The recombination dynamics of Staphylococcus aureus inferred from spA gene.

BACKGROUND: Given the role of spA as a pivotal virulence factor decisive for Staphylococcus aureus ability to escape from innate and adaptive immune responses, one can consider it as an object subject to adaptive evolution and that variations in spA may uncover pathogenicity variations. RESULTS: The population genetic structure was deduced from the extracellular domains of SpA gene sequence (domains A-E and the X-region) and compared to the MLST-analysis of 41 genetically diverse methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains. Incongruence between tree topologies was noticeable and in the inferred spA tree most MSSA isolates were clustered in a distinct group. Conversely, the distribution of strains according to their spA-type was not always congruent with the tree inferred from the complete spA gene foreseeing that spA is a mosaic gene composed of different segments exhibiting different evolutionary histories. Evidences of a network-like organization were identified through several conflicting phylogenetic signals and indeed several intragenic recombination events (within subdomains of the gene) were detected within and between CC's of MRSA strains. The alignment of SpA sequences enabled the clustering of several isoforms as a result of non-randomly distributed amino acid variations, located in two clusters of polymorphic sites in domains D to B and Xr (a). Nevertheless, evidences of cluster specific structural arrangements were detected reflecting alterations on specific residues with potential impact on S. aureus pathogenicity. CONCLUSIONS: The detection of positive selection operating on spA combined with frequent non-synonymous mutations, domain duplication and frequent intragenic recombination events represent important mechanisms acting in the evolutionary adaptive mechanism promoting spA genetic plasticity. These findings argue that crucial allelic forms correlated with pathogenicity can be identified by sequences analysis enabling the design of more robust schemes.

Microbial and functional diversity of a subterrestrial high pH groundwater associated to serpentinization.

Microbial and functional diversity were assessed, from a serpentinization-driven subterrestrial alkaline aquifer - Cabeço de Vide Aquifer (CVA) in Portugal. DGGE analyses revealed the presence of a stable microbial community. By 16S rRNA gene libraries and pyrosequencing analyses, a diverse bacterial composition was determined, contrasting with low archaeal diversity. Within Bacteria the majority of the populations were related to organisms or sequences affiliated to class Clostridia, but members of classes Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Deinococci, Gammaproteobacteria and of the phyla Bacteroidetes, Chloroflexi and Nitrospira were also detected. Domain Archaea encompassed mainly sequences affiliated to Euryarchaeota. Only form I RuBisCO - cbbL was detected. Autotrophic carbon fixation via the rTCA, 3-HP and 3-HP/4H-B cycles could not be confirmed. The detected APS reductase alpha subunit - aprA sequences were phylogenetically related to sequences of sulfate-reducing bacteria belonging to Clostridia, and also to sequences of chemolithoautothrophic sulfur-oxidizing bacteria belonging to Betaproteobacteria. Sequences of methyl coenzyme M reductase - mcrA were phylogenetically affiliated to sequences belonging to Anaerobic Methanotroph group 1 (ANME-1). The populations found and the functional key markers detected in CVA suggest that metabolisms related to H2 , methane and/or sulfur may be the major driving forces in this environment.

Glaciimonas singularis sp. nov., isolated from a uranium mine wastewater treatment plant.

A bacterial strain, A2-57(T), recovered from a water sample collected in a uranium mine was taxonomically studied in detail. This strain was a Gram-reaction-negative, rod-shaped bacterium that grew optimally at 25 °C and at pH 6.0-7.0 and had a DNA G+C content of 55.0 mol%. Ubiquinone 8 (UQ-8) was the predominant respiratory quinone and the major fatty acids were C16:0, C17:0 cyclo, summed feature 3 (C16:1ω6c and/or ω7c and/or C15:0 iso 2-OH) and C18:1ω7c. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain A2-57(T) belonged to the family Oxalobacteraceae and formed a distinct branch with Glaciimonas immobilis Cr9-30(T). Strain A2-57(T) shared approximately 97.3% 16S rRNA sequence similarity with G. immobilis Cr9-30(T) and also showed high sequence similarity with members of the genera Herbaspirillum (96.3-97.0%) and Collimonas (96.2-97.0%). Although phylogenetically closely related to the type strain of G. immobilis, the low level of DNA-DNA hybridization between the two strains (21.6%) and several physiological and biochemical properties indicated that the novel strain could be clearly distinguished from G. immobilis LMG 25547(T). Therefore, it is concluded that strain A2-57(T) represents a novel species of the genus Glaciimonas, for which the name Glaciimonas singularis sp. nov. is proposed. The type strain is A2-57(T) (=CIP 110539(T)=LMG 27070(T)).

Cecembia calidifontis sp. nov., isolated from a hot spring runoff, and emended description of the genus Cecembia.

Two red-pigmented isolates, designated RQ-33(T) and TU-13, were recovered from hot spring runoffs on the Island of São Miguel in the Azores, Portugal. These organisms have an optimum growth temperature of approximately 45 °C and an optimum pH for growth between 7.5 and 8.5. Based on 16S rRNA gene sequence analysis, the novel isolates were most closely related to the type strain of Cecembia lonarensis at 96.4 % pairwise similarity. Cells of the two isolates were non-motile, rod-shaped, Gram-stain-negative, strictly aerobic, and oxidase- and catalase-positive. The major fatty acids were iso-C15 : 0 and C15 : 0, phosphatidylethanolamine was the major polar lipid, and menaquinone 7 was the major respiratory quinone. Based on phylogenetic analyses, and physiological and biochemical characteristics, these isolates from the Azores represent a novel species of the genus Cecembia for which the name Cecembia calidifontis is proposed with strain RQ-33(T) ( = DSM 21411(T) = LMG 24596(T)) as the type strain. The description of the genus Cecembia is also emended.

Pullulanibacillus uraniitolerans sp. nov., an acidophilic, U(VI)-resistant species isolated from an acid uranium mill tailing effluent and emended description of the genus Pullulanibacillus.

Two Gram-positive-staining, rod-shaped, endospore-forming isolates (UG-2(T) and UG-3), with an optimum growth temperature of around 37 °C and an optimum pH for growth of about 4, were recovered from an acidic effluent of the uranium mill tailing at Urgeiriça in Central Portugal. On the basis of 16S rRNA gene sequence similarity, the strains belonged to the family Sporolactobacillaceae and were closely related to Pullulanibacillus naganoensis ATCC 53909(T) (97.9 %). Unlike P. naganoensis, strains UG-2(T) and UG-3 grew in medium containing up to 5000 p.p.m. U(VI) but did not hydrolyse pullulan. Chemotaxonomic data also supported the affiliation of strains UG-2(T) and UG-3 to the genus Pullulanibacillus. Physiological and biochemical tests along with fatty acid composition allowed differentiation of strains UG-2(T) and UG-3 from P. naganoensis. It is suggested that strains UG-2(T) and UG-3 represent a novel species, for which the name Pullulanibacillus uraniitolerans is proposed; the type strain is UG-2(T) (=DSM 19429(T) = LMG 24205(T)). An emended description of the genus Pullulanibacillus is also proposed.

Molecular evolution of key genes for type II secretion in Legionella pneumophila.

Given the role of type II protein secretion system (T2S) in the ecology and pathogenesis of Legionella pneumophila, it is possible that this system is a target for adaptive evolution. The population genetic structure of L.pneumophila was inferred from the partial sequences of rpoB and from the complete sequence of three T2S structural components (lspD, lspE and pilD) and from two T2S effectors critical for intracellular infection of protozoa (proA and srnA) of 37 strains isolated from natural and man-made environments and disease-related from worldwide sources. A phylogenetic analysis was obtained for the concatenated alignment and for each individual locus. Seven main groups were identified containing the same L.pneumophila strains, suggesting an ancient divergence for each cluster and indicating that the operating selective pressures have equally affected the evolution of the five genes. Although linkage disequilibrium analysis indicate a clonal nature for population structure in this sample, our results indicate that recombination is a common phenomenon among T2S-related genes on this species, as 24 of the 37 L.pneumophila isolates contained at least one locus in which recombination was identified. Furthermore, neutral selection acting on the analysed T2S-related genes emerged as a clear result, namely on T2S effectors, ProA and SrnA, indicating that they are probably implicated in conserved virulence mechanisms through legionellae hosts.

Jeotgalibacillus soli sp. nov., a Gram-stain-positive bacterium isolated from soil.

A Gram-staining-positive, motile, rod-shaped, spore-forming bacterium, designated P9(T), was isolated from soil in Portugal. This organism was aerobic and catalase- and oxidase-positive. It had an optimum growth temperature of about 35 °C and an optimum growth pH of about 8.0-8.5, and grew in medium with 0-9% (w/v) NaCl. The cell-wall peptidoglycan was of the A1α type, with L-lysine as the diagnostic diamino acid. The major respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids were anteiso-C(15:0) (45.4%), iso-C(15:0) (22.0%) and anteiso-C(17:0) (11.2%). The genomic DNA G+C content was about 39.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain P9(T) was most closely related to Jeotgalibacillus campisalis DSM 18983(T) (96.8%) and Jeotgalibacillus marinus DSM 1297(T) (96.5%). These two recognized species formed a coherent cluster with strain P9(T) that was supported by a bootstrap value of 99%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics, strain P9(T) (=DSM 23228(T)=LMG 25523(T)) represents a novel species of the genus Jeotgalibacillus, for which the name Jeotgalibacillus soli sp. nov. is proposed.

Guts Bacterial Communities of Porcellio dilatatus: Symbionts Predominance, Functional Significance and Putative Biotechnological Potential.

Terrestrial isopods are effective herbivorous scavengers with an important ecological role in organic matter cycling. Their guts are considered to be a natural enrichment environment for lignocellulosic biomass (LCB)-degrading bacteria. The main goal of this work was to assess the structural diversity of Porcellio dilatatus gut bacterial communities using NGS technologies, and to predict their functional potential using PICRUSt2 software. Pseudomonadota, Actinomycetota, Bacillota, Cyanobacteria, Mycoplasmatota, Bacteroidota, Candidatus Patescibacteria and Chloroflexota were the most abundant phyla found in P. dilatatus gut bacterial communities. At a family level, we identified the presence of eleven common bacterial families. Functionally, the P. dilatatus gut bacterial communities exhibited enrichment in KEGG pathways related to the functional module of metabolism. With the predicted functional profile of P. dilatatus metagenomes, it was possible to envision putative symbiotic relationships between P. dilatatus gut bacterial communities and their hosts. It was also possible to foresee the presence of a well-adapted bacterial community responsible for nutrient uptake for the host and for maintaining host homeostasis. Genes encoding LCB-degrading enzymes were also predicted in all samples. Therefore, the P. dilatatus digestive tract may be considered a potential source of LCB-degrading enzymes that is not to be neglected.

The gastrointestinal microbiome of browsing goats (Capra hircus).

Despite the growing interest in the ruminants' gastrointestinal tract (GIT) microbiomes' ability to degrade plant materials by animal husbandry and industrial sectors, only a few studies addressed browsing ruminants. The present work describes the taxonomic and functional profile of the bacterial and archaeal communities from five different gastrointestinal sections (rumen, omasum-abomasum, jejunum, cecum and colon) of browsing Capra hircus, by metabarcoding using 16S rRNA genes hypervariable regions. The bacterial communities across the GITs are mainly composed of Bacillota and Bacteroidota. Prevotella was the leading bacterial group found in the stomachs, Romboutsia in the jejuna, and Rikenellaceae_RC9_gut_group, Bacteroides, UCG-010_ge, UCG-005, and Alistipes in large intestines. The archaeal communities in the stomachs and jejuna revealed to be mainly composed of Methanobrevibacter, while in the large intestines its dominance is shared with Methanocorpusculum. Across the GITs, the main metabolic functions were related to carbohydrate, amino acid, and energy metabolisms. Significant differences in the composition and potential biological functions of the bacterial communities were observed among stomachs, jejuna and large intestines. In contrast, significant differences were observed among stomachs and jejuna verse large intestines for archaeal communities. Overall different regions of the GIT are occupied by different microbial communities performing distinct biological functions. A high variety of glycoside hydrolases (GHs) indispensable for degrading plant cell wall materials were predicted to be present in all the GIT sections.

Amplified ribosomal DNA restriction analysis as a routine tool to assess toxicant driven changes in hindgut bacterial populations of Porcellio dilatatus (Crustacea: Isopoda).

Changes in saprophytic organism's gut microbial communities may present a threat to organic matter breakdown which can ultimately lead to soil function impairment. In this study, Amplified Ribosomal DNA Restriction Analysis (ARDRA) was evaluated as a potential simple molecular tool to assess shifts in bacterial community structure in hindgut populations of Porcellio dilatatus exposed to contaminated food. This prospective tool can also be used for a variety of purposes and samples prior to the use of more specific and sophisticated methods.

Molecular evolution of Legionella pneumophila dotA gene, the contribution of natural environmental strains.

Given the role of DotA protein in establishing successful infections and the diversity of host cells interacting with Legionella pneumophila in nature, it is possible that this gene product is a target for adaptive evolution. We investigated the influence of L. pneumophila isolates from natural environments with the molecular evolution of this crucial virulence-related gene. The population genetic structure of L. pneumophila was inferred from the partial sequences of rpoB and dotA of 303 worldwide strains. The topology of the two inferred trees was not congruent and in the inferred dotA tree the vast majority of the natural environmental isolates were clustered in a discrete group. The Ka/Ks ratio demonstrated that this group, contrary to all others, has been under strong diversifying selection. The alignment of all DotA sequences allowed the identification of several alleles and the amino acid variations were not randomly distributed. Moreover, from these results we can conclude that dotA from L. pneumophila clinical and man-made environmental strains belong to a sub-set of all genotypes existing in nature. A split graph analysis showed evidence of a network-like organization and several intergenic recombination events were detected within L. pneumophila strains resulting in mosaic genes in which different gene segments exhibited different evolutionary histories. We have determined that the allelic diversity of dotA is predominantly found in L. pneumophila isolates from natural environments, suggesting that niche-specific selection pressures have been operating on this gene. Indeed, the high level of dotA allelic diversity may reflect fitness variation in the persistence of those strains in distinct environmental niches and/or tropism to various protozoan hosts.

Genetic Diversity of Pseudomonas syringae pv. actinidiae: Seasonal and Spatial Population Dynamics.

Pseudomonassyringae pv. actinidiae (Psa) is a gram-negative bacterium responsible for the bacterial canker in Actinidia chinensis var. deliciosa and A. chinensis var. chinensis, a quarantine organism threatening the kiwifruit industry sustainability. The present study aimed to determine the genetic structure of the endophytic and epiphytic populations of Psa isolated from four different Portuguese orchards with distinct abiotic conditions in two consecutive seasons. The results identified several coexisting and highly heterogeneous Psa populations. Moreover, evident changes in population structure occurred between the epiphytic and endophytic populations, and between seasons with a notable decrease in Psa diversity in autumn. This work provided solid evidence that the initial clonal expansion of Psa in Europe was followed by a wide genomic diversification. This perspective is important for the understanding of kiwifruit bacterial canker disease occurrence and Psa evolution, namely when adopting strategies for management of epidemics.

High-Quality Draft Genome Sequences of Crenobacter cavernae Strain CAVE-375 and Oxalobacteriaceae sp. Strain CAVE-383, Two Bacteria Isolated from Dripping Water in a Karstic Cave in Portugal.

Crenobacter cavernae CAVE-375 and Oxalobacteriaceae sp. strain CAVE-383, two Gram-negative bacteria, were isolated during the first microbiology survey performed in a karst cave in Portugal. We report here the release of their high-quality draft genome sequences, which will be useful for geographic-ecological purposes and the description of novel taxa.

Differences in Virulence Between Legionella pneumophila Isolates From Human and Non-human Sources Determined in Galleria mellonella Infection Model.

Legionella pneumophila is a ubiquitous bacterium in freshwater environments and in many man-made water systems capable of inducing pneumonia in humans. Despite its ubiquitous character most studies on L. pneumophila virulence focused on clinical strains and isolates from man-made environments, so little is known about the nature and extent of virulence variation in strains isolated from natural environments. It has been established that clinical isolates are less diverse than man-made and natural environmental strains, suggesting that only a subset of environmental isolates is specially adapted to infect humans. In this work we intended to determine if unrelated L. pneumophila strains, isolated from different environments and with distinct virulence-related genetic backgrounds, displayed differences in virulence, using the Wax Moth Galleria mellonella infection model. We found that all tested strains were pathogenic in G. mellonella, regardless of their origin. Indeed, a panoply of virulence-related phenotypes was observed sustaining the existence of significant differences on the ability of L. pneumophila strains to induce disease. Taken together our results suggest that the occurrence of human infection is not related with the increased capability of some strains to induce disease since we also found a concentration threshold above which L. pneumophila strains are equally able to cause disease. In addition, no link could be established between the sequence-type (ST) and L. pneumophila pathogenicity. We envision that in man-made water distribution systems environmental filtering selection and biotic competition acts structuring L. pneumophila populations by selecting more resilient and adapted strains that can rise to high concentration if no control measures are implemented. Therefore, public health strategies based on the sequence based typing (STB) scheme analysis should take into account that the major disease-associated clones of L. pneumophila were not related with higher virulence in G. mellonella infection model, and that potential variability of virulence-related phenotypes was found within the same ST.

Antibiotic resistance of enterococci and related bacteria in an urban wastewater treatment plant.

The main objective of this work was to study the ecology of enterococci and related bacteria in raw and treated wastewater from a treatment plant receiving domestic and pretreated industrial effluents in order to assess the influence of treatment on the prevalence of antibiotic resistance phenotypes among this group of bacteria. The predominant species found in the raw wastewater were Entercoccus hirae, Entercoccus faecium and Entercoccus faecalis. Wastewater treatment led to a reduction in E. hirae (alpha<0.1) and an increase in E. faecium (alpha<0.1); the relative proportions of E. faecalis remained the same in the raw and in the treated wastewater. Among the isolates tested, no vancomycin resistance was observed among the enterococci. Entercoccus faecium and E. faecalis showed resistance prevalence values reaching 33%, 40% and 57% for the antibiotics ciprofloxacin, erythromycin and tetracycline, respectively. Antibiotic-resistant strains of enterococci were not eliminated by wastewater treatment. A positive selection of ciprofloxacin-resistant enterococci was indicated by a significant increase in resistance prevalence (alpha<0.02) in treated wastewater compared with the raw wastewater.

Tepidimonas thermarum sp. nov., a new slightly thermophilic betaproteobacterium isolated from the Elisenquelle in Aachen and emended description of the genus Tepidimonas.

Several non-pigmented bacterial isolates, with an optimum growth temperature of about 50-55 degrees C, were recovered from the Elisenquelle at Aachen, Germany. Phylogenetic analysis of the 16S rRNA gene sequence of strains AA-1(T) and AA-2 indicated that these organisms represent a new species of the genus Tepidimonas. The major fatty acids of strains AA-1(T) and AA-2 are 16:0 and 16:1 omega7c. Ubiquinone 8 is the major respiratory quinone, the major polar lipids are phosphotidylethanolamine and phosphotidylglycerol. The new isolates are aerobic; thiosulfate is oxidized to sulfate in the presence of a metabolizable carbon source. The organism assimilated organic acids and amino acids, but did not assimilate carbohydrates or polyols. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strains AA-1(T) (=LMG 23094(T); =CIP 108777(T)) and AA-2 (=LMG 23095; =CIP 108778) represents a new species for which we recommend the name Tepidimonas thermarum.

Chimaereicella alkaliphila gen. nov., sp. nov., a Gram-negative alkaliphilic bacterium isolated from a nonsaline alkaline groundwater.

A Gram-negative bacterium designated AC-74(T) was isolated from a highly alkaline groundwater environment (pH 11.4). This organism formed rod-shaped cells, is strictly aerobic, catalase and oxidase positive, tolerates up to 3.0% NaCl, has an optimum growth temperature of 30 degrees C, but no growth occurs at 10 or 40 degrees C, and an optimum pH value of 8.0, but no growth occurs at pH 7.0 or 11.3. The predominant fatty acids are iso-15:0, iso-17:1 omega9c and 16:1 omega7c and or iso-15:2OH. The G+C content of DNA was 43.5mol%. The phylogenetic analyses of the sequences of the 16s RNA genes indicated that strain AC-74(T) belongs to the family "Flexibacteriaceae" and is phylogenetically equidistant ( approximately 94.5%) from the majority of the species of the genus Algoriphagus and from the genus Hongiella. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strain AC-74(T), represents a new species of the novel genus for which we propose the name Chimaereicella alkaliphila gen. nov., sp. nov.

Dokdonella fugitiva sp. nov., a Gammaproteobacterium isolated from potting soil.

A Gram-negative bacterium designated A3(T) was isolated from a potting soil produced in Portugal. This organism formed rod-shaped cells, was non-motile and strictly aerobic. Strain A3(T) was catalase-negative, weakly oxidase positive, with an optimum growth temperature of 40 degrees C and an optimum pH value for growth of 7.0. The predominant cellular fatty acids detected were iso-17:1 omega9c, iso-17:0, iso-15:0, iso-16:0 and iso-11:0 3OH and the major respiratory quinone was ubiquinone 8. The G+C content of DNA was 68.3%. The phylogenetic analyses based on the 16S rRNA sequencing showed that the closest relative of strain A3(T) was the type strain of Dokdonella koreensis indicating that the organism is a member of the family Xanthomonadaceae, class Gammaproteobacteria. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strain A3(T), represents a novel species of the genus Dokdonella for which we propose the name Dokdonella fugitiva sp.nov.

Meiothermus timidus sp. nov., a new slightly thermophilic yellow-pigmented species.

Several yellow-pigmented isolates, with optimum growth temperatures between 55 and 60 degrees C, were recovered from hot springs in Central Portugal and the Azores. Phylogenetic analysis of the 16S rDNA showed that these organisms represented a new species of the genus Meiothermus. The new isolates could be distinguished from other strains of the species of the genus Meiothermus by biochemical characteristics and the fatty acid composition because they had very high levels of iso C15:0 and iso C17:0 and very low levels of anteiso C17:0 and iso C16:0. On the basis of the results presented here we propose the name Meiothermus timidus for the new species represented by strains SPS-243(T) (=LMG 22897(T)=CIP 108604(T)), RQ-10 and RQ-12.