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High-throughput sequencing studies have shown that diet or antimicrobial treatments impact animal gut microbiota equilibrium. However, properties related to the gut microbial ecosystem stability, such as resilience, resistance, or functional redundancy, must be better understood. To shed light on these ecological processes, we combined advanced statistical methods with 16 S rRNA gene sequencing, functional prediction, and fitness analyses in the gut microbiota of the cockroach Blattella germanica subject to three periodic pulses of the antibiotic (AB) kanamycin (n=512). We first confirmed that AB did not significantly affect cockroaches' biological fitness, and gut microbiota changes were not caused by insect physiology alterations. The sex variable was examined for the first time in this species, and no statistical differences in the gut microbiota diversity or composition were found. The comparison of the gut microbiota dynamics in control and treated populations revealed that (1) AB treatment decreases diversity and completely disrupts the co-occurrence networks between bacteria, significantly altering the gut community structure. (2) Although AB also affected the genetic composition, functional redundancy would explain a smaller effect on the functional potential than on the taxonomic composition. (3) As predicted by Taylor's law, AB generally affected the most abundant taxa to a lesser extent than the less abundant taxa. (4) Taxa follow different trends in response to ABs, highlighting "resistant taxa," which could be critical for community restoration. (5) The gut microbiota recovered faster after the three AB pulses, suggesting that gut microbiota adapts to repeated treatments., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Multiple mutations often have non-additive (epistatic) phenotypic effects. Epistasis is of fundamental biological relevance but is not well understood mechanistically. Adaptive evolution, i.e., the evolution of new biochemical activities, is rich in epistatic interactions. To better understand the principles underlying epistasis during genetic adaptation, we studied the evolution of TEM-1 β-lactamase variants exhibiting cefotaxime resistance. We report the collection of a library of 487 observed evolutionary trajectories for TEM-1 and determine the epistasis status based on cefotaxime resistance phenotype for 206 combinations of 2-3 TEM-1 mutations involving 17 positions under adaptive selective pressure. Gain-of-function (GOF) mutations are gatekeepers for adaptation. To see if GOF phenotypes can be inferred based solely on sequence data, we calculated the enrichment of GOF mutations in the different categories of epistatic pairs. Our results suggest that this is possible because GOF mutations are particularly enriched in sign and reciprocal sign epistasis, which leave a major imprint on the sequence space accessible to evolution. We also used FoldX to explore the relationship between thermodynamic stability and epistasis. We found that mutations in observed evolutionary trajectories tend to destabilize the folded structure of the protein, albeit their cumulative effects are consistently below the protein's free energy of folding. The destabilizing effect is stronger for epistatic pairs, suggesting that modest or local alterations in folding stability can modulate catalysis. Finally, we report a significant relationship between epistasis and the degree to which two protein positions are structurally and dynamically coupled, even in the absence of ligand.

Recent studies show that the salivary microbiome in subjects with obesity differ from those without obesity, but the mechanism of interaction between the salivary microbiome composition and body weight is unclear. Herein we investigate this relation by analyzing saliva samples from 35 adult patients with obesity undergoing bariatric surgery. Our aim was to describe salivary microbiome changes during body weight loss on an individual-specific level, and to elucidate the effect of bariatric surgery on the salivary microbiome which has not been studied before. Analysis of samples collected before and 1 day after surgery, as well as 3 and 12 months after surgery, showed that the salivary microbiome changed in all study participants, but these changes were heterogeneous. In the majority of participants proportions of Gemella species, Granulicatella elegans, Porphyromonas pasteri, Prevotella nanceiensis and Streptococcus oralis decreased, while Veillonella species, Megasphaera micronuciformis and Prevotella saliva increased. Nevertheless, we found participants deviating from this general trend which suggests that a variety of individual-specific factors influence the salivary microbiome composition more effectively than the body weight dynamics alone. The observed microbiome alternations could be related to dietary changes. Therefore, further studies should focus on association with altered taste preferences and potential oral health consequences.

Human lungs harbor a scarce microbial community, requiring to develop methods to enhance the recovery of nucleic acids from bacteria and fungi, leading to a more efficient analysis of the lung tissue microbiota. Here we describe five extraction protocols including pre-treatment, bead-beating and/or Phenol:Chloroform:Isoamyl alcohol steps, applied to lung tissue samples from autopsied individuals. The resulting total DNA yield and quality, bacterial and fungal DNA amount and the microbial community structure were analyzed by qPCR and Illumina sequencing of bacterial 16S rRNA and fungal ITS genes. Bioinformatic modeling revealed that a large part of microbiome from lung tissue is composed of microbial contaminants, although our controls clustered separately from biological samples. After removal of contaminant sequences, the effects of extraction protocols on the microbiota were assessed. The major differences among samples could be attributed to inter-individual variations rather than DNA extraction protocols. However, inclusion of the bead-beater and Phenol:Chloroform:Isoamyl alcohol steps resulted in changes in the relative abundance of some bacterial/fungal taxa. Furthermore, inclusion of a pre-treatment step increased microbial DNA concentration but not diversity and it may contribute to eliminate DNA fragments from dead microorganisms in lung tissue samples, making the microbial profile closer to the actual one.

In this paper, we explore how two discrete and geographically separated populations of the lesser long-nosed bat (Leptonycteris yerbabuenae)-one in central and the other in the Pacific region of Mexico-differ in their fecal microbiota composition. Considering the microbiota-host as a unity, in which extrinsic (as food availability and geography) or intrinsic factors (as physiology) play an important role in the microbiota composition, we would expect differentiation in the microbiota of two geographically separated populations. The Amplicon Sequences Variants (ASVs) of the V4 region of the 16s rRNA gene from 68 individuals were analyzed using alpha and beta diversity metrics. We obtained a total of 11 566 (ASVs). The bacterial communities in the Central and Pacific populations had a diversity of 6,939 and 4,088 ASVs, respectively, sharing a core microbiota of 539 ASVs accounting for 75% of the relative abundance, suggesting stability over evolutionary time. The Weighted UniFrac metrics tested by a PERMANOVA showed that lactating and pregnant females had significant beta diversity differences in the two populations compared with other reproductive stages. This could be a consequence of the increased energy requirements of these physiological stages, more than the variation due to geographical separation. In contrast, a positive correlation of the observed ASVs of fecal microbiota with the observed ASVs of plastids related to the diet was observed in the juveniles and adults, suggesting that in these physiological stages an extrinsic factor as the diet shapes the microbiota composition. The results provide a baseline for future studies of the microbiome in these two wild populations of the lesser long-nosed bat, the main pollinator of the Agaves from which the beverages tequila and mezcal are made., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Background: The chaperone ClpB, a bacterial protein, is a conformational antigen-mimetic of α-melanocyte-stimulating hormone (α-MSH) implicated in body weight regulation in mice. We here investigated the potential associations of gut bacterial ClpB-like gene function with obesity status and gut microbiota in humans., Results: Gut microbiota ClpB KEGG function was negatively associated with body mass index, waist circumference, and total fat mass (DEXA). The relative abundance (RA) of several phyla and families directly associated with ClpB was decreased in subjects with obesity. Specifically, the RA of Rikenellaceae, Clostridiaceae and not assigned Firmicutes were lower in subjects with obesity and positively associated with gut bacterial ClpB-like gene function (not assigned Firmicutes (r = 0.405, FDR = 2.93 × 10-2), Rikenellaceae (r = 0.217, FDR = 0.031), and Clostridiaceae (r = 0.239, FDR = 0.017)). The gut bacterial ClpB-like gene function was also linked to specific plasma metabolites (hippuric acid and 3-indolepropionic acid) and fecal lupeol. The α-MSH-like epitope similar to that of Escherichia coli ClpB was also identified in some sequences of those bacterial families. After fecal transplantation from humans to mice, the families that more contributed to ClpB-like gene function in humans were also associated with ClpB-like gene function in mice after adjusting for the donor's body mass index (not assigned Firmicutes (r = 0.621, p = 0.003), Prevotellaceae (r = 0.725, p = 4.1 × 10 -7 ), Rikenellaceae (r = 0.702, p = 3.9 × 10 -4 ), and Ruminococcaceae (r = 0.526, p = 0.014)). Clostridiaceae (r = - 0.445, p = 0.038) and Prevotellaceae RA (r = - 0.479, p = 0.024) and were also negatively associated with weight gain in mice. The absolute abundance (AA) of Prevotellaceae in mice was also positively associated with the gut bacterial ClpB-like gene function in mice. DESeq2 identified species of Prevotellaceae, both negatively associated with mice' weight gain and positively with gut bacterial ClpB-like gene function., Conclusions: In summary, gut bacterial ClpB-like gene function is associated with obesity status, a specific gut microbiota composition and a plasma metabolomics profile in humans that could be partially transplanted to mice. Video Abstract.

Membrane computing is a bio-inspired computing paradigm whose devices are the so-called membrane systems or P systems. The P system designed in this work reproduces complex biological landscapes in the computer world. It uses nested "membrane-surrounded entities" able to divide, propagate, and die; to be transferred into other membranes; to exchange informative material according to flexible rules; and to mutate and be selected by external agents. This allows the exploration of hierarchical interactive dynamics resulting from the probabilistic interaction of genes (phenotypes), clones, species, hosts, environments, and antibiotic challenges. Our model facilitates analysis of several aspects of the rules that govern the multilevel evolutionary biology of antibiotic resistance. We examined a number of selected landscapes where we predict the effects of different rates of patient flow from hospital to the community and vice versa, the cross-transmission rates between patients with bacterial propagules of different sizes, the proportion of patients treated with antibiotics, and the antibiotics and dosing found in the opening spaces in the microbiota where resistant phenotypes multiply. We also evaluated the selective strengths of some drugs and the influence of the time 0 resistance composition of the species and bacterial clones in the evolution of resistance phenotypes. In summary, we provide case studies analyzing the hierarchical dynamics of antibiotic resistance using a novel computing model with reciprocity within and between levels of biological organization, a type of approach that may be expanded in the multilevel analysis of complex microbial landscapes. IMPORTANCE The work that we present here represents the culmination of many years of investigation in looking for a suitable methodology to simulate the multihierarchical processes involved in antibiotic resistance. Everything started with our early appreciation of the different independent but embedded biological units that shape the biology, ecology, and evolution of antibiotic-resistant microorganisms. Genes, plasmids carrying these genes, cells hosting plasmids, populations of cells, microbial communities, and host's populations constitute a complex system where changes in one component might influence the other ones. How would it be possible to simulate such a complexity of antibiotic resistance as it occurs in the real world? Can the process be predicted, at least at the local level? A few years ago, and because of their structural resemblance to biological systems, we realized that membrane computing procedures could provide a suitable frame to approach these questions. Our manuscript describes the first application of this modeling methodology to the field of antibiotic resistance and offers a bunch of examples-just a limited number of them in comparison with the possible ones to illustrate its unprecedented explanatory power., (Copyright © 2019 Campos et al.)

Fourteen lots of cooked ham in modified atmosphere packaging (CH) were analyzed within a few days from packaging (S) and at the end of the shelf-life (E), after storage at 7 °C to simulate thermal abuse. Five more lots, rejected from the market because spoiled (R), were included in the study. Quality of the products was generally compromised during the shelf life, with only 4 lots remaining unaltered. Analysis of 16S rRNA gene amplicons resulted in 801 OTUs. S samples presented a higher diversity than E and R ones. At the beginning of the shelf life, Proteobacteria and Firmicutes dominated the microbiota, with Acinetobacter, Brochothrix, Carnobacterium, Lactobacillus, Prevotella, Pseudomonas, Psychrobacter, Weissella, Vibrio rumoiensis occurring frequently and/or abundantly. E and R samples were dominated by Firmicutes mostly ascribed to Lactobacillales. It is noteworthy the appearance of abundant Leuconostoc, negligible in S samples, in some E and R samples, while in other LAB were outnumbered by V. rumoiensis or Brochothrix thermosphacta. The microbiota of spoiled and R samples could not be clustered on the basis of specific defects (discoloration, presence of slime, sourness, and swollen packages) or supplemented additives. LAB population of S samples, averaging 2.9 log 10 (cfu/g), increased to 7.7 log 10 (cfu/g) in the E and R samples. Dominant cultivable LAB belonged to the species Lactobacillus sakei and Leuconostoc carnosum. The same biotypes ascribed to different species where often found in the corresponding S and R samples, and sometime in different batches provided from the same producer, suggesting a recurrent contamination from the plant of production. Consistently with growth of LAB, initial pH (6.26) dropped to 5.74 in E samples. Volatiles organic compound (VOCs) analysis revealed that ethanol was the major metabolite produced during the shelf life. The profile of volatile compounds got enriched with other molecules (e.g. 2-butanone, ethyl acetate, acetic acid, acetoin, butanoic acid, ethyl ester, butanoic acid, and 2,3-butanediol) mainly ascribed to microbial metabolism., (Copyright © 2018 Elsevier B.V. All rights reserved.)

HIV infection causes a disruption of gut-associated lymphoid tissue, driving a shift in the composition of gut microbiota. A deeper understanding of the metabolic changes and how they affect the interplay with the host is needed. Here, we assessed functional modifications of HIV-associated microbiota by combining metagenomic and metatranscriptomic analyses. The transcriptionally active microbiota was well-adapted to the inflamed environment, overexpressing pathways related to resistance to oxidative stress. Furthermore, gut inflammation was maintained by the Gram-negative nature of the HIV-associated microbiota and underexpression of anti-inflammatory processes, such as short chain fatty acid biosynthesis or indole production. We performed co-occurrence and metabolic network analyses that showed relevance in the microbiota structure of both taxonomic and metabolic HIV-associated biomarkers. The Bayesian network revealed the most determinant pathways for maintaining the structure stability of the bacterial community. In addition, we identified the taxa's contribution to metabolic activities and their interactions with host health.

Objective: In a recent blinded randomized study, we found that in HIV-infected individuals a short supplementation with prebiotics (scGOS/lcFOS/glutamine) ameliorates dysbiosis of total gut bacteria, particularly among viremic untreated patients. Our study goal was to determine the fraction of the microbiota that becomes active during the intervention and that could provide additional functional information., Design: A total of six healthy individuals, and 16 HIV-infected patients comprising viremic untreated patients (n = 5) and antiretroviral therapy-treated patients that are further divided into immunological responders (n = 7) and immunological nonresponders (n = 4) completed the 6-week course of prebiotic treatment, including six patients receiving a placebo., Methods: Alpha and beta diversity of potentially active and total gut microbiota was evaluated using shotgun proteomics and 16S rRNA gene sequencing., Results: HIV infection decreased dormancy and increased alpha diversity of active bacteria in comparison with the healthy controls, whose richness was not further influenced by the prebiotic intervention. The effect of the prebiotics was most evident at the beta-diversity of active bacteria, particularly within viremic untreated patients. We found that the prebiotics did not only ameliorate dysbiosis of total bacteria in viremic untreated patients but also increased the abundance of active bacteria with strong immunomodulatory properties and amino acids metabolism, namely Bifidobacteriaceae, at similar levels to those in healthy individuals. This effect was attenuated in ART-treated individuals., Conclusion: The effect of prebiotics was greater among ART-naive HIV-infected individuals than in ART-treated patients and healthy controls. This highlights the importance of therapies aimed at manipulating the microbiome in this group of patients.

While changes in gut microbial populations have been described in human immuno-deficiency virus (HIV)-infected patients undergoing antiretroviral therapy (ART), the mechanisms underlying the contributions of gut bacteria and their molecular agents (metabolites and proteins) to immune recovery remain unexplored. To study this, we examined the active fraction of the gut microbiome, through examining protein synthesis and accumulation of metabolites inside gut bacteria and in the bloodstream, in 8 healthy controls and 29 HIV-infected individuals (6 being longitudinally studied). We found that HIV infection is associated to dramatic changes in the active set of gut bacteria simultaneously altering the metabolic outcomes. Effects were accentuated among immunological ART responders, regardless diet, subject characteristics, clinical variables other than immune recovery, the duration and type of ART and sexual preferences. The effect was found at quantitative levels of several molecular agents and active bacteria which were herein identified and whose abundance correlated with HIV immune pathogenesis markers. Although, we cannot rule out the possibility that some changes are partially a random consequence of the disease status, our data suggest that most likely reduced inflammation and immune recovery is a joint solution orchestrated by both the active fraction of the gut microbiota and the host., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Imbalances in gut bacteria have been associated with multiple diseases. However, whether there are disease-specific changes in gut microbial metabolism remains unknown. Here, we demonstrate that human immunodeficiency virus (HIV) infection (n = 33) changes, at quantifiable levels, the metabolism of gut bacteria. These changes are different than those observed in patients with the auto-immune disease systemic lupus erythaematosus (n = 18), and Clostridium difficile-associated diarrhoea (n = 6). Using healthy controls as a baseline (n = 16), we demonstrate that a trend in the nature and directionality of the metabolic changes exists according to the type of the disease. The impact on the gut microbial activity, and thus the metabolite composition and metabolic flux of gut microbes, is therefore disease-dependent. Our data further provide experimental evidence that HIV infection drastically changed the microbial community, and the species responsible for the metabolism of 4 amino acids, in contrast to patients with the other two diseases and healthy controls. The identification in this present work of specific metabolic deficits in HIV-infected patients may define nutritional supplements to improve the health of these patients.

The evolution of eukaryotic organisms is often strongly influenced by microbial symbionts that confer novel traits to their hosts. Here we describe the intracellular Enterobacteriaceae symbiont of the invasive ant Cardiocondyla obscurior, 'Candidatus Westeberhardia cardiocondylae'. Upon metamorphosis, Westeberhardia is found in gut-associated bacteriomes that deteriorate following eclosion. Only queens maintain Westeberhardia in the ovarian nurse cells from where the symbionts are transmitted to late-stage oocytes during nurse cell depletion. Functional analyses of the streamlined genome of Westeberhardia (533 kb, 23.41% GC content) indicate that neither vitamins nor essential amino acids are provided for the host. However, the genome encodes for an almost complete shikimate pathway leading to 4-hydroxyphenylpyruvate, which could be converted into tyrosine by the host. Taken together with increasing titers of Westeberhardia during pupal stage, this suggests a contribution of Westeberhardia to cuticle formation. Despite a widespread occurrence of Westeberhardia across host populations, one ant lineage was found to be naturally symbiont-free, pointing to the loss of an otherwise prevalent endosymbiont. This study yields insights into a novel intracellular mutualist that could play a role in the invasive success of C. obscurior.

Bronchial colonization by potentially pathogenic microorganisms (PPMs) is often demonstrated in chronic obstructive pulmonary disease (COPD), but culture-based techniques identify only a portion of the bacteria in mucosal surfaces. The aim of the study was to determine changes in the bronchial microbiome of COPD associated with the severity of the disease. The bronchial microbiome of COPD patients was analyzed by 16S rRNA gene amplification and pyrosequencing in sputum samples obtained during stable disease. Seventeen COPD patients were studied (forced expiratory volume in the first second expressed as a percentage of the forced vital capacity [FEV1%] median, 35.0%; interquartile range [IQR], 31.5 to 52.0), providing a mean of 4,493 (standard deviation [SD], 2,598) sequences corresponding to 47 operational taxonomic units (OTUs) (SD, 17) at a 97% identity level. Patients were dichotomized according to their lung function as moderate to severe when their FEV1% values were over the median and as advanced when FEV1% values were lower. The most prevalent phyla in sputum were Proteobacteria (44%) and Firmicutes (16%), followed by Actinobacteria (13%). A greater microbial diversity was found in patients with moderate-to-severe disease, and alpha diversity showed a statistically significant decrease in patients with advanced disease when assessed by Shannon (ρ = 0.528; P = 0.029, Spearman correlation coefficient) and Chao1 (ρ = 0.53; P = 0.028, Spearman correlation coefficient) alpha-diversity indexes. The higher severity that characterizes advanced COPD is paralleled by a decrease in the diversity of the bronchial microbiome, with a loss of part of the resident flora that is replaced by a more restricted microbiota that includes PPMs., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The cockroach gut harbors a wide variety of microorganisms that, among other functions, collaborate in digestion and act as a barrier against pathogen colonization. Blattabacterium, a primary endosymbiont, lives in the fat body inside bacteriocytes and plays an important role in nitrogen recycling. Little is known about the mode of acquisition of gut bacteria or their ecological succession throughout the insect life cycle. Here we report on the bacterial taxa isolated from different developmental instars of the cockroach Blattella germanica. The bacterial load in the gut increased two orders of magnitude from the first to the second nymphal stage, coinciding with the incorporation of the majority of bacterial taxa, but remained similar thereafter. Pyrosequencing of the hypervariable regions V1-V3 of the 16S rRNA genes showed that the microbial composition differed significantly between adults and nymphs. Specifically, a succession was observed in which Fusobacterium accumulated with aging, while Bacteroides decreased. Blattabacterium was the only symbiont found in the ootheca, which makes the vertical transmission of gut bacteria an unlikely mode of acquisition. Scanning electron microscopy disclosed a rich bacterial biofilm in third instar nymphs, while filamentous structures were found exclusively in adults., (Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.)

It is known that the gastrointestinal tract (GIT) microbiota responds to different antibiotics in different ways and that while some antibiotics do not induce disturbances of the community, others drastically influence the richness, diversity, and prevalence of bacterial taxa. However, the metabolic consequences thereof, independent of the degree of the community shifts, are not clearly understood. In a recent article, we used an integrative OMICS approach to provide new insights into the metabolic shifts caused by antibiotic disturbance. The study presented here further suggests that specific bacterial lineage blooms occurring at defined stages of antibiotic intervention are mostly associated with organisms that possess improved survival and colonization mechanisms, such as those of the Enterococcus, Blautia, Faecalibacterium, and Akkermansia genera. The study also provides an overview of the most variable metabolic functions affected as a consequence of a β-lactam antibiotic intervention. Thus, we observed that anabolic sugar metabolism, the production of acetyl donors and the synthesis and degradation of intestinal/colonic epithelium components were among the most variable functions during the intervention. We are aware that these results have been established with a single patient and will require further confirmation with a larger group of individuals and with other antibiotics. Future directions for exploration of the effects of antibiotic interventions are discussed.

Host-associated microbiota varies in distribution depending on the body area inhabited. Gut microbes are known to interact with the human immune system, maintaining gut homoeostasis. Thus, we studied whether secreted-IgA (S-IgA) coat specific microbial taxa without inducing strong immune responses. To do so, we fractionated gut microbiota by flow cytometry. We found that active and S-IgA-coated bacterial fractions were characterized by a higher diversity than those observed in raw faecal suspensions. A long-tail effect was observed in family distribution, revealing that rare bacteria represent up to 20% of total diversity. While Firmicutes was the most abundant phylum, the majority of its sequences were not assigned at the genus level. Finally, the single-cell-based approach enabled us to focus on active and S-IgA-coated bacteria. Thus, we revealed a microbiota core common to the healthy volunteers participating in the study. Interestingly, this core was composed mainly of low frequency taxa (e.g. Sphingomonadaceae).

The irritable bowel syndrome (IBS) is a functional gastrointestinal disorder with a largely unknown aetiology and a wide range of symptoms. Most cross-sectional studies carried out so far suggest subtle alterations in the structure of the intestinal microbiota that are barely reproduced, partly because of the high inter-subject variation in the community composition and disorder-specific features. We performed a longitudinal study to explore the within-subject variation in the faecal microbiota in two patients with IBS classified into the diarrhoea subtype and the healthy spouse of one of them. Faecal communities were monitored over 6-8 weeks and analysed through metagenomic and metatranscriptomic approaches. We found a higher temporal instability in the fraction of active microbiota related to the IBS condition and fluctuating symptoms. Strong and quick shifts in the distribution of the active microbiota and changes in the global pattern of gene expression were detected in association with acute diarrhoea, whereas microbial composition and encoded functions were more stable. The specific alterations in the microbiota were barely reproduced within and between patients. Further research is needed to assess whether these changes are a consequence of the abnormal gut function in acute diarrhoeic episodes and the potential usefulness of tackling them., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Objective: Antibiotic (AB) usage strongly affects microbial intestinal metabolism and thereby impacts human health. Understanding this process and the underlying mechanisms remains a major research goal. Accordingly, we conducted the first comparative omic investigation of gut microbial communities in faecal samples taken at multiple time points from an individual subjected to β-lactam therapy., Methods: The total (16S rDNA) and active (16S rRNA) microbiota, metagenome, metatranscriptome (mRNAs), metametabolome (high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry) and metaproteome (ultra high performing liquid chromatography coupled to an Orbitrap MS(2) instrument [UPLC-LTQ Orbitrap-MS/MS]) of a patient undergoing AB therapy for 14 days were evaluated., Results: Apparently oscillatory population dynamics were observed, with an early reduction in Gram-negative organisms (day 6) and an overall collapse in diversity and possible further colonisation by 'presumptive' naturally resistant bacteria (day 11), followed by the re-growth of Gram-positive species (day 14). During this process, the maximum imbalance in the active microbial fraction occurred later (day 14) than the greatest change in the total microbial fraction, which reached a minimum biodiversity and richness on day 11; additionally, major metabolic changes occurred at day 6. Gut bacteria respond to ABs early by activating systems to avoid the antimicrobial effects of the drugs, while 'presumptively' attenuating their overall energetic metabolic status and the capacity to transport and metabolise bile acid, cholesterol, hormones and vitamins; host-microbial interactions significantly improved after treatment cessation., Conclusions: This proof-of-concept study provides an extensive description of gut microbiota responses to follow-up β-lactam therapy. The results demonstrate that ABs targeting specific pathogenic infections and diseases may alter gut microbial ecology and interactions with host metabolism at a much higher level than previously assumed.

The ecological relationships that organisms establish with others can be considered as broad and diverse as the forms of life that inhabit and interact in our planet. Those interactions can be considered as a continuum spectrum, ranging from beneficial to detrimental outcomes. However, this picture has revealed as more complex and dynamic than previously thought, involving not only factors that affect the two or more members that interact, but also external forces, with chance playing a crucial role in this interplay. Thus, defining a particular symbiont as mutualist or pathogen in an exclusive way, based on simple rules of classification is increasingly challenging if not unfeasible, since new methodologies are providing more evidences that depict exceptions, reversions and transitions within either side of this continuum, especially evident at early stages of symbiotic associations. This imposes a wider and more dynamic view of a complex landscape of interactions.

To assess the effect that the rotavirus vaccine RotaTeq may have on the gut microbiota, this study searched for differences in intestinal bacterial composition between vaccinated and unvaccinated children. Bacterial diversity in fecal samples was evaluated by pyrosequencing of the 16S rRNA gene and taxonomic analyses using bioinformatics tools. No evidence of such differences was observed.

Culture of bacteria from bronchial secretions in respiratory patients has low sensitivity and does not allow for complete assessment of microbial diversity across different bronchial compartments. In addition, a significant number of clinical studies are based on sputum samples, and it is not known to what extent they describe the real diversity of the mucosa. In order to identify previously unrecognized lower airway bacteria and to investigate the complexity and distribution of microbiota in patients with chronic obstructive pulmonary disease (COPD), we performed PCR amplification and pyrosequencing of the 16S rRNA gene in patients not showing signs or symptoms of infection. Four types of respiratory samples (sputum, bronchial aspirate, bronchoalveolar lavage, and bronchial mucosa) were taken from each individual, obtaining on average >1,000 16S rRNA sequences per sample. The total number of genera per patient was >100, showing a high diversity, with Streptococcus, Prevotella, Moraxella, Haemophilus, Acinetobacter, Fusobacterium, and Neisseria being the most commonly identified. Sputum samples showed significantly lower diversity than the other three sample types. Lower-bronchial-tree samples, i.e., bronchoalveolar lavage and bronchial mucosa, showed a very similar bacterial compositions in contrast to sputum and bronchial aspirate samples. Thus, sputum and bronchial aspirate samples are upper bronchial tree samples that are not representative of the lower bronchial mucosa flora, and bronchoalveolar lavage samples showed the results closest to those for the bronchial mucosa. Our data confirm that the bronchial tree is not sterile in COPD patients and support the existence a different microbiota in the upper and lower compartments.

In the last decade, an extensive effort has been made to characterize the human intestinal microbiota by means of SSU rRNA gene sequence and metagenomic analysis. Relatively few studies have followed intestinal bacterial communities over time to assess their stability in the absence of perturbation. In this study, we have monitored the faecal bacteria of three healthy subjects during 15 consecutive days. The global community structure was analysed through SSU rRNA gene sequencing. In agreement with previous studies, we found that the between-subject variation in community structure was larger than within. The composition was fairly stable throughout, although daily fluctuations were detected for all genera and phylotypes at 97% of sequence identity. While the core shared between subjects was very small, each subject harboured a stable high-abundance core composed of a small number of bacterial groups (9% of the phylotypes accounted for between 74% and 93% of the sequences). This may suggest that studies aimed at linking the microbiota composition with disease risk should be limited to the numerically most dominant phylotypes, as the rest appears transient. Networks of potential interactions between co-occurring genera were also subject-specific, even for the same bacterial genus, which might be reflecting host-specific selective pressures and historic events., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

When a bacterial genome is compared to the metagenome of an environment it inhabits, most genes recruit at high sequence identity. In free-living bacteria (for instance marine bacteria compared against the ocean metagenome) certain genomic regions are totally absent in recruitment plots, representing therefore genes unique to individual bacterial isolates. We show that these Metagenomic Islands (MIs) are also visible in bacteria living in human hosts when their genomes are compared to sequences from the human microbiome, despite the compartmentalized structure of human-related environments such as the gut. From an applied point of view, MIs of human pathogens (e.g. those identified in enterohaemorragic Escherichia coli against the gut metagenome or in pathogenic Neisseria meningitidis against the oral metagenome) include virulence genes that appear to be absent in related strains or species present in the microbiome of healthy individuals. We propose that this strategy (i.e. recruitment analysis of pathogenic bacteria against the metagenome of healthy subjects) can be used to detect pathogenicity regions in species where the genes involved in virulence are poorly characterized. Using this approach, we detect well-known pathogenicity islands and identify new potential virulence genes in several human pathogens.

The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between 400 to 1000 bacterial species distributed among nine phyla with Firmicutes, Bacteroidetes and Actinobacteria representing around 75% of the diversity. One of the most intriguing issues relates to understanding which microbial groups are active players in the maintenance of the microbiota homeostasis.Here, we describe the diversity of active microbial fractions compared with the whole community from raw human fecal samples. We studied four healthy volunteers by 16S rDNA gene pyrosequencing. The fractions were obtained by cell sorting based on bacterial RNA concentration. Bacterial families were observed to appear or disappear on applying a cell sorting method in which flow cytometry was used to evaluate the active cells by pyronin-Y staining of RNA. This method was able to detect active bacteria, indicating that the active players differed from that observed in raw fecal material. Generally, observations showed that in the active fractions, the number of reads related to Bacteroidetes decreased whereas several families from Clostridiales (Firmicutes) were more highly represented. Moreover, a huge number of families appeared as part of the active fraction when cell sorting was applied, indicating reads that are simply statistically hidden by the total reads.

Background: Insects are associated with microorganisms that contribute to the digestion and processing of nutrients. The European Corn Borer (ECB) is a moth present world-wide, causing severe economical damage as a pest on corn and other crops. In the present work, we give a detailed view of the complexity of the microorganisms forming the ECB midgut microbiota with the objective of comparing the biodiversity of the midgut-associated microbiota and explore their potential as a source of genes and enzymes with biotechnological applications., Methodological/principal Findings: A high-throughput sequencing approach has been used to identify bacterial species, genes and metabolic pathways, particularly those involved in plant-matter degradation, in two different ECB populations (field-collected vs. lab-reared population with artificial diet). Analysis of the resulting sequences revealed the massive presence of Staphylococcus warneri and Weissella paramesenteroides in the lab-reared sample. This enabled us to reconstruct both genomes almost completely. Despite the apparently low diversity, 208 different genera were detected in the sample, although most of them at very low frequency. By contrast, the natural population exhibited an even higher taxonomic diversity along with a wider array of cellulolytic enzyme families. However, in spite of the differences in relative abundance of major taxonomic groups, not only did both metagenomes share a similar functional profile but also a similar distribution of non-redundant genes in different functional categories., Conclusions/significance: Our results reveal a highly diverse pool of bacterial species in both O. nubilalis populations, with major differences: The lab-reared sample is rich in gram-positive species (two of which have almost fully sequenced genomes) while the field sample harbors mainly gram-negative species and has a larger set of cellulolytic enzymes. We have found a clear relationship between the diet and the midgut microbiota, which reveals the selection pressure of food on the community of intestinal bacteria.

Endosymbiotic bacteria play a vital role in the evolution of many insect species. For instance, endosymbionts have evolved metabolically to complement their host's natural diet, thereby enabling them to explore new habitats. In this paper, we will review and give some examples of the nature of the metabolic coupling of different primary and secondary endosymbionts that have evolved in hosts with different nutritional diets (i.e., phloem, xylem, blood, omnivores, and grain). Particular emphasis is given to the evolutionary functional convergence of phylogenetically distant endosymbionts, which are evolving in hosts with similar diets., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Background: The increasing availability of gene sequences of prokaryotic species in samples extracted from all kind of locations allows addressing the study of the influence of environmental patterns in prokaryotic biodiversity. We present a comprehensive study to address the potential existence of environmental preferences of prokaryotic taxa and the commonness of the specialist and generalist strategies. We also assessed the most significant environmental factors shaping the environmental distribution of taxa., Results: We used 16S rDNA sequences from 3,502 sampling experiments in natural and artificial sources. These sequences were taxonomically assigned, and the corresponding samples were also classified into a hierarchical classification of environments. We used several statistical methods to analyze the environmental distribution of taxa. Our results indicate that environmental specificity is not very common at the higher taxonomic levels (phylum to family), but emerges at lower taxonomic levels (genus and species). The most selective environmental characteristics are those of animal tissues and thermal locations. Salinity is another very important factor for constraining prokaryotic diversity. On the other hand, soil and freshwater habitats are the less restrictive environments, harboring the largest number of prokaryotic taxa. All information on taxa, samples and environments is provided at the envDB online database, http://metagenomics.uv.es/envDB., Conclusions: This is, as far as we know, the most comprehensive assessment of the distribution and diversity of prokaryotic taxa and their associations with different environments. Our data indicate that we are still far from characterizing prokaryotic diversity in any environment, except, perhaps, for human tissues such as the oral cavity and the vagina.

Recent technical and conceptual advances in the biological sciences opened the possibility of the construction of newly designed cells. In this paper we review the state of the art of cell engineering in the context of genome research, paying particular attention to what we can learn on naturally reduced genomes from either symbiotic or free living bacteria. Different minimal hypothetically viable cells can be defined on the basis of several computational and experimental approaches. Projects aiming at simplifying living cells converge with efforts to make synthetic genomes for minimal cells. The panorama of this particular view of synthetic biology lead us to consider the use of defined minimal cells to be applied in biomedical, bioremediation, or bioenergy application by taking advantage of existing naturally minimized cells.

Our understanding of prokaryote-eukaryote symbioses as a source of evolutionary innovation has been rapidly increased by the advent of genomics, which has made possible the biological study of uncultivable endosymbionts. Genomics is allowing the dissection of the evolutionary process that starts with host invasion then progresses from facultative to obligate symbiosis and ends with replacement by, or coexistence with, new symbionts. Moreover, genomics has provided important clues on the mechanisms driving the genome-reduction process, the functions that are retained by the endosymbionts, the role of the host, and the factors that might determine whether the association will become parasitic or mutualistic.

Most endosymbiotic bacteria have extremely reduced genomes, accelerated evolutionary rates, and strong AT base compositional bias thought to reflect reduced efficacy of selection and increased mutational pressure. Here, we present a comparative study of evolutionary forces shaping five fully sequenced bacterial endosymbionts of insects. The results of this study were three-fold: (i) Stronger conservation of high expression genes at not just nonsynonymous, but also synonymous, sites. (ii) Variation in amino acid usage strongly correlates with GC content and expression level of genes. This pattern is largely explained by greater conservation of high expression genes, leading to their higher GC content. However, we also found indication of selection favoring GC-rich amino acids that contrasts with former studies. (iii) Although the specific nutritional requirements of the insect host are known to affect gene content of endosymbionts, we found no detectable influence on substitution rates, amino acid usage, or codon usage of bacterial genes involved in host nutrition.

Here we investigate how thermodynamic properties of orthologous proteins are influenced by the genomic environment in which they evolve. We performed a comparative computational study of 21 protein families in 73 prokaryotic species and obtained the following main results. (i) Protein stability with respect to the unfolded state and with respect to misfolding are anticorrelated. There appears to be a trade-off between these two properties, which cannot be optimized simultaneously. (ii) Folding thermodynamic parameters are strongly correlated with two genomic features, genome size and G+C composition. In particular, the normalized energy gap, an indicator of folding efficiency in statistical mechanical models of protein folding, is smaller in proteins of organisms with a small genome size and a compositional bias towards A+T. Such genomic features are characteristic for bacteria with an intracellular lifestyle. We interpret these correlations in light of mutation pressure and natural selection. A mutational bias toward A+T at the DNA level translates into a mutational bias toward more hydrophobic (and in general more interactive) proteins, a consequence of the structure of the genetic code. Increased hydrophobicity renders proteins more stable against unfolding but less stable against misfolding. Proteins with high hydrophobicity and low stability against misfolding occur in organisms with reduced genomes, like obligate intracellular bacteria. We argue that they are fixed because these organisms experience weaker purifying selection due to their small effective population sizes. This interpretation is supported by the observation of a high expression level of chaperones in these bacteria. Our results indicate that the mutational spectrum of a genome and the strength of selection significantly influence protein folding thermodynamics.

The development of molecular techniques for the study of uncultured bacteria allowed the extensive study of the widespread association between insects and intracellular symbiotic bacteria. Most of the bacterial endosymbionts involved in such associations are gamma-proteobacteria, closely related to Escherichia coli. In recent years, five genomes from insect endosymbionts have been sequenced, allowing the performance of extensive genome comparative analysis that, as a complement of phylogenetic studies, and analysis on individual genes, can help to understand the different traits of this particular association, including how the symbiotic process is established, the explanation of the special features of these microbial genomes, the bases of this intimate association and the possible future that awaits the endosymbionts with extremely reduced genomes.

The availability of a large number of complete genome sequences raises the question of how many genes are essential for cellular life. Trying to reconstruct the core of the protein-coding gene set for a hypothetical minimal bacterial cell, we have performed a computational comparative analysis of eight bacterial genomes. Six of the analyzed genomes are very small due to a dramatic genome size reduction process, while the other two, corresponding to free-living relatives, are larger. The available data from several systematic experimental approaches to define all the essential genes in some completely sequenced bacterial genomes were also considered, and a reconstruction of a minimal metabolic machinery necessary to sustain life was carried out. The proposed minimal genome contains 206 protein-coding genes with all the genetic information necessary for self-maintenance and reproduction in the presence of a full complement of essential nutrients and in the absence of environmental stress. The main features of such a minimal gene set, as well as the metabolic functions that must be present in the hypothetical minimal cell, are discussed.

We have analysed oral wash samples from 72 healthy young adults in Valencia (Spain) for a metagenomic analysis through the construction of shotgun libraries and high-throughput-sequencing. The oral viral communities have been taxonomically characterised as well as and the gene content from the latter. The majority of viruses are found in few individuals, with single occurrences being the most widespread ones, whereas universally distributed viruses, while present, are relatively rare, with bacteriophages from families Siphoviridae and Myoviridae, and Streptococcus phages, as well as the eukaryotic viral family Herpesviridae amongst the most widespread viruses. No significant differences were found between females and males for either viruses and bacteria in abundance and alpha and beta diversity. The virome show similarities with other oral viromes previously reported for healthy individuals, suggesting the existence of a universal core of oral viruses, at least in the Western society, regardless of the geographical location. [ABSTRACT FROM AUTHOR]

Dysbalance in gut microbiota has been linked to increased microbial translocation, leading to chronic inflammation in HIV-patients, even under effective HAART. Moreover, microbial translocation is associated with insufficient reconstitution of CD4+T cells, and contributes to the pathogenesis of immunologic non-response. In a double-blind, randomised, placebo-controlled trial, we recently showed that, compared to placebo, 12 weeks treatment with probiotic Saccharomyces boulardii significantly reduced plasma levels of bacterial translocation (Lipopolysaccharide-binding protein or LBP) and systemic inflammation (IL-6) in 44 HIV virologically suppressed patients, half of whom (n = 22) had immunologic non-response to antiretroviral therapy (<270 CD4+Tcells/μL despite long-term suppressed viral load). The aim of the present study was to investigate if this beneficial effect of the probiotic Saccharomyces boulardii is due to modified gut microbiome composition, with a decrease of some species associated with higher systemic levels of microbial translocation and inflammation. In this study, we used 16S rDNA gene amplification and parallel sequencing to analyze the probiotic impact on the composition of the gut microbiome (faecal samples) in these 44 patients randomized to receive oral supplementation with probiotic or placebo for 12 weeks. Compared to the placebo group, in individuals treated with probiotic we observed lower concentrations of some gut species, such as those of the Clostridiaceae family, which were correlated with systemic levels of bacterial translocation and inflammation markers. In a sub-study of these patients, we observed significantly higher parameters of microbial translocation (LBP, soluble CD14) and systemic inflammation in immunologic non-responders than in immunologic responders, which was correlated with a relative abundance of specific gut bacterial groups (Lachnospiraceae genus and Proteobacteria). Thus, in this work, we propose a new therapeutic strategy using the probiotic yeast S. boulardii to modify gut microbiome composition. Identifying pro-inflammatory species in the gut microbiome could also be a useful new marker of poor immune response and a new therapeutic target. [ABSTRACT FROM AUTHOR]

Background: In all branches of life there are plenty of symbiotic associations. Insects are particularly well suited to establishing intracellular symbiosis with bacteria, providing them with metabolic capabilities they lack. Essential primary endosymbionts can coexist with facultative secondary symbionts which can, eventually, establish metabolic complementation with the primary endosymbiont, becoming a co-primary. Usually, both endosymbionts maintain their cellular identity. An exception is the endosymbiosis found in mealybugs of the subfamily Pseudoccinae, such as Planococcus citri, with Moranella endobia located inside Tremblaya princeps. Results: We report the genome sequencing of M. endobia str. PCVAL and the comparative genomic analyses of the genomes of strains PCVAL and PCIT of both consortium partners. A comprehensive analysis of their functional capabilities and interactions reveals their functional coupling, with many cases of metabolic and informational complementation. Using comparative genomics, we confirm that both genomes have undergone a reductive evolution, although with some unusual genomic features as a consequence of coevolving in an exceptional compartmentalized organization. Conclusions: M. endobia seems to be responsible for the biosynthesis of most cellular components and energy provision, and controls most informational processes for the consortium, while T. princeps appears to be a mere factory for amino acid synthesis, and translating proteins, using the precursors provided by M. endobia. In this scenario, we propose that both entities should be considered part of a composite organism whose compartmentalized scheme (somehow) resembles a eukaryotic cell [ABSTRACT FROM AUTHOR]

Background: Cockroaches are terrestrial insects that strikingly eliminate waste nitrogen as ammonia instead of uric acid. Blattabacterium cuenoti (Mercier 1906) strains Bge and Pam are the obligate primary endosymbionts of the cockroaches Blattella germanica and Periplaneta americana, respectively. The genomes of both bacterial endosymbionts have recently been sequenced, making possible a genome-scale constraint-based reconstruction of their metabolic networks. The mathematical expression of a metabolic network and the subsequent quantitative studies of phenotypic features by Flux Balance Analysis (FBA) represent an efficient functional approach to these uncultivable bacteria. Results: We report the metabolic models of Blattabacterium strains Bge (iCG238) and Pam (iCG230), comprising 296 and 289 biochemical reactions, associated with 238 and 230 genes, and 364 and 358 metabolites, respectively. Both models reflect both the striking similarities and the singularities of these microorganisms. FBA was used to analyze the properties, potential and limits of the models, assuming some environmental constraints such as aerobic conditions and the net production of ammonia from these bacterial systems, as has been experimentally observed. In addition, in silico simulations with the iCG238 model have enabled a set of carbon and nitrogen sources to be defined, which would also support a viable phenotype in terms of biomass production in the strain Pam, which lacks the first three steps of the tricarboxylic acid cycle. FBA reveals a metabolic condition that renders these enzymatic steps dispensable, thus offering a possible evolutionary explanation for their elimination. We also confirm, by computational simulations, the fragility of the metabolic networks and their host dependence. Conclusions: The minimized Blattabacterium metabolic networks are surprisingly similar in strains Bge and Pam, after 140 million years of evolution of these endosymbionts in separate cockroach lineages. FBA performed on the reconstructed networks from the two bacteria helps to refine the functional analysis of the genomes enabling us to postulate how slightly different host metabolic contexts drove their parallel evolution. [ABSTRACT FROM AUTHOR]