1. How low can we go?
Implications of low bacterial biomass in otitis media microbiota studies
R Marsh, C Pope, M Nelson, H Smith-Vaughan, A Chang,
L Hoffman, A Leach
Aim is to highlight we are studying low biomass and that methods developed for poop may not be generalisable

2. Microbiota methods developed for high biomass environments
Poop at 1:1000 dilution
Nasal discharge during URTI
Noting that methods were developed for high biomass environments, which in humans has largely beeen poop. So the question we have been interested in exploring is how well do the methods developed for poop etc translate to the types of specimens we analyse in OM research.
So, when we think about whether these methods can be translated from poop to noses and middle ears, one of the first things we need to contend with is that these environments are really really different in terms of the bacterial biomass that is present at homeostasis. So, if you consider the gut, where bacterial colonistaion is critical to health and well being, there is a really high bacterial biomass. In contrast the NP and middle ear have much lower biomasses at homeostasis.
These differences in biomass are probably best visualised – so what we have here is a couple of gram stains the first one is a picture that was prepared from poop after it was diluted 1:1000 with saline, and you can see that even in this type of heavy dilution there are clearly loads and loads of microbiota present. In contrast this is gram stain of some snot my son sneezed out when he had a respiratory infection and you can see that even when infected, there is a substantially lower bacterial load
Ending with:
the middle ear at homeostasis is sterile/low biomass
So, the question is, do these differences matter in microbiota analyses? Especially when we consider that 16S seq is a DNA based method that involves PCR and so it should be super sensitive and so these load issues shouldn’t matter – right?

3. Low biomass affects microbiota profiles
Spurious reads increase as bacterial biomass
decreases <103 GE
Note errors in the literature – e.g. the placenta stuff
Need to note the starting concentration
Salter et al BMC Biology 12:87

4. Is this a problem in otitis media?
105
104
103
102
101
100
Bacterial biomass/uL DNA
11 children with OME
Jervis-Bardy et al. Int J Pediatr Otorhinolaryngol 2015
Describe the study
Note that 4/11 NP swabs, all of the MEF samples, and 8/11 adenoid samples had low bacterial biomass
Ok, so this is a small cohort, could this just be something unique to this small study?

5. Low bacterial biomass affects:
Study Design
DNA extraction
Library preparation
Data analysis

6. Bacterial biomass affects study design
What is the expected bacterial biomass?
Estimation by 16S qPCR is essential
“Sterile” vs infected environments?
Well, first up we need to consider bacterial biomass in our study design
Regardless of the environment being studied, we should always commence a microbiota analysis by asking “what is the expected bacterial biomass”?
If you are studying poop or throat samples – environments that at homeostasis are expected to have a dense resident mucosal flora – then it is probably ok to proceed directly to sequencing.
If you are studying environments that are expected to be sterile or near sterile at homeostasis, then it’s critical that bacterial biomass is determined as part of the microbiota analysis.
We also need to consider the biomass in the environments being compared – for example, if the study aims to compare mucosal environments in healthy controls with patients having acute infections, then it is critical that we consider whether differences in bacterial biomass would be expected in that environment during health compared with disease (and that the middle ear and lower airways are classic environments which have evolved to have minimal, if any, resident bacterial flora. If the study will compared healthy controls and cases, then it is critical that differences in bacterial biomass be tested as potential confounders of any significant differences detected in the microbiota – are you looking at dysbiosys or differences between low and high bacterial load?

7. DNA extraction method matters
Silica column-based DNA extraction
MoBio 5.8X more DNA than Qiagen
Silica column vs Magnetic bead-based extraction
LGC MagMini 6.7X more DNA than MoBio
Thin down….....
Equivalent to a 9.6X increase in yield over our standard method (QE)
We extracted DNA from oropharyngeal swabs using silica column and magnetic bead-based methods and found that DNA yield from a magnetic bead method was up to 6X higher than
We extracted DNA from oropharyngeal swabs using silica column and magnetic bead-based methods and found that DNA yield from a magnetic bead method was up to 6X higher than other methods. other methods.

8. Bacterial biomass/uL DNANP
Bacterial biomass/uL DNA
105
104
103
102
101
100
106
107
532 NP swabs
Even with optimised DNA extraction conditions, 39% of 532 NP swabs had low bacterial biomass
Noting that even with the DNA extraction optimised around 40% of endv samples were still low biomass
This may reflect a high use of antibiotics in this patients in this particular study, but still demonstrates that if the bacteria aren’t there then even the best DNA extraction method in the world won’t be able to increase the DNA yield.
So, what else needs to be done?

9. Library preparation Should all samples be run?
Prepare library with 40 ng amplicons
Run samples with <40 ng as negative controls
EMP: add 240 ng amplicons from each sample
Low biomass NP swabs (n=209)
1-67 ng/uL (median 3.5, IQR )
Negative controls (n=25)
Amplicon concn 1-6 ng/uL (median 2.2, IQR )
centile read if rep<1000, centile ( ) /* median 3490 IQR */
centile read if rep>=1000, centile ( ) /* median 29074, IQR */ noting pre remsingOTUs and contams
Noting upping the volumes may decrease the overall library concentration, and this will need to be corrected for
All samples must be run – noting at this point, you’ll see papers where the low samples are arbitrarily excluded from the analysis; however, this is not current recommended best practice (referring to the placenta paper)
Neg Control" /*1-5.5 */
centile ngul if site=="Exn Neg Control", centile( ) /* med 2.23 IQR */
Modification of standard library preparation protocols is required for low bacterial biomass specimens, including increasing template volume and replicate numbers when preparing individual libraries. Preparation of the pooled sequencing library is routinely done using a standardized DNA mass. This was not always possible for low bacterial biomass specimens that were instead added based on a standardized volume.
For study of 532 NP swabs
All samples returned reads
Range 4 - >50000 reads/sample
54 samples returned <1000 reads
qPCR - 3/54 had bacteria

10. Essential to test for spurious reads
Remove Spn
Add rhos and p’s
?ENDV %singleton reads vs load
Please come see me if you’d like to know how to do this.
Jervis-Bardy et al. Microbiome 2014

11. Conclusions
Low bacterial biomass affects all stages of microbiota analysis
16S qPCR is essential in OM microbiota studies
Failure to remove spurious reads risks false positive results
Noting not unique to OM but to study of any mucosal site that doesn’t have a dense resident mucosal flora

12. Acknowledgements Supported by: Child Health Amanda Leach Peter Morris
Anne Chang
Heidi Smith-Vaughan
Jemima Beissbarth
Cain Hendy
Gabrielle MacCallum
Global + Tropical Health
Mirjam Kaestli
Hoffman Lab
Luke Hoffman
Chris Pope
Maria Nelson
Dan Wolter
CF Sequencing Core
Kyle Hager
Alaska
Ros Singleton