“Lots of countries rely on wastewater treatment plants to purify water and turn sewage water into water that is safe for the environment.

Traditional wastewater treatment requires a lot of personnel, money and energy. Some countries are looking for alternative and more sustainable ways to sanitise water.

We’re working as part of an international project, called PhageLand. PhageLand is investigating the potential of constructed wetlands and phage treatment as a sustainable wastewater treatment. The project is funded by the Joint Programming Initiative on Antimicrobial Resistance( JPIAMR) which is an international collaborative platform engaging 29 nations and the European Commission to curb antimicrobial resistance.

Using wetlands to sustainably sanitise water

Constructed wetlands offer a more natural way of treating wastewater using plants, rocks, natural materials and environmental microorganisms to treat wastewater.

The PhageLand project is seeing if adding phages can make them extra efficient by helping to remove harmful bacteria.

Bacteriophages, or “phages” for short, are viruses that kill bacteria. It’s important to remember that phages don’t infect humans and are specific to the bacteria they target. This means that they can be a safe way of killing harmful bacteria like E. coli and Salmonella.

The main idea behind PhageLand is to use phages to remove antibiotic-resistant bacterial pathogens and prevent the spread of antimicrobial resistance from wastewater into surface water, by using phages alongside constructed wetland technology.

International collaboration

The PhageLand project brings together international and interdisciplinary expertise. The constructed wetland treatment site is in Poland.

The project is led by Carles Borrego at the Catalan Institute for Water Research in Spain collaborating with two teams in Poland, a public health focused team in Moldova, two phage-expert teams (one here at the Quadram Institute and one in Belgium) and an engineering team in the Netherlands. The team in Poland are experts in the construction side of the wetland and have constructed a pilot-scale wetland which we will be using to test the efficacy of our phage cocktail in eradicating disease-causing microorganisms. The team in Moldova have isolated antimicrobial resistant bacterial isolates in the clinic and are conducting a patient review, interviewing both clinicians and patients about antibiotics and antimicrobial resistance in the Republic of Moldova.

It’s exciting to see lots of different disciplines coming together on the project.

Finding phages

Firstly, we looked for phages in the natural environment that can target five key bacteria. The five target bacteria are part of the ESKAPE pathogens group that the World Health Organisation (WHO) have identified as the most priority concern and have the highest rates of antimicrobial resistance.

I took wastewater and river water samples and enriched these with the five key bacteria to isolate phages. After spotting the phage lysate onto a lawn of bacteria on an agar plate, any zones of clearing indicated that there is a phage that could kill the bacteria.

Then we can extract DNA from the phage and find out what it is. Sometimes we find phages we already know exist, but we find lots of new, previously undiscovered phages too.

The technique of isolating the phages is fun. You never know if you’ll find a specific phage that can kill your bacteria of interest. It’s exciting to discover a clearing zone which indicates there’s a phage.

For the PhageLand project, we isolated over 100 phages. From this we then had to narrow down ones to use in the constructed wetland treatment.

We looked for phages that are tolerant to UV because the constructed wetland is open to the sun. Another key selection criteria were phages that can cope with low temperatures of 10°C.

We’ve developed a mix of phages, which we call a phage cocktail. The phage cocktail consists of five different phages targeting different ESKAPE pathogens. We are hoping this selection of phages will reduce the amount of ESKAPE pathogens in the wastewater that flows through the constructed wetland.

One of the main challenges I’ve had is making such a large stock of phages. We normally use 5 to 10 mL volumes of phage in the lab, but for this pilot project we have to scale up to 8 litres to use in the wetland.

It’s not possible to ship 8 litres to Poland in liquid form, so I’m working on concentrating it into a smaller volume using anion exchange chromatography, that can then be diluted at the wetland.

Piloting phages in the real world

We’re going to see whether we scale up the findings we’ve found in the lab to the real world.

We’re initially trialing a pilot scheme so the water then will flow back into the traditional wastewater treatment so nothing will flow out into the environment.

We plan to do a two-week run with no phages, monitoring each stage of the treatment plant. We’ll be looking at what microbes are present at each stage of the treatment tanks and which antimicrobial genes are there. We’ll use qPCR and metagenomics at each stage to answer those questions. That will be our control trial.

Then we’ll do the same thing with phages added and compare these results to the control trial to see the effects of the phages in the real world.

I like the impact side of working on the PhageLand project. The technology has the potential to benefit low and middle-income countries the most because they might not have the funding or infrastructure for big wastewater treatment plants. But there’s no reason phages and constructed wetland wastewater treatment couldn’t be used in other countries if we find the technology is effective.”