Over ten years ago, I was going down one of the many rivers branching off the Ganges to get to the village of Mathbaria in Southern Bangladesh. Mathbaria has long had issues with cholera, a serious diarrheal disease which can be fatal if not treated rapidly and that has plagued humanity for centuries. On this trip I wanted to isolate environmental strains of its bacterial causative agent, Vibrio cholerae, to compare them to those I had from North America. On the boat, I remember Tareq, a research officer who was accompanying me, asking about doing graduate school in Canada, where my lab was based. Little did I know he would later become Dr. Tarequl Islam, a postdoctoral fellow leading on-the-ground efforts of a large project looking at the effect of drinking polluted water on the gut microbiome and health of Mathbaria residents. About a year earlier, I had contacted Dr. Munirul Alam, a well-known cholera researcher at the International Center for Diarrheal Disease Research (icddr,b) in Dhaka, who promptly invited me to visit Bangladesh. He chose Tareq to accompany us on our sampling trip downriver to Mathbaria, as he was his top research officer at the time. We have been collaborating ever since.

This project, funded by RGHI, was one of the first ones I worked on after moving to Singapore from Canada at the beginning of COVID. I could never have done something at this scale if I had not been in Asia. The idea came after I spoke with my friend and colleague Maurizio Labbate, who at the time was an Associate Professor at the University of Technology Sydney. He had done a study of the gut microbiome of avid water users such as surfers and swimmers on a polluted beach in Australia, finding that they had more drug-resistant pathogens in their gut compared to people who did little swimming. After hearing that, I thought if the Australian surfers carry pathogens from just accidental swallowing of water, then this must surely be the case for people in Bangladesh with no access to clean water. Bangladesh is a unique country, most of it situated around sea level in the delta of the Ganges and Brahmaputra rivers. This means that if you dig a well, there will likely be salt intrusion in the well water. Not only that, but the soil is also often rich in arsenic, which leaches in the water, making it quite toxic. Bangladeshis found this out the hard way when international organizations who were trying to solve the problem of enteric diseases dug millions of tube wells in the 1970s, leading to one of the largest public health crises in modern history, and remains ongoing. This left little choice for the local residents but to harvest rainwater in barrels or obtain water from shallow ponds dug to accumulate rainwater. Unfortunately, barrels have limited capacity and shallow ponds are prone to contamination from animals or nearby latrines, leading to exposure to enteric pathogens. Some of these ponds have sand filters connected to hand pumps to clean the water. These are good at filtering particles but are limited in efficacy at removing bacteria. On previous visits to Mathbaria, we had also seen fancy filtration systems powered by solar energy that had likely been installed by foreign aid agencies. But speaking to residents, these clogged rapidly as the ponds are filled with algae, and the solar power was insufficient to run the pumps properly. We also observed that, lacking the money to buy fuel to boil the water, locals removed particulate matter making the water turbid using alum (aluminium sulphate), showing us that this agent made the water clear. We tested it for bacteria, and alum treatment removed most of them, but it gave a strong taste to the water and its long-term effects on health are not well known at such concentrations (aluminum can be toxic). These observations made us decide on rainwater harvesting systems as a simple and proven solution to improve water sanitation. No electricity, no filter, just a water tank connected to a rooftop to harvest rainwater. We aimed to test if pathogen exposure and carriage would be reduced in families using these for drinking water.

Although the idea is simple, the implementation was not so. Choosing volunteer households was complex.  We had to balance locations so the control group families would not share water with the experimental group families. We also needed to ensure fairness in the community and balance in the diversity of families chosen. Our fantastic team at icddr,b worked with local Mathbaria contractors not only to transport the water tanks to the rural area but also install them with care. The community was involved, because installing these for dozens of families (35 at the beginning of the seven months study and 35 at the end for the control group) is no mean feat in a village with little infrastructure and in a hard-to-reach location between numerous rivers. This included finding suitable clean roof space, connecting it with pipes to the tank, and pouring a cement base to keep the tank safely off the ground. Although the large size (2m wide and 3m tall) made the task difficult, it ended up being a blessing. There was some rain after installation, but it was followed by several months of drought and we were afraid that people would run out of tank water, jeopardizing the whole study. Fortunately, they did not, partly because most volunteers took very good care of the tanks, using the water sparingly. One family even covered the tank with a tarp to avoid exposure to the elements and animals. In future planning, we do need to consider that these dry inter-monsoon seasons are getting longer, and rain is more intense (with corresponding flooding) during the monsoon due to climate change.

The icddr,b team had a field office in Mathbaria, basically a small apartment with a lab on a kitchen bench. This gave the medical technicians a base from which to operate when doing monthly rounds to collect stool samples from volunteers. They maintained constant communication with the volunteers, on some occasions having to be clear they should only drink water from the tank for safety, as some would still use some pond water for the “taste” to cook rice or to save the precious tank water.

Capturing a time series was critical to the success of the project, but because of resource limitations we could only run the study for seven months. We also collected drinking water and water from local ponds on each visit to look at the source of pathogens. We ended up with over 5,000 samples, around ~1,300 of these being stool samples from volunteers. We performed culture enrichment for bacterial pathogens on all samples. At first, DNA from both enriched and raw samples were screened at icddr,b by quantitative PCR for the presence of major enteric pathogens, such as enterohaemorragic E. coli, Shigella and Vibrio cholerae, revealing their recurrent presence in a small proportion of the population, despite the absence of any outbreak or significant symptoms. Every individual positive for a pathogen in any given month had all their samples sequenced by metagenomics. This allows us to determine not only the genotypes of bacterial pathogens they carry, but also the duration of carriage (whether it is temporary or long term). Preservation of samples and shipping of either microbial isolates or their DNA is always a problem when the location lacks developed infrastructure. In our case, we could only obtain a government permit for the shipping of DNA (not live strains) and the number of samples allowed to be shipped was limited, requiring multiple shipments, which took over a year.

The work to liaise with the Bangladesh team and organize the shipment of samples was taken on by a new Ph.D. student who had come from South Korea to work with my team. I knew this project was very promising and I wanted top talent working on it, and it was clear to me Deborah was up to the task. After tireless work from her and the icddr,b team, she received the first batch of samples, sequencing their metagenomes and revealing several interesting leads through her analytical skills. First, the proportion of people carrying enteric pathogens such as Klebisella pneumonia in their guts increased progressively from January to June, closely matching increases in outdoor temperatures, but not rain patterns. These bacteria can cause opportunistic wounds and lung infections, and carriers have increased risk. It is known that enteric infections increase in warm seasons in temperate countries, but not that temperature affects the actual carriage. Klebsiella acquisition is also somewhat of a mystery. This relation with temperature hints at an environmental source or spread, since bacteria grows more easily at higher temperatures.

Another intriguing finding was that two novel species of bacteria absent from western microbiomes, Elusimicrobia, were commonly found in the gut of volunteers, sometimes for long periods of time and occasionally at very high abundance. These are ultramicrobacteria, so small they can pass through a 0.2 micron filter we use to sterilize solutions in hospitals. Elusimicrobia is extremely rare in human guts, usually found in freshwater or other animals, such as insects or ruminants. We hypothesized these might be acquired from the cows who live in close contact with their owners, and Deborah went to Bangladesh to work with Tareq, Munir and their team to sample the cows’ gut microbiome to test this hypothesis. There were other environmental and animal bacteria found in our volunteers’ microbiomes in high abundance, such as gut adapted Melainabacteria (ancestral relatives of cyanobacteria lacking photosynthetic capacity) as well as some Treponema peruense and succinifaciens (spirochaetes relatives of Treponema pallidum which causes syphilis). This suggests a close connection between their microbiome to the environment and animals, which is lacking in more urban dwellers. As the data was overwhelming for Deborah alone to analyze, a postdoctoral fellow, Dr. Winona Wiyaja, joined us and is helping to look into the diet of volunteers from the food DNA present in their stool, looking for associations between these foods and certain bacteria (pathogenic and non-pathogenic) in the gut microbiome.

Fortuitously, my colleague Maurizio, from whom I originally got the idea for the project, had moved out of academia and was now working for a diagnostics company. He generously offered to screen all our stool samples using their gastrointestinal multiplex qPCR panels, used to diagnose people with gastrointestinal tract infections in hospitals and clinics around the world. Because of its cost of around two hundred dollars per sample, this is almost never done with people not suffering from an acute infection, which none of our volunteers were. Amazingly, we are just beginning to screen samples, but this extremely sensitive method is detecting widespread carriage of Vibrio cholerae and of the intestinal parasite Giardia, suggesting a heavy pathogen burden in the gut microbiomes of “healthy” volunteers. This is a mutually beneficial collaboration, saving the company a lot of money by offering free samples on which to test their products and giving us a unique chance to look for over twenty pathogens in our entire time series.

Despite the enormous talent of Deborah and Winona at bioinformatics, analyzing a complex dataset such as this is a challenge, requiring significant computer infrastructure that is difficult to obtain even in well-resourced Singapore. It will take time to properly analyse and investigate microbial links between humans, their drinking water, their animals and their foods. There are many discoveries to come from this work, well worth the effort, which will hopefully positively impact the health of volunteers in the long run.  Aside from the science, we already know that the participants have been positively impacted.  We can see and hear their joy about their new water tank, how it is making their life easier and telling us they don’t have diarrhea as often as they used to. It proved so successful that some of their neighbors who saw the benefits that the water tanks could bring but who did not or could not participate in the study bought similar water tanks with their own funds. This “snowball” effect is very interesting, yielding additional positive impact on the community beyond what was funded in the study. Some of them said to us “Any new study you have, I am a volunteer!”. Hearing this makes all the effort worth it.

Funder: Reckitt Global Hygiene Foundation (RGHI)