Will It Sequence? Hunting drug resistant bugs

Today I’m taking a step outside the office to get some fresh air. It’s a lovely spring day here, and I need no excuse to take a walk after the winter we’ve just had. Rubbish weather and, of course, the various winter illnesses. No-one likes being ill, and even some of the more ubiquitous illnesses can pack a punch. It’s not uncommon or particularly unreasonable to seek out a doctor and get a course of antibiotics. But, as I’m sure you well know, antimicrobial resistance, when a bacteria, fungi, virus or what have you becomes immune to classical treatments, is fast becoming a point of serious discussion.

Courtney - The latest estimates put that close to 5 million deaths annually, you know, attributed to drug resistant infections and that's projected to skyrocket over the next 30 years. And, you know, if we don't do anything about it you know, and on top of that antimicrobial resistance threatens, you know, animal health, the environment and food security, the economic equity within societies because it disproportionately impacts low-income countries.

Will - That’s Illumina’s Courtney Gonzalez painting a grim picture. So, how does Illumina’s genetic sequencing come into this? And why am I walking outside for seemingly no reason? Well, on this genetics programme, it’s time to talk about wastewater. That’s why I’m outside of one of Cambridge’s wastewater treatment plants, to really soak up the atmosphere. <Breathe in> hmmm.

Scott - Wastewater is everything that goes into your toilet and in through the sewage system and to the sewage treatment plants where the material is processed and cleaned up so that infectious diseases and other nasties don't get out into the environment. We looked at a variety of different sources of wastewater. So that can be things like a manhole in a city someplace. It could also be the wastewater coming directly out of, say, a dormitory at a university campus or a building of some sort. So, all of those different sources will have sort of like different concentrations of material. If it's a manhole cover that's, you know, servicing, let's say, you know, 50 to a hundred thousand people, then it's going to be a very different composition than something coming straight out of a building.

Will - That’s Illumina’s Scott Kuersten, and I’m now very aware that I’m standing near other people’s waste. I’m going back inside.

That’s better. So, why sift through wastewater? What secrets does it hold?

Courtney - Wastewater surveillance I think is done for, like Scott said, you know, three main reasons to monitor, track and screen. So yeah, you want to monitor for evidence of infection within a community, track trends in those infections within the community and screen for infections that could trigger like, you know, additional testing or mitigation measures.

Scott - My work in wastewater started out at the beginning of the pandemic for the detection of covid. It was a very convenient way to be able to non-invasively test a large number of people and track the presence or absence of covid in samples and what kind of strains that are in there.

Will - Since you do end up passing a lot of the microbes in your gut out when you do your business, monitoring wastewater is a passive, but very insightful cross section of the disease causing microbes found in a population. And amongst these nasty bugs are the aforementioned resistant ones.

Courtney - Agencies like the CDC and the World World Health Organization issue these sort of priority lists of microorganisms with drug resistance that are particularly threatening to human health. So things like methicillin resistant staph aureus or MRSA, for example. So since we're not actually isolating and growing these pathogens in a lab, we're just looking at the genetic sequences. We have to, you know, rely on the markers of drug resistance, so these antimicrobial resistance genes and variants and at the same time evidence of the bacteria that can carry those markers.

Will - So what was limiting about the previous methods of sequencing when it comes to tracking antimicrobial resistant species?

Courtney - Wastewater particularly is really interesting because as Scott mentioned, it is a really complex, or potentially a really complex sample type compared to say, you know, like a nasal swab from a sick patient where you might have at least some idea of what's likely to be in there, you know, like one or if you're unlucky, a couple respiratory pathogens, whereas a single wastewater sample can have genetic material from hundreds of bacteria, viruses, fungi, plasmids, antimicrobial resistance genes, all at really low concentration, which presents a, a challenge for a lot of technologies. So, you know, on one hand you, you kind of have PCR, which can enrich and pull out signal from these low concentrations, but you have to have an idea upfront what you're looking for, and it's maybe gonna be limited to 10 or 20 things, you know, in a typical multiplex PCR. And then on the other hand you have shotgun metagenomic sequencing, which can, you know, sequence everything in the sample. So it's hypothesis free, which is good for surveillance, but you're potentially gonna miss a lot because it's at that really low concentration.

Will - Multiplex PCR, where you amplify a select few sequences of DNA isn’t all that helpful because wastewater surveillance requires you to cast a very wide microbial net. And shotgun sequencing had a broader range of genetic material to identify, but often wastewater concentrations were too low. The old options were either a mile wide or an inch deep. But maybe a sweet spot could be found.

Scott - So in the lab with the nucleic acids, we convert those nucleic acids into libraries as we call them, DNA and or RNA libraries that will then go on to an Illumina sequencer and be sequenced. Part of those library preps. There are various ways that you can manipulate those library preps to, to benefit certain types of organisms. So for example, we do hybrid capture enrichment assays that fish out certain sequences out of the material and enrich them. So then we don't have to sequence nearly so many, and we can sequence a lot more samples out of that after the sequencing.

Courtney - So what we found in this study is that we have this sort of sweet spot of precision metagenomic sequencing, which like Scott said, includes this step where probes go in and hybrid capture targets of interest for hundreds of pathogens and thousands of AMR genes. So you get that sensitivity for low concentration targets, but you also get the breadth that you need to really see what's in the sample without knowing exactly what you're looking for upfront.

Scott - And by doing that kind of enrichment, you can also process far more samples than you could in the past for a lot cheaper cost. And that's, that's really key for this, this, this field is to keep the cost as low as we can so we can keep monitoring and, and checking different sites and sources of wastewater.

Will - So a cheaper, more precise means of tracking antimicrobial resistance in populations. And the lower cost means it can be deployed more often, which gives you greater resolution of data across, say, a year long time frame.

Scott - Yeah, exactly. And fact, at the beginning of the pandemic, I was on a paper that we were able to track variants over the course of time as well. So you can kind of see the viral evolution taking place over time.

Will - Indeed, it has already spotted times in which resistance to prescription drugs was rising in certain populations.

Courtney - Yes, actually in the pandemic there was a lot of speculation early on that the drug prescribing practices were, you know, nobody knew exactly what sick patients were coming in with. So they would be prescribed drugs just based on, you know, having a cold. And it turned out to be COD, which an antibiotic would not be the right thing to prescribe for that. And so you were seeing more resistance in the wastewater as a result of those drug prescribing practices.

Will - So with an eagle eye being placed on the particularly harmful microorganisms, what can actually be done with this data to reduce them as a problem?

Courtney - One thing we can do with AMR surveillance data in general is use it to inform empirical treatment decisions. So for example, you know, in the absence of surveillance data, physicians are going to rely on their own experience of what drugs do and don't work for say, a given bacterial infection. But if you know from surveillance data that 90% of those bacteria in your community are resistant to the first line drug, then you can potentially use a different drug and save lives. And especially in countries without, you know, a robust clinical surveillance network, I think wastewater surveillance has a lot of promise to fill that gap.

Scott - And at least for the infectious disease type fields, monitoring wastewater gives you a little bit of a headstart as they basically say that when you start seeing say COVID in, in a wastewater sample then it might mean that in three or four days then patients are gonna start showing up in the hospital with certain symptoms. So if you can get ahead of the curve a little bit and understand if some new infection is popping up in the population, then you can better inform hospitals about what to expect as far as symptoms or how to treat the patients.

Will - So it's an early warning system and it can show you what drugs might be more effective. Is there anything this can't do?

Scott - Cure the disease <laugh>

Courtney - <Laugh>

Will - Well, you can’t have everything. But a fascinating insight into the world of sequencing either way. Thanks very much to Illumina’s Courtney Gonzalez and Scott Kuersten. That certainly wasn’t a waste.