How bacteria and HA interact: A Live chat with Elaine Berry and Greg Siragusa

In this live chat we spoke to Elaine Berry and Greg Siragusa about their work using hydroxl apatite to concentrate bacteria. 

Elaine is a Research Microbiologist at the U.S. Meat Animal Research Center.
Greg is a Senior Principal Scientist at Eurofins Microbiology Laboratories.

In 1997 they wrote a paper about using hydroxyl apatite to concentrate bacteria. And they seem like the best people we could find to answer some questions about how bacteria and HA might interact (eg inside smelly nappies!)

Below is the Live chat log from 22nd November 2016, 9pm. (Experts are in bold) We apologise for the slightly raw feel of it – people are typing fast in live chats, mistyping is common, and there can be delays between questions and their answers. But we don’t have the staff time to edit it!
[If you would like to volunteer to tidy it up a bit, please get in touch with Sophia]

Hello
E: @all hello, nice to be here
Hi, thank you so much for coming!
It usually takes a few minutes for people to log in and stuff.
Could you tell us a little about your work and what you found out?
@Elaine Hi Elaine!
G: Sure: I am aSenior Principal Scientist with Eurofins Microbiology Laboratories a global biosciences analytical services lab. My specific role is that I direct a new Microbiome division in North America.
E: Hi Greg! The objective of the work we did was to develop a way to rapidly concentrate foodborne pathogens from food homogenates. We found that bacteria adhered rapidly to hydroxyapatite and we could use this to concentrate them and speed their detection.
E: I am a Research Microbiologist with the US Dept. of Agriculture.
G: Elaine is a microiblogical genius!
Could you explain the process by which this happens in the simplest terms possible please
E: Greg was my mentor in the work. A tolerant guy 🙂
G: In clearer terms; my work uses DNA sequencing to determine the names of bacteria in a sample and their proportion. That sample might be feces or it might be food or a whole list of things.
@Greg and can you explain how the bacteria are adhered to HA?
I don’t know how much Laura explained about our situation. But we are interested in cloth nappies. We got some ‘problem’ nappies tested last year and two of them had very high levels of hydroxyl apatite in them.
E: The interaction between hydroxyapatite (HA) and the bacteria is non-specific and we think is due to electrostatic interactions. As in “opposites attract”–bacteria tend to be negatively charged and may be attracted to HA.
G: I will defer to Elaine.
We read something about HA having crevices that are a good size to fit bacteria within, does that sound plausible?
Hi @all. Are all bacteria negatively charged or just some?
E: I read about your research and hypotheses online, and while I am not so familiar with diapers, I think you are right in that HA may play a role in increasing bad diaper odor, by attracting bacteria.
(just wondering if some types more likely to adhere to HA)
E: In the work we did, a variety of bacteria adhered to HA, so quite likely that bacteria involved in odor do too.
@Elaine @Greg We are about to start our next sets of experiments. I only managed to read the abstract of your paper. What form was the HA? A pressed pellet? A crystal?
So does HA attract bacteria more than other surfaces do?
@Greg can you give us an idea of the bacteria you c
E: It was crystalline HA. Yes, I think HA may attract them quite strongly, though we didn’t compare with other compounds.
Sorry, commonly find in feaces
E: E. coli and Enterococcus are common in feces, but there are many, many others.
@Elaine, why were you testing HA then? Do you believe it’s just the positive charge that attracts the bacteria? Or something else too?
@all Hi! Sorry just catching up following trying to get sproglet in bed. I haven’t read your research (sorry), but I was wondering if HA can help bacteria ‘survive’ e.g. does it make nice colonies that are harder to get rid of? (Completely off topic now..!)
Hi Ellie
E: We were testing HA to find a way to rapidly concentrate pathogenic bacteria. Detection methods require a threshold amount of bacteria typically to detect them. We wanted to improve sensitivity of the methods to detect bacteria, by concentrating more of them from a larger sample.
E: Greg has emailed to say that his internet connection keeps failing but he’s trying to get back in
G: Depending on pH; most tend to be negatively charged.
G: Elaine might know if there are any major differences between Gram pos and neg.
E: both Gram pos and Gram neg bacteria adhered to the HA.
@Elaine, what was it made you think that HA would do this?
@Greg, we appreciate you wrestling with your internet:-)
@Elaine we’ve done a bit of reading about biofilms (colonies of bacteria, is that right?) – are these likely to adhere to the HA any differently than bacteria not in a biofilm?
G: @all I am back ………… for time being.
E: I also wonder if HA might be helping the bacteria survive on the diapers. I saw that you guys had a discussion about biofilms with some other scientists, and they probably told you that biofilms are harder to clean or remove than “free-floating” bacteria. The HA may be helping the bacteria form a biofilm on the diapers so that they are harder to clean off.
@all Sorry I’m late, just catching up with the chat now 🙂
@Elaine how might that work?
G: Does anyone know if HA is intentionally coated onto diaper cloth (fabric)?
E: We thought HA might do this after we read about how HA is an important part of tooth enamel. People studying dental plaque formation use HA as a model, and dental plaque starts forming as soon as you finish brushing your teeth!
@helen-johnston just to highlught the questio from earlier,what form did the HA take? Crystal, pellet, other form?
@Greg there was no HA on the control nappy tested.
E: Crystalline HA is what we used. If the HA has built up on the diaper, and bacteria adheres to the HA, that would be kinda like a biofilm.
@Greg why would people put HA on things like cloth?
@Elaine Aha! So teeth are actually a perfect surface for growing biofilms on?
@Greg, no, we don’t believe any of the HA is put their deliberately.
E: Yes on teeth!
Is that why teeth can feel a bit fuzzy if you don’t have time to brush them in the morning?! 😮
E: I read your hypotheses, and agree that the HA might form from calcium and phosphate from many sources. Calcium is abundant in urine, and both calcium and phosphate are important dietary minerals. Important for teeth and bones. HA is also a critical component of bones. So it makes sense that we excrete them in urine and they could build up on a diaper.
E: yes, that fuzz is bacterial biofilm!
@Elaine That might suggest it being deposited between when the nappy is used and when it is washed.
@Elaine its great to have some enciurageement that we are heading down the right road.
I think most of us had been assuming that it was formed during the wash.
is there anything else that would need to be present to cause HA to form from calcium in the urine, or would this alone be enough to ‘grow’ HA?
We also talked abiut urine beung the source, the wash is just faillng to remove or halt it
sorry, and phosphate
So if the HA had built up a lot during the use of the nappy, say at night, could the washing be not getting rid of it fully.
E: I also agree that washing in hard water may aggravate the odor problem. When we did our experiments, increasing the concentration of calcium made even more bacteria adhere.
@Elaine that’s really interesting, why is that the case?
@Elaine interesting. Is there a sweet spot or ideal concemtration
E: Yes, I think that if the HA with the adhered bacteria aren’t being completely removed, so they are ready and waiting to make more smells again when the diaper gets wet.
E: Can a water softener be used? I am not sure if there are products like that to add to the wash?
@Elaine so, theoretically if it isn’t washing off and then you are adding more phosphate etc you are perpetuating the issue even more
@all is calgon a water softener?
@Sarah yes it is
@sarah-west yes
I don’t know about Calgon but I have heard of people using soda crystals in their wash in hard water areas
G: laundry soap makers have tried to reduce or remove phosphate from their products. I don’t know if there are such limits in the UK; but is it possible that the phosphate species identified was actually trisodium phosphate?
E: Not sure if adding more phos/calcium is perpetuating the issue, but I do think that if the HA with bacteria are there already, then the biofilm is poised to grow, ferment, and produce odors when the diaper is wetted again. Urine and feces provide food for the bacteria.
Yum.
@Elaine Washing detergents have water softening components added (I’m sure I read about zeolites as an example). However there is a lot of concern and confusion about if laundry detergents are good or bad for skin and if they are fully washed out.
Therefore, some people use very limited doses of detergent, make their own from simple soap or use other products like soapnuts or washing balls.
@Greg Phosphates in detergents have been banned in the EU for a few years. Although although they can still contain up to 1% I think. We don’t know anything about trisodium phosphate I don’t think.
@Elaine would it be fair to say that the presence of HA would suggest an improved environment for bacteria and that more HA would encourage more bacteria.
Aha!! this explains why the stinky nappies are generally reported as “smelling clean until they are on the bottom”
E: What about using bleach to inactivate bacteria? I understand the concern with bleach not being fully removed.
E: Yes, Ellie, that is it!
@Greg i wondered whether the phosphate might be coming from the water: my aquarium supplier said we get algae problems in our tank because we have high phosphate levels in the water due to intensive agriculture. No idea if any of that is true!!
I’m still interested to try to understand why bacteria like HA so much- appols if I missed it, had to tend to my microwave cake in a mug!!
@sarah-west There are areas of the UK that are designated as nitrate/phosphate sensitive due to farming practice (non-point pollution source) the EA produce maps/ monitoring data on this
E: We are not 100% sure but it just seems to be electrostatic interactions, diffs in charges.
G: Phosphates enhance algal growth for sure. Aquariums are no exception. If the water used for washing was softened with hexametaphosphate(s) (i.e. Calgon) it might have high levels of general ‘phosphates’ .
@sarah-west you may also be able to get the data for your water source from your water company.
Thanks @ellie-marland will go and have a look!
E: It looked like in your research that the odor problem was greater on the viscose diapers. is that right?
@Elaine Yes- bamboo viscose
@Elaine correct although a small sample size
E: That is interesting that HA built up more on those fibers. No idea why that would be.
@all didn’t someone think it might be due to the structure of the fibres?
Could drying time have anything to do with HA formation? or the ‘absorbency’? these nappies are generally used as they are more absorbent and can hold more water/ wee!
@Elaine there was a suggestion that it is due to how much water its retained in the fibre after washing and that helps to build the hA
@Elaine have you tested HA on differnt materials to see if it grows more on certain ?
sorry – certain ones?
@Elaine can you tell us what the phosphate / calcium dose levels waste that you found most effective for building the ha
Our Ha looked like this – https://nappysciencegang.files.wordpress.com/2016/02/12669508_10154112431112150_6487019597232002442_n.jpg is that in anyway similar to the crystals you were using?
E: No, we have not done that, but I think there are lots of medical applications for putting HA on things like dental implants and hip replacements.
So it definitely sounds like a HA coating on your nappies will make them hold onto more bacteria. (Or maybe attract more bacteria?) But does it go the other way too? Do loads of bacteria affect the HA building up?
E: We didn’t build the HA, but purchased it. this is a guess, but I think it forms quite naturally at physiological conditions. Meaning it might form quite naturally at urine pH in a wet diaper as the urine dries. Not sure how having greater loads of bacteria would affect the buildup.
@Elaine – is there anyway to know if our nappies contain HA or not?
E: Sorry, nappies! I don’t think there is an easy way to find out if the nappies have HA or not, without sending it to the lab for analysis.
@Elaine, it seems like you’ve had a look at some of our findings and thoughts so far. If you were us, what experiment(s) would you do next?
E: I would do an experiment to see if using chlorine bleach in the wash might help. It wouldn’t likely get rid of the HA, but it might kill some of the bacteria.
Sorry in so late, poorly baby and poor connection. Trying to catch up, amazing conversations
E: WHat would you do, greg?
G: A medical diagnostics lab ought to be able to tell the difference between the two major types of kidney stones [1] Calcium oxalate stones and [2] HA stones; so they might be able to test; but it is the lab route.
Sorry if I’m way out of it – wouldn’t bleach deteriorate the fabrics?
@Greg Kidney stones can be made of HA??
@Greg ah ha!! Sooooo kidney stones can be made of HA! THAT is interesting! It indicates that all the ingredients are present in the urological (?) system and that the water etc may have less to do with it than we thought.
Does the drying time effect ha build up? I. E bamboo takes a long time compared to cotton – is ha building up as they dry?
so if HA might form readily with urine drying, perhaps rinsing a nappy or wet pailing might discourage it.
Or does a solution of urine in wet pailing increase it?
E: Yes, bleach use can wear out the fabric faster. Do you put wet nappies straight in the nappy pail as is? Wet pailing, Kate just said, that was what I was going to ask about.
So HA may be forming during the ‘storage’ time BEFORE the wash. We should make sure we collect ‘time between washes’ in our nappy survey data.
@Greg So if HA can be formed entirely within the body would it theoretically be possible to find HA ina ddisposable nappy too?
I think most people ‘dry pail’ these days – no water or chemicals, just soiled nappies in a bucket and then transfer to machine.
Wet pailing is not generally advised, I understand. I wet pailed first time around and then was warned off.
E: Yes, the HA could be forming then, while waiting to be washed. Wet pailing would be a good experiment, compare to dry pailing.
@Elaine dry pail its most common nowadays, wet pail its considered too have a detrimental effect on the fabric, and be a bit grim
But interesting that my mum wet paired,
@iona – I hope not as some of mine go 3-4 days now!!! Might explain crunchy nappies
E: Yes, it does sound like it would create a separate odor problem.
Could be an easy experiment!
So the HA could be there even before the nappy goes into the washing machine? My mind is blown.
G: As far as a chemical solution; I recall someone mentioning that ozonation is a good way to control diaper odors.
ozonation?
@Greg, ozonation? Can you explain?
G: Elaine, you might recall the ozone generator people trying to use ozone cabinets for beef carcass sanitizing)
@aj-ridout – mine too!
G: Thats a whole different kettle of fish. But ozone can work in dry conditions like, I would imagine dry pailing.
Elaine, the last person we spoke to was an expert in biofilms but felt that bacteria in a biofilm would be unlikely to survive a washing machine and then being dried. do you think The HA would increase their chances?
Yes, i think that they are probably forming a biofilm on the HA, thereby improving their survival.
What temperature do you think would kill the bacteria?
If I’m understanding this right… even if the bacteria are thoroughly washed away, while there is that HA surface there then new bacteria will be attracted to it and hang out there.
E: Most probably don’t survive, but some always will. Not sure about the drying temperature, that might be another experiment for you!
@sophiacollins that sounds really plausible
Ooo exciting, lots of new leads.
@Everyone, new theory. The HA is forming while the nappy sits in the pail. Historic practices like wet pailing and boil washing may have helped deal with it. ALSO the hexametaphosphate used as the main stabiliser in detergents before it was outlaw…
ed in 2011 may also have dealt with it.
Both, Sophia. If the HA remains, new bacteria from urine or feces could adhere. And some bacteria may also have survived washing and drying.
Wasn’t one of the test nappies tumbled? I think it had some HA but not stat. significant amounts.
I love tumbling my nappies once in a while to make them soft- wonder if this is breaking something down as it increases the drying speed (as well as applying heat). Hrm
I’m aware that we’ve been here well over an hour now. We should probably let Greg and Elaine go! Does anyone have a burning last question?
I washed my nappies daily first time around though and still had stinky nappies, used non. Bio. This time 3-4 days to a wash and use bio. Confused
G: This ia really fascinating but releuctantly I must go to another meeting. I will check in to see the conlcusions. In the meantime I would suggest aartricle by the guru of washing machine microbiology Dr. Charles “Chcuck” Gerba. See: http://www.theatlantic.com/magazine/archive/1997/09/something-in-the-water/376950/ Cheers everyone.
E: Do you usually hang dry the nappies?
Thank you so much Elaine and Greg. You have really given us a lot of food for thought!
Nope. I’m just going to go to bed to dream of HA (probably for about 45 minutes before the small wakes@)
Thanks!
Thank you both so much!
@Elaine, new question. if the attraction between the bacteria and HA is electrostatic, what effect would bacteria already on the HA have on the building on of more apatite? might it help it along?
@Greg @Elaine if you think of anything else that might be interesting we would love to hear it. Keep in touch and follow us if you can, we may need to chat again!! Thank you! So incredibly interesting!!!
oh sorry, too late! Thanks so much 🙂
Thanks Greg. We really appreciate you talking to us.
Brilliant chat, sorry to be late, you guys are great x
E: Thanks for the opportunity! Never knew that our old HA work would attract attention from the Nappy Science Gang!
@elaine, yes, most people hang dry. Various nappy materials can’t be tumble-dried.

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