16S rRNA High-throughput Sequencing Research Articles

Co-extraction of DNA and PLFA from soil samples

Lipid/DNA co-extraction from one sample is attractive in limiting biases associated with microbial community analysis from separate extractions. We sought to enhance established co-extraction methods and use high-throughput 16S rRNA sequencing to identify preferentially extracted taxa from co-extracted DNA. Co-extraction results in low DNA yields and distinct community structure changes.

Skin-to-Skin Care and the Development of the Preterm Infant Oral Microbiome.

The oral cavity represents an initial entry way for oral and gut indigenous colonization. Skin-to-skin (STS) care, in which the mother holds the diaper clad naked preterm (PT) infant between her breasts, is associated with improved digestive function, decreased stress, and improved survival. This study evaluated the development of oral microbial colonization repertoires and health characteristics in PT infants with or without STS exposure. Saliva from 42 PT infants (<32 weeks of gestation at birth) was collected prospectively at 1 month and/or at discharge. High-throughput 16S rRNA sequencing identified microbial diversity and prevalence of bacterial signatures correlated with clinical STS or non-STS care. Corrected for gestational age (CGA) at sampling, bacterial taxa demonstrated increased Streptococcus as a signature of oral repertoire maturation. STS was associated with increased Streptococcus (p < 0.024), while non-STS was associated with greater Corynebacterium (p < 0.023) and Pseudomonas (p < 0.019) in infants ≤ 32 weeks CGA. In infants > 32 weeks CGA, Neisseria and Acinetobacter were more prevalent, 50 vs. 16.7% and 40 vs. 0%, respectively. STS care was associated with shorter hospitalization (p < 0.039). STS care during earlier gestation was associated with a distinct microbial pattern and an accelerated pace of oral microbial repertoire maturity.

The unique chemistry of Eastern Mediterranean water masses selects for distinct microbial communities by depth.

The waters of the Eastern Mediterranean are characterized by unique physical and chemical properties within separate water masses occupying different depths. Distinct water masses are present throughout the oceans, which drive thermohaline circulation. These water masses may contain specific microbial assemblages. The goal of this study was to examine the effect of physical and geological phenomena on the microbial community of the Eastern Mediterranean water column. Chemical measurements were combined with phospholipid fatty acid (PLFA) analysis and high-throughput 16S rRNA sequencing to characterize the microbial community in the water column at five sites. We demonstrate that the chemistry and microbial community of the water column were stratified into three distinct water masses. The salinity and nutrient concentrations vary between these water masses. Nutrient concentrations increased with depth, and salinity was highest in the intermediate water mass. Our PLFA analysis indicated different lipid classes were abundant in each water mass, suggesting that distinct groups of microbes inhabit these water masses. 16S rRNA gene sequencing confirmed the presence of distinct microbial communities in each water mass. Taxa involved in autotrophic nitrogen cycling were enriched in the intermediate water mass suggesting that microbes in this water mass may be important to the nitrogen cycle of the Eastern Mediterranean. The Eastern Mediterranean also contains numerous active hydrocarbon seeps. We sampled above the North Alex Mud Volcano, in order to test the effect of these geological features on the microbial community in the adjacent water column. The community in the waters overlaying the mud volcano was distinct from other communities collected at similar depths and was enriched in known hydrocarbon degrading taxa. Our results demonstrate that physical phenomena such stratification as well as geological phenomena such as mud volcanoes strongly affect microbial community structure in the Eastern Mediterranean water column.

Predicting Microbial Interactions Using Vector Autoregressive Model with Graph Regularization.

Microbial interactions play important roles on the structure and function of complex microbial communities. With the rapid accumulation of high-throughput metagenomic or 16S rRNA sequencing data, it is possible to infer complex microbial interactions. Co-occurrence patterns of microbial species among multiple samples are often utilized to infer interactions. There are few methods to consider the temporally interacting patterns among microbial species. In this paper, we present a Graph-regularized Vector Autoregressive (GVAR) model to infer causal relationships among microbial entities. The new model has advantage comparing to the original vector autoregressive (VAR) model. Specifically, GVAR can incorporate similarity information for microbial interaction inference--i.e., GVAR assumed that if two species are similar in the previous stage, they tend to have similar influence on the other species in the next stage. We apply the model on a time series dataset of human gut microbiome which was treated with repeated antibiotics. The experimental results indicate that the new approach has better performance than several other VAR-based models and demonstrate its capability of extracting relevant microbial interactions.

CO2 sequestration by methanogens in activated sludge for methane production

Carbon dioxide (CO2) is the main greenhouse gas; hence, processes are needed to remove it from the environment. Here, CO2 was used as the substrate to generate methane (CH4) by using enriched methanogens after anaerobic enrichment of waste activated sludge (WAS); therefore, we demonstrate that methanogens from WAS have significant potential for converting the greenhouse gas CO2 into the fuel methane. Methane production was found to increase 70 fold by active methanogens in the enriched methanogens culture after 3days in the presence of H2 and CO2. Throughout the process, CO2 was completely consumed after 4days of incubation in the vials after sparging with a mixture of H2 and CO2, resulting in significant biological CO2 sequestration by methanogens. Using a mixture of H2 and 13CO2, we also demonstrated that the methane produced is due to the utilization of CO2. Microbial community studies via by quantitative real time PCR (qRT-PCR) indicate the dominance of archaea in the enriched methanogens culture of WAS. Archaeal community studies of the enriched methanogens via high-throughput 16S rRNA sequencing also showed that the archaea consist mainly of hydrogenotrophic and aceticlastic methanogens such as Methanobacteriaceae, Methanospirillaceae and Methanosarcinaceae spp. which are actively grown in H2 and CO2. We envision that CO2 gas from power plants can be directed to enriched methanogens of WAS to prevent release of this greenhouse gas while generating a useful biofuel (methane) or other valuable products using this single carbon atom.

Streptozotocin-induced type-1-diabetes disease onset in Sprague-Dawley rats is associated with an altered intestinal microbiota composition and decreased diversity.

There is a growing appreciation that microbiota composition can significantly affect host health and play a role in disease onset and progression. This study assessed the impact of streptozotocin (STZ)-induced type-1-diabetes (T1D) on intestinal microbiota composition and diversity in Sprague-Dawley rats, compared with healthy controls over time. T1D was induced by injection of a single dose (60 mg STZ kg(-1)) of STZ, administered via the intraperitoneal cavity. Total DNA was isolated from faecal pellets at weeks 0 (pre-STZ injection), 1, 2 and 4 and from caecal content at week 5 from both healthy and T1D groups. High-throughput 16S rRNA sequencing was employed to investigate intestinal microbiota composition. The data revealed that although intestinal microbiota composition between the groups was similar at week 0, a dramatic impact of T1D development on the microbiota was apparent post-STZ injection and for up to 5 weeks. Most notably, T1D onset was associated with a shift in the Bacteroidetes : Firmicutes ratio (P<0.05), while at the genus level, increased proportions of lactic acid producing bacteria such as Lactobacillus and Bifidobacterium were associated with the later stages of T1D progression (P<0.05). Coincidently, T1D increased caecal lactate levels (P<0.05). Microbial diversity was also reduced following T1D (P<0.05). Principle co-ordinate analyses demonstrated temporal clustering in T1D and control groups with distinct separation between groups. The results provide a comprehensive account of how T1D is associated with an altered intestinal microbiota composition and reduced microbial diversity over time.

Impact of dietary fatty acids on metabolic activity and host intestinal microbiota composition in C57BL/6J mice

Different dietary fat and energy subtypes have an impact on both the metabolic health and the intestinal microbiota population of the host. The present study assessed the impact of dietary fat quality, with a focus on dietary fatty acid compositions of varying saturation, on the metabolic health status and the intestinal microbiota composition of the host. C57BL/6J mice (n 9-10 mice per group) were fed high-fat (HF) diets containing either (1) palm oil, (2) olive oil, (3) safflower oil or (4) flaxseed/fish oil for 16 weeks and compared with mice fed low-fat (LF) diets supplemented with either high maize starch or high sucrose. Tissue fatty acid compositions were assessed by GLC, and the impact of the diet on host intestinal microbiota populations was investigated using high-throughput 16S rRNA sequencing. Compositional sequencing analysis revealed that dietary palm oil supplementation resulted in significantly lower populations of Bacteroidetes at the phylum level compared with dietary olive oil supplementation (P< 0·05). Dietary supplementation with olive oil was associated with an increase in the population of the family Bacteroidaceae compared with dietary supplementation of palm oil, flaxseed/fish oil and high sucrose (P< 0·05). Ingestion of the HF-flaxseed/fish oil diet for 16 weeks led to significantly increased tissue concentrations of EPA, docosapentaenoic acid and DHA compared with ingestion of all the other diets (P< 0·05); furthermore, the diet significantly increased the intestinal population of Bifidobacterium at the genus level compared with the LF-high-maize starch diet (P< 0·05). These data indicate that both the quantity and quality of fat have an impact on host physiology with further downstream alterations to the intestinal microbiota population, with a HF diet supplemented with flaxseed/fish oil positively shaping the host microbial ecosystem.

Molecular investigation of bacterial communities on intravascular catheters: no longer just Staphylococcus

Intravascular catheter-related bloodstream infections (IVC-BSIs) are associated with significant morbidity and mortality. Culture-independent molecular approaches can reveal and capture the composition of complex microbial communities, and are now being used to reveal "new" pathogens as well as the polymicrobial nature of some infections. Patients with concurrently sited arterial and central venous catheters who had clinically suspected IVC-BSIs, were examined by the high-throughput sequencing of microbial 16S rRNA. An average of 100 operational taxonomic units (OTUs, phylotypes) was observed on each IVC, indicating that IVCs were colonised by complex and diverse bacterial communities. Ralstonia (53 % of 16S rRNA sequences), Escherichia group (16 %), Propionibacterium (5 %), Staphylococcus (5 %), and Streptococcus (2 %) were the most abundant genera. There was no statistically significant difference in the bacterial communities examined from arterial and central venous catheters; from those with and without systemic antibiotic treatment; or from conventionally colonised and uncolonised IVCs. The genome of the predominant bacteria, R. pickettii AU12-08, was found to encode resistance to antimicrobial drugs of different classes. In addition, many encoded gene products are involved in quorum sensing and biofilm formation that would further contribute to increased antimicrobial drug resistance. Our results highlight the complex diversity of microbial ecosystems on vascular devices. High-throughput sequencing of 16S rRNA offers an insight into the pathogenesis of IVC-related infections, and opens up the scope for improving diagnosis and patient management.

Quantitation and Composition of Cutaneous Microbiota in Diabetic and Nondiabetic Men

Diabetic foot infections are a leading cause of lower extremity amputations. Our study examines the microbiota of diabetic skin prior to ulcer development or infection. In a case-control study, outpatient males were recruited at a veterans hospital. Subjects were swabbed at 4 cutaneous sites, 1 on the forearm and 3 on the foot. Quantitative polymerase chain reaction (qPCR) with primers and probes specific for bacteria, Staphylococcus species, Staphylococcus aureus, and fungi were performed on all samples. High-throughput 16S ribosomal RNA (rRNA) sequencing was performed on samples from the forearm and the plantar aspect of the foot. qPCR analysis of swab specimens from 30 diabetic subjects and 30 control subjects showed no differences in total numbers of bacteria or fungi at any sampled site. Increased log concentrations of Staphylococcus aureus, quantified by the number of nuc gene copies, were present in diabetic men on the plantar aspect of the foot. High-throughput 16S rRNA sequencing found that, on the foot, the microbiota in controls (n = 24) was dominated by Staphylococcus species, whereas the microbiota in diabetics (n = 23) was more diverse at the genus level. The forearm microbiota had similar diversity in diabetic and control groups. The feet of diabetic men had decreased populations of Staphylococcus species, increased populations of S. aureus, and increased bacterial diversity, compared with the feet of controls. These ecologic changes may affect the risk for wound infections.

The use of microbial and chemical analyses to characterize the variations in fouling profile of seawater reverse osmosis (SWRO) membrane

Biofouling of reverse osmosis (RO) membranes is one of the most common problems in desalinations plants reducing the efficiency of the water production process. The characterization of bacterial community composition from fouling layers as well as detailed analysis of surrounding chemical environment might reveal process specific bacterial groups/species that are involved in RO biofouling. In this study, advanced organics analytic methods (elemental analysis, FTIR, and ICP-OES) were combined with high-throughput 16S rRNA (pyro) sequencing to assess in parallel, the chemical properties and the active microbial community composition of SWRO membranes from a pilot desalination plant (MFT, Tarragona) in February 2011 and July 2011. Prefiltered ultrafiltration. waters fed SWRO membranes during third and fifth month of operation, respectively. SWRO samples were taken from three modules at different positions (first, fourth, and sixth) in order to investigate the spatial changes in fouling layers’ chemical and microbiological composition. The overall assessment of chemical parameters revealed that fouling layers were mainly composed by bio and organic material (proteins and lipids). Ca and Fe were found to be the most abundant elements having an increasing concentration gradient according to the module position. Bacterial community composition of SWRO membranes is mostly represented by the Gammaproteobacteria class with interesting differences in genera/species spatial and temporal distribution. This preliminary result suggests that pretreatments and/or operational conditions might have selected different bacterial groups more adapted to colonize SWRO membranes.

An eight-year investigation of bovine livestock fecal microbiota

Cattle represent a major source of fecal contamination worldwide. Understanding the natural variation of the bovine livestock fecal microbiota is therefore important. For this reason we addressed the yearly differences of the fecal microbiota for bovine livestock reared in the same geographical region from 1999 to 2007 – analyzing a total of 300 samples representing a range of experimental regimes. The aim of our work was to determine the effect of year visa experimental regime of the bovine livestock fecal microbiota. We used a newly developed high-throughput 16S rRNA sequencing approach (a single mixed Sanger sequence was generated per sample) in combination with deep pyrosequencing. We found that similar feeding and treatment regimes for different years showed major differences in the fecal microbiota, suggesting other factors important for shaping the fecal microbiota than those experimentally controlled. Ruminococcaeae, Peptostreptococcaceae, Acinetobacter, Escherichia/Shigella, Lachnospiraceae and Lactobacillales were the main taxa associated with the yearly fluctuations. Furthermore, we found that fecal samples with high levels of Lactobacillales, Ruminococcaea and Lachnospiraceae had the most even species distributions. The Peptostreptococcaceae and Acinetobacter dominated samples, on the other hand, showed a few highly dominant taxa. Testing of neutrality showed that the evenly distributed samples were explained by a neutral mode (that the assembly of the microbiota was random), while for the other samples there were overrepresentation of the dominant species (indicating bacterial-bacterial nice competition). We therefore propose that there are natural yearly fluctuations of the bovine livestock microbiota – both with respect to ecology and composition. This knowledge will have impact on our management of fecal bacteria in the environment, since it is very difficult to predict risk based on historical data.

Characterization of the Relative Abundance of the Citrus Pathogen Ca. Liberibacter Asiaticus in the Microbiome of Its Insect Vector, Diaphorina citri, using High Throughput 16S rRNA Sequencing

The relationship between the causal agent of Huanglongbing (HLB), Ca. Liberibacter asiaticus(Las), and the naturally occurring endosymbiotic community of its insect vector, the Asian citrus psyllid (ACP), Diaphorina citri, was studied. Variation was observed in the titer of Las within an ACP population feeding on the same material. The cause of this disparity is unknown, and has implications for Las transmission and the spread of HLB. This study utilizes culture independent methods to establish the relationship between the ACP’s microbial community and Las acquisition. DNA from 21 psyllids was amplified using universal 16S rRNA primers with Illumina adaptor regions and a sample-specific 7- base identifier. These amplicons were then batch-sequenced on the Illumina platform. The resulting sequences were separated by the identifier, and compared to known sequences in a 16S rRNA database. The microbial communities of each psyllid were compared to determine whether a correlation exists between the ACP’s endosymbionts and level of Las acquisition.ACPs were dominated by the same four bacterialgenera regardless of the abundance of Ca.Liberibacter. A combination of qPCR and Illumina sequencing was used to establish an infection gradient among the sampled ACPs. The Ca. Liberibacter titer within the insect was found to have a strong negative relationship with an endosymbiont residing in the syncytium of the mycetocyte and a positive relationship with Wolbachia. These correlations have implications in the acquisition of Las by the ACP as well as the activities of Las within this vector.

Fecal Microbiota in Premature Infants Prior to Necrotizing Enterocolitis

Intestinal luminal microbiota likely contribute to the etiology of necrotizing enterocolitis (NEC), a common disease in preterm infants. Microbiota development, a cascade of initial colonization events leading to the establishment of a diverse commensal microbiota, can now be studied in preterm infants using powerful molecular tools. Starting with the first stool and continuing until discharge, weekly stool specimens were collected prospectively from infants with gestational ages ≤32 completed weeks or birth weights≤1250 g. High throughput 16S rRNA sequencing was used to compare the diversity of microbiota and the prevalence of specific bacterial signatures in nine NEC infants and in nine matched controls. After removal of short and low quality reads we retained a total of 110,021 sequences. Microbiota composition differed in the matched samples collected 1 week but not <72 hours prior to NEC diagnosis. We detected a bloom (34% increase) of Proteobacteria and a decrease (32%) in Firmicutes in NEC cases between the 1 week and <72 hour samples. No significant change was identified in the controls. At both time points, molecular signatures were identified that were increased in NEC cases. One of the bacterial signatures detected more frequently in NEC cases (p<0.01) matched closest to γ-Proteobacteria. Although this sequence grouped to the well-studied Enterobacteriaceae family, it did not match any sequence in Genbank by more than 97%. Our observations suggest that abnormal patterns of microbiota and potentially a novel pathogen contribute to the etiology of NEC.