The rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and ...error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments.
We obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.
The pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.
Secondary bacterial infections often complicate viral respiratory infections. We hypothesize that perturbation of the gut microbiota during influenza A virus (IAV) infection might favor respiratory ...bacterial superinfection. Sublethal infection with influenza transiently alters the composition and fermentative activity of the gut microbiota in mice. These changes are attributed in part to reduced food consumption. Fecal transfer experiments demonstrate that the IAV-conditioned microbiota compromises lung defenses against pneumococcal infection. In mechanistic terms, reduced production of the predominant short-chain fatty acid (SCFA) acetate affects the bactericidal activity of alveolar macrophages. Following treatment with acetate, mice colonized with the IAV-conditioned microbiota display reduced bacterial loads. In the context of influenza infection, acetate supplementation reduces, in a free fatty acid receptor 2 (FFAR2)-dependent manner, local and systemic bacterial loads. This translates into reduced lung pathology and improved survival rates of double-infected mice. Lastly, pharmacological activation of the SCFA receptor FFAR2 during influenza reduces bacterial superinfection.
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•Influenza alters the production of SCFAs by the gut microbiota•The dysbiotic microbiota transfers susceptibility to respiratory bacterial infection•Supplementation with acetate restores the killing activity of alveolar macrophages•Activation of the SCFA receptor FFAR2 protects against bacterial superinfection
Sencio et al. provide insights into the mechanisms that underlie bacterial superinfection post-influenza. The authors demonstrate that influenza infection remotely alters the production of short-chain fatty acids (SCFAs) by the gut microbiota. Supplementation with acetate or pharmacological activation of the SCFA receptor FFAR2 reduces susceptibility to secondary bacterial infection.
Surface-ripened cheeses host complex microbial communities responsible for the transformation of milk into cheese as well as the development of important properties in terms of texture, color and ...sensory perception. In this study, we used high-throughput amplicon sequencing to decipher the bacterial and fungal diversity of 60 cheeses belonging to 12 popular French cheese varieties. Using this approach, 76 bacterial and 44 fungal phylotypes were identified. Major differences were observed between rind and core samples and also according to cheese varieties and manufacturing processes. Occurrence analysis revealed the presence of widespread taxa as well as operational taxonomic units (OTUs) specific to one or several cheese varieties. Finally, we observed patterns specific to the cheese production facility, supporting the importance of indigenous microorganisms for the microbial assemblage of cheese microbiota.
•Deciphering the microbial diversity of 12 French cheese varieties.•Sample clustering according to cheese varieties and manufacturing processes.•Patterns specific to the cheese production facility.
The links between microbial environmental exposures and asthma are well documented, but no study has combined deep sequencing results from pulmonary and indoor microbiomes of patients with asthma ...with spirometry, clinical, and endotype parameters.
The goal of this study was to investigate the links between indoor microbial exposures and pulmonary microbial communities and to document the role of microbial exposures on inflammatory and clinical outcomes of patients with severe asthma (SA).
A total of 55 patients with SA from the national Cohort of Bronchial Obstruction and Asthma cohort were enrolled for analyzing their indoor microbial flora through the use of electrostatic dust collectors (EDCs). Among these patients, 22 were able to produce sputum during “stable” or pulmonary “exacerbation” periods and had complete pairs of EDC and sputum samples, both collected and analyzed. We used amplicon targeted metagenomics to compare microbial communities from EDC and sputum samples of patients according to type 2 (T2)-asthma endotypes.
Compared with patients with T2-low SA, patients with T2-high SA exhibited an increase in bacterial α-diversity and a decrease in fungal α-diversity of their indoor microbial florae, the latter being significantly correlated with fraction of exhaled nitric oxide levels. The β-diversity of the EDC mycobiome clustered significantly according to T2 endotypes. Moreover, the proportion of fungal taxa in common between the sputum and EDC samples was significantly higher when patients exhibited acute exacerbation.
These results illustrated, for the first time, a potential association between the indoor mycobiome and clinical features of patients with SA, which should renew interest in deciphering the interactions between indoor environment, fungi, and host in asthma.
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Chlorinated ethenes (CEs) are most problematic pollutants in groundwater. Dehalogenating bacteria, and in particular organohalide-respiring bacteria (OHRB), can transform PCE to ethene under ...anaerobic conditions, and thus contribute to bioremediation of contaminated sites. Current approaches to characterize in situ biodegradation of CEs include hydrochemical analyses, quantification of the abundance of key species (e.g. Dehalococcoides mccartyi) and dehalogenase genes (pceA, vcrA, bvcA and tceA) involved in different steps of organohalide respiration (OHR) by qPCR, and compound-specific isotope analysis (CSIA) of CEs. Here we combined these approaches with sequencing of 16S rRNA gene amplicons to consider both OHRB and bacterial taxa involved in CE transformation at a multi-contaminated site. Integrated analysis of hydrogeochemical characteristics, gene abundances and bacterial diversity shows that bacterial diversity and OHRB mainly correlated with hydrogeochemical conditions, suggesting that pollutant exposure acts as a central driver of bacterial diversity. CSIA, abundances of four reductive dehalogenase encoding genes and the prevalence of Dehalococcoides highlighted sustained PCE, DCE and VC degradation in several wells of the polluted plume. These results suggest that bacterial taxa associated with OHR play an essential role in natural attenuation of CEs, and that representatives of taxa including Dehalobacterium and Desulfosporosinus co-occur with Dehalococcoides. Overall, our study emphasizes the benefits of combining several approaches to evaluate the interplay between the dynamics of bacterial diversity in CE-polluted plumes and in situ degradation of CEs, and to contribute to a more robust assessment of natural attenuation at multi-polluted sites.
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•Pollutants drove bacterial diversity distribution at the multi-contaminated site.•Bacterial diversity and dehalogenase gene abundances did not change over time.•Compound specific isotope analysis evidenced biodegradation of chloroethenes.•Bacterial diversity reinforces natural attenuation assessment at polluted sites.•Combining biomarkers improves natural attenuation assessment of chloroethenes.
The current pandemic of coronavirus disease (COVID) 2019 constitutes a global public health issue. Regarding the emerging importance of the gut-lung axis in viral respiratory infections, analysis of ...the gut microbiota's composition and functional activity during a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection might be instrumental in understanding and controling COVID 19. We used a nonhuman primate model (the macaque), that recapitulates mild COVID-19 symptoms, to analyze the effects of a SARS-CoV-2 infection on dynamic changes of the gut microbiota. 16S rRNA gene profiling and analysis of β diversity indicated significant changes in the composition of the gut microbiota with a peak at 10-13 days post-infection (dpi). Analysis of bacterial abundance correlation networks confirmed disruption of the bacterial community at 10-13 dpi. Some alterations in microbiota persisted after the resolution of the infection until day 26. Some changes in the relative bacterial taxon abundance associated with infectious parameters. Interestingly, the relative abundance of Acinetobacter (Proteobacteria) and some genera of the Ruminococcaceae family (Firmicutes) was positively correlated with the presence of SARS-CoV-2 in the upper respiratory tract. Targeted quantitative metabolomics indicated a drop in short-chain fatty acids (SCFAs) and changes in several bile acids and tryptophan metabolites in infected animals. The relative abundance of several taxa known to be SCFA producers (mostly from the Ruminococcaceae family) was negatively correlated with systemic inflammatory markers while the opposite correlation was seen with several members of the genus Streptococcus. Collectively, SARS-CoV-2 infection in a nonhuman primate is associated with changes in the gut microbiota's composition and functional activity.
Microcosm experiments with CE-contaminated groundwater from a former industrial site were set-up to evaluate the relationships between biological CE dissipation, dehalogenase genes abundance and ...bacterial genera diversity. Impact of high concentrations of PCE on organohalide respiration was also evaluated. Complete or partial dechlorination of PCE, TCE,
-DCE and VC was observed independently of the addition of a reducing agent (Na
S) or an electron donor (acetate). The addition of either 10 or 100 μM PCE had no effect on organohalide respiration. qPCR analysis of reductive dehalogenases genes (
, and
) indicated that the version of
gene found in the genus
hereafter named
(Dhc) and
gene increased in abundance by one order of magnitude during the first 10 days of incubation. The version of the
gene found, among others, in the genus
, and
hereafter named
(Dhb) and
gene showed very low abundance. The
gene was not detected throughout the experiment. The proportion of
(Dhc) or
genes relative to the universal 16S ribosomal RNA (16S rRNA) gene increased by up to 6-fold upon completion of
DCE dissipation. Sequencing of 16S rRNA amplicons indicated that the abundance of Operational Taxonomic Units (OTUs) affiliated to dehalogenating genera
, and
represented more than 20% sequence abundance in the microcosms. Among organohalide respiration associated genera, only abundance of
spp. increased up to fourfold upon complete dissipation of PCE and
-DCE, suggesting a major implication of
in CEs organohalide respiration. The relative abundance of
and
genes correlated with the occurrence of
and with dissipation extent of PCE,
-DCE and CV. A new type of dehalogenating
sp. phylotype affiliated to the Pinellas group, and suggested to contain both
(Dhc) and
genes, may be involved in organohalide respiration of CEs in groundwater of the study site. Overall, the results demonstrate
dechlorination potential of CE in the plume, and suggest that taxonomic and functional biomarkers in laboratory microcosms of contaminated groundwater following pollutant exposure can help predict bioremediation potential at contaminated industrial sites.
Previous microscopy-based studies in the eastern English Channel have revealed it to be a productive meso-eutrophic coastal ecosystem, characterized by strong repeating patterns in microplankton ...succession. The present study examines the seasonal structure of the entire protistan community from March 2011 to July 2013, using tag pyrosequencing of the V2–V3 hypervariable region of the 18S rRNA gene. A total of 1242 OTUs and 28 high-level taxonomic groups, which included previously undetected taxa in the area, were identified. The detected OTUs were considered according to taxon-specific traits, which included their trophic role, abundance and specialization level. Taxa differentiation based on specialization level rather than abundance was more informative in describing community organization. While generalists were always abundant, numerous specialists that were either rare or absent in most samples, increased in abundance for short periods, appearing to be overall abundant. Statistical and network analyses showed that the protistan seasonal organization was influenced by environmental parameters. It also highlighted that in addition to grazers, fungi and parasites played potentially significant roles during phytoplankton blooms. Overall, while the protistan succession was mainly shaped by environmental variations, biotic interactions among co-occurring taxa were the main structural drivers of the temporal assemblages.
The seasonal structure of the protistan community was examined from March 2011 to July 2013 in a temperate meso-eutrophic coastal system, using tag pyrosequencing of the 18S rRNA gene.
Antibiotic disruption of the intestinal microbiota favors colonization by
Using a charcoal-based adsorbent to decrease intestinal antibiotic concentrations, we studied the relationship between ...antibiotic concentrations in feces and the intensity of dysbiosis and quantified the link between this intensity and mortality. We administered either moxifloxacin (
= 70) or clindamycin (
= 60) to hamsters by subcutaneous injection from day 1 (D
) to D
and challenged them with a
toxigenic strain at D
Hamsters received various doses of a charcoal-based adsorbent, DAV131A, to modulate intestinal antibiotic concentrations. Gut dysbiosis was evaluated at D
and D
using diversity indices determined from 16S rRNA gene profiling. Survival was monitored until D
We analyzed the relationship between fecal antibiotic concentrations and dysbiosis at the time of
challenge and studied their capacity to predict subsequent death of the animals. Increasing doses of DAV131A reduced fecal concentrations of both antibiotics, lowered dysbiosis, and increased survival from 0% to 100%. Mortality was related to the level of dysbiosis (
< 10
for the change of Shannon index in moxifloxacin-treated animals and
< 10
in clindamycin-treated animals). The Shannon diversity index and unweighted UniFrac distance best predicted death, with areas under the receiver operating curve (ROC) of 0.89 (95% confidence interval CI, 0.82, 0.95) and 0.95 (0.90, 0.98), respectively. Altogether, moxifloxacin and clindamycin disrupted the diversity of the intestinal microbiota with a dependency on the DAV131A dose; mortality after
challenge was related to the intensity of dysbiosis in similar manners with the two antibiotics.
Although it is known that zinc has several beneficial roles in the context of gut inflammation, the underlying mechanisms have not been extensively characterized. Zinc (Zn) is known to be the primary ...physiological inducer of the expression of the metallothionein (MT) superfamily of small stress‐responsive proteins. The expression of MTs in various tissues is induced or enhanced (including the gastrointestinal tract (GIT)) by a variety of stimuli, including infection and inflammation. However, the MTs’ exact role in inflammation is still subject to debate. In order to establish whether or not MTs are the sole vectors in the Zn‐based modulation of intestinal inflammation, we used transcriptomic and metagenomic approaches to assess the potential effect of dietary Zn, the mechanisms underlying the MTs’ beneficial effects, and the induction of previously unidentified mediators. We found that the expression of endogenous MTs in the mouse GIT was stimulated by an optimized dietary supplementation with Zn. The protective effects of dietary supplementation with Zn were then evaluated in mouse models of chemically induced colitis. The potential contribution of MTs and other pathways was explored via transcriptomic analyses of the ileum and colon in Zn‐treated mice. The microbiota’s role was also assessed via fecal 16S rRNA sequencing. We found that high‐dose dietary supplementation with Zn induced the expression of MT‐encoding genes in the colon of healthy mice. We next demonstrated that the Zn diet significantly protected mice in the two models of induced colitis. When comparing Zn‐treated and control mice, various genes were found to be differentially expressed in the colon and the ileum. Finally, we found that Zn supplementation did not modify the overall structure of the fecal microbiota, with the exception of (i) a significant increase in endogenous Clostridiaceae, and (ii) some subtle but specific changes at the family and genus levels. Our results emphasize the beneficial effects of excess dietary Zn on the prevention of colitis and inflammatory events in mouse models. The main underlying mechanisms were driven by the multifaceted roles of MTs and the other potential molecular mediators highlighted by our transcriptomic analyses although we cannot rule out contributions by other factors from the host and/or the microbiota.
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