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The genomic era has resulted in the generation of a massive amount of genetic data concerning the genomic diversity of bacterial taxa. As a result, the microbiological community is increasingly looking for ways to define reference bacterial strains to perform experiments that are representative of the entire bacterial species. Despite this, there is currently no established approach allowing a reliable identification of reference strains based on a comprehensive genomic, ecological, and functional context. In the current study, we developed a comprehensive multi-omics approach that will allow the identification of the optimal reference strains using the Bifidobacterium genus as test case. Strain tracking analysis based on 1664 shotgun metagenomics datasets of healthy infant faecal samples were employed to identify bifidobacterial strains suitable for in silico and in vitro analyses. Subsequently, an ad hoc bioinformatic tool was developed to screen local strain collections for the most suitable species-representative strain alternative. The here presented approach was validated using in vitro trials followed by metagenomics and metatranscriptomics analyses. Altogether, these results demonstrated the validity of the proposed model for reference strain selection, thus allowing improved in silico and in vitro investigations both in terms of cross-laboratory reproducibility and relevance of research findings.

Bifidobacteria have been shown to produce exopolysaccharides (EPS), which are polymeric structures composed of various carbohydrates, commonly containing glucose, galactose, and rhamnose. EPS are produced by different bifidobacterial taxa commonly identified in the human gut, such as Bifidobacterium breve and Bifidobacterium longum subsp. longum, and have been suggested to modulate the interaction of bifidobacterial cells with other members of the human gut microbiota as well as with their host. In this study, we evaluated if bifidobacterial EPS production of four selected EPS-producing strains is associated with enhanced resistance to antibiotic treatments through MIC analysis when compared to bacterial cultures that do not produce exopolysaccharides. Our results showed that an increase in EPS production by modifying the growth medium with different carbon sources, i.e. glucose, galactose or lactose and/or by applying stressful conditions, such as bile salts and acidity, is associated with a tolerance enhancement of bifidobacterial cells toward various beta-lactam antibiotics. In addition, after analyzing the production of EPS at the phenotypic level, we explored the genes involved in the production of these structures and evaluated their expression, in presence of various carbon sources, using RNAseq. Overall, this study provides preliminary experimental evidence showing how bifidobacterial EPS modifies the level of susceptibility of these bacteria towards antibiotics.

Fresh potable water is an indispensable drink which humans consume daily in substantial amounts. Nonetheless, very little is known about the composition of the microbial community inhabiting drinking water or its impact on our gut microbiota. In the current study, an exhaustive shotgun metagenomics analysis of the tap water microbiome highlighted the occurrence of a highly genetic biodiversity of the microbial communities residing in fresh water and the existence of a conserved core tap water microbiota largely represented by novel microbial species, representing microbial dark matter. Furthermore, genome reconstruction of this microbial dark matter from water samples unveiled homologous sequences present in the faecal microbiome of humans from various geographical locations. Accordingly, investigation of the faecal microbiota content of a subject that daily consumed tap water for 3 years provides proof for horizontal transmission and colonization of water bacteria in the human gut.

Amoxicillin-clavulanic acid (AMC) is the most widely used antibiotic, being frequently prescribed to infants. Particular members of the genus Bifidobacterium are among the first microbial colonizers of the infant gut, and it has been demonstrated that they exhibit various activities beneficial for their human host, including promotion/maintenance of the human gut microbiota homeostasis. It has been shown that natural resistance of bifidobacteria to AMC is limited to a small number of strains. In the current study, we investigated the mitigation effects of AMC-resistant bifidobacteria in diversity preservation of the gut microbiota during AMC treatment. To this end, an in vitro coculture experiment based on infant fecal samples and an in vivo study employing a rodent model were performed. The results confirmed the ability of AMC-resistant bifidobacterial strains to bolster gut microbiota resilience, while specific covariance analysis revealed strain-specific and variable impacts on the microbiota composition by individual bifidobacterial taxa. IMPORTANCE The first microbial colonizers of the infant gut are members of the genus Bifidobacterium, which exhibit different activities beneficial to their host. Amoxicillin-clavulanic acid (AMC) is the most frequently prescribed antibiotic during infancy, and few strains of bifidobacteria are known to show a natural resistance to this antibiotic. In the present work, we evaluated the possible positive effects of AMC-resistant bifidobacterial strains in maintaining gut microbiota diversity during AMC exposure, performing an in vitro and in vivo experiment based on an infant gut model and a rodent model, respectively. Our results suggested the ability of AMC-resistant bifidobacterial strains to support gut microbiota restoration.

TwoBifidobacteriumstrains, i.e., 2176BTand 2177BT, were isolated from Golden-Headed Lion Tamarin (Leontopithecus chrysomelas) and Goeldi's monkey (Callimico goeldii). Isolates were shown to be Gram-positive, non-motile, non-sporulating, facultative anaerobic andd-fructose 6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA sequences, multilocus sequences (includinghsp60,rpoB,dnaJ,dnaG andclpC genes) and the core genome revealed that bifidobacterial strains 2176BTand 2177BTexhibit close phylogenetic relatedness toBifidobacterium felsineumDSM 103139TandBifidobacterium bifidumLMG 11041T, respectively. Further genotyping based on the genome sequence of the isolated strains combined with phenotypic analyses, clearly show that these strains are distinct from each of the type strains of the so far recognizedBifidobacteriumspecies. Thus,Bifidobacterium cebidarumsp. nov. (2176BT=LMG 31469T=CCUG 73785T) andBifidobacterium leontopithecisp. nov. (2177BT=LMG 31471T=CCUG 73786Tare proposed as novelBifidobacteriumspecies.

Members of the genus Lactobacillus represent the most common colonizers of the human vagina and are well-known for preserving vaginal health and contrasting the colonization of opportunistic pathogens. Remarkably, high abundance of Lactobacillus crispatus in the vaginal environment has been linked to vaginal health, leading to the widespread use of many L. crispatus strains as probiotics. Nevertheless, despite the scientific and industrial relevance of this species, a comprehensive investigation of the genomics of L. crispatus taxon is still missing. For this reason, we have performed a comparative genomics analysis of 97 L. crispatus strains, encompassing 16 strains sequenced in the framework of this study alongside 81 additional publicly available genome sequences. Thus, allowing the dissection of the L.crispatus pan-genome and core-genome followed by a comprehensive phylogenetic analysis based on the predicted core genes that revealed clustering based on ecological origin. Subsequently, a genomics-targeted approach, i.e., probiogenomics analysis, was applied for in-depth analysis of the eight L. crispatus strains of human origin sequenced in this study. In detail their genetic repertoire was screened for strain-specific genes responsible for phenotypic features that may guide the identification of optimal candidates for next-generation probiotics. The latter includes bacteriocin production, carbohydrates transport and metabolism, as well as a range of features that may be responsible for improved ecological fitness. In silico results regarding the genetic repertoire involved in carbohydrate metabolism were also validated by growth assays on a range of sugars, leading to the selection of putative novel probiotic strains.

Probiotic bacteria are widely administered as dietary supplements and incorporated as active ingredients in a variety of functional foods due to their purported health-promoting features. Currently available probiotic products may have issues with regards to their formulation, such as insufficient levels of viable probiotic bacteria, complete lack of probiotic strains that are stated to be present in the product, and the presence of microbial contaminants. To avoid the distribution of such unsuitable or misleading products, we propose here a novel approach named Probiotic Identity Card (PIC), involving a combination of shotgun metagenomic sequencing and bacterial cell enumeration by flow cytometry. PIC was tested on 12 commercial probiotic supplements revealing several inconsistencies in the formulation of five such products based on their stated microbial composition and viability.

International audience; Necrotizing enterocolitis (NEC) is among the most relevant gastrointestinal diseases affecting mostly prematurely born infants with low birth weight. While intestinal dysbiosis has been proposed as one of the possible factors involved in NEC pathogenesis, the role of the gut microbiota remains poorly understood. In this study, the gut microbiota of preterm infants was explored to highlight differences in the composition between infants affected by NEC and infants prior to NEC development. A large-scale gut microbiome analysis was performed, including 47 shotgun sequencing data sets generated in the framework of this study, along with 124 retrieved from publicly available repositories. Meta-analysis led to the identification of preterm community state types (PT- CSTs), which recur in healthy controls and NEC infants. Such analyses revealed an overgrowth of a range of opportunistic microbial species accompanying the loss of gut microbial biodiversity in NEC subjects. Moreover, longitudinal insights into preterm infants prior to NEC development indicated Clostridium neonatale and Clostridium perfringens species as potential biomarkers for predictive early diagnosis of this disease. Furthermore, functional investigation of the enzymatic reaction profiles associated with pre-NEC condition suggested DL-lactate as a putative metabolic biomarker for early detection of NEC onset.IMPORTANCE Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease occurring predominantly in premature infants whose etiology is still not fully understood. In this study, the analysis of infant fecal samples through shotgun metagenomics approaches revealed a marked reduction of the intestinal (bio)diversity and an overgrowth of (opportunistic) pathogens associated with the NEC development. In particular, dissection of the infant's gut microbiome before NEC diagnosis high-lighted the potential involvement of Clostridium genus members in the progression of NEC. Remarkably, our analyses highlighted a gastrointestinal DL-lactate accumulation among NEC patients that might represent a novel potential functional biomarker for the early diagnosis of NEC.

Members of the Bifidobacterium longum species have been shown to possess adaptive abilities to allow colonization of different mammalian hosts, including humans, primates and domesticated mammalian species, such as dogs, horses, cattle and pigs. To date, three subspecies have formally been recognized to belong to this bifidobacterial taxon, i.e. B. longum subsp. longum , B. longum subsp. infantis and B. longum subsp. suis . Although B. longum subsp. longum is widely distributed in the human gut irrespective of host age, B. longum subsp. infantis appears to play a significant role as a prominent member of the gut microbiota of breast-fed infants. Nevertheless, despite the considerable scientific relevance of these taxa and the vast body of genomic data now available, an accurate dissection of the genetic features that comprehensively characterize the B. longum species and its subspecies is still missing. In the current study, we employed 261 publicly available B. longum genome sequences, combined with those of 11 new isolates, to investigate genomic diversity of this taxon through comparative genomic and phylogenomic approaches. These analyses allowed us to highlight a remarkable intra-species genetic and physiological diversity. Notably, characterization of the genome content of members of B. longum subsp. infantis subspecies suggested that this taxon may have acquired genetic features for increased competitiveness in the gut environment of suckling hosts. Furthermore, specific B. longum subsp. infantis genomic features appear to be responsible for enhanced horizontal gene transfer (HGT) occurrences, underpinning an intriguing dedication toward acquisition of foreign DNA by HGT events.

Members of the Bifidobacterium longum species have been shown to possess adaptive abilities to allow colonization of different mammalian hosts, including humans, primates and domesticated mammalian species, such as dogs, horses, cattle and pigs. To date, three subspecies have formally been recognized to belong to this bifidobacterial taxon, i.e. B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis. Although B. longum subsp. longum is widely distributed in the human gut irrespective of host age, B. longum subsp. infantis appears to play a significant role as a prominent member of the gut microbiota of breast-fed infants. Nevertheless, despite the considerable scientific relevance of these taxa and the vast body of genomic data now available, an accurate dissection of the genetic features that comprehensively characterize the B. longum species and its subspecies is still missing. In the current study, we employed 261 publicly available B. longum genome sequences, combined with those of 11 new isolates, to investigate genomic diversity of this taxon through comparative genomic and phylogenomic approaches. These analyses allowed us to highlight a remarkable intra-species genetic and physiological diversity. Notably, characterization of the genome content of members of B. longum subsp. infantis subspecies suggested that this taxon may have acquired genetic features for increased competitiveness in the gut environment of suckling hosts. Furthermore, specific B. longum subsp. infantis genomic features appear to be responsible for enhanced horizontal gene transfer (HGT) occurrences, underpinning an intriguing dedication toward acquisition of foreign DNA by HGT events.

The use of bioinformatic tools for read-based taxonomic and functional analyses of metagenomic data sets, including their assembly and management, is rather fragmentary due to the absence of an accepted gold standard. Moreover, most currently available software tools need input of millions of reads and rely on approximations in data analysis in order to reduce computing times. These issues result in suboptimal results in terms of accuracy, sensitivity, and specificity when used either for the reconstruction of taxonomic or functional profiles through read analysis or analysis of genomes reconstructed by metagenomic assembly. Moreover, the recent introduction of novel DNA sequencing technologies that generate long reads, such as Nanopore and PacBio, represent a valuable data resource that still suffers from a lack of dedicated tools to perform integrated hybrid analysis alongside short read data. In order to overcome these limitations, here we describe a comprehensive bioinformatic platform, METAnnotatorX2, aimed at providing an optimized user-friendly resource which maximizes output quality, while also allowing user-specific adaptation of the pipeline and straightforward integrated analysis of both short and long read data. To further improve performance quality and accuracy of taxonomic assignment of reads and contigs, custom preprocessed and taxonomically revised genomic databases for viruses, prokaryotes, and various eukaryotes were developed. The performance of METAnnotatorX2 was tested by analysis of artificial data sets encompassing viral, archaeal, bacterial, and eukaryotic (fungal) sequence reads that simulate different biological matrices. Moreover, real biological samples were employed to validate in silico results. IMPORTANCE We developed a novel tool, i.e., METAnnotatorX2, that includes a number of new advanced features for analysis of deep and shallow metagenomic data sets and is accompanied by (regularly updated) customized databases for archaea, bacteria, fungi, protists, and viruses. Both software and databases were developed so as to maximize sensitivity and specificity while including support for shallow metagenomic data sets. Through extensive tests performed on Illumina and Nanopore artificial data sets, we demonstrated the high performance of the software to not only extract taxonomic and functional information from sequence reads but also to assemble and process genomes from metagenomic data. The robustness of these functionalities was validated using “real-life” data sets obtained from Illumina and Nanopore sequencing of biological samples. Furthermore, the performance of METAnnotatorX2 was compared to other available software tools for analysis of shotgun metagenomics data.

The abilities of certain microorganisms to be transferred across the food production chain, persist in the final product and, potentially, colonize the human gut are poorly understood. Here, we provide strain-level evidence supporting that dairy cattle-associated bacteria can be transferred to the human gut via consumption of Parmesan cheese. We characterize the microbial communities in samples taken from five different locations across the Parmesan cheese production chain, confirming that the final product contains microorganisms derived from cattle gut, milk, and the nearby environment. In addition, we carry out a human pilot study showing that Bifidobacterium mongoliense strains from cheese can transiently colonize the human gut, a process that can be enhanced by cow milk consumption., Some microorganisms may be transferred across the food production chain and, potentially, colonize the human gut. Here, Milani et al. provide strain-level evidence supporting that dairy cattle-associated bacteria can be transferred to the human gut via consumption of Parmesan cheese.

In recent decades, infants' gut microbiota has aroused constant scientific interest, primarily due to early‐ and long‐term repercussions on the host health. In this context, nutritional challenges such as those found in less developed countries can influence infants' gut microbiota development, thus generating potentially critical health outcomes. However, comprehensive investigations regarding species‐level differences in the infant gut microbiota's composition between urbanized and rural countries are still missing. In this study, 16S rRNA and Shallow Shotgun metagenomics sequencing were exploited to dissect the microbial community's species‐level composition of 11 faecal samples collected from infants living in a semi‐urban area of Sub‐Saharan Africa, i.e. Côte d'Ivoire. Moreover, the generated data were coupled with those retrieved from public available metagenomic repositories, including two rural communities and 13 urban communities of industrialized countries. The meta‐analysis led to the identification of Infant Species Community States Type (ISCSTs) and microbial species covariances, which were exploited to reveal key signatures of infants living in rural and semi‐urban societies. Remarkably, analysis of rural and semi‐urban datasets revealed shifts from ISCSTs prevalent in urbanized populations with putative health implications. Thus, indicating the need for population‐wide investigations aimed to define the factors determining such potentially harmful gut microbial communities' signatures.

The vaginal microbiota is defined as the community of bacteria residing in the human vaginal tract. Recent studies have demonstrated that the vaginal microbiota is dominated by members of the Lactobacillus genus, whose relative abundance and microbial taxon composition are dependent on the healthy status of this human body site. Particularly, among members of this genus, the high prevalence of Lactobacillus crispatus is commonly associated with a healthy vaginal environment. In the current study, we assessed the microbial composition of 94 healthy vaginal microbiome samples through shotgun metagenomics analyses. Based on our results, we observed that L. crispatus was the most representative species and correlated negatively with bacteria involved in vaginal infections. Therefore, we isolated 15 L. crispatus strains from different environments in which this species abounds, ranging from vaginal swabs of healthy women to chicken fecal samples. The genomes of these strains were decoded and their genetic content was analyzed and correlated with their physiological features. An extensive comparative genomic analysis encompassing all publicly available genome sequences of L. crispatus and combined with those decoded in this study revealed a genetic adaptation of strains to their respective ecological niche. In addition, in vitro growth experiments involving all isolated L. crispatus strains, together with a synthetic vaginal microbiota, reveal how this species is able to modulate the composition of the vaginal microbial consortia at the strain level. Overall, our findings suggest that L. crispatus plays an important ecological role in reducing the complexity of the vaginal microbiota by depleting pathogenic bacteria. IMPORTANCE The vaginal microbiota is defined as the community of bacteria residing in the human vaginal tract. Recent studies have demonstrated that the high prevalence of Lactobacillus crispatus strains is commonly associated with a healthy vaginal environment. In the current study, we assessed the microbial composition of 94 public healthy vaginal samples through shotgun metagenomics analyses. Results showed that L. crispatus was the most representative species and correlated negatively with bacteria involved in vaginal infections. Moreover, we isolated and sequenced the genomes of new L. crispatus strains from different environments, and the comparative genomics analysis revealed a genetic adaptation of strains to their ecological niche. In addition, in vitro growth experiments display the capability of this species to modulate the composition of the vaginal microbial consortia. Overall, our findings suggest an ecological role exploited by L. crispatus in reducing the complexity of the vaginal microbiota toward a depletion of pathogenic bacteria.

Amoxicillin-clavulanic acid (AMC) is one of the most frequently prescribed antibiotic formulations in the Western world. Extensive oral use of this antimicrobial combination influences the gut microbiota. One of the most abundant early colonizers of the human gut microbiota is represented by different taxa of the Bifidobacterium genus, which include many members that are considered to bestow beneficial effects upon their host. In the current study, we investigated the impact of AMC administration on the gut microbiota composition, comparing the gut microbiota of 23 children that had undergone AMC antibiotic therapy to that of 19 children that had not been treated with antibiotics during the preceding 6 months. Moreover, we evaluated AMC sensitivity by MIC test of 261 bifidobacterial strains, including reference strains for the currently recognized 64 bifidobacterial (sub)species, as well as 197 bifidobacterial isolates of human origin. These assessments allowed the identification of four bifidobacterial strains that exhibit a high level of AMC insensitivity, which were subjected to genomic and transcriptomic analyses to identify the putative genetic determinants responsible for this AMC insensitivity. Furthermore, we investigated the ecological role of AMC-resistant bifidobacterial strains by in vitro batch cultures. IMPORTANCE Based on our results, we observed a drastic reduction in gut microbiota diversity of children treated with antibiotics, which also affected the abundance of Bifidobacterium, a bacterial genus commonly found in the infant gut. MIC experiments revealed that more than 98% of bifidobacterial strains tested were shown to be inhibited by the AMC antibiotic. Isolation of four insensitive strains and sequencing of their genomes revealed the identity of possible genes involved in AMC resistance mechanisms. Moreover, gut-simulating in vitro experiments revealed that one strain, i.e., Bifidobacterium breve PRL2020, is able to persist in the presence of a complex microbiota combined with AMC antibiotic.

Five Bifidobacterium strains, VB23T, VB24T, VB25T, VB26T and VB31T, were isolated from chimpanzee (Pan troglodytes), cotton-top tamarin (Saguinus oedipus), Goeldi’s marmoset (Callimico goeldii), moustached tamarin (Saguinus mystax) and patas monkey (Erythrocebus patas), respectively, which were kept in two Czech zoos. These strains were isolated from faecal samples and were Gram-positive, non-motile, non-sporulating, anaerobic and fructose-6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA revealed close relatedness between VB23T and Bifidobacterium angulatum LMG 11039T (96.0 %), VB24T and Bifidobacterium pullorum subsp. pullorum DSM 20433T (96.1 %), VB25T and Bifidobacterium goeldii LMG 30939T (96.5 %), VB26T and Bifidobacterium imperatoris LMG 30297T (98.1 %), and VB31T and B . angulatum LMG 11039T (99.40 %). Internal transcribed spacer profiling revealed that VB23T, VB24T, VB25T, VB26T and VB31T had highest similarity to Bifidobacterium breve LMG 13208T (77.2 %), Bifidobacterium longum subsp. infantis ATCC 15697T (85.8 %), Bifidobacterium biavatii DSM 23969T (76.9 %), B. breve LMG 13208T (81.2 %) and B. angulatum LMG 11039T (88.2 %), respectively. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) analyses with their closest neighbours supported the independent phylogenetic positions of the strains with values between 86.3 and 94.3 % for ANI and 25.8 and 54.9 % for dDDH. These genomic and phylogenetic analyses suggested that the evaluated strains were novel Bifidobacterium species named Bifidobacterium erythrocebi sp. nov. (VB31T=DSM 109960T=CCUG 73843T), Bifidobacterium moraviense sp. nov. (VB25T=DSM 109958T=CCUG 73842T), Bifidobacterium oedipodis sp. nov. (VB24T=DSM 109957T=CCUG 73932T), Bifidobacterium olomucense sp. nov. (VB26T=DSM 109959T=CCUG 73845T) and Bifidobacterium panos sp. nov. (VB23T=DSM 109963T=CCUG 73840T).

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In recent years, various studies have demonstrated that the gut microbiota influences host metabolism. However, these studies were focused primarily on a single or a limited range of host species, thus preventing a full exploration of possible taxonomic and functional adaptations by gut microbiota members as a result of host-microbe coevolution events. In the current study, the microbial taxonomic profiles of 250 fecal samples, corresponding to 77 host species that cover the mammalian branch of the tree of life, were reconstructed by 16S rRNA gene-based sequence analysis. Moreover, shotgun metagenomics was employed to investigate the metabolic potential of the fecal microbiomes of 24 mammals, and subsequent statistical analyses were performed to assess the impact of host diet and corresponding physiology of the digestive system on gut microbiota composition and functionality. Functional data were confirmed and extended through metatranscriptome assessment of gut microbial populations of eight animals, thus providing insights into the transcriptional response of gut microbiota to specific dietary lifestyles. Therefore, the analyses performed in this study support the notion that the metabolic features of the mammalian gut microbiota have adapted to maximize energy extraction from the host’s diet. IMPORTANCE Diet and host physiology have been recognized as main factors affecting both taxonomic composition and functional features of the mammalian gut microbiota. However, very few studies have investigated the bacterial biodiversity of mammals by using large sample numbers that correspond to multiple mammalian species, thus resulting in an incomplete understanding of the functional aspects of their microbiome. Therefore, we investigated the bacterial taxonomic composition of 250 fecal samples belonging to 77 host species distributed along the tree of life in order to assess how diet and host physiology impact the intestinal microbial community by selecting specific microbial players. Conversely, the application of shotgun metagenomics and metatranscriptomics approaches to a group of selected fecal samples allowed us to shed light on both metabolic features and transcriptional responses of the intestinal bacterial community based on different diets.

Gamma aminobutyric acid (GABA) is the principal inhibitory neurotransmitter playing a key role in anxiety and depression disorders in mammals. Recent studies revealed that members of the gut microbiota are able to produce GABA modulating the gut–brain axis response. Among members of the human gut microbiota, bifidobacteria are well known to establish many metabolic and physiologic interactions with the host. In this study, we performed genome analyses of more than 1,000 bifidobacterial strains publicly available revealing that Bifidobacterium adolescentis taxon might represent a model GABA producer in human gastrointestinal tract. Moreover, the in silico screening of human/animal metagenomic datasets showed an intriguing association/correlation between B. adolescentis load and mental disorders such as depression and anxiety. Interestingly, in vitro screening of 82 B. adolescentis strains allowed identifying two high GABA producers, i.e. B. adolescentis PRL2019 and B. adolescentis HD17T2H, which were employed in an in vivo trial in rats. Feeding Groningen rats with a supplementation of B. adolescentis strains, confirmed the ability of these microorganisms to stimulate the in vivo production of GABA highlighting their potential implication in gut–brain axis interactions., This work was funded by the EU Joint Programming Initiative—A Healthy Diet for a Healthy Life (JPI HDHL, https://www.healthydietforhealthylife.eu/) to MV (in conjunction with MIUR, Italy). IPLA was funded by the projects RTI2018-096339-B-I00 (MCIU/AEI/FEDER, UE), RTI2018-095021-J-I00 (MCIU/AEI/FEDER, UE) and IDI_2018_000236 (PCTI Govierno Principado de Asturias / FEDER); H. Tamés acknowledges the “Severo Ochoa” Research grant from the Government of “Principado de Asturias”. We thank GenProbio srl for financial support of the Laboratory of Probiogenomics. Part of this research is conducted using the High Performance Computing (HPC) facility of the University of Parma. Thanks are given to Isabel Cuesta, from the Technical Services of IPLA-CSIC, and Diana Luaces for their excellent technical assistance.

We developed a novel method for accurate cataloguing of bacterial communities at (sub)species level involving amplification of the internal transcribed spacer (ITS) region through optimized primers, followed by next-generation sequencing and taxonomic classification of amplicons by means of a comprehensive database of bacterial ITS sequences. Host-associated, food, and environmental matrices were employed to test the performance of the microbial ITS profiling pipeline. Moreover, mucosal biopsy samples from colorectal cancer patients were analyzed to demonstrate the scientific relevance of this profiling approach in a clinical setting through identification of putative novel biomarkers. The results indicate that the ITS-based profiling pipeline proposed here represents a key metagenomic tool with major relevance for research, industrial, and clinical settings., 16S small-subunit (SSU) rRNA gene-based bacterial profiling is the gold standard for cost-effective taxonomic reconstruction of complex bacterial populations down to the genus level. However, it has been proven ineffective in clinical and research settings requiring higher taxonomic resolution. We therefore developed a bacterial profiling method based on the internal transcribed spacer (ITS) region employing optimized primers and a comprehensive ITS database for accurate cataloguing of bacterial communities at (sub)species resolution. Performance of the microbial ITS profiling pipeline was tested through analysis of host-associated, food, and environmental matrices, while its efficacy in clinical settings was assessed through analysis of mucosal biopsy specimens of colorectal cancer, leading to the identification of putative novel biomarkers. The data collected indicate that the proposed pipeline represents a major step forward in cost-effective identification and screening of microbial biomarkers at (sub)species level, with relevant impact in research, industrial, and clinical settings. IMPORTANCE We developed a novel method for accurate cataloguing of bacterial communities at (sub)species level involving amplification of the internal transcribed spacer (ITS) region through optimized primers, followed by next-generation sequencing and taxonomic classification of amplicons by means of a comprehensive database of bacterial ITS sequences. Host-associated, food, and environmental matrices were employed to test the performance of the microbial ITS profiling pipeline. Moreover, mucosal biopsy samples from colorectal cancer patients were analyzed to demonstrate the scientific relevance of this profiling approach in a clinical setting through identification of putative novel biomarkers. The results indicate that the ITS-based profiling pipeline proposed here represents a key metagenomic tool with major relevance for research, industrial, and clinical settings.

Among the bacterial genera that are used for cheese production, Lactobacillus is a key taxon of high industrial relevance that is commonly present in commercial starter cultures for dairy fermentations. Certain lactobacilli play a defining role in the development of the organoleptic features during the ripening stages of particular cheeses. We performed an in-depth 16S rRNA gene-based microbiota analysis coupled with internally transcribed spacer-mediated Lactobacillus compositional profiling of 21 common Italian cheeses produced from raw milk in order to evaluate the ecological distribution of lactobacilli associated with this food matrix. Statistical analysis of the collected data revealed the existence of putative Lactobacillus community state types (LCSTs), which consist of clusters of Lactobacillus (sub)species. Each LCST is dominated by one or two taxa that appear to represent keystone elements of an elaborate network of positive and negative interactions with minor components of the cheese microbiota. The results obtained in this study reveal the existence of peculiar cheese microbiota assemblies that represent intriguing targets for further functional studies aimed at dissecting the species-specific role of bacteria in cheese manufacturing. IMPORTANCE The microbiota is known to play a key role in the development of the organoleptic features of dairy products. Lactobacilli have been reported to represent one of the main components of the nonstarter bacterial population, i.e., bacteria that are not deliberately added to the milk, harbored by cheese, although the species-level composition of this microbial population has never been assessed in detail. In the present study, we applied a recently developed metagenomic approach that employs an internally transcribed spacer to profile the Lactobacillus population harbored by cheese produced from raw milk at the (sub)species level. The obtained data revealed the existence of particular Lactobacillus community state types consisting of clusters of Lactobacillus (sub)species that tend to cooccur in the screened cheeses. Moreover, analysis of covariances between members of this genus indicate that these taxa form an elaborate network of positive and negative interactions that define specific clusters of covariant lactobacilli.

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IMPARARE L’ITALIANO. UN BISOGNO EDUCATIVO SPECIALE DA GIULIO TARRA A OGGIAtti delle Giornate di Studio dedicate a Giulio Tarra, Milano 2016-2017a cura di Giuseppe Polimeni, Massimo Prada Contributi di Massimo Prada, Matteo Grassano, Giuseppe Polimeni, Michela Dota, MariaGiulia Longhi, Giulia Galbiati, Francesca Almini e Federica Venier, Cecilia Demuru, ElisaMarazzi, Aldo Roma, Daniela Notarbartolo

IMPARARE L’ITALIANO. UN BISOGNO EDUCATIVO SPECIALE DA GIULIO TARRA A OGGI Atti delle Giornate di Studio dedicate a Giulio Tarra, Milano 2016-2017 a cura di Giuseppe Polimeni, Massimo Prada Contributi di Massimo Prada, Matteo Grassano, Giuseppe Polimeni, Michela Dota, Maria Giulia Longhi, Giulia Galbiati, Francesca Almini e Federica Venier, Cecilia Demuru, Elisa Marazzi, Aldo Roma, Daniela Notarbartolo

During the course of evolution, dogs and cats have been subjected to extensive domestication, becoming the principal companion animals for humans. For this reason, their health care, including their intestinal microbiota, is considered of considerable importance. However, the canine and feline gut microbiota still represent a largely unexplored research area. In the present work, we profiled the microbiota of 23 feline fecal samples by 16S rRNA gene and bifidobacterial internally transcribed spacer (ITS) approaches and compared this information with previously reported data from 138 canine fecal samples. The obtained data allowed the reconstruction of the core gut microbiota of the above-mentioned samples coupled with their classification into distinct community state types at both genus and species levels, identifying Bacteroides, Fusobacterium, and Prevotella 9 as the main bacterial components of the canine and feline gut microbiota. At the species level, the intestinal bifidobacterial gut communities of dogs and cats differed in terms of both species number and composition, as emphasized by a covariance analysis. Together, our findings show that the intestinal populations of cats and dogs are similar in terms of genus-level taxonomical composition, while at the bifidobacterial species level, clear differences were observed, indicative of host-specific colonization behavior by particular bifidobacterial taxa. IMPORTANCE Currently, domesticated dogs and cats are the most cherished companion animals for humans, and concerns about their health and well-being are therefore important. In this context, the gut microbiota plays a crucial role in maintaining and promoting host health. However, despite the social relevance of domesticated dogs and cats, their intestinal microbial communities are still far from being completely understood. In this study, the taxonomical composition of canine and feline gut microbiota was explored at genus and bifidobacterial species levels, allowing classification of these microbial populations into distinct gut community state types at either of the two investigated taxonomic levels. Furthermore, the reconstruction of core gut microbiota coupled with covariance network analysis based on bifidobacterial internally transcribed spacer (ITS) profiling revealed differences in the bifidobacterial compositions of canine and feline gut microbiota, suggesting that particular bifidobacterial species have developed a selective ability to colonize a specific host.

Human milk is known to carry its own microbiota, of which the precise origin remains obscure. Breastfeeding allows mother-to-baby transmission of microorganisms as well as the transfer of many other milk components, such as human milk oligosaccharides (HMOs), which act as metabolizable substrates for particular bacteria, such as bifidobacteria, residing in infant intestinal tract. In the current study, we report the HMO composition of 249 human milk samples, in 163 of which we quantified the abundance of members of the Bifidobacterium genus using a combination of metagenomic and flow cytometric approaches. Metagenomic data allowed us to identify four clusters dominated by Bifidobacterium adolescentis and Bifidobacterium pseudolongum, Bifidobacterium crudilactis or Bifidobacterium dentium, as well as a cluster represented by a heterogeneous mix of bifidobacterial species such as Bifidobacterium breve and Bifidobacterium longum. Furthermore, in vitro growth assays on HMOs coupled with in silico glycobiome analyses allowed us to elucidate that members of the Bifidobacterium bifidum and B. breve species exhibit the greatest ability to degrade and grow on HMOs. Altogether, these findings indicate that the bifidobacterial component of the human milk microbiota is not strictly correlated with their ability to metabolize HMOs.

COLOSTRONONI is a dietary supplement consisting of bovine colostrum and Morinda citrifolia fruit (Noni). In this study, we tested the capability of COLOSTRONONI to influence gut microbiota composition using an in vivo evaluation in rats. Furthermore, we analyzed the effect of COLOSTRONONI on the systemic inflammatory responses as well as on the gut permeability of the animals. Altogether, our analyses supported the concept of COLOSTRONONI as a natural food supplement that doesn't affect (neither negatively nor positively) gut microbiota homeostasis in healthy conditions. Moreover, COLOSTRONONI highlighted a lower effect in the expression of genes coding for IL-10, Il-12 and TNF-α response allowing us to hypothesize an immunomodulatory activity of this dietary supplement.

Bifidobacteria are commensals of the animal gut, and are commonly found in mammals, birds, and social insects. Specifically, strains of Bifidobacterium adolescentis , Bifidobacterium bifidum , Bifidobacterium longum and Bifidobacterium pseudolongum are widely distributed in the mammalian gut. In this context, we investigated the genetic variability and metabolic abilities of the B. pseudolongum taxon, whose genomic characterization has so far not received much attention. Phylogenomic analysis of genome sequences of 60 B. pseudolongum strains revealed that B. pseudolongum subsp. globosum and B. pseudolongum subsp. pseudolongum may actually represent two distinct bifidobacterial species. Furthermore, our analysis highlighted metabolic differences between members of these two subspecies. Moreover, comparative analyses of genetic strategies to prevent invasion of foreign DNA revealed that the B. pseudolongum subsp. globosum group exhibits greater genome plasticity. In fact, the obtained findings indicate that B. pseudolongum subsp. globosum is more adaptable to different ecological niches such as the mammalian and avian gut when compared to B. pseudolongum subsp. pseudolongum . Importance Currently little information exists on the genetics of the B. pseudolongum taxon, due to the limited number of sequenced genomes belonging to this species. In order to survey genome variability within this species and explore how members of this taxon evolved as commensals of the animal gut, we isolated and decoded the genomes of 51 newly isolated strains. Comparative genomics coupled with growth profiles on different carbohydrates has further provided insights concerning the genotype and phenotype of members of the B. pseudolongum taxon.

Bifidobacteria are members of the gut microbiota of animals, including mammals, birds, and social insects. In this study, we analyzed and determined the pangenome of Bifidobacterium animalis species, encompassing B. animalis subsp. animalis and the B. animalis subsp. lactis taxon, which is one of the most intensely exploited probiotic bifidobacterial species. In order to reveal differences within the B. animalis species, detailed comparative genomics and phylogenomics analyses were performed, indicating that these two subspecies recently arose through divergent evolutionary events. A subspecies-specific core genome was identified for both B. animalis subspecies, revealing the existence of subspecies-defining genes involved in carbohydrate metabolism. Notably, these in silico analyses coupled with carbohydrate profiling assays suggest genetic adaptations toward a distinct glycan milieu for each member of the B. animalis subspecies, resulting in a divergent evolutionary development of the two subspecies. IMPORTANCE The majority of characterized B. animalis strains have been isolated from human fecal samples. In order to explore genome variability within this species, we isolated 15 novel strains from the gastrointestinal tracts of different animals, including mammals and birds. The present study allowed us to reconstruct the pangenome of this taxon, including the genome contents of 56 B. animalis strains. Through careful assessment of subspecies-specific core genes of the B. animalis subsp. animalis/lactis taxon, we identified genes encoding enzymes involved in carbohydrate transport and metabolism, while unveiling specific gene acquisition and loss events that caused the evolutionary emergence of these two subspecies.

Cheese microbiota is of high industrial relevance due to its crucial role in defining the organoleptic features of the final product. Nevertheless, the composition of and possible microbe&ndash, microbe interactions between these bacterial populations have never been assessed down to the species-level. For this reason, 16S rRNA gene microbial profiling combined with internally transcribed spacer (ITS)-mediated bifidobacterial profiling analyses of various cheeses produced with raw milk were performed in order to achieve an in-depth view of the bifidobacterial populations present in these microbially fermented food matrices. Moreover, statistical elaboration of the data collected in this study revealed the existence of community state types characterized by the dominance of specific microbial genera that appear to shape the overall cheese microbiota through an interactive network responsible for species-specific modulatory effects on the bifidobacterial population.

Mycobacterium tuberculosis is the etiological agent of tuberculosis. It is a pathogen that continues to draw international concerns particularly due to the emergence of multi-drug resistance and to the difficulties associated with the diagnosis and treatment of latent tuberculosis. A key process in the pathogenesis of this disease is the interaction between this pathogen and host macrophages. Invasion of the macrophages protects the pathogen from attack by the immune system, allows it to multiply while protected within the macrophages, and alters the immune response as it influences the profiles of the cytokine and chemokine responses. Key to the intracellular survival of the pathogen within the macrophages is the specific interaction between the pathogen and its virulence factors with the host. This review provides an a summary of pertinent literature on the topic of macrophage receptors utilized by the pathogen, its survival strategies within the macrophage, and the general profile of immune signalling upon exposure to the pathogen. The importance of specific macrophage receptors and certain components of the pathogen to the direction of the immune response are also discussed.