Molecular characterization of enterohemorrhagic E . coli O 157 and O 153 isolated from tissue camel and human stool samples in Al-Diwaniyah , Iraq

The present study aimed to describe the genetic relationships of zoonotic characterization of Escherichia coli isolated from human and livestock camel clinical infection. The study includes collected (50) meat of camel and (50) stool human samples. These samples were foreword to traditional bacterial isolation and identification using enrichment culture method and biochemical tests, then confirmed by PCR technique based on Gyr B gene Escherichia coli and DNA sequencing was done on some positive isolates. The results show that Escherichia coli were isolated from animals at 42 (84%) and 39 (78%) from human infection. The PCR technique was show highly sensitive and specific confirmative detection of Escherichia coli the positive results into 40 (95%) meat sample of camel, and 35 (89.7%) stool sample of a human. To evaluate of Virulence E.coli,we used specific virulence hlyA gene from NCBI-GenBank, published sequence of E. coli hly A gene (Genbank code: X94129.1) and the results show high of presence of virulence gene hly A in camel in percentage (19) 45% than of virulence gene in human (15) 38%. DNA sequencing of a partial sequence of GyrB gene was shown highly homology sequence identity with NCBI-Blast Escherichia coli strain O157H7 isolates from human and O153H3 from the camel. The phylogenetic analysis was shown there is clear genetic similarity at between human and animal’s E. coli isolates and then the gene sequence deposited into NCBI-Genbank accession numbers (MG560867.1, MG560866.1). Also, study design for detection of some virulence gene hly A Escherichia coli. In conclusion, there prevalence E. coli in humans and camel. Therefore, it is essential to define the role of animals as an important source for the distribution of pathogen related to public health. Our study found gyrB gene sequence could be used for identification and making a phylogenetic analysis of gyrB nucleotide. Keyword: E. coli PCR O153H2, O157H7, gyr B, hly A, sequencing and phylogenetic tree Available online at http://www.vetmedmosul.com ةيفزنلا ةينولوقلا ايشيرشيلاا ايرتكبل يئيزجلا فيصوتلا O157 و O153 ةلوزعملا نم لامجلا موحل ةيناويدلا يف ناسنلإا زارب تانيعو ، قارعلا فيلك حلاف ابص ١ ، حلاص داؤف ةنيز ١ ، نيسح بلاط قاتشم ٢ ، كلا دبع ءلاع داوج مظا ١ و داوج يربص محمد ٣ ١ ةكرتشملا ضارملأاو ثوحبلا ةدحو ، ٢ ةيرطيبلا ةيرهجملا ءايحلأا عرف ، ٣ ةجسنلأاو حيرشتلا عرف ، يرطيبلا بطلا ةيلك ، ةيسداقلا ةعماج ، ةيناويدلا ، قارعلا

. The four linked genes hly C, A, B, D products are required for the production of ὰ-hemolysin inactive form (3).
Hly C protein plays an important role in activation of inactive ὰ-hemolysin polypeptides by catalysis add of the fatty acid group, hemolysin of E. coli is the main virulence factor for some strains of the pathogenic bacteria involved in human and animals extraintestinal diseases like urinary tract infections, peritonitis, meningitis and septicemia (4).ὰ-hemolysin secretion is signal peptide-independent and interpose by determinant membranous translocated protocol encoded by hly B, D (5).Enterohemolysin is another hemolysin closely related with E. coli serotype O157, O26, O 111.E. coli O 157:H7 strains caused several types of diseases as hemorrhagic colitis.Cytolysin has activity in several cells of the blood and renal tubular cell (6).Hemolysin owes its name to the fact that it causes hemolysis or lysis of red blood cells, and this is indeed the basis for its most commonly used assay.(The prefix a, that is often omitted nowadays, indicated the extracellular form of the toxin originally).In spite of its name, ὰ-hemolysin is widely believed to act mainly by attacking the immune system cells of the host, usually without inducing cell lysis, yet severely impairing their function (7).
HlyA gene composed of a long chain of polypeptides (1024 amino acids) with 31 and 32 is some carbohydrates which are associated.The polypeptide activity damage membrane directly, since changes as mutations as one or two amino acids changes in specific positions render the inactive of protein (8).The gyr B gene is a coding protein called DNA gyrase, a type II DNA topoisomerase.The protein has a great role in DNA multiplication in the bacteria,Sequences of the gyrB gene used in phylogenetic reports of Salmonella spp, Shigella and E. coli (9).Our study found gyrB gene sequence could be used for identification and making a phylogenetic analysis of gyrB nucleotide, this study aimed to isolate and characterize EHEC strains from animal tissue and stool samples to elucidate the extent of food contaminated by camel tissue in Iraq.

Samples collection
One hundred samples divided into fifty tissues (meat) of camel were collected from slaughterhouse of Al-Diwaniyah city, and fifty samples of human suffered from diarrhea in different sites of Al-Qadisyah province.These samples were placed in a cold container and transported to bacteriology Lab, in Veterinary Medicine College for isolationi and definition of E. coli.

Bacterial isolation
E. coli was isolated from stool samples and many of the tissues by culture on Brain Heart Infusion Broth (BHIB) agar for overnight at 37°C. then the bacterial growth were cultured on MacConkey media, Eosin methylene blue (EMB) media, blood agar, and orientation-CHROM agar for overnight for preparation pure E. coli colony.Biochemical identification tests (IMViC and TSI) were also used for more detection of Escherichia coli isolates agreement with (10).
DNA extraction of bacteria genome E. coli genome was extracted by using a special kit called (PrestoTM Mini gDNA Bacteria Kit.Gene aid. the USA) however, the extraction included 1ml of the new bacterial colony from nutrient broth media and put it in 1.5 ml microcentrifuge tubes.The centrifuge used to run the tubes for one minute at (10000).After centrifuge finished, the supernatant is produced then it removed.The Nanodrop spectrophotometer was used to testing the concentration DNA and keep in the freezer at -20°C.

Polymerase chain reaction (PCR)
The specific primer was used in PCR assay to detect E. coli the housekeeping gene gyrB, F (GAA GTC ATC ATG ACC GTT CTG CA) and R (AGC AGG GTA CGG ATG TGC GAG CC) (11).With product size 1256 bp and specific virulence gene of hemolysin toxin (hlyA).E. coli hlyA-F forward primer (GGAGTTAGTGCAGCCTCCAG) and hlyA-R reverse primer (ACCACTCTGACTGCGATCAG) 360bp.In NCBI-GenBank website used for primers designing under code (Genbank code: X94129.1)through use primer 3 plus design online.Bioneer Company provided the primers at a 360 bp.PCR premix tube consist of Taq DNA polymerase 1U, dNTPs 250 μM, Trisi-HCl (pH 9.0) 10mM, KCl 30 mM, MgCl2 1.5 mM, stabilizer, and tracking dye).PCR reaction was acted by added 5μl of purified genomic DNA and 1.5 μl of 10pmole of forwarding primer and 1.5 μl of 10 pmole of reverse primer to PCR premix tube and complete the volume into the 20 μl total volume by deionizer PCR water according to kit instructions.Briefly, it mixed by Exispini vortex centrifuge (Bioneer.Korea).The PCR thermocycler apparatus (Mygene Bioneer.Korea) used to make a reaction.The reaction consists of 30 cycles.The reaction consists of four stages included the first stage is DNA denaturation for one minute at 94°C, the second stage is primer annealing for one minute at 60°C, and the third stage is primer extension for one minute at 72°C.The last stage is extension stage for ten minutes at 72 °C for gre gene.While in hly A gene was 95°C in the initial denaturation for five minutes, next 30 cycles at 95°C as denaturation for 30 second, annealing for thirteen second at 58°C, and extension stage at 72°C for one minute and finallyi extensioni for ten minute at 72°C.The last step, the electrophoresis used to testing PCR products band at a concentration an agarose gel 1.5%, then stained by ethidiumi bromide stain and watch underi UV light.

DNA sequencing
The sequencing was performed for final diagnosis of E.coli, one isolate was taken from human and one from camel based on Gyr B gene, the PCR product was purified from agarose gel by using (EZ EZ-10 Spin Columni DNA Gel Extraction Kit, Biobasic.Canada).Then purifiedi Gyr B gene was sent to Korea in the Bioneer Company for testing and confirm DNA sequence of the Gyr B gene.The final genomic sequences were submitted in GenBank-NCBI website then alignment was made by search Tool (BLAST) for showing phylogenetic analysis and tree construction by MEGA program.

Results
Cultural characters of E.coli growth on MacConkey agar pink colonies while some strains show zone β-hemolysis on blood agar, the metallic green sheen on Eosin-methylene blue agar and Orientation -CHROM agar showed a metallic pink colour (Figure 1).The results showed that 42 (84%) samples were positive for Escherichia coli from a tissue sample of a camel while positive culture was 39 (78%).Obtain from the stool of human samples (Table 1).The results of PCR technique show 34 (41.9%)positive samples of hemolysin toxin gene, divided into19 (45%) tissue sample of camel, and 15 (38%) stool sample of a human (Table 3).PCR assay results were dependent on the extracted bacterial genomic DNA by using (PrestoTM Mini gDNA Bacteria Kit.Geneaid.USA) this kit was used spin columnbased nucleic acid purification its solid phase extraction method to quickly purify nucleic acids.This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.Therefore this kit was appeared rapid in 1hours extraction and simple method for purification of Escherichia coli bacterial genomic DNA from overnight bacterial growth on enrichment BHI broth media at 37°C and the agarose gel electrophoresis was appeared, there were sharp gyr B bands without nucleic acid lysis (Figure 2).
PCR amplification of hlyA gene in positive samples was shown clear PCR product bands on agarose gel electrophoresis at 360 bp PCR product (Figure 3).
Two isolates, one from camel while the second isolation from human sequence sent for sequencing were after that submission in NCBI-GenBank database to get accession number codes (MG560867.1,MG560866.1)frequently.DNA sequencing method was performed for phylogenetic confirmative of E-coli on Gyr B detection, phylogenetic analysis, and zoonotic importance (Figure 4 and 5).

Discussion
Enterohemorrhagic Escherichia coli (EHEC) is an important emerging zoonotic foodborne pathogen that can cause gastroenteritis in humans as well as in animal (11,12).The Food contaminated with EHEC is a principal source of infection in humans (13,14).
The results of this study had shown the isolation of E. coli hly A gene at percentage 19 (45%) from tissue camel and,15 (38%) stool from human isolates.The high prevalence of EHEC in Camel is dependent on various factors, including the hygienic conditions of the farms and abattoirs.Also, surrounding environments can impact the distribution of EHEC in animal faeces, food products, and water samples respectively so that these results appeared the main role of E.coli; our results showed that the proportion of EHEC was higher in faeces (15)(16)(17).The pathogenesis of EHEC O157 is associated with several virulence factors, such enterohemolysin (encoded by hlyA) (18).
Used PCR methods by using multiplex PCR technique for detection some genes in E. coli in stool samples from many of animals such as sheep, goat, pigs, and cattle.The hemolysin is virulence factor used for cell detachment in vitro (19).The E. coli isolate can production of the hemolysins.Ehx gene or hly gene is very similar to HlyA gene in the function that including calcium ion dependency in the cell (20).
In the phylogenetic-tree analysis of gyrB, E.coli isolated from human and from camel The phylogenetic tree shows high identity of human isolates is similar to O157H7 which consider virulence strain and zoonotic impotence that may transmitted to animal as well as the E. coli isolates show O153H3 high similarity with 99% with AB083866.1,thestrain that coded MG560866.1,a Japan with human resource that mean of zoonotic strain and importance in infection to human, while isolate that coded MG560867.1 and recorded as O157H7 high similarity with 99% with CP014314.1 O157H7 an USA isolated from camel that mean of zoonotic importance and high virulence in transmission disease.
The percentage of changing of the gene gyrB is higher than the 16S rRNA region, and all bacterial carry the gyrB gene.We think gyrB gene have a high capability for identifying microbe generally and bacteria especially.The method is characterized by rapid and high accuracy for identification most genus of the bacteria (21), using of the gyrB gene is considered a useful method for identification and confirmation species of the bacteria (22,23).
In the end, the PCR is technique have rapidly in the detection of (hlyA) gene.Enterohemorrhagic E. coli have hlyA that important virulence factor wherever isolated from human and camel.The PCR has high specific and high sensitive very useful for detection pathogen.

Figure 1 :
Figure 1: Orientation CHROM agar showed colonies have metallic pink colour.

Figure 2 :
Figure 2: The image shows the PCR product band on gel electrophoresis represent of gyr B gene in E. coli isolates.

Figure 3 :
Figure 3: The image was shown the PCR product band that represent a product band of the of Hly A gene in E. coli isolates.Sequencing and phylogenetic analysis.

Figure 4 :
Figure 4: Phylogenetic tree of E.coli strain O157H7which isolated from human.

Figure 5 :
Figure 5: Phylogenetic tree E. coli strain O153H2 which isolated from camel.

Table 1 :
E. coli isolates numbers and percentages from the both samples

Table 2 :
Isolates number of Escherichia coli which have

Table 3 :
Isolates number of Escherichia coli which have hemolysin toxin gene (hlyA)