Biofilms form when bacteria adhere to a surface in a watery environment and secrete a slimy, glue-like substance. This substance forms a matrix that helps the bacteria stick to different types of material, including fabric, and provides them with environmental protection. The bacterial cells then form a colony, or group, within the matrix. Biofilms can be found everywhere; the plaque on your teeth and the pinkish stuff around your bath are biofilms!
Nappies are obviously a prime source of bacteria given the job they are made to do, and sanitising them is the source of many a discussion online – understatement of the year! Naturally, cloth nappy users are eager to ensure their wash routine is adequate to remove bacteria from their nappies for good hygiene, and because build up of bacteria may cause absorbency issues. Indeed, in many of our discussions in Nappy Science Gang, we have hypothesised that some of the issues we have encountered with inadequately washed nappies may be due to the formation of biofilms in the fabric of the nappy thus resulting in the bacteria being more difficult to remove. However, many questions still remain to be answered:
- are biofilms forming in our nappies?
- if they are, is this a problem?
- can we get rid of biofilms or make them less likely to form?
- might biofilms have anything to do with the hydroxylapatite exoskeleton?
To give us an introduction to the fascinating world of biofilms, we invited Rob Hull to chat with us. Rob is currently doing a PhD in Biofilms at Southampton University so he was well placed to answer the many questions we have about them.
Rob: Hi, I work at the University of Southampton and research Streptococcus pneumoniae biofilms. How they grow, develop and evolve over time. A biofilm is a group of bacteria that have grown on a surface, and enclosed themselves in a matrix. This matrix is composed of many things and will vary from species to species, but will generally contain DNA, protein, sugars, and bits of dead cells.
Sophia: So will a biofilm be composed of just one sort of bacteria?
Rob: Yes a biofilm can be formed of just one species, and in fact often under lab conditions we will grow and study a single species biofilm. However, in nature it is unlikely that a biofilm will ever be just one species. Even if a biofilm is formed of a single species they rapidly produce what we call colony variants. A colony variant will be a daughter of the the single species but will display very different characteristics.
Sophia: So kind of whatever bacteria are around will just join in the biofilm?
Rob: Any bacteria might try and join a biofilm, yes. However the resident bacteria there may try and kill any newcomers.
Sophia: So why do bacteria form these biofilms?
Rob: Bacteria form biofilms for a number of reasons. Principally to protect themselves from hostile conditions. For instance in the environment a biofilm protects against dehydration, while inside the human body it protects against the immune system.
Kate: Amazing. Those colony variants: are they evolving because of the new conditions in the biofilm?
Rob: Essentially yes. A biofilm is formed of many gradients of nutrients, oxygen, waste products, acidity, and many other factors. This means that a colony variant may evolve (over a matter of days) to better grow in an evolutionary niche within the biofilm.
Erin: Would bacteria in a biofilm have the same infectious ability as the same bacterium outwith a biofilm or would they have to ‘come out’ to infect? Does the biofilm act more like a defence mechanism once they’ve colonised a host?
Rob: That is a good and somewhat debated question. First of all you have to consider the species that is growing and what it may or may not be capable of infecting. The organism I work on is Streptococcus pneumoniae and the biofilm cells of these are generally less virulent/infectious, however they occasionally disperse more infectious cells.
Kate: Do you think a biofilm could protect against the effects of a washing machine?
Rob: Yes, and no. Most of a biofilm will be destroyed by a washing machine, through the heat and mechanical stresses. However biofilms are known to promote persister cells and some species may survive a 90C wash.
Sophia: What are ‘persister cells’?
Rob: The definition of persister cells is still discussed, but a good one is “Persisters are dormant variants of regular cells that form stochastically in microbial populations and are highly tolerant to antibiotics”. Basically due to the different regions of the biofilm and environmental conditions some cells mostly shut down and are very hard to kill, but our lab in Southampton is working on it.
Kate: Are you describing bacterial resistance, Rob? Something similar? We learned about bacterial resistance from some microbiologists in an earlier chat. I mean resistant bacteria, I think!
Rob: These bacteria are more tolerant to things like antibiotics, because our current antibiotics are mainly targeted towards growing mechanism, and persister cells are not growing and can then ‘wake up’ after the antibiotic concentration has dropped. Biofilms can make their inhabitants much more resistant to antibiotics (up to 1000 times). This is through a number of different mechanisms. The matrix can slow or stop the antibiotics on the way in. Some cells may neutralise it protecting others at the same time, while others may sleep through it.
Sophia: So it’s a bit like in sci-fi films when people survive a nuclear blast, or a thousand years in a space ship, by being in suspended animation?!
Helen: The main other form of resistance, as in MRSA, is the bugs develop enzymes that can break down the antibiotics before they harm the cell.
Rob: Yes they can produce enzymes that break down antibiotics, but other ways might be producing pumps that remove the antibiotic from their cellular body before it can act, or they might change how a pore functions to prevent entry.
Kate: Helen, I’m wondering too if the same sort of interactions between us and the bugs encourage the resistance. Like with superbugs, we unnecessarily stress the population with antimicrobials which kill off weak strains and leave the stronger ones to thrive; if we keep putting a biofilm say through the stress of a wash but fail to shift it all, do we inadvertently help those resister cells to evolve and thrive?
Sophia: What about washing nappies? Do biofilms form on clothes (and nappies) and do they make fabrics harder to clean?
Rob: Biofilms can form on fabric, and clothes would be no different. However due to the dry nature clothes are generally in I think it unlikely that a significant biofilm would form.
Hannah: Would you notice if a biofilm had formed on clothes (nappies) by touch or sight, or would/could it be microscopic?
Rob: Biofilms can grow large enough to see by eye, and certain species can be very smelly, however it is very species dependant.
Kate: Oh they can be smelly. Do you know what any smell like?! We have identified some different stinks in nappies 🙂
Rob: Yes I can identify a few different bacteria by smell, but thankfully mine aren’t too bad.
Kate: Describe the smells, Rob!
Sophia: Rob, would you like to smell our nappies?:-)
Rob: I’m afraid to me it just smells like bacteria! And while I am sure your nappies smell great I have enough small children of my own to smell!
Sophia: How long does a biofilm take to form? Could nappies left 3-4 days before washing grow a biofilm?
Rob: A biofilm can form quite rapidly, but a mature biofilm could generally form within 3 days if the growth conditions for that species were met.
Helen: If you are storing nappies for a few days before a wash, how can we be sure our wash routine will clear any biofilms that may have formed?
Rob: I think it is unlikely that a bacterial strain would develop resistance to wash cycles if it is being dried and generally worn, moved around, and the large changes in temperature are likely to kill any biofilm off. However if you left it running continuously for a very long time something might form. We find biofilms in the harshest of conditions, from hydrothermal vents on the sea floor at very high temperatures, to the international space station. But almost none of these would be any good at
Sophia: So biofilms may form on nappies left a few days without washing. You said most will still be removed by the washing machine? I assume that depends on the wash – I mean will higher temps remove more? Will more or less detergent affect how much is removed? If machines differ in how good their agitation is, will that affect how well the bacteria are removed?
Rob: To be sure you would have to test it in a lab, which you guys have done some of. However if you were to keep cloth dry, after a good wash I would think there would be negligible biofilm present. Higher temperatures are better for killing bacteria; 90C will kill most things. In the lab we use 122C to sterilise our equipment. The mechanical stress of a wash will also help remove the bacterial cells, and a higher concentration of detergent (within reason) will probably remove more cells.
Sophia: The problem is that most nappies can’t be washed at 90. Some people are washing their nappies at 30C. In your opinion, will there be much difference between washing at 30C, 40C, 60C?
Rob: I would think that 60C would be much better for killing bacteria than 30C, quite a few infectious bacteria like 30C to grow happily.
Kate: How well do they like 40C? We did a temperature experiment and it was a bit hand-knitted, but we did see results that we didn’t expect around temperature of wash v. bacteria?
Rob: I would think that you would get greater killing of bacteria at 40C than 30C. The human body heats itself up to 38.5C to kill bacteria.
Sophia: If there’s a little bit of biofilm left after the wash, does that make it easier for a biofilm to form next time that nappy is sitting in the bucket? Or do they start from scratch again?
Rob: If there is biofilm left after a wash, and it is able to survive until there is sufficient food and water to grow then it may help form more biofilm, however I think that the level of biofilm left after a wash is likely to be very small.
Erin: Would that be the same for bacteria not in a biofilm: unlikely to survive but could regrow if nutrients and conditions were good?
Rob: Bacteria not in a biofilm would almost certainly be diluted out, and washed away by the wash cycle, and so would be unlikely to be a problem.
Kate: Some nappies are made with microfibre. Do you think the extensive surface area of those brush-like fibres would make a nice hiding place for bacteria?
Rob: I am not familiar enough with microfibre to make a judgement on that, however we do know that different surface roughnesses result in different levels of biofilm formation. Unfortunately I don’t know if this would be a significant factor for nappies.
Sophia: What about clothes which are wet most of the time, Rob?
Kate: Oh and to link that to Sophia’s question, would the fact that a fabric is difficult to wet make a difference?
Rob: Fabric wetting properties would really have to be tested and varies a great deal from substance to substance, and how this anti-wetting was achieved.
Kate: What’s the trend for roughness? More rough = better for biofilms?
Rob: The trend for roughness is a tricky one. My limited knowledge of this area is that bacteria like a roughness where the grooves in a surface are about as big as they are so they can get nicely stuck in. More or less roughness than this and there is less adherence.
Kate: Oh it would be interesting to know the size of a bacteria then. We have look at electron microscope images of different types of fibres and seen the different shapes of the fibres.
Rob: Bacteria come in many shapes an sizes but a few micrometers (um) in size is the ball park for most bacteria.
Helen: I’ve found another site saying microfibers are normally 3-5 microns. Browser open on another device so I’ll put the link on Facebook.
Kate: What is a micron compared to 1um?
Erin: 1um is a micron or micrometre
Kate: I found a SEM image of some microfibre. I wonder if these gaps are a nice size for bacteria. I think, the way microfibre works, if bacteria filled up all the gaps it might not trap water so well.
Kate: Sophia, I wonder about the roughness of the hydroxyl apatite?
Sophia: We got some problem nappies tested, and two of them had a mineral called hydroxyl apatite coating all the fibres (shown in our scanning electron micrograph image). We’ve been wondering if there might be any interaction between that and biofilms? Might the mineral buildup make biofilms more likely to form? Or might the biofilms promote the mineral forming?
Kate: The exoskeleton looks like it has little cracks in it. It also looks like there is stuff in the cracks.
Rob: Some bacteria will adhere to almost any surface, but as a rule they do all like to be wet, so if after washing a nappy was well dried, I would think that any biofilm formation would be extremely minimal.
Helen: I’m getting the impression that biofilms form and are stable when there are steady conditions. They can resist more than a bug on its own, but dramatic changes in conditions (esp heat) will still kill the bugs. Could biofilms then form in your washing machine especially if you regularly wash at low temp with little or mild detergent?
Rob: Biofilms could form in a washing machine. We know that within water pipes there is biofilm formation, and even within hot water tanks there is biofilm formation. One species that is particularly good at this is Pseudomonas aeruginosa. This is a big problem in hospitals, and our lab is currently working on this problem in partnership with Public Health England.
Sophia: Another of the test nappies had a really high bioburden and was repelling water – might a biofilm make a fabric less absorbent?
Rob: I am not sure whether a high bioburden would effect water absorbance, however generally biofilms have water pores within them to allow water flow, whether this would be enough for your tests I do not know.
Erin: Were the bacteria on the nappies we tested alive?
Sophia: Yes, cos only the alive ones form colonies to count. We don’t know how many dead bacteria there were.
Kate: So Rob you feel that a biofilm is unlikely to survive after washing and drying, basically? What about individual bacteria? Because one of our test nappies had loads of ’em and it had been dry for ages. What a puzzle.
Rob: Yes I think that it would be unlikely that a biofilm would survive a wash and dry. As to why there may still be a lot of bacteria left on a nappy that had been dry for a long time, did your test account for dead bacteria? What was the CFU level?
Kate: 34000CFU on the worst one Rob and I think they shook it in fluid then grew colonies from a specific amount of the nappy juice. The sample was just a little square. Someone else will know how big. I don’t know if that’s relevant to the CFU count.
Rob: 34000 CFU/ml is more than I would expect, but it would really depend what species it was as to whether it is a problem.
Kate: The CFU 34000 was ten times the sort of numbers found in the other stinky nappies.
Emily: Would biofilms be more likely to linger in a nappy bucket than on a nappy? Is there any problem with it? Could someone get infected from the bin?
Rob: If a nappy bucket is full of water, all the time and dirty nappies are going in and out then I would expect a biofilm to form. However if it was cleaned often, occasionally with bleach then that would kill off biofilms well. As for getting infected from the bin, probably unlikely, but poo can contain pathogens.
Emily: At what age would baby poo start to contain pathogens? Would they be more nasty, e.g. resistant to antibiotics, if baby themself had taken antibiotics? Do you know if there are health concerns like build up of pathogens in gut that mean cloth nappies are not used for incontinent old people?
Rob: Babies are colonised pretty quick, but most of these bacteria will pose no issue for healthy individuals.
Sophia: Rob, if we wanted to make a biofilm grow on something, for experimental purposes, how would we do that?
Rob: It would depend what you want to grow on what! However a starting point might be submerging a sterile piece of cloth in sterile LB broth, and inoculating with your species of interest. However, that would not necessarily be a good mimic for a wet/dirty nappy. Kate: Perhaps we could try growing them on different samples, eg. viscose with and without apatite exoskeleton, other fabrics like microfibre.
Helen: Rob, I’m more thinking that if we did as you suggest, then cut a bit off we could test that to see how much biofilm has formed? Then wash the other bit and test again to see how much it has reduced i.e. trying to answer: do biofilms grow more readily on certain fibers and is the selected wash protocol good at getting rid of them? I have no idea how you test for the presence or amount of biofilm on something like fabric though.
Rob: Yes you could do that to test how much biofilm was removed. You might have to control the experiment a little to ensure that the media etc does resemble a nappy bucket though, and that the species are good choices.
Kate: Oh, another question we had was whether bacteria in a biofilm would be protected from swabbing if that method was used.
Rob: A biofilm would not protect against a swab.
Emily: Are bio films just made by bacteria of are viruses, Protozoa, fungi etc involved too? Are there different types of biofilm in different hardness of water?
Rob: Biofilms can be formed by bacteria, and fungi, but not viruses. I know less about protozoa but I do know that they like to eat biofilms!
Helen: I think I heard that the orange slime that you sometimes see in the shower is a biofilm?
Rob: So it depends what that orange is, but it could well be a biofilm, probably a fungus.
Kate: Helen, I think someone described a bacteria more common in rural areas which forms the pink stain in the bathroom?
Emily: Are there bio films on human skin?
Rob: Bacteria grow on human skin, so it likely that there are biofilms there.
Emily: Would the same species forming biofilms on skin also potentially form biofilms on fabric?
Rob: Pseudomonas aeruginosa is probably a species that could grow on both.
Kate: What would a biofilm look like under a microscope?
Rob: It once again depends on the species, however you do get some very cool structures. Some have tall towers like mushrooms, others form long strands, while the species I look at forms honeycomb like structures. Confocal microscopy is a good method for visualising biofilms on all sorts of items, however at Southampton we also have an EDIC microscopy which is designed to look for prions on fabric strands and would be well suited to your experiments.
Kate: Thanks so much for this, Rob. Can we keep you in the cupboard now so we can ask you questions any time?
Sophia: Rob, thank you so much for coming!
Rob: Thank you all, this was fun. If you have more questions in the future I am sure I could do this again.
If you want to learn a little bit more about biofilms you can check out Wikipedia, or for some more detailed information check out Biofilms: Microbial Life on Surfaces (Donlan, 2002) published in the Journal of Emerging Infectious Diseases.