Download O A RIGINAL RTICLE

2681
Advances in Environmental Biology, 7(9): 2681-2688, 2013
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Rapid, specific and sensitive coliforms/Escherichia coli detection in water samples
Through selectively pre enriching and an octaplex PCR
1
Roohollah kheiri, Mahyar Mostaghim, 2Roohollah kheiri, 3Mahyar Mostaghim
1
Water Hygiene and Quality Control Office, Alborz Province Water and Waste Water Company
Water and Waste Water Company, Karaj, Alborz province, IRI.
3
Water and Waste Water Company, Karaj, Alborz province, IRI.
2
Roohollah kheiri, Mahyar Mostaghim, Roohollah kheiri, Mahyar Mostaghim: Rapid, specific and
sensitive coliforms/Escherichia coli detection in water samples Through selectively pre enriching and
an octaplex PCR
ABSTRACT
A selectively enriching medium and octaplex PCR were developed for direct and sensitive detection of 8
specific genes of coliforms/Escherichia coli in water samples within 24 h. Genes traced by octaplex PCR
include 16S rRNA, phoE, mdh, gadA/B, lacZ, uidA, cadA and tnaOP, yielding 348, 392, 544, 670, 876, 1700,
2000 and 3065 bp PCR products, respectively. In this study we tested and compared 5235 water samples both
by current approach and standard method 9221 D. Results gained confirmed molecular (genetical) approaches
are more specific and sensitive rather than the laborious and time consuming biochemical approaches.
Key words: Octaplex PCR; Escherichia coli; xylose broth.
Introduction
WHO and United States federal regulations state
that public water systems must conduct analyses for
Escherichia coli for any routine drinking water
sample that is positive for total coliform bacteria
[12]. Since confirming Escherichia coli as a suitable
marker of water hygiene [29], the microbiologists
tried to find a rapid, sensitive and specific method of
detecting such bacterium in water. Alike other
developments, bacterial detection methods are
developing through the time, it means the first
methods were biochemical (enzymatic) while
nowadays that's the molecular (genetic) ones which
override others. In biochemical assays the enzyme
activity is tested and in molecular assays the genome
is the sole target and not the activity, however both
of them (molecular and biochemical) imparts
advantages and disadvantages. For example
microorganisms in VBNC phase can't be cultured
and a perished cell or a single genome in water may
lead to fake positive result in molecular approaches
[20,9].
Biochemically, the most common feature of
coliforms/Escherichia coli used in detection methods
is the ability of these bacteria to ferment lactose and
producing gas and acid [4]. owever we should
consider some Escherichia coli strains especially
pathogen ones are out of lactose fermenting
capability or may be slow fermenter and some other
bacteria such as some Shigella sonnei strains are
lactose slow fermenter [13]. Considering the
advantages and disadvantages of the mentioned
approaches, we found best to join the advantages of
both biochemical and molecular approaches to detect
coliforms/Escherichia coli rapidly, specifically and
sensitively.
Since in most of the cases especially in treated
water, number of coliforms/Escherichia coli are not
high enough to be detected through molecular
methods, it is necessary to pre enrich the water
samples to increase the number of the cells and
subsequently perform molecular approaches to trace
specific coliforms/Escherichia coli genes [3].
Because of the great similarity between the DNA
sequence of Escherichia coli and Shigella [24], these
bacteria share high percentage of genes and so it is
necessary to screen Shigella. In current study we
developed an enriching and selective culture medium
to multiply coliforms/Escherichia coli and screen
Shigella strains. Inhibiting gram positive and non
microflora bacteria, carbon and energy source of this
culture medium is metabolized by (most of)
Escherichia coli and not by (most of) Shigella, hence
ensures screening of Shigella strains. After being
enriched and multiplied, DNA of the biomass is
extracted and introduced into multiplex PCR to trace
simultaneously
7
specific
genes
of
coliforms/Escherichia coli.
Corresponding Author
Roohollah kheiri, Water and Waste Water Company, Karaj, Alborz province, IRI.
Phone: +982612515103. Fax: +982612502354. E-mail: [email protected]
2682
Adv. Environ. Biol., 7(9): 2681-2688, 2013
Single detection of any gene (single PCR) may
lead to wrong detection of the target bacteria, so we
looked for the best marker genes of E. coli in
different surveys and developed a novel octaplex
PCR by which we were able to detect the following
genes: uidA, tnaA, gadAB, cadA, phoE, lacZ, lamB
and mdh. uidA encodes for <beta>-glucuronidase by
which the transformation of 4-methylumbelliferyl-bD-glucuronide (MUG) to the fluorogenic 4methylumbelliferone is catalyzed [22]. This enzyme
is ubiquitous in most of Escherichia coli strains
however some Shigella strains also carry this gene.
Tryptophanase operon (tnaOP) is a region consisting
of two major structural genes: tnaA, coding for the
tryptophanase which catalyzes the degradation of Ltryptophan to indole, pyruvate, and ammonia and
tnaB coding for a tryptophan permease. This operon
enables the bacterium to metabolize L-tryptophan as
a source of nitrogen [8]. gadA/B encodes for
glutamate decarboxylase. This enzyme catalyzes the
α-decarboxylation of L-glutamic acid to yield γaminobutyric acid (GABA) and carbon dioxide [4].
In Escherichia coli and some other coliforms, lysine
decarboxylase is encoded by cadA which participates
in the decarboxylation of lysine and synthesis of
cadaverine [30]. Producing alkaline GABA and
cadaverine, both glutamate decarboxylase and lysine
decarboxylase enzymes have strategic role in
neutralizing acidic condition. lacZ is one of the lac
operon genes encoding for <beta>-galactosidase by
which lactose is hydrolysed. This enzyme is a
diagnostic feature of coliforms [28]. mdh is the gene
responsible for malate dehydrogenase enzyme, an
important TCA constituents. Malate dehydrogenase
oxidizes malate to form oxaloacetate [15]. phoE
encodes for an outer membrane porin protein, which
forms three separate channels that traverse the width
of the membrane [2]. PhoE is an Anion-selective
diffusion channels induced under phosphate
limitation and finally the 16S rRNA gene [17]. is a
highly conserved sequence in Escherichia coli,
however is not so much specific because it may be
present in some Shigella strains.
Material And Methods
Selectively enriching the coliforms/Escherichia coli:
To enrich coliforms/Escherichia coli selectively
and screen the Shigella strains, we developed a
medium which the ingredients and the corresponding
roles are as follow: peptone from casein and soymeal
provides amino acids and other nitrogenous
substances making it a nutritious medium. Sodium
chloride maintains the osmotic equilibrium, while
dipotassium hydrogen phosphate and potassium
dihydrogen phosphate act as buffers to maintain pH.
Sodium deoxycholate inhibit gram positive and non
flora microorganisms. Since xylose is the carbon and
energy source and most of Shigella strains can't
metabolize it, this medium can selectively and
differentially
enrich
coliforms
and
coliforms/Escherichia
coli
as
we
found
experimentally. To prepare this medium, the
substances listed in Table 1 should be added to 1 liter
deionized water, dissolved and autoclaved (15 min at
121 °C). It is noteworthy that we tried different
weights of the ingredients and checked the OD of
growth so that finally optimized it as shown in
Table1.
To enrich the water samples, 100 ml of sampled
water was inoculated into 100 ml, 2x mentioned
enriching broth (total volume of 200 ml) and
incubated at 35-37°C.
Table 1: Coliforms/ Escherichia coli selective enriching broth typical composition
Ingredients
Company, Cat number
peptone from casein
(Merck Chemicals, 107213)
peptone from meat
(Merck Chemicals, 107212)
sodium chloride
(Merck Chemicals, 106406)
di-potassium hydrogen phosphate
(Merck Chemicals, 105109)
potassium dihydrogen phosphate
(Merck Chemicals, 105108)
sodium deoxycholate
(Merck Chemicals, 106504)
Xylose
(Merck Chemicals, 108689)
Specificity and sensitivity of Coliforms/ Escherichia
coli selective enriching broth:
To determine the specificity of this medium, we
used some standard strains listed in Table 4. These
standard strains were obtained from Iranian Research
Organization for Science and Technology (IROST),
Reference Laboratories of Iran in Ministry of Health
and Pasteur Institute of Iran.
Incubation time determination:
Weight g/l
20
3.0
5.0
2.75
2.75
1.5
5
To optimize the incubation period and
simultaneously caring for the stringency and
limitation of time, we determined the McFarland
turbidity of the inoculated samples hour by hour. To
do
so,
we
used
HACH
DR/4000
U
Spectrophotometer, 115 Vac, applying 4 different
McFarland turbidity unit including 0.5, 1, 2, 3 and 4,
at 600 nm wavelength.
To prepare McFarland turbidity unit,
according to described and confirmed procedures,
different ratio of Sulfuric acid, 1% (vol/vol) (H2SO4)
and anhydrous Barium Chloride, 1% (wt/vol) (BaCl2)
were mixed.
2683
McFarland turbidity unit
Adv. Environ. Biol., 7(9): 2681-2688, 2013
Hours of incubation
Fig. 1: Average McFarland turbidity of 100 samples during hours of incubation.
DNA extraction:
Following incubation at 35-37°C for 15 hours,
190 ml, inoculated water was aliquoted into 4, 50 ml
falcon conical centrifuge tubes, and centrifuged at
1006 ×g for 15 min. The supernatant was discarded
and the cells were suspended in 5 ml PBS. Vortexing
the falcon tubes, the biomass of falcon tubes were
transferred into 1 falcon tube and concentrated at
1006 ×g for 15 min once again. Discarding the
Supernatant and following biomass concentration,
using AccuPrep® Genomic DNA Extraction Kit,
DNA was extracted. To ensure the efficiency and
suitability, we evaluated 5µl of the extracted DNA
by electrophoresis on 1.5% agarose gel in 1x TAE
buffer. To evaluate the quality and quantity of the
DNA, following 1/200 dilution, we determined the
absorbance at 260 nm (A260 for DNA) and 280 nm
(A280 for protein) wavelengths.
PCR condition:
To detect 16S rRNA [17], mdh [9], phoE [2],
gadA/B [21], lacZ [5], uidA ([22], cadA and tnaA
(Camilla et al., 2007) genes, we applied specific
primers listed in Table 2 which all were introduced
by investigators.
The gene amplification protocol was performed
in reaction mixture at a final volume of 25 μl
containing 12.5 μl, 2x QIAGEN Multiplex PCR
Master Mix (containing HotStarTaq® DNA
polymerase, Multiplex PCR Buffer, dNTP Mix), in
addition to 2 μl template DNA and specific pmol of
each pair of primers, listed in Table 3.
Table 2: Specific primers used in this study
gene
forward / reverse
Primer sequence
phoE
mdh
16S rRNA
gadAB
lacZ
uidA
cadA
tnaOP
forward
sequence
reverse sequence
forward
sequence
reverse sequence
forward
sequence
reverse sequence
forward
sequence
reverse sequence
forward
sequence
reverse sequence
forward
sequence
reverse sequence
forward
sequence
reverse sequence
forward
sequence
reverse sequence
AAAGCCGTGGCACAGGCAAGCGT
PCR product
size MW (bp)
Reference
348
Spierings et al., 1993
392
Chen et al., 2001
544
Lane et al., 1991
670
MCdaniels
1996
876
Bej et al, 1990
1700
Lang et al., 1994
2000
Kikuchi et al., 1997
3065
Bernasconi
2007
TCAATTTGTTATCGCTATCCAGTTGG
ACTGAAAGGCAAACAGCCAAG
CGTTCTGTTCAAATGGCCTCAGG
GGAAGAAGCTTGCTTCTTTGCTGAC
AGCCCGGGGATTTCACATCTGACTTA
ACCTGCGTTGCGTAAATA
GGGCGGGAGAAGTTGATG
ATGAAAGCTGGCTACAGGAAGGCC
et
al.,
CACCATGCCGTGGGTTTCAATATT
CCAAAAGCCAGACAGAGT
GCACAGCACATCCCCAAAGAG
TGGATAACCACACCGCGTCT
GGAAGGATCATATTGGCGTT
CGAGGATAAGTGCATTATGAATATCT
TTAGCCAAATTTAGGTAACACGTT
et
al.,
2684
Adv. Environ. Biol., 7(9): 2681-2688, 2013
The optimized thermocycling procedure was
performed in ABI verity 96 well thermal cycler with
an initial denaturation step of 15 min at 95°C
followed by 35 cycles of 94°C for 30 s, annealing at
60°C for 90 s and 72°C for 90 s, and final extension
at 72°C for 8 min.
Simultaneous amplification of all 8 target genes
was performed by comparing presence, intensities
and (non)specificity of bands applying different
annealing temperature such as 59 ºC, 59.5 ºC 60 ºC,
60.5 ºC, 61 ºC, and 61.5 ºC and we found
experimentally, 60 ºC, the best for annealing since we
could visualize 8 distinct and sharp bands.
5 μl of PCR products were electrophoresed on
1.5 % agarose gel, stained with SYBR green, and
visualized by a Gel Doc™ XR+ system (Image
Lab™ 4.0). The gel separated PCR amplicons are
shown in Fig 1.
Octaplex PCR sensitivity determination:
To assess the sensitivity of current octaplex PCR
assay, a 24 hour incubated Escherichia coli ATCC
25922 in lauryl sulphate broth and Shigella flexneri
PTCC 1234 in GN enrichment broth were suspended
in 0.1% sterile peptone water. Using HACH
DR/4000 U Spectrophotometer, they were adjusted
to McFarland standard of 1 (equivalent to 3×108
CFU/ml) separately. A serial 10 fold dilution with
0.1% sterile peptone water was performed for 7
consecutive concentration ranges from 3×107 to
3×101 CFU/ml. Applying the same protocol
described above, DNA was extracted from these
samples and subjected to single PCR and
subsequently the lower detection limits of the 10
target genes by octaplex PCR were evaluated.
Comparison between the introduced approach and
the standard method to verify the efficiency:
To validate the efficiency, we tested all the
samples simultaneously both by standard method
(9221 D. Presence-Absence Coliforms Test) and the
current introduced approach. To do so, after
inoculation 100 ml water into 100 ml, 2x enriching
medium and incubation for 15 h, we submitted 190
ml for DNA extraction but prolonged the incubation
for 24 h for the 10 ml remained of the inoculated
water and assumed it as presumptive phase. We
considered xylose fermenters (acid producers) as
Table 3: specific pmols of the primer sets per reaction
Primer
pmol of sets of primers
phoE
12
malic
15
uspA
8
gadA/B
8.5
lac Z
11
uidA
8
cadA
10
tnaOP
9
positive and submitted the presumptive tubes into
BGBL broth (to detect total coliforms as confirmed
phase) and EC broth (to detect thermotolerant
coliforms as completed phase).
Results:
Results for the standard strains:
Inoculating the listed standard strains and the
results gained, we found such medium would be a
reliable
selective
enriching
broth
for
coliforms/Escherichia coli (Table 4). Determining
the McFarland turbidity of inoculated samples
hourly, as shown in Fig 1, we found, 15 hours
incubation is long enough to multiply the
coliforms/Escherichia coli to extract DNA
efficiently. To check the recovery and growth of the
target microorganisms and also to check the
specificity of the introduced medium, we inoculated
the standard strains listed in Table 4 and checked the
growth status (turbidity) every 15 hours.
At the first 15 hours, coliforms/Escherichia coli
showed notable turbidity however continuing the
incubation period for 24 hours, some Shigella and
Salmonella grew and could increase the turbidity.
Following inoculation, incubation, DNA
extraction and gene amplification, we gained results
listed in Table 5.
Results for environmental samples:
In this study we included water samples from
different sources of Alborz province water supplies
e.g., deep wells, surface and treated (chlorinated) and
private (unknown) sources, during a period of 1 year
(from February 2011 to March 2012). Total number
of samples tested in current study through both
standard method and current approach was 5235 of
which 5025 samples were negative for both methods.
Genetically (octaplex PCR), 280 were positive for
total coliforms (containing lacZ and cadA) of which
130 were positive for Escherichia coli.
Biochemically, of 5235 environmental samples, 210
were positive for total coliforms (confirmed phase)
and just 67 for thermotolerant coliforms. It is
necessary to mention that all environmental samples
were collected from different sources and were tested
just once.
2685
Adv. Environ. Biol., 7(9): 2681-2688, 2013
Fig. 2: Gel agarose analysis of octaplex PCR products. Lane 1, Bioneer Mid-range DNA ladder 100 bp to 3
kb linear scale; lane 2, octaplex PCR products of Escherichia coli ATCC 25922; lane 3, octaplex PCR
products of Escherichia coli ATCC 8739; lane 4, octaplex PCR products of Escherichia coli ATCC
35218; lane 5, octaplex PCR products of Escherichia coli ATCC 15224; lane 6, octaplex PCR
products of Shigella flexneri PTCC 1234 ; lane 7, octaplex PCR products of Salmonella Typhi ATCC
14028; lane 8, octaplex PCR products of Citrobacter freundii ATCC 1600 ; lane 9, octaplex PCR
products of Enterobacter aerogenes ATCC 1221.
Table 4: Standard strain growth during period of incubation
standard strains
Growth (turbidity) at 3537°C for 8 hours
Escherichia coli ATCC 10799
fair
Escherichia coli ATCC 8739
fair
Escherichia coli ATCC 35218
fair
Escherichia coli ATCC 15224
fair
Escherichia coli ATCC 25922
fair
Escherichia coli ATCC 10536
fair
Escherichia coli PTCC 1270
fair
Escherichia coli ATCC 11303
fair
Citrobacter freundii ATCC 8454
fair
Citrobacter freundii ATCC 1600
fair
Citrobacter freundii ATCC 6879
fair
Enterobacter aerogenes ATCC 1221
fair
Enterobacter aerogenes ATCC 13048
fair
Klebsiella pneumoniae ATCC 10031
fair
Klebsiella pneumoniae ATCC 27799
fair
Klebsiella pneumoniae ATCC 13883
fair
Shigella flexneri ATCC 12022
none
Shigella flexneri PTCC 1234
none
Shigella flexneri ATCC 25875
none
Salmonella Typhimurium ATCC 19430
none
Salmonella Typhi ATCC 14028
none
Entrococcus fecalis ATCC 9584
none
Entrococcus fecalis ATCC 11700
none
Vibrio Cholerae PTCC 1611
none
Pseudomonas aeruginosa ATCC 9027
none
Clostridium perfringens ATCC 10873
none
Table 5: Genes detected in standard strains by octaplex PCR
mdh
standard strains
Escherichia coli ATCC 10799
Escherichia coli ATCC 8739
Escherichia coli ATCC 35218
Escherichia coli ATCC 15224
Escherichia coli ATCC 25922
Escherichia coli ATCC 10536
Escherichia coli PTCC 1270
Escherichia coli ATCC 11303
Citrobacter freundii ATCC 8454
Citrobacter freundii ATCC 1600
Citrobacter freundii ATCC 6879
Enterobacter aerogenes ATCC 1221
Enterobacter aerogenes ATCC 13048
+
+
+
+
+
+
+
+
+
+
-
phoE
+
+
+
+
+
+
+
+
-
16S
rRNA
+
+
+
+
+
+
+
+
-
Growth (turbidity) at
35-37°C for 15 hours
good
good
good
good
good
good
good
good
good
good
good
good
good
good
good
good
none
fair
fair
fair
fair
none
none
none
none
none
Growth (turbidity) at 3537°C for 24 hours
very good
very good
very good
very good
very good
very good
very good
very good
very good
very good
very good
very good
very good
very good
very good
very good
fair
fair
fair
fair
fair
none
none
none
none/poor
none
gadA/B
lacZ
uidA
cadA
tnaOP
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
2686
Adv. Environ. Biol., 7(9): 2681-2688, 2013
Klebsiella pneumoniae ATCC 10031
Klebsiella pneumoniae ATCC 27799
Klebsiella pneumoniae ATCC 13883
Shigella flexneri ATCC 12022
Shigella flexneri PTCC 1234
Shigella flexneri ATCC 25875
Salmonella Typhimurium ATCC 19430
Salmonella Typhi ATCC 14028
Entrococcus fecalis ATCC 9584
Entrococcus fecalis ATCC 11700
Vibrio Cholerae PTCC 1611
Pseudomonas aeruginosa ATCC 9027
Clostridium perfringens ATCC 10873
+
+
+
+
+
+
+
-
+
+
+
-
Discussion:
Results gained in this study showed genetical
approaches are much more accurate, rapid, sensitive
and specific rather than the biochemical ones.
However biochemical methods, mostly based on
lactose fermentation, are more emphasized by
Standard Methods for the Examination of Water and
Wastewater, 21st edition. Regarding the results of
study, of 280 samples genetically positive for total
coliforms, 210 samples were biochemically positive
for total coliforms (75%), while 70 of which were
negative biochemically, It means biochemical
methods lost 70 samples imparting coliforms. Of 130
samples containing Escherichia coli genes, just 67
ones could be detected biochemically (51.53%)
which indicates unreliability of biochemical
methods. As we found experimentally, in some cases
Escherichia coli present in water sample could grow
in BGBL but not in EC medium as we found it
through striking BGBL positive on EMB agar and
isolating the Escherichia coli colonies. In some other
cases we found Escherichia coli in consortia of other
coliforms can't grow in EC medium but if isolated
and inoculated, it would [26].
By the way, being time consuming and
laborious, these are the most significant
disadvantages of biochemical methods. As suggested
by standard methods, a typical biochemical method
of total, thermotolerant and Escherichia coli
detection from water samples my last for 4 days
while applying the current method would not last
more than 24 hours.
The results of the current investigation indicate
that genotypic assays should provide superior
confirmational detection of E. coli in drinking water
samples compared with the corresponding
phenotypic assays or standard methods for lactose
fermentation [19].
In contrast to the phenotypic assays, the choice
of the phoE, mdh, 16S rRNA, gadAB, lacZ, uidA,
cadA, tnaOP genes as a target for such genotypic
assays may be relatively unimportant, but the use of
all targets (in a multiplex PCR) could further
increase the specificity and detect the widest range of
E. coli strains possible.
In conclusion, the use of PCR and gene probes
permits both the specificity and sensitivity necessary
+
+
+
+
-
+
+
+
-
+
+
+
+
-
+
+
+
-
+
+
+
+
-
-
for monitoring coliforms/Escherichia as indicators of
human fecal contamination of waters.
With some simplification and optimization of
the DNA extraction and primer designing, PCRbased methods can permit a rapid and reliable means
of assessing the bacteriological safety of waters and
should provide an effective alternative methodology
to conventional viable culture methods. PCR-based
methods may also permit sufficient sensitivity and
specificity for the direct detection of pathogens in
environmental samples, rather than the current
practice of relying on the indirect detection of
indicator organisms [5].
Applying the current approach, not only we can
detect coliforms/Escherichia coli but also we can
perform analytical surveys to find the best genes for
molecular detection of coliforms/Escherichia.
Acknowledgements
This study was completely funded and supported
by Alborz province water and waste water Quality
Control Office.
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