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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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A COMPARATIVE STUDY OF CULTURE METHODS, API SYSTEM AND
PCR ASSAY FOR Salmonella DETECTION ISOLATED FROM
HUMAN, COWS AND POULTRY IN IRAQ
Mitham S.S. , Rasha M. O.
Department of Microbiology, College of Veterinary Medicine, University of Basrah, Iraq
Keywords: Salmonella enterica, PCR, API System.
correspond Author e.mail:[email protected]
ABSTRACT
The genus Salmonella is one of the most important enteric pathogens, over the world
Salmonella Entritidis and Salmonella Typhimurium are the two most widespread serotypes that
lead to salmonellosis in human and animals and are often transferred to humans by infected
animals and their products. The present study was conducted to compare the culture methods, API
20E test and PCR assays for detection of Salmonella spp. isolated from 300 different samples
collected from various sources included healthy and infected human, cows ,chickens, chickens
eggs and other sources. To achieve this goal three selective media were used included (XLD, S.S.
and KIA agar) in addition to using the chromogenic salmonella media and the result of culturing
was compared with the results of API 20E test and PCR assays . We find that the using of
chromogenic media and PCR assay for detection of Salmonella spp. is more current and efficient
than using selective media and/or API 20E.
INTRODUCTION
Salmonellosis is an important infection disease in human and animals (1,2). Salmonella are
inhabitant in the gastrointestinal lumen of a broad range of vertebrates (3, 4). Over 2610 variant
serotypes (serovars) have been recognized by Kaufmann-White-Le minor scheme (5). Over the
world Salmonella Entritidis and Salmonella Typhimurium are the two most commonly serotypes
that led to salmonellosis in human and animals and are often transferred by infected animals and
their products to humans (6, 7).Culture methods for detecting Salmonella are well established and
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are routinely used for food testing, clinical diagnosis, and surveillance (8). In spite of the
upgrading in the culture of Salmonella as a result of many years of productive research work, the
isolation procedures currently in use are not ideal: false-positive and false-negative colonies are
familiar. Both Citrobacter and Proteus spp. are commonly misidentified as Salmonella because of
similar colonial features on selective xylose lysine desoxycholate (9). because the increased
diffusion of Salmonella serovar Enteritidis, and its complicated life cycle, a lot of researchers
emphasize the requirement and importance of finding a more quickly and effective detection
technique as a basis of control (10,11). Now, Salmonella is find out by standard bacteriological,
biochemical and serological method. These method are generally time-consuming, boring and
expensive as they require hundreds of antisera as well as well-trained technicians (12,13). Many
rapid and sensitive methods have been developed for conformity of Salmonella serotypes from
clinical samples (14). These methods, however, still loss the required sensitivity and specificity.
In recent times the amplification of DNA by the polymerase chain reaction (PCR) technique is
consider a strong equipment in microbiological diagnostics. Bacterial PCRs target evolutionarily
highly preserved genetic elements, e.g., bacterial 16S rRNA genes , Such PCRs with subsequent
sequence analysis are very good confirmed techniques for the identification of bacterial pathogens
(15), permitting for the find of a wide range of strains (16). Several protocols of 16S rRNA genebased
wide range PCRs for the diagnosis of infections have been suggested (17). PCR of short
fragments give a higher sensitivity than PCR of longer fragments of the 16S rRNA gene, while
longer fragments supply better differentiation in sequence analysis (18). Genotypic identification
methods are appear as an alternative or complement to confirmed phenotypic identification
process. For bacteria, 16S rRNA gene sequence analysis is a very accepted factor for molecular
identification (19). The present study was aimed to find the most rapid and accurate method for
detection of salmonella from different sources.
MATERIALS AND METHODS
Samples collection and preparations:
The period of the study was extending 10 months started from 2017 and involve both Basra and
Baghdad province. The total collected samples was (300) distributed as following: The human
samples were collected from two sources the first source was the healthy workers in the field
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who was in contact with the domestic animals, the second human samples were collected from
the diarrheic patient who attended the hospital. The number of samples for each type were 50
sample, a 5 g of the stool was taken and it was added to 10 ml of selenite broth to be mixed. And
then was transported to the laboratory of microbiology in icebox to be incubated at 37o C for
18-24 h. Cows samples: A fresh fecal samples from 50 cows were taken from the bowel as soon
as possible after the animals were butchered. A 5g of feces were mixed with 10 ml of selenite
broth and was transported to the laboratory of microbiology in icebox to be incubated at 37 oC
for18 -24 h. The chicken samples: The 50 chicken swabs samples were done directly from the
rectum by soft insertion of the swab stick after wetting it with selenite broth, then it was
inoculated in 5 ml of selenite broth, and then it was transported immediately to the laboratory by
icebox to be incubated at 37o C for 18-24 h. The eggs samples: A 50 eggs were collected from
irregular local house chicken and put immediately in sterile jar and transported to the laboratory
for more processing. The eggs shells were cleaned thoroughly and wiped by cotton with 70%
ethanol. Then two type of samples were collected first one included swab samples collected
directly after remove the shell membrane of the eggs. While the second type of samples were
included the taken whole eggs content and put directly in sterile container with 25 ml of peptone
water and mixed well, then 3 ml was taken from the mixture and was added to 7 ml of selenite
broth. The last Samples from other sources: included 50 swab samples were collected from
drainage water and the ground of the field of domestic animals and slaughter tools, the swab
samples were transported by a test tube containing selenite broth to the laboratory. All samples
were transported immediately to the laboratory by icebox and incubated with selenite broth at 37o
C for 18-24 h., except the eggs samples were incubated for two days the first day with peptone
water and the second day was incubated with selenite broth.
Isolation and identification:
Identification of Salmonella spp. was carried out according to the method of (20,21). After
incubation of sample in selenite broth for 24h., a loopful of the selenite broth streaked on XLD,
S.S. and KIA agar and were incubated at 37oC for 24 to 48h and the dishes were examined for the
morphology of Salmonella colonies. Positive samples were subsequently culture on Brian heart
infusion broth for other biochemical tests and for streaking on chromogenic agar plates and
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incubated at 37oC for the 24 to 48h. the plates were examined for the colour of colonies.
Salmonella (including S.typhi, S.paratyphi and lactose positive salmonella) was appeared mauve
colour. E.coli and Proteus, etc. was colorless or inhibited and Coliforms, etc. was blue colour.
Another Conventional tests were done like Gram's stain, Motility test, Oxidase test and Urease
test. Moreover the result was confirmed by inoculation the API 20 E system (BioMérieux, Inc.,
France).
API 20E
API 20E test is a plastic strips holding twenty mini-test tubes were inoculated with the distilled
water suspensions of the cultures from nutrient agar that was compared with the McFarland
standard solution for the density. Then the suspension was distributed in each tube. Some of tubes
were completely filled (CIT,VP and GEL), and other tubes were overlaid with mineral oil for
isolation from air reactions (ADH, LDC, ODC, H2S, URE). After incubation in a wet chamber for
24 hours at 37°C, the color reactions were read (some with the aid of added reagents as supplied
by the kit). The data were analyzed by using the indicator book.
Bacterial DNA Extraction and PCR Analysis
DNA Extraction
Because Salmonella is Gram negative bacteria the boiling extraction method was used in DNA
extraction. The procedure of this extraction was done by picked a five colonies from the XLD agar
plate of the suspected bacteria, and then it was transferred into Eppendorf tubes(1.5 or 2 ml)
containing 200 μl of distilled water. Before incubated at 100oC for 15 min in water bath the tubes
was vortexed. Then 800 μl of distilled water was added to get 1ml and remixed well by vortex
until the solution was homogeneous. Then the solution for 10 min was centrifuged at 12000 rpm
in cool centrifuge. The last step was the taken the supernatant which contain the genomic DNA
and transferred into an new Eppendorf tubes to be ready for PCR technique.
PCR technique:
For detection of Salmonella species a specific set of oligonucleotide primer were used in
polymerase chain reaction (F:5′TGTTGTGG TTAATAACCGCA-3′, R:5′-
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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CACAAATCCATCTCTGGA-3′) which amplify 572bp specific region of 16rRNA (21). The
PCR reactions were conducted in a total volume of 25μl,consisted of 5μl of master mix (Bioneer
/Korea), 10μl of genomic DNA , 1μl of each primer and 8μl of nucleus free water. Amplification
condition was obtained with an initial denaturation step at 95C° for 5 min followed by 35 cycle
each at 95C° for 5min , 55C° for 30sec and 72C° for 1 min , with final extension period of 10 min
at 72C°(21). The products amplified sizes were identified using 100 base pair DNA ladder
(Bioneer/Korea). Five μl of PCR products were directly loaded in a 1.5% agarose gel
electrophoresis and visualized by UV trans illuminator.
Statistical analysis:
Statistical analysis was performed by ANOVA test by Minitab 17, using the Fisher LSD method.
P-value less than 0.05 was considered as statistically significant and P-value less than 0.01
considered as highly significant .
Ethical Responsibilities
Protection of human and animal subjects: The authors declare that the procedures followed were
in accordance with the Animal Welfare Regulations and Ethics.
RESULTS
Table and figure (1) show the difference among the results of using three selective media for
isolate Salmonella sp. and use of conventional PCR as indicator for specific detection of
salmonella species. Alternatively table and figure (2) reveal the differences among the results of
using the Kligler iron, API20E, chromogenic media and PCR for diagnosis of salmonella spp.
The result of PCR amplification that performed on the extracted DNA was confirmed by
electrophoresis as the strands of the DNA which are resulted from successful binding between
primers and the extracted DNA. These successful binding appear as a single band under U.V
illuminator using ethidium bromide as a specific DNA stain. Bands with expected size (572bp)
were observed figure (3).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
Table 1 : Comparative between
according to the source of samples
Test
Source
XLD agar %
Human 37/100(37
Cows 11/50(22%)
Egg 25/50(50%)
Chicken 15/50(30%)
Other 23/50(46%)
P value
Figure 1 : Show the variation
215
the XLD, S.S, chromogenic and PCR positive result
samples.
S.S agar%
Chromogenic agar%
37%) 35/100(35%) 8/100(8%)
(12/50(24%) 3/50(6%)
(28/50(56%) 13/50(26%)
(15/50(30%) 4/50(8%)
(24/50(48%) 11/50(22%)
0.005
how between the XLD, S.S, chromogenic
according to the source of samples
results
% PCR %
8/100(8%)
3/50(6%)
13/50(26%)
4/50(8%)
11/50(22%)
and PCR results
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
Table 2: Comparative between
according to the source of samples
Test
Source
Kligler iron
Human 20/100(20%)
Cows 10/50(20%)
Egg 22/50(44%)
Chicken 13/50(26%)
Other 19/50(38%)
P value
Figure 2 : Show the variation
according to the source of samples
216
the KIA, API 20, Chromogenic and PCR
agar% API20E% Chromogenic media
%
%) 16/100(16%) 8/100(8%)
9/50(18%) 3/50(6%)
17/50(34%) 13/50(26%)
9/50(18%) 4/50(8%)
16/50(32%) 11/50(22%)
0.013
how between the KIA, API 20, chromogenic and
samples.
positive results
PCR%
8/100(8%)
3/50(6%)
13/50(26%)
4/50(8%)
11/50(22%)
, PCR results
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Figure. 3. Agarose gel electrophoresis reveals the PCR products of : Lane M: 100 bp
DNA Ladder. Lanes 1- 8: PCR Products of 572bp region of salmonella spp. 16S rRNA
gene and Lane c: control negative.
DISCUSSION
Understanding the limits of presently used selective and differential media, it is request to improve
the specificity and the sensitivity of the medium while keeping cost-effectiveness. Particularly, it
is favorite to differentiate Salmonella spp. from Proteus spp., as well as from Citrobacter spp.
(22). Many quick methods have been developed to detect pathogens (23). However, traditional
selective and differential media are still remarkable, due to many advantages, including costeffectiveness,
ease of use, and knowing among users (24). In the present study table and figure (1)
show the difference among the results of using three selective media for isolate Salmonella sp.
and use of conventional PCR as indicator for specific detection of salmonella species, we find that
the chromogenic media result was similar to the result of (PCR) while the XLD and S.S. agar
culturing result was show highly significant different( p-value < 0.008 and 0.006 respectively) if
compared with chromogenic, this due to the growth of some type of bacteria that have similar
characters of salmonella the led to false diagnosis. And this finding agree with the previous study
that say, both Citrobacter and Proteus spp. are commonly misidentified as Salmonella because of
similar colonial features on selective xylose lysine desoxycholate (9). Moreover XLD agar are the
most widespread media for finding of Salmonella spp., and their recognition abilities depend on
characteristics of Salmonella, such as hydrogen sulfide generating and the nonfermentation of
lactose (22). However, these features are shared with other bacteria, such as Proteus and
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Citrobacter (25). Although XLD has a high sensitivity and specificity, Proteus and Citrobacter
give colonies indistinguishable from those of Salmonella on this agar ( 22). On the other hand
table (2) and figure (2) reveals that chromogenic media culturing and (PCR) show the same
results in compare with two other test KIA and API 20E, we find that the KIA test (p-value
<0.006) is highly significant different in compare with chromogenic agar this was due to
salmonella and Proteus cause glucose fermented with H2S this may led to false result, and some
type of salmonella may not give black or weak black result, while the API 20 was less different in
the result (p-value 0.064), in spied of the interpretation of the test is fixed on two option positive
or negative sometime it is difficult to recognize between colorless and weak pale color this may
led to mistake in the result, it was more fixed and specific than KIA test. Additionally the acid
formation as outcome of carbohydrate fermentation may affect hydrogen sulfide production (22).
Under the acid conditions of carbohydrate metabolism, H2S-positive Enterobacteriaceae were
unable to produce the black precipitate of iron sulfide. S. Gallinarum and S. Pullorum rarely
generate hydrogen sulfide, and the reaction occurred slowly (22). Also the hydrogen sulfide
producing S. Typhi is weak or negative (26). Although the chromogenic media culturing result was
similar to PCR result (100%) this was due to inhibit other type of bacteria or appeared in another
color that make the detection of salmonella more easy and faster. Over the last 30 years, a range of
chromogenic media has been developed that are designed to target patho-genes with high
specificity. Such media take advantage of enzyme substrates that release colored dyes at
hydrolysis, thus resulting in pathogens forming colored colonies that can easily be recognize from
other bacteria. Ideally, other bacteria should either be inhibited completely by selective agents or
give colorless colonies to allowing pathogens to emerge against background .This make easy
differentiation of microbes have the enzyme from those that do not. This is very important when
trying to detect specific pathogens within polymicrobial cultures. The substrate and products of
hydrolysis should not restrained microbial growth (27).We concluded that using of chromogenic
media and PCR assay for detection of Salmonella spp. is more current and efficient than using
selective media and API 20E test.
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ACKNOWLEDGMENTS
We are grateful to the Departments of Microbiology, College of Veterinary Medicine, Basrah
University, Iraq for providing the laboratory facilities.
للکشف عن السالمونیلا المعزولة من الانسان PCR و اختبار API دراسة مقارنة لطرق الزرع و،نظام
،الابقار والدواجن.
میثم صباح صادق، رشا منذر عثمان
فرع الاحیاء المجھریة، کلیة الطب البیطری، جامعة البصرة، البصرة، العراق
الخلاصة
و Salmonella Entritidis جنس السالمونیلا واحد من أھم مسببات الأمراض المعویة، وعالمیا تعتبر کل من
من الانواع المصلیة الأکثر انتشارا والمسببان للسالمونیلا فی الإنسان والحیوان، وکثیر اً ما Salmonella Typhimurium
تنتقل إلى البشر من قبل خلال الحیوانات المصابة بھا ومنتجاتھا. أجریت الدراسة الحالیة للمقارنة بین استخدام الاوساط الزرعیة
فی الکشف عن أنواع السالمونیلا المعزولة من ٣٠٠ عینة جمعت من مصادر مختلفة شملت PCR وال API 20E واختبار
عینات من اشخاص اصحاء ومصابین من الأبقار المصابة، الدجاج ، بیض الدجاج، ومصادر أخرى. ولتحقیق ھذا الھدف
chromogenic بالإضافة إلى استخدام وسط (XLD, S.S. and KIA agar) استخدمت ثلاث اوساط انتقائیة شملت
وقد وجدنا من خلال النتائج .PCR و فحص API 20E وقورنت نتائج التشخیص بالزرع مع نتائج اختبار salmonella agar
للکشف عن السالمونیلا ھو أکثر دقة وکفاءة من استخدام PCR وفحص chromogenic salmonella agar بان استخدام
.API 20E الاوساط الانتقائیة واختبار
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