PMA-PhyloChip

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:

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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

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

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.

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

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

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.

Microbes in Toxic Mud

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.

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

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.