Characterization of urinary
microbiota for its possible role as a
biomarker in urolithiasis
Introduction
The term microbiota refers to the microbial taxa
that are associated with an environment.
Bacteria, archaea, viruses and fungi- found in the
gastrointestinal tract, but also in other exposed
tissues, such as the skin, upper respiratory and
urogenital tracts [1].
Some of these organisms perform tasks that are
useful for the human host.
[1].Gajer,
P., Brotman, R. M., Bai, G., Sakamoto, J., Schütte, U. M., Zhong, X., & Ravel, J. (2012). Temporal
dynamics of the human vaginal microbiota. Science translational medicine, 4(132), 132ra52-132ra52.
However, the majority have been too poorly
researched for us to understand the role they
play, however communities of microflora have
been shown to change their behavior in diseased
individuals.
A number of recent studies suggest that the
urinary tract harbors a unique urinary microbiota
(Fricke et al., 2014 and Hilt et al,. 2014) which is
substantially different from the populations of
the gut and vagina.
1. Fricke, W. F., Maddox, C., Song, Y., & Bromberg, J. S. (2014). Human microbiota characterization in the course
of renal transplantation. American Journal of Transplantation, 14(2), 416-427.
2. Hilt, E. E., Rosenfeld, A. B., Zilliox, M. J., Thomas-White, K., Fok, C.,& Wolfe, A. J. (2014). The female urinary
microbiome: a comparison of women with and without urgency urinary incontinence. MBio, 5(4), e01283-14.
 Urolithiasis denotes stones originating anywhere in
the urinary tract is an ailment that has afflicted
human kind for many centuries.
Up till now, urinary stones were a major health
problem.
A large number of patients required extensive
surgical procedures and a significant minority had
their kidney removed.
Recurrent stone formation and progressive
pyelonephritis occur in those who are not cured.
The morbidity and expense that result from this
disease are great.
 Urolithiasis is a complex disease, thus an
understanding of the epidemiology, particularly the
interactions among different factors, may help
leading to approaches that reduce the risk of stone
formation.
Urinary calculi may harbor bacteria, and this may
lead to deleterious events during stone
fragmentation and removal.
Performing a stone culture may identify organisms
that are not covered by initial antibiotic therapy,
providing an opportunity to clinician’s earlier
targeted therapy.
Undoubtedly, the microbiota is linked to urological
health and disease, but the extent of this
relationship is still unclear.
Contrary to doctrine, the urinary tract is inhabited
by a unique urinary microbiota; further research is
needed to characterize this microbial community in
health and disease.
The present study will focus on the composition of
urinary microbiota that may be altered with
urolithiasis, thereby help in understanding the role
of microbiota in urolithiasis.
Objectives
 To study the urinary microbiota in urolithiasis
and normal cohorts by culture based techniques.
 To compare composition of urinary microbiota in
urolithiasis and normal cohorts by sequence
based (16S rRNA gene sequencing) molecular
techniques.
 To establish the relation between urolithiasis and
urinary microbiota.
 To identify the possible indicator organisms to
establish as biomarker for urolithiasis.
Research plan/Methodology
Study Design: Hospital based experimental study.
Study Population: A total of 60 kidney stone cases
(irrespective of age/ gender), Dept. Urology, YMCHEthical approval & informed consent.
Control group 60 healthy urine donors.
Exclusion Criteria: a) Underlying systemic diseases
and secondary causes of urolithiasis, b) Active UTIs or
other infections within 1 year prior to admission c)
History of antibiotic treatment for the last two weeks
& d) Patients who are not willing to consent.
Milestones
6
12
18
24
30
months
Urine, stone samples collection
√
√
√
Blood and urine parameters
√
√
√
Isolation and maintenance of
bacterial and fungal cultures
√
√
√
√
√
√
ELISA and electron microscopy
√
√
Chemical analysis of urinary
stones
√
√
DNA extraction, PCR and gene
sequencing
Data Analysis and Publication
√
√
Work elements and detailed plan
Work element 1: Sample collection, blood and urine
parameters, Microbiological culturing Scheduled
time: 6-18 months
Work element 2: Nucleic acid extractions. PCR and
sequencing. PCR, Oxalobacter formigenes Scheduled
time: 12-24 months
Work element 3: Chemical analysis of urinary stones,
ELISA and electron microscopy for nanobacterial
detection. Scheduled time: 18- 26 months.
Work element 4: Data analysis and publications
Scheduled time: 24-30months
 Isolation, biochemical, characterization/ identification
of bacteria/ fungi: Genomic DNA will be isolated, PCR
and I6Sr RNA sequencing, Sequence analysis .
 Determination of O. formigenes in feces/ urine: PCR
(Sidhu et al., 1998)
 Plasma oxalate concentration: HPLC method (Ho¨now
, et al., 2002).
 Detection of nanobacteria: Nanobacterial detection
will be done by ELISA (Kajander et al., 1998).
 Chemical composition of stones: FTIR spectroscopy
Statistical analysis
SPSS software ( Version 17) continuous variables,
Student’s t tests.
Categorical variables,
Fisher’s exact tests.
Pearson
chi-square
and
 Wilcoxon rank sum tests: median abundances of
most abundant sequenced taxa .
Frequencies of detected genera: chi-square or
Fisher’s exact test
Facilities available- equipments
Real Time PCR ,Electrophoresis units/power
packs , Ultra low Deep Freezer (-80 ºC), HPLCHitachi, Thermal Cycler.
UV-VIS Spectrophotometer,
Biosafety Cabinet, Ice Flaker.
ELISA
reader,
UVP Gel Documentation & Analysis System ,
Fluorescent Microscope.
Budget requirements
Sl.
No
1
2
3
4
5
Items
1st year
(lacks)
2nd
year
(lacks)
3rd
year(l
acks)
Glass wares, plastic wares and stationary
1.0
0.5
0.5
Chemicals and enzymes
Culture media, Molecular biology reagents,
PCR chemicals, Primers, Electrophoresis
reagents, Nucleic acid stains
3.0
2.0
1.0
Micropipettes
0.5
Isolation and purification kits,
DNA isolation kits
PCR purification kits, RTPCR kits,
Commercial kits-bacteria detection, Urine
DNA Isolation kit Gel extraction kits,
Sequencing kits, ELISA kits
2.0
2.0
Total
6.5
4.5
1.5
Non-recurring (equipment)
spectroscopy- 6.00 lacks
Travel:
FT-IR
Travel charges: 1.5 lacks (FTIR analysis, electron
microscopy analysis, sequence analysis, to
attend conference, seminars, workshops etc)
Overhead charges: As per the norms
Expected outcome
Undoubtedly, the microbiota is linked to urological
health and disease.
Urinary microbiota may be altered with
urolithiasis, thereby help in understanding the role
of microbiota in urinary stone formation:
diagnosis/ Prognosis
Alternative treatment strategies focused on
reconstituting the microbiota should be explored.
 Microbiota-targeted treatment strategies