P851 Effects of conservation time on the diversity and relative abundance of intestinal microbiome in samples of faecal matters

Abstract Background Given the complexity and diversity of the intestinal microbiome, there is a technical challenge of finding the best way to study the factors that affect the quality of the sample to obtain results in a precise an complete way. Objective To establish the effect of storage time under constant cryopreservation conditions (−20°C) in stool samples for the study of the gastrointestinal microbiome Methods A sample of stool was distributed in 3 fractions (1, 2 and 3). Time 0 without cryopreservation and immediate DNA extraction (1). The samples of 15 (2) and 30 (3) days were cryopreserved at −20°C before the extraction of the genetic material. After ultracentrifugation at 4 degrees Celsius, the precipitate was subjected to enzymatic and mechanical lysis to obtain the total DNA. The DNA quality was evaluated techniques to ensure the quality and concentration of genetic material. Once the DNA of the three samples was obtained, they were subjected to the latest generation of a variable region of the 16S gene, using MiSeq technology (Illuminates). Data in relative abundances and frequencies were recorded. Project supported by the Administrative Department of Science, Technology and Innovation-Colciencias Health Call 2017, Code 130877757442 Results About 140 thousand readings were obtained for samples 1 (day 0) and 2 (day 15) and 160 thousand for sample 3 (day 30), with a reading quality Q20% between 97 and 98 and Q30% around 94, indicating a high-reliability value for each of the samples. Regarding the classification from the Operating Taxonomic Unit (OTU), 119, 99 and 106 were reported for samples 1, 2 and 3, respectively. The data obtained revealed changes over time at the level of the main edges reported for normal microbiome (Bacteroidetes, Firmicutes, Actinobacteria) between 1 and 2 vs. sample 3. For Firmicutes and Actinobacteria, decreases in abundance of 34% were observed (samples 1 and 2) at 20% for Firmicutes in sample 3 and from 4% (samples 1 and 2) to 0.8% (sample 3) for Actinobacteria. On the contrary, Bacteroidetes presented an increase in its abundance from 58% (Samples 1 and 2) to 76% (sample 3). The Proteobacterium edge did not show significant changes in its abundance Conclusion It was possible to demonstrate that the cryopreservation time before DNA extraction is an important variable that influences the percentage and integrity of the microbiota from faecal matter. Confirming that the maximum reliable shelf life at −20° in stool samples is up to 15 days, suggesting that for longer storage the temperature decrease up to −80° should be taken into account to maintain stability in the results of relative abundance and bacterial diversity

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Comparison of Two DNA Extraction Methods and Two PCRs for Detection of Echinococcus multilocularis in the Stool Samples of Naturally Infected Red Foxes

Animals ◽

10.3390/ani10122381 ◽

2020 ◽

Vol 10 (12) ◽

pp. 2381

Author(s):

Katarzyna Skrzypek ◽

Jacek Karamon ◽

Małgorzata Samorek-Pieróg ◽

Joanna Dąbrowska ◽

Maciej Kochanowski ◽

...

Keyword(s):

Positive Result ◽

Multiplex Pcr ◽

Dna Extraction ◽

Echinococcus Multilocularis ◽

Nested Pcr ◽

Genetic Material ◽

Extraction Methods ◽

Red Foxes ◽

Stool Samples ◽

Positive Results

(1) Background: Due to the increasing distribution of Echinococcus multilocularis infections in final hosts, epidemiological investigations are important for recognizing the spreading pattern of this parasite and also to estimate risk infection for humans. (2) Methods: Investigations were conducted with two commercial kits dedicated for DNA extraction from feces: ZR Fecal DNA Mini Prep (Zymo Research, Freiburg, Germany) and QIAamp FAST DNA Stool Mini Kit (Qiagen, Hilden, Germany) (marked as Z and Q), together with two common PCR protocols (nested PCR and multiplex PCR). The goal was to compare their efficiency in detecting the genetic material of E. multilocularis in the samples of feces. Stool samples from red foxes were collected in a highly endemic area in Poland. Sedimentation and counting technique (SCT) was used as a reference method. (3) Results: From 48 samples, 35 were positive in SCT. Further investigations showed that 40.0% of samples (from those with SCT positive result) after Z-DNA extraction and 45.7% after Q-DNA extraction gave positive results in nested PCR. In multiplex PCR, positive results were obtained in 54.3% of samples after Z isolation and 48.6% of samples after Q. Additionally, one sample that resulted in being negative in SCT gave a positive result in PCR. The number of worms detected in the intestines had no influence on PCR results. (4) Conclusions: Both of the extraction methods showed similar efficiency in DNA isolation and dealing with inhibitors; however, they showed relatively low sensitivity. This was probably caused by degradation of genetic material in the field-collected samples.

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Gut Microbiome Composition Remains Stable in Individuals with Diabetes-Related Early to Late Stage Chronic Kidney Disease

Biomedicines ◽

10.3390/biomedicines9010019 ◽

2020 ◽

Vol 9 (1) ◽

pp. 19

Author(s):

Ashani Lecamwasam ◽

Tiffanie M. Nelson ◽

Leni Rivera ◽

Elif I. Ekinci ◽

Richard Saffery ◽

...

Keyword(s):

Chronic Kidney Disease ◽

Kidney Disease ◽

Relative Abundance ◽

Cross Sectional Study ◽

Early Event ◽

Sectional Study ◽

Cross Sectional ◽

Microbiome Composition ◽

Stool Samples ◽

Late Stages

(1) Background: Individuals with diabetes and chronic kidney disease display gut dysbiosis when compared to healthy controls. However, it is unknown whether there is a change in dysbiosis across the stages of diabetic chronic kidney disease. We investigated a cross-sectional study of patients with early and late diabetes associated chronic kidney disease to identify possible microbial differences between these two groups and across each of the stages of diabetic chronic kidney disease. (2) Methods: This cross-sectional study recruited 95 adults. DNA extracted from collected stool samples were used for 16S rRNA sequencing to identify the bacterial community in the gut. (3) Results: The phylum Firmicutes was the most abundant and its mean relative abundance was similar in the early and late chronic kidney disease group, 45.99 ± 0.58% and 49.39 ± 0.55%, respectively. The mean relative abundance for family Bacteroidaceae, was also similar in the early and late group, 29.15 ± 2.02% and 29.16 ± 1.70%, respectively. The lower abundance of Prevotellaceae remained similar across both the early 3.87 ± 1.66% and late 3.36 ± 0.98% diabetic chronic kidney disease groups. (4) Conclusions: The data arising from our cohort of individuals with diabetes associated chronic kidney disease show a predominance of phyla Firmicutes and Bacteroidetes. The families Ruminococcaceae and Bacteroidaceae represent the highest abundance, while the beneficial Prevotellaceae family were reduced in abundance. The most interesting observation is that the relative abundance of these gut microbes does not change across the early and late stages of diabetic chronic kidney disease, suggesting that this is an early event in the development of diabetes associated chronic kidney disease. We hypothesise that the dysbiotic microbiome acquired during the early stages of diabetic chronic kidney disease remains relatively stable and is only one of many risk factors that influence progressive kidney dysfunction.

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Conventional myelosuppressive chemotherapy for non-haematological malignancy disrupts the intestinal microbiome

BMC Cancer ◽

10.1186/s12885-021-08296-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Lito E. Papanicolas ◽

Sarah K. Sims ◽

Steven L. Taylor ◽

Sophie J. Miller ◽

Christos S. Karapetis ◽

...

Keyword(s):

Gut Microbiota ◽

Relative Abundance ◽

Haematological Malignancy ◽

Amplicon Sequencing ◽

Intestinal Microbiome ◽

Rrna Gene ◽

Conventional Chemotherapy ◽

Myelosuppressive Chemotherapy ◽

Gram Positive Bacteria ◽

Faecal Samples

Abstract Background The gut microbiota influences many aspects of host physiology, including immune regulation, and is predictive of outcomes in cancer patients. However, whether conventional myelosuppressive chemotherapy affects the gut microbiota in humans with non-haematological malignancy, independent of antibiotic exposure, is unknown. Methods Faecal samples from 19 participants with non-haematological malignancy, who were receiving conventional chemotherapy regimens but not antibiotics, were examined prior to chemotherapy, 7–12 days after chemotherapy, and at the end of the first cycle of treatment. Gut microbiota diversity and composition was determined by 16S rRNA gene amplicon sequencing. Results Compared to pre-chemotherapy samples, samples collected 7–12 days following chemotherapy exhibited increased richness (mean 120 observed species ± SD 38 vs 134 ± 40; p = 0.007) and diversity (Shannon diversity: mean 6.4 ± 0.43 vs 6.6 ± 0.41; p = 0.02). Composition was significantly altered, with a significant decrease in the relative abundance of gram-positive bacteria in the phylum Firmicutes (pre-chemotherapy median relative abundance [IQR] 0.78 [0.11] vs 0.75 [0.11]; p = 0.003), and an increase in the relative abundance of gram-negative bacteria (Bacteroidetes: median [IQR] 0.16 [0.13] vs 0.21 [0.13]; p = 0.01 and Proteobacteria: 0.015 [0.018] vs 0.03 [0.03]; p = 0.02). Differences in microbiota characteristics from baseline were no longer significant at the end of the chemotherapy cycle. Conclusions Conventional chemotherapy results in significant changes in gut microbiota characteristics during the period of predicted myelosuppression post-chemotherapy. Further study is indicated to link microbiome changes during chemotherapy to clinical outcomes.

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DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid

mSystems ◽

10.1128/msystems.00619-21 ◽

2021 ◽

Author(s):

Erika Ganda ◽

Kristen L. Beck ◽

Niina Haiminen ◽

Justin D. Silverman ◽

Ban Kawas ◽

...

Keyword(s):

Food Safety ◽

Dna Extraction ◽

Bacterial Communities ◽

Biological Fluid ◽

Genetic Material ◽

Next Generation ◽

Microbial Composition ◽

Chemical Methods ◽

Bacterial Detection ◽

Do So

Tracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of the microbial composition.

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Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples

Diagnostics ◽

10.3390/diagnostics9020036 ◽

2019 ◽

Vol 9 (2) ◽

pp. 36 ◽

Cited By ~ 4

Author(s):

Sören Hansen ◽

Marco Roller ◽

Lamia Alslim ◽

Susanne Böhlken-Fascher ◽

Kim Fechner ◽

...

Keyword(s):

Dna Extraction ◽

Mycobacterium Avium ◽

Extraction Method ◽

Magnetic Beads ◽

Mycobacterium Avium Subspecies Paratuberculosis ◽

Rapid Identification ◽

Cold Chain ◽

Silica Membrane ◽

Molecular Assays ◽

Stool Samples

The rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) infected animals within the herd is essential for preventing the spread of the disease as well as avoiding human exposure. Although culture is seen as the gold standard, there are various molecular assays available i.e., polymerase chain reaction (PCR) or isothermal amplification technique (recombinase polymerase amplification (RPA)) for the detection of MAP. The accuracy of the molecular assays is highly dependent on the DNA extraction method. In order to establish a rapid point of need system for the detection of MAP DNA from stool samples, we developed a rapid DNA extraction protocol (MAP DNA SpeedXtract) specified for use in combination with the RPA. The whole procedure from “sample in” to “result out” was conducted in a mobile suitcase laboratory. The DNA extraction is based on reverse purification by magnetic beads, which reduces the required technical demand. The MAP DNA SpeedXtract was performed within 25 min and only three pipetting steps were needed. The amplification and detection time were 20 min in RPA. The sensitivity and specificity of the developed protocol in comparison with the lab-based silica membrane column extraction and real-time PCR were 90.9% (n = 22) and 100% (n = 23), respectively. In conclusion, we established a rapid and reliable protocol for the extraction and detection of MAP DNA. All reagents are cold chain independent. The entire setup is ideal for point of need identification of MAP infected cases.

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Antiamoebic Chemoprophylaxis Using Quinfamide in Children: A Comparative Study

The Scientific World JOURNAL ◽

10.1100/tsw.2002.174 ◽

2002 ◽

Vol 2 ◽

pp. 1070-1078 ◽

Cited By ~ 4

Author(s):

Nicolas Padilla ◽

Rosalinda Diaz ◽

Alfonso Alarcon ◽

Roberto Barreda

Keyword(s):

Primary Schools ◽

Personal Hygiene ◽

Study Group ◽

Three Samples ◽

Stool Samples ◽

Group 3 ◽

Group 2 ◽

Study Participants ◽

Prospective Longitudinal ◽

Group 1

This study sought to examine whether the administration of quinfamide at 3- or 6-month intervals diminished the frequency ofEntamoeba histolyticacysts in stool samples compared to controls. The prospective, longitudinal, randomized, single-blind study examined children from six primary schools in Celaya and Neutla, Guanajuato. Of the 1,524 students in these schools, we selected participants for the study as follows: Children were included in the study if their parents agreed in writing to the study and if the children demonstrated evidence ofE. histolyticacysts after a parasitoscopic analysis by concentration (PSC) in three samples over consecutive days using Faust’s method. Those included in the study received a single 4.3-g/kg dose of quinfamide, and we performed PSC on days 5, 6, and 7 following dose administration to examine whether quinfamide had affected the presence of the cysts. The study participants who tested negative for cysts were divided into three groups: Group 1 had 102 patients who underwent quinfamide treatment and three CPS analyses after the 12 months of the study; Group 2 had 98 subjects who underwent the quinfamide treatment and three CPS analyses at months 3, 6, 9, and 12 after their entrance into the study; and Group 3 had 102 patients, who underwent the quinfamide treatment and series of three CPS analyses at months 6 and 12 of the study. All participants received the dose of quinfamide after providing stool samples and after a clinical gastrointestinal history was obtained. Further clinical gastrointestinal data were collected 5 days after the quintamide dose was administered. We used EpiInfo 6.0 for statistical analysis, calculatingX2andpvalues for the clinical data and the CPS data after the 12 months concluded. Of the initial samples of 1,524 subjects, 308 (20.2%) had Entamoebic cysts. Of these, six were further eliminated because they did not meet the inclusion requirements. At the conclusion of the study, Group 1 presented with 37.6% of subjects still testing positive for cysts; of Group 2, 12.5% tested positive; and in Group 3, 23.5% of participants tested positive for cysts (X2= 16.8; df = 2;p= 0.0002). For comparisons of groups 1 and 2 and 1 and 3,p> 0.05. We conclude that antiamoebic chemoprophylaxis can be a choice for control of amoebic infection where personal hygiene and food consumption habits are not improving.

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Detection and Characterization of Human Enteroviruses, Human Cosaviruses, and a New Human Parechovirus Type in Healthy Individuals in Osun State, Nigeria, 2016/2017

Viruses ◽

10.3390/v11111037 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1037 ◽

Cited By ~ 3

Author(s):

Folakemi Abiodun Osundare ◽

Oladele Oluyinka Opaleye ◽

Akeem Abiodun Akindele ◽

Samuel Adeyinka Adedokun ◽

Olusola Anuoluwapo Akanbi ◽

...

Keyword(s):

Healthy Individuals ◽

Coding Region ◽

Rt Pcr ◽

Human Parechovirus ◽

Full Genome Sequencing ◽

Three Samples ◽

Two Samples ◽

Stool Samples ◽

Pcr Assays ◽

Species Specific

Human enteroviruses and human parechoviruses are associated with a broad range of diseases and even severe and fatal conditions. For human cosaviruses, the etiological role is yet unknown. Little is known about the circulation of non-polio enteroviruses, human parechoviruses, and human cosaviruses in Nigeria. A total of 113 stool samples were collected from healthy individuals in Osun State between February 2016 and May 2017. RT-PCR assays targeting the 5′ non-coding region (5′ -NCR) were used to screen for human enteroviruses, human parechoviruses, and human cosaviruses. For human enteroviruses, species-specific RT-PCR assays targeting the VP1 regions were used for molecular typing. Inoculation was carried out on RD-A, CaCo-2, HEp-2C, and L20B cell lines to compare molecular and virological assays. Ten samples tested positive for enterovirus RNA with 11 strains detected, including CV-A13 (n = 3), E-18 (n = 2), CV-A20 (n = 1), CV-A24 (n = 1), EV-C99 (n = 1), and EV-C116 (n = 2). Three samples tested positive for human parechovirus RNA, and full genome sequencing on two samples allowed assignment to a new Parechovirus A type (HPeV-19). Thirty-three samples tested positive for cosavirus with assignment to species Cosavirus D and Cosavirus A based on the 5′-NCR region. Screening of stool samples collected from healthy individuals in Nigeria in 2016 and 2017 revealed a high diversity of circulating human enteroviruses, human parechoviruses, and human cosaviruses. Molecular assays for genotyping showed substantial benefits compared with those of cell-culture assays.

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670. VRE Clearance in Patients with Recurrent Clostridium difficile Infection Following Treatment with Microbiota-Based Drug RBX2660

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.738 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S306-S307

Author(s):

Heidi Hau ◽

Sarah Mische ◽

Sarah Klein ◽

Ken Blount

Keyword(s):

Clostridium Difficile ◽

Clostridium Difficile Infection ◽

Cohort Analysis ◽

Intestinal Microbiome ◽

Phase 2 ◽

Open Label ◽

Recurrent Clostridium Difficile Infection ◽

Gram Staining ◽

Increased Risk ◽

Stool Samples

Abstract Background Vancomycin-resistant Enterococcus (VRE) infection is frequently associated with immunocompromised and critically ill patients. VRE carriers are at increased risk for infection due to VRE colonization and they pose a risk as a transmission source. VRE infection and Clostridium difficile infection (CDI) share common risk factors, including disruption of the intestinal microbiome. Thus, therapeutic approaches that decolonize VRE would be valuable. Herein, we report on stool VRE clearance in a cohort analysis from a Phase 2 open-label study of RBX2660, standardized microbiota-based drug, for recurrent CDI. Methods This prospective, multicenter, open-label Phase 2 study enrolled subjects with recurrent CDI. Participants received up to 2 doses of RBX2660 delivered via enema with doses 7 days apart. Patients were requested to voluntarily submit stool samples at baseline and at 7, 30 and 60 days, 6, 12, and 24 months after the last administration of RBX2660. Stool samples were tested for VRE using bile esculin azide agar with 6 µg/mL vancomycin and gram staining. Vancomycin resistance was confirmed via blood agar and etest. Results Stool samples were available for 143 patients. Twenty-one patients were VRE-positive at the first test (baseline or 7 day). Of the 19 VRE-positive patients that provided additional samples at later timepoints, 18 (94.7%) converted to negative as of the last available follow-up (30 or 60 days and 6, 12, or 24 months). The remaining patient remained positive at all follow-ups. Conclusion This cohort analysis of VRE-positive patients within an rCDI population provides additional support that microbiota-based formulations, such as RBX2660, may have additional benefit beyond reducing the recurrence of CDI. Additional study is needed to confirm the role of microbiome restoration on VRE clearance. Disclosures All authors: No reported disclosures

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A Novel Prebiotic Supplement Increases Bifidobacteria Abundance in Participants Consuming Low-Fiber Diets

Current Developments in Nutrition ◽

10.1093/cdn/nzab054_018 ◽

2021 ◽

Vol 5 (Supplement_2) ◽

pp. 1163-1163

Author(s):

Jea Woo Kang ◽

Xinyu Tang ◽

Angela Zivkovic

Keyword(s):

Relative Abundance ◽

Lactate Production ◽

Random Order ◽

Daily Basis ◽

Metagenomic Sequencing ◽

Funding Sources ◽

Diet Records ◽

Stool Samples ◽

Double Blinded ◽

Usual Diet

Abstract Objectives The objective of this study was to determine whether a novel fiber supplement consumed by healthy individuals with a habitual diet low in fiber (<15 g/day) increases the proportion of saccharolytic gut microbiota which is associated with the increase in the production of SCFA and their related genes in stool without changing their usual diet. Methods Twenty individuals were enrolled in this double-blinded, randomized order, placebo-controlled, cross-over study. Participants were young, healthy, normal to overweight (BMI 23.0–32.0) and consumed < 15 g/day of fiber. All participants consumed a fiber and placebo supplement for a period of 4 weeks each, with a 4-week washout between intervention arms in random order. Participants recorded their diet for 3 days using 24-hour diet record at each 2-week segment. The diet was patternized each week (i.e., participants were asked to consume the same meals and foods for the 3 days prior to each test day) without significantly changing the participants’ usual diet. The fiber packets contained 12 g/serving per day as a powder containing resistant starch, fructooligosaccharide, sugarcane fiber, and inulin while the placebo packets contained 12 g/serving per day of a powder that matched the fiber supplement in taste and appearance. The powder packet was mixed with water for consumption. Stool samples were collected every 2 weeks throughout the study, and metagenomic sequencing and SCFA analysis was performed. Results The concentration of SCFA measured in the stool sample did not change after the intervention. However, the relative abundance of one of the well known saccharolytic bacteria, Bifidobacterium, increased after the fiber supplementation. Genes related to acetate and lactate production, poxB (P = 0.04) and ldh (P = 0.07) respectively, showed tendency to increase which aligns with the increase in the relative abundance of Bifidobacterium in stool samples. No significant changes and correlations were found with anthropometrics and diet records. Conclusions A small amount of fiber supplemented on a daily basis to individuals consuming low fiber diets resulted in an increase in the relative abundance of the beneficial gut microbial genus, Bifidobacterium. Funding Sources I would like to acknowledge Usana Health Sciences, Inc. for the support in this research.

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Intermixing of Commercial Pure Breed Chickens with Indigenous (Sakini) Breed of Nepal

Journal of Nepal Agricultural Research Council ◽

10.3126/jnarc.v7i1.36927 ◽

2021 ◽

Vol 7 ◽

pp. 92-96

Author(s):

Neena Amatya Gorkhali ◽

Chhiring Sherpa ◽

Mana Raj Kolachhapati ◽

Bhoj Raj Pokharel ◽

Nirajan Bhattarai ◽

...

Keyword(s):

Rhode Island ◽

Genetic Erosion ◽

Variable Region ◽

Production Performance ◽

Indigenous Chicken ◽

Three Samples ◽

The Status ◽

D Loop ◽

The Government ◽

Commercial Chickens

The intermixing of pure breeds of commercial chickens with Nepalese indigenous flock of chickens has not been explored yet genetically. The study aims to investigate the status of interbreeding of Nepalese indigenous chickens with exotic breeds. Thirty three samples of indigenous chicken, Sakini, from three different ecological regions, Terai, mid-hill and high hill, were taken for the study. The 522 bp hyper-variable region of D loop mtDNA of each sampled population was PCR amplified and sequenced. The Neighbor joining tree revealed that most of Nepalese Sakini and commercial chickens such as New Hampshire, Rhode Island Red, White Leghorn, White Plymouth Rock and Barred Plymouth Rock lied in same clade. Our study provides first direct evidence that the Sakini was already bred with commercial breeds for the sake of enhancing production performance and also alarms the government to introduce proper strategies and policies to avoid such genetic erosion.

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