Revealing the genetic traits of the foodborne microbial genus hafnia: Implications for the human gut microbiome.

The bacterial genus Hafnia has recently attracted attention due to its complex metabolic features and host-interaction capabilities, which are associated with health benefits, primarily weight loss. However, significant gaps remain in our understanding of the genomic characteristics of this emerging microbial group. In this study, we utilized all available high-quality genomes of Hafnia alvei and Hafnia paralvei to uncover the broad distribution of Hafnia in human and honeybee guts, as well as in dairy products, by analysing 1068 metagenomic datasets. We then investigated the genetic traits related to Hafnia's production of vitamins and short-chain fatty acids (SCFAs) through a comparative genomics analysis that included all dominant bacterial species in the three environments under study. Our findings underscore the extensive metabolic capabilities of Hafnia, particularly in the production of vitamins such as thiamine (B1), nicotinate (B3), pyridoxine (B6), biotin (B7), folate (B9), cobalamin (B12), and menaquinone (K2). Additionally, Hafnia demonstrated a conserved genetic makeup associated with SCFA production, including acetate, propanoate, and butanoate. These metabolic traits were further confirmed using RNAseq analyses of a newly isolated H. paralvei strain T10. Overall, our study illuminates the ecological distribution and genetic attributes of this bacterial genus, which is of increasing scientific and industrial relevance.

Genomic and ecological approaches to identify the Bifidobacterium breve prototype of the healthy human gut microbiota.

Members of the genus Bifidobacterium are among the first microorganisms colonizing the human gut. Among these species, strains of Bifidobacterium breve are known to be commonly transmitted from mother to her newborn, while this species has also been linked with activities supporting human wellbeing. In the current study, an in silico approach, guided by ecology- and phylogenome-based analyses, was employed to identify a representative strain of B. breve to be exploited as a novel health-promoting candidate. The selected strain, i.e., B. breve PRL2012, was found to well represent the genetic content and functional genomic features of the B. breve taxon. We evaluated the ability of PRL2012 to survive in the gastrointestinal tract and to interact with other human gut commensal microbes. When co-cultivated with various human gut commensals, B. breve PRL2012 revealed an enhancement of its metabolic activity coupled with the activation of cellular defense mechanisms to apparently improve its survivability in a simulated ecosystem resembling the human microbiome.

Saponin treatment for eukaryotic DNA depletion alters the microbial DNA profiles by reducing the abundance of Gram-negative bacteria in metagenomics analyses.

Background: Recent advances in microbiome sequencing techniques have provided new insights into the role of the microbiome on human health with potential diagnostic implications. However, these developments are often hampered by the presence of a large amount of human DNA interfering with the analysis of the bacterial content. Nowadays, extensive scientific literature focuses on eukaryotic DNA depletion methods, which successfully remove host DNA in microbiome studies, even if a precise assessment of the impact on bacterial DNA is often missing. Methods: Here, we have investigated a saponin-based DNA isolation protocol commonly applied to different biological matrices to deplete the released host DNA. Results: The bacterial DNA obtained was used to assess the relative abundance of bacterial and human DNA, revealing that the inclusion of 2.5% wt/vol saponin allowed the depletion of most of the host's DNA in favor of bacterial DNA enrichment. However, shotgun metagenomic sequencing showed inaccurate microbial profiles of the DNA samples, highlighting an erroneous increase in Gram-positive DNA. Even the application of 0.0125% wt/vol saponin altered the bacterial profile by depleting Gram-negative bacteria, resulting in an overall increase of Gram-positive bacterial DNA. Conclusion: The application of the saponin-based protocol drastically changes the detection of the microbial composition of human-related biological specimens. In this context, we revealed that saponin targets not only host cells but also specific bacterial cells, thus inducing a drastic reduction in the profiling of Gram-negative bacterial DNA.

Social stress-induced depressive-like symptoms and changes in gut microbial and lipidomic profiles are prevented by pharmacological inhibition of FAAH activity in male rats.

Pharmacological inhibition of fatty acid amide hydrolase (FAAH) activity has antidepressant-like effects in preclinical models of stress. In this study, we investigated whether the antidepressant-like effects of FAAH inhibition are associated with corresponding changes in gut microbial and lipidomic profiles, which are emerging as critical components in the pathophysiology of depression. Adult male Wistar rats experienced five weeks of repeated social defeat or control procedure and were treated with the FAAH inhibitor URB694 (0.3 mg/kg/day, i.p.) or vehicle starting from the third week. Repeated social defeat induced the emergence of depressive-like behavioral (sucrose preference reduction and passive coping behaviors in the forced swim test) and neuroendocrine (increased corticosterone levels) changes, which were prevented by URB694 treatment. Repeated social defeat also provoked a significant variation in gut microbiota (changes in the relative abundance of 14 bacterial taxa) and lipidic (e.g., glycerophospholipids) composition. These stress-induced changes were prevented by URB694 treatment. These findings indicate that inhibition of FAAH activity with URB694 blocks the co-occurrence of depressive-like behavioral and neuroendocrine changes and alterations in gut microbial and lipid composition in rats exposed to repeated social defeat. In conclusion, these results suggest that the gut microbiota-lipid crosstalk may represent a novel biological target for FAAH inhibitors to enhance stress resilience.

Amorphous silica nanoparticles and the human gut microbiota: a relationship with multiple implications.

Amorphous silica nanoparticles (ASNP) are among the nanomaterials that are produced in large quantities. ASNP have been present for a long time in several fast-moving consumer products, several of which imply exposure of the gastrointestinal tract, such as toothpastes, food additives, drug excipients, and carriers. Consolidated use and experimental evidence have consistently pointed to the very low acute toxicity and limited absorption of ASNP. However, slow absorption implies prolonged exposure of the intestinal epithelium to ASNP, with documented effects on intestinal permeability and immune gut homeostasis. These effects could explain the hepatic toxicity observed after oral administration of ASNP in animals. More recently, the role of microbiota in these and other ASNP effects has attracted increasing interest in parallel with the recognition of the role of microbiota in a variety of conditions. Although evidence for nanomaterial effects on microbiota is particularly abundant for materials endowed with bactericidal activities, a growing body of recent experimental data indicates that ASNPs also modify microbiota. The implications of these effects are recounted in this contribution, along with a discussion of the more important open issues and recommendations for future research.

Genetic strategies for sex-biased persistence of gut microbes across human life.

Although compositional variation in the gut microbiome during human development has been extensively investigated, strain-resolved dynamic changes remain to be fully uncovered. In the current study, shotgun metagenomic sequencing data of 12,415 fecal microbiomes from healthy individuals are employed for strain-level tracking of gut microbiota members to elucidate its evolving biodiversity across the human life span. This detailed longitudinal meta-analysis reveals host sex-related persistence of strains belonging to common, maternally-inherited species, such as Bifidobacterium bifidum and Bifidobacterium longum subsp. longum. Comparative genome analyses, coupled with experiments including intimate interaction between microbes and human intestinal cells, show that specific bacterial glycosyl hydrolases related to host-glycan metabolism may contribute to more efficient colonization in females compared to males. These findings point to an intriguing ancient sex-specific host-microbe coevolution driving the selective persistence in women of key microbial taxa that may be vertically passed on to the next generation.

Contribution of the capsular polysaccharide layer to antibiotic resistance in bifidobacteria.

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.

The human gut microbiome of athletes: metagenomic and metabolic insights.

BACKGROUND: The correlation between the physical performance of athletes and their gut microbiota has become of growing interest in the past years, since new evidences have emerged regarding the importance of the gut microbiota as a main driver of the health status of athletes. In addition, it has been postulated that the metabolic activity of the microbial population harbored by the large intestine of athletes might influence their physical performances. Here, we analyzed 418 publicly available shotgun metagenomics datasets obtained from fecal samples of healthy athletes and healthy sedentary adults. RESULTS: This study evidenced how agonistic physical activity and related lifestyle can be associated with the modulation of the gut microbiota composition, inducing modifications of the taxonomic profiles with an enhancement of gut microbes able to produce short-fatty acid (SCFAs). In addition, our analyses revealed a correlation between specific bacterial species and high impact biological synthases (HIBSs) responsible for the generation of a range of microbially driven compounds such vitamin B12, amino acidic derivatives, and other molecules linked to cardiovascular and age-related health-risk reduction. CONCLUSIONS: Notably, our findings show how subsist an association between competitive athletes, and modulation of the gut microbiota, and how this modulation is reflected in the potential production of microbial metabolites that can lead to beneficial effects on human physical performance and health conditions. Video Abstract.

Identification of a prototype human gut Bifidobacterium longum subsp. longum strain based on comparative and functional genomic approaches.

Bifidobacteria are extensively exploited for the formulation of probiotic food supplements due to their claimed ability to exert health-beneficial effects upon their host. However, most commercialized probiotics are tested and selected for their safety features rather than for their effective abilities to interact with the host and/or other intestinal microbial players. In this study, we applied an ecological and phylogenomic-driven selection to identify novel B. longum subsp. longum strains with a presumed high fitness in the human gut. Such analyses allowed the identification of a prototype microorganism to investigate the genetic traits encompassed by the autochthonous bifidobacterial human gut communities. B. longum subsp. longum PRL2022 was selected due to its close genomic relationship with the calculated model representative of the adult human-gut associated B. longum subsp. longum taxon. The interactomic features of PRL2022 with the human host as well as with key representative intestinal microbial members were assayed using in vitro models, revealing how this bifidobacterial gut strain is able to establish extensive cross-talk with both the host and other microbial residents of the human intestine.

Multifactorial Microvariability of the Italian Raw Milk Cheese Microbiota and Implication for Current Regulatory Scheme.

Raw milk cheese manufactory is strictly regulated in Europe by the Protected Designation of Origin (PDO) quality scheme, which protects indigenous food products based on geographical and biotechnological features. This study encompassed the collection of 128 raw milk cheese samples across Italy to investigate the resident microbiome correlated to current PDO specifications. Shotgun metagenomic approaches highlighted how the microbial communities are primarily linked to each cheesemaking site and consequently to the use of site-specific Natural Whey Cultures (NWCs), defined by a multifactorial set of local environmental factors rather than solely by cheese type or geographical origin that guide the current PDO specification. Moreover, in-depth functional characterization of Cheese Community State Types (CCSTs) and comparative genomics efforts, including metagenomically assembled genomes (MAGs) of the dominant microbial taxa, revealed NWCs-related unique enzymatic profiles impacting the organoleptic features of the produced cheeses and availability of bioactive compounds to consumers, with putative health implications. Thus, these results highlighted the need for a profound rethinking of the current PDO designation with a focus on the production site-specific microbial metabolism to understand and guarantee the organoleptic features of the final product recognized as PDO. IMPORTANCE The Protected Designation of Origin (PDO) guarantees the traceability of food production processes, and that the production takes place in a well-defined restricted geographical area. Nevertheless, the organoleptic qualities of the same dairy products, i.e., cheeses under the same PDO denomination, differ between manufacturers. The final product's flavor and qualitative aspects can be related to the resident microbial population, not considered by the PDO denomination. Here, we analyzed a complete set of different Italian cheeses produced from raw milk through shotgun sequencing in order to study the variability of the different microbial profiles resident in Italian PDO cheeses. Furthermore, an in-depth functional analysis, along with a comparative genomic analysis, was performed in order to correlate the taxonomic information with the organoleptic properties of the final product. This analysis made it possible to highlight how the PDO denomination should be revisited to understand the effect that Natural Whey Cultures (NWCs), used in the traditional production of raw milk cheese and unique to each manufacturer, impacts on the organoleptic features of the final product.

Exploring species-level infant gut bacterial biodiversity by meta-analysis and formulation of an optimized cultivation medium.

In vitro gut cultivation models provide host-uncoupled, fast, and cost-efficient solutions to investigate the effects of intrinsic and extrinsic factors impacting on both composition and functionality of the intestinal microbial ecosystem. However, to ensure the maintenance and survival of gut microbial players and preserve their functions, these systems require close monitoring of several variables, including oxygen concentration, pH, and temperature, as well as the use of a culture medium satisfying the microbial nutritional requirements. In this context, in order to identify the macro- and micro-nutrients necessary for in vitro cultivation of the infant gut microbiota, a meta-analysis based on 1669 publicly available shotgun metagenomic samples corresponding to fecal samples of healthy, full-term infants aged from a few days to three years was performed to define the predominant species characterizing the "infant-like" gut microbial ecosystem. A subsequent comparison of growth performances was made using infant fecal samples that contained the most abundant bacterial taxa of the infant gut microbiota, when cultivated on 18 different culture media. This growth analysis was performed by means of flow cytometry-based bacterial cell enumeration and shallow shotgun sequencing, which allowed the formulation of an optimized growth medium, i.e., Infant Gut Super Medium (IGSM), which maintains and sustains the infant gut microbial biodiversity under in vitro growth conditions. Furthermore, this formulation was used to evaluate the in vitro effect of two drugs commonly used in pediatrics, i.e., acetaminophen and simethicone, on the taxonomic composition of the infant gut microbiota.

Designation of optimal reference strains representing the infant gut bifidobacterial species through a comprehensive multi-omics approach.

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.

Exploring the Ecological Effects of Naturally Antibiotic-Insensitive Bifidobacteria in the Recovery of the Resilience of the Gut Microbiota during and after Antibiotic Treatment.

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.

Tap water as a natural vehicle for microorganisms shaping the human gut microbiome.

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.

Unraveling the Microbiome of Necrotizing Enterocolitis: Insights in Novel Microbial and Metabolomic Biomarkers.

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 highlighted 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.

Investigating the infant gut microbiota in developing countries: worldwide metagenomic meta-analysis involving infants living in sub-urban areas of Côte d'Ivoire.

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.

METAnnotatorX2: a Comprehensive Tool for Deep and Shallow Metagenomic Data Set Analyses.

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.

Comparative genome analyses of Lactobacillus crispatus isolated from different ecological niches reveal an environmental adaptation of this species to the human vaginal environment.

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 taxa 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 fifteen L. crispatus strains from different environments in which this species is abounding, 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 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 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 species 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 genome 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 Resistance in the Genus Bifidobacterium.

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.