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

BCasasolaR@iingen.unam.mx

Bety.casasola@gmail.com

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