Detection of VBNC Vibrio cholerae byrt-qPCR based on
transcriptome analysis (RNA-seq)
M. Eng. Beatriz Casasola Rodríguez
PhD at Engineering Institute UNAM
México City
HDID VHow dead is dead?
Conference on exploring the edge of bacterial life
Luis Losanob , Ignacio Monje Ramíreza, Isaura Yáñez Noguéza, María Teresa Orta de
Velásqueza*, Guillermo Ruiz Palaciosc*
a) Engineering Institute UNAM
b) Center for Genomic Sciences UNAM
c) Medical Sciences National Institute
2
VBNC
Viability
- Culture
- (1) Fluorescent dyes (qPCR-PMA, LIVE/DEAD, CTC, Flow cytometry )
- (3) Molecular Viability Testing (pre-rRNA)
- (4) rt-qPCR (based on? )
Detect and quantify VIABLE microorganisms
rt-qPCRRNA-seq Differential gene
expression
analysis
Upregulated sequence?
OBJECTIVES
Principal
Develop a molecular technique, capable of detecting the
VBNC state in Vibrio cholerae based on transcriptome
analysis
1. Induce the VBNC state in V.cholerae
2. Determine a genome sequence upregulated in the VBNC state
3. Evaluate the induction process by culture, qPCR-PMA, and rt-qPCR
4. Compare the resistance of V. cholerae against chlorine and ozone
disinfection in the VBNC state and the viable culturable form
4
CULTURE
INDUCTION SEQUENCING1) CONTROL (Exp culture)
2) ASW @4 °C
3) ASW + c-DI-GMP
Vibrio cholerae O1 El tor
1) ASW and 4 °C
2) ASW + c-DI-GMP
1 3
9
MICROBExpress™ RiboPure ™
ION PROTON
DAY 1
DAY 10-
15
Culture + LIVE/DEAD
CULTURE AND INDUCTION
- Exponential phase ✔
- Stationary phase ✖
- c-DI-GMP ✔
Loss of culturability and viability of V.cholerae
SEQUENCING (NOIseq)
- Total reads
- Usable reads (mRNA)
- Upregulated
B_mean
Control_mea
n log2FC
g184 98.3 18.4 2.4
g478 491.8 217.2 1.1
g756 187.1 25.8 2.8
g939 351.9 84.3 2.0
g1997 1042.0 394.1 1.4
g2359 686.9 214.8 1.6
p571 135.4 4.17 5.0
g258 9409.3 2202.1 2.0
g589 1036.8 426.8 1.2
p648 163.9 40.3 2.0
g588 410.3 185.7 1.1
g703 122.5 39.6 1.6
p699 185.6 60.2 1.6
g2023 117.4 26.4 2.1
g2197 466.2 310.0 0.5
g408 113.3 50.9 1.1
g2248 326.36 149.9 1.1
A_mean
Control_me
an log2FC
g1226 308.8 2.1 7.1
g1711 371.8 5.3 6.1
g684 295.3 34.8 3.0
g1997 1768.1 477.1 1.8
g682 141.1 10.7 3.7
g653 2425.8 897.4 1.4
g683 375.6 79.9 2.2
g1952 490.6 69.5 2.8
p635 86.4 6.8 3.6
g1945 479.6 74.9 2.6
g1893 318.9 84.4 1.9
g2258 195.8 33.9 2.5
g956 66.0 6.84 3.2
g2005 948.7 223.5 2.0
g1785 59.9 2.9 4.3
g2014 283.1 46.9 2.5
g148 171.8 19.4 3.1
g2283 233.0 22.5 3.3
p803 151.3 16.1 3.2
g1556 68.9 8.7 2.9
g157 113.8 15.5 2.8
g1816 193.2 30.3 2.6
Condition 117 Upregulated genes
(Control and ASW+4 °C)
Condition 222 Upregulated genes (Control and c-DI-GMP and ASW+4
METABOLISM
ID Log2fc Product Function
G1842.41 Hemolysin secretion ATP-binding protein
Aminobenzoate Pathway related with aerobic and
anaerobic degradation
G9392.06 Inosine monophosphate dehydrogenase-like protein
The proteins responsible for the synthesis and
degradation of c-di-GMP
G199
7 1.4 Flagellin Flagellar assembly
P5715.01 Aminoimidazole riboside kinase
Amino sugar and nucleotide sugar metabolism - Vibrio
cholerae O1 biovar El Tor N16961
G2582.09 Lysine/cadaverine antiporter
Excretion and uptake of cadaverine by CadB , maintains
the integrity of cellular surface
P6482.01 Glycerol kinase
Glycerolipid metabolism, the majority of membrane
phospholipids are glycerolipids
g202
3 2.15 Glutamyl-tRNA synthetase concerning structure and catalytic properties.
0
100
200
300
400
500
600
0 10 20 30 40
Bacte
rial
Un
its
Days
Detection of bacterial units in the VBNC
stateRiboside Kinase Ctx Lol B
DETECTION OF THE VBNC
STATE
- Expression close to zero
from pure culture to day 5
(CT >38) with Riboside
kinase was detected
- After day 15th the
expression of Lolb
decreases while the
Riboside kinase increased
End of culture
DETECTION OF THE VBNC
STATE
- Bacterial counts decreased
along the induction process
- Bacterial counts were
higher that the ones
obtained by rt-qPCR
0.00E+00
2.00E+03
4.00E+03
6.00E+03
8.00E+03
1.00E+04
1.20E+04
1.40E+04
0 10 20 30 40B
acte
rial
Un
its
Days
qPCR-PMA
DETECTION OF THE VBNC
STATE
-6
-5
-4
-3
-2
-1
0
1
0 5 10 15 20 25 30 35
Lo
g r
ed
uctio
n
(Nt/
N0
)
CT(mg min/L)
Effects of chlorine in V.cholerae (Viable cultivable y VBNC)
VC
VBNC
-4
-3
-2
-1
0
0 2 4 6 8 10 12 14 16 18 20
Lo
g r
ed
uctio
n (
Nt/
N0
)
CT(mg min/L)
Effects of ozone in V. cholerae (Viable culturable and y VBNC)
VBNC
VC
- Biological systems are complex, and reproducibility of results do not
follow a pattern
- The transcriptomic analysis is a useful and powerful technique to
recognize upregulated regions that can be used to detect the VBNC
state
- This technique can be used as a method to detect metabolic activity
and to evaluate the particular resistance of the VBNC state against
disinfection
- I would like to thank people from three different
institutes that helped in this Research (Engineering
Institute, Institute of medical sciences and the genomic
institute)
- This study was supported by the National Commission of
Science and Technology of Mexico to solve National
problems
- The committee of the How dead is dead conference V