PowerSoil Kit Research Articles

Optimized protocol proposal to extract eDNA from oligotrophic and degraded water samples

Aquifer represents an oligotrophic environment that sustains a relatively small amount of microbial cells, mostly non-culturable. Due to this dominance of unculturable microorganisms in natural ecosystems, studying microbial communities and their functionality should include culture-independent approaches based on molecular techniques using DNA analysis (Purswani et al. 2011). For practical reasons, aquifer routine analyses focus on groundwater samples, while solid aquifer samples are typically not included (Ritalahti et al. 2010). The amount of groundwater collected, together with the types and concentrations of inhibitory compounds if present, determine the abundance of the target biomarker(s) available for subsequent analyses. Hence, filtering large volumes of groundwater seems beneficial, but for practical purposes, 0.5–2 L of water are typically collected, depending on groundwater characteristics (Ritalahti et al. 2010). In this work, environmental DNA (eDNA) was extracted from groundwater samples, filtering three different initial volumes (1000 ml, 500 ml, and 250 ml) of water samples, using 0.22 µm membranes. Also, two DNA extraction commercial kits were tested, DNeasy PowerWater Kit and DNeasy PowerSoil kit (Qiagen, Germantown, MD) specific for water samples and solid matrix, respectively. For DNeasy PowerWater Kit the standard protocol was carried out, whereas a modified protocol equipped with the Inhibitor Removal Technology® (IRT) was selected for testing DNeasy PowerSoil kit. In order to minimize operator bias, both the protocols were made semi-automated by using a QIAcube provided by Qiagen for genomic DNA extraction. Additional steps to the PowerSoil IRT protocol were performed for optimizing chemical and mechanical cell lysis processes and facilitating the sample dispersion into the buffer solutions. eDNA was verified via electrophoresis and quantified fluorometrically. eDNA extracted from 250 ml of groundwater sample using the DNeasy PowerSoil kit with modified IRT protocol was also tested in downstream applications, including Polymerase Chain Reactions (PCRs) with specific primer pairs for the identification of microbial targets. Results suggest that the PowerSoil modified IRT protocol was the best performing one, allowing a higher eDNA yield from all the water sample volumes tested. In addition, plotting on a graph eDNA concentration values against sample volumes filtered, the yield of PowerSoil modified IRT protocol appeared more similar to an ideal direct proportionality than the yield of PowerWater standard protocol. eDNA quality was suitable for PCR analyses and the identification of bacterial targets, including bacterial subgroups (α and β-proteobacteria) and single species of interest, such as Shewanella oneidensis capable of hexavalent chromium reduction (Tumolo et al. 2020). Under these good performances, the PowerSoil optimized IRT protocol was also applied in a further experiment about bioremediation to extract eDNA from 50 ml of water spiked with 1000 µg/l of hexavalent chromium. The resulting genomic material was successfully used in quantitative PCR (qPCR) assays for monitoring the relative abundance of Shewanella oneidensis during the bioremediation process, allowing to highlight the hexavalent chromium inhibitor effect on the selected microbial target (Ancona et al. 2020). Downstream applications of eDNA obtained using DNeasy PowerSoil kit resulted in positive outcomes for both the experiments previously described. In light of this, it is possible to conclude that this kit combined with the protocol adjustments proposed in this work, can be a performing tool for eDNA extraction, also from small amounts of water sample collected from oligotrophic or degraded environments. Further investigations will be oriented to optimize eDNA extraction from the aquifer solid portion characterized by few nutrients and microbial cells to better understand how microbial populations can distribute themselves between solid and aqueous phases.

A new and straightforward protocol for microplastic's DNA extraction from freshwater environments

Microplastics (< 5 mm particles) are ubiquitous environmental contaminants, capable of providing an artificial substrate for the development of microbial aggregates distinguished between water column, seston, and sediments distinct in taxonomic composition from those on natural surfaces such as water column, seston, and sediment. These complex communities, constituted by prokaryotic and eukaryotic microorganisms, represent a peculiar micro-ecosystem, identified only recently as "plastisphere". The microbial composition of the plastisphere associated with microplastics in surface waters is poorly known. Therefore, there is a need for a thorough investigation into the possible role of microplastics as a transport vector for biological contaminants of hygienic-sanitary interest. Several studies have described molecular techniques used to extract nucleic acids from aquatic environments and environmental matrices. However, little is known about the DNA extraction methods used for plastic. This study's main objective is to develop a protocol for microplastic's DNA extraction, improving the genetic material’s quantity and quality necessary for future applications such as gene amplification and sequencing. DNA was extracted from microplastic and environmental samples of a freshwater ecosystem. Two different microplastics were analyzed: a) environmental particles collected from an Italian river (Ofanto river) composed prevalently by black and transparent fragments of 5-1mm in size, b) virgin microplastics purchased by local industry used as controls and composed by PE transparent pellets of 5mm and green PE particles of 100µm. DNA extraction was also performed from superficial river water, suspended organic matter (OM), and sediment of the river taking care to process three replicates for each type. Starting from the commercial DNeasy PowerSoil Kit, the method was optimized testing the minimum microplastic's amount, incubation parameters, time of cell lysis, and final elution volumes. We collected material with forceps from the glass containers for the environmental microplastics and OM, placing about 250mg of each replicate into 2-mL microcentrifuge tubes. We separated the 5 L water samples into three 500 mL aliquots and filtered each one with Whatman 0.2-μm filters. The filters were cut with sterilized forceps and placed into 2-mL microcentrifuge tubes (Debeljak et al., 2017). The main modifications made to the extractive protocol have been a method alternative of cell lysis including a preliminary incubation at 60 °C for 20 minutes and a reduction of the final elution volume to 70 µl in order to concentrate the extracted DNA. The quantity and quality of the extracted DNA were obtained respectively through the use of Qubit™ dsDNA HS Assay Kit (ThermoFisher), and agarose gel electrophoresis. Our results showed that the minimum amount of microplastics useful to obtain a valid genomic extract is about 120 mg. The modifies applied to the commercial kit resulted in significantly improving the material extracted more than eight times. The nucleic acids extracted from environmental microplastics applying the improved protocol ranged from 7.97 to 14.1 ng µl-1 with a mean value of 10.5 ± 3.2 ng µl-1 respect a medium value of 1 ± 0.1 ng µl-1 of material extracted from the same samples using the unmodified protocol of the commercial kit. As expected, the virgin microplastic samples did not show a measurable amount of DNA (< 0.2 ng µl-1). Concentrations of DNA extracted from aqueous samples resulted in a mean value of 19.53 ± 6.47 ng µl-1, while the amount of genetic material attached to sediment and OM samples revealed a concentration of 35.1 ± 2.19ng µl-1 and a value above the detection limit of 100 ng µl-1 respectively.

207 ESOPHAGEAL BIOPSY AND SWAB MICROBIAL CHARACTERIZATION WITH HEALTH AND ESOPHAGEAL DISEASE IN CHINA

Abstract Little is known about the esophageal microbiota and esophageal health or disease in China, especially esophageal squamous cell precancerous lesions. And there is currently no optimal sampling method for esophageal microbiota. Consequently, we compared microbial signature between and within esophageal biopsy and swab specimens from participants in healthy or with esophageal diseases. Methods A total of 236 subjects were recruited in the Linxian Cancer Hospital (Henan, China), including 5 groups as 70 healthy cases, 70 esophagitis cases, 70 low grade intraepithelial neoplasia (LGIN) cases, 19 high grade intraepithelial neoplasia (HGIN) cases and 7 esophageal squamous cell carcinoma (ESCC) cases. Sterile biopsies and swabs were used to collect paired samples from the same participants under the endoscopy examination. DNA was extracted with the MoBio PowerSoil Kit. Data from 16S rRNA gene sequencing were processed using QIIME2 and R to evaluate differences in alpha and beta diversity and taxonomic relative abundances. Results Alpha diversity was not significantly different between specimens (biopsy = 116.2, swab = 118.0, P > 0.05). In swabs, there were differences of observed OTUs among groups but not Shannon index, detailly, there was an inconspicuous decreasing from Health (125.0) to ESCC (114.0). About beta diversity, no distinct clustering by specimen was observed for weighted Unifrac distance and unweighted Unifrac distance. The 10 dominant genera were similar between specimens and among groups. Specially, Streptococcus was the most abundant except ESCCs; Fusobacterium was in the top ten only in ESCCs. And there was a decreasing of Prevotella 7 and an increasing of Haemophilus as progression. Conclusion Esophageal biopsy and swab specimens could yield similar bacterial composition. Both can be used to find clues of microbiota and disease. And esophageal microbial composition and progression characterizations could provide new idea and basis for large scale, prospective cohort studies in the future.

Biases in bulk: DNA metabarcoding of marine communities and the methodology involved.

With the growing anthropogenic pressure on marine ecosystems, the need for efficient monitoring of biodiversity grows stronger. DNA metabarcoding of bulk samples is increasingly being implemented in ecosystem assessments and is more cost‐efficient and less time‐consuming than monitoring based on morphology. However, before raw sequences are obtained from bulk samples, a profound number of methodological choices must be made. Here, we critically review the recent methods used for metabarcoding of marine bulk samples (including benthic, plankton and diet samples) and indicate how potential biases can be introduced throughout sampling, preprocessing, DNA extraction, marker and primer selection, PCR amplification and sequencing. From a total of 64 studies evaluated, our recommendations for best practices include to (a) consider DESS as a fixative instead of ethanol, (b) use the DNeasy PowerSoil kit for any samples containing traces of sediment, (c) not limit the marker selection to COI only, but preferably include multiple markers for higher taxonomic resolution, (d) avoid touchdown PCR profiles, (e) use a fixed annealing temperature for each primer pair when comparing across studies or institutes, (f) use a minimum of three PCR replicates, and (g) include both negative and positive controls. Although the implementation of DNA metabarcoding still faces several technical complexities, we foresee wide‐ranging advances in the near future, including improved bioinformatics for taxonomic assignment, sequencing of longer fragments and the use of whole‐genome information. Despite the bulk of biases involved in metabarcoding of bulk samples, if appropriate controls are included along the data generation process, it is clear that DNA metabarcoding provides a valuable tool in ecosystem assessments.

Abstract 6097: Alterations of the pancreatic tumor microbiome: the role of biliary stents

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers, with an average five-year survival of 9% across all stages. This dismal prognosis can be attributed to advanced stage at diagnosis and poorly effective treatment options. Growing evidence has shown that commensal microbiota play a pivotal role in the oncogenicity of a number of malignancies. Traditionally, the pancreas has been considered to be a sterile organ, with an environment that could not promote microbial colonization. However, advances in metagenomics and next-generation sequencing have enabled researchers to identify that bacterial DNA can be detected in PDAC tissue. Moreover, Geller et al. showed that presence of intratumor Gammaproteobacteria conferred resistance to the chemotherapy agent gemcitabine, further emphasizing the importance of understanding the role of these microbiota. Bacterial colonization of the pancreas in PDAC is thought to be due to migration of intestinal microbiota that thrive in the pancreatic tumor microenvironment, however there is still much unknown about the factors that promote growth of these bacteria. It is imperative to address these gaps in knowledge to enhance the efficacy of anti-tumor drug therapies. We hypothesized that biliary stent placement contributes to the increased abundance of bacterial signatures seen in PDAC. We obtained matched tumor and normal pancreatic tissue specimens from 27 patients who had undergone surgical resection for PDAC from the University of Minnesota biorepository. DNA extraction of these tissue specimens was performed with DNeasy PowerSoil Kit (Qiagen) in accordance to manufacturer instructions. Out of the 27 patients examined, microbial abundance was detected in samples from 18 patients (67%) via qPCR targeting the 16S rRNA gene. The V4 hypervariable region of the 16S rRNA gene was then amplified and pair-ended sequenced on the Illumina MiSeq platform by the University of Minnesota Genomics Core. We found that 26 (48.1%) out of 54 pancreatic tissue samples harbored detectable bacterial communities. This comprised of 13 (50%) healthy and 13 (50%) neoplastic tissue samples. The most abundant families of bacteria included Ruminococcaceae, Enterobacteriaceae, and Staphylococcaceae. Bacterial DNA was identified in 19 (73%) samples from patients with biliary stents and 7 (27%) samples from patients without biliary stents. Our results confirmed that microbial signatures were more detectable in patients who had undergone biliary stent placement (χ2 = 7.21, p = 0.007). This finding suggests that biliary stents lead to specific alterations in pancreatic microbial floral in the setting of PDAC that encourage bacterial growth. Further studies addressing whether these compositional alterations could contribute to increased chemoresistance will be essential for optimizing medical therapies in the future. Citation Format: Harika Nalluri, Christopher Staley, Eric H. Jensen. Alterations of the pancreatic tumor microbiome: the role of biliary stents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6097.

Assessment of fecal DNA extraction protocols for metagenomic studies.

BackgroundShotgun metagenomic sequencing has improved our understanding of the human gut microbiota. Various DNA extraction methods have been compared to find protocols that robustly and most accurately reflect the original microbial community structures. However, these recommendations can be further refined by considering the time and cost demands in dealing with samples from very large human cohorts. Additionally, fungal DNA extraction performance has so far been little investigated.ResultsWe compared 6 DNA extraction protocols, MagPure Fast Stool DNA KF Kit B, Macherey Nagel™ NucleoSpin™®Soil kit, Zymo Research Quick-DNA™ Fecal/Soil Microbe kit, MOBIO DNeasy PowerSoil kit, the manual non-commercial protocol MetaHIT, and the recently published protocol Q using 1 microbial mock community (MMC) (containing 8 bacterial and 2 fungal strains) and fecal samples. All samples were manually extracted and subjected to shotgun metagenomics sequencing. Extracting DNA revealed high reproducibility within all 6 protocols, but microbial extraction efficiencies varied. The MMC results demonstrated that bead size was a determining factor for fungal and bacterial DNA yields. In human fecal samples, the MagPure bacterial extraction performed as well as the standardized protocol Q but was faster and more cost-effective. Extraction using the PowerSoil protocol resulted in a significantly higher ratio of gram-negative to gram-positive bacteria than other protocols, which might contribute to reported gut microbial differences between healthy adults.ConclusionsWe emphasize the importance of bead size selection for bacterial and fungal DNA extraction. More importantly, the performance of the novel protocol MP matched that of the recommended standardized protocol Q but consumed less time, was more cost-effective, and is recommended for further large-scale human gut metagenomic studies.

Elimination of \u201ckitome\u201d and \u201csplashome\u201d contamination results in lack of detection of a unique placental microbiome

BackgroundA placental microbiome, which may be altered in gestational diabetes mellitus (GDM), has been described. However, publications raising doubts about the existence of a placental microbiome that is different than contaminants in DNA extraction kits and reagents (“kitomes”) have emerged. The aims of this study were to confirm the existence of a placental microbiome distinct from contaminants and determine if it is altered in GDM mothers.ResultsWe first enrolled normal weight, obese and GDM mothers (N = 17) at term elective cesarean section delivery in a pilot case control study. Bacterial DNA was extracted from placental parenchyma, maternal and cord blood, maternal vaginal-rectal swabs, and positive and negative controls with the standard Qiagen/MoBio Power Soil kit. Placentas had significantly higher copies of bacterial 16S rRNA genes than negative controls, but the placental microbiome was similar in all three groups and could not be distinguished from contaminants in blank controls. To determine the source and composition of the putative placental bacterial community identified in the pilot study, we expanded the study to 10 subjects per group (N = 30) and increased the number and variety of negative controls (N = 53). We modified our protocol to use an ultraclean DNA extraction kit (Qiagen QIAamp UCP with Pathogen Lysis Tube S), which reduced the “kitome” contamination, but we were still unable to distinguish a placental microbiome from contaminants in negative controls. We noted microbial DNA from the high biomass vaginal-rectal swabs and positive controls in placental and negative control samples and determined that this resulted from close proximity well-to-well cross contamination or “splashome”. We eliminated this source of contamination by repeating the sequencing run with a minimum of four wells separating high biomass from low biomass samples. This reduced the reads of bacterial 16S rRNA genes in placental samples to insignificant numbers.ConclusionsWe identified the problem of well-to-well contamination (“splashome”) as an additional source of error in microbiome studies of low biomass samples and found a method of eliminating it. Once “kitome” and “splashome” contaminants were eliminated, we were unable to identify a unique placental microbiome.

4444 The effect of early life antibiotics on gut microbiome and fecal bile acid concentrations in children

OBJECTIVES/GOALS: The current proposal seeks to investigate the effect of early life antibiotic use in the development of functional gastrointestinal (GI) disorders. We propose that infants exposed to antibiotics will present with gut microbial dysbiosis, changes in fecal bile acid concentrations and develop more GI symptoms compared to unexposed children. METHODS/STUDY POPULATION: We analyzed fecal samples from 174 subjects at 12 months of age, of whom 52 were exposed to antibiotics in their first year of life. Of these, 33 subjects were sampled again at 24 months of age. DNA from 200mg of frozen stool (−80C) was isolated with the Qiagen DNeasy PowerSoil kit. Shotgun libraries were generated using the NexteraXT kit and sequenced on the Illumina HiSeq 2500 using 2x125 bp chemistry. Sequence data were analyzed using the Sunbeam metagenomics pipeline. The abundance of bacteria was estimated using Kraken version 2.0.8. Fecal bile acids will be quantified by liquid chromatography–mass spectrometry (LC-MS). RESULTS/ANTICIPATED RESULTS: Overall bacterial community composition at 12 or 24 months was not associated with antibiotic exposure (PERMANOVA test, Bray-Curtis distance). An increase in Enterobacteriaceae, in particular Escherichia coli, is a signature of antibiotic-induced dysbiosis, but also of early infant gut. Children with antibiotic exposure had slightly higher abundance of Escherichia coli compared to those with no exposure (p = 0.03). At 24 months, the abundance of Bacteroides caccae, a commensal gut species, was decreased for children exposed to antibiotics in the first year of life (fdr = 0.02). We will perform further analysis of bile acid modifying bacteria, fecal bile acid concentrations and correlate to GI symptoms. DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest a significant but nuanced impact of early life antibiotic use on the composition of the gut microbiota. The association of antibiotic exposure with B. caccae and E. coli warrant further attention in the context of the rapidly developing early-life microbiome. CONFLICT OF INTEREST DESCRIPTION: The authors declare no conflicts of interest relevant to this work.

Comparative evaluation of UNEX-based DNA extraction for molecular detection of Cyclospora cayetanensis, Toxoplasma gondii, and Cryptosporidium parvum as contaminants of berries

The potential public health impact of foodborne parasites (FBP) transmitted via contaminated fresh produces indicates the necessity for robust and reliable laboratory methods for their detection and identification on this infection vehicle. Standardization of methods for detection of common FBP in fresh produce is to be expected and ensuring that the DNA extraction approach is most appropriate for the FBP of interest and for the matrix being analyzed is also important. Therefore, the aim of the present study was to compare the efficacy of two commercially available DNA extraction procedures, the UNEX-based method and DNeasy PowerSoil kit in the detection of three protozoan parasites, C. cayetanensis, C. parvum, and T. gondii, on contaminated berries. Oocysts of each parasite were spiked into the pellets of raspberry and blueberry washes. The spiked pellets were then randomly assigned to DNA extraction using either the PowerSoil or UNEX method, with DNA extraction with both methods performed by two independent analysts. The detection rate when berry washes were spiked with 20 oocysts of C. cayetanensis, T. gondii, and C. parvum was 95%, 85%, and 40%, respectively, when using the PowerSoil kit; whereas the equivalent results using the UNEX method were 55%, 60%, and 5%, respectively. In addition, significantly lower Cq values were achieved for each parasite in the samples spiked with 500 oocysts when the PowerSoil kit was used. Possible reasons for these results are discussed, and include the composition of both the beads and the buffers in each method.

Comparison of Swabbing Solution Volume and gDNA Extraction Kits on DNA Recovery from Rigid Surface.

For bacteria sampling studies, various collection methods have been used to identify bacteria. To obtain accurate information about bacteria, high quality samples should be obtained. In order to obtain a high quality sample, a stable and large number of DNA copies must be collected. This study compared the efficiency of different methods of bacterial gDNA extraction and bacteria collection according to swabbing solution volumes and types. The efficiency of bacterial genomic DNA extraction was compared using a AccuPrep® Genomic DNA Extraction kit, a QIAamp® DNA Mini kit, and a MOBIO® DNeasy PowerSoil kit. The DNA Mini kit was shown to extract the highest amount of gDNA, and sub-experiments were conducted using this kit. Phosphate-buffered saline and phosphate-buffered saline with 0.1% Tween 20 were used as collection solutions of various volumes (0, 40, 50, 60, 70, 80, 90, 100, 110, and 120μL) using cotton swabs. Bacteria collection efficiency was highest when 70μL PBS was used. The target strains collected in this experiment were Staphylococcus aureus and Escherichia coli, and these were quantified using the number of colony-forming units, DNA concentrations, and the number of DNA copies. These results can be used to efficiently bacterial collection for experiments in various fields.

Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics.

Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.

First Report of Serratia marcescens Causing a Leaf Spot Disease on Industrial Hemp (Cannabis sativa)

In the 2017 and 2018 growing seasons (between May and October), several cultivars of industrial hemp plants grown for flowers in greenhouse production from four North Carolina counties showed symptoms of an angular leaf spot on leaves, stems, and flower parts. Lesions were initially small, dark brown, 1 to 3 mm in size, and vein-limited. As the disease progressed, lesions coalesced to form larger regions of necrosis that engulfed large portions of leaves, and whole plants were lost to disease. Red bacterial ooze was observed streaming from the plant tissues. Bacterial colonies growing on potato dextrose agar (PDA) were raised and dark-pink colored. The Gram stain indicated gram-negative bacilli. A representative isolate (called strain T2) was used to inoculate Cannabis sativa L. ‘Carmagnola’. Two-week-old seedlings (n = 6) were inoculated with a bacterial suspension (OD₆₀₀ = 0.1, approximately 10⁸ CFU/ml) using a PreVal hand sprayer; additional seedlings (n = 6) were inoculated with sterile water to serve as the negative control. Plants were incubated at 23°C for 21 days in a growth chamber with a 12-h photoperiod. After 8 days, dark brown-black lesions similar to those described above were observed on inoculated leaves. Control plants remained symptomless. The pathogen of interest was the only microorganism reisolated from lesions, and after transferring onto PDA, colonies were identical in morphology to those isolated from the original diseased plants. Molecular identification was conducted by first extracting DNA from the representative isolate T2 using the DNeasy Powersoil kit (Qiagen, Hilden, Germany). The 16S ribosomal RNA region (Klindworth et al. 2013) and the RNA polymerase β-subunit (rpoB) gene (Mollet et al. 1997) were PCR amplified. Amplicons were sequenced at the North Carolina State Genomic Sciences Laboratory, and sequences for each gene from the isolate assessed (T2) were deposited to GenBank (accessions MK598699 and MN400982 for 16S rRNA and rpoB, respectively). NCBI-BLAST searches (Altschul et al. 1997) showed the highest similarity (99.6% identity) with 16S rRNA of Serratia marcescens subsp. sakuensis and 99.6% pairwise identity to rpoB of S. marcescens strain B3R3 (GenBank accession KU894791). Based on pathogenicity, morphology, and molecular identification, the unknown bacterial isolate was identified as S. marcescens. Because industrial hemp is increasingly grown across the United States for cannabidiol production, this disease could pose significant challenges and cause yield reduction in affected plants. Additionally, this pathogen could be of human health concern should it survive hemp processing practices, because there are strains that affect humans.

Indoor Plumbing Drains Are Important Sources for Invasive Fusariosis in Patients with Hematological Disorders: Culture-Based and Metagenomic Analysis

Despite the advances in antifungal drugs, the number of invasive fusariosis (IF) has been increasing in patients with hematological disorders, especially hematopoietic stem cell transplantation (HSCT) patients. IF is still a severe infection and refractory to treat because Fusarium is naturally resistant to most of the antifungal drugs. Therefore, prior to infection, avoidance of contact with Fusarium would be critical in immunocompromised patients. However, little is known about the source of Fusarium infection. The purpose of this study is to investigate the epidemiology of IF in patients with hematological disorders in Japan and to find out the infectious source of Fusarium and the relationship between Fusarium species isolated from clinical samples and the ones from environment using culture-based and metagenomic methods. In our center, a total of 99 clinically isolated Fusarium were stored and maintained. They were obtained from hospitals in 33 prefectures in Japan. 50 and 49 strains were recovered from IF patients and superficial fusariosis (SF) patients, respectively. In this study, we analyzed 42 IF cases which developed in patients with hematological disorders. The most common underlying disease was AML (35.7%), followed by ALL (23.8%). About 60% of the patients received HSCT, and most of them became infected with Fusarium in the early phase after HSCT (within eight weeks). This result indicated that IF infection mostly occurred in indoor environments. As the next step, we performed the surveys in indoor environments such as air and plumbing drains in medical institutions and residences to figure out the source of infection. Few Fusarium species were collected from air samples. From the indoor air, we collected 142 filamentous fungi isolates. Only 2/142 strains (1.4%) were Fusarium species (Fusarium fujikuroi species complex 1, F. nisikadoi 1). In contrast, among the 72 drain port samples collected from 6 medical institutions (hematology wards, including bone marrow transplantation units, and ICU/NICU) and 15 residences, Fusarium species were positive at 26 (36.1%) sampling points. One hundred and eight filamentous fungi were isolated from the drain samples, and 29/108 isolates (27%) were Fusarium species. In details, 20 strains were Fusarium solani species complex (F. petroliphilum 7, F. keratoplasticum 4, F. sensu stricto 4, F. falciforme 1, others 4) and nine strains were Fusarium oxysporum species complex. Interestingly, the FSSC compositions of drain port samples were similar to those of IF samples from patients with hematological disorders and different from those of SF samples. In contrast, no Fusarium strains were detected in the places where the regular sink-cleaning was performed. Likewise, the samples for the metagenomic analysis were obtained in the places where the culture samples were collected. DNA extraction from samples was performed by the PowerLyzer Powersoil Kit (QIAGEN, Hilden, Germany). Fungal ITS regions were amplified with modified ITS1F and ITS2 primer set and sequenced with Illumina Miseq platform. After filtering and denoising, we were able to analyze 8 air samples and 23 drain samples (10 culture-positive samples and 13 culture-negative samples). It is also revealed that a lot of Fusarium live indoor plumbing drains but few Fusarium spp. inhabit indoor air (less than 0.1%). In summary, we investigate the epidemiology of IF in patients with hematological disorders in Japan and the distribution of Fusarium species in indoor environments. Our survey has demonstrated that a lot of Fusarium inhabits the indoor plumbing drains, not the indoor air using the culture-based and metagenomic methods. The similarity of FSSC distribution between the drain samples and causative species of IF cases indicated that the indoor plumbing drains would be potential sources of IF infection. We suggested that regular sink-cleaning was critical to prevent patients with hematological disorders from Fusarium infection. Disclosures No relevant conflicts of interest to declare.

Storage media and not extraction method has the biggest impact on recovery of bacteria from the oral microbiome

Next Generation sequencing has greatly progressed the exploration of the oral microbiome’s role in dental diseases, however, there has been little focus on the effect of sample storage conditions and their interaction with DNA extraction method. Dental plaque samples collected from 20 healthy participants were pooled and stored in either 75% ethanol or Bead solution for up to 6-months at −80 °C, prior to DNA extraction with either QIAamp (non-bead beating) or PowerSoil (bead-beating) kit, followed by Illumina sequencing of 16S rRNA gene. We found that storage media and not extraction method had the biggest influence on the diversity and abundance of the oral microbiota recovered. Samples stored in Bead solution, independent of the extraction kit, retrieved higher diversity (PowerSoil p = 1.64E-07, QIAamp p = 0.0085) and had dissimilar overall ecologies as indicated by lower level of shared diversity (PowerSoil p = 0.0000237, QIAamp p = 0.0088). Comparatively, samples stored in Bead solution and extracted with PowerSoil recovered a higher abundance of Streptococcus species. These data indicate that Bead solution can preserve the oral microbiome in dental plaque reliably, for periods of up to 6-months at −80 °C, and is compatible, with either a bead-beating or non-bead beating DNA extraction method.

Microbial characterization of esophageal squamous cell carcinoma and gastric cardia adenocarcinoma from a high-risk region of China.

Little is known about the microbiota and upper gastrointestinal tumors. Esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA) occur in adjacent organs, co-occur geographically, and share many risk factors despite being of different tissue types. This study characterized the microbial communities of paired tumor and nontumor samples from 67 patients with ESCC and 36 patients with GCA in Henan, China. DNA was extracted with the MoBio PowerSoil kit. The V4 region of the 16S ribosomal RNA gene was sequenced with MiniSeq and was processed with Quantitative Insights Into Microbial Ecology 1. The linear discriminant analysis effect size method was used to identify differentially abundant microbes, the Wilcoxon rank-sum test was used to test α diversity differences, and permutational multivariate analysis of variance was used to test for differences in β diversity. The microbial environments of ESCC and GCA tissues were all composed primarily of Firmicutes, Bacteroidetes, and Proteobacteria. ESCC tumor tissues contained more Fusobacterium (3.2% vs 1.3%) and less Streptococcus (12.0% vs 30.2%) than nontumor tissues. GCA nontumor tissues had a greater abundance of Helicobacter (60.5% vs 11.8%), which may have been linked to the lower α diversity (58.0 vs 102.5; P=.0012) in comparison with tumor tissues. A comparison of ESCC and GCA nontumor tissues showed that the microbial composition (P=.0040) and the α diversity (87.0 vs 58.0; P=.00052) were significantly different. No significant differences were detected for α diversity within ESCC and GCA tumor tissues. This study showed differences in the microbial compositions of paired ESCC and GCA tumor and nontumor tissues and differences by organ site. Large-scale, prospective cohort studies are needed to confirm these findings.

PSII-16 Evidence for stratification of rumen wall microbial communities revealed by 16S rRNA based amplicon sequencing

Abstract Rumen content (RC) stratifies based on particle size and density consisting of the less dense forage within the dorsal and the denser particles in the ventral portions of the rumen and is in constant contact with the microbial communities present on the rumen wall (RW) epithelium. Little is known about the nutrient requirements and functional processes of RW microbial communities. Our hypothesis is that the RW microbial communities stratify mirroring the stratification of RC due to different available nutrients. Five fistulated, milking Holstein cows of the same management conditions were sampled at four rumen layers corresponding to the RC stratification. Epithelial biopsies were taken through the fistula; the uppermost aligned with the dorsal portion of the RC (A), and three other sites, each 10 cm ventral to the previous (B, C and D). Each cow and stratification layer was sampled five times over four months to analyze temporal stability of the RW microbial communities. DNA was extracted using the Qiagen Powerlyzer Powersoil kit and used for 16S rRNA gene Illumina MiSeq sequencing. Sequences were clustered into operational taxonomic units (OTU) based on a 99% similarity cutoff using MOTHUR. After quality control, 2.0 million reads remained for 90 samples which were clustered into 5,016 OTUs with 10 or more reads. 99.2% of the reads were bacterial, whereas 0.8% affiliated to Archaea. Statistical analysis revealed that among the 20 most abundant OTUs, phylotypes classified as Desulfobulbus, unclassified_Cardiobacteraceae, Mogibacterium, Lachnospiraceae-UCG008 and Methanobrevibacter were significantly different in abundance between sites A compared to D. On a whole community level, analysis of molecular variance (AMOVA) revealed significant differences between groups A, C and D. Our data reveal first evidence that a stratification of RW microbiota is present in dairy cattle and also reveal high temporal stability of RW microbiota.

Abstract 3326: Mucositis, candidiasis, and associations with the oral microbiome in treatment naive patients with oropharyngeal cancer

Abstract Purpose: Oropharyngeal cancer (OPC) incidence continues to increase dramatically in the US. Accumulating evidence suggests that oral microbiota (communities of microorganisms that reside in the oral cavity) may influence cancer treatment-related toxicities. We sought to examine the composition and diversity of oral microbiota in OPC patients prior to treatment, and identify associations between oral microbiota and subsequent development of oral mucositis (inflammation of mucous membranes) and candidiasis (fungal infection with Candida yeast). Methods: 60 newly diagnosed, treatment naïve, OPC patients were recruited from Moffitt Cancer Center (2015-2017). Patients provided mouthwash-based oral gargles before starting chemoradiation or radiation. DNA was isolated using the QIAGEN DNeasy PowerSoil kit, and the V1-V3 region of the bacterial 16S rRNA gene was sequenced. An operational taxonomic unit table was generated and Ribosomal Database Project Classifier used to assign taxonomy. The development of oral mucositis (no/mild vs. severe) and candidiasis (no vs. yes) during treatment was abstracted from medical records. Comparisons of bacterial relative abundance (Mann Whitney U), alpha diversity (Chao1, Shannon, and Simpson), and beta diversity (Bray-Curtis) were performed in R and its extension (Phyloseq). Results: Of 60 OPC patients, 65% were stage IV, 48% tonsillar and 42% base of tongue, and 40% never smokers. Pre-treatment levels of genus Klebsiella and class Gammaproteobacteria were significantly greater in the oral cavity of patients who had no/mild mucositis vs. those who developed severe mucositis (p<0.04). Genera Atopobium and Mogibacterium, as well as families Coriobacteriaceae and Clostridiales Incertae Sedis, were significantly more abundant in patients who did not develop oral candidiasis compared to those who did (p<0.04). Oral bacterial alpha diversity (intra-subject) was higher in patients who developed vs. did not develop severe mucositis, and lower in patients who developed vs. did not develop candidiasis during treatment. Analyses of beta diversity (inter-subject) showed that the microbial community structures were not significantly different by mucositis or candidiasis development. Conclusions: Microbiome profiling may hold promise as a prognostic biomarker of treatment-related toxicities. Several potentially predictive taxa were identified to be differentially abundant in OPC patients who subsequently developed oral mucositis or candidiasis. Future analyses will evaluate the role of oral microbial resilience (the rate of recovery after disturbance) using oral specimens collected 3 mo. after treatment. Translational research focused on understanding how oral microbiota influence local immune and inflammatory responses may aid in the development of microbiota-based interventions to minimize adverse events. Citation Format: Christine M. Pierce, Stephanie Hogue, Shirlene Paul, Bo-Young Hong, Wildson Vieira da Silva, Maria F. Gomez, Anna R. Giuliano, Jimmy J. Caudell, George M. Weinstock. Mucositis, candidiasis, and associations with the oral microbiome in treatment naive patients with oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3326.

Evaluation of Methods for the Extraction of Microbial DNA From Vaginal Swabs Used for Microbiome Studies.

Background: The composition of the microbiome in human body sites plays an important role in health. The vaginal environment is colonized by several species of bacteria that have a major influence on reproductive health. The advancement of sequencing technologies has made the assessment of the composition of the microbiota possible through microbial DNA extraction and sequencing. Therefore, it is of a paramount importance to select a sensitive and reproducible DNA extraction method, that facilitates isolation of microbial DNA with a sufficient quantity and purity, from microbial species living in the vaginal environment. Here, we have evaluated four different DNA extraction protocols from self-collected vaginal swabs.Methods: Five healthy female volunteers were enrolled in the study. Each donor was asked to self-collect 4 samples using Copan ESwab. DNA was extracted using Qiagen DNeasy kit and three modified protocols of the MoBio PowerSoil kit (“DNeasy PowerSoil” after acquisition from Qiagen). DNA quantity and integrity was checked through Nanodrop and LabChip GX. DNA was further tested through quantitative real-time PCR (qPCR) and 16S sequencing. Vaginal microbiota diversities were determined using MiSeq-Illumina high-throughput sequencing of bacterial 16S rDNA V1–V3 fingerprint. Sequencing data were analyzed using QIIME pipeline.Results: Qiagen DNeasy protocol resulted in the highest DNA yield as compared to the modified protocols of MoBio Powersoil kit. The size of the DNA extracted using each protocol was ~40 kb. Qiagen DNeasy protocol gave the highest Genomic Quality Score (average ± standard deviation: 4.24 ± 0.36), followed by the different MoBio Powersoil protocols. A substantial variability in microbial DNA abundance was found across the protocols. The vaginal microbiota of the healthy volunteers was dominated by Lactobacillus species. MoBio Powersoil kit provided a significantly higher alpha diversity as compared to the Qiagen DNeasy kit, while beta diversity measures did not reveal any significant cluster changes, except when the Bray-Curtis method was applied.Conclusion: We were able to isolate microbial DNA from the vaginal swabs. Qiagen DNeasy method gave the highest DNA yield and quality but was not optimal in detecting microbial diversity. The modified MoBio PowerSoil protocols showed higher microbial diversities as compared to the standard protocol.

Use of a filter cartridge combined with intra‐cartridge bead‐beating improves detection of microbial DNA from water samples

Abstract Microbial communities play an important role in driving the dynamics of aquatic ecosystems. As difficulties in DNA sequencing faced by microbial ecologists are continuously being reduced, sample collection methods and DNA extraction protocols are becoming more critical to the outcome of any sequencing study. In the present study, I added a manual, intra‐cartridge, bead‐beating step in the protocol using a DNeasy® Blood & Tissue kit for DNA extraction from a filter cartridge without breaking the cartridge unit (“Beads” method) and compared its performance with those of two other protocols (“NoBeads” method, which was similar to the Beads method but without the bead‐beating step and “PowerSoil” method, which followed the manual of the DNeasy® PowerSoil kit after breaking apart the filter cartridge). Water samples were collected from lake, river, pond and coastal ecosystems in Japan and DNA was extracted using the three protocols. Then, the V4 region of prokaryotic 16S rRNA genes was amplified. In the library preparation process, internal standard DNAs were included to estimate the number of DNA copies. The DNA library was sequenced using Illumina MiSeq and sequences were analysed using the amplicon sequence variant (ASV) approach. I found that, (a) the total prokaryotic DNA yields were highest with the Beads method, (b) the number of ASVs (a proxy for species richness) was also highest with the Beads method, (c) overall community compositions were significantly different among the three methods and (d) the number of method‐specific ASVs was highest with the Beads method. These results were generally robust across samples from all aquatic ecosystems examined. In conclusion, the inclusion of a bead‐beating step performed inside the filter cartridge increased the DNA yield as well as the number of prokaryotic ASVs detected compared with the other two methods. Performing the bead‐beating step inside the filter cartridge causes no dramatic increase in either handling time or processing cost and it can reduce the potential contamination risk from the ambient air and/or other samples. Therefore, this method has the potential to become one of the major choices when one aims to extract aquatic microbial DNAs.

Impacts of Genistein on the Gut Microbiota of a Mouse Model of Cystic Fibrosis (P06-070-19)

ObjectivesDysbiosis of the gut microbiota (GM) is observed in both murine models and humans with cystic fibrosis (CF). Laxative treatments are typically used to improve gastric motility and alleviate gastrointestinal symptoms associated with CF. Genistein, a phytochemical in soy, exerts therapeutic benefits by activating intestinal CFTR ion channels but whether genistein affects the GM in CF is unknown. This study aimed to establish whether dietary genistein alters the GM community structure in a murine model of the most common CF-associated mutation, DF508. MethodsIn this 3-arm parallel randomized intervention, 21-day old DF508 mice (n = 24) were fed standard chow (CHOW; n = 11), CHOW plus Colyte (laxative; n = 7), or CHOW plus 600 mg of dietary genistein (GEN) per kg of diet (n = 6) for 45 days. Fecal pellets from day 45 were frozen at –80°C and microbial genomic DNA extracted using a DNeasy PowerSoil Kit. The 16S rRNA gene was sequenced via Illumina MiSeq. Sequences were quality checked and denoised using DADA2 and taxonomy determined using the GreenGenes 13.8 database. Treatment differences in the GM were compared using both alpha (within-sample) and beta (between-sample) diversity metrics (Kruskal-Wallis and PERMANOVA tests). ResultsBoth observed operational taxonomic units (OTUs) and Shannon’s diversity index (alpha diversity richness measures) were greater for CHOW (OTUs: p = < 0.003; Shannon’s: p < 0.003) and Colyte (OTUs: p = 0.012; Shannon’s: p = 0.006) compared to GEN animals. Colyte resulted in significantly lower species richness (OTUs: p < 0.005) compared to CHOW. Species evenness (Pielou’s) did not differ suggesting the importance of species richness over evenness for group differences. Jaccard, Bray-Curtis, unweighted and weighted UniFrac metrics (beta diversity measures) revealed significant group differences (GEN vs. CHOW: p’s < 0.004; GEN vs. Colyte: p’s < 0.005; CHOW vs. Colyte: p’s < 0.006) suggesting that differences in GM community structure were driven by presence/absence, abundance, and phylogeny of taxa. ConclusionsColyte and GEN resulted in lower within-sample diversity and significant differences in beta diversity compared to CHOW mice. Species abundance differences between Colyte and GEN groups and how disease prognosis is impacted require further investigation. Funding SourcesMidwestern University intramural funds (LA) and Arizona State University internal funds (CMW).