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Copyright © 2016, Avicenna Journal of Medical Biotechnology. All rights reserved. Vol. 8, No. 1, January-March 2016
Short Communication
42
Outer Membrane Protein C (
ompC
) Gene as the Target for Diagnosis of
Salmonella
Species Isolated from Human and Animal Sources
Alaa Abdel-Kadhim Jawad 1 and Alaa H. Al-Charrakh 2*
1. College of Veterinary Medicine, Al-Qadisiya University, Al Diwaniyah, Iraq
2. Department of Microbiology, College of Medicine, Babylon University, Hilla, Babylon Governorate, Iraq
Abstract
Background: The use of selective and differential plating media is a simple method
for the isolation of
Salmonella
spp. Recently, there has been a general move toward
molecular methods of
Salmonella
detection and typing.
Methods: A total of 1200 different specimens collected from human and animal
sources were involved in his study. 600 stool specimens from patients suffering from
diarrhea and 600 specimens from gall bladder (bile) of cattle from Al-Diwaniya
slaughter house, Iraq were used.
Salmonella
spp. were isolated and identified using
bacterial culturing on selective media and colonies were tested by API 20Eand then
serotyping through polyvalent antisera and conformation by Polymerase Chain Reac-
tion (PCR). PCR was used to detect
ompC
gene encoding biosynthesis of outer mem-
brane protein C of
Salmonella
genus.
Results: The results revealed that the rate of
Salmonella
isolates was 0.5% (3/600)
from human and 1% (6/600) from animals. The PCR technique revealed that 9 iso-
lates of
Salmonella
spp
.
harbored
ompC
gene. The results of this study revealed that
the PCR technique had a high specificity in detection of
Salmonella
spp.,
in compari-
son to culture and biochemical test, Mini API 20 E and serological tests. The present
study found no significant differences between human and animal isolates.
Conclusion: Detection of
ompC
gene is a good method for detection of
Salmonella
species isolated from clinical specimens. It has a high specificity in comparison with
other tests, with its advantages of greater speed and effectiveness than conventional
detection methods.
Keywords: Detection, Gene, Polymerase chain reaction,
Salmonella
Introduction
Enteric pathogens such as Salmonella enterica (S.
enterica) cause significant morbidity and mortality. S.
enterica serovars are a diverse group of pathogens that
have evolved to survive in a wide range of environ-
ments and across multiple hosts 1. Infection begins with
the ingestion of contaminated food or water so that
salmonellae reach the intestinal epithelium and trigger
gastrointestinal disease. In some patients, the infection
spreads upon invasion of the intestinal epithelium, in-
ternalization within phagocytes, and subsequent dis-
semination 2. It has been estimated that there are more
than 3 million deaths associated with Gram-negative
enteric pathogens worldwide due to diarrhea and enter-
ic fever each year 3. The use of selective and differen-
tial plating media is a simple method for the isolation
of Salmonella spp. A wide variety of selective and dif-
ferential media has been developed for this purpose,
including xylose lysine desoxycholate agar (XLD),
Hektoen Enteric (HE) agar, and bismuth sulfite (BS)
agar 4. There has been a general move toward molecu-
lar methods of Salmonella detection and typing and
PCR has become a potentially powerful alternative in
microbiological diagnostics. While it is easy to amplify
DNA derived from pure cultures, problems arise if the
sample investigated is as complex as clinical specimen
or food, since the PCR is easily inhibited by numerous
substances, including humic acids, fats, and proteins.
The ompC gene contains sequences unique to Salmo-
nella isolates and demonstrates that this gene is a suit-
able PCR target for detection of Salmonella strains 5.
The aim of this study was to evaluate the rapid de-
tection test to identify salmonellosis using the PCR
technique for the detection of ompC gene of Salmonel-
la genus, and to compare the PCR with traditional iso-
* Corresponding author:
Alaa H. Al-Charrakh, Ph.D.,
Department of Microbiology
College of Medicine, Babylon
University, Hilla, Babylon
Governorate, Iraq
Tel: +96 47813216822
E-mail:
ahani67@gmail.com;
aalcharrakh@yahoo.com
Received: 1 May 2015
Accepted: 13 Sept 2015
Avicenna J Med Biotech 2016; 8(1): 42-45
Abdel-Kadhim Jawad A and Al-Charrakh AH
Avicenna Journal of Medical Biotechnology, Vol. 8, No. 1, January-March 2016
43
lation and characterization methods currently used in
diagnostic laboratories.
Materials and Methods
Samples collection
A total of 1200 different specimens collected from
human and animal sources were involved in this study,
of which 600 stool specimens from patients suffering
from diarrhea admitted to Al-Diwaniya Teaching Hos-
pital for Maternity and Children (Al-Diwaniya prov-
ince, Iraq) and 600 specimens from gall bladder (bile)
of cattle from Al-Diwaniya slaughter house were col-
lected. This study was conducted during the period that
extended from May 2013 to April 2014.
From each specimen, 25 gm of human stool with a
sterile wood spatula or animal bile were directly inject-
ed into an Erlenmeyer flask and 225 ml of buffered
peptone water were added to obtain 1 part sample+9
part buffer, then mixed and incubated at 37°C over-
night (16-20 hr). After that, 1 ml of the pre enrichment
broth was transferred to 10 ml Tetrathionate broth and
incubated at 37°C overnight (18-24 hr). Moreover,
0.1 ml (100 ul) of the pre-enrichment broth was also
transferred to 10 ml Rappaport-Vassiliadis soy peptone
(RVS) broth and incubated at 41°C overnight.
Isolation and identification of Salmonella isolates
For each specimen, a loopful (10 µl) from the incu-
bated Tetrathionate broth (I) and RVS broth (II) was
spread on XLD and on BGA agar plates and incubated
at 37°C overnight (18-24 hr). For biochemical confir-
mation and serotyping, two suspected colonies (grown
on XLD and BGA agar) were plated onto non-selective
medium (nutrient agar) 6. Colonies that showed bio-
chemical characteristics similar to that of Salmonella
spp. were tested by Mini API 20E then serotyping. The
identified isolates were then confirmed by PCR with
ompC primers for detection of Salmonella genus 7.
DNA extraction and purification
One colony of each strain (cultured on solid medi-
um) was inoculated into 5 ml of BHI (Broth Heart In-
fusion) and grown overnight at 37°C. From these cul-
tures, DNA was purified from bacterial cells using Ge-
nomic DNA Mini kit (Geneaid, UK). Chromosomal
DNAs obtained were used as templates for all PCR
experiments. The PCR reactions were carried out in a
thermal cycler (Clever, U.K). Before PCR assay, and in
order to quantify the DNA concentration (ngμl-1), the
quantification of DNA samples was carried out by
means of a spectrophotometric reading using 1 μl ali-
quots of Genomic DNA with a NanoDropTM spec-
trometer (NanoDrop Technologies), adopting the man-
ufacturer’s recommendations. The concentration of
DNA was estimated from absorbance at 260 nm. DNA
profiles were performed using bacterial DNA and load-
ing buffer according to the manufacturer’s instructions
(Bioneer, Korea).
Detection of ompC gene by PCR
The ompC gene was detected in the present study.
This gene is encoding biosynthesis of outer membrane
protein C of Salmonella genus. The primer sequence of
this gene (provided by Bioneer Co., S. Korea) was
(ompC F: ATC GCT GAC TTA TGC AAT CG, ompC
R: CGG GTT GCG TTA TAG GTC TG) 204 bp.
Temperature for ompC F was 49.7ºC, and for ompC R
53.8ºC. PCR was performed at the following condi-
tions: 95ºC 2 min 1, 95ºC 60 s, 57ºC 60 s, 72ºC, 120 s
30, and 72ºC 5 min 1 8. Each primer was prepared
by dissolving it in 1000 µl of deionized distilled water
to obtain stocks concentration of 12 picomole/µl. Green
master mix, 2 (Promega, USA) includes: 1: Taq DNA
polymerase, 2: dNTPs which include 400 µM of each
dATP, dGTP, dCTP, dTTP, 3: 3 mM of MgCl2 (mag-
nesium chloride), and 4: Yellow and blue dyes as the
loading dye. After PCR, the profiles of amplification
products were detected by gel electrophoresis. Five
l
of total reaction mixture was loaded on a 1.5% agarose
gel and electrophoresed at 100 V at 70 mA for 60 min.
The amplified DNA fragments were visualized by UV
illumination after agarose gel electrophoresis and ethi-
dium bromide staining by standard procedures.
Statistical analysis
Statistical analysis was carried out using SPSS ver-
sion 17. Variables were presented as frequencies and
percentages. Pearson’s chi square (
2) test was applied
and p-value was considered significant at p<0.01 9.
Results
The isolation rates of Salmonella spp. were 0.5%
(3/600) from human and 1% (6/600) from animals us-
ing the conventional culture methods on enrichment
and selective media (Table 1).
The confirmed diagnosis of Salmonella spp. was
performed by Mini API 20 E system and the results
were obtained based on interpretation kit chart and
results of Mini API 20 E system. The isolates showed
positive results for arginine dihydrolase, lysine decar-
boxylase, ornithine decarboxylase, citrate utilization
and H2S production. Sugar fermentations showed posi-
tive results for glucose, mannose, inositol, sorbitol,
rhamnose, melibiose, arabinose, while they were nega-
tive for beta-galactosidase (ONPG), urease, tryptophan
deaminase, indole production, acetoin production, and
gelatin hydrolysis. They were also negative to sucrose
and amygdalin.
Table 1. Isolation rates of Salmonella spp. from human and animals
sources
Isolate
source No. Positive
result
Negative
result
2 value
Human 600 3 597 Calculated
2=1.008
Tabulated
2=6.6349
df= 1
No significant (p<0.01)
Animal 600 6 594
Total 1200 9 1191
44
ompC
as the Target for Diagnosis of
Salmonella
Avicenna Journal of Medical Biotechnology, Vol. 8, No. 1, January-March 2016
44
Validity of different techniques in diagnosis of salmonella
isolates
Nine isolates gave positive results for culturing and
biochemical tests, Mini API 20E (at 99.9% likelihood),
serotyping by polyvalent antisera (unpublished data)
and gave positive results for PCR tests (Table 2).
Amplification of target DNA (ompC gene)
The results of PCR amplification (performed on all
DNA samples extracted from the isolates) were con-
firmed by electrophoresis analysis. Depending on DNA
marker (8000 bp DNA ladder), the result of this esti-
mation revealed that the amplified DNA was 204 bp
for ompC gene (Figure 1).
Discussion
In this study, it was found that Salmonella spp. in-
fection is one of the causes of diarrhea. This may re-
flect the fact that Salmonella spp. is one of etiologic
agents of diarrhea that infect human and animals espe-
cially during the summer, and that Salmonella is a zo-
onotic bacterial agent, and Salmonella typhimurium
was the most common serovar found in animals and in
human specimens. In this study, 3 isolates from human
(0.5%) and six (1%) from animals were recovered us-
ing the conventional culture methods on enrichment
and selective media. Results revealed no significant
differences between human and animal isolates (p<
0.01). Traditional Salmonella detection methods are
based on cultures using selective media and characteri-
zation of suspicious colonies by biochemical and sero-
logical tests 7. Other studies conducted in Iraq, also
revealed prevalence of Salmonella spp. at different
rates of 7.9, 8.47 and 10%, 10-12 respectively. Salmonel-
la detection by using conventional media, such as Sal-
monella-Shigella agar (SS), is based on lactose fermen-
tation and H2S production and the number of false-
positive results with these media that may occur neces-
sitates conducting time-consuming and expensive addi-
tional testing 13. According to Mini API 20E results, all
the 9 isolates (by culture and biochemical tests) were
detected as Salmonella spp. at 99.9% likelihood level.
Nucera et al 14 evaluated API 20E and PCR for the
identification of Salmonella spp. isolates. They found
that API 20E had the highest similarity with PCR tests
at the 99.9% likelihood level and the validation of both
PCR and API 20E (at the 99.9% likelihood level)
showed that they are accurate diagnostic tests. Results
also revealed that the isolates (No=9) were confidently
identified as Salmonella spp. by detection of ompC
gene. The specific PCR product (ompC gene) is a 204
bp fragment which was visualized by gel electrophore-
sis. All Salmonella isolates (detected by phenotypical
tests) gave positive results with the PCR. The ompC
gene contains sequences unique to Salmonella isolates
and demonstrates that this gene is a suitable PCR target
for detection of Salmonella strains.
Conclusion
PCR technique has a high specificity in comparison
with other tests, with its advantages of greater speed
and effectiveness than conventional detection methods.
Detection of ompC gene is a good method for detection
of Salmonella species isolated from clinical specimens.
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