SUMFIARY

Summarv and Conclusions

The consumption of unsafe water and food in developing countries causes a

number of disease outbreaks. Salmonella and Escherichia coU pathotypes viz.

Enterotoxigenic E. coli (ETEC) and Enteroiiemorrhagic E. coli (EHEC) are among

the prevalent pathogens in water and food which often escape by conventional

methods of detection. The detection of target pathogens needs improvements to

overcome existing drawbacks and should be based on simple, rapid, sensitive and

specific methodologies. In view of this, the present study aims to exploit current

understanding of microbial genomics and ecology, and molecular techniques to

explore;

• Real-Time PCR probe chemistries in detection and tracking microbial

contaminants in low doses in water and food.

® Nanoprobes in the detection of nucleic acids of microbial origin in water and food.

® Development o f physico-chemical techniques for concentration-reconcentration of

microbial contaminants in water and food for increased sensitivity in tracking.

The study design required the computation of real time PCR primers and

probes like Molecular beacon (MB) and Scorpion using dedicated and web-based

bioinformatics tools. The MB and Scorpion primers were computed for the virulent

genes of Salmonellae (invA and Ur) and ETEC (LTl and STl) in highly conserved

regions using Beacon Designer software. In-silico PCR simulation was used to

validate computed primers for the anticipated amplification products. Further, the

formation of a unique probe structure for each gene was confirmed in mFold server.

The computed primers and probes were found to be highly specific towards target

genes on NCBI-BLAST analysis.

A study explored the culture-independent quantification of Salmonellae in

surface and potable water, riverine sediments and food by targeting invA gene using

MB based real time PCR assay. The assay could detect IGE/PCR of reference strain

S. Typhimurium ATCC 14028. The assay was 100 times more sensitive than

conventional PCR and could detect 10 CFU/PCR of Salmonella in the presence of 10̂

CFCJ/mi of E. coli DH5a. The assay could detect Salmonellae in surface waters of

river Gomti and Ganga, potable water (at two sampling locations.) and food samples

(vegetables, street foods, fioiit juices) collected from Lucknow city .The developed

assay was further explored for the detection of viable Salmonellae in food samples in144

Siimmarv and Conclusions

conjunction with the Propidium Mono-Azide (PMA) that inhibits PCR ampHfication.

irom dead cells due to covalent binding to DNA upon exposure to bright visible light.

The viability of Salmonellcie in pine apple and citrus juices were 36.land 19.7%,

respectively as per PMA assisted real time PCR.

In the present dissertation, ETEC strains harboring LTl and STl genes were

detected in food samples and riverine sediments using MB based real time PCR. The

present assay could detect ICFU/PCR of E. coli MTCC 723 as reference DNA.

Prevalence of ETEC strains (exhibiting LTl and STl) was observed in food samples

and street fruit juices. Sediments collected from river Gomti were heavily

contaminated with ETEC. The level o f contamination was highest at Bhaisakund, a

cremation point. A significant variation in distribution of ETEC strains exhibiting

LTl or ST 1 gene was observed between the sampling locations

In the present study, Scorpion uni-probe based real time PCR assays targeting

LTl and STl genes were developed for the specific detection of ETEC. The assay

could quantify as low as ICFU/PCR of ETEC in the presence of high background

flora (4x10^ CFU/ml). The developed assay was assessed for detection of ETEC in

surface water and sediments of river Gomti as well as food samples. Water and

sediment samples collected from Indirajal setu exhibited maximum number of ETEC.

High concentrations of ETEC strains exhibiting LTl and STl genes were observed in

vegetables, street foods and fruit juices.

Identification of environmental reservoirs of Salmonella enterica serovar

Typhimurium in Gangetic riverine was carried out by adopting two step strategy.

Stepl comprised a selective serovar specific capture of Salmonella enterica serovar

Typhimurium from potential reservoirs. Step 2 involved culture-free detection of

selectively captured S. Typhimurium by ttr gene specific MB based real time PCR.

The ttr gene specific MB designed in this study could detect I CFU/PCRcaptured by

serovar specific DNA aptamer. Sediments, water and aquatic flora collected from the

river Ganga were highly contaminated with S. Typhimurium. The pre-analytical step

in the form of serovar specific DNA aptamer based bio-capture of the bacterial cell

was found to enhance the sensitivity of the fluorescent probe in the presence of

nonspecific DNA.

Although, the real time PCR probes enable pathogen detection in extremely

low-doses, but the methodology requires trained personnel and expensive

145

Summary and Conclusions

instrumentation. Tlierefore, we have explored the development of a simple gold nano­

particle (GNP) based colorinietric detection system for two pathotyj^es o f E. coli

(ETEC and EHEC) for on-site diagnostics to enable protection of public health.

Under this study the stx2 (PCR product) gene of EHEC and LTl (complete operon)

gene of ETEC were detected using GNP probes. The assay was capable to detect 10̂

copies of PCR product of stx2 and lO'* copies o f LTl gene as observed in the UV

visible spectra and Transmission Electron Microscopic analysis. The sensitivity o f the

assay was further improved with the use of nucleic acid hybridization enhancers. The

GNP probes could detect as low as 10̂ copies of the LTl gene. The assay was further

explored for the development o f ‘spot and read test’ ofDNA hybridization.

In conclusion, the observations presented in dissertation demonstrate the

presence of Salmonellae and ETEC in aquatic environments of two Indian rivers and

a variety of food samples. The culture independent real time PCR assays developed

here are highly specific and sensitive which can also detect viable pathogens. The

samples can be concentrated by aptamer for detection of a specific pathogen. In

addition, the present study also concludes that the hybridization induced changes in

optical properties o f GNPs could lead to a rapid and simple colorimetric ‘spot and

read’ test detection method for ETEC and EHEC. The assays presented in this

dissertation open a new possibility of rapid, easy and reliable genetic diagnosis of

potential pathogens present in environment for public health protection.

146

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165

RESEARCH PUBLICATIONS

Research Piihiications

Research Articles Published in Journals

1.^Anurag Jyoti, S. P. Singh, Madhu Yashpal, P. D. Dwivedi and Rishi Shanker.

Rapid Detection o f Enterotoxigenic Escherichia coll Gene Using Bio-Conjugated

Gold Nano-Particles. (2011). Journai ofBiomedical Nanotechnology 7: 170-171.

2. Anurag Jyotl, Siya Ram, Poornima Vajpayee, Gulshan Singh, Premendra D.

Dwivedi, Swatantra K. Jain and Rishi Shanker. Contamination o f surface and

potable water in South Asia by Salmonellae: Culture-Independent quantification

with Molecular Beacon real-time PCR. (2010). Science of the Total Environment

408: 1256-1263.

3. Anurag Jyoti, Pratibha Pandey, Surinder Pal Singh, Svi^atantra Kumar Jain, and

Rishi Shanker. Colorimetric Detection o f Nucleic Acid Signature o f Shiga Toxin

Producing Escherichia coli using Gold Nanoparticles. (2010). Journal of

Nanoscience and Nanotechnology 10; 4154-4158.

4. Gulshan Singh, Poomima Vajpayee, Imrana Khatoon, Anurag Jyoti, Alok

Dhawan, K. C. Gupta, and Rishi Shanker. Chromium Oxide Nano-Particles

Induce Stress in Bacteria: Probing Cell Viability. (2011). Journal of Biomedical

Nanotechnology. 7: 166-167.

5. C B Patel, P Vajpayee, G Singh, A Jyoti, R S Upadhyay and R Shanker.

Computation and in-silico Validation o f a Real-Time PCR Array for Quantitative

Detection o f Fecal Coliforms in Surface and Potable Water. (2011). International

Journal of Bioscience and Biotechnology (In Press).

6. Ghanshyara Upadhyay, Manindra N. Tiwari, Om Prakash, Anurag Jyoti, Rishi

Shanker and Mahendra P. Singh. Involvement o f multiple molecular events in

pyrogallol-induced hepatotoxicity and silymarin-mediated protection: Evidence

from gene expression profiles. (2010). Food and Chemical Toxicology 48: 1660-

1670.

Abstracts Published in Journals

1. Anurag Jyoti, Pratibha Pandey, S.P. Singh S.K. Jain, P. D. Dwivedi and Rishi

Shanker. Detection of nucleic acid of diarrheagenic Escherichia coli by gold

nanoparticle probes. (2008). Nanotoxicology, 2: 1 S-85.

2. Rishi Shanker, Siya Ram, Pushpa Lata, Poomima Vajpayee, Anurag Jyoti,

C.B. Patel and P.D. Dwivedi. Pathogen Detection: PCR Probes to Nano-Probes!

(2008). Nanotoxicology, 2 :1 S-42.

166

Research Publications

Papers presented in National/International Coeferences and Symposia

1. Amsrag Jyoti, Poornima Vajpayee, Gulshan Singh, Siya Ram, Premendra Dhar

Dwivedi atid Rishi Shankar Detection of Nucleic Acid Signatures of

Dian'heagenic Escherichia coli : Real-Time PCR Probes to Gold Nanoparticle

Probes at International Conference on Nanomaterials and Nanotechnology,

NANO-2010 to be held at K. S. Rangasamy College of Technology,

Timchengode, Tamil Nadu, India. December 13-16, 2010. (Oral presentation)

2. Aniirag Jyoti, Poornima Vajpayee, Siya Ram and Rishi Shanker

Enviromnental reservoirs of Enterotoxigenic Escherichia coli: Molecular beacon

based Detection and quantificadon in river sediments at “International Conference

on Genomic sciences” recent trends (ICGS - 2010) VII Convention of The

Biotech Research Society, India (BRSl) held at Madurai Kamaraj University,

Madurai, India. November 12-14, 2010.

3. Atiurag Jyoti, Surinder Pal Singh and Rishi Shanker. Gold Nanoparticle-based

colorimetric assay for the detection of nucleic acid signature of shiga toxin

producing Escherichia coli at “Golden Jubilee International Conference on Nano

Sensors & Technology, (ICNST-2010)” held at Central Scientific Instruments

Organization (CSIO), Chandigarh. October 28-30, 2010

4. Anurag Jyoti and Rishi Shanker. Culture Plate to Nano-Probes: A novel

approach for detection of shiga toxin producing Escherichia coli. Invited Plenary

Lecture in the 2"‘' National Conference on Nanomaterials & Nanotechnology, held

at University of Lucknow, Lucknow. December 21-23, 2009

5. Anurag Jyoti, Gulshan Singh, Poornima Vajpayee and Rishi Shanker.

Contamination of surface and potable water by Salmonella spp.: Rapid detection

and enumeration by Molecular Beacon Probes at “International Symposium on

Environmental Pollution, Ecology and Human Health”, held at Sri Venkateswara

University, Timpati. July, 25-27, 2009.

6. Anurag Jyoti, Gulshan Singh, Poornima Vajpayee and Rishi Shanker. Rapid

Culture Independent Quantitative Enumeration of Salmonella in Water and Food

by Molecular Beacon based Real Time PCR. 77̂ '* Annual Meeting of The Society

of Biological Chemists (India). I IT Madras, December 18-20, 2008.

7. Anurag Jyoti, Pratibha Pandey, S.P. Singh S.K. Jain, P. D. Dwivedi and Rishi

Shanker. Detecdon of nucleic acid of diarrheagenic Escherichia coli by gold167

Research Publications

nanoparticle probes. International Conference on Nanomaterial Toxicology,

ICONTOX 2008, held at IITR, Lucknow. February 5-7, 2008.

8. Rishi Shanker, Siya Ram, Pushpa Lata, Poornima Vajpayee, Aiiurag .lyotl,

Chandra Bali Patel and Premendra D. Dwivedi. Pathogen Detection: PCR Probes

to Nano-Probes! International Conference on Nanomaterial Toxicology,

ICONTOX 2008, held at IITR, Lucknow. February 5-7, 2008.

9. Aimrag Jyoti, Pratibha Fandey, S.P. Singh and Rishi Shanker. Detection of

diarrheagenic Escherichia coll using gold nanoparticle probes. Indo-Australia

Symposium of MultifLmctional Nanomaterials, Nanostructvires and Applications.

Department of Physics and Astrophysics, University of Delhi, December 19-21,

2007

10. Anurag Jyoti, Pratibha Pandey, S.P. Singh, P.D. Dwivedi and Rishi Shanker.

Nano-Probes for Detection of Pathogenic Bacteria. National Workshop on

Nanomaterials and Nanotechnology, Lucknow University, Lucknow. March 24-

25, 2007.

11. Ram, S., Jyoti, A., Lata, P., Vajpayee, P. and Shanker, R. Probing pathogens

in waste waters: Real-Time PCR Probes. National conference on “Scope &

Applications of Microbes in Agriculture and Environment: New Horizons &

Technologies” (Session IV: Microbes in Waste Management), Institute of

Biosciences & Biotechnology, C.S. J. M. University, Kanpur. February 19-21,

2007

12. Anurag Jyoti, Pratibha Pandey, B.D. Malhotra, S.P. Singh and Rishi Shanker.

Detection of Water and Food-Bome Pathogenic Bacteria Using Gold Nano-

Particles. National Seminar on Mutifunctional Nanomaterials, Nanostructures and

Applications. Department of Physics and Astrophysics, University of Delhi.

December, 22-23, 2006.

168

AWARDS

> Youiig Scientist Award. “Contamination of surface and potable water by

Salmonella spp.: Rapid detection and enumeration by Molecular Beacon

Probes”. Aimrag Jyotl, Gulshan Singh, Poornima Vajpayee and Rishi

Shanker at “International Symposium on Environmental Pollution, Ecology

and Human Health”, held at Sri Venkateswara University, Tirupati. July, 25-

27, 2009.

> Best paper award, “Detection of nucleic acid of diarrheagenic Escherichia

coli by gold nanoparticle probes.” Aniirag Jyoti, Pratibha Pandey, S.P. Singh,

S.K. Jain, P. D. Dwivedi and Rishi Shanker at the International Conference of

Nanomaterial Toxicology, held at Indian Institute of Toxicology Research,

Lucknow. February 5-7, 2008.

169