Next-generation Sequencing Of 16S rRNA Gene Research Articles

Taxonomic profiling of Nasutitermes takasagoensis microbiota to investigate the role of termites as vectors of bacteria linked to ironwood tree decline in Guam.

The ironwood tree (Casuarina equisetifolia, family Casuarinaceae), an indigenous agroforestry species in Guam, has been threatened by ironwood tree decline (IWTD) since 2002. Formation of bacterial ooze by the wilt pathogen from the Ralstonia solanacearum species complex and wetwood bacteria (primarily Klebsiella species) has been linked to IWTD. In addition, termite infestation of trees was statistically associated with IWTD. Termites are known carriers of a diverse microbiome. Therefore, we hypothesized that termites could be vectors of bacteria linked to IWTD. To investigate the potential role of termites as pathogen vectors, we employed next-generation 16S rRNA gene sequencing to describe the bacteria diversity of Nasutitermes takasagoensis (Family Termitidae) workers collected from 42 ironwood trees of different disease stages in Guam in association with tree-, plot-, and location-related factors. Nasutitermes takasagoensis workers account for the majority of termite infestations of ironwood trees. The bacterial phyla composition of N. takasagoensis workers was typical for wood-feeding higher termites consisting mainly of Spirochaetes and Fibrobacteres. However, Ralstonia species were not detected and Klebsiella species were rare even in termites collected from trees infected with Ralstonia and wetwood bacteria. Feeding experiments suggested that termites prefer to consume wood with low pathogen content over wood with high pathogen load. Termites were able to ingest Ralstonia but Ralstonia could not establish itself in healthy termite bodies. We concluded that N. takasagoensis workers are not vectors for Ralstonia spp. or the bacterial endophytes associated with wetwood (Klebsiella, Pantoea, Enterobacter, Citrobacter, and Erwinia) that were previously observed in IWTD-infested trees. The bacterial diversity in termite samples was significantly influenced by various factors, including Tree Health, Site Management, Plot Average Decline Severity, Proportion of Dead Trees in the Plot, Proportion of Trees with Termite Damage in the Plot, Presence of Ralstonia, and Altitude.

Gallbladder microbiota in healthy dogs and dogs with mucocele formation.

To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass.

Effects of Alcohol Binge Drinking and Oleoylethanolamide Pretreatment in the Gut Microbiota.

IntroductionChronic alcohol consumption is known to cause gut dysbiosis (changes in microbiota composition and/or function, disruptive of the normal host–microbiota interactions). However, little is known about the changes that alcohol binge drinking induces in the gut microbiota. Here, we have tested the hypothesis that a protocol of alcohol binge drinking, known to induce neuroinflammation in previous studies, also promotes intestinal dysbiosis, and we explored how oleoylethanolamide (OEA, an acylethanolamide proven to counteract alcohol binge drinking-induced neuroinflammation) pretreatment modulates alcohol-induced dysbiosis.MethodsAlcohol binges were forced by gavage three times per day during 4 consecutive days; OEA pretreatment (intraperitoneal or intragastric) was administered before each alcohol gavage. Stool microbiota composition was assessed by next-generation 16S rRNA gene sequencing, prior and after the 4-day alcohol binge protocol.ResultsAlcohol binge drinking reduced the richness of the gut microbiota and changed the microbial community, reducing Lactobacillus among other genera. Pretreatment with OEA in the alcohol-administered rats decreased the richness, evenness, and Shannon indices to a greater extent with respect to alcohol alone, also changing the community structure. Microbial interactions in the association network were further decreased following OEA administration in the alcohol group, with respect to the water administration. The synergistic interaction between alcohol binge and OEA was affected by the route of administration of OEA, since oral and i.p. administrations differently changed the community structure.ConclusionResults suggest that alcohol binge drinking produces a clear dysbiosis in animals; we observed that the well-known protective actions of OEA in the context of alcohol abuse might not be related to OEA-induced changes in alcohol-induced dysbiosis. These are observational results, and thus, further research will be needed for a complete understanding of the biological significance of the observed changes.

Endometrial microbiota is more diverse in people with endometriosis than symptomatic controls

Endometriosis is a chronic, estrogen-dependent gynecological condition affecting approximately 10% of reproductive age women. The most widely accepted theory of its etiology includes retrograde menstruation. Recent reports suggest the uterus is not sterile. Thus, the refluxed menstrual effluent may carry bacteria, and contribute to inflammation, the establishment and growth of endometriotic lesions. Here, we compared and contrasted uterine bacteria (endometrial microbiota) in people with surgically confirmed presence (N = 12) or absence of endometriosis (N = 9) using next-generation 16S rRNA gene sequencing. We obtained an average of > 9000 sequence reads per endometrial biopsy, and found the endometrial microbiota of people with endometriosis was more diverse (greater Shannon Diversity Index and proportion of ‘Other’ taxa) than symptomatic controls (with pelvic pain, surgically confirmed absence of endometriosis; diagnosed with other benign gynecological conditions). The relative abundance of bacterial taxa enriched in the endometrial microbiota of people with endometriosis belonged to the Actinobacteria phylum (Gram-positive), Oxalobacteraceae (Gram-negative) and Streptococcaceae (Gram-positive) families, and Tepidimonas (Gram-negative) genus, while those enriched in the symptomatic controls belonged to the Burkholderiaceae (Gram-negative) family, and Ralstonia (Gram-negative) genus. Taken together, results suggest the endometrial microbiota is perturbed in people with endometriosis.

Designing specific bacterial 16S primers to sequence and quantitate plant endo-bacteriome.

Plant endophytic bacteria colonize the internal tissues of plants and interact with plants closely. The past two decades have witnessed the increasing application of next-generation 16S rRNA gene sequencing in the investigation of bacterial communities. However, deciphering plant endo-bacterial communities by this method is difficult because of the co-amplification of massive plant organellar DNAs with bacterial 16S. Here, we designed polymerase chain reaction (PCR) primer sets, including 799F/1107R, 322F/796R, and 322F-Dr/796Rs (primer pair 322F/796R with a penultimate-base substitution in 322F), that can specifically amplify bacterial 16S from plant total DNAs. We computationally and experimentally evaluated the specificity, coverage, and accuracy of the newly designed primer sets. Both 799F/1107R and 322F-Dr/796Rs produced plant DNA-free 16S amplicon libraries or reduced plant DNA contamination to lower than 5% for the plant materials with extremely-low-abundance bacterial communities. The primer set 322F-A/796R was used through absolute quantitative PCR to quantitate the population size of rice leaf or root endo-bacteriome, which revealed 106-107 and 109-1010 bacteria per gram fresh weight, respectively. These 16S primer sets and amplification methods enable the simple and inexpensive next-generation sequencing and quantification of plant endo-bacteriome, which will significantly advance studies on the plant-related microbiome.

Environment and Diet Influence the Bacterial Microbiome of Ambigolimax valentianus, an Invasive Slug in California.

Simple SummarySlugs are significant pests, physically damaging plants from their voracious appetite as well as dispersing bacteria which could be harmful to plants and humans. They produce substantial economic costs, both as a direct result of plant destruction, and indirectly through attempts of pest control. This study explored the ecological aspects of the bacterial microbiome of Ambigolimax valentianus, a slug invasive to California. We identified a core microbiome in A. valentianus and found that their bacterial microbiome can be influenced and may depend substantially on both diet and environment. We also found that A. valentianus slugs harbor ecologically important bacteria, therefore their dispersal could have environmental and agricultural implications for both crop health and plant science. Future studies that provide a better understanding of the slug bacterial microbiome may be an important step in the development of comprehensive slug management. Ambigolimax valentianus is an invasive European terrestrial gastropod distributed throughout California. It is a serious pest of gardens, plant nurseries, and greenhouses. We evaluated the bacterial microbiome of whole slugs to capture a more detailed picture of bacterial diversity and composition in this host. We concentrated on the influences of diet and environment on the Ambigolimax valentianus core bacterial microbiome as a starting point for obtaining valuable information to aid in future slug microbiome studies. Ambigolimax valentianus were collected from two environments (gardens or reared from eggs in a laboratory). DNA from whole slugs were extracted and next-generation 16S rRNA gene sequencing was performed. Slug microbiomes differed between environmental sources (garden- vs. lab-reared) and were influenced by a sterile diet. Lab-reared slugs fed an unsterile diet harbored greater bacterial species than garden-reared slugs. A small core microbiome was present that was shared across all slug treatments. This is consistent with our hypothesis that a core microbiome is present and will not change due to these treatments. Findings from this study will help elucidate the impacts of slug-assisted bacterial dispersal on soils and plants, while providing valuable information about the slug microbiome for potential integrated pest research applications.

Factors Related to Outcomes of Fecal Microbiota Transplantation in Patients with Clostridioides difficile Infection

Background/AimsThe aim of this study was to evaluate factors related to outcomes of fecal microbiota transplantation (FMT) in patients with Clostridioides difficile infection (CDI) and viability of frozen stock for FMT.MethodsClinical data of patients who had received FMT for CDI were prospectively collected. Next-generation 16S rRNA gene sequencing of bacteria was performed from donors’ and recipients’ stool. Colony-forming units (CFUs) of cultures from frozen stock solutions for FMT were measured at 0, 4, 8, 12, 24, 48 weeks after preparation of the solutions.ResultsIn total, 25 FMT procedures were performed in 20 cases (14 fresh and 11 frozen FMT). Forty-five percent of cases involved fulminant CDI. The overall success rate was 55% after the 1st FMT and 75% after the 2nd FMT. The success rate was significantly higher in partially treated CDI than in refractory CDI (100% vs 71.4%; p=0.001). In successful cases only, the decrease in alpha-diversity in the recipient stool microbiomes was recovered after FMT to a level similar to that in donor stools. There was a significant difference in the microbiome composition in pre-FMT recipients’ stool between successful and failed cases (p=0.001). The CFUs of frozen solution for FMT did not decrease for 48 weeks in both aerobic and anaerobic cultures.ConclusionsFMT is highly effective in partially treated CDI but not in refractory CDI. The microbiome differs between failed and successful cases. Frozen stock for FMT is viable up to 48 weeks.

Controlled ovarian stimulation and progesterone supplementation affect vaginal and endometrial microbiota in IVF cycles: a pilot study

PurposeDoes controlled ovarian stimulation (COS) and progesterone (P) luteal supplementation modify the vaginal and endometrial microbiota of women undergoing in vitro fertilization?MethodsFifteen women underwent microbiota analysis at two time points: during a mock transfer performed in the luteal phase of the cycle preceding COS, and at the time of fresh embryo transfer (ET). A vaginal swab and the distal extremity of the ET catheter tip were analyzed using next-generation 16SrRNA gene sequencing. Heterogeneity of the bacterial microbiota was assessed according to both the Bray-Curtis similarity index and the Shannon diversity index.ResultsLactobacillus was the most prevalent genus in the vaginal samples, although its relative proportion was reduced by COS plus P supplementation (71.5 ± 40.6% vs. 61.1 ± 44.2%). In the vagina, an increase in pathogenic species was observed, involving Prevotella (3.5 ± 8.9% vs. 12.0 ± 19.4%), and Escherichia coli-Shigella spp. (1.4 ± 5.6% vs. 2.0 ± 7.8%). In the endometrium, the proportion of Lactobacilli slightly decreased (27.4 ± 34.5% vs. 25.0 ± 29.9%); differently, both Prevotella and Atopobium increased (3.4 ± 9.5% vs. 4.7 ± 7.4% and 0.7 ± 1.5% vs. 5.8 ± 12.0%). In both sites, biodiversity was greater after COS (p < 0.05), particularly in the endometrial microbiota, as confirmed by Bray-Curtis analysis of the phylogenetic distance among bacteria genera. Bray-Curtis analysis confirmed significant differences also for the paired endometrium-vagina samples at each time point.ConclusionsOur findings suggest that COS and P supplementation significantly change the composition of vaginal and endometrial microbiota. The greater instability could affect both endometrial receptivity and placentation. If our findings are confirmed, they may provide a further reason to encourage the freeze-all strategy.

Comprehensive evaluation of complex polymicrobial specimens using next generation sequencing and standard microbiological culture

Optimal clinical decision-making depends on identification of clinically relevant organisms present in a sample. Standard microbiological culture may fail to identify unusual or fastidious organisms and can misrepresent relative abundance of sample constituents. Culture-independent methods have improved our ability to deconvolute polymicrobial patient samples. We used next-generation 16S rRNA gene sequencing (NGS16S) to determine how often cultivatable organisms in complex polymicrobial samples are not reported by standard culture. Twenty consecutive bronchoalveolar lavage (BAL) samples were plated to standard and additional media; bacteria were identified by NGS16S analysis of DNA extracted directly from samples or from washed culture plates. 96% of organisms identified were cultivable, but only 21% were reported by standard culture, indicating that standard work-up provides an incomplete assessment of microbial constituents. Direct NGS16S correlated well with standard culture, identifying the same predominant organism in 50% of samples. When predominant organisms differed, NGS16S most often detected anaerobes, whose growth is unsupported by standard culture conditions for this specimen. NGS16S identified more organisms per sample and allowed identification of fastidious organisms, while culture was better at capturing organisms when bacterial load was low, and allowed incidental recovery of non-bacterial pathogens. Molecular and culture-based methods together detect more organisms than either method alone.

The Positive Effects of Grifola frondosa Heteropolysaccharide on NAFLD and Regulation of the Gut Microbiota.

Non-alcoholic fatty liver disease (NAFLD) is a major public health problem in many countries. In this study, the ability of Grifola frondosa heteropolysaccharide (GFP) to ameliorate NAFLD was investigated in rats fed a high-fat diet (HFD). The molecular mechanisms modulating the expression of specific gene members related to lipid synthesis and conversion, cholesterol metabolism, and inflammation pathways were determined. The components of the intestinal microflora in rats were analyzed by high-throughput next-generation 16S rRNA gene sequencing. Supplementation with GFP significantly increased the proportions of Allobaculum, Bacteroides, and Bifidobacterium and decreased the proportions of Acetatifactor, Alistipes, Flavonifractor, Paraprevotella, and Oscillibacter. In addition, Alistipes, Flavonifractor, and Oscillibacter were shown to be significant cecal microbiota according to the Spearman’s correlation test between the gut microbiota and biomedical assays (|r| > 0.7). Histological analysis and biomedical assays showed that GFP treatments could significantly protect against NAFLD. In addition, Alistipes, Flavonifractor, and Oscillibacter may play vital roles in the prevention of NAFLD. These results suggest that GFP could be used as a functional material to regulate the gut microbiota of NAFLD individuals.

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome's role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Effect of Chlorella Pyrenoidosa Protein Hydrolysate-Calcium Chelate on Calcium Absorption Metabolism and Gut Microbiota Composition in Low-Calcium Diet-Fed Rats.

In our current investigation, we evaluated the effect of Chlorella pyrenoidosa protein hydrolysate (CPPH) and Chlorella pyrenoidosa protein hydrolysate-calcium chelate (CPPH-Ca) on calcium absorption and gut microbiota composition, as well as their in vivo regulatory mechanism in SD rats fed low-calcium diets. Potent major compounds in CPPH were characterized by HPLC-MS/MS, and the calcium-binding mechanism was investigated through ultraviolet and infrared spectroscopy. Using high-throughput next-generation 16S rRNA gene sequencing, we analyzed the composition of gut microbiota in rats. Our study showed that HCPPH-Ca increased the levels of body weight gain, serum Ca, bone activity, bone mineral density (BMD) and bone mineral content (BMC), while decreased serum alkaline phosphatase (ALP) and inhibited the morphological changes of bone. HCPPH-Ca up-regulated the gene expressions of transient receptor potential cation V5 (TRPV5), TRPV6, calcium-binding protein-D9k (CaBP-D9k) and a calcium pump (plasma membrane Ca-ATPase, PMCA1b). It also improved the abundances of Firmicutes and Lactobacillus. Bifidobacterium and Sutterella were both positively correlated with calcium absorption. Collectively, these findings illustrate the potential of HCPPH-Ca as an effective calcium supplement.

Faecal microbiota from patients with cirrhosis has a low capacity to ferment non-digestible carbohydrates into short-chain fatty acids.

Cirrhosis is associated with dysbiosis, but its functional consequences are still largely unknown. Short-chain fatty acids (SCFAs) account for physiological interactions between the gut microbiota and host. Our aim was to assess the impact of cirrhotic dysbiosis on the production of SCFAs. Seventeen patients with cirrhosis and 17 controls were selected. Microbiota composition in faecal samples was assessed by next-generation 16S rRNA gene sequencing. SCFAs were measured with GC-MS in faecal samples and after in vitro batch fermentations using arabinoxylan, resistant starch, pectin, and lactulose as substrates. Among the 17 cirrhotic patients (mean age 58, eight males), six, nine and two were, respectively, Child-Pugh class A, B and C. Eleven patients were on oral antibiotics, 11 on lactulose and 13 on proton pump inhibitors. Cirrhotic patients showed marked differences in the composition and diversity of gut microbiome when compared to controls, that were more pronounced with increased severity. Stool samples from cirrhotic patients showed lower SCFAs content and reduced capacity to produce SCFAs in batch fermentations, with butyrate production being the most abnormal. These functional aberrancies were more pronounced with greater liver disease severity. Abundance of Ruminococcus faecis (in family Ruminococcaceae), Faecalicatena fissicatena and Fusicatenibacter saccharivorans (in family Lachnospiraceae) was positively correlated with the SCFAs production. Cirrhotic dysbiosis is associated with a decreased capacity to ferment non-digestible carbohydrates into SCFAs, especially into butyrate. These functional abnormalities are more pronounced as disease progresses. These results might inform the design of gut-targeted therapies for cirrhosis.

Effect of Marine Microalga Chlorella pyrenoidosa Ethanol Extract on Lipid Metabolism and Gut Microbiota Composition in High-Fat Diet-Fed Rats.

Effects of marine microalga Chlorella pyrenoidosa 55% ethanol extract (CPE55) on lipid metabolism, gut microbiota and regulation mechanism in high fat diet-fed induced hyperlipidaemia rats were investigated. Structure characterizations of major compounds in CPE55 were determined by ultra-performance liquid chromatography-quadrupole/time of flight mass spectrometry (UPLC-Q-TOF-MS/MS). The compositions of gut microbiota in rats were analyzed by high-throughput next-generation 16S rRNA gene sequencing. Oral administration with CPE55 markedly alleviated dyslipidemia through improving adverse blood lipid profile and inhibiting hepatic lipid accumulation and steatosis. CPE55 has downregulated the gene expression levels of acetyl CoA carboxylase, sterol regulatory element-binding transcription factor-1c, and 3-hydroxy-3-methyl glutaryl coenzyme A reductase and upregulated adenosine 5′-monophosphate-activated protein kinase-α. It has also improved the abundance of bacteria Alistipes, Prevotella, Alloprevotella, and Ruminococcus1 and decreased the abundances of Turicibacter and Lachnospira. Turicibacter and Lachnospira were both positive correlations of metabolic phenotypes. The findings above illustrated that CPE55 might be developed as food ingredients to ameliorate lipid metabolic disorders and hyperlipidaemia.

Regulatory Efficacy of Brown Seaweed Lessonia nigrescens Extract on the Gene Expression Profile and Intestinal Microflora in Type 2 Diabetic Mice.

In this study, the antidiabetic activity of Lessonia nigrescens ethanolic extract (LNE) is investigated in streptozotocin (SZT)-induced type 2 diabetic mice fed with a high-sucrose/high-fat diet. Ultra high performance liquid chromatography coupled with photo-DAD and electospray ionization-mass spectrometry (ESI-MS) is employed to analyze the major compounds in LNE. The components of the intestinal microflora in type 2 diabetic mice are analyzed by high-throughput next-generation 16S rRNA gene sequencing. Fasting blood glucose levels in diabetic mice are significantly decreased after LNE administration. The histology reveals that LNE could protect the cellular architecture of liver and kidney. LNE treatment significantly increases Bacteroidetes and decreases Firmicutes populations in intestinal microflora. Specifically, It could selectively enrich the amounts of beneficial bacteria, Barnesiella, as well as reduce the abundances of Clostridium and Alistipes. The increased gene and protein expression levels of phosphatidylinositol 3-kinase (PI3K) in the liver are observed in LNE treatment groups, while the expressions of c-Jun N-terminal kinase (JNK) are significantly downregulated. The above findings suggest that LNE could be considered as a functional food for reducing blood glucose and regulating intestinal microflora.

231 - Antidiabetic Activity of Lessonia nigrescens Extract in Type 2 Diabetic Mice

Lessonia nigrescens inhabits rocky intertidal and shallow subtidal zone, being a crucial brown algae to ecology and economy. L. nigrescens contains a broad range of bioactive compounds, such as fucans and phlorotannins. In this study, the antidiabetic activity of Lessonia nigrescens ethanolic extract was investigated in streptozotocin (SZT)-induced type 2 diabetic mice fed with a high-sucrose/high-fat diet. The components of the intestinal microflora in diabetic mice were analyzed by high-throughput next-generation 16S rRNA gene sequencing. Fasting blood glucose levels in type 2 diabetic mice were significantly decreased after administration of the extract. The histology revealed that the extract could protect the cellular architecture of liver and kidney. The extract significantly increased Bacteroidetes bacteria and decreased Firmicutes populations in intestinal microflora. Specifically, the extract can increase Barnesiella and reduce Clostridium and Alistipes. The increased gene and protein expression levels of PI3K in liver were observed in the extract groups, while the expressions of JNK1 were significantly downregulated by the extract.

Corexit 9500 Enhances Oil Biodegradation and Changes Active Bacterial Community Structure of Oil-Enriched Microcosms.

To better understand the impacts of Corexit 9500 on the structure and activity levels of hydrocarbon-degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at 5 and 25°C using both DNA and RNA extracts as the sequencing templates. Oil biodegradation patterns in both 5 and 25°C enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). Slight increases in biodegradation were observed in the presence of Corexit at both temperatures. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25°C consortia were dominated by Vibrio, Idiomarina, Marinobacter, Alcanivorax, and Thalassospira species, while the 5°C consortia were dominated by several species of the genera Flavobacterium, Alcanivorax, and Oleispira Most of these genera have been linked to hydrocarbon degradation and have been observed after oil spills. Colwellia and Cycloclasticus, known aromatic degraders, were also found in these enrichments. The addition of Corexit did not have an effect on the active bacterial community structure of the 5°C consortia, while at 25°C, a decrease in the relative abundance of Marinobacter was observed. At 25°C, Thalassospira, Marinobacter, and Idiomarina were present at higher relative abundances in the RNA than DNA libraries, suggesting that they were active in degradation. Similarly, Oleispira was greatly stimulated by the addition of oil at 5°C.IMPORTANCE While dispersants such as Corexit 9500 can be used to treat oil spills, there is still debate on the effectiveness on enhancing oil biodegradation and its potential toxic effect on oil-degrading microbial communities. The results of this study provide some insights on the microbial dynamics of hydrocarbon-degrading bacterial populations in the presence of Corexit 9500. Operational taxonomic unit (OTU) analyses indicated that several OTUs were inhibited by the addition of Corexit. Conversely, a number of OTUs were stimulated by the addition of the dispersant, many of which were identified as known hydrocarbon-degrading bacteria. The results highlight the value of using RNA-based methods to further understand the impact of dispersant on the overall activity of different hydrocarbon-degrading bacterial groups.

Effect of humic acid on anaerobic digestion of cellulose and xylan in completely stirred tank reactors: inhibitory effect, mitigation of the inhibition and the dynamics of the microbial communities.

Inhibition effect of humic acid (HA) on anaerobic digestion of cellulose and xylan and the mitigation potential of the inhibition were evaluated in controlled fed batch reactors at 30 °C and a hydraulic retention time (HRT) of 20 days. Reactor performances were evaluated by biogas production and metabolite measurements for 220 days. Microbial population dynamics of the reactors were monitored with next-generation 16S rRNA gene sequencing at nine different sampling times. Our results showed that increasing levels of HA inhibited the hydrolysis efficiency of the digestion by 40% and concomitantly reduced the methane yield. Addition of hydrolytic enzymes helped to reverse the negative effects of HA, whereas calcium addition did not reverse HA inhibition. Microbiological analyses showed that the relative abundance of hydrolytic/fermentative bacterial groups such as Clostridiales, Bacteroidales and Anaerolineales was significantly lowered by the presence of HA. HA also affected the archaeal populations. Mostly hydrogenotrophic methanogens were negatively affected by HA. The relative abundance of Methanobacteriaceae, Methanomicrobiales-WCHA208 and Unassigned Thermoplasmata WCHA1-57 were negatively affected by the presence of HA, whereas Methanosaetacea was not affected.

Emerging Perspectives on the Natural Microbiome of Fresh Produce Vegetables

Plants harbor a diverse microbiome existing as bacterial populations on the leaf surface (the phyllosphere) and within plant tissues (endophytes). The composition of this microbiome has been largely unexplored in fresh produce vegetables, where studies have tended to focus on pathogen detection and survival. However, the application of next-generation 16S rRNA gene sequencing approaches is beginning to reveal the diversity of this produce-associated bacterial community. In this article we review what is known about the composition of the microbiome of fresh produce vegetables, placing it in the context of general phyllosphere research. We also demonstrate how next-generation sequencing can be used to assess the bacterial assemblages present on fresh produce, using fresh herbs as an example. That data shows how the use of such culture-independent approaches can detect groups of taxa (anaerobes, psychrophiles) that may be missed by traditional culture-based techniques. Other issues discussed include questions as to whether to determine the microbiome during plant growth or at point of purchase or consumption, and the potential role of the natural bacterial community in mitigating pathogen survival.