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The human gut microbiota has co-evolved with humans by exchanging bidirectional signals. This study aims at deepening the knowledge of this crucial relationship by analyzing phenotypic and interactive responses of the probiotic Enterococcus faecium NCIMB10415 (E. faecium SF68) to the top-down signals norepinephrine (NE) and serotonin (5HT), two neuroactive molecules abundant in the gut. We treated E. faecium NCIMB10415 with 100 µM NE and 50 µM 5HT and tested its ability to form static biofilm (Confocal Laser Scanning Microscopy), adhere to the Caco-2/TC7 monolayer, affect the epithelial barrier function (Transepithelial Electrical Resistance) and human dendritic cells (DC) maturation, differentiation, and cytokines production. Finally, we evaluated the presence of a putative hormone sensor through in silico (whole genome sequence and protein modelling) and in vitro (Micro-Scale Thermophoresis) analyses. The hormone treatments increase biofilm formation and adhesion on Caco-2/TC7, as well as the epithelial barrier function. No differences concerning DC differentiation and maturation between stimulated and control bacteria were detected, while an enhanced TNF-α production was observed in NE-treated bacteria. Investigations on the sensor support the hypothesis that a two-component system on the bacterial surface can sense 5HT and NE. Overall, the data demonstrate that E. faecium NCIMB10415 can sense both NE and 5HT and respond accordingly.

We previously showed that the physiological concentration of 17β-estradiol in the vaginal environment is sufficient to affect the membrane dynamics and adhesion phenotype of the Lactobacillus crispatus strain CIP104459. However, L. crispatus is a heterogeneous species. Here, we investigated the effect of 17β-estradiol on the recently isolated L. crispatus vaginal strain V4, related to a cluster distant from CIP104459 and at the limit of being a different subspecies. Grown in the same medium, the two strains expressed a highly similar pool of proteins. However, in contrast to CIP104459, L. crispatus V4 showed high aggregation potential and 17β-estradiol promoted this phenotype. This effect was associated with large changes in cell-surface polarity and Lewis acid/base properties. In addition, we observed no effect on the membrane dynamics, contrary to CIP104459. These results can be explained by differences in the properties and organization of the S layer between the two strains. However, as for CIP104459, 17β-estradiol increased biosurfactant production of L. crispatus V4 and their adhesion to vaginal cells. This suggests that 17β-estradiol agonists would be valuable tools to favor a stable re-implantation of L. crispatus in the vaginal mucosa.

Three bacterial strains, named hOe-66T, hOe-124 and hOe-125, were isolated from the haemolymph of different specimens of the flat oyster Ostrea edulis collected in Concarneau bay (Finistère, France). These strains were characterized by a polyphasic approach, including (i) whole genome analyses with 16S rRNA gene sequence alignment and pangenome analysis, determination of the G+C content, average nucleotide identity (ANI), and in silico DNA–DNA hybridization (isDDH), and (ii) fatty acid methyl ester and other phenotypic analyses. Strains hOe-66T, hOe-124 and hOe-125 were closely related to both type strains Pseudoalteromonas rhizosphaerae RA15T and Pseudoalteromonas neustonica PAMC 28425T with less than 93.3% ANI and 52.3% isDDH values. Regarding their phenotypic traits, the three strains were Gram-negative, 1–2 µm rod-shaped, aerobic, motile and non-spore-forming bacteria. Cells grew optimally at 25 °C in 2.5% NaCl and at 7–8 pH. The most abundant fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0 and C17:1 ω8c. The strains carried a genome average size of 4.64 Mb and a G+C content of 40.28 mol%. The genetic and phenotypic results suggested that strains hOe-66T, hOe-124 and hOe-125 belong to a new species of the genus Pseudoalteromonas . In this context, we propose the name Pseudoalteromonas ostreae sp. nov. The type strain is hOe-66T (=CECT 30303T=CIP 111911T).

Prokaryotes and eukaryotes have coexisted for millions of years. The hormonal communication between microorganisms and their hosts, dubbed inter-kingdom signaling, is a recent field of research. Eukaryotic signals such as hormones, neurotransmitters or immune system molecules have been shown to modulate bacterial physiology. Among them, catecholamines hormones epinephrine/norepinephrine, released during stress and physical effort, or used therapeutically as inotropes have been described to affect bacterial behaviors (i.e., motility, biofilm formation, virulence) of various Gram-negative bacteria (e.g., Escherichia coli, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Vibrio sp.). More recently, these molecules were also shown to influence the physiology of some Gram-positive bacteria like Enterococcus faecalis. In E. coli and S. enterica, the stress-associated mammalian hormones epinephrine and norepinephrine trigger a signaling cascade by interacting with the QseC histidine sensor kinase protein. No catecholamine sensors have been well described yet in other bacteria. This review aims to provide an up to date report on catecholamine sensors in eukaryotes and prokaryotes, their transport, and known effects on bacteria.

Since antiquity, the ubiquitous lactic acid bacteria (LAB) Enterococci, which are just as predominant in both human and animal intestinal commensal flora, have been used (and still are) as probiotics in food and feed production. Their qualities encounter several hurdles, particularly in terms of the array of virulence determinants, reflecting a notorious reputation that nearly prevents their use as probiotics. Additionally, representatives of the Enterococcus spp. genus showed intrinsic resistance to several antimicrobial agents, and flexibility to acquire resistance determinants encoded on a broad array of conjugative plasmids, transposons, and bacteriophages. The presence of such pathogenic aspects among some species represents a critical barrier compromising their use as probiotics in food. Thus, the genus neither has Generally Recognized as Safe (GRAS) status nor has it been included in the Qualified Presumption of Safety (QPS) list implying drastic legislation towards these microorganisms. To date, the knowledge of the virulence factors and the genetic structure of foodborne enterococcal strains is rather limited. Although enterococcal infections originating from food have never been reported, the consumption of food carrying virulence enterococci seems to be a risky path of transfer, and hence, it renders them poor choices as probiotics. Auspiciously, enterococcal virulence factors seem to be strain specific suggesting that clinical isolates carry much more determinants that food isolates. The latter remain widely susceptible to clinically relevant antibiotics and subsequently, have a lower potential for pathogenicity. In terms of the ideal enterococcal candidate, selected strains deemed for use in foods should not possess any virulence genes and should be susceptible to clinically relevant antibiotics. Overall, implementation of an appropriate risk/benefit analysis, in addition to the case-by-case assessment, the establishment of a strain’s innocuity, and consideration for relevant guidelines, legislation, and regulatory aspects surrounding functional food development seem to be the crucial elements for industries, health-staff and consumers to accept enterococci, like other LAB, as important candidates for useful and beneficial applications in food industry and food biotechnology. The present review aims at shedding light on the world of hurdles and limitations that hampers the Enterococcus spp. genus and its representatives from being used or proposed for use as probiotics. The future of enterococci use as probiotics and legislation in this field are also discussed.

Staphylococcus spp. and Pseudomonas spp. are widely distributed bacteria in the environment and are found in association with animals and humans. Here, we present the draft genome sequence data of the healthy human skin commensal strains Staphylococcus aureus MFP03, Staphylococcus epidermidis MFP04, Staphylococcus capitis MFP08, and Pseudomonas fluorescens MFP05.

The skin constitutes with its microbiota the first line of body defense against exogenous stress including air pollution. Especially in urban or sub-urban areas, it is continuously exposed to many environmental pollutants including gaseous nitrogen dioxide (gNO2). Nowadays, it is well established that air pollution has major effects on the human skin, inducing various diseases often associated with microbial dysbiosis. However, very few is known about the impact of pollutants on skin microbiota. In this study, a new approach was adopted, by considering the alteration of the cutaneous microbiota by air pollutants as an indirect action of the harmful molecules on the skin. The effects of gNO2 on this bacterial skin microbiota was investigated using a device developed to mimic the real-life contact of the gNO2 with bacteria on the surface of the skin. Five strains of human skin commensal bacteria were considered, namely Staphylococcus aureus MFP03, Staphylococcus epidermidis MFP04, Staphylococcus capitis MFP08, Pseudomonas fluorescens MFP05, and Corynebacterium tuberculostearicum CIP102622. Bacteria were exposed to high concentration of gNO2 (10 or 80 ppm) over a short period of 2 h inside the gas exposure device. The physiological, morphological, and molecular responses of the bacteria after the gas exposure were assessed and compared between the different strains and the two gNO2 concentrations. A highly significant deleterious effect of gNO2 was highlighted, particularly for S. capitis MFP08 and C. tuberculostearicum CIP102622, while S. aureus MFP03 seems to be the less sensitive strain. It appeared that the impact of this nitrosative stress differs according to the bacterial species and the gNO2 concentration. Thus the exposition to gNO2 as an air pollutant could contribute to dysbiosis, which would affect skin homeostasis. The response of the microbiota to the nitrosative stress could be involved in some pathologies such as atopic dermatitis.

Pseudomonas aeruginosa PAO1 has an integrated Pf4 prophage in its genome, encoding a relatively well-characterized filamentous phage, which contributes to the bacterial biofilm organization and maturation. Pf4 variants are considered as superinfectives when they can re-infect and kill the prophage-carrying host. Herein, the response of P. aeruginosa H103 to Pf4 variant infection was investigated. This phage variant caused partial lysis of the bacterial population and modulated H103 physiology. We show by confocal laser scanning microscopy that a Pf4 variant-infection altered P. aeruginosa H103 biofilm architecture either in static or dynamic conditions. Interestingly, in the latter condition, numerous cells displayed a filamentous morphology, suggesting a link between this phenotype and flow-related forces. In addition, Pf4 variant-infection resulted in cell envelope stress response, mostly mediated by the AlgU and SigX extracytoplasmic function sigma factors (ECF&sigma, ). AlgU and SigX involvement may account, at least partly, for the enhanced expression level of genes involved in the biosynthesis pathways of two matrix exopolysaccharides (Pel and alginates) and bis-(3&rsquo, 5&rsquo, )-cyclic dimeric guanosine monophosphate (c-di-GMP) metabolism.

Biofilms produced by Pseudomonas aeruginosa present a serious threat to cystic fibrosis patients. Here, we report the draft genome sequences of four cystic fibrosis isolates displaying various mucoid and biofilm phenotypes. The estimated average genome size was about 6,255,986 ± 50,202 bp with a mean G+C content of 66.52 ± 0.06%.

Lactobacillus crispatus strain V4 was isolated from a vaginal swab from a healthy nonmenopausal 35-year-old French woman. We report here its draft genome sequence of 2,091,889 bp, with an average G+C content of 37.02%.

Lactic acid bacteria (LAB) strains OB14 and OB15 were isolated from traditional Tunisian fermented dairy products, Testouri cheese and Rigouta, respectively. They were identified as Enterococcus faecalis by the MALDI TOF-MS (matrix assisted laser desorption-ionization time of flight mass spectrometry) biotyper system and molecular assays (species-specific PCR). These new isolates were evaluated for probiotic properties, compared to E. faecalis Symbioflor 1 clone DSM 16431, as reference. The bacteria were found to be tolerant to the harsh conditions of the gastrointestinal tract (acidity and bile salt). They were low to moderate biofilm producers, can adhere to Caco-2/TC7 intestinal cells and strengthen the intestinal barrier through the increase of the transepithelial electrical resistance (TER). Susceptibility to ampicillin, vancomycin, gentamicin and erythromycin has been tested using the broth microdilutions method. The results demonstrated that E. faecalis OB14 and OB15 were sensitive to the clinically important ampicillin (MIC = 1 μg/mL) and vancomycin (MIC = 2 μg/mL) antibiotics. However, Whole Genome Sequencing (WGS) showed the presence of tetracycline resistance and cytolysin genes in E. faecalis OB14, and this led to high mortality of Galleria Mellonella larvae in the virulence test. Hierarchical cluster analysis by MALDI TOF-MS biotyper showed that E. faecalis OB15 was closely related to the E. faecalis Symbioflor 1 probiotic strain than to OB14, and this has been confirmed by WGS using the average nucleotide identity (ANI) and Genome-to-Genome Hybridization similarity methods. According to these results, E. faecalis OB15 seems to be reliable for future development as probiotic, in food or feed industry.

International audience; LecA and LecB tetrameric lectins take part in oligosaccharide-mediated adhesion-processes of Pseudomonas aeruginosa. Glycomimetics have been designed to block these interactions. The great versatility of P. aeruginosa suggests that the range of application of these glycomimetics could be restricted to genotypes with particular lectin types. The likelihood of having genomic and genetic changes impacting LecA and LecB interactions with glycomimetics such as galactosylated and fucosylated calix[4]arene was investigated over a collection of strains from the main clades of P. aeruginosa. Lectin types were defined, and their ligand specificities were inferred. These analyses showed a loss of lecA among the PA7 clade. Genomic changes impacting lec loci were thus assessed using strains of this clade, and by making comparisons with the PAO1 genome. The lecA regions were found challenged by phage attacks and PAGI-2 (genomic island) integrations. A prophage was linked to the loss of lecA. The lecB regions were found less impacted by such rearrangements but greater lecB than lecA genetic divergences were recorded. Sixteen combinations of LecA and LecB types were observed. Amino acid variations were mapped on PAO1 crystal structures. Most significant changes were observed on LecBPA7, and found close to the fucose binding site. Glycan array analyses were performed with purified LecBPA7. LecBPA7 was found less specific for fucosylated oligosaccharides than LecBPAO1, with a preference for H type 2 rather than type 1, and Lewis(a) rather than Lewis(x). Comparison of the crystal structures of LecBPA7 and LecBPAO1 in complex with Lewis(a) showed these changes in specificity to have resulted from a modification of the water network between the lectin, galactose and GlcNAc residues. Incidence of these modifications on the interactions with calix[4]arene glycomimetics at the cell level was investigated. An aggregation test was used to establish the efficacy of these ligands. Great variations in the responses were observed. Glycomimetics directed against LecB yielded the highest numbers of aggregates for strains from all clades. The use of a PAO1ΔlecB strain confirmed a role of LecB in this aggregation phenotype. Fucosylated calix[4]arene showed the greatest potential for a use in the prevention of P. aeruginosa infections.

Enterotoxigenic Escherichia coli (ETEC) has consistently been the predominant bacterial cause of diarrhea in many birth cohort- and hospital-based studies conducted in Egypt. We evaluated the pathogenicity of ETEC isolates in a birth cohort of children living in a rural community in Egypt. Between 2004 and 2007, we enrolled and followed 348 children starting at birth until their second year of life. A stool sample and two rectal swabs were collected from children during twice-weekly visits when they presented with diarrhea and were collected every 2 weeks if no diarrhea was reported. From routine stool cultures, five E. coli -like colonies were screened for ETEC enterotoxins using a GM1 enzyme-linked immunosorbent assay (ELISA). The isolates were screened against a panel of 12 colonization factor antigens (CFAs) by a dot blot assay. A nested case-control study evaluated the association between initial or repeat excretion of ETEC and the occurrences of diarrhea. The pathogenicity of ETEC was estimated in symptomatic children compared to that in asymptomatic controls. ETEC was significantly associated with diarrhea (crude odds ratio, 1.37; 95% confidence interval [CI], 1.24 to 1.52). The distribution of ETEC enterotoxins varied between the symptomatic children (44.2% heat-labile toxin [LT], 38.5% heat-stable toxin [ST], and 17.3% LT/ST) and asymptomatic children (55.5% LT, 34.6% ST, and 9.9% LT/ST) ( P < 0.001). The CFAs CFA/I ( n = 61), CS3 ( n = 8), CS1 plus CS3 ( n = 24), CS2 plus CS3 ( n = 18), CS6 ( n = 45), CS5 plus CS6 ( n = 11), CS7 ( n = 25), and CS14 ( n = 32) were frequently detected in symptomatic children, while CS6 ( n = 66), CS12 ( n = 51), CFA/I ( n = 43), and CS14 ( n = 20) were detected at higher frequencies among asymptomatic children. While all toxin phenotypes were associated with diarrheal disease after the initial exposure, only ST and LT/ST-expressing ETEC isolates ( P < 0.0001) were associated with disease in repeat infections. The role of enterotoxins and pathogenicity during repeat ETEC infections appears to be variable and dependent on the coexpression of specific CFAs.

Introduction: The present study, conducted between January 2004 and April 2007, explored the impact of household hygiene on the risk of bacterial diarrhea, using data from a prospective birth cohort of 348 infants in five villages in the Nile Delta in Egypt. Methodology: Neonates were enrolled at birth and followed up until 24 months of age. Children were visited twice a week to survey them for acute diarrhea. A detailed observational household hygiene survey was completed in-house every six months. Adjusted relative risk (aRR) of developing bacterial diarrhea was calculated for exposure to different hygiene variables and examined for specific bacterial pathogens. Results: Exclusive breastfeeding reduced the risk of bacterial diarrhea by 70%, while bacterial diarrhea cases were 2.6 times higher in the warm season. Risk of Campylobacter diarrhea increased with the absence of barriers to keep birds and animals out of the eating area, the presence of garbage containers and a bathing facility within the compound, and the presence of feces on the floor of the bathing facility. Use of municipal water for drinking and cooking was associated with a lower risk of Campylobacter diarrhea. Risk of enterotoxigenic Escherichia coli diarrhea increased with uncovered garbage containers and the presence of liquid materials in the garbage containers, but decreased with the use of tap water in the washing facility. Conclusion: The results highlight some potential targets for interventions, such as expanding municipal water supply to all houses and comprehensive mass-media awareness programs to change hygiene-promoting behaviors and practices.

International audience; The significance of wastewater treatment lagoons (WWTLs) as point sources of clinically relevant Pseudomonas aeruginosa that can disseminate through rural and peri-urban catchments was investigated. A panel of P. aeruginosa strains collected over three years from WWTLs and community-acquired infections was compared by pulsed field gel electrophoresis (PFGE) DNA fingerprinting and multilocus sequence typing (MLST). Forty-four distantly related PFGE profiles and four clonal complexes were found among the WWTL strains analyzed. Some genotypes were repeatedly detected from different parts of WWTLs, including the influent, suggesting an ability to migrate and persist over time. MLST showed all investigated lineages to match sequence types described in other countries and strains from major clinical clones such as PA14 of ST253 and "C" of ST17 were observed. Some of these genotypes matched isolates from community-acquired infections recorded in the WWTL geographic area. Most WWTL strains harbored the main P. aeruginosa virulence genes; 13% harbored exoU-encoded cytoxins, but on at least six different genomic islands, with some of these showing signs of genomic instability. P. aeruginosa appeared to be highly successful opportunistic colonizers of WWTLs. Lagooning of wastewaters was found to favor dissemination of clinically relevant P. aeruginosa among peri-urban watersheds.