R&D - SEAC Safety & Environmental Assurance Centre

Source/ raw water samples (1Lx 3) were collected from 6 sites

of river Gomati flowing across Lucknow, a major city in Northern

India.

Further potable water samples were collected (10 L x 3) from

12 sites in the piped distribution network including one sample

from water works.

Each sample was filtered through 0.22 μm membrane filter.

Bacteria released from membrane by vigorous shaking,

supernatant concentrated to 500 μl by repeated centrifugation.

PMA treatment was given to Set 2 (Set SW2 and PW 2).

DNA was extracted by boiling from all sample.

Extracted DNA (5 μl) was used as template in the ttr gene-

specific molecular beacon- based qPCR assay.

SAFE DRINKING WATER- A CHALLENGE FOR THE DEVELOPING WORLD DETECTION OF VIABLE SALMONELLAE IN POTABLE WATER

2013 Water and Health Conference

Gulshan Singh1, Poornima Vajpayee1, Saurabh Bhatti1, Nirmala Ronnie2*, Nimish Shah2 Peter McClure2 and Rishi Shanker1 1 Environmental Microbiology, CSIR-Indian Institute of Toxicology Research, Post Box 80, M. G. Marg, Lucknow-226001, India; 2 Safety and Environmental Assurance Centre, Unilever R&D Bangalore, India & Colworth, UK

BACKGROUND

APPROACH & WORKING PRINCIPLES

Primers and Probes

Target genes: The ttr locus, comprising five genes,

ttrA, ttrB, ttrC, ttrS and ttrR.

Responsible for tetrathionate reduction.

Genetically stable in all Salmonella strains3.

Found only in Salmonella and in all major serovars and subspecies of Salmonella2.

Primer Design2, 3: (Forward, ttr 6:5′-CTCACCAGGAGATTACAACATGG-3′ position: 4287–4309 bp and ttr 4:5′-

AGCTCAGACCAAAAGTGACCATC-3′, position: 4359–4381 bp; product length: 95bp)

Probe Design2, 3: Molecular beacon probes corresponding to the primers were used (HEX 5’-

CCAGGCGACCGACTTTTAGCCACTGACGAGCCTGG-3’ DABCYL, position 4323-4345 bp in ttr loci)

STUDY GOALS & OBJECTIVES

Development of culture-independent method for salmonellae

Rapid, sensitive and specific detection

Quantitative enumeration

Viability assessment

In developing countries, such as India water scarcity, insecure pipe

networks, compromised storage and handling, poor sanitation infrastructure

compound the problem of drinking water contamination.

India along with other countries in Asia is identified as high risk site for

infections caused by Salmonella species1.

Some regions in India are recognized as endemic zones for Salmonella

infections.

Technical Challenges for Microbiological Risk Assessments of Drinking

Water

Current methods for exposure assessment target indicator bacteria

Indicator bacteria do not correlate well with presence or levels of all

pathogens

Some pathogens may be present in low concentrations, but are infectious

at low doses.

Conventional culture based methods for pathogens are time consuming,

lack specificity and fail to detect viable but non culturable bacteria.

Molecular methods offer advantages in terms of speed & sensitive

detection, but do not provide information on infectivity.

Existing nucleic acid-based viability assays (RNA based)

have limitations

• mRNA targets can survive long even after cell death

• Stressed/VBNC forms may have very low levels of target RNA

Molecular beacon, is a hybridisation probe

Reports presence of specific nucleic acids.

Hairpin shaped, with internally quenched fluorophore

Fluorescence restored when binds to target sequence DNA

Propidium Monoazide for live & dead differentiation PMA a photo-reactive dye, and has high affinity for DNA.

It intercalates with double stranded DNA to form covalent linkage upon

exposure to visible light, resulting in chemically modified DNA , which cannot

be amplified by PCR.

PMA is cell membrane-impermeable, when treated with PMA only dead

bacteria are susceptible to DNA modification due to compromised cell

membranes.

Subsequent lysis of live bacteria followed by qPCR permits selective

detection of the live cells.

1. Discrimination between viable and non-viable salmonellae

2. Efficacy of PMA assisted qPCR in quantifying viable salmonellae (spiked samples)

3. Quantitative enumeration of viable salmonellae in source and potable (treated) waters

CONCLUSIONS

METHODS & RESULTS

REFERENCES

Salmonella typhimurium ATCC 14028, grown in LB broth at

370C for 12 hrs to reach an OD of 0.8 at 600 nm.

Set A Set B

107 CFU/ 500μl

Cells killed by boiling

(1000C X 10 min

Exposure to 1.25μl of 20mM of PMA dye

5 min at RT

Exposed to visible light 500W X 2 min

DNA isolation & qPCR using ttr gene- specific primers and

Molecular beacon probe

In the qPCR, PMA-modified DNA extracted from heat inactivated cultures (Set A), exhibiting 107 CFU/ml (105

CFU/PCR) behaved like a no template control.

In contrast, amplification signals were observed for Salmonella not exposed to heat killing (Set B) , indicating that

DNA was not modified by PMA.

Set A : 10ml sterile water + 10-fold serially diluted cultures of ref. strain

Set B: 10ml sterile water + 10-fold serially diluted cultures of ref. strain+ 106 CFU/ml heat inactivated ref. strain.

Set C: 10ml sterile water + 10-fold serially diluted cultures of reference strain+ 106 CFU/ml heat inactivated culture

of reference strain. All dilutions in Set C were treated with PMA prior to DNA extraction.

All three sets (A, B, C) subjected to Molecular beacon assisted qPCR.

ttr gene-specific MB based qPCR could not

discriminate between viable and non-viable

cells. This is evident from lowering of CT in

the presence of DNA from heat inactivated

cells (Set B).

While PMA assisted MB- based qPCR could

discriminate between viable and non-viable

salmonellae (Set C)

qPCR assay reported in this study is highly efficient in quantitatively

detecting viable salmonellae and differentiating these from non-viable

cells.

This assay is highly sensitive, and can detect up to 1 viable Salmonella

cell/PCR or 10 Salmonella cells/ml from 104 CFU/PCR nonviable

Salmonella cells.

This method can be used for surveillance of both source and potable

waters in Salmonella endemic regions to inform QMRA and derive

microbial reduction targets

This strategy can be used by municipal distribution networks to map

contamination points for better risk management.

This approach can also be used to evaluate the treatment efficacy of

water disinfection options which inactivate microbes by membrane

damage

This detection strategy can be applied for detection of other waterborne

pathogens of public health significance.

3. Quantitative enumeration in source & potable waters

(Results)

Standard curve for reference strain Salmonella typhimurium ATCC 14028

using Molecular Beacon based qPCR

Source Waters

Potable water

Water sampled at the treatment plant showed presence of dead cells

indicating effectiveness of treatment.

Water samples drawn across various sampling points showed presence of

high numbers of salmonellae.

It appears that the water was subjected to contamination during

distribution possibly due to faulty/ damaged pipes.

Greater number of salmonellae in the same sample (Set SW1 & PW1)

indicates that ttr-gene MB qPCR could detect only cumulative salmonellae,

while PMA assisted MB qPCR could differentially detect live cells.

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