Gut microbiota and physical activity level: characterization from sedentary to soccer players.

Evidence of the relationship between physical activity and gut microbiota composition is steadily increasing. The purpose of the study is to compare the gut microbiota composition of a group of elite male soccer players with a group of subjects with different physical activity levels. Cross-sectional studies were performed on 91 healthy young males, in detail: 17 elite soccer players (23.7 ± 4.2 yrs, BMI 23.2 ± 1.2 kg/m2); 14 with high levels of physical training (24.5 ± 5.6 yrs, BMI 22.7 ± 0.8 kg/m2); 23 with moderate levels of physical training (29.3 ± 3.9 yrs, BMI 22.5 ± 0.8 kg/m2); and 37 healthy men without exercise habits (28.1 ± 5.9 yrs, BMI 22.4 ± 1.0 kg/m2). Relative microbiota composition was determined by analyzing DNA extracted from stool samples. The quality and quantity of extracted DNA were assessed using a Qubit Fluorometer. Differences between subjects' populations were analyzed using a one-way ANOVA, and Bonferroni's post-hoc test was employed to identify localized effects. Elite soccer players and subjects with high physical activity levels showed a significantly higher prevalence of the nine microbiota populations analyzed than subjects with moderate physical training or who were sedentary. No differences were found in the Firmicutes to Bacteroidetes ratio among the different study populations. This study reports the gut microbiota parameters of elite footballers for the first time. In addition, it brings new insights into the effects of different levels of physical activity on the composition of the gut microbiota.

Corrigendum: Characterization of the "gut microbiota-immunity axis" and microbial lipid metabolites in atrophic and potential celiac disease.

[This corrects the article DOI: 10.3389/fmicb.2022.886008.].

Facial Skin Microbiome: Aging-Related Changes and Exploratory Functional Associations with Host Genetic Factors, a Pilot Study.

In this exploratory study, we investigate the variation in the facial skin microbiome architecture through aging and their functional association with host genetic factors in a cohort of healthy women, living in the same area and without cutaneous diseases. Notably, facial skin microbiota (SM) samples were collected from a cohort of 15 healthy Caucasian females, firstly divided into three age groups (younger women aged 20-35 years old; middle aged women of 36-52 years old; and older women aged 53-68 years old). Then, the recruited cohort was divided into two groups based on their facial hydration level (dry and normal skin). The facial SM revealed a different composition in the three analyzed aging groups and between normal and dry skins. The middle-aged women also revealed functional variations associated with collagen biosynthesis and oxidative stress damage repair. Otherwise, the association between selected host SNPs (single nucleotide polymorphisms) and the facial SM profile showed significant associations, suggesting a negative correlation with collagen metabolism and ROS damage protection. Finally, the composition and functionality of the facial SM seemed to affect the aging process through the two aging-correlated pathways of host ROS damage repair and collagen metabolism. Our exploratory data could be useful for future studies characterizing the structure, function, and dynamics of the SM in the aging process to design personalized therapeutic agents focusing on potential genomic targets, microbes, and their metabolites.

A MATE Transporter is Involved in Pathogenicity and IAA Homeostasis in the Hyperplastic Plant Pathogen Pseudomonas savastanoi pv. nerii.

During the last years, many evidences have been accumulating about the phytohormone indole-3-acetic acid (IAA) as a multifaceted compound in the microbial world, with IAA playing a role as a bacterial intra and intercellular signaling molecule or as an effector during pathogenic or beneficial plant-bacteria interactions. However, pretty much nothing is known on the mechanisms that bacteria use to modulate IAA homeostasis, in particular on IAA active transport systems. Here, by an approach combining in silico three-dimensional (3D) structural modeling and docking, mutagenesis, quantitative gene expression analysis, and HPLC FLD auxin quantitative detection, for the first time a bacterial multidrug and toxic compound extrusion (MATE) transporter was demonstrated to be involved in the efflux of IAA, as well as of its conjugate IAA-Lysine, in the plant pathogenic hyperplastic bacterium Pseudomonas savastanoi pv. nerii strain Psn23. Furthermore, according to the role proved to be played by Psn23 MatE in the development of plant disease, and to the presence of Psn23 MatE homologs in all the genomospecies of the P. syringae complex, this membrane transporter could likely represent a promising target for the design of novel and selective anti-infective molecules for plant disease control.

Phenotypic and Molecular-Phylogenetic Analysis Provide Novel Insights into the Diversity of Curtobacterium flaccumfaciens.

A multiphasic approach was used to decipher the phenotypic features, genetic diversity, and phylogenetic position of 46 Curtobacterium spp. strains isolated from dry beans and other annual crops in Iran and Spain. Pathogenicity tests, resistance to arsenic compounds, plasmid profiling and BOX-PCR were performed on the strains. Multilocus sequence analysis (MLSA) was also performed on five housekeeping genes (i.e., atpD, gyrB, ppk, recA, and rpoB) of all the strains, as well as five pathotype strains of the species. Pathogenicity test showed that six out of 42 strains isolated in Iran were nonpathogenic on common bean. Despite no differences found between pathogenic and nonpathogenic strains in their plasmid profiling, the former were resistant to different concentrations of arsenic, while the latter were sensitive to the same concentrations. Strains pathogenic on common bean were polyphyletic with at least two evolutionary lineages (i.e., yellow-pigmented strains versus red/orange-pigmented strains). Nonpathogenic strains isolated from solanaceous vegetables were clustered within either the strains of C. flaccumfaciens pv. flaccumfaciens or different pathovars of the species. The results of MLSA and BOX-PCR analysis were similar to each other and both methods were able to discriminate the yellow-pigmented strains from the red/orange-pigmented strains. A comprehensive study of a worldwide collection representing all five pathovars as well as nonpathogenic strains of C. flaccumfaciens is warranted for a better understanding of the diversity within this phytopathogenic bacterium.

Global Analysis of Type Three Secretion System and Quorum Sensing Inhibition of Pseudomonas savastanoi by Polyphenols Extracts from Vegetable Residues.

Protection of plants against bacterial diseases still mainly relies on the use of chemical pesticides, which in Europe correspond essentially to copper-based compounds. However, recently plant diseases control is oriented towards a rational use of molecules and extracts, generally with natural origin, with lower intrinsic toxicity and a reduced negative environmental impact. In this work, polyphenolic extracts from vegetable no food/feed residues of typical Mediterranean crops, as Olea europaea, Cynara scolymus, and Vitis vinifera were obtained and their inhibitory activity on the Type Three Secretion System (TTSS) and the Quorum Sensing (QS) of the Gram-negative phytopathogenic bacterium Pseudomonas savastanoi pv. nerii strain Psn23 was assessed. Extract from green tea (Camellia sinensis) was used as a positive control. Collectively, the data obtained through gfp-promoter fusion system and real-time PCR show that all the polyphenolic extracts here studied have a high inhibitory activity on both the TTSS and QS of Psn23, without any depressing effect on bacterial viability. Extracts from green tea and grape seeds were shown to be the most active. Such activity was confirmed in planta by a strong reduction in the ability of Psn23 to develop hyperplastic galls on explants from adult oleander plants, as well as to elicit hypersensitive response on tobacco. By using a newly developed Congo red assay and an ELISA test, we demonstrated that the TTSS-targeted activity of these polyphenolic extracts also affects the TTSS pilus assembly. In consideration of the potential application of polyphenolic extracts in plant protection, the absence of any toxicity of these polyphenolic compounds was also assessed. A widely and evolutionary conserved molecular target such as Ca2+-ATPase, essential for the survival of any living organism, was used for the toxicity assessment.

Indole-3-acetic acid in plant-pathogen interactions: a key molecule for in planta bacterial virulence and fitness.

The plant pathogenic bacterium Pseudomonas savastanoi, the causal agent of olive and oleander knot disease, uses the so-called "indole-3-acetamide pathway" to convert tryptophan to indole-3-acetic acid (IAA) via a two-step pathway catalyzed by enzymes encoded by the genes in the iaaM/iaaH operon. Moreover, pathovar nerii of P. savastanoi is able to conjugate IAA to lysine to generate the less biologically active compound IAA-Lys via the enzyme IAA-lysine synthase encoded by the iaaL gene. Interestingly, iaaL is now known to be widespread in many Pseudomonas syringae pathovars, even in the absence of the iaaM and iaaH genes for IAA biosynthesis. Here, two knockout mutants, ΔiaaL and ΔiaaM, of strain Psn23 of P. savastanoi pv. nerii were produced. Pathogenicity tests using the host plant Nerium oleander showed that ΔiaaL and ΔiaaM were hypervirulent and hypovirulent, respectively and these features appeared to be related to their differential production of free IAA. Using the Phenotype Microarray approach, the chemical sensitivity of these mutants was shown to be comparable to that of wild-type Psn23. The main exception was 8 hydroxyquinoline, a toxic compound that is naturally present in plant exudates and is used as a biocide, which severely impaired the growth of ΔiaaL and ΔiaaM, as well as growth of the non-pathogenic mutant ΔhrpA, which lacks a functional Type Three Secretion System (TTSS). According to bioinformatics analysis of the Psn23 genome, a gene encoding a putative Multidrug and Toxic compound Extrusion (MATE) transporter, was found upstream of iaaL. Similarly to iaaL and iaaM, its expression appeared to be TTSS-dependent. Moreover, auxin-responsive elements were identified for the first time in the modular promoters of both the iaaL gene and the iaaM/iaaH operon of P. savastanoi, suggesting their IAA-inducible transcription. Gene expression analysis of several genes related to TTSS, IAA metabolism and drug resistance confirmed the presence of a concerted regulatory network in this phytopathogen among virulence, fitness and drug efflux.

Decolorization of acid and basic dyes: understanding the metabolic degradation and cell-induced adsorption/precipitation by Escherichia coli.

Escherichia coli strain DH5α was successfully employed in the decolorization of commercial anthraquinone and azo dyes, belonging to the general classes of acid or basic dyes. The bacteria showed an aptitude to survive at different pH values on any dye solution tested, and a rapid decolorization was obtained under aerobic conditions for the whole collection of dyes. A deep investigation about the mode of action of E. coli was carried out to demonstrate that dye decolorization mainly occurred via three different pathways, specifically bacterial induced precipitation, cell wall adsorption, and metabolism, whose weight was correlated with the chemical nature of the dye. In the case of basic azo dyes, an unexpected fast decolorization was observed after just 2-h postinoculation under aerobic conditions, suggesting that metabolism was the main mechanism involved in basic azo dye degradation, as unequivocally demonstrated by mass spectrometric analysis. The reductive cleavage of the azo group by E. coli on basic azo dyes was also further demonstrated by the inhibition of decolorization occurring when glucose was added to the dye solution. Moreover, no residual toxicity was found in the E. coli-treated basic azo dye solutions by performing Daphnia magna acute toxicity assays. The results of the present study demonstrated that E. coli can be simply exploited for its natural metabolic pathways, without applying any recombinant technology. The high versatility and adaptability of this bacterium could encourage its involvement in industrial bioremediation of textile and leather dyeing wastewaters.

Water recycle as a must: decolorization of textile wastewaters by plant-associated fungi.

Textile dye effluents are among the most problematic pollutants because of their toxicity on several organisms and ecosystems. Low cost and ecocompatible bioremediation processes offer a promising alternative to the conventional and aspecific physico-chemical procedures adopted so far. Here, microorganisms resident on three real textile dyeing effluent were isolated, characterized, and tested for their decolorizing performances. Although able to survive on these real textile-dyeing wastewaters, they always showed a very low decolorizing activity. On the contrary, several plant-associated fungi (Bjerkandera adusta, Funalia trogii, Irpex lacteus, Pleurotus ostreatus, Trametes hirsuta, Trichoderma viride, and Aspergillus nidulans) were also assayed and demonstrated to be able both to survive and to decolorize to various extents the three effluents, used as such in liquid cultures. The decolorizing potential of these fungi was demonstrated to be influenced by nutrient availability and pH. Best performances were constantly obtained using B. adusta and A. nidulans, relying on two strongly different mechanisms for their decolorizing activities: degradation for B. adusta and biosorption for A. nidulans. Acute toxicity tests using Daphnia magna showed a substantial reduction in toxicity of the three textile dyeing effluents when treated with B. adusta and A. nidulans, as suggested by mass spectrometric analysis as well.

High-resolution melting analysis as a powerful tool to discriminate and genotype Pseudomonas savastanoi pathovars and strains.

Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants.

Type Three Secretion System in Pseudomonas savastanoi Pathovars: Does Timing Matter?

Pseudomonas savastanoi pv. savastanoi is the causal agent of Olive knot disease, relying on the Type Three Secretion System (TTSS) for its pathogenicity. In this regard, nothing was known about the two other pathovars belonging to this species, pv. nerii and pv. fraxini, characterized by a different host range. Here we report on the organization of the entire TTSS cluster on the three pathovars, and a phylogenetic analysis including the TTSS of those bacteria belonging to the P. syringae complex sequenced so far, highlighting the evolution of each operon (hrpC, hrpJ, hrpRS, hrpU and hrpZ). Moreover, by Real-Time PCR we analyzed the in vitro expression of four main TTSS genes, revealing different activation patterns in the three pathovars, hypothetically related to their diverse virulence behaviors.

Development of a versatile tool for the simultaneous differential detection of Pseudomonas savastanoi pathovars by End Point and Real-Time PCR.

BACKGROUND: Pseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars nerii and fraxini, respectively. When artificially inoculated, pv. savastanoi causes disease also on ash, and pv. nerii attacks also olive and ash. Surprisingly nothing is known yet about their distribution in nature on these hosts and if spontaneous cross-infections occur. On the other hand sanitary certification programs for olive plants, also including P. savastanoi, were launched in many countries. The aim of this work was to develop several PCR-based tools for the rapid, simultaneous, differential and quantitative detection of these P. savastanoi pathovars, in multiplex and in planta. RESULTS: Specific PCR primers and probes for the pathovars savastanoi, nerii and fraxini of P. savastanoi were designed to be used in End Point and Real-Time PCR, both with SYBR Green or TaqMan chemistries. The specificity of all these assays was 100%, as assessed by testing forty-four P. savastanoi strains, belonging to the three pathovars and having different geographical origins. For comparison strains from the pathovars phaseolicola and glycinea of P. savastanoi and bacterial epiphytes from P. savastanoi host plants were also assayed, and all of them tested always negative. The analytical detection limits were about 5 - 0.5 pg of pure genomic DNA and about 102 genome equivalents per reaction. Similar analytical thresholds were achieved in Multiplex Real-Time PCR experiments, even on artificially inoculated olive plants. CONCLUSIONS: Here for the first time a complex of PCR-based assays were developed for the simultaneous discrimination and detection of P. savastanoi pv. savastanoi, pv. nerii and pv. fraxini. These tests were shown to be highly reliable, pathovar-specific, sensitive, rapid and able to quantify these pathogens, both in multiplex reactions and in vivo. Compared with the other methods already available for P. savastanoi, the identification procedures here reported provide a versatile tool both for epidemiological and ecological studies on these pathovars, and for diagnostic procedures monitoring the asymptomatic presence of P. savastanoi on olive and oleander propagation materials.