A new project called PathoMap is exploring the microbiome of public spaces in New York City, starting with the subway system.
The long-term goal of the effort, which is spearheaded by a research group at Weill Cornell Medical College and is still looking for funding, is to establish infrastructure for monitoring high-traffic areas in the city for potentially pathogenic microbes using next-gen sequencing, and to use those data in combination with other information to react quickly to public health threats.
"I envision these types of data, and automating these types of analyses to make them happen overnight, would create a very smart city," said Christopher Mason, an assistant professor at the Institute for Computational Biomedicine at Weill Cornell, who is leading the project.
For a pilot study, a team of undergraduate volunteers swarmed the city this summer collecting samples from almost 500 subway stations across New York, which Mason's team is in the process of sequencing and analyzing. The project, which the researchers plan to expand by collecting additional samples from the same sites in the fall, winter, and spring, will establish a baseline of microbes present in the New York subway system.
From the first 24 samples analyzed, they already learned "a variety of very interesting things," he said, finding some bacteria "that you would not necessary want to find on a turnstile or kiosk."
However, Mason is quick to point out that the project – which was almost named "Microbe Map" instead of "PathoMap" – will not focus on pathogens alone. "We don't want everyone to be scared to ride the subway," he said. "The most important thing to note is that these species [of microbes] have always been around us for millions of years, and we're still here."
Even with volunteers collecting samples, the four-seasons project will likely cost on the order of $1 million, including sample prep, sequencing, data analysis, and data storage. The team's plan is to use the pilot data, which is being generated with support from collaborators, such as the American Museum of Natural History, as well as from Illumina, to apply for a grant, for example from the National Institutes of Health. In addition, the project is seeking crowdfunding through a website, an effort that will run until Sept. 24.
The researchers chose the subway for their pilot study because "it's something we use every day," Mason explained, and because every rider has wondered before if they can catch a cold from hanging onto a railing. "We know implicitly the answer is 'yes', and that we all exchange microbes with each other every time we shake hands, but there is no establishment or understanding of the baseline microbiome of public surfaces − there is almost zero data," he said.
For the pilot project, a "swab squad" of five undergraduate students from Cornell, Hunter College, and Queens College collected 1,404 surface samples from 468 subway stations this August. Each station was swabbed at a turnstile, a ticket vending kiosk, and inside a train stopped at that station, either from the railing, seat, or handlebar. In addition, the researchers took what they called "sporadic swabs" from various places in the city, including airplanes, taxis, boats, a petting zoo, park benches, and a garbage can.
Prior to going into the field, the researchers tested different swabbing kits and techniques and found that using Copan Diagnostics' ESwabs and swabbing for three minutes worked best. "It doesn't sound that long, but if you have a Qtip and people are looking at you on the subway, three minutes is actually a long time," Mason said.
Besides samples to analyze, the collection effort resulted in some interesting – and mostly positive – responses from the New York public, he added, including expressions of gratitude for "cleaning the subway."
To log the samples, the team has been using the GIS Cloud Mobile Data Collection app, a freely available program for mobile devices that allows users to record the location of a sample, along with photographs and other information.
The researchers have started to extract DNA from the swabs, using a PowerSoil DNA isolation kit from Mo Bio Laboratories, which they found provides the highest yield, about 200 to 300 nanograms on average per surface.
So far, they have sequenced a few dozen samples at Weill Cornell, using both the Illumina HiSeq and MiSeq, and hope to complete the entire set of 1,469 samples by the end of the year, with a publication following soon afterwards. They are using metagenomic sequencing, not 16S RNA sequencing, in order to be able to pick up any type of DNA, be it from bacteria, viruses, cockroaches, or bed bugs. "We want to be able to see it when it shows up," Mason said.
The use of 300x300-base paired-end reads on the MiSeq in particular has provided good discrimination for a variety of different organisms, down to the species level, he said, and the researchers are hoping to use the long MiSeq reads in the future, though they are open to other platforms as well. So far, they have analyzed the samples for bacteria but are starting to look at viruses now.
While they have identified "hundreds of species" so far, and even sub-strains in some cases, their analysis sometimes goes as far as the genus level or even taxonomic groups.
Among the species they found in the subway samples were Enterococcus bacteria, which are often found in fecal material. "That basically means that not everyone is washing their hands, but we kind of knew that already," Mason said.
Also present were Acinetobacter bacteria, which are associated with skin, as well as – at low frequency – Streptococcus.
In addition, they found bacteria known to be helpful in cleaning up toxins, as well as radiation-resistant species. Interestingly, some surfaces harbored subsets of bacteria that the researchers have not found at other sites so far.
Besides the microbial population of the subway, the analysis might also provide insights into subway riders. About 0.01 percent of the sequence reads are human, providing low-coverage metagenomic data on the human population using the subway. "So far, it looks like different subway lines have different numbers of ancestry-informative markers, which could indicate the ethnographic and ancestral genetic background diversity of each subway line," Mason said. He added that based on the first 24 samples, the number 7 train, which runs between Manhattan and Queens, appears to have the most diverse ridership.
Apart from swabbing surfaces, the PathoMap team also recently started to collect air samples, experimenting with both home-made and professional samplers. Earlier this year, a team from the University of Colorado in Boulder published a study in Applied and Environmental Microbiology in which they analyzed the microbial diversity in air from the New York subway system, using both Sanger and 454 sequencing, but Mason said PathoMap's study will be different, for example, in their sample collection approach.
Also, eight years ago, the J. Craig Venter Institute started sequencing microbes from Manhattan air as part of a project called the Air Genome Project (GWDN 10/17/2005), but it is unclear whether the results were ever published in a peer-reviewed journal.
Going forward, PathoMap might team up with efforts to study the microbiome in other places in New York City. Mason's group is already working with Mount Sinai School of Medicine and New York University's Center for Genomics and Systems Biology, and has begun talking with others, including Rockefeller University, which plans to implement an educational microbiome project for high school students; NYU's Center for Urban Science and Progress; the State University of New York; the City University of New York; and the Hospital for Special Surgery.