Chromium Oxide Nano-Particles Induce Stress in Bacteria: Probing Cell Viability

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J Biomed Nanotechnol. 2011 Feb;7(1):166-7
Chromium Oxide nano-particles induce stress in bacteria:
Probing cell viability

Gulshan Singh, Poornima Vajpayee, Imrana Khatoon, Anurag Jyoti, Alok
Dhawan1,
K. C. Gupta and Rishi Shanker.
Environmental Toxicology Group and 1Nanomaterial Toxicology Group,
Indian Institute of Toxicology Research, (CSIR)
M. G. Marg, Lucknow 226 001, India.



1. Introduction

Engineered metallic nanoparticles have applications in a variety of
nanoproducts. This will lead to an increased release of nanomaterials in
the environment that may initiate adverse impact in ecosystems. Escherichia
coli, an environmentally relevant bacterium plays an important role in
decomposition of organic matter in pristine and polluted habitats. The
pathogenic and commensal variants are prevalent in the environment. It is
understood that under various abiotic stresses including heavy metal stress
bacterial cells reach viable but not culturable (VBNC) state. However most
of the studies conducted on impact of nanoparticles on bacterial cell
viability do not account for VBNC cells. The conventional standard plate
count assay fails to detect VBNC and is usually inconclusive. Most Q-PCR
assays do not discriminate between DNA arising from dead or live cells.
Propidium mono-azide (PMA), a DNA intercalating dye exhibiting azide group
strongly inhibits PCR amplification from dead cells due to covalent binding
to DNA upon exposure to bright visible light1. The dye can easily penetrate
damaged membrane and dead cells. Therefore, in this study, viability of an
environmentally relevant bacterium, E. coli exposed to varying
concentrations of Chromium Oxide nanoparticles (Cr2O3 NPs) was evaluated
PMA assisted Q-PCR.

Keywords: Chromium Oxide Nano-Particles, Cell Viability, Molecular Beacon,
Q-PCR, Propidium Mono-azide

2. Materials and Method

In this study a Molecular Beacon based Q-PCR assay targeting 16-23S rRNA
intergenic spacer region of E. coli was designed for the quantitative
enumeration of E. coli cells






exposed to Cr2O3 NPs. The coding sequences of 16-23S rRNA intergenic spacer
region of E.
coli were retrieved from NCBI GenBank database
(http://www.ncbi.nlm.nih.gov) and alignments were created using ClustalW
programme (www.ebi.ac.uk/clustalW) to determine conserved sequences. A set
of Primers (Forward Primer 5'- AGAAACATCTTCGGGTTGTGAGG-3', and Reverse
Primer 5'-TACCGACGCTTATCGCAGATTAG-3') and Molecular Beacon (5-'HEX
CCGCGCGGCAGTCAGAGGCGATGAAGGACGGCGCGG-Dabcyl.3') were computed in the
conserved region of the target gene by dedicated software Beacon Designer
version, 5. The specificity of primers and designed Molecular Beacon (MB)
was determined by BLAST (Basic Local Alignment Search Tool) programme
(http://www.ncbi.nlm.nih.gov/ BLAST/). Cr2O3 NPs (TEM size ~100 nm) were
procured from Sigma-Aldrich (USA).The particle size characterization was
done by Dynamic Light Scattering through Zetasizer Nano ZS, Zen 3600,
Malvern Instruments Limited2. The E. coli culture grown to 0.6 O.D at
35±10C was treated with varying concentrations of Cr2O3 NPs (5 -,100 µg/ml)
in Luria Bertani broth for 120 min. Bacterial cultures in LB broth
omitting NPs served as negative control. The viability of NPs treated and
untreated E. coli cultures were determined by Standard Plate count method.
Further for quantification of live cells by PMA assisted MB based Q- PCR,
500µl treated or untreated cultures were exposed to 50 µM PMA3. After 2 min
incubation in dark at 4o C, cultures were illuminated by 500 W halogen lamp
for 2 min (4o C). DNA was extracted as per Singh et al (2010). Q-PCR
(cycling conditions: 45 cycles of 940C for 20 sec, 55.80C for 30 sec and
720C for 30 sec) was conducted from DNA extracted from PMA treated and
untreated bacterial cultures using iCycler (Bio-Rad). The quantification
of dead cells was done by excluding the number of viable cells quantified
by PMA assisted Q-PCR from counts retrieved by Q-PCR not involving PMA. To
find out relationship between Cr2O3 NPs concentration in growth medium
and cell viability correlation analysis was carried out. Further, one way
analysis in complete randomized block design was carried out to test the
significance of the data. All the statistical methods were adopted from
Gomez and Gomez (1984)4. All assays were done in triplicate.

3. Results and Discussion

The lowest detection limit of the MB based Q-PCR assay used for
quantitative enumeration of E. coli was 1 CFU/PCR in 10-fold serially
diluted culture of the reference strain (E. coli DH5α, ATCC 35218).
Viability of bacterial culture was analyzed by Plate count method as well
as by PMA assisted Molecular Beacon based Q-PCR. It was observed that as
the concentration of Cr2O3 NPs in culture medium increases, the
percentage of live cell decreases (one way ANOVA, p
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