At the summit of an extinct volcano in Oregon, a research team from the University of Washington was able to collect enough microbial biomass from two trans-Pacific air plumes to permit a microarray analysis.
Using the PhyloChip, a tool developed at Lawrence Berkeley National Laboratory, the UW scientists were able to identify the presence and abundance of microbes in those samples. In total, 2,100 species were detected using the array, compared to the 18 species the same team had detected in samples from the same air plumes in an earlier study that relied on traditional culturing.
"The long-range transport and surprising level of species richness in the upper atmosphere overturns traditional paradigms in aerobiology," said David Smith, a UW biologist and corresponding author on a recent Applied and Environmental Microbiology paper detailing this work.
"Just like other types of pollution, microbes from Asia have no problem leaping across the biggest gap on our planet, the Pacific Ocean," Smith told BioArray News. "Why [does] everything seem to be everywhere in microbial biogeography? Probably because everything is circulating in the atmosphere."
Smith and his coauthors said that the PhyloChip could eventually be used by environmental monitoring stations to trace the origin of specific microbes, as well as to monitor the impact of the airborne populations on epidemiology and climate patterns.
"It seems plausible, when coupled with atmospheric modeling and chemical analysis, that microbial biogeography can be used to pinpoint the source of intercontinental dust plumes," Smith told BioArray News. Given that aerobiology is currently "suffering from a lack of standardized methods," Smith said that international researchers "should agree upon a standardized platform that permits cross-lab analysis", and that the PhyloChip "might be that solution."
Smith's team is just one of several using phylogenetic microarrays that have recently published their results. Researchers at the University of Oklahoma have developed an array called the GeoChip for monitoring soil microbial communities. GeoChip is sold by Glomics, a Norman, Okla.-based firm. Another research team at the University of Bologna in Italy has developed a phylogenetic chip called the HTF-Microbi.Array, which has been used mainly to study human intestinal microbiota. A literature review shows that PhyloChip has been used in similar studies in the past, and that such phylogenetic chips are being used for multiple purposes, including public health issues.
Second Genome
This month, researchers from LBNL, the nonprofit research organization MITRE, and the Bay Area company Second Genome published a paper discussing the use of the PhyloChip to characterize the bacterial diversity of the interior air of commercial airplanes, with the longer-term goal of developing biosensors to detect the presence of infectious organisms in aircrafts. And last fall, studies were published that described the use of the PhyloChip to monitor environmental microbiota related to asthma development in inner city children and to analyze changes in microbial communities related to the Deepwater Horizon oil spill in the Gulf of Mexico.
These are just a few of many studies — about 70 to date — that have relied on the PhyloChip since its development eight years ago, according to Yvette Piceno, an LBNL research associate who has worked with the array.
Piceno told BioArray News that, given the scope of its coverage, the Affymetrix-manufactured, 1.1 million-probe chip "provides a starting point for assessing which microbial populations may be of greatest interest for ongoing monitoring," allowing application-specific arrays to be developed later for "more routine monitoring and field deployment." Currently, the chip targets about 60,000 bacterial and archael taxa.
And even though the technology landscape has evolved since the array was used to test for pathogenic organisms in New Orleans floodwater residue following Hurricane Katrina (BAN 10/5/2005), Piceno said that the PhyloChip remains "complementary" to next-generation sequencing-based approaches, as sequencing "can identify sequences not currently in databases" while microarrays "can provide the equivalent of deep-sequencing [by] detecting sequences in small proportions in a sample in one run."
One of the advantages of the PhyloChip compared to sequencing is its "accuracy in the identification of quantitative differences of specific [operational taxonomic units] over the course of a study," said Gary Andersen, head of the ecology department at LBNL and one of the creators of the PhyloChip.
Andersen told BioArray News that the quantitative controls placed on every microarray and the "reproducible nature" of the response of specific probes to different concentrations of target enables users to "get an accurate assessment" of how the relative concentration of a specific organism changes over the course of a study.
Because of these capabilities, Andersen said that the PhyloChip "pairs well with NGS," and, depending on the questions asked in a specific study, one would be preferred over the other or both may be used to increase the power for identifying specific organisms that are responsible for a particular condition or responsive to a treatment.
He noted that Second Genome uses both the PhyloChip and sequencing in its efforts. However, Second Genome CEO Peter DiLaura told BioArray News that while the firm does work with researchers like Smith's lab at UW, it is focused more on opportunities in the pharmaceutical arena and conducting human health studies than on environmental monitoring as part of public health initiatives. In DiLaura's words, Second Genome is "leveraging the microbiome for the discovery of novel therapies," with an "emphasis in inflammation and metabolic disease."
Second Genome, formerly known as PhyloTech, licensed the PhyloChip from LBNL a few years ago (BAN 7/6/2010). Andersen is a cofounder of the company and sits on its scientific advisory board.
Actionable Information
PhyloChip may enable researchers to detect microbial populations at an extent and throughput that they had never been able to before. But can information obtained from the chip be used to make direct decisions that benefit public health?
According to Piceno, there are two separate issues related to the chip's possible use in public health. One is the array's ability to be applied to many systems, including "human or environment." Piceno maintained that arrays are "useful" in this regard and "could be deployed more widely to speed assessments in some settings."
The second issue is the acceptance of the technology by the public health community, where it competes against more established methods, like culturing and quantitative PCR. Piceno said that gaining acceptance for array-based monitoring programs takes time, as there are "many organizational layers and stakeholders involved in setting policies." These policy-setting stakeholders must weigh the acceptable methods for monitoring, and "maintaining public safety, in human and environmental health, is paramount during any transition" to a new approach, such as array technology, he said.
As part of these deliberations, any method's limitations must be taken into consideration, Piceno said, including the potential for the technology to produce false positives or false negatives. At the moment, Piceno said that arrays are "used best for comparing relative abundance differences across [and] among samples," and that "they are not as well suited to presence/absence calls as other technologies."
When asked about the actionability of PhyloChip results, Andersen referred to an ongoing collaboration between LBNL and the Southern California Coastal Water Research Project, a research institute based in Cosa Mesa, Calif. In a paper published last year in Environmental Science & Technology, the joint research team showed how the PhyloChip was used to characterize microbial communities in coastal California water samples to identify the source of fecal pollution. The current monitoring method is qPCR, Andersen noted.
As detailed in that paper, fecal samples were collected from 42 different populations of humans, birds, cows, horses, elk, and pinnipeds, and analyzed with the PhyloChip. According to the paper, cluster analyses revealed "strong differences" in community composition among the different source organisms. The researchers were able to identify 1,058 different bacterial taxa that were unique to humans, grazing mammals, and bird fecal wastes, operational taxonomic units that they said could serve as specific identifiers for the taxa.
Andersen said that while sources obtained from the ocean are "greatly diluted," the PhyloChip can "accurately detect" the taxa at "very low quantities" and that the power of detecting multiple, confirmatory organisms increases users' confidence that they identified the source of the fecal pollution. The information obtained from the PhyloChip can also be used to guide the development of other array-based tools.
"Knowledge of which probes have the greatest power to discriminate between sources gives us the ability to select a fraction of the original PhyloChip probes for a smaller, cheaper array for routine monitoring and we have already validated that the probes work," Andersen said.
In a separate project, the LBNL researchers are working with partners to investigate the thermophilic composting of human waste from vault toilets and and more sophisticated composting toilets to address concerns that the human compost contains pathogenic organisms. Temperatures reached during the compositing process in such toilets can be between 70 °C and 80 °C for extended periods, Andersen said, noting that the selection pressure of high heat "does interesting things to the microbial community composition."
At the end of an effective composting process, the researchers can see an entirely different community than the one that was present at its beginning, with no organisms that are human-specific in the compost, Andersen said. He added that by using the PhyloChip to monitor microbial communities during the composting process, the efficiency of thermophilic toilets can be improved, and public acceptance of using compost from such toilets could also increase, as confidence is established that the final compositing material is "safe and pathogen free."
A Complementary Tool?
While the PhyloChip is being used increasingly in public health-related projects, some researchers who use the tool doubt that it will completely replace current methods, citing the hurdles mentioned by Piceno of gaining the consent of the regulatory community.
"The biggest challenge for using PhyloChip, or other array technology, to water quality management is adapting it to the regulatory context," said Eric Dubinsky, an LBNL scientist involved with the Southern California Coastal Waters Research Project. Dubinsky told BioArray News that qPCR is "only just now achieving widespread acceptance by the regulatory community," and that he does not think array technology will replace qPCR for water monitoring "any time soon," as regulators are "bound" to state and US Environmental Protection Agency water quality criteria that are based on most probable number or qPCR counts of single indicator organisms like Escherichia coli or Enterococcus.
Given these factors, Dubinsky said that PhyloChip is "complementary" to these methods, especially in the context of bacterial source identification.
"When the standard assays indicate there is a high level of indicator bacteria, the source must be identified," Dubinsky said. "This is where qPCR tests are often inadequate because most sources of contamination do not have unique markers to target with qPCR, and the sensitivity and specificity varies wildly among available tests."
According to Dubinsky, both the State of California and the EPA have expressed interest in using array technologies for this purpose, but resistance comes from the perception that the technology is "too expensive and too complicated for widespread use." Dubinsky said that he and fellow researchers are working to alleviate the regulators' concerns by developing a downscaled array that targets only the important sequences for source identification.