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PrSlists: Elsevier Bv. Pt&li.nfist r}?er -hr8els. ISSN: 2221t691
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submitvour PaperAsian
Pacific
Journal
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Tropical
Biomedicine
p-ISSN:
[Asian
Pac
J
Trop
Biomedl
2221-r6e1
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2014:122.47
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*::t
Pacific
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tJlomeotclneAbbreviated
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Asian Pac J Trop Biomed
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ilChina
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Original article http://dx.doi.org/10.1016/j.apjtb.2015.07.023
Evaluation of zoonotic potency of
Escherichia coli
O157:H7 through arbitrarily primed
PCR methods
I Wayan Suardana1*, Dyah Ayu Widiasih2, I Gusti Ngurah Kade Mahardika3, Komang Januartha Putra Pinatih4, Budi Setiadi Daryono5
1Department of Veterinary Public Health, Faculty of Veterinary Medicine, Udayana University, Jl. PB. Sudirman Denpasar,
Bali, Indonesia
2Department of Veterinary Public Health, Faculty of Veterinary Medicine, Gadjah Mada University, Jl.Fauna 2, Karang
Malang, Yogyakarta 55281, Indonesia
3Department of Virology, Faculty of Veterinary Medicine, Udayana University, Jl. PB. Sudirman Denpasar, Bali, Indonesia 4Department of Clinical Microbiology, Faculty of Medicine, Udayana University, Jl. PB. Sudirman Denpasar, Bali, Indonesia 5Department of Genetic, Faculty of Biology, Gadjah Mada University, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281,
Indonesia
A R T I C L E I N F O
Article history: Received 25 May 2015
Received in revised form 24 Jun, 2nd revised form 20 Jul 2015
Accepted 26 Jul 2015 Available online 30 Aug 2015
Keywords:
E. coliO157:H7
Zoonoses Animals Human AP-PCR
A B S T R A C T
Objective: To evaluate the zoonotic potency of Escherichia coli O157:H7 through arbitrarily primed-PCR (AP-PCR) methods as one of the DNAfingerprinting methods. Methods: A total of 14 isolates consisted of 11 isolates originated from human feces with renal failure symptoms, 2 isolates originated from cattle feces, and 1 control isolate were used in this study. DNA of each isolate was extracted, and their profiles were studied by using AP-PCR method with M13 F and M13 R arbitrary primers.
Results: The results founded that all of 14 isolates had similarity range from 54.6% to 88.5%. Isolates KL-106(3) and KL-55(6) originated from humans showed the degree of similarity with isolates SM-25(1) and SM-7(1) originated from cattle as high as 85% and 77%, respectively.
Conclusions: The high degree of similarity between isolates originated from cattle and human indicated the high potency of zoonoses. The results also concluded AP-PCR method as a briefly fingerprinting method in order to trace the epidemiological of
E. coliO157:H7.
1. Introduction
Escherichia coli(E. coli) O157:H7 is a zoonotic agent of the
type of Shiga toxin-producingE. colithat can cause disease in humans at some parts of the world[1–4], and cattle are known as the
main reservoir of these bacteria[5,6]. Researchers had succeeded in isolating these bacteria from pigs, beef, pork, water or human[7,8]
and proved the food products obtained from supermarkets were contaminated withE. coliO157:H7[9,10].
The infection by these bacteria in animals is usually asymptomatic, whereas these bacterial infections in humans usually show clinical symptoms of diarrhea and hemolytic ure-mic syndrome [1,11,12]. The human infection usually occurred
when proper hygiene is not strictly implemented especially in many developing countries and human consumed undercooked food products[2,13].
In Indonesia, several studies related toE. coliO157:H7, have been carried out by researchers. The study of Drastini in 2007 had found that 59% of vero toxinE. coliconsisting of vero toxin
E. coli O157 and non-O157 can be isolated from all types of
livestock namely, dairy cattle, beef, pigs and goats/sheep [14]. Other researchers reported theirfindings ofE. coliO157:H7 in Badung, Bali in beef and in healthy human feces were 5.62% and 1.30%, respectively [15]. The slight titer toxins of Stx1
and Stx2 resulted from local isolates ofE. coliO157:H7 were
*Corresponding author: I Wayan Suardana, Department of Veterinary Public Health, Faculty of Veterinary Medicine, Udayana University, Jl. PB. Sudirman Denpasar, Bali, Indonesia.
E-mails:iwayansuardana22@yahoo.com,iwayan_suardana@unud.ac.id
Peer review under responsibility of Hainan Medical University.
Foundation Project: Supported by the Directorate of Research and Community Services, Directorate General of Higher Education through Udayana Research Grants with contract No. 21.34/UN14/SBRC/2012.
H O S T E D B Y
Contents lists available atScienceDirect
Asian Paci
fi
c Journal of Tropical Biomedicine
journal homepage:www.elsevier.com/locate/apjtb
Asian Pac J Trop Biomed2015; 5(11): 915–920 915
originated from cattle feces and beef had been reported [16]. Moreover, the pheno-genotypic adherence ofE. coli O157:H7 isolated from beef, feces of cattle, chicken and human had also been studied[17,18].
According to theory, the detection of pathogenic microor-ganisms including E. coli O157:H7 was preferable to using molecular methods, considering the highly sensitivity and specificity of these methods. Molecular method in order to study the epidemiology of food-borne diseases is usually using DNA subtyping. Genomic DNA profiles in DNA subtyping are divided according to the size, and the resulting size will form the patterns which can be used as a characterization of each isolate
[19]. Some DNA subtyping methods are commonly used for the
characterization of bacterial genomes including pulsed-field gel electrophoresis, ribotyping, and random amplified polymorphic DNA (RAPD)[20,21].
Pulsed-field gel electrophoresis subtyping as one of the
fingerprinting methods usually use restriction enzymes that cut genomic DNA randomly to generate approximately 20–30
fragments. The size of the fragments formed quite varied be-tween 10 and 1 000 kbp and this method requires a separation method with special electrophoresis[19,20]. On the other hand, ribotyping method also uses restriction enzymes. Restriction enzyme will cut the DNA fragment in the gene encoding ribosomal RNA. The number of fragments produced will vary ranges from 500 to 1 000 fragments with measure between 1 and 20 kbp. This method has limitation because the results can only be detected using a specific probe for the gene encoding ribosomal RNA from bacteria[22].
RAPD method was developed by researcher using short ol-igonucleotides (10 bases) as random primers. This method was successfully performed by researchers to differentiate the bac-terial genome with only a small amount of genomic DNA[23,24]. RAPD method had been successfully used in an effort to study the zoonotic potency ofE. coliO157:H7[25,26].
In the development of this method, the other researchers developed a similar method that used approximately 15 nucle-otides as random primers with different amplification conditions with RAPD which is known as the method of arbitrarily primed PCR (AP-PCR). The use of AP-PCR method as a variant of RAPD method for epidemiological investigation has been widely used by researchers[27,28].
AP-PCR method is known to have some advantagesi.e. it can be applied to a wide range of organisms, fast and simple to generate fingerprint products. This method also used the selected primers without needing the initial information about the organism to be tested. Besides those, the data generated would be able to provide information about the difference or similarity of strains originated from the same species [29,30]. This method has been success to identify the number and the distribution of genotypes of Streptococcus mutans and
Streptococcus sobrinus [31], to predict the degree of
malignant cholangiocarcinoma as familiarly as the malignant neoplasm of biliary epithelium [32], and to describe an outbreak caused by extended-spectrum beta-lactamase pro-ducing Klebsiella pneumoniae [33]. Furthermore, this method
also proved in good agreement with the results obtained for the 16S rRNA[34].
Based on these facts, in this study we reported the application of AP-PCR method in order to study the zoonotic potency of
E. coli O157:H7 from animals to human and all at once as a
clarification of previous study, which analyzed the zoonotic
aspect of E. coliO157:H7 both animals and human origin by analysis of protein profile and 16S rRNA gene[35,36].
2. Materials and methods
2.1. Bacterial strains
Bacterial strains ofE. coliO157:H7 that used in this study were the same as those used previously[25,36]. Fourteen isolates
consisted of 11 isolates originated from human feces with renal failure symptomsi.e.KL-52(7), KL-87(7), KL-30(4), KL-45(1), 48(2), 85(1), 83(5), 24(5), 68(1), KL-106(3) and KL-55(6), 2 isolates originated from cattle fecesi.e. SM-25(1) and SM-7(1), and 1 control isolatei.e.ATCC 43894 were used in this study.
2.2. Cultivation of bacterial strains
Cultivation of the bacterial isolates was done identically with previous study [35]. Fourteen isolates ofE. coliO157:H7 were taken from stock (stored in 30% glycerol with a storage temperature of −20 C) to subsequent cultivate on lactose broth medium at 37 C for overnight. Isolates were
re-confirmed by culturing on selective medium sorbitol MacCon-key agar, followed by testing on latex agglutination test O157 and testing on H7 antiserum as afinal confirmation.
2.3. Extraction of DNA and PCR
Bacterial DNA was extracted using QIAamp DNA mini kits (Qiagen) according to supplier's instruction as described previ-ously [25,36]. The concentration of DNA was determined by
spectrophotometer. Amplification of genomic DNA by using AP-PCR method was carried out in 40
m
L reaction volumes.These reactions contained 2
m
L DNA template (200 ng/m
L),36
m
L PCR Supermix 2× (Invitrogen), and 2m
L (20 pmol/m
L) of each primer. The primers used were M13F: 50-TGTAAAAC-GACGGCCAG-30and M13R: 50
-CAGGAAACAGCTATGAC-30[37]. PCR program was performed on Eppendorf mastercycler personal. The PCR amplification had initial DNA denaturation at 94C for 5 min, followed by 39 cycles of denaturation at 94C
for 1 min, annealing at 35C for 1 min, and extension at 72C
for 1 min;finished by afinal extension at 72C for 5 min at the
end of the amplification. About 5
m
L of PCR product was analyzed by electrophoresis (Bio-Rad) in 1.5% agarose (Gibco BRL) gel, at 90 v for 45 min. The gel then stained by 1% solution of ethidium bromide (50m
L/L) and destained with tetrabromoethane 1× for 20 min. Gel was visualized by UV transillumination and it was recorded by digital camera FE-270 7.1 megapixels.2.4. Data analysis
The amplified fragments were scored in a descending manner from higher to lower molecular weight in a binary code. The appearance of product was designed as 1 and its absence as 0[38].
Evolutionary distance was measured with algorithm of
unweighted pair group method using arithmetic averages (UPGMA) and similarity coefficients of each cluster were showed near the branch of phenogram. The data were processed using multivariate statistical package 3.1 program[35,39]. I Wayan Suardana et al./Asian Pac J Trop Biomed 2015; 5(11): 915–920
3. Results
3.1. AP-PCR profile
The purified DNA of 14 isolates of E. coli O157:H7 including control isolate was subjected AP-PCR and different patterns of genomic DNA were obtained. The DNA profiles of 14 isolates generated using primer M13F were shown in
Figure 1.
The use of primer M13R showed different patterns. The complete AP-PCR profiles of 14 isolates of E. coli O157:H7 including control isolate ATCC 43894 using primer M13R were shown inFigure 2.
According toFigures 1 and 2, the bands that were obtained from 14 isolates spread out the size range from 300 to 2 000 bp. Total bands and fragment variations of the 14 isolates ofE. coli O157:H7 generated by AP-PCR using both primers M13F and M13R were summarized inTable 1.
3.2. Phenogram analysis
Total bands and fragments of all isolates obtained using AP-PCR method in Table 1 showed variation among isolates. These variations were specific for each isolate so that they can be used to differentiate between one isolate to others. Further analysis of the data in Table 1showed the genetic relatedness among isolates. Genetic relatedness described as a tree-like structure was known as phenogram presented inFigure 3.
Figure 3 shows that E. coliO157:H7 strains were divided into some clades with each coefficient similarities. Isolate KL-87(7) showed closely related to ATCC 43894 with a
coef-ficient similarity of 69.2 or having similarity of 69.2%. More-over, both KL-45(1) and KL-30(4) isolates shared clade with coefficient similarity of 80.8 or having similarity of 80.8%, as well as the others. The complete matrix of similarity coefficient among isolates ofE. coliO157:H7 was presented inTable 2.
Figure 1. AP-PCR profile of genomic DNA ofE. coliO157:H7 by using primer M13F on 1.5% agarose gel.
Line 1: ATCC 43894 (positive control); Line 2: KL52(7); Line 3: KL87(7); Line 4: KL30(4); Line 5: KL45(1); Line 6: KL(48(2); Line 7: KL85(1); Line 8: KL83(5); Line 9: KL24(5); Line 10: KL68(1); Line 11: KL-106(3); Line 12: KL-55(6); Line 13: SM-25(1); Line 14: SM-7(1); M: Marker 100 bp DNA ladder.
Figure 2. AP-PCR profile of genomic DNA ofE. coliO157:H7 by using primer M13R on 1.5% agarose gel.
Line 1: ATCC 43894 (positive control); Line 2: KL52(7); Line 3: KL87(7); Line 4: KL30(4); Line 5: KL45(1); Line 6: KL(48(2); Line 7: KL85(1); Line 8: KL83(5); Line 9: KL24(5); Line 10: KL68(1); Line 11: KL-106(3); Line 12: KL-55(6); Line 13: SM-25(1); Line 14: SM-7(1); M: Marker 100 bp DNA ladder.
Table 1
Fragment and total band of 14 isolates ofE. coliO157:H7 generated by AP-PCR using primers M13F and M13R.
Isolates Source Fragments with primer M13F (bp) Fragments with primer M13R (bp) Total bands ATCC 43894 Human feces control 300; 450; 550; 600; 700; 2 000 750 7 KL-52(7) Human feces 300; 400; 450; 550; 750; 1 400 400; 450; 550; 800; 850 11
KL-87(7) Human feces 300; 400; 500; 700; 800 – 5
KL-30(4) Human feces 300; 400; 450; 750 400; 450; 500; 550; 700; 850; 1 200 11 KL-45(1) Human feces 300; 400; 450; 600; 750; 1 400; 2 000 400; 450; 500; 550; 850 12 KL-48(2) Human feces 300; 500 400; 450; 500; 550; 700; 800; 1 000 9 KL-85(1) Human feces 400; 500; 600; 800; 1 400; 2 000 400; 450; 500; 550; 900 11 KL-83(5) Human feces 400; 500; 600; 800; 1 400; 2 000 400; 550; 700; 900; 1 200 11
KL-24(5) Human feces – 400; 550 2
KL-68(1) Human feces 400; 500; 600; 800; 1 400 350; 550; 900 8 KL-106(3) Human feces 400; 700; 800; 1 400 350; 550; 900 7 KL-55(6) Human feces 400; 700; 900; 1 400 400; 550; 700; 900 8 SM-25(1) Cattle feces 400; 700; 900 550; 600; 700; 1 000; 1 200 8 SM-7(1) Cattle feces 700 300; 400; 550; 700; 1 200 6
Total general 116
Figure 3. Phenogram ofE. coliO157:H7.
Phenogram was constructed using simple matching coefficient (Ssm) and algorithm UPGMA based on 116 AP-PCR fragments generated by arbitrary primer M13F and M13R.
4. Discussion
AP-PCR as one of thefingerprinting techniques was known to have many advantages such as its simplicity and shorter time consumption. This technique allows genetically different bac-terial strains to be distinguished with great sensitivity and effi -ciency [28]. Sensitive and efficient technique for typing
pathogenic microbes was important for tracing routes of infection and for understanding the spread and evolution of virulence agent [28,34]. In our study, AP-PCR was used as a technique to study zoonotic potency of E. coli O157:H7 in humans from animals' origin.
Our study showed AP-PCR technique as a powerful discrimination method to distinguish each isolate characterized by many bands or fragments that were amplified. Either primer M13F or M13R produced a number of lengthy fragments (Table 1). Total amplification produced by this methodi.e.116 bands consisting of 59 amplicons for primer M13F and 57 amplicons for primer M13R. The lengthy fragment resulted from both primers also showed variation. As many as 12 fragments were detected from primer M13F consist of 300, 400, 450, 500, 550, 600, 700, 750, 800, 900, 1 400 and 2 000 bp. Moreover, primer M13R detected 13 variations consisting of 300, 350, 400, 450, 500, 550, 700, 750, 800, 850, 900, 1 000 and 1 200 bp. The variation in the length and number of fragments was amplified from both primers for each isolate indicated the highly power discrimination of this method as one of the DNAfingerprinting methods to describe the genetic variation for each isolate.
Glick and Pasternak revealed that the primer or short oligo-nucleotides used in genomic analysis would form pairs in many places with chromosomal DNA. The number of amplified DNA fragments would depend on the primers used, and the result could be used to determine the characteristic of the entire genome or the chromosome of an individual[19]. Accuracy of
AP-PCR as one of the fingerprinting methods with a great sensitivity and efficiency had proved by researcher[40]. Their research reported this method was successfully used to distinguishE. coli O157:H7 strain using template from boiled stationary-phase cultures, without the need for time-consuming phenol extraction[41].
The use of AP-PCR method also successfully investigated clonal relatedness among the strains of epidemic isolates of
Vibrio cholerae O1 recovered from an outbreak occurring in
different parts of Iran [42] and to type Clostridium difficile
isolated from different sources of Imam Reza hospital, Tabriz, Iran in order to control nosocomial infections produced by these bacteria [28].
Further analysis of the data in Table 1 showed the genetic relatedness among isolates of E. coli O157:H7. Genetic relat-edness were described as a phenogram in Figure 3showed 14 isolates tested exhibiting clustering into 13 clades. Clade 1 was formed by isolates KL-106(3) and KL-68(1) with 88.5% simi-larity; clade 2 was formed by isolates KL-85(1) and KL-83(5) with 84.6% similarity; as well as clade 3 was formed by iso-lates KL-24(5) and SM-7(1) with 84.6% similarity; clade 4 which was formed by isolates KL-45(1) and KL-30(4) was known to have similarity value of 80.8%; while clade 5 was formed as a joint of clade 4 with isolates KL-52(7) having 78.8% similarity and so on to clade 13 with a value of similarity 54.6%.
Phenogram on Figure 3, which was clarified by similarity values of each isolate that was showed inTable 2exhibiting 2 out of 13 local isolates ofE. coliO157:H7i.e.isolates KL-24(5) and KL-87(7), respectively were known to have lower similarity i.e. 65% and 69%, respectively against control isolates ATCC 43894, which was known as the cause of humans' outbreaks in Japan. On the other hand, some isolates of human were also known to have a high similarity against isolates of cattle. Isolate SM-7(1) showed high similarity against isolates of human i.e. KL-24(5) and KL-55(6) as high as 85% and 77%, respectively. Isolate SM-25(1) also showed high similarity against isolate KL-55(6) originated from human with the degree of similarity 77%. The high degree of similarity between isolates of human and cattle origin, especially for isolates with degree of similarity 70% or more, it could be categorized as the same strains. This view was supported by Rosello-Mora and Amann who revealed in generally, organisms in prokaryotic species could be grouped into the same strain if they have similar genome as high as 70% or greater[43].
The high similarity of genomic DNA between isolates of
E. coliO157:H7 in human and cattle origin indicated its potency
as a zoonotic agent which transmitted from animals especially cattle as a main reservoir to humans. Researchers stated that cattle as a natural reservoir of E. coli O157:H7 could be
Table 2
Similarity coefficient among isolates ofE. coliO157:H7. ATCC 43894 KL-52(7) KL-87(7) KL-30(4) KL-45(1) KL-48(2) KL-85(1) KL-83(5) KL-24(5) KL-68(1) KL-106(3) KL-55(6) SM-25(1) SM-7(1) ATCC 43894 1.00
KL-52(7) 0.54 1.00 KL-87(7) 0.69 0.54 1.00 KL-30(4) 0.46 0.77 0.54 1.00 KL-45(1) 0.58 0.81 0.50 0.81 1.00 KL-48(2) 0.46 0.62 0.62 0.69 0.58 1.00 KL-85(1) 0.46 0.54 0.62 0.54 0.73 0.62 1.00 KL-83(5) 0.46 0.46 0.62 0.54 0.58 0.54 0.85 1.00 KL-24(5) 0.65 0.65 0.73 0.65 0.62 0.73 0.65 0.65 1.00 KL-68(1) 0.50 0.50 0.73 0.42 0.54 0.50 0.81 0.81 0.69 1.00 KL-106(3) 0.54 0.54 0.77 0.46 0.50 0.46 0.69 0.69 0.73 0.89 1.00 KL-55(6) 0.50 0.58 0.65 0.58 0.54 0.58 0.65 0.73 0.77 0.69 0.81 1.00 SM-25(1) 0.50 0.42 0.65 0.58 0.39 0.58 0.42 0.58 0.69 0.54 0.65 0.77 1.00 SM-7(1) 0.58 0.50 0.65 0.65 0.46 0.65 0.50 0.65 0.85 0.54 0.65 0.77 0.77 1.00 Similarity matrix was generated using value of simple matching coefficient (Ssm) and algorithm UPGMA.
I Wayan Suardana et al./Asian Pac J Trop Biomed 2015; 5(11): 915–920
transferred to humans through beef or contaminated environ-ment[3,5,6]. Other researches also reportedE. coliO157:H7 as
one strain of pathogenic E. coli known as a zoonotic agent could be detected in animals or humans [44,45] and the transmission usually associated with a complex factors involved environmental-host ecology that directly affects the likelihood of enterohemorrhagicE. coliO157[8,46,47].
Zoonotic potency of E. coli O157:H7 local isolates was supported by closely contact between human and cattle. In the area of study, cattle were not always cared by farmers in their cage, but also the farmer brought them outside, and cattle got feed or drinking water from their environment. On the other hand, the beef sold in traditional market was generally with poor hygiene so that the bacteria could be transmitted from animals to humans, according to the statement of previously researchers
[48,49]. In conclusion, the high degree of similarity of E. coli O157:H7 strains between isolates originated from cattle and human indicated the high potency of zoonoses, and AP-PCR method was proved to be a simple and rapid method to iden-tify the zoonoses agent ofE. coliO157:H7.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgments
The authors would like thank Prof. Dr. Supar MS for his kindness to give control isolates of E. coli O157:H7 ATCC 43894. The authors also wish to thank the Directorate of Research and Community Services, Directorate General of Higher Education which funded this research project through Udayana Research Grants with contract No.21.34/UN14/SBRC/ 2012, January 19th, 2012.
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