originalarticle openaccess rapd-eaea marker for...

Haque et al. Journal of Nature and Natural Sciences. 2:2, 1-13, 2017.

Original Article Open Access

RAPD-eaeA marker for detection ofEnteropathogenic Escherichia coli from stoolsamples of north Indian patientsShafiul Haque1, Zahida Qamri1 and Arif Ali1*1 Gene Expression Laboratory, Department of Biosciences, Faculty of Natural Sciences, Jamia Millia Islamia (A Central University), NewDelhi, India

Received February 2017, Accepted April 2017, Published Online May 2017

AbstractEnteropathogenic Escherichia coli (EPEC) are among the most important causes of acute enteritis andsubsequent morbidity and mortality in children. This study aims to check the practicability of making use ofRandom Amplified Polymorphic DNA (RAPD) technique for the differentiation of various serotypes/ strains ofEPEC by targeting eaeA gene. RAPD was used to fingerprint EPEC isolates and eaeA detection was done toconfirm that the selected isolates harbored the "locus of enterocyte effacement" pathogenicity island. Usingspecific primers, we were able to get the band of 300-bp of eaeA gene coding for an outer membrane protein,intimin, and the presence of intimin among isolates was endorsed by immunoblotting. A consistent profile ofRAPD was obtained with 90% polymorphism observed among the isolates. From this data, 6 unique strains ofE. coli were identified and similarly, the constructed phylogenetic tree demonstrated the variation between thepathogenic and the non-pathogenic strains, suggesting genetic drift of these strains from each other. However,one pathogenic strain ZQA5 demonstrated close similarity with the non-pathogenic ZQA6 strain andsuggesting the former to be a modem version of the ZQA6 by recently acquiring its virulent nature. Hence, itmight be suggested that RAPD-eaeA marker can be utilized as a potential molecular tool for the identificationof EPEC isolates.Keywords: EPEC; Serotype; RAPD-PCR; Pathogenicity Island LEE; EAF

1 IntroductionDiarrheal diseases are second only to cardiovascu-lar diseases as a cause of death worldwide (WHO,2009). In India, diarrheal disease is a major publichealth problem among children (WHO, 2009). En-teropathogenic Escherichia coli (EPEC) were the firstrecognized diarrheagenic class of E. coli having cometo light in the 1940s as the main cause of diarrheal out-breaks in infant nurseries and cause of sporadic infantdiarrhea. The status of EPEC strains as pathogens wasdebated since they lack recognizable virulence traitssuch as heat-labile and heat-stable enterotoxins or ep-ithelial cell invasiveness. Though, volunteer studies inadults inequitably confirmed EPEC as pathogenic bac-terial agents (Levine et al., 1987). Generally, EPEC*Corresponding Author: [email protected] Gene Expression Laboratory, Department of Biosciences, Faculty ofNatural Sciences, Jamia Millia Islamia (A Central University), New Delhi,India

strains have a pathogenicity island, called as Locusof Enterocyte Effacement (LEE) and a 50-70 MDaplasmid, labeled as EAF (EPEC Adherence Factor).The typical mechanism of EPEC pathogenesis is a le-sion termed as ’attaching and effacing’ (A/E), thatis technically characterized by intimate adherence ofEPEC bacteria to the intestinal epithelium of the host(Nougayrede et al., 2003). The EPEC bacteria havebeen classified into typical and atypical strain group onthe basis of the eaeA gene located in the LEE patho-genecity island, the bfpA gene accountable for BFPfimbriae (bundle-forming pilus), and several regulatoryproteins located on a plasmid, called the EAF (Gironet al., 1991). The ’typical EPEC’ are those E. colistrains of the A/E genotype (eaeA+) that harbors theEAF plasmid (bfpA+) and most such strains pertainto certain O:H serotypes (Trabusi et al., 2002). While,EPEC strains of the A/E genotype, which do not har-bor the EAF plasmid (bfpA-), have been designated

Haque et al. Journal of Nature and Natural Sciences. 2:2, 1-13, 2017. of 13

as ’atypical EPEC’ (Trabusi et al., 2002). Similarly,the eaeA- positive E. coli strains possess Shiga toxingenes (stx1 and/or stx2 ) are classified into enterohe-morrhagic E. coli (EHEC) pathogenic group bacteria.Usually, the identification of LEE and EAF harboringE. coli are mostly done by eaeA gene probe and EAFprobe, respectively (Nataro and Kaper, 1998). It hasbeen reported that the EAF plasmid can be lost duringstorage or even during infection ( Levine et al., 1985),but the absence of this plasmid in certain serotypes(i.e., O111:H9 and O26:H11) seems to be a natural oc-currence and distinguished by electrophoretic types orclones.For infection, EPEC strains adhere to the enterocyte

and produce a characteristic "attaching and effacing"(A/E) lesions in the brush border membrane of thehost (Giron et al., 1991; Hicks et al., 1998). In addition,other adherence factors such as, the bundle forming pilistarts adherence by serving as a long-range adhesion,since initial adherence helps to bring EPEC bacteriain close contact with the host cell (Giron et al., 1991;Hicks et al., 1998). The chromosomal genes are presentin a 35 kb pathogenicity LEE island and facilitates theactin condensation (McDaniel et al., 1995; McDanieland Kaper, 1991), which leads to localized elevationand invagination of the epithelial cell plasma mem-brane (i.e. pedestal formation), and represents a his-tological characteristic of EPEC infection in children.Earlier studies have reported that intimate attachmentof EPEC strain is facilitated by a 94 kDa outer mem-brane intimin protein (encoded by eaeA gene) thatbinds to a 90 kDa translocated intimin receptor protein(Tir), present in the host cell (Jerse and Kaper, 1991;Kenny et al., 1997). The Tir protein is translocated bythe EPEC cell into the host membrane and there itacts as a receptor for outer membrane intimin protein(Jerse and Kaper, 1991; Kenny et al., 1997; Rosenshineet al., 1996). Frankel et al., (1996) reported that puri-fied intimin also binds to β1 integrins, which indicatesthat intimin may be binding more than one receptoron the epithelial cell (Freankel et al., 1996). Basically,integrins are located on the apical surface of micro-fold cells found in the Peyer’s patches along the in-testinal lumen, not on the apical surface of enterocytes(Sanger et al., 1996). Typically, the intimin polypep-tides are classified into four serological distinct typesand called as intimin α, β, δ, λ and a non-typable group(Adu-Bobie et al., 1998). The attachment of EPEC tothe enterocyte follows the signaling events of bacteria-to-cell communication and triggers actin condensation,which ultimately results into cellular dysfunction lead-ing to occurrence of diarrhea (Finlay et al., 1992).Gomez-Duarte and Kaper (1995), reported that thechromosomal genes (including eaeA) of EPEC present

on the LEE region are implanted/inserted into the ex-act chromosomal site where a block of virulence genesof uropathogenic E. coli (UPEC) is present (Gomez-Duarte and Kaper, 1995; McDaniel et al., 1995). Inter-estingly, this entire region is of great importance be-cause it is highly conserved among the A/E pathogens,but it is absent from bacteria that do not produce A/Elesions. It is possible that LEE region were introducedinto EPEC bacteria by some mobile genetic elements.Many PCR based detection assays have been de-

veloped in the past few years for the rapid identifi-cation of pathogenic E. coli (Kaper et al., 2004). Incase of pathogenic E. coli, most of the PCR primershave been designed for the precise amplification ofvirulence genes, including haemolysin gene (hlyA),E. coli attaching-effacing intimin gene (eaeA), andgenes encoding for Shiga-like toxin I or II (stx1 andstx2 ). In the recent years, RAPD has been fully estab-lished and employed for the characterization of bac-teria for their classifications, groupings, identificationand even placement on the level of strains (Novo etal., 1996). Besides, RAPD is grounded upon a verysensitive and reproducible technique of PCR. Basedupon above, it can be conceived that RAPD-PCR rep-resents an alternative approach for the molecular typ-ing and probe designing and that no prior DNA se-quence is needed. RAPD-PCR methodology may leadto the identification of a unique and exclusive frag-ment among the isolates tested. The nucleotide se-quence of this unique fragment must be determined ifa probe is designed by using this fragment. Kobayashiet al., (2000) reported that the prevalence of EPECin children in Londrina-PR, Brazil, was evaluated bymeans of digoxigenin-labeled DNA probes which iden-tify the plasmid responsible for EPEC adherence fac-tor (EAF), and virulence genes for EPEC as bundle-forming pilus (bfp) and E. coli attaching-effacing fac-tor (eaeA) (Kobayashi et al., 2000). Even after greatsophistication and high automation of different bio-logical techniques, still the RAPD technique is greatlyfamous among scientists, as proved through recent ar-ticles (AL-Haj et al., 2008a; AL-Haj et al., 2008b).Indian isolates that have been phenotypically char-acterized by our group for antimicrobial resistancewere genotypically characterized using RAPD analy-sis targeting eaeA gene (Ali et al., 2008). During thisstudy we have reported the different EPEC strains,isolated from stool samples of diarrheal patients ofnorthern India, distinguished by RAPD analysis fol-lowing the eaeA detection to confirm the presence ofLEE pathogenicity island among selected isolates. ThisRAPD-eaeA marker can be utilized as a strong molec-ular tool for the rapid identification of EPEC isolates.


Based on the findings of this PCR based rapid identi-fication of diarrheagenic E. coli from north Indian iso-lates, the applicability of RAPD-eaeA technique hasbeen proved for the characterization of different iso-lates of EPEC causing diarrhea in infants.

2 MATERIALS AND METHODS2.1 Bacterial strainsA total of 18 clinical bacterial strains were collectedfrom stool samples of patients admitted to the Pedi-atrics Department of All India Institute of Medical Sci-ences (AIIMS), New Delhi, India. A non-clinical, envi-ronmental E. coli, un/non-serotyped isolate (ZQA6)from Yamuna river was used as a negative controlfor the study. All the strains were already availablein author’s lab (Ali et al., 2008). Strains were storedat -70oC in 50% Luria Bertani (LB) broth - 50%(v/v) glycerol and grown on LB agar plates or in LBbroth with ampicillin (200 µg/mL). Gram staining wasperformed for the identification of bacterial strains;and all Gram-negative bacilli were subjected to var-ious biochemical tests performed through HiAssortedBiochemical test kit (HiMedia, Mumbai-India). Theclinical E. coli isolates were confirmed and serotypedthrough Latex Agglutination test kit (HiLatex kit,HiMedia, Mumbai-India). The clinical isolates wereserotyped at AIIMS, New Delhi and after serotyp-ing of E. coli clinical strains, only EPEC serotypeswere utilized for further study. The details of differ-ent serotypes of EPEC strains used in this study weregiven into Table 1 (Ali et al., 2008). During amplifi-cation, restriction and southern blotting studies a wellcharacterized EPEC positive strain (gift by Dr. M. K.Bhan) was also included.

2.2 DNA preparationPlasmid DNA was prepared by the method of Birn-boim and Dolly (1979), and purified by extractingtwice with equal volumes of phenol chloroform (1:1)followed by washing with chloroform. The purifiedDNA was precipitated by 5M ammonium acetate andchilled absolute alcohol. Afterwards, the contents wereincubated on ice for 10 min and precipitated by cen-trifugation at 12,000 rpm for 30 min. The collectedpellet was washed with 70% ethanol, air dried andsuspended in TE buffer. The concentration of theDNA was checked by observing the ratio O.D 260/280following the method described by Sambrook et al.,(2001). and lastly the DNA was electrophoresed on0.8% agarose gel in TBE buffer. Bacterial genomicDNA was extracted using ROSE (Rapid One Step Ex-traction) method of Steiner et al., (1995), and purifiedand estimated as mentioned earlier.

2.3 PCR amplification and elution of the DNA bandfrom agarose gel

The eaeA gene from each clinical isolate was amplifiedby Polymerase Chain Reaction using eaeA specific syn-thetic sense (ZAJ1) oligonucleotide primer having thesequence 5/ (ATGATTACTCATGGTTGTTATACC-CGG) 3/ and an antisense (ZAJ2) primer with se-quence as 5/ (TTCGCTTTCGGAACTGTATAAAT-GTTT) 3/. The primers capable of amplifying an in-ternal 300 bp region of the eaeA gene encoding themature 94 kD outer membrane intimin protein weredesigned based on the already published intimin genesequence by Beebalkhee et al., (1992). The PCR reac-tion mixture was prepared (50 ng of template DNA,10 mM each of dNTPs, 25 mM MgCl2, 20 pmol eachof forward and reverse primers, 1X buffer and 1U ofTaq DNA polymerase) and total volume of the reac-tion mixture was made 25 µL by adding sterile water.The DNA was amplified for 29 cycles after an initialdenaturation at 94oC for 1 min. Each cycle consisted of30 s of denaturation at 94oC, 32 s of annealing at 68oCand 1 min of extension at 72oC. Final extension of 15min was given at 72oC. The amplified product was vi-sualized on a 2% agarose gel. Molecular size marker(Lambda/ HindIII) was included in each gel. For theelution of DNA from agarose, the band of interest wasexcised, minced and transferred to a microfuge tube.It was suspended in sufficient quantity of equilibratedphenol and incubated at -70oC for 1 h. The frozen mixwas immediately centrifuged at 12,000 rpm for 10 minat 4oC. The aqueous phase containing the eluted DNAwas extracted twice with an equal volume of chloro-form. The total DNA was precipitated by addition of2.5 volumes of pre-chilled ethanol and overnight incu-bation at -20oC. The DNA was rinsed once with 70%ethanol, dried and suspended in TE buffer.

2.4 RAPD analysisFifteen primers of 10 bases each (OPERON technolo-gies, Inc. Alameda, CA) were used in this study (Table2). PCR was carried out in a total volume of 25 µL con-taining 10 ng of E. coli genomic DNA, 3 mMMgCl2, 20pmoles of primer, 1U of Taq DNA Polymerase, 250 µMeach of dCTP, dGTP, dATP and dTTP in 10 mM TrisHCl pH 8.3, 50 mM KCl. Amplification was carried outon Techne thermal cycler with cycling program of 40cycles of 94oC, 1 min; 37oC, 1 min; 72oC, 1 min; andfinal extension of 72oC for 6 min. Amplified productswere elecrophoresed on 1.2% agarose gel with 100 bpDNA ladder and Lambda/HindIII (MBI, Fermentas)marker.

2.5 Scoring and data analysisEach amplification product was scored as present (1)or absent (0) across all the lanes. Molecular weights


of the bands were estimated by using 100 bp DNAladder and LambdaHindIII (MBI, Fermentas) as stan-dards. The two-way data matrix of strains x ampliconswas used to calculate pair-wise similarity coefficientsas described by Jaccard (1908). This matrix of sim-ilarity coefficients was subjected to Unweighed Pair-Group Method of Arithmetic Averages (UPGMA) togenerate a dendrogram using average linkage proce-dure. All the numerical taxonomic statistical analyseswere performed using the computer program NTSYS-PC (Numerical Taxonomy and Multivariate AnalysisSystem), version 1.80 (Exter Software, New York).

2.6 Southern blottingThe purified genomic DNA was digested with HindIIIrestriction endonuclease. In a typical digestion reac-tion, about 15 - 25 µg of genomic DNA was digestedwith 80 - 150 U of the enzyme and incubated for 12- 18 h at 37oC. The digestion reaction was stoppedby heat-inactivation of the enzyme, and digested DNAwas analyzed on 0.8 % agarose gel. The gel was stainedwith ethidium bromide (5 µg/mL) for 10 min and pho-tographed. For blotting purpose the digested genomicDNA, plasmid DNA and the RAPD amplicons fromtheir respective agarose gels were transferred sepa-rately on to the nylon membranes by Southern transfertechnique. The genomic DNA gels and the RAPD am-plicon gels were depurinated with 0.25 N HCl at roomtemperature for 45 min. Then the gels were transferredto denaturation solution containing 1.5 M NaCl and0.5 M NaOH for 30 min at room temperature. Theplasmid DNA gel was directly kept in the denaturingsolution. Again the gels were neutralized by treatingwith neutralization solution [1 M Tris HCl (pH 8.0)and 1.5 M NaCl] for 45 min.

2.6.1 Nucleic acid blottingThe denatured gels were washed with 1XSSC (10XSSC solution: 1.5 M NaCl, 0.15 M Sodium citrate, pHwas adjusted at 7.5 with 10 N NaOH and stored at4oC) and placed on 3 MM Whatmann paper dippedin 6X SSC. The same size 6X SSC soaked nylon mem-brane was placed on top of the each gel. Few pieces of6X SSC soaked 3 MM Whatmann papers were placedon nylon membrane followed by stack of 3 - 4 inchespaper towels for each gel. Finally, a 500 g glass platewas kept on the top of each gel and left for 18 h. Aftercompletion, nylon membranes were removed and gelswere seen under UV to make sure that the DNA hasbeen transferred. The nylon membranes were washedwith 1X SSC, dried and baked at 80oC for 2 h. Andmembranes were kept at room temperature till furtheruse.

2.6.2 Preparation of probe and hybridizationThe 300-bp PCR amplified DNA fragment probe wasradiolabeled with [λ32P] by using nick translation kit(Sigma). The reaction was set up by taking 2 µL (500ng/µL) of sample DNA, 10 µL of dNTPs, 5 µL of10X nick translation buffer, 7 µL [α32P] dCTP (10mCi/mL), 5 µL nick translation enzyme and nucleasefree water in a 50 µL reaction mix. The reaction mixwas incubated at 15oC for 1 h and at the end µÎĳL ofnick translation stop mix was added. The radiolabeledDNA probe was purified by column chromatography toremove the unincorporated nucleotides. A sterile 2 mLsyringe plugged at the lower end with siliconized glasswool was filled with equilibrated Sephadex G-50. Thenick translated reaction mixture was diluted to 500µL with TE buffer and loaded on the column. It waswashed with TE and 500 µL fractions were collected.The fractions of first peak were mixed and total ra-dioactivity of the probe was calculated. For hybridiza-tion purpose the blots were kept in pre-hybridizationbuffer (10X SSC, 87.65 gm/L of NaCl, 44.1 gm/L ofSodium citrate, 0.5 % of SDS, pH was adjusted at7.0 with 10 N NaOH and autoclaved) at 65oC for 4-6h in a hybridization oven (Techne). After that blotswere kept under hybridization buffer [6X SSC, 0.5%SDS, (α32p) dCTP labeled probe (1X107 cpm/mL),5X Denhardt’s solution (Ficoll 0.1 g, Poly vinyl py-rollidone 0.1 g, BSA 0.1 mg/mL, denatured salmonsperm DNA 100 µg/mL)]. The filters were kept at65oC overnight. Finally, the probe was removed andthe blots were washed twice with 2X SSC buffer con-taining 0.5% SDS at 65oC for about 15 min each. Theblots were then autoradiographed with X-ray films.

2.7 Isolation of outer membrane proteins (OMPs) andimmunoblotting

Bacterial strains were grown overnight in LB, diluted(1:100) in DMEM, and grown with continuous shak-ing at 37oC for an additional 3 h. The bacterial pel-let was collected and washed with 10 mM Tris HCl(pH 7.5). About 1 g wet weight of the bacterial pel-let was extracted with 20 mL extracting buffer [10mM Tris HCl (pH 7.5), 6 M Urea] at 4oC and after1 h it was subjected to dialysis against distilled wa-ter for 72 h. Afterwards, the dialyzed material wascentrifuged at 8,000 rpm for 1 h at 4oC and super-natant was collected, checked on 12% SDS-PAGE fol-lowing the method of sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE) given byLaemmli (1970) and staining with Coomassie brilliantblue R-250. The sample was lyophilized and storedat -20oC. Following SDS-PAGE, the proteins weretransferred to nitrocellulose membrane (Amersham)by the method of Towbin et al., (1979). Membrane


was blocked overnight with 3% bovine serum albu-min (BSA) in phosphate-buffer saline (PBS) havingpH of 7.2, washed with PBS containing 0.05% Tween-20 (PBST), and finally probed with anti-intimin an-tisera (α and β, courtesy Gad Frankel, UK), diluted1:750 in 0.1% BSA-PBST, for 2 h at room temper-ature. Membrane was washed thrice with PBST, in-cubated with horseradish peroxidase (HRP) - conju-gated anti-rabbit immunoglobulin (Sigma) in 1:1,000dilutions for 2 h and the reaction was visualized withsolution containing O’Phenylenediamine dihydrochlo-ride and 30% H2O2.

3 Results3.1 DNA extraction and primer specific amplification of

eaeA geneThe plasmid profiles of all the pathogenic bacteriumincluding non-pathogenic negative control strain (ZQA6)were found similar as our research group reported ear-lier (Ali et al., 1998). Using specific primers ZAJ1 andZAJ2, we were able to get an expected band of 300-bpof eaeA gene coding for an outer membrane protein,intimin in all the bacterial isolates including positivestrain except the non-pathogenic environmental E. coliisolate which was used as negative control during thestudy (Fig. 1).

3.2 Southern blot analysisIn order to check the presence of pathogenicity islandLEE, and to confirm the amplification of the righteaeA gene sequence in EPEC strains, the amplifiedproduct was further radiolabeled and hybridized to theHindIII-digested genomic DNA of all the five selectedstrains along with the positive and negative control(environmental E. coli) (Fig. 2 and Fig. 3). All theEPEC strains including positive strain produced pos-itive signal, whereas, the non-pathogenic environmen-tal E. coli strain, ZQA6, had not not given any signalof hybridization (Fig. 3). The results obtained thusconfirmed the amplification of right sequence in thoseselected EPEC strains. However, the RAPD ampliconsas well as the plasmid DNA were unable to show anysign of hybridization.

3.3 RAPD analysisOut of 15 decamer primers (Table 2) used in thisstudy, good and consistent profiles were obtained with5 primers only, named as OPB-01, OPB-12, OPB-14,OPC-16 and OPC-17. It was interesting to note thatall these primers gave about 90% polymorphism (Fig.4). Number of polymorphic products ranged from 18 to28. Out of the total 128 amplification products formed,111 were found to be polymorphic with respect to each

other and indicated a varied divergence of these iso-lates from each other at DNA level. Thus, suggestingthem to be different strains. It has been reported thattwo or more band difference in RAPD should be takenas the basis for labeling strains as different (Power,1996), considering the existence of 90% polymorphismamong the E. coli isolates studied we considered themas six different strains of E. coli. Therefore this tech-nique can be used as a strong genetic tool to differen-tiate strains of bacteria at molecular levels.

3.4 DendrogramThe RAPD profiles of the genomic DNA generatedby the five-decamer primers were used to constructa dendrogram by NTSYS-PC program and the Jac-card’s similarity co-efficient values were calculatedwhich ranged between 0.143 and 0.543. The mean simi-larity co-efficient was found to be 0.26. The closest sim-ilarity value was exhibited between ZQA5 and ZQA6,and the lowest similarity value was noticed betweenZQA1 and ZQA5 (Fig. 5). The non-pathogenic en-vironmental strain, ZQA6 showed highest homologywith ZQA5 at a distance of 0.54 but it was quite dis-tant from other four pathogenic strains at a distanceof 0.16, 0.29, 0.23, and 0.19 with respect to ZQA1,ZQA2, ZQA3 and ZQA4.

3.5 Analysis of OMPs and immunoblottingThe OMPs profile of all the strains on SDS-PAGEshowed (Fig. 6a) that there were more than 10 pro-teins clearly present corresponding to molecular weightrange of greater than 20 to less than 100 kD. Interest-ingly, at the same time predominance of low molecularweight proteins (>20 kD) was noticed in all the strains(Fig. 6a) with distinct variation in the OMPs profile ofthe pathogenic and the non-pathogenic strain. A clearband of 94 kD protein proved the presence of intiminprotein in all the EPEC pathogenic strains which wasabsent in non-pathogenic negative control. In addition,immunoblot analysis proved that the 94 kD band is ofintimin protein (Fig. 6b). However, the proteins iso-lated from the strains ZQA2, ZQA3, ZQA4 and ZQA5strongly reacted only with intimin antibodies, whereas,in case of strains ZQA1 and ZQA6, there was no pos-itive signal observed, neither with nor with antibodies(Fig. 6b).

4 DiscussionTill now, a number of PCR based bacterial identifica-tion assays have been developed for the detection ofpathogenic E. coli (Kaper et al., 2004), and most ofthe PCR primers were designed for the specific ampli-fication of virulence genes, including genes encodingShiga-like toxin I or II (stx1 and stx2), haemolysin


(hlyA) and intimin (AL-Haj et al., 2007a; AL-Hajet al., 2007b; Ellingson et al., 2005; Willshaw et al.,1994). Earlier studies have already proved that RAPDis a powerful tool for genetic studies (Williams et al.,1990), and widely used to investigate variability amongmicroorganisms (Welsh and McClelland, 1990). So, forthe rapid screening of isolates of various medically im-portant bacterial species and others, a single RAPD-PCR assay can be used as a primary typing screen forgenetic relatedness (Welsh and McClelland, 1990). Inour study, the results of RAPD-PCR showed variablepatterns with different strains as per their serotypes.We were successful in getting a good and consistentprofile of RAPD and about 90% polymorphism wasobserved among the isolates (Fig. 4). Out of 128 am-plified products visualized as bands, 111 were foundto be polymorphic which indicated a varied divergenceof these isolates from each other at DNA level. Thus,suggesting them to be different strains. It has beenreported that two or more band difference in RAPDshould be taken as the basis for labeling strains as dif-ferent (Power, 1996), considering the existence of 90%polymorphism among the E. coli isolates included inour study; we considered them as six different strainsof E. coli. Therefore this technique can be used as astrong genetic tool to differentiate the strains of bacte-ria at molecular level. A phylogenetic tree constructedalso showed the variation of strains from each otherat molecular level. Some of the strains showed closesimilarity with each other, though, two of the strainsnamed ZQA1 and ZQA5 showed remarkable variationwith respect to each other (Fig. 5), therefore indicat-ing the genetic drifting of above strains from eachother. Also, the phylogenetic tree showed the signif-icant difference between the pathogenic and the non-pathogenic strains, and predicted the genetic driftingof these strains from each other. But, one pathogenicstrain (ZQA5) showed close similarity with the non-pathogenic strain (ZQA6), hence it can be speculatedthat the former (ZQA5) to be a modem version ofthe ZQA6 strain by recently acquiring its virulent na-ture. In this study emphasis was laid on using a DNAprobe which relies on a proven virulence factor forthe pathogenesis of EPEC strains rather than a DNAprobe which comprises sequences of undetermined im-portance. The identification of EPEC by agglutinationwith antisera is difficult to perform correctly, requiressuperior reagents and technical experience, and at thesame time it is available in limited central referencelaboratories only, hence warrants for more sophisti-cated identification test (Nataro et al., 1998). Adhe-sion to tissue culture cells has proved to be a usefultest for many EPEC and so has a gene probe for EAF,which is plasmid encoded (Nataro et al., 1998). How-ever, not all EPEC strains adhere to tissue culture cells

or possess EAF plasmids (Ali et al., 1998). Likewise,Willshaw et al., (1994) reported the hybridization ofstrains of E. coli O157 with probes derived from theeaeA gene of EPEC and eaeA homolog from a Verocytotoxin-producing strain of E. coli O157. Therefore,eaeA gene, which encodes for a outer membrane pro-tein, intimin (94 kDa) is responsible for the intimateattachment of the bacterium with the host was am-plified by PCR and used as a DNA probe (Jerse andKaper, 1991). Our hybridization results clearly showedthat all the strains with the exception of the controlnon-pathogenic environmental E. coli strain (ZQA6),hybridized with the probe and thus showing that eaeAgene was present in all the strains (Fig. 3). On basis ofthe current findings it can also be concluded that thePCR amplified fragment of the eaeA gene can be usedas a DNA probe for the fast detection of the epidemi-ologically important pathogenic E. coli strains. Henceit can be suggested that the presence of eaeA gene onthe chromosomal DNA of these E. coli strains confirmtheir virulence and thus pathogenicity. However, it wasinteresting to note that no sign of hybridization wasobserved in RAPD amplicons as well as with plasmidDNA (data not shown) thus suggesting the absence ofeaeA gene from plasmids and non-amplification of thisregion of eaeA gene while using RAPD technique. Ad-ditional experiments were also performed to confirmthe RAPD-eaeA results and utility for the discrimi-nation of protein profiles of the selected isolates andexpression of intimin protein among the pathogenicstrains. As reported earlier that protein profiling bySDS-PAGE is also a very reliable and reproduciblemolecular method which has been used by many work-ers to type various microorganisms of epidemiologic in-terest. This technique gives high resolution, 100% ty-peability, high reproducibility and high discriminatorypower (Costas et al., 1994; Tabaqchali et al., 1986), andhigh resolution SDS-PAGE of bacterial proteins canbe utilized for identification at the species, subspeciesand intra-species levels (Jackman, 1985). During thisstudy, outer membrane protein intimin expressed byenteric bacterial pathogens capable of inducing intesti-nal A/E lesions have been used to type the differentserotypes of the isolated strains. Different types of in-timins designated as and a non-typable have been de-tected by Adu-Bobie et al., (1998) and Pelayo et al.,(1999) by PCR and immunoblotting. EPEC serotypesO142: H34, O127:H6 and O55:H6 express intimin-while as O128:H2, O119:H2, O119:H6, O111:H2, O111:H-, O26: H- and O26:H11 express intimin-. The otherintimins- and are expressed by O55: H- and O157: H7and O86: H34 serotypes, respectively. The other EPECserotypes O127: H40, O86: H34, O55:H7 and O55: H-do not react or react very faintly with any of the


intimins, hence they are designated as non-typeable(NT). While studying the OMPs, more than 10 pro-teins were observed in all the strains. A clear band of94 kD suggested the protein to be intimin as it wasabsent in non-pathogenic strain, ZQA6. Here in thisstudy, high resolution SDS-PAGE was performed forall of the five serotypes of EPEC, including positivestrain (data not shown here) and one non-EPEC strainusing two anti-intimins designated as and . The strainsbelonging to O142: H6 (ZQA2), O127: H6 (ZQA3),O55: H6 (ZQA4) and O126: H6 (ZQA5) including wellcharacterized positive strain (data not shown) reactedvery well with only with -intimin but not at all with the-intimin. However, serotype ZQA1 (O86: H34) and thenon-pathogenic strain, ZQA6, were not able to reactwith any of the intimins used in this study. The rea-son behind this may be that the former expresses theother or non-typeable intimin while as the later doesnot expresses the intimin protein at all. Hence, it canbe suggested that the 94 kD protein present in ZQA1is not intimin, but some other protein as confirmedby immunoblotting. The outcomes of immunoblottingshowed that due to the presence of intimin protein, thestrains were showing their pathogenic nature. Since in-timin is highly immunogenic, hence, it might be animportant component to be considered in the devel-opment of EPEC vaccine in near future. The overallobservations recorded in this investigation provide ev-idence that RAPD is an informative and simple toolthat can be applied for epidemiological studies. Aswe are aware that RAPD markers may be used as aquick and reliable alternative for establishing differ-ences among different species as well as among strainsof the same species. Many reports have already beenpublished on usage of eaeA gene as a marker gene forthe detection of diarrheagenic E. coli, but this is thefirst study, in which, we performed a series of RAPD-PCR in conjunction with eaeA amplification assays ofE. coli isolates from clinical diarrheal samples collectedfrom north Indian patients and the outcomes were en-dorsed by expressed immunogenic proteins. The keyfinding of this study was the generation of RAPD fin-gerprint of the isolates using 15 decamer primers andamplification of one common fragment of eaeA genewith 300 bp length in all the EPEC strains, and possi-ble utilization of this RAPD-eaeA marker as a strongmolecular probe for the rapid identification of EPECisolates in near future. Although, a less number of sam-ples were taken during RAPD analysis, but, this is avery preliminary step towards the long term goal ofdevelopment of rapid molecular detection method forEPEC. In a hospital setting, this type of amplifica-tion based test can be used for the timely detectionof ongoing nosocomial outbreaks. Currently our group

is aggressively working on various clinical studies re-lated to enteropathogenic and enterohemorrhagic E.coli and trying to develop novel molecular diagnostictools for the fast detection and categorization of dif-ferent diarrheagenic E. coli.

5 AcknowledgmentThe authors are grateful to Jamia Millia Islamia (ACentral University), New Delhi, India for providing thenecessary laboratory facilities for this study. Also, wethank Pediatrics Department of All India Institute ofMedical Sciences (AIIMS), New Delhi, India for theircooperation in providing the samples for this study.

6 Statement of conflicts of interestThe authors declare that they have no conflict of in-terests.

Author details1 Gene Expression Laboratory, Department of Biosciences, Faculty ofNatural Sciences, Jamia Millia Islamia (A Central University), New Delhi,India. 2.

References

1 Adu-Bobie, J., Frankel, G., Bain, C., et al., 1998. Detection ofintimins α, β, λ, andδ, four intimin derivatives expressed byattaching and effacing microbial pathogens. J. Clin. Microbiol. 36(3),662-668.

2 AL-Haj, A.N., Mariana, N.S., Raha, A.R., Ishak, Z., 2008a.Detection of diarrheagenic Escherichia coli isolated using molecularapproaches. J. Res. Microbiol. 3(5), 359-367.

3 AL-Haj, A.N., Mariana, N.S., Raha, A.R., Zamri, I., 2008b.Development of a PCR primer and a marker band detection of E. colifrom various sources based on arbitrary primer set. Res. J. Biol. Sci.3(11), 1327-1332.

4 AL-Haj, A.N., Mariana, N.S., Raha, A.R., Zamri, I., 2007b.Development of polymerase chain reaction and dot blot hybridizationto detect Escherichia coli isolates from various sources. J. Res.Microbiol. 2(12), 926-932.

5 AL-Haj, A.N., Mariana, N.S., Raha, A.R., Zamri, I., 2007a.Simultaneously detection of Enteropathogenic E. coli and Shigatoxin-producing E. coli by Polymerase Chain Reaction. J. Appl. Sci.7(17), 2531-2534.

6 Ali, A., Afshan, A., Qamri, Z., 1998. Drug resistance in Indianisolates of enteropathogenic Escherichia coli. World J. Microbiol.Biotechnol. 24, 2633-2638.

7 Beebalkhee, G., Louie, M., de-Azavedo, J., Brunton, J., 1992.Cloning and nucleotide sequence of the eae gene homologoue fromenterohemorrhagic E. coli serotype O157:H7. FEMS Microbiol. Lett.91, 63-68.

8 Birnboim, H., Dolly, S., 1979. A rapid alkaline extraction procedurefor screening recombinant Plasmid DNA. Nucleic Acids Res. 7,1513-1517. doi: 10.1093/nar/7.6.1513

9 Costas, M., Holmes, B., On, S.L.W., Garner, M., Kelly, M.C., Nath,S.K., 1994. Investigation of an outbreak of Clostridium difficileinfection in a general hospital by numerical analysis of proteinpatterns by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. J. Clin. Microbiol. 32, 759-765.

10 Ellingson, L.E., Koziczkowski, J.J., Anderson, J.L., Carlson, S.A.,Sharma, V.K., 2005. Rapid PCR detection of enterohaemorrhagicEscherichia coli (EHEC) in bovine food products and feces. Mol. CellProbes 19, 213-217.

11 Finlay, B.B., Rosenshine, I., Donnenberg, M.S., Kaper, J.B., 1992.Cytoskeletal composition of attaching and effacing lesions associated


with enteropathogenic E. coli adherence to HeLa cells. Infect.Immun. 60, 2541-2543.

12 Frankel, G., Lider, O., Hershkonz, R., Mould, A.P., Kachalsky, S.G.,Candy, D.C.A., 1996. The cell-binding domain of intimin fromenteropathogenic Escherichia coli binds to -1 integrins. J. Biol.Chem. 271, 20359-20364.

13 Ghosh, P K, Arif Ali. 2010. Isolation of atypical enteropathogenicEscherichia coli from children with and without diarrhea in Delhi andNCR, India. J Med Microbiol 59, 1156-1162; DOI:10.1099/jmm.0.014530-0

14 Giron, J.A., Ho, A.S.Y., Schoolnik, G.K., 1991. An inducible bundleforming pilus of enteropathogenic E. coli. Science 254, 710-713.

15 Gomez-Duarte, O.G., Kaper, J.B., 1995. A plasmid encodedregulatory region activates chromosomal eaeA expression inenteropathogenic E. coli. Infect. Immun. 63, 1767-1776

16 Hicks, S., Frankel, G., Kaper, J.B., Dougan, G., Phillips, A.D., 1998.Role of intimin and bundle-forming pili in enteropathogenic E. coliadhesion to pediatric intestinal tissue in vitro. Infect. Immun. 66,1570-1578.

17 Jaccard, P., 1908. Nouvelles recherches sur la distribution florale.Bul. Soc. Vaudoise Sci. Nat. 44, 223-270.

18 Jackman, P.J.H., 1985. Bacterial taxonomy based on electrophoreticwhole-cell protein patterns. In Goodfellow M, Mnnikin DE (Eds.)Chemical methods in bacterial systematic. London Academic Press,pp 115.

19 Jerse, A.E., Kaper, J.B., 1991. The eae gene of enteropathogenic E.coli encodes a 94 kDa membrane protein, the expression of which isinfluenced by the EAF plasmid. Infect. Immun. 59, 4302-4309.

20 Kaper, J.B., Nataro, J.P., Mobley, H.L.T., 2004. PathogenicEscherichia coli. Natl. Rev. Microbiol. 2, 123-140.

21 Kenny, B., De-Vinney, R., Stein, M., Reinscheid, D.J., Frey, E.A.,Finlay, B.B., 1997. Enteropathogenic E. coli (EPEC) transfers itsreceptor for intimate adherence into mammalian cells. Cell 9,511-520.

22 Kobayashi, R.K.T., Saridakis, H.O., Dias, A.M.G., Vidotto, M.C.,2000. Molecular identification of enteropathogenic Escherichia coli(EPEC) associated with infant diarrhea in Londrina, Parana, Brazil.Bra. J. Microbiol. 31, 275-280.

23 Laemmli, U.K., 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage. Nature 227, 680-685.

24 Levine, M.M., Nataro, J.P., Karch, H., et al., 1985. The diarrhealresponse of humans to some classical serotype enteropathogenic E.coli is dependent on a plasmid encoding an enteroadhesivenessfactor. J. Infect. Dis.152, 550-559.

25 Levine, M.M., Xu, J.G., Kaper, J.B., et al., 1987. A DNA probe toidentify enterohemorrhagic E. coli O157:H7 and other serotypes thatcause hemorrhagic colitis and hemolytic uremic syndrome. J. Infect.Dis. 156, 175-182.

26 McDaniel, T.K., Jarvis, K.G., Donnenberg, M.S., Kaper, J.B., 1995.A genetic locus of enterocyte effacement conserved among diverseenterobacterial pathogens. Proc. Natl. Acad. Sci., USA 92,1664-1668.

27 McDaniel, T.K., Kaper, J.B., 1991. A cloned pathogenicity islandfrom enteropathogenic E. coli confers the attaching and effacingphenotype on E. coli K-12. Mol. Microbiol. 23, 399-407.

28 Nataro, J.P., Baldini, M.M., Kaper, J.B., Black, R.E., Bravo, N.,Levine, M.M., 1998. Detection of an adherence factor ofenteropathogenic E. coli with a DNA probe. J. Infect. Dis. 152,560-565.

29 Nataro, J.P., Kaper, J.B., 1998. Diarrheagenic E. coli. Clin.Microbiol. Rev. 11, 142-201.

30 Nougayrede, J.P., Fernandes, P.J., Donnenberg, M.S., 2003.Adhesion of Enteropathogenic E. coli to host cells. Cell Microbiol.5(6), 359-372.

31 Novo, M.T.M., De’sauza, A.P., Garcia, O., Ottoboni, L.M.M., 1996.RAPD genomic fingerprinting differentiates Thiobacillus ferrooxidansstrains. Syst. and Appl. Microbiol. 19, 91-95.

32 Pelayo, J.S., Scaltetsky, I.C.A., Pedroso, M.Z., et al., 1999. Virulenceproperties of atypical EPEC strains. J. Med. Microbiol. 48, 41-49.

33 Power, E.G., 1996. RAPD typing in microbiology - A technicalreview. J. Hosp. Infect. 33, 247-265.

34 Rosenshine, I., Rusckowski, S., Stein, M., Rienschield, D.J., Mills,S.D., Finlay, B.B., 1996. A pathogenic bacterium triggers epithelialsignals to form a functional bacterial receptor that mediates actinpseudopod formation. EMBO J. 15, 2613-2624.

35 Sambrook, J., Russell, D.W., 2001. Molecular cloning: a laboratorymanual 3rd Ed. Cold Spring Harbor, New York

36 Sanger, J.M., Chang, R., Ashton, F., Kaper, J.B., Sanger, J.W.,1996. Novel form of actin-based motility transport bacteria on thesurface of infected cells. Cell Motil. Cytoskeleton 34, 279-287.

37 Steiner, J.J., Poklemba, C.J., Fjellstrom, R.G., Elliot, L.F., 1995. Arapid one-tube genomic DNA extraction process for PCR and RAPDanalyses. Nucl. Acids. Res. 23, 2569-2570.

38 Tabaqchali, S., OâĂŹfarrell, S., Holland, D., Silman, R., 1986.Method for the typing of Colostridium difficile based on PAGE ofS35- methionine labeled proteins. J. Clin. Microbiol. 23, 197-198.

39 Towbin, H., Staehelint, T., Gordon, J., 1979. Electrophoretic transferof proteins from polyacrylamide gels to Nitrocellulose sheets:Procedure and some applications. Proc. Natl. Acad. Sci. USA 76(9),4350-4354.

40 Trabusi, L.R., Keller, R., Gomes, T.A., 2002. Typical and atypicalEnteropathogenic E. coli. Emerg. Infect. Dis. 8(5), 508-513.

41 Welsh, J., McClelland, M., 1990. Fingerprinting genome using PCRwith arbitrary primers. Nucl. Acids Res. 19, 303-306.

42 Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey,S.V., 1990. DNA polymorphisms amplified by arbitrary primers areuseful as genetic markers. Nucl. Acids Res. 18, 6531-6535.

43 Willshaw, G.A., Scotland, S.M., Smith, H.R., Cheasty, T., Thomas,A., Rowe, B., 1994. Hybridization of strains of Escherichia coli O157with probes derived from the eaeA gene of enteropathogenic E. coliand eaeA homolog from a Vero cytotoxin-producing strain of E. coliO157. J. Clin. Microbiol. 32, 897-902.

44 World Health Organization (WHO)/ United Nations Children’s Fund(UNICEF), 2009. Diarrhoea: Why children are still dying and whatcan be done.


Figure 1 Ethidium bromide stained agarose gel (1.2%) showing PCR amplification of 300 bp eaeA gene from selected isolates;Lane M: Molecular weight marker; Lanes 1 - 6: Amplified eaeA gene from positive control, isolates ZQA1, ZQA2, ZQA3, ZQA4and ZQA5; Lane 7: Negative control (Environmental E. coli, isolated from Yamuna river, New Delhi, India) ZQA6

Figure 2 DNA digested with HindIII restriction enzyme and resolved on 0.7% agarose gel for southern blot analysis from allselected (pathogenic and non-pathogenic) strains; and DNA smear available on the gel showing the restriction pattern of HindIIIfor different strains. Lane M: Molecular weight λ/ EcoRI + HindIII marker; Lanes 1 - 7: showing the digestion pattern ofpositive control, and selected strains ZQA1, ZQA2, ZQA3, ZQA4, ZQA5 and ZQA6


Figure 3 Southern blot analysis of genomic DNA of all the selected strains (pathogenic and non-pathogenic). Autoradiogramshowing hybridization of digested DNA from all the selected strains with [λ32] dCTP labelled DNA probe; Lane M: Molecularweight λ/ EcoRI + HindIII marker, Lanes 1 - 6: showing hybridization of radiolabeled probe with HindIII digested genomic DNAof positive control and selected strains ZQA1, ZQA2, ZQA3, ZQA4, ZQA5; Lane 6: No hybridization signal was noticed in caseof ZQA6

Figure 4 RAPD amplification of genomic DNA from different E. coli isolates with random decamer primers OPB-1, OPB-12,OPB-14, OPC-16 and OPC-17. Lane M1: Molecular weight λ/ HindIII marker; Lane M2: Molecular weight marker 100 bp DNAladder; Lanes 1, 7, 13, 20, 26: Isolate ZQA1; Lanes 2, 8, 14, 21, 27: Isolate ZQA2; Lanes 3, 9, 15, 22, 28: Isolate ZQA3; Lanes4, 10, 16, 23, 29: Isolate ZQA4; Lanes 5, 11, 17, 24, 30: Isolate ZQA5; Lanes 6. 12, 18, 25, 31: Isolate ZQA6


Figure 5 Jaccard’s similarity coefficient between different E. coli strains (UPGMA cluster dendrogram): UPGMA clusteringdendrogram indicating percentage similarity between the RAPD amplicons of E. coli isolates of ZQA1, ZQA2, ZQA3, ZQA4,ZQA5 and ZQA6

Figure 6 Coomassie blue stained 12% SDS PAGE showing outer membrane proteins (OMPs) of different E. coli isolates andidentical immunoblot probed with anti-intimin immunoglobulins. Lane M: Mid-range protein marker; Lane 1: ZQA1; Lane 2:ZQA2; Lane 3: ZQA3; Lane 4: ZQA4; Lane 5: ZQA5; Lane 6: ZQA6


Serotype Name used in this studyO86:H34 ZQA1O142:H6 ZQA2O127:H6 ZQA3O55:H6 ZQA4O126:H6 ZQA5Un or non-serotyped ZQA6

Table 1 Details of the strains used in this study

Primer (Reference) Sequence (5âĂŹ to 3âĂŹ) %G+C MW PicomolesOPA-01 CAGGCCCTTC 70 2955 6014OPA-04 AATCGGGCTG 60 3059 5088OPA-07 GAAACGGGTG 60 3108 4625OPA-20 GTTGCGATCC 60 3010 5654OPB-01 GTTTCGCTCC 60 2961 6363OPB-02 TGATCCCTGG 60 3010 5654OPB-12 CCTTGACGCA 60 2979 5531OPB-14 TCCGCTCTGG 70 2986 6159OPB-15 GGAGGGTGTT 60 3130 4988OPC-01 TTCGAGCCAG 60 3019 5300OPC-04 CCGCATCTAC 60 2939 5783OPC-08 TGGACCGGTG 70 3075 5265OPC-16 CACACTCCAG 60 2948 5413OPC-17 TTCCCCCCAG 70 2915 6313OPC-20 ACTTCGCCAC 60 2939 5783

Table 2 Fifteen primers tested for generation of RAPD profileswith purified genomic DNA from different of E. colistrains


How to cite this article:Haque, S., Qamri, Z., Ali, A., RAPD-eaeA marker for detection of Enteropathogenic Escherichiacoli from stool samples of north Indian patients, Journal of Nature and Natural Sciences, 2:2,1-13, 2017.

Journal of Nature and Natural SciencesISSN: 2456-9488Received: May 2017, Accepted: October 2017Published Online: December 2017DOI Prefix: 10.26859Published by: Barkat Ali Firaq Trust for Education and Research (BAFTER) (Regd.)

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