The PhyloChip Wins a 2008 R&D 100 Award12 min read

The Berkeley Lab Phylochip

Bacteria never live alone. Instead, they grow in complex communities, made up of hundreds of different species. Bacterial communities are found in familiar environments, such as water, soil, food, air, and within our own bodies. Knowledge about these bacterial communities—including the predominant species among them, their interaction, and their changes over time—is essential for understanding the effect of any disturbance within natural ecosystems. Deep, sudden changes in the structure of a bacterial community could represent a danger to us. These changes could take the form of an airborne biological terrorist attack, or an epidemic caused by contaminated water, or soil, or hazardous atmospheric alterations caused by climate change. But how do we know “what’s there,” hidden in those ecosystems? Until now, scientists have had no fully accurate, comprehensive way of detecting the presence, quantity, and diversity of bacteria (including disease-causing microbes or pathogens) in an air, water, soil, or clinical sample. Researchers relied on bacterial cultures to identify what was present in such a sample: The problem with this method, in addition to the time (often days or weeks) that growing the culture requires, is that it leaves out all of the organisms that can’t survive in the culture, which could be as much as 99% of the bacteria in a sample. Moreover, while recent advances in genetic detection technology (for example, PCR assays that detect the presence of a specific gene sequence) have been effective in identifying discrete organisms in samples, they have only been able to test for individual bacteria that researchers anticipate being present in the sample. As in the child’s card game Go Fish, this technology could determine only whether specific bacteria were present, not which bacteria were present. We still would have little idea of “what’s there.” The Berkeley Lab PhyloChip, developed by Gary Andersen, Todd DeSantis, Eoin Brodie, Yvette Piceno, and their colleagues at Berkeley Lab, provides the best answer yet to “what’s there.” The PhyloChip is a microarray unique in its ability to quickly and comprehensively identify multiple bacteria and archaea within microbial DNA samples. Capable of analyzing samples from any environmental source—air, water, soil, blood, tissue—the PhyloChip is unprecedented in its ability to accurately test such samples without any culturing required, and without prior knowledge of a sample’s microbial composition, all in a single test. Easily fitting into a person’s hand, the PhyloChip can simultaneously (i.e., within one testing sample) detect most known microorganisms—testing for over 8,000 bacterial species, a microbial detection power previously unknown. Also, its ability to produce useful results in a matter of hours means that numerous samplings of a specific environment can be conducted virtually on a daily basis, enabling scientists to track, as never before, the progress (i.e., appearance/disappearance, increase/decrease) of a certain microorganism over a short period of time.

Berkeley Lab PhyloChip Operation

Inside the PhyloChip’s disposable cartridge is a glass surface (called a microarray) divided into a grid of 356 rows × 712 columns, resulting in 253,472 separate tests, each capable of capturing a specific nucleic acid. When the DNA or RNA molecules from the sample (soil, blood, etc.) come into contact with the short pieces of DNA bound to the glass surface, each molecule adheres only to the appropriate location (refer to image describing the PhyloChip Operation). The Berkeley Lab PhyloChip can distinguish microbes based on how well their ribosomal DNA or RNA anneals, or “sticks,” to each of the many test sites (refer to image showing bacteria types).

PhyloChip operation: (a) Multiple tests conducted on a single glass surface; (b) DNA from a sample (blood, soil, water, etc.) adheres where a match is found (“hybridization”); (c) Laser scanning reveals which tests were positive (i.e., which microbes are present). In this way, PhyloChip quickly and accurately identifies microbes in complex samples. (Images provided by Affymetrix, Inc., Santa Clara, CA.)

Bacterial types (a) contain ribosomes (b) that are composed of a specific sequence of DNA/RNA bases (c), allowing them to be differentiated. The PhyloChip detects these differences, and thus is the first tool able to classify all types of DNA/RNA sequences in complex mixtures. (Images provided by Affymetrix, Inc., Santa Clara, CA.)

The cartridge is inserted into a scanner that detects which tests are positive by the emission of fluorescent light only from the test sites bound by nucleic acids in the sample. The intensity of the fluorescence from each test corresponds to the quantity of organisms in the sample—allowing ecosystem comparison over time to determine which bacterial populations are changing. All microorganisms have ribosomal genes because these genes (as protein generators) are essential for life. The small sequence differences within the ribosomal gene are what distinguish different species. The PhyloChip has divided all known sequence variations from bacterial and archaeal ribosomal genes into over 8,000 distinctive groupings, each representing a specific microbial genus or species identified from many different sampled environments—including clinical, air, soil, and water.

Given its ability to perform over 250,000 tests simultaneously, the PhyloChip does not require customization for different types of samples, since the total diversity of all known ribosomal genes that could be present within any media is represented within the 250,000 tests. With a comprehensive assay for all currently identified bacteria, the PhyloChip eliminates before-the-sampling guesswork as to what bacteria exist within a sample, taking advantage of (and incorporating into the PhyloChip) the vast amount of ribosomal sequence data available in public databases. As these databases grow, updated versions of the PhyloChip can easily include novel diversity, because room exists on the chip surface for over 1 million tests. Moreover, the PhyloChip’s high-density format, combining tests for each species  with paired mismatch-control tests, significantly reduces the chances of misidentifying a specific microorganism. These PhyloChip capabilities allow for unprecedented accuracy in characterizing a microbial sample—all within a day.

Berkeley Lab PhyloChip Validation, Accomplishments, and Contributions

“The PhyloChip has given us an unparalleled view of the bacterial community…there is nothing else as comprehensive or sensitive.”—Dr. Kasthuri Venkateswaran, Jet Propulsion Laboratory In its short existence, the Berkeley Lab PhyloChip has already achieved considerable success. It has been validated (refer to next image) through an extensive collection of air samples obtained for identifying microbial communities typically inhaled by inhabitants of U.S. metropolitan cities. In addition, preliminary PhyloChip testing of water and soil samples has shown that this technology is feasible in those environments as well. Findings made possible by the PhyloChip have led to publications in a number of scientific journals (jump to list of peer-reviewed articles) and attracted extensive press coverage.

Validation of PhyloChip sensitivity. The figure shows how mixtures of nine bacteria (shown in the legend) in varying concentrations are accurately resolved by the PhyloChip. Fluorescence intensity correlates well with the quantity of bacteria in the sample, revealing the bacterial community structure. The PhyloChip is sensitive over a wide dynamic range—it can simultaneously detect large amounts of one bacterial species and small amounts of another. No other device has the ability to profile both the predominant and the minority bacterial populations.

In 2004, Berkeley Lab researchers conducted (for the U.S. Department of Homeland Security) a first-of-its-kind cataloguing of microbes taken from air samples above the Texas cities of San Antonio and Austin. Before this study, no one had a sense of the diversity of microbes in the air. Investigators found over 1,800 diverse bacterial types, a much richer and more varied population than anyone expected (Figure 5), rivaling that of soil microbial diversity. This research, described in a paper published in the Proceedings of the National Academy of Sciences and reported on in Scientific American in December 2006 (jump to list of peer-reviewed articles), marks the beginning of a regional bacterial census that will help the Department of Homeland Security differentiate between normal and suspicious fluctuations in airborne microbes. It also helps to establish a baseline background of airborne bacteria, which scientists can now use to track how climate change affects bacterial populations.

Closely related bacterial species can be tracked spatially and temporally. This figure shows 13 of the greater than 8,000 bacteria (left axis) profiled from 16 samples (bottom axis) from above San Antonio and Austin. Some bacteria were never found; others were detected in many samples. The height of each bar represents the abundance of each species, while the color indicates detection likelihood (in descending order from red, to orange, to yellow, to white). The PhyloChip allows rapid, inexpensive comparison among samples to reveal community differences, regardless of bacterial culturability.

In addition, the PhyloChip has already been an important part of recent critical medical studies. Respiratory infections caused by the ambient environment are a major problem in hospitals. As reported in the Journal of Clinical Microbiology (2007) (jump to list of peer-reviewed articles), the PhyloChip was used to analyze the microbial environment within respiratory tube airways. It was the key to discovering that a loss of bacterial diversity (resulting from antibiotic treatments) was directly associated with the development of pneumonia in ventilated patients exposed to a certain common strain of bacteria. The potential life-saving possibilities of this finding are obvious.

The PhyloChip has also shown great value in bioremediation cleanup efforts at contaminated sites. When uranium mining and processing for nuclear weapons and fuel were at their peak, in the 1950s and 1960s, uranium-containing wastes accumulated, resulting in a multitude of contaminated sites in the U.S. and worldwide. One promising approach to containing uranium migration is to catalyze the reduction of soluble U(VI) to the less-soluble U(IV). As described in a paper published in Applied and Environmental Microbiology (2006) (jump to list of peer-reviewed articles), the PhyloChip was central in identifying (from soil samples) those bacteria that could, through ingestion of the uranium, prevent U(IV) from converting to soluble uranium U(VI), thus forestalling the migration of this radioactive material and optimizing site remediation efforts. Degraded water quality is also a growing environmental problem. The PhyloChip allows researchers and environmental managers to gain unprecedented knowledge about water-borne microbes to rapidly distinguish between harmful and beneficial species. The PhyloChip has empowered water-resource administrators to assert proper corrective actions within days after a pollution episode (as opposed to months). For instance, in an ongoing study, the PhyloChip was recently used to monitor California creek water suspected to be contaminated by sewage. The PhyloChip allowed researchers to pinpoint the creek locations associated with specific types of human fecal bacteria. Evidence was produced to demonstrate that untreated sewage was entering the creek. Using the results from PhyloChip sampling, public health officials will be able to specify break points in the creek to divert and sanitize the water before it reaches the ocean. Knowing that what we can’t see can affect us in profound ways, we need to be able to detect what was undetectable before now, at a speed inconceivable before now. The Berkeley Lab PhyloChip provides this ability, and in the process changes the way scientists conduct certain basic, essential investigations. The PhyloChip packs an enormous amount of analytical power into a device not much larger than a quarter. Its ability to test all manner of environmental samples for their microbial content is unprecedented. The information that it has already provided about the airborne bacterial content above American cities is a first step in distinguishing between a climate-related bacterial change and a real bioterrorist threat. Moreover, the PhyloChip’s contributions to public health, medical diagnostics, and environmental cleanup projects have already paid large dividends. It promises even more advances in the development of biofuels and carbon sequestration. In short, scientists are continually finding new ways to use the PhyloChip, and finding things that they could not have found by any other means.

Peer-reviewed articles describing or utilizing the PhyloChip

• Wilson, K. H., W. J. Wilson, et al. (2002). "High-density microarray of small-subunit ribosomal DNA probes." Appl Environ Microbiol 68(5): 2535-41.
• DeSantis, T. Z., I. Dubosarskiy, et al. (2003). "Comprehensive aligned sequence construction for automated design of effective probes (CASCADE-P) using 16S rDNA." Bioinformatics 19(12): 1461-8.
• DeSantis, T. Z., C. E. Stone, et al. (2005). "Rapid quantification and taxonomic classification of environmental DNA from both prokaryotic and eukaryotic origins using a microarray." FEMS Microbiol Lett 245(2): 271-8.
• Brodie, E. L., T. Z. DeSantis, et al. (2006). "Application of a High-Density Oligonucleotide Microarray Approach To Study Bacterial Population Dynamics during Uranium Reduction and Reoxidation." Appl Environ Microbiol 72(9): 6288-98.
• Lin, L. H., P. L. Wang, et al. (2006). "Long-term sustainability of a high-energy, low-diversity crustal biome." Science 314(5798): 479-82. Brodie, E. L., T. Z. DeSantis, et al. (2007). "Urban aerosols harbor diverse and dynamic bacterial populations." Proc Natl Acad Sci U S A 104(1): 299-304.
• DeSantis, T. Z., E. L. Brodie, et al. (2007). "High-density universal 16S rRNA microarray analysis reveals broader diversity than typical clone library when sampling the environment." Microb Ecol 53(3): 371-83.
• Flanagan, J. L., E. L. Brodie, et al. (2007). "Loss of bacterial diversity during antibiotic treatment of intubated patients colonized with Pseudomonas aeruginosa." J Clin Microbiol 45(6): 1954-62.
• Wrighton, K. C., P. Agbo, et al. (2008). "A novel ecological role of the Firmicutes identified in thermophilic microbial fuel cells." Isme J 2(11): 1146-56.
• Chivian, D., E. L. Brodie, et al. (2008). "Environmental genomics reveals a single-species ecosystem deep within Earth." Science 322(5899): 275-8.
• Tokunaga, T. K., J. Wan, et al. (2008). "Influences of Organic Carbon Supply Rate on Uranium Bioreduction in Initially Oxidizing, Contaminated Sediment ." Environ Sci Technol in press.
• Yergeau, E., S. Kang, et al. (2008). "Functional microarray analysis of nitrogen and carbon cycling genes across an Antarctic latitudinal transect." Isme J in press.
• DeAngelis, K. M., E. L. Brodie, et al. (2008). "Selective progressive response of soil microbial community to wild oat roots." Isme J in revision.
• Kuramae, E. E., H. Gamper, et al. (2008). "Prokaryotic succession in chalk grasslands after field abandonment."  in preparation.