Pure Science: Bleach Study’s Author Discusses Research on Eliminating C difficile on Soft Surfaces

A bottle of bleach (Adobe Stock 413542793 by FARBAI)

Recently, Infection Control Today^® (ICT^®) posted an article on a peer-reviewed study on the effectiveness of bleach on Clostridrioides difficile. To clarify why the investigation was done, what the parameters were, and how this investigation applies to infection prevention personnel, ICT spoke with the author, Loveleen Tina Joshi, PhD. Joshi is an associate professor of molecular microbiology at the University of Plymouth, UK. She specializes in clinical microbiology and infectious diseases and provides advice to key national groups in these areas. Her research is focused on infection prevention and control, as well as molecular diagnostic testing to help tackle the global health care challenge of antimicrobial resistance (AMR).

ICT: Would you explain your background that led to this study?

Lovleen Tina Joshi, PhD: I am an academic researcher. I'm a scientist, and I'm interested in the pursuit of knowledge out of curiosity and to tackle some of the world's greatest challenges like antimicrobial resistance (AMR). So, the whole rationale for doing research like this is to understand whether disinfection is being conducted appropriately and whether there is triggering AMR in microorganisms.

So I've done research in C difficile since 2008. I'm an expert and have done a PhD in the area. In terms of disinfection, for a very long time, I've been very interested in understanding where the C difficile spores survive certain chemical treatments; it goes back to a paper I published in 2012, looking at how spores sit on surfaces and whether they bind to inorganic surfaces or organic surfaces better.

In that study that we published in [American Society for Microbiology’s Applied and Environmental Microbiology], what we looked at was trying to understand whether spores of C difficile band to stainless steel surfaces, for example, or when they bound better to the apical microvilli of human epithelial cells (Caco-2 and MRC-5 cells) or HT-29 cells.

All the work we do is in the laboratory; it’s in vitro. And it's for the pursuit of general academic knowledge. We were very clear about that. And we found very interesting differences in the way spores stick to surfaces.

What's important about the work that we did there was we realized that spores from trying different strains behave differently. You can't take 1 microorganism and say every single strain of one microorganism behaves the same way. I think that's a crucial problem and a big disconnect between the academic scientific world and the infection prevention and control (IPC) world.

In terms of practice, we're seeing things in the laboratory that don't translate very well into IPC practice because, by the sounds of it, IPC treats all microorganisms in the same way. In [many] tests, like the environmental monitoring (EM) tests and the United States Environmental Protection Agency [EPA] test, 1 strain represents [all the strains of a pathogen].

When you look at a whole range of different strains across 1 species, [many] have different strains. We found that with that [2012] study, and then it moved on, and it's several other studies, and what we're examining was the ability of these spores from different strains to interact with NaDCC. So that's a chlorine-releasing agent. In 2017 [we] published that data, we found that suboptimal levels of NaDCC didn’t work on C difficile, maybe actually exacerbating their ability to adhere to surfaces. This is a small form of the organism.

A spore is not the same as a vegetative bacterium. Many studies that people have done are with vegetative C difficile, which is irrelevant; it leaves [the body] as a spore, which is dormant, and it stays on a surface as a spore. These spores are made of calcium dipicolinic acid, very, very strong and have an exosporium layer on the outside. And that allows it to stick to surfaces.

It's an interesting thing where we find that adherence levels are a bit off C difficile because strains may change in response to biocide exposure. And that feeling is back then after that, in 2019, a published study that did go quite big in America and, and it was NaDCC, versus C difficile, in liquid, and again, across surgical gowns and scrubs. In that study, we found NaDCC was not efficacious at all.

We were approached by a hospital in America, who said they thought that their single-use gowns were transmitting C difficile. And I [thought], why is that even relevant? When a single-use gown is supposed to be a single-use gown, don't you throw it away? Well, their practitioners weren't throwing it away. To save money, they were wearing them again and again.

We did 2 different studies, though; we looked at how C difficile transmitted on those surgical gowns, and we published a small study there. Then, we published a big study with C difficile of biocides and the NaDCC. We tried to disinfect the gown to see if that would work; a quick spray on the gown would get rid of the C difficile. And that didn't [work] either.

There was all this as a huge problem. We tried to help that hospital manage their C difficile and give them a bit of advice. Then, in response to that study, because we proved that C difficile had a high resistance level to NaDCC, we got a huge backlash from the Americans. Many people said to me your studies are irrelevant. The EPA testing you've done isn't doesn't match the EPA; the EPA doesn't think your standards are correct.

ICT: For this study, what was your goal, and what were the parameters?

LTJ: I did this in the pursuit of knowledge, not as an applicable test to the EPA and not to verify a product; it was to try and understand whether the spores are resistant or not.

It's a bigger question beyond whether your product works or not, and it is a question about AMR. and whether the spores are contributing to it. And I got a lot of pushback and people telling me to go and do a study on hypochlorite. Because hypochlorite is the thing that everyone wants to use or everyone does use in America.

I went on almost on my own and continued impartially in a neutral way to test sodium hypochlorite. So we did that in the laboratory. And that's the study that [ICT wrote about].

We were looking at the Department of Health UK guidelines. And we were looking at what they recommended for C difficile, and whether it works. So 1000 parts per million of chlorine-releasing agent versus C difficile and 5000 parts per million, the contact time of 10 minutes. We replicated those types of testing using biocides, susceptibility testing, or suspension tests in liquid using purified spores of varying strains that were clinically relevant. That's where it differs.

So many companies use this kind of EN testing methodology with 1 strain on 1 type of C difficile. Then they try and apply it to every C difficile, which may not be may not necessarily be representative of the microorganism itself.

In this particular study, we used a very high concentration of C difficile spores because we wanted to replicate and see what could be the highest toleration amount of C difficile in an environment. So, we put 1 x 108 spores in our test; we didn't look at varying other things.

(So what we could have looked at and, and that's work that we're doing right now is looking at pH, looking at temperature, and time-kill assays over a range of time to understand a bit more about the organism.)

We wanted to do a straight test of C difficile, 3 clinically relevant strains, and sodium hypochlorite. What we found was that with high concentrations of high C difficile, the hypochlorite didn't even penetrate.

Why is this happening? You've got 10⁸ spores purified in a solution, and you add varying concentrations of hypochlorite. And it's not even penetrating the spore. And you're only seeing 1 log reduction, if that. And there are so many questions scientifically about this.

From my end, it's how the exosporium works. And whether biocide is binding to different C difficile exosporium layers, creating almost like a shield. We don't know what it's doing in that scenario. What we do know is that based on all the previous research and evidence that I've done, it's becoming quite clear that this tolerance of C difficile to chlorine-releasing agents, there are questions to be asked and laboratory testing to be done to see if resistance is emerging.

We did these experiments, and then we thought to replicate the previous study; let's try and get some patient gowns, and since surgical scrubs, spike them or deposit them with C difficile spores, and just see that they're transferred over. And then once you apply biocide to it, whether it causes a change in the transferability of the spores, and it causes some change. It just showed us that result that the spores still survived in these fabrics. There's something at play there that we don't understand.

It's great that these personal protective equipment (PPE) fabrics can retain spores, but it's not very great if autoclaving (disinfecting) them at 121 degrees in a steamer doesn't work because there's research that shows that. And it's also not great if a biocide can't penetrate the fibers of those surgical scrubs. You've got a process during laundering where the spores are still in the laundry process too. And when we're trying to address those kinds of questions as well.

It's like a big picture, and we don't know the full answer, but that's what we were doing and these tests that we were doing laboratory, again, as I said, it was the pursuit of scientific knowledge, but we were neutralizing biocide activity after 10 minutes.

Now, there are so many scientific publications that I've seen where people don't neutralize biocide activity. So how do you know whether your results are correct? You need to have a set contact time, shut off the activity of the biocide, and have a definitive cut-off where [you put in] that biocide in with the C difficile for 5 minutes, and it neutralizes and sees how many C difficile has left. And you can have a real true determination of sporicidal activity at that point.

ICT: What should IPs and other IPC personnel take away from this study?

LTJ: There are several things here. The first thing is about practice. What's done in the laboratory is potentially reflective of what can hit your clinical practice in the next 4 to 5 years. If we're seeing resistance emerging in laboratory environments, it's very likely to emerge across the board. And we've already seen that.

I know in the UK, we're seeing that there's chlorine resistance and tolerance emerging in practice, and many people have come to me after the study and said, we're seeing the same thing. And I'd say, Okay, if you, if you choose to accept that what we've said is real. We know it's real. But if it's different when people do it, IPC practitioners accept this is coming on the horizon; they need to start seeking alternative disinfection measures to ensure that C difficile if that's a problem in their clinical environment, is dealt with efficaciously.

But then, if you want to go bigger and higher level to AMR, IPC clinicians have a massive part to play in ensuring patient safety and ensuring that the appropriate antimicrobials or appropriate disinfection is used and patient or patients aren't exposed to dangerous microorganisms in a clinical environment, and hospital-acquired infections are persisting.

IPs are recognizing that they have a role to play and can be fantastic stewards. So, antimicrobial stewards help us save antibiotics and stop the infection from spreading. I would like to see them empowered to do something in an ideal world. I don't think there's a need to panic yet. Because we have many other biocides available, many of the technologies available can do the job.

Please read the literature before making comments and try to understand that there is a gap between academic scientific research and clinical practice. Try to bridge those gaps and be informed of those changes in research.

ICT: It is well known that soft surfaces cannot be disinfected. In fact, there is no EPA registration for soft surfaces. However, if I understand correctly, this study was pure research.

LTJ: It wasn't associated with EPA. It was a different set of questions that were in pursuit of trying to get rid of the microorganism from the environment, understanding more about [spores'] ability, physiochemically, to adhere to surfaces, not replicating something that disproved [how] a product works. That's not what we were doing here. I hoped the chlorine would work.

if you read the paper, I did 2 things. I did a liquid suspension study, a biocide test, and a soft surface study if you want to pass it as that, in IPC and people who are in disinfection and class it in that way, then those are the 2 things that I did. But from a scientific point of view, I did a susceptibility test using a biocide and then tested for adherence ability. So, again, is a different question for me, but that's how it translated to people. And I just think it's interesting that sort of like the misinterpretation of everything. AMR is a real thing. People need to realize that it's bigger than climate change.

We need to be able to disinfect, and that's the first pillar of getting rid of infections.

The best infection is the one that didn't happen. The first thing is to clean properly. Because years and years ago, people used antibiotics like [candy]. At the time, I was like, oh, no, I think we'll be fine; antibiotics are available. And then what now? We've got our last lines of antibiotics only, and we're all desperately searching for new antimicrobials. And I'm sitting there saying biocide resistance is probably a thing, and we want to reconsider how we're using our biocides effectively. Are we using them at the right time, at the right concentration, and at the right dose for the right microorganism? And if we're not, we're contributing to that problem.

There will be a time when these microorganisms do what they did with AMR. They will become resistant to these disinfection processes. And where does that leave us? If we bury our heads in the sand now and continue to use things that don't work properly, we're going to be in trouble, and we will be scrambling for new disinfectants like we are antimicrobials.

I am on antimicrobials. I'm going to go in and work on hypochlorite and figure out whether it is efficacious or not, or we'll work on multidrug-resistant pathogens and try and find the best way to kill them to try and save people's lives in the long run. That's my moral duty as a scientist to try and find solutions.

Please read the literature. Understand that [AMR] is an important issue that affects all of us as a species. It's important for you to understand that genuinely and for the public to understand it. Again, it shows a lot of myths about communication from the academic and scientific worlds to the public. Again, I think there's a disconnect, and we've got to do more to get that message out about antimicrobial resistance.

ICT: In your study, sodium hypochlorite was diluted using sterile water. The point was made that this is not how it's done in the real world.

LTJ: That’s an interesting comment that’s not necessarily true. I would like anyone who comments on that to go and watch [an EVS], disinfect, and clean in practice. You can't take a bottle of EPA-registered chlorine bleach and spray it; it has to be diluted, or if it isn't diluted, then it has to be made fresh, no matter if it's EPA-registered or not.

So we got [the bleach] from a commercial company, a big scientific company. It had a certain amount of active chlorine in it. When we opened the bottle, we made the sodium hypochlorite fresh in terms of dilution to the parts per million that we wanted, and then we checked it.

We could just get through bottle after bottle when we need to do it at the time; we won't use the same bottle again and again. And I wonder, for cost-cutting measures, do [EVS] do that? What do cleaners actually do? We just don't know what's going on in practice, where the cost-cutting measures are put in place, or how effective their training is.