1. INTRODUCTION:

E. coli O157:H7(EHEC) is one of the most important pathogen that transmit by food, it causes a different signs ranged from reasonable diarrhoea to haemorrhagic colitis and dangerous problem in other body systems or organs. E. coli O157:H7 have several virulence factors, the main important one were Shiga toxins (Stxs) (1), it is some time termed as STEC (2). It can cause a disease not only in calves, but it can also infect adult cattle (Schaut et al., 2018); human (Tarr et al., 2018); sheep (Cornick et al., 2017); dogs (Yousif et al., 2017 ; Hasan et al., 2016 ; Yousif et al., 2016) and other animals.

Lupindu et al. (2018) reported that cattle were regarded as the natural reservoir for E. coli O157:H7.

This bacteria (E. coli O157:H7) usually present in the GIT, especially the terminal recto-anal junction, without causing disease, and the reservoir animals may shed this bacteria to the environment and transmitted to the other animal species (Stein and Katz, 2017) by the way of oral fecal route (Schildgen, 2018).

The detection of pathogenic E. coli strains has been greatly improved by using conventional PCR in the multiplex format (Aranda et al., 2007) or by real-time PCR (Stefan et al., 2007).

Ruminants are the primary reservoir of Shiga toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans (Martorellie et al., 2017). A study was done by Osman et al. (2013) to isolate E. coli from diarrheic ruminants, They found that the prevalence rate were 27.3% in goat; 9.1% in sheep and 63.6% in calves, and these isolates were belonged to different serotypes, the most one O157 serotype.

Also, these bacteria causes a serious disease in other animals, Yousif et al. (2016) concluded that E. coli O157:H7 is an significant doge pathogen and the real time PCR is approved as best, reliable method for confirmatory diagnosis of these strains.

In a study of Dunn (2003) found that the isolates of E. coli were positive for fliCH7and eaeA genes by using PCR.

Realtime PCR techniques have many advantages which invites the researchers to prefer this type such as decrease the contamination of the samples by reduction opening the test tubes which seen in conventional method of PCR, also this type do not need ethidium bromide which is dangerous for researchers, all these advantages made the real time PCR overcomes on the conventional PCR.

The main aims for using antimicrobial susceptibility testing are for detection the resistance of pathogens to antimicrobials and to help in choosing the effective antimicrobial (Jorgensen and Ferraro, 2009).

The useful of antimicrobial therapy for Shiga Toxin E. coli infections is not of value, Because tat antimicrobials may damage the cell wall of bacteria and then liberation of Stxs (Wong et al., 2000) and/ or cause increased expression of Shiga toxin genes in vivo (Zhang et al., 2000).

The antimicrobial tests of E. coli O157:H7 showed an extensive resistance to neomycin, erythromycin and vancomycin and high sensitivity to ampicillin, gentamicin, ciprofloxacin, tetracycline and kanamycin (Osaili et al., 2013).

Cho et al. (2006) conclude that 25% of Escherichia coli O157 were resist to sulfadimethoxine and thirteen percent isolates were resist for tetracycline.

Vidovic and Korber, (2006) found the antimicrobial susceptibility testing of the E. coli O157 isolates revealed that ten from 194 were multidrug resistant and that 5 and73 were resistant to tetracycline and sulfisoxazole, respectively.

Osek, (2004) showed that Most of the isolates of E. coli O157:H7 isolated from cattle were resistant only to rifampicin and sensitive to Amoxicillin, Amoxicillin-Clavulanic Acid, Cephalothin, Colistin, Cefoperazone, Cotrimoxazole, Streptomycin, Sulfamethizole, Spectinomycin, Flumequine, Kanamicin, Oxolinic Acid, Gentamicin, Enrofloxacin, Apramycin, Nitrofurantoin, Chloramphenicol, Tetracycline and Doxycycline.

Stanford et al . (2012), compare between the low shedders and super shedders, Resistance detected for 17/474 isolates for antimicrobials including neomycin, ampicillin, sulfasoxazole, streptomycin, tetracycline and sulfamethoxazole/trimethroprim and they showed that these isolates were have multi-drug resistance characteristic.

So that this study was aimed to identifying the rfb_O157 and fliC_H7genes of Escherichia coli O157:H7 between diarrhoeic and non diarrhoeic calves by using real time PCR and to detect the susceptibility of this bacteria to many antimicrobials .

2. MATERIAL AND METHODS:

2.1. Bacterial Strain:

Thirty two strains of E. coli O157:H7 were obtained from Dept. of Internal and Preventive Medicine/ College of Veterinary Medicine / University of Baghdad, which isolated from 350 faecal samples collected from non diarrheic and diarrheic calves which found in different places in Baghdad Province (Yousif and Hussein, 2015).

2.2. Bacteriological Methods:

All methods were done as recorded in a previous study by (Yousif and Hussein, 2015) and according to (Tahamtan et al., 2011) to insured the bacteria before doing PCR assay. Including culturing on enrichment media, Gram stain and biochemical test for detection and confirmation the suspected isolates. All suspected isolates of E. coli were cultured on Chrom agar specific for E. coli O157 [Himedia] and Sorbitol MacConkey agar (CT-SMAC)(England) supplemented with 0.05 mg/L Cefixime and 2.5 mg/L potassium tellurite to increase the selectivity for E. coli O157:H7.

Serotyping was made by using Latex agglutination test kit (Remel, wellcolex) for detection both somatic O157 and flagellar H7 antigens (Marky et al., 2013), these strains were possess aea A gene as reported previously (Yousif and Hussein, 2015) and also possessed Stx1 and Stx2 (Hussein and Yousif, 2015)

2.3. Real Time PCR Assay:

This test was performed in the laboratories of University of Baghdad, College of Vet. Medicine Department of Internal and Preventive Veterinary medicine, this assay was done on the 32 isolates of E. coli O157:H7 by the following methods:

1. DNA Extraction:

NA were extracted from all isolates by using (DNA extraction kit of Bacteria, Geneaid, USA).

2. Measuring The Purity and Concentration of DNA: The purity and concentration of extracting DNA were measured using a Nanodrop spectrophotometer.

3. Molecular Characterization of E. coli rfbO157 and flicH7 genes by using Real time PCR:

Real-time PCR was performed in a 96-well plate using Applied Biosystems (USA) Sacace Real-Time PCR System by using kit from MicroSEQ ® E. coli O157:H7 Applied biosystem (USA). The FAM and VIC dyes are used to detect the targets; the NED dye is used to detect the internal positive control (IPC). 2 µ of template DNA was used and water free nuclease was added to reach a volume of 30 µ for reaction. The real-time PCR conditions were ﬁrst optimized and were set as in (Table 1).

Table (1) Real time PCR program

step		temperature	time
Enzyme activation/ Hold		95°C	2minut
PCR (40 cycles)	Denature	95°C	1 second
PCR (40 cycles)	Anneal/ extend	60°C	20 second

2.4. Antibiotic susceptibility test (Disk diffusion test):

The disk diffusion susceptibility method is simple and practical and has been well-standardized (Baure et al., 1966). At least four to five colonies of the same morphology were selected from chromeagar, they were transferred to a tube containing 4-5ml of nutrient broth. This broth containing bacteria incubated at 37C°for 18 hrs until the appearance of visible turbidity.

A swab was soaked into the broth of bacteria and excess fluid was get rid of by pressing the swab firmly at inside of the tube.

Then, the soaked swab streaked in all directions over the surface of Muller Hinton agar. The inoculated plates were allowed to dry for 5 minutes. Then the discs of antibiotic were placed onto agar surface using a sterile forceps. After that the plates were inverted, placed at 37C° incubator aerobically for 18-24hrs (Markey et al., 2013).

The zones inhibition around each antimicrobial disks were measured by using a digital caliper. The measured zone of inhibition of each drug were interpreted by (NCCLS, 1990, 1997, CLSI, 2011 and Andrews, 2012).

3. RESULTS:

3.1. Results of real time PCR:

The confirmation process of the 32 isolates revealed that bacteria was given the characteristic feature of E. coli O157:H7 by cultural and serotyping methods. Then the results of testing by real time PCR showed that all these isolates were possessed rfbO157 and flicH7 genes (Figure 1).

Figure (1) Real time PCR results, positive samples were CT less than 35

3.2. Antimicrobial susceptibility test:

The isolated E. coli O157:H7 strains showed different percentage of resistance and sensitivity to different antimicrobials. Figure (2). The isolated organism showed high sensitivity to Gentamicin (100%), Enrofloxacin (100%), Trimethoprim- sulfamethoxazole (87.5%), chloramphenicol (87.5%) and tetracycline (65.62%). Two isolates only showed moderate susceptibility to Tetracycline and chloramphenicol.

All isolates were resistance to Erythromycin (100%), while (96.87%), (31.25%) , (12.5%) and (9.37%) were resistance to Cephalothin, Tetracycline , Trimethoprim-sulfamethoxazole and chloramphenicol respectively (Table 2).Statistically, There is significant difference among different antimicrobials used in susceptibility test for E. coli O157:H7 at level of (P<0.05).

Figure (2) susceptibility test of different antibacterial disc.

4. DISCUSSION:

Jenkins et al. (2012) reported that the real-time PCR is effective, screening method for detecting STEC from stool specimens.

In this study, this assay was used to confirm diagnosis of E. coli O157:H7 by using two genes (rfbO157 and flic H7) and the results of PCR assay coincide with those of bacterial culturing and latex agglutination test, which indicated that PCR assay was more sensitive and trusted method for detecting of this organism. This result is in agreement with result of Rebekka et al. (2006) who reported a sensitivity and accuracy of real-time PCR for detection of E. coli O157:H7 and they concluded that this assay was quick diagnostic methods for the presence or absence of E. coli O157:H7.

Chui et al. (2013) reported that real-time PCR method may use as the "gold standard" for diagnosis of E. coli O157:H7. Other studies reported similar results with this study, Abbasi et al. (2014) used RT- PCR as a replacement of conventional type to detect the frequency and presence of virulence genes of Shiga toxigenic E. coli and Enteropathogenic E. coli from diarrheic children. They found that this assay is being faster, more confirmable assay, because it doesn't demand any additive procedures for confirmation the amplificated products.

Among bacterial pathogens involved in food-illnesses STEC has been frequently identiﬁed in a study of Mondani et al. (2016) who found that using of real time PCR for diagnosis is reducing the time and decreasing the limitations which accompanying the other traditional tests.

A study performed of Qin et al. (2015) performed to estimate the real-time PCR assay in confirming the presence of Stx1,Stx2 and rfbO157 with 2,386 stool samples in microbiology laboratory of Pediatric center. Among the 59 positive PCR samples, 29 (54.7%) specimens only were positive to toxin. They recommended that PCR is rapid method for diagnosis of pediatric shigatoxigenic E. coli infections.

Our results showed that all bacterial isolates were highly sensitive to Gentamicin and Enrofloxacin this agreement with Osek, (2004) who found the total isolates of E. coli O157:H7 are sensitive to Gentamicin and Enrofloxacin also they found the isolates 100% sensitive to Cephalothin, Chloramphenicol and Tetracycline. And with results of Galland et al ., (2001), that recorded most isolates of E. coli O157:H7 were susceptible to antibiotics used at feedlots, such as trimethoprim-sulfamethoxazole . Also Schroeder et al., (2002) found that (20%) of the isolates, however, were resistant to tetracycline. A much smaller proportion of cattle isolates were resistant to chloramphenicol, cephalothin (2.3% each).

The study of Mora et al. (2005) concluded that E. coli O157:H7 showed a higher degree of resistant which isolated from cattle (53%) and beef (57%) than from human being (23%) and sheep (20%) sources, they found that Sulfisoxazole (36%) was the most antimicrobial resistance, followed by tetracycline, streptomycin, ampicillin, trimethoprim, cotrimoxazole, chloramphenicol, kanamycin, piperacillin and neomycin.

Our results were similar to study of Osaili et al. (2013) who showed that E. coli O157:H7 isolates were extensively resistant to erythromycin and high susceptibility to Gentamicin, Tetracycline, Ampicillin, Ciprofloxacin, and Kanamycin.

The different results of resistance may be due to the use of non-specific antibiotic and random administration of antibiotic and this will make persistent strains have ability to resist a large number of antibiotics, some of these resistance appeared in calves introduced from other country.

5. CONCLUSIONS:

In conclusion, the Real time PCR assay described in this study is a sensitive and accurate method for specific detection and profiling of EHEC O157:H7 and this bacteria can be referred as Multi drug resistant bacteria due to its ability to resist most antimicrobial that used in this study.

6. ACKNOWLEDGMENT:

This work was supported by College of Veterinary Medicine, Department of Internal and Preventive Veterinary Medicine, University of Baghdad, Iraq.

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Received on 19.05.2018 Modified on 10.07.2018

Research J. Pharm. and Tech 2019; 12(1): 245-250.

DOI: 10.5958/0974-360X.2019.00046.5

Total No of Isolates	Resistant	Intermediate	Susceptible	Antimicrobial
32	10(31.25%)	1(3.12%)	21(65.62%)	Tetracycline
	32(100%)	0(0%)	0(0%)	Erythromycin
	0(0%)	0(0%)	32(100%)	Gentamicin
	31(96.87%)	0(0%)	1(3.12%)	Cephalothin
	4(12.5%)	0(0%)	28(87.5%)	Trimethoprim-sulfamethoxazole
	0(0%)	0(0%)	32(100%)	Enrofloxacin
	3(9.37%)	1(3.12%)	28(87.5%)	Chloramphenicol