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ISSN: 0975 -8542
Journal of Global Pharma Technology
www.jgpt.co.inAvailable Online at
RESEARCH ARTICLE
©2009-2018, JGPT. All Rights Reserved 90
Confirmatory Detection of Escherichia Coli O157:H7 in Diarrheic
and Non-Diarrheic Calves by using Real Time PCR with Studying
the Antimicrobial Susceptibility of these Bacteria
Mustafa Salah Hasan1*, Mohammed Ali Hussein1, Afaf Abdul Rahman Yousif2
Department of Internal and Preventive Vet. Medicine, College of Vet. Med., University of Fallujah, Iraq.
*Corresponding Author Email:, dr.mustafa.salah@uofallujah.edu.iq
Abstract
Shiga toxigenic E. coliO157:H7 is one of the Shiga-toxin producing types of E. coli, which can produce
disease in man and animals. The aims of this study were to detect O gene (rfbO157) and H gene (fliCH7)
and determine the pattern of antimicrobial susceptibility of 32 E. coli O157:H7 isolates. The isolates
isolated by conventional culture methods and latex test from faecal samples of 4 calves suffering from
diarrhoea and 28 calves’ apparent normal. DNA was extracted from these isolates and the real time PCR
was applied on the extract of DNA for confirming the diagnosis ofrfbO157 and flic H7 genes. Seven
antimicrobial discs were used in this study. The result presented that all the (32) isolates were possessed
the rfbO157 and flics H7 genes. The susceptibility test of the isolates revealed that all isolates were
highly resistant to Erythromycin (100%) and Cephalothin (96.87 %). Less resistance to Tetracycline,
Trimethoprim-Sulfamethoxazole and Chloramphenicol at different percentage (31.25%), (12.5%) and
(9.37%) respectively and all isolates showed high sensitivity to Gentamicin (100 %), Enrofloxacin (100 %).
In conclusion, the real time PCR can be used as a sensitive and reliable tool for diagnosis of E. coli
O157:H7 and these bacteria showed high resistance to antibiotic.
Keywords: E. coli O157:H7, real time PCR, rfbO157, flic H7, antibiotic susceptibility.
Introduction
E. coli O157: H7 (EHEC) is one of the most
important pathogen that transmit by food, it
causes a different signs ranged from
reasonable diarrhoea to hemorrhagic colitis
and dangerous problem in other body
systems or organs. E. coli O157:H7 have
several virulence factors, the main important
one were Shiga toxins (Stxs) [1], it is some
time termed as STEC [2].
It can cause a disease not only in calves, but
it can also infect adult cattle [3]; human [4];
sheep [5]; dogs [6, 7, 8] and other animals.
Lupindu et al. [9] reported that cattle were
regarded as the natural reservoir for E. coli
O157: H7.
This bacteria (E. coli O157:H7) usually
present in the GIT, especially the terminal
recto-anal junction, without causing disease,
and the reservoir animals may shed this
bacteria to the environment and transmitted
to the other animal species [10] by the way of
oral fecal route[11]. The detection of
pathogenic E. coli strains has been greatly
improved by using conventional PCR in the
multiplex format [12] or by real-time PCR
[13]. Ruminants are the primary reservoir of
Shiga toxin producing Escherichia coli
(STEC) O157:H7 and the main source of
infection for humans [14].
A study was done by Osman et al. [15] to
isolate E. coli from diarrheic ruminants, They
found that the prevalence rate were 27.3 %
in goat; 9.1 % in sheep and 63.6 % in calves,
and these isolates were belonged to different
serotypes, the most one O157 serotype. Also,
these bacteria causes a serious disease in
other animals. Yousif et al. [8] concluded that
E. coli O157:H7 is an significant doge
pathogen and the real time PCR is approved
as best, reliable method for confirmatory
diagnosis of these strains.
In a study of Dunn [16] found that the
isolates of E. coli were positive for
fliCH7andeae A genes by using PCR. Real
time PCR techniques have many advantages
which invites the researchers to prefer this
Mustafa Salah Hasan et. al.| Journal of Global Pharma Technology| 2018: 10(08):90-96
©2009-2018, JGPT. All Rights Reserved 91
type such as decrease the contamination of
the samples by reduction opening the test
tubes which seen in conventional method of
PCR, also this type do not need ethidium
bromide which is dangerous for researchers,
all these advantages made the real time PCR
overcomes on the conventional PCR. The
main aims for using antimicrobial
susceptibility testing are for detection the
resistance of pathogens to antimicrobials and
to help in choosing the effective antimicrobial
[17].
The useful of antimicrobial therapy for Shiga
Toxin E. coli infections is not of value,
because that antimicrobials may damage the
cell wall of bacteria and then liberation of
Stxs [18] and/ or cause increased expression
of Shiga toxin genes in vivo [19]. The
antimicrobial tests of E. coli O157:H7 showed
an extensive resistance to neomycin,
erythromycin and vancomycin and high
sensitivity to ampicillin, gentamicin,
ciprofloxacin, tetracycline and kanamycin
[20].
Cho et al. [21] conclude that 25% of
Escherichia coli O157 were resisting to
sulfadimethoxine and thirteen percent
isolates were resisting for tetracycline.
Vidovic and Korber, [22] found the
antimicrobial susceptibility testing of the E.
coli O157 isolates revealed that ten from 194
were multidrug resistant and that 5 and73
were resistant to tetracycline and
sulfisoxazole, respectively.
Osek, [23] showed that Most of the isolates of
E. coli O157:H7 isolated from cattle were
resistant only to rifampicin and sensitive to
Amoxicillin, Amoxicillin-Clavulanic Acid,
Cephalothin, Colistin, Cefoperazone,
Cotrimoxazole, Streptomycin,
Sulfamethizole, Spectinomycin, Flumequine,
Kanamicin, Oxolinic Acid, Gentamicin,
Enrofloxacin, Apramycin, Nitrofurantoin,
Chloramphenicol, Tetracycline and
Doxycycline.
Stanford et al. [24], compare between the low
shedders and super shedders, Resistance
detected for 17/474 isolates for antimicrobials
including neomycin, ampicillin,
sulfasoxazole, streptomycin, tetracycline and
sulfamethoxazole/ trimethroprim and they
showed that these isolates were have multi-
drug resistance characteristic. So that this
study was aimed to identifying the rfbO157
and fliCH7 genes of Escherichia coli O157:H7
between diarrhoeic and non diarrhoeic calves
by using real time PCR and to detect the
susceptibility of this bacterium to many
antimicrobials.
Material and Methods
Bacterial Strain
Thirty two strains of E. coli O157:H7 were
obtained from Dept. of Internal and
Preventive Medicine/ College of Veterinary
Medicine/ University of Baghdad, which
isolated from 350 faecal samples collected
from non-diarrheic and diarrheic calves
which found in different places in Baghdad
Province [25].
Bacteriological Methods
All methods were done as recorded in a
previous study by [25] and according to [26]
to insured the bacteria before doing PCR
assay. Including culturing on enrichment
media, Gram stain and biochemical test for
detection and confirmation the suspected
isolates.
All suspected isolates of E. coli were cultured
on Chrome agar specific for E. coli O157
[Himedia] and Sorbitol Mac Conkey agar
(CT-SMAC) (England) supplemented with
0.05 mg/L Cefixime and 2.5 mg/L potassium
tell urite to increase the selectivity forE. Coli
O157:H7. Serotyping was made by using
Latex agglutination test kit (Remel,
wellcolex) for detection both somatic O157
and flagellar H7 antigens [27], these strains
were possess aeaA gene as reported
previously [25] and also possessed Stx1 and
Stx2 [28]
Real Time PCR Assay
This test was performed in the laboratories of
University of Baghdad, College of Vet.
Medicine Department of Internal and
Preventive Veterinary medicine, this assay
was done on the 32 isolates of E. coli
O157:H7 by the following methods:
DNA Extraction
DNA was extracted from all isolates by using
(DNA extraction kit of Bacteria, Gene aid,
USA).
Measuring the Purity and
Concentration of DNA
The purity and concentration of extracting
DNA were measured using a Nanodrop
spectrophotometer.
Mustafa Salah Hasan et. al.| Journal of Global Pharma Technology| 2018: 10(08):90-96
©2009-2018, JGPT. All Rights Reserved 92
Molecular Characterization of E.
colirfbO157 and flicH7 Genes by using
Real time PCR:
Real-time PCR was performed in a 96-well
plate using Applied Bio systems (USA)
Sacace Real-Time PCR System by using kit
from Micro SEQ® E. coli O157:H7 Applied
bio system (USA).
The FAM and VIC dyes are used to detect
the targets; the NED dye is used to detect the
internal positive control (IPC). 2 µ of
template DNA was used and water free
nuclease was added to reach a volume of 30 µ
for reaction. The real-time PCR conditions
were first optimized and were set as in (Table
1).
Table 1:Real time PCR program
Step
Temperature
Time
Enzyme activation/ Hold
95°C
2minut
PCR
(40 cycles)
Denature
95°C
1 second
Anneal/ extend
60°C
20 second
Antibiotic Susceptibility Test (Disk
Diffusion Test)
The disk diffusion susceptibility method is
simple and practical and has been well-
standardized [29]. At least four to five
colonies of the same morphology were
selected from chrome agar; they were
transferred to a tube containing 4-5 ml of
nutrient broth.
This broth containing bacteria incubated at
37 C° for 18 hrs until the appearance of
visible turbidity. A swab was soaked into the
broth of bacteria and excess fluid was get rid
of by pressing the swab firmly at inside of the
tube. Then, the soaked swab streaked in all
directions over the surface of Muller Hinton
agar. The inoculated plates were allowed to
dry for 5 minutes.
Then the discs of antibiotic were placed onto
agar surface using a sterile forceps. After
that the plates were inverted, placed at 37C°
incubator aerobically for 18-24hrs [27]. The
zones inhibitions around each antimicrobial
disk were measured by using a digital
caliper. The measured zones of inhibition of
each drug were interpreted by [30, 31, 32 and
33].
Results and Discussion
The confirmation process of the 32 isolates
revealed that a bacterium was given the
characteristic feature of E. coli O157:H7 by
cultural and serotyping methods. Then the
results of testing by real time PCR showed
that all these isolates were possessed
rfbO157 and flicH7 genes (Figure 1).
Figure 1: Real time PCR results, positive samples were CT less than 35
Jenkins et al. [34] reported that the real-time
PCR is effective, screening method for
detecting STEC from stool specimens. In this
study, this assay was used to confirm
diagnosis of E. coli O157:H7 by using two
genes (rfbO157 and flic H7) and the results
of PCR assay coincide with those of
bacterial culturing and latex agglutination
test, which indicated that PCR assay was
more sensitive and trusted method for
Mustafa Salah Hasan et. al.| Journal of Global Pharma Technology| 2018: 10(08):90-96
©2009-2018, JGPT. All Rights Reserved 93
detecting of this organism. This result is in
agreement with result of Rebekka et al. [35]
who reported a sensitivity and accuracy of
real-time PCR for detection of E. coli
O157:H7 and they concluded that this assay
was quick diagnostic methods for the
presence or absence of E. coli O157:H7.
Chui et al. [36] reported that real-time PCR
method may use as the "gold standard" for
diagnosis of E. coli O157:H7. Other studies
reported similar results with this study;
Abbasi et al. [37] used RT- PCR as a
replacement of conventional type to detect
the frequency and presence of virulence genes
of Shiga toxigenic E. coli and
Enteropathogenic E. coli from diarrheic
children. They found that this assay is being
faster, more confirmable assay, because it
doesn't demand any additive procedures for
confirmation the amplificated products.
Among bacterial pathogens involved in food-
illnesses STEC has been frequently identified
in a study of Mondani et al. [38] who found
that using of real time PCR for diagnosis is
reducing the time and decreasing the
limitations which accompanying the other
traditional tests. A study performed of Qin et
al. [39] performed to estimate the real-time
PCR assay in confirming the presence of
Stx1,Stx2 and rfbO157 with 2,386 stool
samples in microbiology laboratory of
Pediatric center. Among the 59 positive PCR
samples, 29 (54.7%) specimens only were
positive to toxin. They recommended that
PCR is rapid method for diagnosis of
pediatric shigatoxigenic E. coli infections.
Antimicrobial Susceptibility Test
The isolated E. coli O157:H7 strains showed
different percentage of resistance and
sensitivity to different antimicrobials Figure
(2). The isolated organism showed high
sensitivity to Gentamicin (100%),
Enrofloxacin (100%), Trimethoprim-
sulfamethoxazole (87.5%), chloramphenicol
(87.5%) and tetracycline (65.62%).
Two isolates only showed moderate
susceptibility to Tetracycline and
chloramphenicol. All isolates were resistance
to Erythromycin (100%), while (96.87%),
(31.25%), (12.5%) and (9.37%) were
resistance to Cephalothin, Tetracycline,
Trimethoprim-sulfamethoxazole and
chloramphenicol respectively (Table 2).
Statistically, There is significant difference
among different antimicrobials used in
susceptibility test for E. coli O157:H7 at level
of (P<0.05).
Table 2: Susceptibility test to E. coli O157:H7 isolated from calves
Total NO
of isolates
Resistant
Intermediate
Susceptible
Antimicrobial
32
10(31.25%)
1(3.12%)
21(65.62%)
Tetracycline
32(100%)
0(0%)
0(0%)
Erythromycin
0(0%)
0(0%)
32(100%)
Gentamicin
31(96.87%)
0(0%)
1(3.12%)
Cephalothin
4(12.5%)
0(0%)
28(87.5%)
Trimethoprim-
sulfamethoxazol
0(0%)
0(0%)
32(100%)
Enrofloxacin
3(9.37%)
1(3.12%)
28(87.5%)
Chloramphenicol
*There is significant difference among many antimicrobials used in susceptibility test for E. coli O157:H7 at level of (P<0.05).
Figure 2: Susceptibility test of different antibacterial disc.
Mustafa Salah Hasan et. al.| Journal of Global Pharma Technology| 2018: 10(08):90-96
©2009-2018, JGPT. All Rights Reserved 94
Our results showed that all bacterial isolates
were highly sensitive to Gentamicin and
Enrofloxacin this agreement with Osek [23]
who found the total isolates of E. coli
O157:H7 are sensitive to Gentamicin and
Enrofloxacin also they found the isolates
100% sensitive to Cephalothin,
Chloramphenicol and Tetracycline and with
results of Galland et al. [40], that recorded
most isolates of E. coli O157:H7 were
susceptible to antibiotics used at feedlots,
such as trimethoprim-sulfamethoxazole. Also
Schroeder et al., [41] found that (20 %) of the
isolates, however, were resistant to
tetracycline. A much smaller proportion of
cattle isolates were resistant to
chloramphenicol, cephalothin (2.3% each).
The study of Mora et. al. [42]. Concluded that
E. coli O157:H7 showed a higher degree of
resistant which isolated from cattle (53%)
and beef (57%) than from human being (23%)
and sheep (20%) sources, they found that
Sulfisoxazole (36%) was the most
antimicrobial resistance, followed by
tetracycline, streptomycin, ampicillin,
trimethoprim, cotrimoxazole,
chloramphenicol, kanamycin, piperacillin
and neomycin. Our results were similar to
study of Osaili et. al. [20]. Who showed that
E. coli O157:H7 isolates were extensively
resistant to erythromycin and high
susceptibility to Gentamicin, Tetracycline,
Ampicillin, Ciprofloxacin, and Kanamycin.
The different results of resistance may be
due to the use of non-specific antibiotic and
random administration of antibiotic and this
will make persistent strains have ability to
resist a large number of antibiotics, some of
these resistance appeared in calves
introduced from other country.
Conclusion
In conclusion, the Real time PCR assay
described in this study is a sensitive and
accurate method for specific detection and
profiling of EHEC O157:H7 and these
bacteria can be referred as Multi drug
resistant bacteria due to its ability to resist
most antimicrobial that used in this study.
Acknowledgment
This work was supported by College of
Veterinary Medicine, Department of Internal
and Preventive Veterinary Medicine,
University of Fallujah, Iraq.
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