Webinar Q&A — Cyclical Bias


     
In a recent webinar, Dr. Alex Rodriguez-Palacios of Case Western Reserve University School of Medicine discussed a confounding influence on many common microbiome study endpoints uncovered in studies using the NesTiso system.

The NesTiso system has been described in a previous publication1 and examined in an on-demand webinar.

Due to time constraints, some of the questions you submitted to our Q&A session went unanswered. We present here the full Q&A, as well as several poll questions posed during the webinar.

Cyclical Bias Webinar Q&A

  • Q: I would like to know how cyclical bias affects uncultivable microbes. Do you have 16S microbiome data documenting this form of bias?

Dr. Alex Rodriguez-Palacios (ARP): Yes, we do. We are conducting carefully planned experiments to validate what I described as "cyclical bias" and spiral dynamics of microbiome divergence.
  • Q: Don't you think wire floors could introduce more bias by causing animal stress?

ARP: Living in a dirty cage is also stressful. The use of wire floors in mice has been mostly restricted nowadays to studies that require metabolic cages. But, in the past, they were more common in nutritional studies.

We are testing different types of floors that could be useful and not stressful to mice. Extrapolating from domestic animals, slatted floors can be a reasonable alternative for synchronizing the sampling time.
  • Q: What is the best practical method to control for cage-cage variability?

ARP: In 2015, we reported the Inter-Subject Pre-Experimental Fecal Exposure Homogenization (IsPrefeH) method as a community-contributed manuscript for the community to comment on. Then, we gavaged mice with feces and bedding microbiota. We have had since then feedback and more data and experience, including our publication.

With the discovery of selection bias in the bedding, we now conduct the same protocol using only fresh fecal samples. We do not gavage dirty bedding material to the mice. We know that spores are present in feces. No need for biased bedding microbiota.
  • Q: When is the best time to collect samples from mice for analysis?

ARP: Because I work Monday-Friday, the best that works for me is Tuesday Morning. We ask for supplies, new cages on Friday, change cages ourselves on Monday morning, and sample the mice early the following morning on Tuesday. If Tuesday doesn't work for us or the experiment, we just rotate this approach over the calendar.

Most people argue that it is too much work. I suggest them to take their time and think about what is best for them, but encourage them to report what they do in this regard. It will become important in the future, given the NIH funding agency directions.
  • Q: What type of IVC cage is being used for the thermal imaging pics? IVCs vary in technical function and air entry.

ARP: The thermal images shown in the webinar correspond to mice housed in Allentown MicroVent units, which have one single port of air entry at one end of the cage. The cages also had Reemay filter top lids.

Further specific details can be found on the manufacturer's website.
  • Q: Have you only looked at corncob bedding? What type of corn cob have you tested?

ARP: Correct, thus far we have only focused on corncob bedding since it is a very popular material in research facilities due to its great moisture retention and the potential to inhibit ammonia production, especially if autoclaved (see ref PMID: 22776115).

Although we do not endorse any manufacturer, at the time the experiments were conducted our cages contained Bed-o' Cobs (The Andersons, Maumee, OH).

Expanding on this answer, I would like to add that although our original goal was to test the hypothesis we generated from 16s rRNA sequencing and mathematical modeling using corncob, we anticipate that different bedding materials would have different effects on the fecal microbiome outside of the animal, possibly confounding research. Regardless of what type of bedding is used and the important effect that its nutrients and chemicals may variably have influencing the animal microbiota (e.g. inhibit coliform or promote Enterococcus), room air is the most substantial driver for selection bias of microorganisms outside of the mouse body, both in the feces and in/on organic matter. The rapid or steady growth of aerobes paired with the negative selection that oxygen imposes on strict anaerobes is what likely makes the bias so strong favoring Bacillales, Pseudomonadales and Burkholderiales (see details in Fig 4 in ref PMID: 29491439).
  • Q: Could you provide more information on fecal homogenization?

ARP: Thanks for asking. For clarity, first I would say as a reminder that 'Fecal Flora Homogenization' refers to a protocol better defined as "Inter-Subject Pre-Experimental Fecal Exposure Homogenization" (IsPreFeH), where animals needed for an experiment are gavaged with a pooled homogenate of their collective feces to even the risk of exposure to the microbes present across all mice, which itself may vary from cage-to-cage by microbiome drifting since birth.

The detailed protocol was initially reported in 2015 as an article entitled: Stereomicroscopy and 3D-target myeloperoxidase intestinal phenotyping following a fecal flora homogenization protocol which we published accompanying an article we published in Nature Communications in 2015.

However, in response to this question and to reflect our novel understanding of cyclical bias in animal microbiome research, I suggest obtaining a version for printing which I have deposited with an update note emphasizing the need to avoid the inclusion of the soiled bedding material as part of the protocol, as well as a reference to this webinar. This PDF version can be reached directly from Google Scholar or from ResearchGate by browsing the article title.
  • Q: What is the culture media used for culture — red color plates?

ARP: Trypticase Soy Blood agar supplemented with 5% defibrinated sheep blood (BBL Catalog# 221261)

Webinar Poll Questions

We asked three poll questions during the cyclical bias webinar. Here are the results submitted by our audience:

  • Q: In a 1-month diet experiment with 5 mice/group, which housing option do you believe is financially preferable?

80% of respondents stated that five mice housed together in one cage (one cage total) was the most financially preferable. The other choices were three mice housed in one cage and two mice in another cage (two cages total), or one mouse per cage (five cages total).
  • Q: How do you control for cage-to-cage microbiome variability in your experiments?

33% of respondents do not control for it, while 34% use either fecal homogenization or mixed bedding protocols across cages.
  • Q: Do you collect fecal or other samples from animals in experiments when the cages are clean?

14% of respondents stated yes and 29% stated sometimes, while 36% of respondents do not monitor how dirty the cages are when samples are collected.
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Reference:
Rodriguez-Palacios, A., Aladyshkina, N., Ezeji, J.C., Erkkila, H.L., Conger, M., Ward, J., Webster, J., Cominelli, F., 2018. 'Cyclical Bias' in Microbiome Research Revealed by A Portable Germ-Free Housing System Using Nested Isolation. Sci. Rep. 8, 3801.

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