Effects of Comano Spring Water-derived Bacterial Lysates on Skin Regeneration: An Ex-vivo Study.

BACKGROUND/AIM: A native non-pathogenic bacterial microflora was identified in Comano (TN, Italy) spring water. The aim of this study was to investigate the regenerative effects of some of the bacterial lysates extracted from this water in a human ex-vivo skin experimental wound model. MATERIALS AND METHODS: Bacterial lysates were extracted from four new isolates: lysate 1 (L1) - closest relative Rudaea cellulosilytica, phylum Proteobacteria; lysate 2 (L2) - closest relative Mesorhizobium erdmanii, phylum Proteobacteria; lysate 3 (L3) - closest relative Herbiconiux ginseng, phylum Actinobacteria; lysate 4 (L4) - closest relative Fictibacillus phosphorivorans, phylum Firmicutes. Their regenerative effects were investigated in a human ex-vivo skin experimental wound healing model at 3 (T1), 5 (T2), and 10 days (T3). RESULTS: The samples cultured with the L2 lysate displayed both an earlier and complete restoration of all the skin layers and their features were the closest to the normal skin. The regenerated epidermis demonstrated a complete maturation as the normal epidermis. The papillary dermis appeared mature, and the reticular dermis displayed both collagen and elastic fibres regularly parallel to the skin surface. An anti-inflammatory effect was displayed by the L1 lysate, but this action did not constitute a regenerative effect, suggesting that pathways for inflammation and regeneration might be different. CONCLUSION: The therapeutic power of spring waters is not exclusively related to their mineral composition, but it may also be attributable to their native non-pathogenic bacterial microflora.

Microbiome characterization of alpine water springs for human consumption reveals site- and usage-specific microbial signatures.

The microbiome of water springs is gaining increasing interest, especially in water intended for human consumption. However, the knowledge about large-scale patterns in water springs microbiome is still incomplete. The presence of bacteria in water sources used for human consumption is a major concern for health authorities; nonetheless, the standard microbiological quality checks are focused only on pathogenic species and total microbial load. Using 16S rRNA high throughput sequencing, we characterized the microbiome from 38 water springs in Trentino (Northern Italy) for 2 consecutive years in order to gain precious insights on the microbiome composition of these unexplored yet hardly exploited environments. The microbiological studies were integrated with standard measurements of physico-chemical parameters performed by the Provincial Office for Environmental Monitoring in order to highlight some of the dynamics influencing the microbial communities of these waters. We found that alpha diversity showed consistent patterns of variation overtime, and showed a strong positive correlation with the water nitrate concentration and negatively with fixed residue, electrical conductivity, and calcium concentration. Surprisingly, alpha diversity did not show any significant correlation with neither pH nor temperature. We found that despite their remarkable stability, different water springs display different coefficients of variation in alpha diversity, and that springs used for similar purposes showed similar microbiomes. Furthermore, the springs could be grouped according to the number of shared species into three major groups: low, mid, and high number of shared taxa, and those three groups of springs were consistent with the spring usage. Species belonging to the phyla Planctomycetes and Verrucomicrobia were prevalent and at relatively high abundance in springs classified as low number of shared species, whereas the phylum Lentisphaerae and the Candidate Phyla radiation were prevalent at higher abundance in the mineral and potable springs. The present study constitutes an example for standard water spring monitoring integrated with microbial community composition on a regional scale, and provides information which could be useful in the design and application of future water management policies in Trentino.

Mesorhizobium comanense sp. nov., isolated from groundwater.

Strain 3P27G6T was isolated from an artesian well connected to the thermal water basin of Comano Terme, Province of Trento, Italy. In phylogenetic analyses based on multilocus sequence analysis, strain 3P27G6T clustered together with Mesorhizobium australicum WSM2073T. Genome sequencing produced a 99.51 % complete genome, with a length of 7 363 057 bp and G+C content of 63.53 mol%, containing 6897 coding sequences, 55 tRNA and three rRNA. Average nucleotide identity analysis revealed that all distances calculated between strain 3P27G6T and the other Mesorhizobium genomes were below 0.9, indicating that strain 3P27G6T represents a new species. Therefore, we propose the name Mesorhizobium comanense sp. nov. with the type strain 3P27G6T (=DSM 110654T=CECT 30067T). Strain 3P27G6T is a Gram-negative, rod-shaped, aerobic bacterium. Growth condition, antibiotic susceptibility, metabolic and fatty acid-methyl esters profiles of the strain were determined. Only few nodulation and nitrogen fixation genes were found in the genome, suggesting that this strain may not be specialized in nodulation or in nitrogen fixation.

Insights into the genome structure of four acetogenic bacteria with specific reference to the Wood-Ljungdahl pathway.

Acetogenic bacteria are obligate anaerobes with the ability of converting carbon dioxide and other one-carbon substrates into acetate through the Wood-Ljungdahl (WL) pathway. These substrates are becoming increasingly important feedstock in industrial microbiology. The main potential industrial application of acetogenic bacteria is the production of metabolites that constitute renewable energy sources (biofuel); such bacteria are of particular interest for this purpose thanks to their low energy requirements for large-scale cultivation. Here, we report new genome sequences for four species, three of them are reported for the first time, namely Acetobacterium paludosum DSM 8237, Acetobacterium tundrae DSM 917, Acetobacterium bakii DSM 8239, and Alkalibaculum bacchi DSM 221123. We performed a comparative genomic analysis focused on the WL pathway's genes and their encoded proteins, using Acetobacterium woodii as a reference genome. The Average Nucleotide Identity (ANI) values ranged from 70% to 95% over an alignment length of 5.4-6.5 Mbp. The core genome consisted of 363 genes, whereas the number of unique genes in a single genome ranged from 486 in A. tundrae to 2360 in A.bacchi. No significant rearrangements were detected in the gene order for the Wood-Ljungdahl pathway however, two species showed variations in genes involved in formate metabolism: A. paludosum harbor two copies of fhs1, and A. bakii a truncated fdhF1. The analysis of protein networks highlighted the expansion of protein orthologues in A. woodii compared to A. bacchi, whereas protein networks involved in the WL pathway were more conserved. This study has increased our understanding on the evolution of the WL pathway in acetogenic bacteria.

Genomic and metagenomic insights into the microbial community of a thermal spring.

BACKGROUND: Water springs provide important ecosystem services including drinking water supply, recreation, and balneotherapy, but their microbial communities remain largely unknown. In this study, we characterized the spring water microbiome of Comano Terme (Italy) at four sampling points of the thermal spa, including natural (spring and well) and human-built (storage tank, bathtubs) environments. We integrated large-scale culturing and metagenomic approaches, with the aim of comprehensively determining the spring water taxonomic composition and functional potential. RESULTS: The groundwater feeding the spring hosted the most atypical microbiome, including many taxa known to be recalcitrant to cultivation. The core microbiome included the orders Sphingomonadales, Rhizobiales, and Caulobacterales, and the families Bradyrhizobiaceae and Moraxellaceae. A comparative genomic analysis of 72 isolates and 30 metagenome-assembled genomes (MAGs) revealed that most isolates and MAGs belonged to new species or higher taxonomic ranks widely distributed in the microbial tree of life. Average nucleotide identity (ANI) values calculated for each isolated or assembled genome showed that 10 genomes belonged to known bacterial species (> 95% ANI), 36 genomes (including 1 MAG) had ANI values ranging 85-92.5% and could be assigned as undescribed species belonging to known genera, while the remaining 55 genomes had lower ANI values (< 85%). A number of functional features were significantly over- or underrepresented in genomes derived from the four sampling sites. Functional specialization was found between sites, with for example methanogenesis being unique to groundwater whereas methanotrophy was found in all samples. CONCLUSIONS: Current knowledge on aquatic microbiomes is essentially based on surface or human-associated environments. We started uncovering the spring water microbiome, highlighting an unexpected diversity that should be further investigated. This study confirms that groundwater environments host highly adapted, stable microbial communities composed of many unknown taxa, even among the culturable fraction.

Pseudomonas aeruginosa mutants defective in glucose uptake have pleiotropic phenotype and altered virulence in non-mammal infection models.

Pseudomonas spp. are endowed with a complex pathway for glucose uptake that relies on multiple transporters. In this work we report the construction and characterization of Pseudomonas aeruginosa single and multiple mutants with unmarked deletions of genes encoding outer membrane (OM) and inner membrane (IM) proteins involved in glucose uptake. We found that a triple ΔgltKGF ΔgntP ΔkguT mutant lacking all known IM transporters (named GUN for Glucose Uptake Null) is unable to grow on glucose as unique carbon source. More than 500 genes controlling both metabolic functions and virulence traits show differential expression in GUN relative to the parental strain. Consistent with transcriptomic data, the GUN mutant displays a pleiotropic phenotype. Notably, the genome-wide transcriptional profile and most phenotypic traits differ between the GUN mutant and the wild type strain irrespective of the presence of glucose, suggesting that the investigated genes may have additional roles besides glucose transport. Finally, mutants carrying single or multiple deletions in the glucose uptake genes showed attenuated virulence relative to the wild type strain in Galleria mellonella, but not in Caenorhabditis elegans infection model, supporting the notion that metabolic functions may deeply impact P. aeruginosa adaptation to specific environments found inside the host.

Air and waterborne microbiome of a pharmaceutical plant provide insights on spatiotemporal variations and community resilience after disturbance.

BACKGROUND: The presence of microrganisms in pharmaceutical production plant environments is typically monitored by cultural methods, however these cannot detect the unculturable fraction of the microbial community. To get more accurate information on the composition of these indoor microbial communities, both water and air microbiome from a pharmaceutical production plant were profiled by 16S amplicon sequencing. RESULTS: In the water system, we found taxa which typically characterize surface freshwater, groundwater and oligotrophic environments. The airborne microbiome resulted dominated by taxa usually found in outdoor air in combination with human-associated taxa. The alpha- and beta- diversity values showed that the heat-based sanitization process of the water plant affects the composition of the water microbiome by transiently increasing both diversity and evenness. Taxonomic compositional shifts were also detected in response to sanitization, consisting in an increase of Firmicutes and α-Proteobacteria. On the other hand, seasonality seems to be the main driver of bacterial community composition in air of this work environment. CONCLUSIONS: This approach resulted useful to describe the taxonomy of these indoor microbiomes and could be further applied to other built environments, in which the knowledge of the microbiome composition is of relevance. In addition, this study could assist in the design of new guidelines to improve microbiological quality control in indoor work environments.

Intestinal Candida parapsilosis isolates from Rett syndrome subjects bear potential virulent traits and capacity to persist within the host.

BACKGROUND: Rett syndrome (RTT) is a neurological disorder mainly caused by mutations in MeCP2 gene. It has been shown that MeCP2 impairments can lead to cytokine dysregulation due to MeCP2 regulatory role in T-helper and T-reg mediated responses, thus contributing to the pro-inflammatory status associated with RTT. Furthermore, RTT subjects suffer from an intestinal dysbiosis characterized by an abnormal expansion of the Candida population, a known factor responsible for the hyper-activation of pro-inflammatory immune responses. Therefore, we asked whether the intestinal fungal population of RTT subjects might contribute the sub-inflammatory status triggered by MeCP2 deficiency. METHODS: We evaluated the cultivable gut mycobiota from a cohort of 50 RTT patients and 29 healthy controls characterizing the faecal fungal isolates for their virulence-related traits, antifungal resistance and immune reactivity in order to elucidate the role of fungi in RTT's intestinal dysbiosis and gastrointestinal physiology. RESULTS: Candida parapsilosis, the most abundant yeast species in RTT subjects, showed distinct genotypic profiles if compared to healthy controls' isolates as measured by hierarchical clustering analysis from RAPD genotyping. Their phenotypical analysis revealed that RTT's isolates produced more biofilm and were significantly more resistant to azole antifungals compared to the isolates from the healthy controls. In addition, the high levels of IL-1ß and IL-10 produced by peripheral blood mononuclear cells and the mixed Th1/Th17 cells population induced by RTT C. parapsilosis isolates suggest the capacity of these intestinal fungi to persist within the host, being potentially involved in chronic, pro-inflammatory responses. CONCLUSIONS: Here we demonstrated that intestinal C. parapsilosis isolates from RTT subjects hold phenotypic traits that might favour the previously observed low-grade intestinal inflammatory status associated with RTT. Therefore, the presence of putative virulent, pro-inflammatory C. parapsilosis strains in RTT could represent an additional factor in RTT's gastrointestinal pathophysiology, whose mechanisms are not yet clearly understood.

Persistence and Microevolution of Pseudomonas aeruginosa in the Cystic Fibrosis Lung: A Single-Patient Longitudinal Genomic Study.

Background: During its persistence in cystic fibrosis (CF) airways, P. aeruginosa develops a series of phenotypic changes by the accumulation of pathoadaptive mutations. A better understanding of the role of these mutations in the adaptive process, with particular reference to the development of multidrug resistance (MDR), is essential for future development of novel therapeutic approaches, including the identification of new drug targets and the implementation of more efficient antibiotic therapy. Although several whole-genome sequencing studies on P. aeruginosa CF lineages have been published, the evolutionary trajectories in relation to the development of antimicrobial resistance remain mostly unexplored to date. In this study, we monitored the adaptive changes of P. aeruginosa during its microevolution in the CF airways to provide an innovative, genome-wide picture of mutations and persistent phenotypes and to point out potential novel mechanisms allowing survival in CF patients under antibiotic therapy. Results: We obtained whole genome sequences of 40 P. aeruginosa clinical CF strains isolated at Trentino Regional Support CF Centre (Rovereto, Italy) from a single CF patient over an 8-year period (2007-2014). Genotypic analysis of the P. aeruginosa isolates revealed a clonal population dominated by the Sequence Type 390 and three closely related variants, indicating that all members of the population likely belong to the same clonal lineage and evolved from a common ancestor. While the majority of early isolates were susceptible to most antibiotics tested, over time resistant phenotypes increased in the persistent population. Genomic analyses of the population indicated a correlation between the evolution of antibiotic resistance profiles and phylogenetic relationships, and a number of putative pathoadaptive variations were identified. Conclusion: This study provides valuable insights into the within-host adaptation and microevolution of P. aeruginosa in the CF lung and revealed the emergence of an MDR phenotype over time, which could not be comprehensively explained by the variations found in known resistance genes. Further investigations on uncharacterized variations disclosed in this study should help to increase our understanding of the development of MDR phenotype and the poor outcome of antibiotic therapies in many CF patients.

The new phylogeny of the genus Mycobacterium: The old and the news.

BACKGROUND: Phylogenetic studies of bacteria have been based so far either on a single gene (usually the 16S rRNA) or on concatenated housekeeping genes. For what concerns the genus Mycobacterium these approaches support the separation of rapidly and slowly growing species and the clustering of most species in well-defined phylogenetic groups. The advent of high-throughput shotgun sequencing leads us to revise conventional taxonomy of mycobacteria on the light of genomic data. For this purpose we investigated 88 newly sequenced species in addition to 60 retrieved from GenBank and used the Average Nucleotide Identity pairwise scores to reconstruct phylogenetic relationships within this genus. RESULTS: Our analysis confirmed the separation of slow and rapid growers and the intermediate position occupied by the M. terrae complex. Among the rapid growers, the species of the M. chelonae-abscessus complex belonged to the most ancestral cluster. Other major clades of rapid growers included the species related to M. fortuitum and M. smegmatis and a large grouping containing mostly environmental species rarely isolated from humans. The members of the M. terrae complex appeared as the most ancestral slow growers. Among slow growers two deep branches led to the clusters of species related to M. celatum and M. xenopi and to a large group harboring most of the species more frequently responsible of disease in humans, including the major pathogenic mycobacteria (M. tuberculosis, M. leprae, M. ulcerans). The species previously grouped in the M. simiae complex were allocated in a number of sub-clades; of them, only the one including the species M. simiae identified the real members of this complex. The other clades included also species previously not considered related to M. simiae. The ANI analysis, in most cases supported by Genome to Genome Distance and by Genomic Signature-Delta Difference, showed that a number of species with standing in literature were indeed synonymous. CONCLUSIONS: Genomic data revealed to be much more informative in comparison with phenotype. We believe that the genomic revolution enabled by high-throughput shotgun sequencing should now be considered in order to revise the conservative approaches still informing taxonomic sciences.

Evolution of Stenotrophomonas maltophilia in Cystic Fibrosis Lung over Chronic Infection: A Genomic and Phenotypic Population Study.

Stenotrophomonas maltophilia has been recognized as an emerging multi-drug resistant opportunistic pathogen in cystic fibrosis (CF) patients. We report a comparative genomic and phenotypic analysis of 91 S. maltophilia strains from 10 CF patients over a 12-year period. Draft genome analyses included in silico Multi-Locus Sequence Typing (MLST), Single-Nucleotide Polymorphisms (SNPs), and pangenome characterization. Growth rate, biofilm formation, motility, mutation frequency, in vivo virulence, and in vitro antibiotic susceptibility were determined and compared with population structure over time. The population consisted of 20 different sequence types (STs), 11 of which are new ones. Pangenome and SNPs data showed that this population is composed of three major phylogenetic lineages. All patients were colonized by multiple STs, although most of them were found in a single patient and showed persistence over years. Only few phenotypes showed some correlation with population phylogenetic structure. Our results show that S. maltophilia adaptation to CF lung is associated with consistent genotypic and phenotypic heterogeneity. Stenotrophomonas maltophilia infecting multiple hosts likely experiences different selection pressures depending on the host environment. The poor genotype-phenotype correlation suggests the existence of complex regulatory mechanisms that need to be explored in order to better design therapeutic strategies.

Unexplored diversity and strain-level structure of the skin microbiome associated with psoriasis.

Psoriasis is an immune-mediated inflammatory skin disease that has been associated with cutaneous microbial dysbiosis by culture-dependent investigations and rRNA community profiling. We applied, for the first time, high-resolution shotgun metagenomics to characterise the microbiome of psoriatic and unaffected skin from 28 individuals. We demonstrate psoriatic ear sites have a decreased diversity and psoriasis is associated with an increase in Staphylococcus, but overall the microbiomes of psoriatic and unaffected sites display few discriminative features at the species level. Finer strain-level analysis reveals strain heterogeneity colonisation and functional variability providing the intriguing hypothesis of psoriatic niche-specific strain adaptation or selection. Furthermore, we accessed the poorly characterised, but abundant, clades with limited sequence information in public databases, including uncharacterised Malassezia spp. These results highlight the skins hidden diversity and suggests strain-level variations could be key determinants of the psoriatic microbiome. This illustrates the need for high-resolution analyses, particularly when identifying therapeutic targets. This work provides a baseline for microbiome studies in relation to the pathogenesis of psoriasis.

Two-Way Chemical Communication between Artificial and Natural Cells.

Artificial cells capable of both sensing and sending chemical messages to bacteria have yet to be built. Here we show that artificial cells that are able to sense and synthesize quorum signaling molecules can chemically communicate with V. fischeri, V. harveyi, E. coli, and P. aeruginosa. Activity was assessed by fluorescence, luminescence, RT-qPCR, and RNA-seq. Two potential applications for this technology were demonstrated. First, the extent to which artificial cells could imitate natural cells was quantified by a type of cellular Turing test. Artificial cells capable of sensing and in response synthesizing and releasing N-3-(oxohexanoyl)homoserine lactone showed a high degree of likeness to natural V. fischeri under specific test conditions. Second, artificial cells that sensed V. fischeri and in response degraded a quorum signaling molecule of P. aeruginosa (N-(3-oxododecanoyl)homoserine lactone) were constructed, laying the foundation for future technologies that control complex networks of natural cells.

Genomic characterization of Nontuberculous Mycobacteria.

Mycobacterium tuberculosis and Mycobacterium leprae have remained, for many years, the primary species of the genus Mycobacterium of clinical and microbiological interest. The other members of the genus, referred to as nontuberculous mycobacteria (NTM), have long been underinvestigated. In the last decades, however, the number of reports linking various NTM species with human diseases has steadily increased and treatment difficulties have emerged. Despite the availability of whole genome sequencing technologies, limited effort has been devoted to the genetic characterization of NTM species. As a consequence, the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting. In this work, we widen the knowledge of NTMs by reconstructing and analyzing the genomes of 41 previously uncharacterized NTM species. We provide the first comprehensive characterization of the genomic diversity of NTMs and open new venues for the clinical identification of opportunistic pathogens from this genus.

New evidences on the altered gut microbiota in autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASDs) are neurodevelopmental conditions characterized by social and behavioural impairments. In addition to neurological symptoms, ASD subjects frequently suffer from gastrointestinal abnormalities, thus implying a role of the gut microbiota in ASD gastrointestinal pathophysiology. RESULTS: Here, we characterized the bacterial and fungal gut microbiota in a cohort of autistic individuals demonstrating the presence of an altered microbial community structure. A fraction of 90% of the autistic subjects were classified as severe ASDs. We found a significant increase in the Firmicutes/Bacteroidetes ratio in autistic subjects due to a reduction of the Bacteroidetes relative abundance. At the genus level, we observed a decrease in the relative abundance of Alistipes, Bilophila, Dialister, Parabacteroides, and Veillonella in the ASD cohort, while Collinsella, Corynebacterium, Dorea, and Lactobacillus were significantly increased. Constipation has been then associated with different bacterial patterns in autistic and neurotypical subjects, with constipated autistic individuals characterized by high levels of bacterial taxa belonging to Escherichia/Shigella and Clostridium cluster XVIII. We also observed that the relative abundance of the fungal genus Candida was more than double in the autistic than neurotypical subjects, yet due to a larger dispersion of values, this difference was only partially significant. CONCLUSIONS: The finding that, besides the bacterial gut microbiota, also the gut mycobiota contributes to the alteration of the intestinal microbial community structure in ASDs opens the possibility for new potential intervention strategies aimed at the relief of gastrointestinal symptoms in ASDs.

AoS28D, a proline-Xaa carboxypeptidase secreted by Aspergillus oryzae.

Prolyl peptidases of the MEROPS S28 family are of particular interest because they are key enzymes in the digestion of proline-rich peptides. A BLAST analysis of the Aspergillus oryzae genome revealed sequences coding for four proteases of the S28 family. Three of these proteases, AoS28A, AoS28B, and AoS28C, were previously characterized as acidic prolyl endopeptidases. The fourth protease, AoS28D, showed high sequence divergence with other S28 proteases and belongs to a phylogenetically distinct cluster together with orthologous proteases from other Aspergillus species. The objective of the present paper was to characterize AoS28D protease in terms of substrate specificity and activity. AoS28D produced by gene overexpression in A. oryzae and in Pichia pastoris was a 70-kDa glycoprotein with a 10-kDa sugar moiety. In contrast with other S28 proteases, AoS28D did not hydrolyze internal Pro-Xaa bonds of several tested peptides. Similarly, to human lysosomal Pro-Xaa carboxypeptidase, AoS28D demonstrated selectivity for cleaving C-terminal Pro-Xaa bonds which are resistant to carboxypeptidases of the S10 family concomitantly secreted by A. oryzae. Therefore, AoS28D could act in synergy with these enzymes during sequential degradation of a peptide from its C-terminus.

Conjugative type IVb pilus recognizes lipopolysaccharide of recipient cells to initiate PAPI-1 pathogenicity island transfer in Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1) is one of the largest genomic islands of this important opportunistic human pathogen. Previous studies have shown that PAPI-1 encodes several putative virulence factors, including a major regulator of biofilm formation and antibiotic-resistance traits. PAPI-1 is horizontally transferable into recipient strains lacking this island via conjugation mediated by the specialized type IV pilus. The PAPI-1 encodes a cluster of ten genes associated with the synthesis and assembly of the type IV pilus. The PAPI-1 acquisition mechanism is currently not well understood. RESULTS: In this study, we performed a series of conjugation experiments and identified determinants of PAPI-1 acquisition by analyzing transfer efficiency between the donor and a series of mutant recipient strains. Our data show that common polysaccharide antigen (CPA) lipopolysaccharide (LPS), a homopolymer of D-rhamnose, is required for initiating PAPI-1 transfer, suggesting that this structure acts as a receptor for conjugative type IV pilus in recipient strains. These results were substantiated by experimental evidence from PAPI-1 transfer assay experiments, in which outer membrane or LPS preparations from well-defined LPS mutants were added to the transfer mix to assess the role of P. aeruginosa LPS in PAPI-1 transfer and in vitro binding experiments between pilin fusion protein GST-pilV2' and immobilized LPS molecules were performed. Our data also showed that P. aeruginosa strains that had already acquired a copy of PAPI-1 were unable to import additional copies of the island, and that such strains produced proportionally lower amounts of CPA LPS compared to the strains lacking PAPI-1. CONCLUSIONS: These results suggest that a PAPI-1 exclusion mechanism exists in P. aeruginosa that might serve to regulate the avoidance of uncontrolled expansions of the bacterial genome.

MetaMLST: multi-locus strain-level bacterial typing from metagenomic samples.

Metagenomic characterization of microbial communities has the potential to become a tool to identify pathogens in human samples. However, software tools able to extract strain-level typing information from metagenomic data are needed. Low-throughput molecular typing schema such as Multilocus Sequence Typing (MLST) are still widely used and provide a wealth of strain-level information that is currently not exploited by metagenomic methods. We introduce MetaMLST, a software tool that reconstructs the MLST loci of microorganisms present in microbial communities from metagenomic data. Tested on synthetic and spiked-in real metagenomes, the pipeline was able to reconstruct the MLST sequences with >98.5% accuracy at coverages as low as 1×. On real samples, the pipeline showed higher sensitivity than assembly-based approaches and it proved successful in identifying strains in epidemic outbreaks as well as in intestinal, skin and gastrointestinal microbiome samples.

Draft Genome Sequences of 40 Pseudomonas aeruginosa Clinical Strains Isolated from the Sputum of a Single Cystic Fibrosis Patient Over an 8-Year Period.

We report draft genome sequences of 40 Pseudomonas aeruginosa strains, isolated from the sputum of a single cystic fibrosis patient over eight years. Analyses indicated a correlation between multidrug-resistant phenotypes and population structure. Our data provide new insights into the mechanisms leading to acquisition of antibiotic resistance in P. aeruginosa.

Age and Gender Affect the Composition of Fungal Population of the Human Gastrointestinal Tract.

The fungal component of the human gut microbiota has been neglected for long time due to the low relative abundance of fungi with respect to bacteria, and only recently few reports have explored its composition and dynamics in health or disease. The application of metagenomics methods to the full understanding of fungal communities is currently limited by the under representation of fungal DNA with respect to the bacterial one, as well as by the limited ability to discriminate passengers from colonizers. Here, we investigated the gut mycobiota of a cohort of healthy subjects in order to reduce the gap of knowledge concerning fungal intestinal communities in the healthy status further screening for phenotypical traits that could reflect fungi adaptation to the host. We studied the fecal fungal populations of 111 healthy subjects by means of cultivation on fungal selective media and by amplicon-based ITS1 metagenomics analysis on a subset of 57 individuals. We then characterized the isolated fungi for their tolerance to gastrointestinal (GI) tract-like challenges and their susceptibility to antifungals. A total of 34 different fungal species were isolated showing several phenotypic characteristics associated with intestinal environment such as tolerance to body temperature (37°C), to acidic and oxidative stress, and to bile salts exposure. We found a high frequency of azoles resistance in fungal isolates, with potential and significant clinical impact. Analyses of fungal communities revealed that the human gut mycobiota differs in function of individuals' life stage in a gender-related fashion. The combination of metagenomics and fungal cultivation allowed an in-depth understanding of the fungal intestinal community structure associated to the healthy status and the commensalism-related traits of isolated fungi. We further discussed comparatively the results of sequencing and cultivation to critically evaluate the application of metagenomics-based approaches to fungal gut populations.