BMC Microbiology

The gut microbiome plays a vital role in host homeostasis and understanding of its biology is essential for a better comprehension of the etiology of disorders such as foetal alcohol spectrum disorders. Here we assessed the effectiveness of targeted and untargeted (metagenomic) nanopore sequencing approaches to profile the gut microbiota of infant mice exposed to ethanol in utero. DNA extracts from the gut content of 12 infant mice exposed to ethanol in utero were analysed using one untargeted and two targeted (full-length 16S rRNA gene and the 16S-ITS-23S region of the ribosomal RNA operon) nanopore sequencing approaches. The targeting of the full-length 16S rRNA gene provided the most comprehensive analysis of the mouse gut microbiota. The differences in diversity between approaches were accounted by the sequencing target (p-value < 0.001). Faecalibaculum rodentium and Duncaniella sp. were the two most prevalent taxa detected using targeted sequencing approaches, while bacterial taxa were more evenly represented when using the metagenomic approach. Full-length 16S rRNA gene nanopore sequencing provides the most discriminatory microbiota compositional analysis of mice faecal samples. However, using nanopore sequencing approaches targeting the metagenome or different taxonomically-informative DNA region appears to introduce significant target-related biases. ImportanceCurrent nanopore approaches have not been standardized which may confound the biological interpretations of hight-throughput sequencing datasets. Additionally, nanopore sequencing still present a high error-rate compared to other more mature sequencing technologies, such as Illumina sequencing. These technological handicaps create the need to study and optimize nanopore sequencing approaches to answer biological questions, such as interrogations of the microbial composition and abundance of clinical and environmental samples. In this work, three nanopore sequencing approaches were designed and attempted to optimize fungal and bacterial profiling sequencing methodologies. Two targeted methods based on the bacterial 16S rRNA gene, and 16S-ITS-23S rrn operon region, and one untargeted shotgun/metagenomic approach were tested. Despite potential experimental and/or bioinformatical biases were found, the 16S rRNA gene-targeted nanopore sequencing was the most comprehensive approach to study the microbial composition of the infant mice gut microbiotas.

Gut microbiome is a group of microorganisms that plays important roles in contributing to health and diseases. These bacterial compositions have been demonstrated to impact bile acids (BAs) profiles, either by directly metabolizing primary BAs to secondary BAs or indirect ways through host metabolism by influencing BAs synthesis, transportation and conjugation in liver. It has been observed sexually dimorphic gut microbiome and bile acids composition, with variations in expression levels of bile acid metabolizing genes in the liver. However, associations betweensex-specific differences in gut microbiome and BAs profiles are not well understood. This study aimed to investigate whether gut microbiome could influence BAs profiles in host in a sex-specific manner. We transplanted cecum feces of male and female C57BL/6 mice to male mice and measured BAs concentrations in feces, serum and liver samples 7 days after fecal transplantation. We found different BAs profiles between mice with male and female gut microbiome, including altering levels and proportions of secondary BAs. We also observed varied expression levels of genes related to bile acid metabolism in the liver and distal ileum.Our results highlight sex-specific effects of gut microbiome on shaping bile acid metabolism through gut bacteria and regulation of host genes.

In the present era, emergence of next generation sequencing approaches has revolutionized the field of gut microbiome study. However, the adopted DNA extraction step used in metagenomics experiments and its efficiency may play a critical role in their reproducibility and outcome. In this study, fecal samples from active and non-tuberculosis subjects (ATB/NTB, n=7) were used. Fecal samples of a subgroup of these subjects were subjected to Mechanical enzymatic lysis (MEL) and Phenol: Chloroform: Isoamyl Alcohol (PCIA) methods of DNA extraction and a third-generation sequencing platform i.e. MinION was employed for microbiome profiling. Findings of this study demonstrated that DNA extraction method significantly impacts the DNA yield and microbial diversity. Irrespective of the adopted method of DNA extraction, ATB patients showed altered microbial diversity compared to NTB controls. Also, the fecal microbial diversity details are better captured in samples processed by MEL method and may be suitable to be adopted for high-throughput gut microbiome studies.

In vitro gut microbiota models are often used to study drug-microbiome interaction. Similar to culturing individual microbial strains, the biomass accumulation of in vitro gut microbiota follows a logistic growth curve. Current studies on in vitro gut microbiome responses introduce drug stimulation during different growth stages, e.g. lag phase or stationary phase. However, in vitro gut microbiota in different growth phases may respond differently to same stimuli. Therefore, in this study, we used a 96-deep well plate-based culturing model (MiPro) to culture the human gut microbiota. Metformin, as the stimulus, was added at the lag, log and stationary phases of growth. Microbiome samples were collected at different time points for optical density and metaproteomic functional analysis. Results show that in vitro gut microbiota responded differently to metformin added during different growth phases, in terms of the growth curve, alterations of taxonomic and functional compositions. The addition of drugs at log phase leads to the greatest decline of bacterial growth. Metaproteomic analysis suggested that the strength of the metformin effect on the gut microbiome functional profile was ranked as lag phase > log phase > stationary phase. Our results showed that metformin added at lag phase resulted in a significantly reduced abundance of the Clostridiales order as well as an increased abundance of the Bacteroides genus, which was different from stimulation during the rest of the growth phase. Metformin also resulted in alterations of several pathways, including energy production and conversion, lipid transport and metabolism, translation, ribosomal structure and biogenesis. Our results indicate that the timing for drug stimulation should be considered when studying drug-microbiome interactions in vitro.

The gut microbiome plays a vital role in host homeostasis and understanding of its biology is essential for a better comprehension of the etiology of disorders such as Foetal Alcohol Spectrum Disorder. Foetal Alcohol Spectrum Disorder represents a cluster of abnormalities including growth deficiencies and neurological impairments, which are not easily diagnosed nor treated. Here the effect of ethanol exposure in utero on the gut microbial profiles of 16 infant mice (nine exposed in utero and seven non-exposed) was assessed by targeted nanopore sequencing and Illumina sequencing approaches. The nanopore sequencing was implemented using MinION system targeting PCR-amplified amplicons made from the full-length 16S rRNA gene. The Illumina sequencing was performed using Miseq system targeting the V3-V4 region of the 16S rRNA gene. Ethanol exposure did not affect the microbial profiles. Several low prevalent taxa, like Akkermansia muciniphila, were detected but further studies must be performed to detail the effect of ethanol exposure to these taxa since no clear pattern was detected throughout this study. ImportanceDetailed knowledge about the interactions between gut microbes and the developing nervous system is still scarce. Foetal Alcohol Spectrum Disorder represents a clinically relevant set of conditions with cumbersome diagnostic and treatment. In this work the microbial profiles of infant mice gut exposed to ethanol in utero were analysed through third-generation Illumina and optimized next-generation nanopore sequencing technologies. The fungal (albeit not detected) and bacterial microbial profiles here obtained through nanopore and Illumina sequencing represent a technological and biological advancement towards a better comprehension of the microbial landscape in Foetal Alcohol Spectrum Disorder at early post-natal periods.

DNA extraction methods play an important role in the acquisition of accurate and reproducible 16S sequencing data in microbiome studies. In this study, we assessed the impact of bead-beating intensity during DNA extraction on microbiome recovery in mouse and human stool. We observed a higher DNA yield, better DNA integrity, higher Shannons entropy and Simpsons index in samples beaten for 4 and 9 minutes as compared to unbeaten samples. 16S sequencing data showed that bead beating has a statistically-significant (p<0.05) impact on the recovery of many clinically relevant microbes that live in the mouse and human gut, including Bifidobacterium, Sutterella and Veillonella. It was observed that 4 minutes of bead beating promotes recovery of about 70% of OTUs in mouse and human stool, while the remaining 30% requires longer bead beating. In conclusion, our study indicates adjustments in bead beating treatment based on the composition of the specimen and the targeted bacteria.

The Escherichia coli Nissle 1917 strain (EcN) has shown its probiotic efficacy against many enteric pathogenic bacteria infecting human, including Vibrio cholerae, either alone or in combination with prebiotics. Understanding of these mechanisms of infection control requires the basic knowledge of probiotic mediated gut microbial community alterations especially in presence of different prebiotics. The present study has used the ex-vivo microbiota model and Next Generation Sequencing techniques to demonstrate the effect of EcN along with different sugars, namely glucose, galactose and starch, on the human gut microbiome community composition. The microbiome compositional changes have been observed at two different time-points, set one and a half years apart, in fecal slurries obtained from two donors. The study has indicated that the extent of microbiome alterations varies with different carbohydrate prebiotics and EcN probiotic and most of the alterations are broadly dependent upon the existing gut microbial community structure of the donors. The major distinct compositional changes have been found in the conditions where glucose and starch were administered, both with and without EcN, in spite of the inter-donor microbial community variation. Several of these microbiome component variations also remain consistent for both the time-points, including genus like Bacteroides, Prevotella and Lactobacillus. Altogether, the present study has shown the effectiveness of EcN along with glucose and starch towards specific changes of microbial community alterations independent of initial microbial composition. This type of model study can be implemented for hypothesis testing in case of therapeutic and prophylactic use of probiotic and prebiotic combinations.

The human gut microbiome undergoes transformation due to various exogenous and endogenous influences. Lifestyle has been one of the major contributors of gut microbiome composition. Our study investigated the effect of migration on the gut microbiome of the Mishing ethnic community in India associated with urban migration from their pastural lifestyle. Gut bacterial diversity was profiled using 16S rRNA amplicon sequencing. We observed notable alterations in microbial diversity and composition, including changes in the core microbiome. While Bifidobacterium, Lactococcus and Streptococcus significantly increased (p < 0.01) among the migrant population there was depletion in bacteria such as Succinivibrio and Prevotella. This change in core microbiome was further reflected on the functional profile of the bacterial community harbored by the urban and rural dwellers. While the rural population was enriched in fatty acid biosynthesis pathways, amino acid and Vitamin B2 biosynthesis pathways were more prominent in the microbiome of the urban dwellers. We further observed increase in diversity and richness of the microbiome following migration. Co-analysis with ASVs from food samples consumed by the Mishing rural population revealed that 51 ASVs among the rural dwellers were from a widely consumed beverage, Apong which were completely absent among the migrants. Our analyses highlight the dietary preferences, particularly abstinence from specific foods and beverages, may modulate the alterations in gut microbial profiles. While the cross-sectional design and amplicon sequencing limited our ability to assess temporal and functional changes, our findings highlight a measurable impact of lifestyle and dietary transitions on the gut microbiome.

BackgroundLiterature has highlighted the gut microbiotas role in metabolic functions, suggesting a potential link between gut microbiota composition and T2DM. The purpose of the study was to identify microbial signatures unique to T2DM patients and non-diabetic individuals, to compare microbial profiles between the two groups and to investigate how gut microbiota may be related to inflammation associated with T2DM. MethodsA cross-sectional study was conducted involving 51 T2DM patients and 99 non-diabetic South African individuals. Faecal samples were collected and analysed using 16S rRNA gene sequencing to characterize the gut microbiota. Blood samples were obtained to perform HbA1c, CRP and ferritin tests. Bioinformatic and statistical analyses were performed to identify differences in microbial composition and diversity between the two groups. ResultsThe gut microbiota in T2DM patients was predominantly composed of Firmicutes (47.7%), Bacteroidota (37.5%), and Proteobacteria (11.4%), while the non-diabetic group showed a slightly different microbial profile with higher Bacteroidota (41.9%) and a notable presence of Actinobacteriota (4.5%). Abundant families in the T2DM group included Bacteroidaceae (22.8%), Prevotellaceae (7.4%), Enterobacteriaceae (7.4%), Erysipelotrichaceae (6.0%) and Lachnospiraceae (5.2%). The non-diabetic group exhibited dominant families such as Lachnospiraceae 26.7%, Prevotellaceae (25.3%), Bacteroidaceae (12.7%), Ruminococcaceae (9.5%) and Oscillospiraceae (3.8%). At the genus level, Bacteroides (22.8%), Escherichia-Shigella (5.0%), Holdemanella (4.8%), Phascolarctobacterium (3.2%) and Blautia (2.8%) were prevalent in the T2DM group, while Prevotella_9 (22.1%), Bacteroides (12.7%), Agathobacter (6.7%), Blautia (6.3%) and Faecalibacterium (5.1%) were dominant in the non-diabetic group. Differential abundance testing revealed 5 phyla, 16 families, and 25 genera that were either enriched/depleted in T2DM patients relative to non-diabetic individuals. The comparison of alpha diversity metrics between the two groups revealed significant differences across all four measures (P < 0.001), with non-diabetic individuals showing higher values than T2DM patients. HbA1c and CRP levels showed correlations with the relative abundance of various gut microbes at various phyla, family, and genus levels, as well as with all alpha diversity metrics. ConclusionThe study revealed distinct differences in gut microbiota composition between T2DM patients and non-diabetic individuals, with T2DM patients showing a higher prevalence of certain phyla, families, and genera linked to metabolic dysregulation. Non-diabetic individuals exhibited greater microbial diversity and beneficial taxa, highlighting a potential protective microbial profile.

BackgroundAltered bacterial translocation is associated with transitioning from compensated to decompensated cirrhosis. Thus, we aimed to study differences in the blood microbiome of HCV-infected patients with and without hepatic decompensation. MethodsWe conducted a cross-sectional study in patients with advanced HCV-related cirrhosis with or without human immunodeficiency virus (HIV) infection (n=88). MiSeq Illumina technology for bacterial 16S rRNA sequencing was used. Non-targeted metabolomics was performed by GC-MS and LC-MS ESI+ and ESI-. ResultsPatients with decompensated cirrhosis had lower levels of richness (Chao1), and alpha diversity (Shannon and Simpson indexes) at phylum level, than patients without decompensation. Likewise, we observed significant differences in beta diversity between groups at phylum, class and order levels, being lower in decompensated cirrhotic patients. Higher relative abundance of Proteobacteria (Fold Change (FC)=1.54, p=0.012), Alphaproteobacteria (FC=1.57, p=0.016) and Sphingomonadales (FC=1.61, p=0.050) were significantly associated with hepatic decompensation. The phylum Proteobacteria was positively correlated with ethanolamine and oleic acid (p=0.005 and p=0.004, respectively) and negatively with p-cresol (p=0.006). In addition, the order Sphingomonadales was also negatively correlated with p-cresol (p=0.001). ConclusionsBlood microbial diversity was significantly decreased in patients with decompensated cirrhosis, who presented an enrichment of Proteobacteria, Alphaproteobacteria, and Sphingomonadales, compared to patients with compensated cirrhosis.

Antimicrobial resistance (AMR) alleviation warrants antimicrobial stewardship (AS) entailing indispensability of epidemiological surveillance. We undertook a small-scale surveillance in Kolkata to detect the presence of antimicrobial resistance genes (ARGs) in the healthy gut microbiome. We found that it was a reservoir of ARGs against common antibiotics. A targeted PCR and sequencing-based ARGs detection against tetracyclines, macrolides, trimethoprim, sulfamethoxazole, aminoglycosides, amphenicol and mobile genetic element (MGE) markers was deployed in twenty-five fecal samples. Relative abundance and frequency of ARGs was calculated. We detected markers against all these classes of antibiotics. 100% samples carried aminoglycoside resistance marker and int1U. A comparison with our previously published diarrheal resistome from the same spatial and temporal frame revealed that a higher diversity of ARGs were detected in the community and a higher rate of isolation of tetC, msrA, tmp and sul-2 was found. The presence of common markers in the two cohorts proves that the gut microbiome has been contaminated with ARGs and which are being disseminated among different ecosystems. This is an issue of discerning concern for public health. The study raises an alarming picture of the AMR crisis in low-middle and emergent economies. It emphasizes the strict enforcement of AS in the community.

ObjectiveEscherichia coli ST410 with blaNDM-5 has been increasingly detected as multidrug resistance pathogens globally, even though there are very few reports of infections caused by blaNDM-5 producing E. coli in Singapore[1]. And significantly limit sequencing information of blaNDM-5 carried E. coli strain from Singapore. In 2018, our group obtained a carbapenem resistance E. coli ST410 strain SrichA-1 isolated from reservoir water in Singapore, determined to harbor the NDM-5 gene. (BioSample Accession: SAMN18579051). MethodsThe susceptibility test to antimicrobials was performed with microdilution minimum inhibitory concentration (MIC) test and interpreted according to the Clinical And Laboratory Standards Institute (CLSI) -M100 standards. The genomic DNA of this strain was extracted and send for Whole-genome sequencing(WGS) with the Illumina platform. The WGS analysis was processed with the Center for Genomic Epidemiology (CGE, DTU) server. ResultsDuring the minimum inhibitory concentration (MIC) test, SrichA-1 has shown strong resistance to all the beta-lactams, including cephalosporin and carbapenem, which can not be inhibited by the clavulanic acid. Further whole genome sequencing analysis has shown that the strain harboring five beta-lactamase genes covers all class A to D, including the carbapenemase genes as blaNDM-5. ConclusionHere, we reported the complete chromosome sequence of this isolate as well as the sequence of a cycler plasmid. The pSGNDM-5 plasmid was furtherly identified to carry four beta-lactamase genes, including blaNDM-5, blaCTX-M-15, blaTEM-1B, blaOXA-1, while a blaCMY-2 was detected to be located on the chromosome.

Ribosomal RNA gene amplicon sequencing is commonly used to evaluate microbiome profiles in health and disease and document the impact of interventional treatments. Long-read nanopore sequencing is attractive since it can provide greater classification at the species level. However, optimised protocols to target marker genes for bacterial and fungal profiling are needed. To achieve an increased taxonomic resolution, we developed extraction and long-amplicon PCR-based approaches using Nanopore sequencing. Three sample lysis conditions were applied to a mock microbial community, including known bacterial and fungal species; the 96 MagBead DNA lysis buffer (ML) alone, incorporating bead-beating (MLB) or bead-beating plus MetaPolyzyme enzymatic treatment (MLBE). Profiling of bacterial comparison, MLB had more statistically different bacterial phyla and genera than the others. For fungal profiling, MLB had a significant increase of Ascomycota and a decline of Basidiomycota, subsequently failing to detect Malassezia and Cryptococcus. Also, the principal coordinates analysis (PCoA) plot by the Bray-Curtis index showed a significant difference among groups for bacterial (p = 0.033) and fungal (p = 0.012) profiles. Overall, the microbial profiling and diversity analysis revealed that ML and MLBE have more similarity than MLB for both bacteria and fungi, therefore, bead-beating is not recommended for long-read amplicon sequencing. However, ML alone was suggested as an optimal approach considering DNA yield, classification, reagent cost and hands-on time. This could be an initial proof-of-concept study for simultaneous human microbiome and mycobiome studies.

Clostridium scindens is a commensal gut bacterium capable of forming the secondary bile acids deoxycholic acid and lithocholic acid from the primary bile acids cholic acid and chenodeoxycholic acid, respectively, as well as converting glucocorticoids to androgens. Historically, only two strains, C. scindens ATCC 35704 and C. scindens VPI 12708, have been characterized in vitro and in vivo to any significant extent. The formation of secondary bile acids is important in maintaining normal gastrointestinal function, in regulating the structure of the gut microbiome, in the etiology of such diseases such as cancers of the GI tract, and in the prevention of Clostridium difficile infection. We therefore wanted to determine the pangenome of 34 cultured strains of C. scindens and a set of 200 metagenome-assembled genomes (MAGs) to understand the variability among strains. The results indicate that the 34 strains of C. scindens have an open pangenome with 12,720 orthologous gene groups, and a core genome with 1,630 gene families, in addition to 7,051 and 4,039 gene families in the accessory and unique (i.e., strain-exclusive) genomes, respectively. The core genome contains 39% of the proteins with predicted metabolic function, and, in the unique genome, the function of storage and processing of information prevails, with 34% of the proteins being in that category. The pangenome profile including the MAGs also proved to be open. The presence of bile acid inducible (bai) and steroid-17,20-desmolase (des) genes was identified among groups of strains. The analysis reveals that C. scindens strains are distributed into two clades, indicating the possible onset of C. scindens separation into two species, confirmed by gene content, phylogenomic, and average nucleotide identity (ANI) analyses. This study provides insight into the structure and function of the C. scindens pangenome, offering a genetic foundation of significance for many aspects of research on the intestinal microbiota and bile acid metabolism.

Bacillus subtilis is widely studied in the microbial secondary metabolite (SM) field due to its rich variety of important natural products and genetic tractability. However, identification of novel SMs and their biosynthetic gene cluster (BGCs) has become increasingly difficult, especially in Bacilli, as the tools for screening and genome mining are dependent on clear function or similarity to already known BGCs. Pigments are SMs identified by their absorption of visible light, resulting in a certain color perceived by our eyes at sufficient concentrations. Thereby, pigments provide the evidence of a BGC without knowing the sequence or function. Expanding the known repertoire of SM BGCs with novel BGCs will further reinforce identification of a broader set of BGCs by mining tools such as antiSMASH. Here, we study a pigment observed in B. subtilis soil isolate MB9_B4 on certain media. We characterize the conditions where this pigment is produced and identify the corresponding BGC using a comparative genomic approach exploiting our strain collection containing other isolates with pigment production ability. The responsible BGC carried several genes, which were annotated as parts of the tryptophan biosynthesis pathway, possibly originating from a duplication and divergence of an originally primary metabolism. Identification of the pigment gene cluster additionally lead to the discovery of additional pigment BGC carrier B. subtilis isolates, some of which were described at the earliest in 1896 under the name Bacillus aterrimus, with a name referring to a dark pigmentation (the Latin "aterrimus" meaning very black). In addition, we employed solid-state nuclear magnetic resonance and Fourier transform infrared spectroscopies to characterize the chemical groups of the pigment. This study describes the chemical and biological features of a new class of SM BGC, which we hope will serve to improve the current BGC discovery pipelines in Bacilli.

This in silico investigation aimed to: 1) evaluate a set of primer pairs with high coverage, including those most commonly used in the literature, to find the different oral species with 16S rRNA gene amplicon similarity/identity (ASI) values [&ge;]97%; and 2) identify oral species that may be erroneously clustered in the same operational taxonomic unit (OTU) and ascertain whether they belong to distinct genera or other higher taxonomic ranks. Thirty-nine primer pairs were employed to obtain amplicon sequence variants (ASVs) from the complete genomes of 186 bacterial and 135 archaeal species. For each primer, ASVs without mismatches were aligned using BLASTN and their similarity values were obtained. Finally, we selected ASVs from different species with an ASI value [&ge;]97% that were covered 100% by the query sequences. For each primer, the percentage of species-level coverage with no ASI[&ge;]97% (SC-NASI[&ge;]97%) was calculated. Based on the SC-NASI[&ge;]97% values, the best primer pairs were OP_F053-KP_R020 for bacteria (65.05%), KP_F018-KP_R002 for archaea (51.11%), and OP_F114-KP_R031 for bacteria and archaea together (52.02%). Eighty percent of the oral-bacteria and oralarchaea species shared an ASI[&ge;]97% with at least one other taxa, including Campylobacter, Rothia, Streptococcus, and Tannerella, which played conflicting roles in the oral microbiota. Moreover, around a quarter and a third of these two-by-two similarity relationships were between species from different bacteria and archaea genera, respectively. Furthermore, even taxa from distinct families, orders, and classes could be grouped in the same cluster. Consequently, irrespective of the primer pair used, OTUs constructed with a 97% similarity provide an inaccurate description of oral-bacterial and oral-archaeal species, greatly affecting microbial diversity parameters. As a result, clustering by OTUs impacts the credibility of the associations between some oral species and certain health and disease conditions. This limits significantly the comparability of the microbial diversity findings reported in oral microbiome literature.

Intestinal microbiome, comprising the whole microbiota, their genes and genomes living in the human gut have significant roles in promoting health or disease status. As many studies showed so far, identifying the bacterial components of the microbiome can reveal important biomarkers to help in the disease comprehension to a further adequate treatment. However, the human nature is quite variable considering the genetic components associated with life styles, directly reflecting on the gut microbiome. Thus, it is extremely important to know the populational microbiome background in order to draw conclusions regarding the health and disease conditions. Also, methodological best practices and knowledge about the methods being used are essential for the results quality and applicability with clinical relevance. In this way, we standardized the sample collection and processing methods used for the Probiome assay, a test developed to identify the Brazilian bacteriome from stool samples. EncodeTools Metabarcode pipeline of analysis was developed to obtain the best result from the samples. This pipeline uses the information of amplicon single variants (ASVs) in 100% identical oligotype clusters, and performs a de novo taxonomical assignment based on similarity for unknown sequences. To better comprehend the results obtained in Probiome assays, is essential to know the intestinal bacteriome diversity of Brazilians. Thus, we applied the standardized methods herein developed and began characterizing our populational data to allow a better understanding of the Brazilian bacteriome profiles and how they can be related to other microbiome studies.

ObjectiveTo semi-quantitively screen filamentous fungi isolated from different habitats for L-asparaginase production by three indicators; phenol red, cresol red and bromothymol blue and to examine the impact of different carbon and nitrogen sources on the enzyme production using different fungal isolates. Materials and methodsFifty-five fungal isolates were tested for L-asparaginase production by plate assay using Modified Czapek-Dox (MCD) medium. The enzyme activity was estimated using the Nessler method which measures the concentration of ammonia formed owing to the enzyme action on the substrate. The impact of nitrogen and carbon sources on the enzyme production was done by using the best three L-asparaginase producers from the semi-quantitative screening. Results and conclusionsA total of 53/55 (96.36%) fungal isolates were L-asparaginase producing strains, of them, Cladosporium tenuissimum, Penicillium camembertii and Aspergillus carneus showed high enzyme production. Production of L-asparaginase was higher with the glucose and urea as carbon and nitrogen sources, respectively. The highest enzyme level (5,558 U/ml) was produced by C. tenuissimum in a glucose-containing medium. This study shows that P. camemberti, A. carneus, and C. tenuissimum are good L-asparaginase producers and thus could be used for L-asparaginase production

Mitochondrial dysfunction lead to a wide group of progressive and fatal pathologies known as mitochondrial diseases (MD). One of the most common pediatric representation of MD is Leigh Syndrome, affecting 1/40.000 births. LS is characterized by neurodegeneration in specific brain areas, such as brainstem and basal ganglia, and by respiratory and motor alterations. However, the results obtained from clinical trials based on antioxidant therapies are controversial. Thus, the development novel antioxidant strategy is required to improve the efficacy of current palliative treatments. In this regard, Ndufs4KO mouse model is a suitable model to test new drugs in the field of MD and LS. Therefore, we set to assess the therapeutic potential of oral administration of Micrococcus luteus, a high-antioxidant content microorganism. Incidentally, we identified that while M. luteus administration did not possess any beneficial actions, the cryopreservant maltodextrin (MDX), included in the preparation, ameliorated the phenotype of Ndufs4KO mice. Our results show that MDX treatment at a concentration of 30 g/L increased lifespan and reduced microglial reaction compared to vehicle-treated Ndufs4KO mice. However, no improvement in locomotion nor respiratory function was observed in MDX-treated mice compared to vehicle-treated Ndufs4KO mice. Metataxonomic characterization of intestinal microbiome identified differential profiles in Ndufs4KO mice at the genus level. Furthermore, MDX treatment increased the variability of the abundance of Akkermansia sp. Thus, this work paves the way for further studies to confirm the therapeutic potential of MDX in mitochondrial disease.

Bile acids (BAs), biological detergents for nutrient digestion, are important local and systemic signalling molecules to interact with a variety of cell receptors central to influence host responses. While BAs are synthesized in the liver, the range and diversity of bile acids available to interact with these receptors is dictated by the gut microbiota. Bile salt hydrolase (BSH) activity is one such function, it is commonly represented and highly conserved across all major bacterial phyla in the gut. Studies relating to the importance of such modifications in early life are scarce. This study highlights BA metabolism diversity by functionally isolating BA metabolizing strains and by characterizing specific classes of BSH from the formula-fed transitioning gut. Isolates were identified to species levels, in silico and in vitro characterisation of their BSH genetic content, enzyme activity and substrate specificity. One of these isolates was identified as Lactobacillus acidophilus, a species frequently applied as a probiotic whereas three of these four isolates were identified as Enterococcus avium. This particular species is not well characterized in the literature and to our knowledge this is the first report of BSH activity and assessment for probiotic potential within this class of microbes. This study indicates that microbial BA altering activity appears functionally reduced, in the formula fed infant gut.