1. Introduction and Purpose References and Acknowledgements
Modified Track-plating: A Rapid, Accurate and Economical Quantitative Culture Method to Measure the Composition and Resistance Profile of the Gut Microbiome Fawcett N1, Mathers A2, Cheruvanky A2, Sifri C2 Nuffield Department of Medicine, University of Oxford, Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia Health System Correspondence: @drnjfawcett
P 1333
Introduction and Purpose
Results
Efficient methods are needed to quantify resistant organisms in the gut microbiota. High level carriage of Extended-Spectrum Beta-Lactamase (ESBL) Resistant Enterobacteriaceae and Vancomycin-resistant Enterococci (VRE) are associated with risk of bloodstream infection1 and ESBL UTI2. Whilst molecular methods are advancing rapidly, selective culture remains the gold standard. Quantification of bacterial Colony Forming Units(CFUs) involves spreading serial dilutions onto individual plates. Other quantitative methods include spiral plating (requiring specialist equipment which can be blocked by faecal samples), the Miles and Misra method3 and a modified track-plating4 method. The relative accuracy of these methods in assessing the composition and resistance profile of the gut microbiota was assessed compared to ‘gold standard’ spread plating.
There was no significant difference in the calculated logCFUs /ml using track plating compared to spread plating. The Miles and Misra method resulted in a significant difference (p<0.01) in estimated logCFUs/ml with over-estimation of the by around 0.25 logCFUs/ml. Track plating was reproducible between operators, significantly faster (average 48sec vs 5min58sec) and required 20 fewer plates.  
In an assessment of modified track-plating to quantitatively assess the resistance profile of 7 faecal samples (isolates growing on ESBL/VRE screening agar compared to sensitive isolate growth), the method fast and cost-efficient. Spread plating these samples (from 102 to 108) would require an estimated 168 plates and £250 material costs. With drip-plating, this was achievable in 18min 30 sec and £30.50, making quantitative resistance screening feasible for multiple samples.
Spread plating Modified Track Plating Miles and Misra
(6 lines)
Estimated LogCFU counts obtained from Track Plating are comparable to Spread Plate estimation when performed by 3 operators on 3 faecal samples
Difference in LogCFU count from the Spread Plate Mean and ANOVA p
Method
Conclusions
3 faecal samples were plated onto chromogenic agar by 1 experienced operator, 1 Biomedical Scientist and 1 Physician with 20 minutes of training. Spread plating, track plating with 6 and 8 lines, and Miles and Misra method were used. Operators recorded time taken to successfully complete plating, including repeat plating when initial attempt unsuccessful. A blinded colony count was performed on all unique colony morphologies. and estimated log10 CFUs/ml calculated. A one-way ANOVA analysis was used to compare the variance between the spread-plate estimate and other methods.  7 faecal samples were subsequently plated onto selective agar using track-plating, and resistance phenotype confirmed using disc-diffusion. Cost estimates were based on up-to-date UK quotations. .
Track plating can be used in quantitative assessment of the faecal microbiota with an accuracy comparable to spread plating. It makes quantitative assessment of the faecal microbiota in multiple samples feasible by greatly reducing the time and number of agar plates required, and can be performed using inexpensive standard laboratory equipment.
References and Acknowledgements
Mean time per sample /min:sec(sd)
5:58 (1:07)
0:38 (0:18)
0:54
(0:39)
1:09
(0:18)
Plates per sample 8 1
Intestinal domination and the risk of bacteremia in patients undergoing allogeneic hematopoietic stem cell transplantation, Taur et al, Clin Infect Dis 2012
Ruppe et al, Relative fecal abundance of extended-spectrum-β-lactamase-producing Escherichia coli strains and their occurrence in urinary tract infections in women. Antimicrob Agents Chemother. 2013
The Estimation of the Bactericidal Power of the Blood, Miles et al, The Journal of Hygiene,1938
Simplified Agar Plate Method for Quantifying Viable Bacteria, Bradley et al, Biotechniques,1997
Quantitative assessment of resistance in 7 faecal samples – based on growth on screening agar and subsequent disc-diffusion testing
identity
Sample 1 2 3 4 5 6 7
E.Coli
7.3
5.8
7.8
7.6
6.0
5.3
7.9
Enterobacter/Klebsiella
4.0
6.6
Enteroccus sp.
5.9
4.5
4.8
7.5
VRE
ESBL Enterobacteriaceae
3.6
3.8
ESBL on direct plating
Yes No
Modified Track-Plating video demonstration
Acknowledgements for Method development: Original Track plating method – Bradley et al Use of multichannel and further development at the Ausubel lab, Massachusetts General Hospital ( C. Sifri, J. Villanueva, P. Yorgey) and University of Virginia (A Cheruvansky, A Mathers ), faecal sample/resistance method – N Stoesser, N Fawcett)
Supplemental data and pdf available online
This work was supported by the Health Protection Research Unit UK. This report is independent research by the National Institute for Health Research. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.. All image rights – Oxford Medical Illustration. And Nicola Fawcett, under Creative Commons Licence for non-commercial use with attribution.