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Main title [English] *::t Pacific Journal of rropical

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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 primedPCR 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, KarangMalang, 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

ARTICLE INFO

Article history:Received 25 May 2015Received in revised form 24 Jun, 2ndrevised form 20 Jul 2015Accepted 26 Jul 2015Available online 30 Aug 2015

Keywords:E. coli O157:H7ZoonosesAnimalsHumanAP-PCR

ABSTRACT

Objective: To evaluate the zoonotic potency of Escherichia coli O157:H7 througharbitrarily primed-PCR (AP-PCR) methods as one of the DNA fingerprinting methods.Methods: A total of 14 isolates consisted of 11 isolates originated from human feceswith renal failure symptoms, 2 isolates originated from cattle feces, and 1 control isolatewere used in this study. DNA of each isolate was extracted, and their profiles werestudied 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% to88.5%. Isolates KL-106(3) and KL-55(6) originated from humans showed the degree ofsimilarity with isolates SM-25(1) and SM-7(1) originated from cattle as high as 85% and77%, respectively.Conclusions: The high degree of similarity between isolates originated from cattle andhuman indicated the high potency of zoonoses. The results also concluded AP-PCRmethod as a briefly fingerprinting method in order to trace the epidemiological ofE. coli O157:H7.

1. Introduction

Escherichia coli (E. coli) O157:H7 is a zoonotic agent of thetype of Shiga toxin-producing E. coli that can cause disease inhumans at some parts of theworld [1–4], and cattle are known as themain reservoir of these bacteria [5,6]. Researchers had succeeded inisolating these bacteria from pigs, beef, pork, water or human [7,8]

and proved the food products obtained from supermarkets werecontaminated with E. coli O157:H7 [9,10].

The infection by these bacteria in animals is usuallyasymptomatic, whereas these bacterial infections in humansusually show clinical symptoms of diarrhea and hemolytic ure-mic syndrome [1,11,12]. The human infection usually occurredwhen proper hygiene is not strictly implemented especially inmany developing countries and human consumed undercookedfood products [2,13].

In Indonesia, several studies related to E. coli O157:H7, havebeen carried out by researchers. The study of Drastini in 2007had found that 59% of vero toxin E. coli consisting of vero toxinE. coli O157 and non-O157 can be isolated from all types oflivestock namely, dairy cattle, beef, pigs and goats/sheep [14].Other researchers reported their findings of E. coli O157:H7 inBadung, Bali in beef and in healthy human feces were 5.62%and 1.30%, respectively [15]. The slight titer toxins of Stx1and Stx2 resulted from local isolates of E. coli O157:H7 were

*Corresponding author: I Wayan Suardana, Department of Veterinary PublicHealth, Faculty of Veterinary Medicine, Udayana University, Jl. PB. SudirmanDenpasar, Bali, Indonesia.

E-mails: iwayansuardana22@yahoo.com, iwayan_suardana@unud.ac.idPeer 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 Grantswith contract No. 21.34/UN14/SBRC/2012.

HOSTED BY Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Biomedicine

journal homepage: www.elsevier.com/locate/apjtb

Asian Pac J Trop Biomed 2015; 5(11): 915–920 915

2221-1691/Copyright © 2015 Hainan Medical University. Production and hosting by Elsevier (Singapore) Pte Ltd. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

originated from cattle feces and beef had been reported [16].Moreover, the pheno-genotypic adherence of E. coli O157:H7isolated from beef, feces of cattle, chicken and human had alsobeen studied [17,18].

According to theory, the detection of pathogenic microor-ganisms including E. coli O157:H7 was preferable to usingmolecular methods, considering the highly sensitivity andspecificity of these methods. Molecular method in order to studythe epidemiology of food-borne diseases is usually using DNAsubtyping. Genomic DNA profiles in DNA subtyping aredivided according to the size, and the resulting size will form thepatterns which can be used as a characterization of each isolate[19]. Some DNA subtyping methods are commonly used for thecharacterization of bacterial genomes including pulsed-field gelelectrophoresis, ribotyping, and random amplified polymorphicDNA (RAPD) [20,21].

Pulsed-field gel electrophoresis subtyping as one of thefingerprinting methods usually use restriction enzymes that cutgenomic DNA randomly to generate approximately 20–30fragments. The size of the fragments formed quite varied be-tween 10 and 1000 kbp and this method requires a separationmethod with special electrophoresis [19,20]. On the other hand,ribotyping method also uses restriction enzymes. Restrictionenzyme will cut the DNA fragment in the gene encodingribosomal RNA. The number of fragments produced will varyranges from 500 to 1000 fragments with measure between 1and 20 kbp. This method has limitation because the resultscan only be detected using a specific probe for the geneencoding ribosomal RNA from bacteria [22].

RAPD method was developed by researcher using short ol-igonucleotides (10 bases) as random primers. This method wassuccessfully 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 studythe zoonotic potency of E. coli O157:H7 [25,26].

In the development of this method, the other researchersdeveloped a similar method that used approximately 15 nucle-otides as random primers with different amplification conditionswith RAPD which is known as the method of arbitrarily primedPCR (AP-PCR). The use of AP-PCR method as a variant ofRAPD method for epidemiological investigation has beenwidely used by researchers [27,28].

AP-PCR method is known to have some advantages i.e. itcan be applied to a wide range of organisms, fast and simple togenerate fingerprint products. This method also used theselected primers without needing the initial information aboutthe organism to be tested. Besides those, the data generatedwould be able to provide information about the difference orsimilarity of strains originated from the same species [29,30].This method has been success to identify the number and thedistribution of genotypes of Streptococcus mutans andStreptococcus sobrinus [31], to predict the degree ofmalignant cholangiocarcinoma as familiarly as the malignantneoplasm of biliary epithelium [32], and to describe anoutbreak caused by extended-spectrum beta-lactamase pro-ducing Klebsiella pneumoniae [33]. Furthermore, this methodalso proved in good agreement with the results obtained forthe 16S rRNA [34].

Based on these facts, in this study we reported the applicationof AP-PCR method in order to study the zoonotic potency ofE. coli O157:H7 from animals to human and all at once as aclarification of previous study, which analyzed the zoonotic

aspect of E. coli O157:H7 both animals and human origin byanalysis of protein profile and 16S rRNA gene [35,36].

2. Materials and methods

2.1. Bacterial strains

Bacterial strains of E. coli O157:H7 that used in this studywere the same as those used previously [25,36]. Fourteen isolatesconsisted of 11 isolates originated from human feces with renalfailure symptoms i.e. 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) and KL-55(6), 2 isolates originated from cattle feces i.e.SM-25(1) and SM-7(1), and 1 control isolate i.e. ATCC 43894were used in this study.

2.2. Cultivation of bacterial strains

Cultivation of the bacterial isolates was done identically withprevious study [35]. Fourteen isolates of E. coli O157:H7 weretaken from stock (stored in 30% glycerol with a storagetemperature of −20 �C) to subsequent cultivate on lactosebroth 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 O157and testing on H7 antiserum as a final 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 byspectrophotometer. Amplification of genomic DNA by usingAP-PCR method was carried out in 40 mL reaction volumes.These reactions contained 2 mL DNA template (200 ng/mL),36 mL PCR Supermix 2× (Invitrogen), and 2 mL (20 pmol/mL) ofeach primer. The primers used were M13F: 50-TGTAAAAC-GACGGCCAG-30 and M13R: 50-CAGGAAACAGCTATGAC-30 [37]. PCR program was performed on Eppendorf mastercyclerpersonal. The PCR amplification had initial DNA denaturation at94 �C for 5 min, followed by 39 cycles of denaturation at 94 �Cfor 1 min, annealing at 35 �C for 1 min, and extension at 72 �Cfor 1 min; finished by a final extension at 72 �C for 5 min at theend of the amplification. About 5 mL of PCR product wasanalyzed by electrophoresis (Bio-Rad) in 1.5% agarose (GibcoBRL) gel, at 90 v for 45 min. The gel then stained by 1%solution of ethidium bromide (50 mL/L) and destained withtetrabromoethane 1× for 20 min. Gel was visualized by UVtransillumination and it was recorded by digital camera FE-270 7.1 megapixels.

2.4. Data analysis

The amplified fragments were scored in a descending mannerfrom higher to lower molecular weight in a binary code. Theappearance of product was designed as 1 and its absence as 0 [38].Evolutionary distance was measured with algorithm ofunweighted pair group method using arithmetic averages(UPGMA) and similarity coefficients of each cluster wereshowed near the branch of phenogram. The data were processedusing multivariate statistical package 3.1 program [35,39].

I Wayan Suardana et al./Asian Pac J Trop Biomed 2015; 5(11): 915–920916

3. Results

3.1. AP-PCR profile

The purified DNA of 14 isolates of E. coli O157:H7including control isolate was subjected AP-PCR and differentpatterns of genomic DNA were obtained. The DNA profiles of14 isolates generated using primer M13F were shown inFigure 1.

The use of primer M13R showed different patterns. Thecomplete AP-PCR profiles of 14 isolates of E. coli O157:H7including control isolate ATCC 43894 using primer M13R wereshown in Figure 2.

According to Figures 1 and 2, the bands that were obtainedfrom 14 isolates spread out the size range from 300 to 2000 bp.Total bands and fragment variations of the 14 isolates of E. coliO157:H7 generated by AP-PCR using both primers M13F andM13R were summarized in Table 1.

3.2. Phenogram analysis

Total bands and fragments of all isolates obtained usingAP-PCR method in Table 1 showed variation among isolates.These variations were specific for each isolate so that they can beused to differentiate between one isolate to others. Furtheranalysis of the data in Table 1 showed the genetic relatednessamong isolates. Genetic relatedness described as a tree-likestructure was known as phenogram presented in Figure 3.

Figure 3 shows that E. coli O157:H7 strains were dividedinto some clades with each coefficient similarities. IsolateKL-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 withcoefficient similarity of 80.8 or having similarity of 80.8%, aswell as the others. The complete matrix of similarity coefficientamong isolates of E. coli O157:H7 was presented in Table 2.

Figure 1. AP-PCR profile of genomic DNA of E. coli O157:H7 by usingprimer 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: Marker100 bp DNA ladder.

Figure 2. AP-PCR profile of genomic DNA of E. coli O157:H7 by usingprimer 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: Marker100 bp DNA ladder.

Table 1

Fragment and total band of 14 isolates of E. coli O157: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; 2000 750 7KL-52(7) Human feces 300; 400; 450; 550; 750; 1400 400; 450; 550; 800; 850 11KL-87(7) Human feces 300; 400; 500; 700; 800 – 5KL-30(4) Human feces 300; 400; 450; 750 400; 450; 500; 550; 700; 850; 1200 11KL-45(1) Human feces 300; 400; 450; 600; 750; 1400; 2000 400; 450; 500; 550; 850 12KL-48(2) Human feces 300; 500 400; 450; 500; 550; 700; 800; 1000 9KL-85(1) Human feces 400; 500; 600; 800; 1 400; 2000 400; 450; 500; 550; 900 11KL-83(5) Human feces 400; 500; 600; 800; 1400; 2000 400; 550; 700; 900; 1 200 11KL-24(5) Human feces – 400; 550 2KL-68(1) Human feces 400; 500; 600; 800; 1400 350; 550; 900 8KL-106(3) Human feces 400; 700; 800; 1400 350; 550; 900 7KL-55(6) Human feces 400; 700; 900; 1400 400; 550; 700; 900 8SM-25(1) Cattle feces 400; 700; 900 550; 600; 700; 1000; 1200 8SM-7(1) Cattle feces 700 300; 400; 550; 700; 1200 6Total general 116

Figure 3. Phenogram of E. coli O157:H7.Phenogram was constructed using simple matching coefficient (Ssm) andalgorithm UPGMA based on 116 AP-PCR fragments generated by arbitraryprimer M13F and M13R.

I Wayan Suardana et al./Asian Pac J Trop Biomed 2015; 5(11): 915–920 917

4. Discussion

AP-PCR as one of the fingerprinting techniques was knownto have many advantages such as its simplicity and shorter timeconsumption. This technique allows genetically different bac-terial strains to be distinguished with great sensitivity and effi-ciency [28]. Sensitive and efficient technique for typingpathogenic microbes was important for tracing routes ofinfection and for understanding the spread and evolution ofvirulence agent [28,34]. In our study, AP-PCR was used as atechnique to study zoonotic potency of E. coli O157:H7 inhumans from animals' origin.

Our study showed AP-PCR technique as a powerfuldiscrimination method to distinguish each isolate characterizedby many bands or fragments that were amplified. Either primerM13F or M13R produced a number of lengthy fragments(Table 1). Total amplification produced by this method i.e. 116bands consisting of 59 amplicons for primer M13F and 57amplicons for primer M13R. The lengthy fragment resulted fromboth primers also showed variation. As many as 12 fragmentswere detected from primer M13F consist of 300, 400, 450, 500,550, 600, 700, 750, 800, 900, 1400 and 2000 bp. Moreover,primer M13R detected 13 variations consisting of 300, 350, 400,450, 500, 550, 700, 750, 800, 850, 900, 1000 and 1200 bp. Thevariation in the length and number of fragments was amplifiedfrom both primers for each isolate indicated the highly powerdiscrimination of this method as one of the DNA fingerprintingmethods 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 manyplaces with chromosomal DNA. The number of amplified DNAfragments would depend on the primers used, and the resultcould be used to determine the characteristic of the entiregenome or the chromosome of an individual [19]. Accuracy ofAP-PCR as one of the fingerprinting methods with a greatsensitivity and efficiency had proved by researcher [40]. Theirresearch reported this method was successfully used todistinguish E. coli O157:H7 strain using template from boiledstationary-phase cultures, without the need for time-consumingphenol extraction [41].

The use of AP-PCR method also successfully investigatedclonal relatedness among the strains of epidemic isolates of

Vibrio cholerae O1 recovered from an outbreak occurring indifferent parts of Iran [42] and to type Clostridium difficileisolated from different sources of Imam Reza hospital, Tabriz,Iran in order to control nosocomial infections produced bythese bacteria [28].

Further analysis of the data in Table 1 showed the geneticrelatedness among isolates of E. coli O157:H7. Genetic relat-edness were described as a phenogram in Figure 3 showed 14isolates tested exhibiting clustering into 13 clades. Clade 1 wasformed 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 4which was formed by isolates KL-45(1) and KL-30(4) wasknown to have similarity value of 80.8%; while clade 5 wasformed as a joint of clade 4 with isolates KL-52(7) having78.8% similarity and so on to clade 13 with a value of similarity54.6%.

Phenogram on Figure 3, which was clarified by similarityvalues of each isolate that was showed in Table 2 exhibiting 2out of 13 local isolates of E. coli O157:H7 i.e. isolates KL-24(5)and KL-87(7), respectively were known to have lower similarityi.e. 65% and 69%, respectively against control isolates ATCC43894, which was known as the cause of humans' outbreaks inJapan. On the other hand, some isolates of human were alsoknown to have a high similarity against isolates of cattle. IsolateSM-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 andcattle origin, especially for isolates with degree of similarity70% or more, it could be categorized as the same strains. Thisview was supported by Rosello-Mora and Amann who revealedin generally, organisms in prokaryotic species could be groupedinto the same strain if they have similar genome as high as 70%or greater [43].

The high similarity of genomic DNA between isolates ofE. coli O157:H7 in human and cattle origin indicated its potencyas a zoonotic agent which transmitted from animals especiallycattle as a main reservoir to humans. Researchers stated thatcattle as a natural reservoir of E. coli O157:H7 could be

Table 2

Similarity coefficient among isolates of E. coli O157:H7.

ATCC43894

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)

ATCC43894

1.00

KL-52(7) 0.54 1.00KL-87(7) 0.69 0.54 1.00KL-30(4) 0.46 0.77 0.54 1.00KL-45(1) 0.58 0.81 0.50 0.81 1.00KL-48(2) 0.46 0.62 0.62 0.69 0.58 1.00KL-85(1) 0.46 0.54 0.62 0.54 0.73 0.62 1.00KL-83(5) 0.46 0.46 0.62 0.54 0.58 0.54 0.85 1.00KL-24(5) 0.65 0.65 0.73 0.65 0.62 0.73 0.65 0.65 1.00KL-68(1) 0.50 0.50 0.73 0.42 0.54 0.50 0.81 0.81 0.69 1.00KL-106(3) 0.54 0.54 0.77 0.46 0.50 0.46 0.69 0.69 0.73 0.89 1.00KL-55(6) 0.50 0.58 0.65 0.58 0.54 0.58 0.65 0.73 0.77 0.69 0.81 1.00SM-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 1.00SM-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): 915–920918

transferred to humans through beef or contaminated environ-ment [3,5,6]. Other researches also reported E. coli O157:H7 asone strain of pathogenic E. coli known as a zoonotic agentcould be detected in animals or humans [44,45] and thetransmission usually associated with a complex factorsinvolved environmental-host ecology that directly affects thelikelihood of enterohemorrhagic E. coli O157 [8,46,47].

Zoonotic potency of E. coli O157:H7 local isolates wassupported by closely contact between human and cattle. In thearea of study, cattle were not always cared by farmers in theircage, but also the farmer brought them outside, and cattle gotfeed or drinking water from their environment. On the otherhand, the beef sold in traditional market was generally with poorhygiene so that the bacteria could be transmitted from animals tohumans, according to the statement of previously researchers[48,49]. In conclusion, the high degree of similarity of E. coliO157:H7 strains between isolates originated from cattle andhuman indicated the high potency of zoonoses, and AP-PCRmethod was proved to be a simple and rapid method to iden-tify the zoonoses agent of E. coli O157: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 hiskindness to give control isolates of E. coli O157:H7 ATCC43894. The authors also wish to thank the Directorate ofResearch and Community Services, Directorate General ofHigher Education which funded this research project throughUdayana Research Grants with contract No.21.34/UN14/SBRC/2012, January 19th, 2012.

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