Second Genome

PMA-PhyloChip

Todd — Wed, 14 Dec 2011 22:15:05 +0000

PMA-PhyloChip DNA Microarray To Elucidate Viable Microbial Community Structure

On December 1st, 2011 NASA’s Jet Propulsion Laboratory in Pasadena California posted this brief on a technique for differentiating live versus dead bacteria in complex samples using a combination of sample treatment with propidium monoazide (PMA) followed by community characterization with Second Genome's PhyloChip assay. The article is reprinted here with permission from JPL-NASA and Tech Briefs magazine.
You can visit the original article at:
http://www.techbriefs.com/component/content/article/12241

Please contact us if you plan to use or test this approach in your research. There may be special boxes to mark on your Specimen Shipment Forms (SSFs) and Data Analysis Plans (DAPs) to inform our lab of the additive. Currently, Second Genome does not perform the PMA step but we do provide the downstream lab operations and analysis from your isolated gDNA.

Tech Briefs Article:

This technology has applications in pharmaceutical and medical equipment manufacturing, and food processing.

Since the Viking missions in the mid- 1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a “true” estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells.

The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method.

DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours.

Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Second Genome: Shaping a Better World

Todd — Wed, 23 Nov 2011 17:23:00 +0000

Every year the Association of University Technology Managers (AUTM) identifies the world’s most innovative technologies for their Better World Report. This year’s report, “Respond, Recover, Restructure: Technologies Helping the World in the Face of Adversity” features 23 innovations that have changed the way we live and includes our PhyloChip technology in the category of “Technologies to Improve Health” section. The report features the story of how PhyloChip was developed at the Lawrence Berkeley National Laboratory based on the potential of the 16S ribosomal gene and the development of DNA micorarrays and how it took a chance conversation to turn an experimental technology into a commercial one. It also details the power and accuracy of PhyloChip and its wide range of uses including detecting oil-digesting bacteria in the Deepwater Horizon spill, assessing the health of coral reefs, and understanding the human microbiome.

PhyloChip’s worthy companions in the report include a device for analyzing the function of knee joints while they are in motion; the first blood test to diagnose brain injuries; a new high-yield wheat strain; and a small, inexpensive water filter developed from a teabag that can be mass produced and shipped to areas of the world stricken by natural disasters and poverty. “Inclusion in this report re-affirms the importance of microbiome research in finding solutions to medical and environmental problems”, says Todd DeSantis, one of the inventors of PhyloChip. “We know that bacteria live all around us and even within our bodies. Understanding the interplay between the microbiome and the human genome is fundamental to understanding human health. Comparing the gut bacteria of patients suffering from Irritable Bowel Syndrome, for instance, with those in healthy subjects reveals that certain, but not all bacteria, exhibit a population increase with the disease. Bacteroides vulgatus was identified with the PhyloChip as one of those bacteria in a study conducted at Baylor College of Medicine (Saulnier et al., Gastroenterology 2011).”

The Better World Report is part of the Better World Project, which aims to promote public understanding of how new technologies improve quality of life for us all. The report was sponsored by AstraZeneca, Lehigh University, and Massachusetts Institute of Technology among others. Copies of the report are available from betterworldproject.net.

About the author: Ruth Warre is a freelance scientific writer and editor currently living in Toronto. She writes on a variety of subjects from microbiomes to neuroscience, in a variety of mediums from blogs to peer-reviewed articles.

Dysbiosis in Autism

Judy — Tue, 01 Nov 2011 06:22:43 +0000

The defining characteristics of autism spectrum disorders (ASD) are impairments in cognition and social function. However, gastrointestinal (GI) symptoms have also been observed to be present in many individuals with ASD and the presence of those symptoms has been correlated with autism severity. Indeed, dietary modification and treatment with vancomycin have been reported to improve the social and cognitive function of autistic children. A recent study by Dr. Brent Williams and colleagues, published in the online journal PLoS One, goes one step further by examining the deficiencies in intestinal gene expression and microbial dysbiosis that underlie the presence of GI problems in children with autism.

Image Credit: Sebastian Kaulitzki, Dreamstime.com

In the study, the research team took ileal and cecal biopsies from 15 children with autism and GI problems and 7 children suffering only from GI problems (control group). All the children were male and aged between 3 and 5 years of age. Human DNA and mRNA was extracted from the biopsies and used to identify the expression levels of various genes involved in carbohydrate digestion and transport. Bacterial 16S rRNA was extracted in order to identify the composition of the bacterial community present in the children’s intestines. The GI problems experienced in the two groups of children were similar with the majority suffering from food allergies including milk-related and/or wheat-related allergies. The GI symptoms were also similar in both groups and included diarrhea and changes in stool frequency and consistency.

Despite the similarities in GI problems between the two groups of children, gene expression analysis indicated that, compared to the control group, 14 out of the 15 autistic children had deficiencies in expression of at least 1 or more of the 5 disaccharidase and hexose transporter genes involved in carbohydrate digestion and transport. The expression of the genes in question is regulated by caudal homeobox 2 (CDX2) and deficiencies in expression of CDX2 were also observed in the autistic children. Changes in carbohydrate digestion and absorption due to deficiencies in gene expression are likely to influence the composition of the gut microbiome. Indeed, 16S rRNA sequencing indicated that although the overall microbiome diversity was similar between the two groups of children, the autistic children had significantly lower abundances of Bacteriodetes, increases in the ratio of Firmicutes to Bacteriodetes, and increases in Betaproteobacteria.

The causes and potential effects of these changes in gut function are difficult to identify but it is likely that the deficiencies in gene expression are likely to lead to maldigestion and malabsorption as well as microbial dysbiosis. There is also some evidence that in addition to causing GI problems, dysbiosis can have system-wide effects including alterations in immune responses and brain development and behavior. The identification of distinct changes that occur in autistic children with GI problems takes one step further towards understanding this multifaceted condition.

About the author: Ruth Warre is a freelance scientific writer and editor currently living in Toronto. She writes on a variety of subjects from microbiomes to neuroscience, in a variety of mediums from blogs to peer-reviewed articles.

Article:

Brent L. Williams, Mady Hornig, Timothy Buie, Margaret L. Bauman, Myunghee Cho Paik, Ivan Wick, Ashlee Bennett, Omar Jabado, David L. Hirschberg, and W. Ian Lipkin. (2011). Impaired Carbohydrate Digestion and Transport and Mucosal Dysbiosis in the Intestines of Children with

Autism and Gastrointestinal Disturbances PLoS ONE 6(9): e24585.

DOI: 10.1371/journal.pone.0024585

Gut microbes in cows and hoatzins

Judy — Thu, 27 Oct 2011 01:15:18 +0000

In order to effectively access the energy stored in vegetation, cows and other ruminants have an enlarged foregut that contains a variety of microbes that aid in the breakdown of the otherwise indigestible plant matter through fermentation. Interestingly this trait is not only common to various mammals including cows, sheep, deer, and sloths but is also present in the hoatzin, a folivorous bird that feeds on young plant leaves. The presence of foregut fermentation in both cows and hoatzins is an example of evolutionary convergence that Dr. Filipa Godoy-Vitorino and her colleagues in a team lead by Dr. Maria Gloria Dominguez-Bello used to answer the question of whether host phylogeny or organ function contributes to the evolution of gut microbial communities.

In their study, published recently in the ISME Journal, Dr. Godoy-Vitorino and her colleagues used PhyloChip to analyze the microbes present in the foreguts and hindguts of four cows and four hoatzins. The team chose to use PhyloChip as it is “a fast and economic way to compare profiles of microbiota between different ecosystems.” The results indicated that there was “a clear 'core' microbiota for the foregut organs and a 'core' microbiota for the hindgut organs, regardless of the independent origin of foregut fermentation in birds and mammals.” This core microbiota for the foregut included populations of Bacteriodetes, Acidobacteria, and Spriochaetes but fewer Proteobacteria and Firmicutes than the hindgut. In addition to the core microbiota there were a variety of other microbes present with the cow digestive organs containing a higher number of species than the hoatzin. There were also microbes that were present only in the foreguts and not in the hindguts including various Bacteriodetes, Cyanobacteria, Lentisphaerae, Planctomycetes, and Spirochaetes species. A broad UniFrac comparison between hoatzins, cows and various other ruminants and birds showed that the hoatzin microbiota is “more similar to that of foregut fermenter mammals than to organs from other birds.”

As Dr. Godoy-Vitorino says “our study clearly shows that organ function is a stronger determinant of microbial community structure than host phylogeny. Despite the phylogenetic distance of the hosts (bird and mammal), the fact that they have a similar digestive strategy, their fermentative organs play similar roles and thus we find 'core' taxa in all of them.” As the next step in their research the team is currently analyzing the metagenome of the hoatzin foregut in order to create an inventory of the genes present. The identification of genes present in foregut fermenters is potentially useful to the biofuel industry. As Dr. Godoy-Vitorino points out, “to stop our dependency in fossil fuels we might be able to modify bacteria to express carbohydrate-active enzymes (found for example in the hoatzin or cows) to degrade, wood, paper, etc., to the ultimate production of alcohol to be used as alternative source of energy.”

Article:

Filipa Godoy-Vitorino, Katherine C Goldfarb, Ulas Karaoz, Sara Leal, Maria A Garcia-Amado, Philip Hugenholtz, Susannah G Tringe, Eoin L Brodie, and Maria Gloria Dominguez-Bello. (2011). Comparative analyses of foregut and hindgut bacterial communities in hoatzins and cows. The ISME Journal, advance online publication, 22 September 2011.

DOI: 10.1038/ismej.2011.131

Microbes in Toxic Mud

Judy — Tue, 18 Oct 2011 02:26:40 +0000

For 125 years the waste water from the sulfidic ore mines of Silver Valley in northern Idaho has drained in to the Coeur d’Alene River. As a result the sediments of the river are heavily enriched with high concentrations of toxic metals such as arsenic, iron, lead, and zinc. Although it is hard to imagine that life might thrive in such conditions a recent study by Dr. Gurdeep Rastogi and colleagues, published in Microbial Ecology, used PhyloChip technology to investigate the diversity of microbes that live within the sediment.

Image Credit: Sharon Lebrun, Dreamstime.com

Dr. Rastogi and his colleagues took sediment cores from the riverbed downstream of the mines, extracted microbial DNA, and then used several techniques to identify the microbes present in the sediment. The use of a bacterial 16S rRNA clone library detected 60 operational taxonomic units (OTUs) with the most common being Proteobacteria (53 clones), followed by Actinobacteria (9 clones) and Bacteriodetes (8 clones). In contrast to these results, the use of PhyloChip identified a total of 1,571 OTUs; again the most common phylum was Proteobacteria (48.5% of total OTUs), followed by Firmicutes (17% of total OUTs) and Actinobacteria (10% of total OTUs). In addition there were 22 OTUs that could not be classified at the phylum level suggesting that there are as yet unclassified microbes living in the river sediments.

Within the highly diverse population of microbes present in the sediment were many bacteria that have adapted to living in such hostile conditions and in some cases may even help in limiting the effects of the contamination. Proteobacteria, such as Pseudomonas and Burkholderia for example, are well known for their ability to survive at extreme pH and also to deal with toxic heavy metals. In addition the PhyloChip data also indicated the presence of Arthrobacter spp. from the phylum Actinobacteria, which are heavy metal resistant, sulfate-reducing bacteria such as Desulfobacterium and Desulfonauticus, Fe(III)-reducing bacteria including Shewanella and Brevibacillus, Fe(II)-oxidizing bacteria such as Leptothrix, as well as ammonia-oxidizing bacteria such as Nitrosospira and Nitrosomonas. Although direct comparisons with uncontaminated river sediment still need to be performed the presence of such a broad community of microbes indicates that heavy-metal contamination may alter but does not necessarily reduce the diversity of microbe populations in river sediment.

Article:

Gurdeep Rastogi, Sutapa Barua, Rajesh K. Sani, and Brent M. Peyton. (2011). Investigation of Microbial Populations in the Extremely Metal-Contaminated Coeur d'Alene River Sediments. Microbial Ecology, 62:1–13.

DOI: 10.1007/s00248-011-9810-2

Microbes and Medicines

Judy — Tue, 11 Oct 2011 00:35:05 +0000

The metabolism of drugs, and therefore their effects, can vary widely from person to person and attempts to understand the genetic and phenotypic reasons behind this have led to the rise of the field of pharmacogenomics. Unfortunately, not only can genetic differences result in altered drug metabolism but also environmental factors such as gut bacterial activity, disease, and age. To counter this fact a pharmacometabonomic approach has been suggested that takes into account measurements of metabolites and biomarkers prior to drug administration. In a recent study, published in PNAS, Dr. Jeremy Nicholson and his team used this approach to demonstrate that a person’s gut microbes can affect their ability to metabolize acetaminophen.

Image Credit: Dana Rothstein, Dreamstime.com

In the study 99 healthy male volunteers provided urine samples before (predose) and after (postdose) being given 1000 mg of acetaminophen. The samples were then analyzed by NMR spectroscopy to identify endogenous metabolites in the predose samples and acetaminophen-related compounds in the postdose samples. Acetaminophen sulfate (S) and acetaminophen glucuronide (G) are two common metabolites of acetaminophen that are produced through two different metabolic processes and accounted for approximately 85% of the acetaminophen-related compounds detected in the urine samples. The relative contribution of either sulfonation or glucuronidation to acetaminophen metabolism is known to vary greatly between people thus the team was interested in any correlations between intersubject variations in the S/G ratio and intersubject variations in any predose metabolites.

In the analysis of the pre- and postdose metabolites only one significantly correlated with the variations in the S/G ratio—p-cresol sulphate (PCS), higher levels of which were associated with a lower S/G ratio. PCS is produced by the sulfonation of p-cresol which is a byproduct of the metabolism of tyrosine by colonic bacteria. The sulfonation of p-cresol occurs not within bacteria but within human tissue, such as the colonic mucosa and the liver, and is mediated by the same enzyme that sulfonates acetaminophen. Thus the results suggest that a high level of circulating PCS significantly decreases the ability of the body to sulfonate acetaminophen which could increase the risk of acetaminophen-induced liver damage.

Although this potential risk would need to be investigated further the finding also has consequences for various processes such as the metabolism of other drugs, the production of biomolecules (e.g. chondroitin sulphate, a component of cartilage), and the sulfonation-related modulation of various hormones and neurotransmitters as well as various diseases including hyperactivity in children and multiple sclerosis, which have both been associated with high levels of p-cresol and/or PCS. Overall, this study indicates that gut microbes could play a significant role in drug efficacy and adverse drug reactions and highlights once again the importance of the gut microbiome to humans in both health and disease. Indeed, the authors conclude that “assessing the effects of microbiome activity should be an integral part of pharmaceutical development and of personalized health care.”

Article:

T. Andrew Clayton, David Baker, John C. Lindon, Jeremy R. Everett, and Jeremy K. Nicholson. (2011). Pharmacometabonomic identification of a significant host-microbiome metabolic interaction affecting human drug metabolism. PNAS, 106(34): 14728–14733.

DOI: 10.1073/pnas.0904489106

Oral microbiome, Periodontitis

Judy — Tue, 27 Sep 2011 23:26:46 +0000

The oral microbiome contains over 600 species of microbiota. Under normal conditions the predominant phyla are Firmicutes, Bacteriodetes, Proteobacteria, Actinobacteria, and Fusobacteria. The most abundant bacterial genera in the oral microbiome are members of the genus Streptococcus, but Abitrophia, Gemella, Granulicatella, and Prophyromonas are also extremely common. P. gingivalis is often present in the oral microbiome where it colonizes the oral epithelium and can exist without causing any overt symptoms of disease. However, the microbe expresses a number of virulence factors including extracellular proteases that can modulate the immune system and affect epithelial cell proteins and receptors, a lipopolysaccharide that also induces a pathogenic immune response, and other proteins that allow the bacteria to invade host cells. Once inside the cells the toxins produced by the infection can cause cell death and tooth decay which eventually spreads to the underlying bone. Despite this, some research suggests that the overall levels of P. gingivalis do not differ between healthy individuals and those with periodontitis. Rather than the simple presence of the bacteria in the epithelial cells it is hypothesized that it is an imbalance in the interaction between the bacteria and the local and systemic environment, possibly mediated by an inappropriate immune system response, that results in disease.

P. gingivalis and systemic disease

Image credit: Sandor Kacso, Dreamstime.com

In addition to being associated with gingivitis and periodontitis the presence of P. gingivialis and the occurrence of periodontitis have been associated with cardiovascular diseases, preterm delivery of low birth weight babies, diabetes mellitus, respiratory diseases, osteoporosis, and rheumatoid arthritis. The mechanisms underlying the connection between the oral presence of the bacteria and systemic disease are thought to involve systemic inflammatory responses, direct infection, and cross-reactivity between bacterial antigens and self-antigens. In addition, the bacteria have been detected in the plaques that are a pathological hallmark of atherosclerotic heart disease. The presence of periodontitis in a patient has also been declared to be a significant risk factor for atherosclerosis along with smoking, high cholesterol, hypertension, and diabetes. Experiments in animal models have shown that the presence of P. gingivalis in the oral cavity accelerates that formation of atherosclerotic plaques. The mechanism behind this is unknown but it indicates that the bacteria can enter the bloodstream through epithelial cells of the mouth that have been weakened by disease to cause system wide effects.

References

The human oral microbiome. Dewhirst FE et al. J Bacteriol. 2010; 192(19): 5002–5017. DOI: 10.1128/JB.00542-10. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944498/?tool=pubmed
Defining the health “core microbiome” of oral microbial communities. Zaura E, Keijser BJ, Huse SM, and Crielaard W. BMC Microbiol. 2009; 9: 259. DOI: 10.1186/1471-2180-9-259. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805672/?tool=pubmed
The chronicles of Porphyromonas gingivalis: the microbium, the human oral epithelium and their interplay. Yilmaz O. Microbiology. 2008; 154(Pt 10): 2897–2903. DOI: 10.1099/mic.0.2008/021220-0. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2639765/?tool=pubmed
Roles of oral bacteria in cardiovascular diseases—from molecular mechanisms to clinical cases: Implication of periodontal diseases in development of systemic diseases. Inaba H and Amano A. J Pharmacol Sci. 2010; 113(2): 103–109. http://www.jstage.jst.go.jp/article/jphs/113/2/113_103/_article
Rheumatoid arthritis is linked to oral bacteria: etiological association. Ogrendik M. Med Rheumatol. 2009; 19(5): 453–456. DOI: 10.1007/s10165-009-0194-9. http://www.springerlink.com/content/u0308p11j2800205/
The American Journal of Cardiology and Journal of Periodontology Editors’ Consensus: periodontitis and atheroscletoric cardivascualr disease. Freidewald VE et al. Am J Cardiol. 2009; 104(1):59–68. http://www.ajconline.org/article/S0002-9149%2809%2901025-X/abstract.

Gut Microbiome, Allergies

Judy — Tue, 13 Sep 2011 05:33:31 +0000

The “hygiene hypothesis” suggests that the increase in allergic and autoimmune diseases such as asthma, allergic rhinitis (hayfever), and multiple sclerosis in developed countries is linked to a decline in infectious burden that causes an immune imbalance. One particular theory is that certain infectious agents that co-evolved with us, including probiotics such as Lactobacillus, are protective against the development of allergic disease. New research into the infant gut microbiome and the development of childhood allergies adds weight to this theory. In a study published online in the Journal of Allergy and Clinical Immunology, Dr. Hans Bisgaard and colleagues show that infants with a less diverse gut microbiome have an increased risk of developing allergic sensitization and allergic rhinitis as they grow up.

Image Credit: Corepics Vof, Dreamstime.com

In the study Dr. Bisgaard and colleagues used 16S rRNA PCR and conventional culturing to identify the fecal bacteria present in 253 infants. Samples were taken when the children were 1 month and 12 months old and the children were then monitored every 6 months until they were 6 years old. In addition to the fecal bacterial analysis IgE levels in response to a panel of allergens were assessed as was the response to a skin prick test for the same allergens and the levels of peripheral blood eosinophils. When the children reached 6 years old the presence or absence of allergic rhinitis, asthma, and atopic dermatitis was identified.

The analysis of fecal bacteria showed that the diversity of the gut microbiome increases between 1 month and 12 months of age. In particular the prevalence of enterobacteriaceae, enterococcaceae, yeast, and fungi increased but the abundance of staphylococcaceae decreased. The level of diversity present was inversely associated with the development of sensitization as measured by serum IgE levels, skin prick test results, peripheral blood eosinophilia, and the presence of allergic rhinitis at 6 years of age. There was, however, no increase in the risk of asthma or atopic dermatitis at the age of 6, possibly indicating differences in the mechanisms of development of these two conditions.

Interestingly, sensitization as measured by IgE levels was associated with the presence of staphylococcaceae at 1 month but not at 12 months and no other single bacterial genera was implicated in the development of allergic sensitization. This suggests that having a diverse range of microbiota in the gut is more important than the presence or absence of a particular bacterial strain. The next step in this research is to identify the mechanisms by which the gut microbiome influences the development of allergic responses.

The Inside Scoop on Poop

Judy — Tue, 13 Sep 2011 01:14:46 +0000

We often receive questions from researchers starting fecal microbiome studies about recommended procedures and expected yields for microbial DNA isolation, as well as proper storage, processing, and shipping conditions.

So, to help you successfully plan your project, we've tried to do the dirty work of gathering tips and tricks from leading experts in fecal DNA isolation and microbiome analysis so that you can get to the…um…dirtier work of starting your own microbiome study.

(To download the full white paper, click here)

To gather the best information out there, we spoke with Joseph Petrosino, Assistant Professor of Molecular Virology & Microbiology at Baylor College of Medicine, who helped lead procedure development for specimen collection and DNA isolation for the Human Microbiome Project (HMP), Suzanne Kennedy, Director of Research and Development at MO BIO Laboratories where they have pioneered tools for microbial DNA isolation, and Kate Patterson, research associate at TGA Sciences where they support the pharmaceutical, biotechnology, and academic communities by providing laboratory services for DNA isolation.

The following 5 hints summarize solutions that have worked in their laboratories and will hopefully extend to your project.

1. Catching the specimen:

For rodents, collecting fecal pellets is relatively straightforward. While there are cages that you can purchase that collect the feces away from the cage floor, they can be expensive and may be overkill for this type of study. Researchers can pick up fecal pellets with sterile gloved hands and place them in a sterile container like a BD Falcon™ or Eppendorf tube.

With human stool, it gets a little messier, so to speak, but talking to our experts, there is a "lot of forgiveness" in the different methods. Many labs provide patients/subjects with a bucket-like tray, sometimes referred to as a “hat” that fits under the toilet seat for catching the specimen. Another alternative is for the subject to use newspaper to catch the specimen, which allows more moisture to be absorbed. It's key that the subject is provided with instructions for avoiding contamination from toilet water or the side of the toilet. These types of instructions should also include a recommendation that the subject urinate first to avoid specimen contamination.

If you are collecting swabs from the anal area, be sure to use a sterile swab, allow it to air dry, and enclose the swab in a capped sterile transport container. Swabs should be allowed to dry for an hour, to avoid microbial DNA degradation upon freezing and storage.

2. Transporting and storing of the specimen:

Human feces collection kit available through Second Genome

Once the specimen is caught it can be kept in the same container and stored prior to processing. Alternatively, the specimen may be transferred into a specific sterile collection container.

If the specimen is collected in the subject's home, the specimen collection container should be frozen in the subject's freezer and shipped on ice packs to the laboratory for processing. For specimens collected in a laboratory environment, specimens should also be frozen immediately. For the HMP project, specimens were frozen in their collection buckets at -80°C and processed for DNA isolation within 24 to 48 hours. One of our experts observed that the microbial community profiles of specimens that were frozen for a year did not exhibit large variation from specimens that were processed immediately.

For microbial DNA profiling, our experts agreed that using a stabilization solution like Qiagen RNAlater for storage is probably not necessary and may interfere with downstream DNA isolation. One of our experts observed that stool samples stored in RNAlater separated into different solution phases when processed and DNA recovery was decreased.

3. Handling human stool from healthy and diseased individuals:

Human fecal samples from healthy or diseased individuals should be processed under BSL-2 conditions, including the use of a facemask, gloves, and lab coat. Basically, treat all samples as if you don't know whether they contain an infectious agent like HIV.

Stool from individuals experiencing constipation may require additional subsequent bead-beating as part of the DNA isolation process and loose stool may require a pre-processing step to spin out excess liquid.

4. Isolating DNA:

While there are many published methods for microbial DNA isolation from fecal samples, all of our experts recommended the use of bead-beating methods and MO BIO PowerSoil® isolation kits. Non-bead beating methods don’t recover gram-positive bacterial DNA as well. With the MO BIO PowerSoil® isolation kits, the final eluted DNA is enriched for microbial DNA, making PCR more sensitive. Human or animal epithelial cells will lyse quickly under the heating and beating conditions used for microbial lysis, releasing the free DNA into the supernatant. The free DNA is subjected to prolonged mechanical breakage resulting in small fragment sizes that are washed out of the column during the binding step. The microbial DNA remains as high molecular weight fragments and is retained on the column.

Our experts observed that bead-beading methods like those used in the PowerSoil® kit generally yield approximately 100 µl of 2 to 20 ng/µl of total genomic DNA from 0.1 g of stool. Starting with a range of 0.1 g to 0.25 g of human feces is ideal. For dry mouse feces, our experts recommended working with less starting material, closer to 0.1 g, or diluting the samples 1:4 or 1:5 so that sufficient lysis solution can be absorbed. They suggest using the beads provided in the kit and found other separately purchased glass or silica bead products to be unnecessary.

One expert has observed that yields from human stool retain approximately 2% human DNA. In contrast, other types of samples, like vaginal swabs, may contain up to 80% human DNA post-isolation.

5. Removing PCR inhibitors

Stool is similar to soil in that it contains a high concentration of PCR inhibitors. Further, these inhibitors will vary between individuals based on their diet. These inhibitors often bind to the silica membranes of spin columns and co-elute with DNA. Samples with this problem will often show a brown or yellow color after purification and low purities when measured with spectrophotometry. Again, because of these properties, our experts recommended using the MO BIO PowerSoil® DNA Isolation kit because it incorporates Inhibitor Removal Technology® (IRT) to remove humic acids, polyphenols, polysaccharides, heme, and dyes prior to binding of lysates to the spin column, resulting in high purity DNA and more accurate microbial profiles after amplification.

Final Thoughts

If you have additional questions about these recommendations, don’t hesitate to contact us at info@secondgenome.com. Or, if you have your own tips or tricks that you'd like to add, please post a comment to this article.

Finally, Second Genome offers DNA isolation services for human stool, animal feces, solid tissue, swabs, blood, and frozen water filters. We also offer specimen collection and transport kits. If you’d like to learn more about these solutions or our microbial profiling services in general, contact us at info@secondgenome.com.

References:

1. Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis. Julia R. deLipthay et al. (2004) Soil Biology and Biochemistry. 36;10:1607-1614.

2. Manual of Procedures for Human Microbiome Project.

Gut Microbes and Colorectal Cancer

Judy — Fri, 02 Sep 2011 21:35:08 +0000

Colorectal cancer (CRC) is the second most common cause of death from cancer in the United States. Early diagnosis is important in successful treatment but detection requires regular screening through the use of colonoscopies in at-risk patients, an unpleasant and lengthy process for the patient. Research suggests that some species of gut bacteria, either acquired as an infection or naturally occurring in the gut microbiome, play a role in the pathogenesis of CRC but there is little clinical data to confirm the particular microbiome composition associated with CRC. An understanding of the CRC microbiome could provide useful information for the development of more efficient, effective, and patient-friendly diagnostic tools and treatments for CRC. In a paper recently published online in PLoS One, Dr. Julian Marchesi and colleagues go one step further towards this goal by providing a high-resolution map of the microbiome of CRC tumor tissue.

In the study, samples of tissue from primary colon tumors and from adjacent non-malignant tissue were removed during surgery in six patients with CRC. Interestingly the microbial communities present in the tumor tissue were significantly different from those present in the normal tissue. In general there were more Bacteriodetes and less Firmicutes in the tumor tissue than in the normal tissue. More specifically there was an overrepresentation of Coriobacteridae in the tumor tissue samples and an underrepresentation of Enterobacteriaceae including Citrobacter, Shigella, Cronobacter, and Salmonella. In addition to the differences between the two types of tissue there was no consistent presence of any potential pathogenic bacteria in the CRC tissue.

Image Credit: Sebastian Kaulitzki, Dreamstime.com

The Coriobacteridae are considered to be gut commensal bacteria with probiotic features. Their enrichment in CRC tumor tissues suggests that as a result of the physiological and metabolic alterations that occur during the process of carcinogenesis certain bacterial species are able to thrive in the tumor microenvironment while others are not. In addition, some of the CRC “passenger” bacteria have anti-tumorigenic and anti-carcinogenic properties which the authors argue may prevent rapid progression of the disease. In contrast the underrepresentation of some of the Endobacteriaceae in the tumor tissue compared to the normal tissue suggests that these bacteria may be a part of the intrinsic microbiome in CRC patients. In general Citrobacter, Shigella, Cronobacter, and Salmonella are pathogenic and are found only in low levels in the gut microbiome. The fact that they were detectable in higher levels in these patients may point to their involvement in the development of CRC. However, without a comprehensive understanding of what constitutes a healthy microbiome this idea is only a hypothesis.

In the future, further study of how the microbiome of CRC tissue changes as the disease progresses could be useful in the development of diagnostic tools, particularly if those changes can be detected in the fecal microbiome, and also perhaps in the development of therapeutic interventions.