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1.
Mucosal Immunol ; 10(5): 1279-1293, 2017 09.
Article in English | MEDLINE | ID: mdl-28000678

ABSTRACT

Altered interactions between the gut mucosa and bacteria during HIV infection seem to contribute to chronic immune dysfunction. A deeper understanding of how nutritional interventions could ameliorate gut dysbiosis is needed. Forty-four subjects, including 12 HIV+ viremic untreated (VU) patients, 23 antiretroviral therapy-treated (ART+) virally suppressed patients (15 immunological responders and 8 non-responders) and 9 HIV- controls (HIV-), were blindly randomized to receive either prebiotics (scGOS/lcFOS/glutamine) or placebo (34/10) over 6 weeks in this pilot study. We assessed fecal microbiota composition using deep 16S rRNA gene sequencing and several immunological and genetic markers involved in HIV immunopathogenesis. The short dietary supplementation attenuated HIV-associated dysbiosis, which was most apparent in VU individuals but less so in ART+ subjects, whose gut microbiota was found more resilient. This compositional shift was not observed in the placebo arm. Significantly, declines in indirect markers of bacterial translocation and T-cell activation, improvement of thymic output, and changes in butyrate production were observed. Increases in the abundance of Faecalibacterium and Lachnospira strongly correlated with moderate but significant increases of butyrate production and amelioration of the inflammatory biomarkers soluble CD14 and high-sensitivity C-reactive protein, especially among VU. Hence, the bacterial butyrate synthesis pathway holds promise as a viable target for interventions.


Subject(s)
Bacteria/genetics , Dysbiosis/prevention & control , Gastrointestinal Microbiome/genetics , HIV Infections/microbiology , HIV-1/immunology , Intestinal Mucosa/immunology , Prebiotics/administration & dosage , RNA, Ribosomal, 16S/analysis , Adult , Butyrates/metabolism , Dietary Supplements , Dysbiosis/etiology , Dysbiosis/microbiology , Feces/microbiology , Female , Gastrointestinal Microbiome/immunology , HIV Infections/complications , HIV Infections/immunology , Host-Pathogen Interactions , Humans , Immunity , Intestinal Mucosa/microbiology , Intestinal Mucosa/virology , Male , Middle Aged , Placebo Effect
2.
J Dev Orig Health Dis ; 7(1): 35-44, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26353938

ABSTRACT

The gastrointestinal tract (GIT) microbiota has been identified as an important reservoir of antibiotic resistance genes (ARGs) that can be horizontally transferred to pathogenic species. Maternal GIT microbes can be transmitted to the offspring, and recent work indicates that such transfer starts before birth. We have used culture-independent genetic screenings to explore whether ARGs are already present in the meconium accumulated in the GIT during fetal life and in feces of 1-week-old infants. We have analyzed resistance to ß-lactam antibiotics (BLr) and tetracycline (Tcr), screening for a variety of genes conferring each. To evaluate whether ARGs could have been inherited by maternal transmission, we have screened perinatal fecal samples of the 1-week-old babies' mothers, as well as a mother-infant series including meconium, fecal samples collected through the infant's 1st year, maternal fecal samples and colostrum. Our results reveal a high prevalence of BLr and Tcr in both meconium and early fecal samples, implying that the GIT resistance reservoir starts to accumulate even before birth. We show that ARGs present in the mother may reach the meconium and colostrum and establish in the infant GIT, but also that some ARGs were likely acquired from other sources. Alarmingly, we identified in both meconium and 1-week-olds' samples a particularly elevated prevalence of mecA (>45%), six-fold higher than that detected in the mothers. The mecA gene confers BLr to methicillin-resistant Staphylococcus aureus, and although its detection does not imply the presence of this pathogen, it does implicate the young infant's GIT as a noteworthy reservoir of this gene.


Subject(s)
Gastrointestinal Microbiome , Meconium/chemistry , Tetracycline Resistance/genetics , beta-Lactam Resistance/genetics , Cohort Studies , Female , Humans , Infant , Infant, Newborn , Meconium/microbiology , Pregnancy
3.
Mucosal Immunol ; 8(4): 760-72, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25407519

ABSTRACT

Altered interplay between gut mucosa and microbiota during treated HIV infection may possibly contribute to increased bacterial translocation and chronic immune activation, both of which are predictors of morbidity and mortality. Although a dysbiotic gut microbiota has recently been reported in HIV+ individuals, the metagenome gene pool associated with HIV infection remains unknown. The aim of this study is to characterize the functional gene content of gut microbiota in HIV+ patients and to define the metabolic pathways of this bacterial community, which is potentially associated with immune dysfunction. We determined systemic markers of innate and adaptive immunity in a cohort of HIV-infected individuals on successful antiretroviral therapy without comorbidities and in healthy non-HIV-infected subjects. Metagenome sequencing revealed an altered functional profile, with enrichment of the genes involved in various pathogenic processes, lipopolysaccharide biosynthesis, bacterial translocation, and other inflammatory pathways. In contrast, we observed depletion of genes involved in amino acid metabolism and energy processes. Bayesian networks showed significant interactions between the bacterial community, their altered metabolic pathways, and systemic markers of immune dysfunction. This study reveals altered metabolic activity of microbiota and provides novel insight into the potential host-microbiota interactions driving the sustained inflammatory state in successfully treated HIV-infected patients.


Subject(s)
Gastrointestinal Microbiome , HIV Infections/immunology , HIV Infections/microbiology , HIV-1/immunology , Adaptive Immunity , Antiretroviral Therapy, Highly Active , Bayes Theorem , Biodiversity , Case-Control Studies , Cluster Analysis , Disease Progression , HIV Infections/drug therapy , HIV Infections/metabolism , Humans , Immunity, Innate , Markov Chains , Metabolome , Metabolomics , Metagenome , RNA, Ribosomal, 16S
4.
Clin Exp Allergy ; 43(2): 198-211, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23331561

ABSTRACT

BACKGROUND: Culture-dependent methods have shown that meconium, the newborn's first intestinal discharge, is not sterile, but the diversity of bacteria present in this material needs to be further characterized by means of more sensitive molecular techniques. OBJECTIVE: Our aims were to characterize molecularly the meconium microbiota in term infants, to assess whether it contributes to the future microbiota of the infants' gastrointestinal tract, and to evaluate how it relates to lifestyle variables and atopy-related conditions. METHODS: We applied high-throughput pyrosequencing of the 16S rRNA gene to study the meconium microbiota in twenty term newborns from a Spanish birth cohort. For comparison, we characterized the microbiota in fecal samples from seven pregnant women days before delivery and in two series of infant samples spanning the first seven months of life. We also compared our data with vaginal and skin microbiota characterized in independent studies. Different types of meconium microbiota were defined based on taxonomic composition and abundance and their associations with different factors were statistically evaluated. RESULTS: The meconium microbiota differs from those in adult feces, vagina and skin, but resembles that of fecal samples from young infants. Meconium samples clustered into two types with different bacterial diversity, richness and composition. One of the types was less diverse, dominated by enteric bacteria and associated with a history of atopic eczema in the mother (P = 0.038), whereas the second type was dominated by lactic acid bacteria and associated with respiratory problems in the infant (P = 0.040). CONCLUSIONS & CLINICAL RELEVANCE: Our findings suggest that the meconium microbiota has an intrauterine origin and participates in gut colonization. Although based on a small population sample, our association analyses also suggest that the type of bacteria detected in meconium is influenced by maternal factors and may have consequences for childhood health.


Subject(s)
Eczema/microbiology , Enterobacteriaceae/classification , Hypersensitivity, Immediate/microbiology , Intestines/microbiology , Lactobacillales/classification , Meconium/microbiology , Metagenome , Adult , DNA, Bacterial , Enterobacteriaceae/genetics , Female , Humans , Infant, Newborn , Lactobacillales/genetics , Life Style , Phylogeny , Pregnancy , RNA, Ribosomal, 16S , Risk Factors
5.
Clin Microbiol Infect ; 18 Suppl 4: 21-6, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22647043

ABSTRACT

The establishment of a balanced intestinal microbiota is essential for numerous aspects of human health, yet the microbial colonization of the gastrointestinal tract of infants is both complex and highly variable among individuals. In addition, the gastrointestinal tract microbiota is often exposed to antibiotics, and may be an important reservoir of resistant strains and of transferable resistance genes from early infancy. We are investigating by means of diverse metagenomic approaches several areas of microbiota development in infants, including the deployment of functional capabilities at the community level, the presence of antibiotic resistances and the population dynamics of the most abundant genera.


Subject(s)
Biota , Gastrointestinal Tract/microbiology , Metagenome , Metagenomics/methods , Adult , Drug Resistance, Bacterial , Female , Humans , Infant , Infant, Newborn
6.
Clin Microbiol Infect ; 18 Suppl 4: 47-9, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22647049

ABSTRACT

The gut microbiota presents a symbiotic relationship with the human host playing a beneficial role in human health. Since its establishment, the bacterial community is subjected to the influence of many different factors that shape its composition within each individual. However, an important convergence is observed at functional level in the gut microbiota. A metatranscriptomic study of healthy individuals showed homogeneity in the composition of the active microbiota that increased further at functional level.


Subject(s)
Metagenome , Metagenomics/methods , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Humans , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA/methods , Transcriptome
7.
Appl Environ Microbiol ; 66(11): 4822-8, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11055930

ABSTRACT

The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L. casei. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3' end of lacG and the complete lacF gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of L. casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the E. coli gusA gene and the L. lactis ilvBN genes in L. casei. Following the same expression pattern as that for the lactose genes, beta-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in L. casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.


Subject(s)
Genetic Vectors/genetics , Lacticaseibacillus casei/genetics , Lacticaseibacillus casei/metabolism , Lactose/metabolism , Recombination, Genetic/genetics , Regulon/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Food Microbiology , Glucuronidase/genetics , Glucuronidase/metabolism , Lactose/genetics , Plasmids/genetics , Transformation, Bacterial
8.
J Bacteriol ; 181(13): 3928-34, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10383959

ABSTRACT

In Lactobacillus casei ATCC 393, the chromosomally encoded lactose operon, lacTEGF, encodes an antiterminator protein (LacT), lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) elements (LacE and LacF), and a phospho-beta-galactosidase. lacT, lacE, and lacF mutant strains were constructed by double crossover. The lacT strain displayed constitutive termination at a ribonucleic antiterminator (RAT) site, whereas lacE and lacF mutants showed an inducer-independent antiterminator activity, as shown analysis of enzyme activity obtained from transcriptional fusions of lac promoter (lacp) and lacpDeltaRAT with the Escherichia coli gusA gene in the different lac mutants. These results strongly suggest that in vivo under noninducing conditions, the lactose-specific PTS elements negatively modulate LacT activity. Northern blot analysis detected a 100-nucleotide transcript starting at the transcription start site and ending a consensus RAT sequence and terminator region. In a ccpA mutant, transcription initiation was derepressed but no elongation through the terminator was observed in the presence of glucose and the inducing sugar, lactose. Full expression of lacTEGF was found only in a man ccpA double mutant, indicating that PTS elements are involved in the CcpA-independent catabolite repression mechanism probably via LacT.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Glycoside Hydrolases , Lac Operon , Lacticaseibacillus casei/genetics , RNA-Binding Proteins/metabolism , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Enzyme Induction , Enzyme Repression , Genes, Reporter , Glucose/pharmacology , Glucuronidase/biosynthesis , Glucuronidase/genetics , Lacticaseibacillus casei/drug effects , Lacticaseibacillus casei/enzymology , Lactose/pharmacology , Mutagenesis , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , RNA-Binding Proteins/genetics , Repressor Proteins/genetics , Transcription, Genetic , beta-Galactosidase/genetics
9.
J Bacteriol ; 179(21): 6657-64, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9352913

ABSTRACT

The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria.


Subject(s)
Bacterial Proteins , DNA-Binding Proteins/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Glycoside Hydrolases , Lacticaseibacillus casei/genetics , Repressor Proteins/genetics , Acetylglucosaminidase/biosynthesis , Amino Acid Sequence , Bacillus subtilis/genetics , Base Sequence , Cloning, Molecular , Enzyme Repression , Genetic Complementation Test , Gram-Positive Bacteria/genetics , Lac Operon/genetics , Molecular Sequence Data , Mutagenesis , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription, Genetic , beta-Galactosidase/biosynthesis
10.
FEMS Microbiol Lett ; 148(1): 83-9, 1997 Mar 01.
Article in English | MEDLINE | ID: mdl-9066115

ABSTRACT

The chromosomally encoded lactose-specific phosphoenol pyruvate-dependent phosphotransferase system (PTS) has been investigated in Lactobacillus casei ATCC 393 [pLZ15-] and it was considered an excellent system to study the regulation of the lactose operon. This chromosomal operon has been cloned and sequenced, being 99% homologous to that encoded on the plasmid pLZ64. Expression of the lactose operon in different mutants of L. casei ATCC 393 [pLZ15-] and primer extension analysis revealed that it is subject to a dual regulation: (i) glucose repression possibly mediated by CcpA and PTS elements, and (ii) induction by lactose through transcriptional antitermination.


Subject(s)
Lac Operon/physiology , Lacticaseibacillus casei/genetics , Transcription, Genetic/genetics , Base Sequence , Cloning, Molecular , Gene Expression Regulation, Bacterial/physiology , Genes, Bacterial/physiology , Genes, Reporter , Lacticaseibacillus casei/chemistry , Lacticaseibacillus casei/metabolism , Molecular Sequence Data , Mutation/physiology , Sequence Analysis, DNA , beta-Galactosidase
11.
Microbiology (Reading) ; 142 ( Pt 12): 3469-77, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9004509

ABSTRACT

In Lactobacillus curvatus, a phosphoenolpyruvate:mannose phosphotransferase system (mannose-PTS) has been characterized and it was shown to be involved in glucose and mannose transport, but no glucose-specific PTS activity could be detected. A 2.1 kb DNA fragment amplified by PCR from the L. curvatus genome was sequenced. Sequence analysis showed four ORFs which could encode proteins similar to PTS transporters EIIA, EIIB, EIIC and EIID of the mannose class. The expression of the manB gene (encoding EIIB) from L. curvatus in a mutant of Lactobacillus sake impaired in EIIMan activity restored this activity. Furthermore, this DNA fragment complemented the regulatory function of LevE (EIIB) in a Bacillus subtilis levE-deficient mutant, suggesting that the protein encoded by manB could also play a regulatory role in L. curvatus.


Subject(s)
Lactobacillus/enzymology , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Chromosomes, Bacterial , Cloning, Molecular , DNA Primers , Genes, Bacterial , Lactobacillus/genetics , Molecular Sequence Data , Open Reading Frames , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Phosphorylation , Polymerase Chain Reaction , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
12.
J Bacteriol ; 173(23): 7705-10, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1938968

ABSTRACT

Two genes, xynD and gluB, encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis. A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp. The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB). Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E. coli and culture supernatants of B. subtilis clones containing the xynD gene. Both peptides show xylanase activity in zymogram analysis. The XYND enzyme also shows alpha-L-arabinofuranosidase activity. The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (O. Grépinet, M. C. Chebrou, and P. Béguin, J. Bacteriol. 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids.


Subject(s)
Bacillus subtilis/genetics , Bacillus/genetics , Escherichia coli/genetics , Genes, Bacterial , Glycoside Hydrolases/genetics , Isoenzymes/genetics , Multigene Family , Amino Acid Sequence , Bacillus/enzymology , Base Sequence , Cloning, Molecular , Clostridium/enzymology , Clostridium/genetics , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Gene Expression , Glycoside Hydrolases/metabolism , Isoenzymes/metabolism , Molecular Sequence Data , Molecular Weight , Plasmids , Recombinant Proteins/metabolism , Restriction Mapping , Sequence Homology, Nucleic Acid , Xylan Endo-1,3-beta-Xylosidase
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