbatim, then used as a basis for analysis and synthesis of the areas where improvement seems possible for the health care workers interviewed., Results: Data saturation was obtained after 10 interviews. Analysis of the verbatim allowed the classification of the leads proposed by health care workers in three main themes: improvement of transparency and restoration of the trust for vaccine policy, improved initial and further training of health care workers, and a better communication towards the population., Conclusion: Results provide helpful insights into practical avenues to improve the vaccination confidence. If some of them need an in-depth reflection, others could be easily implemented in order to increase the adhesion of the population to vaccination, and consequently the vaccination coverage., (Copyright © 2020 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.)

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K D of 108 ± 2 nM and has a high thermostability (T m  = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications., (© 2019 Wiley Periodicals, Inc.)

Treatment with antibiotics leads to the selection of isolates with increased resistance. We investigated if evolution towards resistance was associated with virulence changes, in the context of P. aeruginosa ventilator-associated pneumonia (VAP). Four patients were selected because they had multiple VAP episodes during short periods (12 days to 5 weeks), with emergence of resistance. We performed whole-genome sequencing of 12 P. aeruginosa from bronchoalveolar lavages or blood culture (3 isolates per patient). Production of quorum sensing-dependent virulence factors, serum resistance, cytotoxicity against A549 cells, biofilm production, and twitching motility were studied. Each patient was infected with a unique strain. For all patients, resistance development was explained by genetic events in ampD, mexR or oprD. Additional variations were detected in virulence- and/or fitness-associated genes (algB, gacA, groEL, lasR, mpl, pilE, pilM, rhlR) depending on the strain. We noticed a convergence towards quorum sensing deficiency, correlated with a decrease of pyocyanin and protease production, survival in serum, twitching motility and cytotoxicity. In one patient, changes in pilM and pilE were related to enhanced twitching. We show that the emergence of resistance in P. aeruginosa is associated with virulence modification, even in acute infections. The consequences of this short-term pathoadaptation need to be explored.

Six long-chain peptaibols, 1 - 6, were identified from agar cultures of a marine-derived Trichoderma longibrachiatum Rifai strain (MMS151) isolated from blue mussels. The structure elucidation was carried out using electrospray ionization ion trap mass spectrometry (ESI-IT-MS) and GC/EI-MS. The long-chain peptaibols exhibited the general building scheme Ac-Aib-Ala-Aib-Ala-Aib-XXX-Gln-Aib-Vxx-Aib-Gly-XXX-Aib-Pro-Vxx-Aib-XXX-Gln-Gln-Pheol and were similar or identical to recurrent 20-residue peptaibols produced by Trichoderma spp. Three new sequences were identified and were called longibrachins A-0, A-II-a, and A-IV-b. The isolated peptaibols were assayed for cytotoxic, antibacterial, and antifungal activities, and acute toxicity on Dipteran larvae., (© 2016 Verlag Helvetica Chimica Acta AG, Zürich.)

Escherichia coli is one of the first causes of Gram-negative orthopedic implant infections (OII), but little is known about the pathogenicity of this species in such infections that are increasing due to the ageing of the population. We report how this pathogen interacts with human osteoblastic MG-63 cells in vitro, by comparing 20 OII E. coli strains to two Staphylococcus aureus and two Pseudomonas aeruginosa strains. LDH release assay revealed that 6/20 (30%) OII E. coli induced MG-63 cell lysis whereas none of the four control strains was cytotoxic after 4 h of coculture. This high cytotoxicity was associated with hemolytic properties and linked to hlyA gene expression. We further showed by gentamicin protection assay and confocal microscopy that the non-cytotoxic E. coli were not able to invade MG-63 cells unlike S. aureus strains (internalization rate <0.01% for the non-cytotoxic E. coli versus 8.88 ± 2.31% and 4.60 ± 0.42% for both S. aureus). The non-cytotoxic E. coli also demonstrated low adherence rates (<7%), the most adherent E. coli eliciting higher IL-6 and TNF-α mRNA expression in the osteoblastic cells. Either highly cytotoxic or slightly invasive OII E. coli do not show the same infection strategies as S. aureus towards osteoblasts., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Gastrointestinal disturbances are a side-effect frequently associated with haematological malignancies due to the intensive cytotoxic treatment given in connection with bone marrow transplantation (BMT). However, intestinal microbiota changes during chemotherapy remain poorly described, probably due to the use of culture-based and low-resolution molecular methods in previous studies. The objective of our study was to apply a next generation DNA sequencing technology to analyse chemotherapy-induced changes in faecal microbiota. We included eight patients with non-Hodgkin's lymphoma undergoing one course of BMT conditioning chemotherapy. We collected a prechemotherapy faecal sample, the day before chemotherapy was initiated, and a postchemotherapy sample, collected 1 week after the initiation of chemotherapy. Total DNA was extracted from faecal samples, denaturing high-performance liquid chromatography based on amplification of the V6 to V8 region of the 16S ribosomal RNA (rRNA) gene, and 454-pyrosequencing of the 16 S rRNA gene, using PCR primers targeting the V5 and V6 hypervariable 16S rRNA gene regions were performed. Raw sequence data were screened, trimmed, and filtered using the QIIME pipeline. We observed a steep reduction in alpha diversity and significant differences in the composition of the intestinal microbiota in response to chemotherapy. Chemotherapy was associated with a drastic drop in Faecalibacterium and accompanied by an increase of Escherichia. The chemotherapy-induced shift in the intestinal microbiota could induce severe side effects in immunocompromised cancer patients. Our study is a first step in identifying patients at risk for gastrointestinal disturbances and to promote strategies to prevent this drastic shift in intestinal microbiota.

Objectives: This study reports details on Escherichia coli isolates recovered from a cystic fibrosis (CF) patient in order to understand how this pathogen adapts to and resists broad-spectrum antipseudomonal therapy in this context., Methods: Five E. coli isolates were obtained from various clinical samples (airways, urine or dialysis catheter) over a 7 month period covering a double-lung transplantation. All isolates were analysed in terms of clonality [enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing], virulence profiles (phylogroup and search for 15 virulence genes), growth patterns (morphotype, biofilm-forming ability and growth rate), hypermutability and antimicrobial susceptibility, with molecular characterization of β-lactamases and porins., Results: The five isolates shared similar ERIC-PCR profiles and sequence types (ST1193) and exhibited the same virulence profile. The respiratory isolates were strong mutators, exhibited mucoid or small-colony morphotypes, exhibited strong biofilm-forming ability and grew slowly compared with the others. All isolates were highly resistant to ceftazidime. The respiratory isolates showed reduced susceptibility to cefepime and high resistance to aztreonam. The patient had received a 31 day course of ceftazidime/aztreonam until transplantation. All isolates harboured the same wild-type chromosomal AmpC. A CMY-2 enzyme was detected in the non-respiratory isolates. The respiratory isolates harboured L293S and V211A/L293S new CMY-2 variants, which were designated CMY-94 and CMY-95, respectively. OmpF porin loss was observed in the non-respiratory isolates., Conclusions: Our study shows that, similarly to Pseudomonas aeruginosa, E. coli can undergo phenotypic and genomic changes in the CF context. For the first time, we identified an in vivo expanded-spectrum evolution of the CMY-2 β-lactamase, during bacterial persistence in the CF lung.