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Brazilian Researchers Profile Microbiome of Rio Before, During, After Olympic Games

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NEW YORK (GenomeWeb) – As gymnasts tumble, runners hurdle, and divers twist in the air at the Olympic Games in Rio de Janeiro, researchers are dashing around the city in matching T-shirts to collect microbial samples.

They hope that by comparing samples they obtained before the athletes and international delegates arrived in Rio for the 2016 Summer Olympic Games to those they are now collecting and will collect after the last athletes go home, they'll be able to see how the microbiome of a city changes when there's a large influx of visitors from around the globe. About half a million visitors are expected to travel to Rio for the Games, according to the International Olympic Committee.

The effort, dubbed the Olympiome, is a side project spun off of the International MetaSub Consortium project. That group, led by Weill Cornell Medicine's Christopher Mason, banded researchers from 45 cities together to study their urban microbiomes, with a focus on their transit systems. Mason previously conducted a microbial analysis of the New York City subway system.

The A. C. Camargo Cancer Center in São Paulo's Emmanuel Dias-Neto, who studies the connection between bacteria and cancer, had already been thinking about what microbes are present on the metro when he learned about Mason's project and the MetaSub project, which he then joined and attended its first meeting.

"When we worked together there came this idea of this study for the first time: What would be the impact of a big mass of people coming to one city and leaving after some time?" Dias-Neto, who came up with the idea for the Olympiome project, told GenomeWeb. "Are they going to leave their bugs behind? Will these bugs be able to persist and survive in this new environment?"

By swabbing various parts of Rio for DNA before, during, and after the Olympics, he hopes to answer that question. In addition, by collecting RNA and mosquitoes, he also hopes to examine the prevalence of Zika and other viruses in the city.

For the New York City Subway system project, details of which were published in Cell Systems last summer, Mason and colleagues swabbed more than 450 subway stations and other sites in the city. While they found that nearly half the DNA they isolated couldn't be traced back to any known organisms, the ones they could identify belonged to 1,688 different taxa, spanning bacterial, viral, archaeal, and eukaryotic groups. Most of what they could assign were bacteria, largely skin-associated bacteria like Acinetobacter or Staphylococcous.

"There was a lot of excitement initially," Mason told GenomeWeb. Other researchers got in touch with him to say they wanted to take a similar look at the microbiomes of their own cities. Some 45 cities have now signed up to be part of the MetaSub consortium, including Rio de Janeiro, Tokyo, Barcelona, Johannesburg, and Sacramento.

The consortium just held its first Global Sampling Day on June 21, coinciding with Ocean Sampling Day, sending out researchers with swabs to collect samples from their cities' transit systems and other sites. In total, the MetaSub website says more than 5,600 samples were collected that day.

Each of the MetaSub cites, Mason added, has been encouraged to pursue its own side project. In New York, for instance, the investigators are monitoring the Gowanus Canal, a US Environmental Protection Agency-designated Superfund site in Brooklyn. They hope to be able to trace changes as the canal gets cleaned up.

And Rio de Janeiro is doing the Olympiome. "We will actually catch the shifting in the microbiome that may be impacted by so many people coming to Rio," added Milton Ozório Moraes from FIOCRUZ, a public health institution in Rio de Janeiro, where he studies leprosy. Moraes is overseeing the project in Rio. According to Moraes, they are currently swabbing nine different metro stations three times a week, gathering samples from a range of surfaces in the trains and stations, including seats, hand rails, and kiosks.

He noted that the metro isn't Rio's biggest transportation system — the buses are — but the metro followed by a bus is the best route to get to Olympic Park.

Still, they are not focusing on Olympic arenas and sites, but on places where people ordinarily go, Dias-Neto said. The researchers are also sampling parks and squares in the city. "We'd like to capture the regular microbiome that we have in the city," he said.

Mason added that the researchers have been able to obtain some samples from within the Olympic Village as well.

In addition to isolating DNA from the samples they collect, the researchers will also be examining RNA from their swab samples. That way, they might be able to pick up traces of RNA-based organisms like the Zika virus. Mason noted, though, that they are unlikely to pick up any RNA viruses, as they don't live that long on surfaces.

Dias-Neto predicted that any changes to Rio's microbiome with the influx of visitors are likely to be subtle. The metro and people's hands already have their permanent microbial residents and it can be difficult for new ones to get a foothold to colonize, he said. Moraes envisaged, meanwhile, that there might be a few new species circulating in Rio, while perhaps a few bacterial or fungal species typically there might be lost. Nancy Merino, an environmental microbiologist at the Tokyo Institute of Technology, noted in an email to GenomeWeb that the New York subway study picked up a microbial shift in a station flooded by Hurricane Sandy some two years after the storm. This suggested to her that some changes that occur during the Olympics might persist.

"We don't know, but my guess is it's going to be a subtle alteration and eventually some of them are going to be able to persist on those surfaces," Dias-Neto said. "But let's see."

As other cities around the world participated in June's Global Sampling Day, Dias-Neto said that that data could be used to try to trace any persistent or interesting bug that does pop up back to its origin. Mason noted that when he looked at the MetaSub pilot data, there were species that appeared endemic to certain areas.

"For example, if we find a strain of a different bacteria that's not common here in Brazil, but it's common, for example, in Barcelona or any other city that is part of the consortium, we could actually compare the data because we are doing the collective sampling day every year," Moraes said.

This could be the start of metagenomic forensics "where you could see where are you in the world based on what species you are finding," Mason added.

At the same time that they are examining Rio's microbiome and how it may change, the researchers will also be analyzing human DNA that their swabs pick up for ancestry and other markers. Dias-Neto said there are limited privacy issues because the reads they'll have will be too short to trace back to individuals.

With such samples, Dias-Neto said he hopes to get a sense of the baseline ethnic mixture of Brazil — which is a highly diverse nation — while Moraes added that it also gives them the opportunity to estimate the frequency of rare polymorphisms linked to disease among Brazilians.

A similar ancestry analysis was done as part of the New York City subway study. Mason and his colleagues compared the ancestry markers they found to census data to find that that markers that traced to Mexican, Colombian, and Puerto Rican populations were found in a section of the Bronx with mostly Hispanic and Amerindian residents, while Yoruban ancestry markers were identified in the Canarsie neighborhood of Brooklyn that has mostly African-American residents.

But in Rio, the researchers will also then see how these data points change with the arrival of Olympic visitors and when they revert to baseline.

In addition to collecting swab samples the metro and parks, investigators in Rio are trapping mosquitoes in and near the city to tease apart their gut microbiomes. Mosquitoes in Brazil can transmit a handful of infectious diseases like Zika, dengue, and chikungunya. But as Dias-Neto noted, it's currently winter there and the mosquitoes are less plentiful.

The MetaSub researchers partnered with another group that was doing its own Olympics-related study of mosquitoes. Originally, the mosquito researchers were investigating the genetics of the mosquito population and how visitors from around the world might affect its dynamics and mobility.

"[But,] we decided that that would be very interesting to also do the microbiome of the mosquitoes' guts," Moraes said.

The researchers are also collecting swab samples from public areas near where the mosquitoes are captured for comparison. The insects will be divided by sex and then characterized based on whether or not they'd recently had a blood meal, drawing blood from people or other hosts. They plan to examine the gut microbiomes of some 30 female mosquitoes.

From this, the researchers might be able to detect viruses or other organisms within the trapped mosquitoes. "We'll see what percentage of the population is actually carrying things in and nearby the city," Mason said.

Even after the Games end, the researchers will continue to collect samples. If they do uncover any interesting microorganisms, Dias-Neto plans to follow them over time to see how long they last in Rio's hustle and bustle.

Once they have all the samples, Moraes said they will be extracting DNA and RNA from them in Brazil and then likely will be sending those samples to New York for sequencing because it's cheaper to do there. Mason noted that Qiagen is supporting the extraction and library preparation of these samples.

The data will then return to Brazil for analysis in Rio and São Paulo. The researchers estimated that they would have some 1,500 samples. "That's a lot data that we are going to need to process," Moraes said.

Collecting each of these samples requires a dance of swabbing and documentation. Each collection site is not only swabbed, but it is also photographed, geotagged, and uploaded via a smartphone app to the MetaSub consortium website. A map there shows where in each MetaSub city samples have been collected.

In a paper appearing in Microbiome in June that summarized one of their meetings, the larger MetaSub consortium laid out its thinking for how such samples should be handled and analyzed. It, for instance, noted the importance of positive, negative, and laboratory controls and called for developing and testing methods and standards for metagenomic research, including bioinformatics.

The New York City subway microbiome project ran into some difficulties in its analysis and interpretation. In their Cell Systems paper, Mason and his colleagues also reported that they uncovered DNA fragments that matched that of Yersinia pestis, Bacillus anthracis, and other potential pathogens. But critics pointed out — and Mason and his colleagues acknowledged in an erratum — it was highly unlikely that Y. pestis or B. anthracis were actually in the subway.

The University of California, Davis' Jonathan Eisen told GenomeWeb that the informatics pipeline used was overly generous and assigned reads to groups where they didn't actually belong. He said that to say that an organism is present, researchers have to not only find reads that match that organism, but also find that they aren't a better match to any other organism. Further, he added, to say a pathogen is present, pathogenic or virulence factors need to be found. And none of that, he added, means the organism is alive.

Eisen added that he thinks Mason and the consortium will be more careful going forward. Indeed, an undergraduate in Eisen's lab is involved in MetaSub and Eisen is involved in Global Sampling Day. Still, he sent a tweet to underscore his expectations.

"I wanted to publicly express my hope that they would be more careful this time, to put them on record," Eisen told GenomeWeb.

Moraes added that if they do find anything like anthrax or multi-drug resistant bacteria, they would comb through the data carefully. "[Metagenomics] is the first level of evaluation," he said. "And then if we have positive results, we need to go deep and do extra analysis to be sure that actually have this kind of [bacteria] in the system."

In particular, Mason said they will be relying on 11 computational tools, rather than three used in the Cell Systems paper, and will be using computational marker-based panels. Then, he added, if they find any species that are associated with disease, they'd do follow-up PCR and other analyses as well as turn to the MetaSub advisory board and other researchers in the consortium for their input. And, he added, they'll release the data for anyone to see.

Much as how the Olympic torch will be passed on at the end of the Games, so too will the Olympiome project. Researchers in Tokyo, the next host of the 2020 Summer Games, are planning a similar project.

"Eventually, of course, we are going to transfer to them every thing that we learned here," Dias-Neto said.

The preparations are already underway in Tokyo. Tokyo's Merino, who is heading up that effort along with Keio University's Haruo Suzuki, said that they have started some pilot projects there and are performing time series studies to guide their selection of sites to study for the Tokyo Olympiome.

With the time series, Merino said they'll be investigating whether seasons and other events affect the stability of the Tokyo microbiome, while a separate preliminary study will examine the microbiome before, during, and after an international conference takes place at the Tokyo Institute of Technology.

While they are still developing their experimental design, Merino said she expects the Tokyo 2020 Olympiome project to be similar to what's being done in Rio. She predicted that they would see a "very international microbial community" when it is their turn to host the Games.

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