EVALUATION OF THE RELATIONSHIP BETWEEN VIRULENCE, ANTIBIOTIC RESISTANCE GENES AND DEVELOPMENT OF BIOFILM IN ESCHERICHIA COLI ISOLATED FROM BROILER CHICKEN

Assiut University web-site: www.aun.edu.eg

EVALUATION OF THE RELATIONSHIP BETWEEN VIRULENCE, ANTIBIOTIC RESISTANCE GENES AND DEVELOPMENT OF BIOFILM IN ESCHERICHIA COLI ISOLATED FROM BROILER CHICKEN

MARAM, M. TAWAKOL¹ and AHLAM, E. YOUNIS. ²

¹ Reference Laboratory for Veterinary Quality Control on Poultry Production, Gamasa,

 Animal Health Research Institute.

²Reference Laboratory for Veterinary Quality Control on Poultry Production, Damanhour,

 Animal Health Research Institute.

Received: 31 March 2019;     Accepted: 9 April 2019

INTRODUCTION

Avian colibacillosis caused by E. coli is serious infectious disease occurring in different types of chicken resulting in a significance losses in poultry industry. Escherichia coli (E. coli) is one of the normal bacterial flora in the gastrointestinal tract of poultry. About 10-15% of the intestinal coliforms in chickens are of pathogenic serotypes. Colisepticemia, respiratory tract infections, poultry cellulitis, swollen head syndrome, omphalitis/yolk-sac infection, pericarditis, peritonitis and salpangitis are important diseases caused by E. coli in birds Barnes et al. )1999). Escherichia coli recognized as major pathogen for public health problems in developing countries and represents leading etiological agent of diarrhea where several classes of enterovirulent E. coli, namely enterotoxigenic E. coli

Corresponding author: Dr. MARAM, M. TAWAKOL

E-mail address: maram_salah82@hotmail.com

Present address: Reference Laboratory for Veterinary Quality Control on Poultry Production,Gamasa, Animal Health Research Institute.

(ETEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), diarrhoea-associated haemolytic E. coli and cytolethal distending toxin (CLDT)-producing        E. coli have been recognized by Nataro and Kaper (1998). The term biofilm is used to describe matrix-enclosed bacterial population adherent to each other and/or to surfaces Costerton et al. (1995).E. coli is one of many bacteria that can switch between planktonic form and biofilm form. Several reasons can explain the need of bacteria to create biofilm, in this way bacteria can avoid being washed away by water flow or, cells in biofilms are about 1000 times more resistant than their planktonic Jefferson, )2004). Several surface organelles, including various types of fimbriae, autotransporter proteins and extracellular polysaccharides, have been found to facilitate or enhance biofilm formation of E. coli, largely depending on the environmental conditions and the particular strains studied Schembri et al. )2002) which may be resistant to antibiotic or not. The aim of this study is to evaluate the relationship between virulence, antibiotic resistance genes and development of biofilm in E. coli isolated from broiler chicken farms in Dakahlia Governorate in Egypt.

MATERIALS AND METHODS

A total of 100 diseased broiler chickens of average age 22-28 days old were collected from different farms located in Dakahlia Governorate were subjected to clinical and postmortem (P.M) examination as well as for isolation and identification of E. coli from tissue samples including liver, caecum, spleen, lungs, kidneys and heart according to Maram, )2014(.

1. Detection of Escherichia coli by conventional method: was done according to Swayne et al. (1998) and Quinn et al. (2002).

1.1. Selective enrichment of E. coli in broth:

Each sample was inoculated separately into buffer peptone water were incubated at 37 ^oC for 24 hrs under aerobic condition.

1.2. Colonization of E. coli on selective differential solid media:

A loopful from the broth of each sample was streaked onto MacConkey's agar and Eosin Methylene Blue agar. The inoculated plates were incubated at 37 ^oC for 24 hours. Suspected E. coli colonies were purified and kept for further identification.

1.3. Identification of suspected E. coli colonies was done according to Quinn et al. (2002):

Culture characters, Microscopic examination and motility: were done according to Cruickshank et al. (1975)

2. Biochemical Identification of E. coli:

It was done according to Quinn et al. (2002) on indole reaction, methyl red test, voges proskauer test, citrate utilization test, catales test, sugar fermentation test, oxidase test, triple sugar iron and christener's urea agar test.

3. Serological identification of E. coli isolates was carried out according Edwards and Ewing (1972):

The obtained isolates were serogrouped in Animal Health Research Institute, Dokki, Giza using: Sifinantisera "Berlin, Germany" Polyvalent and monvalent diagnostic E. coli antisera.

4. Sensitivity of E. coli isolates to antimicrobial agents:

E. coli strains were tested for their antimicrobial sensitivity to various antibiograms (Amoxicillin, Enrofloxacin, Tetracycline, Doxycycline, Ampicillin, Flumequine, Gentamycin, Naldixic acid, Chloramphenicol, Erythromycin, Ciprofloxacin, Lincomycin and Colistin) by the agar disc diffusion method according to finegold and martin (1982) and interpretation of the results according to CLSI (2016)

5. Biofilm formation of E. coli:

According to Maram, (2011); it was done on 10 serotyped E. coli isolates represents to serotypes detected. A loopful of tested organisms was inoculated in 10 mL of trypticase soya broth with 1% glucose in test tubes. The tubes were incubated at 37^oC for 24 hours. One ml from the inoculated broth was transferred into another tube containing 4 ml trypticase soy broth with 1% glucose, one tube used as a control negative (not inoculated) and another tube was inoculated with E. coli (positive control).All test tubes were incubated at 37 ^oC for 5 days. After incubation, tubes were decanted and washed with phosphate buffer saline (pH 7.3) and dried. Tubes were then stained with crystal violet (0.1%). Excess stain was washed with deionized water. Tubes were dried in inverted position. The results of tube method were compared with the control positive strain. Biofilm formation was considered positive when a visible film lined the wall and the bottom of the tube. The experiment was performed in triplicate and repeated three times.

6. Molecular detection of E. coli resistant and virulence genes using Polymerase chain reaction (PCR):

6.1. DNA extraction:

DNA extraction from 10 E. coli isolates represented 10 serotypes detected was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56 ^oC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

6.2.Oligonucleotide Primer: Primers used were supplied from Metabion (Germany) are listed in Table (1).

6.3. PCR amplification:Primers were utilized in a 25 µl reaction containing 12.5 µl of DreamTaq Green PCR Master Mix (2X) (Thermo Scientific), 1 µl of each primer of 20 pmol concentration, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an Applied biosystem 2720 thermal cycler.

6.4. Analysis of the PCR Products:

The products of PCR were separated by electrophoresis on 1% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 20 µl of the PCR products were loaded in each gel slot. Gelpilot 100 bp, 100 bp plus plus DNA Ladders (Qiagen, Germany, GmbH), generuler 100 bp ladder (Fermentas, Thermo) and Genedirex 100 bp DNA ladder H3 RTU, Cat. No. DM003-R500 were used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

Table 1: Primers sequence, target genes, amplicon sizes and cycling conditions.

7. In vitro sensitivity testing using Olive Leaf Extract on isolated E. coli:

All E. coli isolates that showed resistance to antibiotics were subjected for testing the effect of Olive Leaf Extract.

7.1. Extraction of Olive Leaf:

The leaves were purchased from herbal market, cleaned from extraneous matter and properly washed then dried in hot air-oven for 24 h at 40 °C. The dried leaves were ground in a blender to form powder. Thereafter, 100 g of the powder were macerated in 1000 ml absolute ethanol (10% concentration) and allowed to extract for 48 h Ahmed and Abolghait (2014) with some modification. The resultant (dark green-brown mixture) was filtered and the filtrate was concentrated in a rotary evaporator under reduced pressure.

7.2. Preparation of filter paper discs from olive leaf extract:

Six millimeter filter paper was used to prepare discs. The discs were then sterilized in by autoclaving. The extract was diluted at different concentrations. A total volume of 250 μl was used to soak 50 discs without over or under wetting those Hannan et al. (2008).

Discs with concentration 10.0 mg per discs were obtained. Prepared discs were stored at 4 °C in the refrigerator till use. The discs were kept at room temperature for one hour to before use.

Antimicrobial susceptibility testing using filter paper discs from olive leaf extracts according to Finegold and Martin (1982).

RESULTS

1. Prevalence rate of E. coli isolated from examined broilers:

In 100 broiler chickens suffering from ruffled feathers, depression, off food were subjected for P.M. examination, revealed caseous masses on internal organs, cellulitis, entero-colitis and damage intestinal mucosa then  examined by bacteriological methods during different seasons of the year, E. coli was recovered from 37 samples with a prevalence rate 37 % (37 out of 100) as shown in Table (2).

Table 2: Incidence of E. coli infection in broiler chickens.

2. Prevalence of E coli in different chicken organs:

The internal organs of each chicken were examined by bacteriological examination to determine the prevalence of Escherichia coli in each chicken organ as shown in Table (3) where E. coli was isolated as the following 30% (30 out of 100) from liver; 24% (24 out of 100) from lung; 20% (20 out of 100) from caecum; 14% (14 out of 100) from spleen; 11% (11 out of 100) from kidney and 9% (9 out of 100) from heart.

Table 3: Rate of E coli recovery from internal organs.

* calculated according to the number of tested birds (100).

** calculated according to the number of examined samples (600).

3. E. coli serotypes isolated from examined chickens:

The isolated E. coli were serotyped using polyvalent and monovalent E. coli antisera to determine the E. coli serotype.

The serotyping of isolated biochemically identified E. coli revealed that the most predominant serotypes and the most predominant serogroup were O₁₂₅ 6%& O₉₁ 5%& O₁ 4%& O₂₆ K₆₀ 4% & O₈₆ K₆₄ 3% & O₁₂₈ 2% & O₅₅ K₅₉ 1% & O₁₆₆ 1% & O₁₀₃ 1% & O₁₄₄ 1% and 9 strains untypable E. coli (9 %) as shown in Table (4).

Table 4: E. coli serotypes recovered from bacteriologically examined chickens.

4. Sensitivity of E. coli serotypes to different antibiotic agents: 

As shown in Table (5), E. coli O groups (28) were found to be 100% resistant to Lincomycin antibiotic followed by Amoxicillin (82.14), Enrofloxacin (71.73%), Tetracycline (67.86%), Doxycycline (60.71%), Ampicillin (57.14%), Flumequine (42.86%), Gentamycin (32.14%), Erythromycin (32.14%), Naldixic acid (28.27%), Chloramphenicol (25%), Ciprofloxacin (10.7%) and Colistin (7.14%).

Table 5: Sensitivity of E. coli serotypes and to different antibiotic agents.

R: resistant.                      S: sensitive.                     I: intermediate.

CL: Colistin.                    L: Lincomycine.              C: Chloramphenicol.      

CF: Ciprofloxacine.        ENR: Enrofloxacine.      UB: Flomequine.         

NA: Nalidixic acid.         DO: Doxycycline.            T: Tetracycline.       

A: Amoxycilline              E: Erythromycin.             G: Gentamycine.

AM: Ampicilline.

5. PCR Detection of resistant Genes of E. coli:

PCR using primers fragments listed in materials and methods for amplification of bla_TEM and tetA(A) genes from the isolated E. coli strains in this study.

5.1. Detection of bla_TEM gene of E coli:

bla_TEM gene responsible for resistant of the isolated E. coli strains to Beta-lactames antibiotics. Our results showed amplification of 516 bp of (bla_TEM) gene from the extracted DNA of all tested E. coli strains (10) as shown in Figure (1)

Figure (1) amplification of bla_TEM gene of Escherichia coli strains: Amplification of 516 bp was observed in the extracted DNA of O₁, O₂₆, O₅₅, O₈₆, O₉₁, O₁₀₃, O₁₂₅, O₁₂₈, O₁₄₄ and O₁₆₆, (in lane number 1, 2,3, 4, 5, 6, 7, 8, 9, 10, respectively).

5.2. Detection of tet (A) gene of E. coli:

tetA (A)gene responsible for resistant of the isolated E. coli strains to tetracycline antibiotics. Our results showed amplification of 576 bp of tetA(A) gene from the extracted DNA of all isolated E. coli strains except O₅₅ (No. 3) not have this gene as shown inFigure (2)

Figure (2) amplification of tetA(A) gene of Escherichia coli strains: Amplification of 576 bp was observed in the extracted DNA of O₁, O₂₆, O₈₆, O₉₁, O₁₀₃, O₁₂₅, O₁₂₈, O₁₄₄ and O₁₆₆ (in lane number 1, 2, 4, 5, 6, 7, 8, 9 and 10, respectively). No amplificationin O₅₅ (in lane number 3).

6. PCR for Detection of virulence Genes of E. coli:

PCR using primers fragments listed in materials and methods for amplification of eaeA, fimH, csgD and adrA from the isolated E coli strains in this study.

6.1. Detection of eaeA gene of E. coli:

Ettaching and effacing mechanisms gene (eae A)is responsible for attachement and effacing of E. coli to the enterocytes of the intestine of chicken. Our results showed amplification of 248 bp of (eae A) gene of all isolated serotypes except O₁₂₅ (in lane No. 7) from the extracted DNA of E coli strains as shown in Figure (3)

Figure (3) amplification eaeA gene of Escherichia coli strains: Amplification of 248 bp was observed in the extracted DNA of O₁, O₂₆, O₅₅, O₈₆, O₉₁, O₁₀₃, O₁₂₈, O₁₄₄ and O₁₆₆ (in lane number 1, 2,3, 4, 5, 6, 8, 9 and 10, respectively). No amplificationin O₁₂₅ (in lane number 7).

6.2. Detection of fimH gene of E. coli:

fimH gene which responsible for adhesion of E. coli. The results showed amplification of 508 bp of fimH gene of all isolated serotypes from the extracted DNA of E. coli strains as shown in Figure (4).

Figure (4) amplification of fimH gene of Escherichia coli strains: Amplification of 508 bp was observed in the extracted DNA of O₁, O₂₆, O₅₅, O₈₆, O₉₁, O₁₀₃, O₁₂₅, O₁₂₈, O₁₄₄ and O₁₆₆, (in lane number 1, 2,3, 4, 5, 6, 7, 8, 9, 10, respectively).

6.2.Detection of csgD gene of E. coli:

csgD gene which is the master regulator for adhesive curli fimbriae expression, plays a positive role in biofilm formation of E. coli. The results showed amplification of 651 bp of csgD gene of all isolated serotypes from the extracted DNA of E. coli strains as shown in Figure (5).

Figure (5) amplification of csgD gene of Escherichia coli strains: Amplification of 651 bp was observed in the extracted DNA of O₁, O₂₆, O₅₅, O₈₆, O₉₁, O₁₀₃, O₁₂₅, O₁₂₈, O₁₄₄ and O₁₆₆, (in lane number 1, 2,3, 4, 5, 6, 7, 8, 9, 10, respectively).

6.3. Detection of adrA gene of E. coli:

adrA gene which responsible for cellulose synthesis. The results showed amplification of 1113 bp of adrA gene of all isolated serotypes from the extracted DNA of E. coli strains except O₁₀₃ as shown in Figure (6).

Figure (6) amplification adrA gene of Escherichia coli strains: Amplification of 1113 bp was observed in the extracted DNA of O₁, O₂₆, O₅₅, O₈₆, O₉₁, O₁₂₅, O₁₂₈, O₁₄₄ and O₁₆₆ (in lane number 1, 2,3, 4, 5, 7, 8, 9 and 10, respectively). No amplificationin O₁₀₃ (in lane number 6).

7. Biofilm formation:

PCR amplifications of eaeA, fimH, csgD and adrA genes from the isolated E. coli strains in this study were assayed for biofilm formation in vitro using tube biofilm assay. Result revealed that, all the examined serogroups (O₁, O₂₆, O₅₅, O₈₆, O₉₁,O₁₀₃, O₁₂₅, O₁₂₈, O₁₄₄ and O₁₆₆ have the ability to make biofilms on the inner walls of the glass tubes after crystal violet staining. On the other hand, no biofilm was observed with negative uninoculated tube. These results might point to the role that these genes play during expression of proteins involved in biofilm formation.

8. Effect of olive leaf extract on the multiresistant strains:

Results of antimicrobial susceptibility testing for 10 E. coli serotypes revealed that olive leaf extracts had inhibitory effect at a concentration of (10 mg) on tested serotypes as shown inFigure (7).

Figure (7): effect of olive leaf extraction E. coli multiresistant strains.

DISCUSSION

In this study, the incidence of E. coli in broiler chickens from Dakahlia governorate was 37 %. These results were agreed with that of Robert et al. (2002)and Ružauskas et al. (2010), who isolated    E. coli with percentage of 36.8% and 41.7%, respectively. Higher rates were recorded by           El-Sukhon et al. (2002) and Alimehr et al. (1999) who recovered E. coli in 88.2% and 100% of the examined samples, respectively.

The obtained results of this study revealed that all the most E. coli isolates obtained from liver of the examined chickens followed by lung, caecum, spleen, kidney and heart 30%, 24%, 20%, 14%, 11% and 9%, respectively which agreed with Otaki, 1995 and the explanation of these results is due to infection with APEC generally begins as a localized infection of the air sacs commonly referred as airsacculitis or the air sac disease which in turn may spread to other internal organs resulting in systemic infection Barnes et al. (1999). These results were agreed with Ogunleye et al. (2008) who reported that, the most Escherichia coli isolates obtained from liver 67% then lung and intestine10%. Also, Sharada et al. (2010) recovered highest percent of isolates from cases of hepatitis 44.6%, enteritis 33.8%, pericarditis 16.9% followed by air saculitis 7.7%.

More than 1000 E. coli serotypes have been reported but only small percentages have been implicated in poultry diseases Cloud et al. )1985). In this study, 10 E. coli serogroupes were identified in 28 positive samples and the most predominant serogroup were O₁₂₅ 16%, O₉₁ 14%, O₁ 11%, O₂₆ K₆₀ 11% & O₈₆ K₆₄ 8 & O₁₂₈ 5% & O₅₅ K₅₉ 3% & O₁₆₆ 3% & O₁₀₃ 3% & O₁₄₄ 3% and untypable E.coli (24 %). These results nearly go hand to hand with the previous studies of Abd El Tawab et al. )2015). The occurrence of a specific serotype and its role in disease production depends upon the health status of the birds, climatic conditions, geographical situations and managemental strategies Srinivasan et al. (2013).

The Results for Antibiotic sensitivity showed that most of the isolates were multidrug resistant as they resist at least 3 antibiotics as these results agreed with that reported by Momtaz et al. )2012(. E. coli isolates were found to be 100% resistant to Lincomycin almost similar resistance were detected by Ngeleka et al. (1996) who reported 100% resistant to lincomycin. Lower rate 39.50% were detected by Sharada et al. (2010).

The Amoxicillin resistance of the E. coli isolates in the present study were 82.14%, these results go hand to hand with the previous studies of Anthonia)2012( who showed 80% resistant to amoxicillin in free range chickens.On the other handSalehi and Bonab )2006( reported medium resistance AM 53% in      E. coli isolates from chickens of Colisepticemia.

The present study showed resistance percentages to Enrofloxacin (71.73%). Almost similar resistance were detected bySalehi andBonab )2006(, 76% however less percentages 23%, 34.8 were detected byAmara et al. (1995) and Alimehr et al. (1999), respectively.

In this study, there was high resistance rate of Tetracycline (67.86%) in the isolated E. coli which agreed with earlier reports of Alhaj et al.  (2007) and Morad )2013( to these antibiotics 81.4%, 85.1%, respectively in chicken isolates and disagreed with those of Kolar et al. (2005) who showed less resistant to tetracycline  about 48%.

Also E. coli isolates were found to be resistant to Doxycycline with a percentage of (60.71%) which similar to that of Ngeleka et al. (1996) (more than 50%) but disagree with the results of Salehi and Bonab )2006(, who showed high resistant to Doxycycline (88%).

The E. coli isolates in this study expressed resistance to ampicillin at (57.14%) percentage, these results go hand to hand with the previous studies of Akond et al. (2009) who reported that 58% of E. coli strains isolated from poultry and poultry environment in Bangladesh were resistant to ampicillin on the other hand resistance rate to ampiclllin in this study was higher than those reported byIdrees et al. (2011) from poultry in Pakistan.

In this study, about (42.86%) of the isolated E.coli were resistant to Flumequine which represent a lower percentage than those reported byMorad, 2013 and Alimehr et al. (1999) who reported percentage of 81.8% and 67.5%, respectively.

It was also reported that 32.14% resistant rate was found to Gentamycin. Lower rate (0%) was detected by Momtaz et al.(2012). Higher resistant rates 74.3% were detected by Alhaj et al.  (2007).

This result revealed Erythromycin resistant rate (32.14%) in the isolated E. coli however higher level detected in previous studies by Salehi and Bonab (2006) who reported 97% resistant to Erythromycine.

The present study revealed that 28.27% of the E. coli isolates were resistant to Nalidixic acid. This finding was agreed with those of Johnson et al. (2003) who recorded resistance rates of (37%) and disagreed with the results reported by KmetandKmetova(2010), (87/85/67 %) from Escherichia coli isolatedfrom healthy chicken broilers during (2006/2007/2008).

The present study showed intermediate percentage of Chloramphenicol resistant (25%) between those of Alhaj et al. (2007) who reported high percentage of resistant 75.7% and  Miles et al. (2006) with low percentage rate 2.9% from broiler chichens.

However, the level of resistance to ciprofloxacin (10.7%) of isolated serotypes observed in this study was similar to that reported by Anthonia, (2012),12 %in free range chicken. This low resistance rate may also be associated with the low usage of this drug by poultry farmers.

The highest sensitivity rate detected in this study was to colistin 89.28%. This result agreed with that of Filali et al. (1988) and Amara et al. (1995) who reported that colistin exhibited excellent activity against Escherichia coli isolates.

Among food animals that act as reservoirs of ESBL-producing E. coli, broilers were considered to be the most potent reservoir Pacholewicz et al. (2015).In this study the percentage of bla_TEM gene from the isolated E .coli strains was 100 % (10 out of 10 strains) which nearly go hand with the results of Maram (2014) who detected bla_TEMgene in 18 out of 19 E.coli isolates with 94.73%. But these results disagreed with Ghosh et al. (2017) who obtained lower percentage about 10%from isolated E. coli.

In recent years, tetracycline resistance has emerged among many pathogenic and nonpathogenic species of bacteria. This resistance is mainly due to different efflux pump and ribosomal protection genes, mostly associated with mobile components such as plasmids or transposons Roberts (2012). Screening of the tetracycline resistance gene showed that tet(A) gene was detected in all isolated E. coli strains except O₅₅ not have the gene with a percentage 90% which nearly agreed with the results reported by Zibandeh et al. (2016), 72.5% of E. coli isolated from the chickens on the day before slaughter.

virulence-associated genes and pathogenicity islands of bacteria play an important role in the pathogenicity of bacteria and that they are important parameters to clarify the mechanism of bacterial pathogenicity Vandekerchove et al. (2005) and Cheng et al. (2006).

This study showed many different genes which are responsible for virulence and biofilm formation of   E. coli. Intimin, an outer membrane protein, encoded by eaeA, is a bacterial adhesion molecule that mediates the intimate bacterium host cell interaction characteristic of A/E (attaching and effacing) lesions of avian pathogenic E. coli Kilicet al. (2007). In this study, high incidence rate (90%) of eaeAgene detection was recorded, as it was detected by PCR in 9 out of the 10 tested isolates and these high results agreed withRamadan et al. (2016) and disagreed with Kilicet al. (2007) who reported the incidence rate 48% of the E. coli isolates.

Surface virulence factors of the pathogens including different adhesion factors may promote bacterial adhesion and biofilm development Schembri et al. (2003). fimH consists of a fimbria- associated pilin domin and a mannose binding lactin domin, receptor-ligand specific adhesion is among the most fundamental of biological phenomena in nature. This phenomenon underlies eukaryotic cell-cell or cell- surface attachment, initiates recognition and signaling events, binds bacteria to target cells and mediates biofilm formation on medical implants Aprikian et al. (2007). In this study the prevalence of fim H gene in the isolated E. coli strains was    100 % (10 out of 10 strains) and these results were agreed with the results reported by Trkov et al. (2014) as seventy-four (88.1 %) isolates carried the type 1 fimbriae gene fimH and Ghanbarpour et al. (2011) also reported 96.4% of fecal isolates positive for fimH compared to 95% of isolated E. coli from cases of colibacillosis. The results were disagreed with the results ofEftekharian et al. (2016) who detected less percentage 41.7% of the intestinal isolates only positive for fim H gene.

Curli fibers (also known as thin aggregative fimbriae) are a major factor in adhesion to surfaces, cell aggregation, and biofilm formation in many enterobacteria Cookson et al. (2002) and Prigent-Combaret et al. (2001). Expression of both curli and cellulose depends on the csgD proteinArnqvist et al. (1994) in this study, 100% of the tested strains have the csgD gene which go hand with the results of Wang et al. (2016) in 36 non-O157 Shiga toxin–producing Escherichia coli (STEC) strains.

Cellulose synthesis regulation is a very complex phenomenon. In enterobacteria, this phenomenon involves a AgfD-regulated protein (adrA) that contains four N-terminal units of the GGDEF domain. These domains may be involved in the regulation of a second messenger molecule, called cyclic di-guanosine mono phosphate (c-di-GMP), the interaction between the GGDEF domain of adrA and the c-di-GMP molecule could initiate cellulose production Romling(2002). In the present study PCR detection of adr A gene in the isolates revealed that, all strains had the gene except O ₁₀₃ and this result agreed with that reported by Yin et al. (2018) as present exceeded 75% among all biofilm producer strains.

The result of in vitro tube biofilm assay revealed that, all the examined serogroups (O₁, O₂₆, O₅₅, O₈₆, O₉₁, O₁₀₃, O₁₂₅, O₁₂₈, O₁₄₄ and O₁₆₆ have the ability to make biofilms on the inner walls of the glass tubes after crystal violet staining and these results were similar to that of Kot et al. (2016) who reported the ability of E. coli strains to make biofilm in 81.1% of the isolates and differ from the results of Marhova   et al. (2010) as biofilms were detected in vitro from 24% of investigated E.coli strains only.

From the results of PCR for detection of virulence and resistant genes we found that, the more resistant and more virulent strains and biofilm forming strains which disagreed with the results of Pavlickova et al. (2017) who reported that, the highest prevalence of antibiotic resistance was observed in weak biofilm producers. Biofilm formation was not statistically associated with any virulence determinant. In this study we found that, all multi resistant strain were sensitive to olive leaf extract and these results were agreed with the results reported byLiu et al. (2017) who demonstrated that at a concentration of 62.5mg/ml, OLE almost completely inhibited the growth of E. coli, so it will be good for controlling the resistant strains of E. coli.

CONCLUSION

In this study we found that, all multi resistant strain were more virulent strains also more biofilm forming strains. Moreover, all multi resistant strain were sensitive to olive leaf extract.

REFERENCES

Abd El Tawab, A.; Ammar, A.; Soad, N. and Reem, M. (2015): Prevalence of E. coli in diseased chickens with its antibiogram pattern. Benha Veterinary Medical Journal, Vol. 28, NO. 2: 224‐230

Ahmed, A. and Abolghait, S. (2014): Antibacterial effect of olive (Olea europaea L.) leaves extract in raw peeled undeveined shrimp (Penaeus semisulcatus). International vet. Journal, 2(1): 53-56.

Akond, M.; Hassan, S.; Alam, S. and Shirin, M. (2009): Antibiotic resistance of Escherichia coli isolated from poultry and poultry environment of Bangladesh. American J. Environ. Sci. 5(1): 47-52.

Alhaj, N.; Mariana1, N.; Raha, A. and Ishak, Z. (2007): Prevalence of Antibiotic Resistance among Escherichia coli from Different Sources in Malaysia. International Journal of Poultry Science 6 (4): 293-297.

Alimehr, M.; Sadeghi-Hashjin, G.; Pourbakhsh, A. and Nofouzi, K. (1999): Isolation, Identification and in vitroSusceptibility of Avian Escherichia coli to Selected Fluoroquinolones. Arch. Razi Ins. 50, 77-82.

Amara, A.; Ziani, Z. and Bouzoubaa, K. (1995): Antibiotic resistance of Escherichia coli strains isolated in Morocco from chickens with colibacillosis. Vet. Microbiol., 43: (4) 325-330.

Anthonia Olufunke Oluduro (2012): Antibiotic Resistant Commensal Escherichia coli in Faecal Droplets from Bats and Poultry in Nigeria. Veterinaria Italiana 48(3), 297-308.

Aprikian, P.; Tchesnokova, V.; Kidd, B.; Yakovenko, O.; Yarovoy, V.; Trinchina, E.; Vogel, V.; Thomas, W. and Sokurenko, E. (2007): interdomain interaction in fimh adhesion of Escherichia coli regulates the affinity to mannose, j boil chem, 282, 23437.

Arnqvist, A.; Olse´n, A. and Normark, S. (1994): Dependent growth-phase induction of the csgBApromoter in Escherichia coli can be achieved in vivo by _70 in the absence of the nucleoid-associated protein H-NS. Mol. Microbiol. 13:1021–1032.

Barnes. J. and Gross, B. Colibacillosis. In: Calnek, W.; Barnes, J.; Beard, W.; McDougald. M. and Saifin, M. (1999): editors. Diseases of poultry. Ames: Iowa State University Press. p. 131–41.

Cheng, R.; Sun, C.; Xu, S. and Gao, S. (2006): Prevalence of LEE and HPI Pathogenicity Islands of Escherichia coli Isolates from Weaned Piglets in China. Acta Microbiologica Sinica, 46, 368-372.

Cloud, S.; Rosenberger, K.; Fries, A., Wilson, A., Wilson, A. and Odor, M. (1985): In vitro and in vivo characterization of avian E. coli 1-serotypes, metabolic activity and antibiotic sensitivity. Avian Dis., 29(4):1084-1093.

CLSI (2016): Clinical and Laboratory standard Institute, M02- A12, M07- A10, and M11- A8. 

Colom, K.; Pèrez, J.; Alonso, R.; Fernández-Aranguiz, A.; Lariňo, E. and Cisterna, R. (2003): Simple and reliable multiplex PCR assay for detection of bla_TEM,bla_SHV and bla_OXA-1 genes in Enterobacteriaceae. FEMS Microbiology Letters 223, 147-151.

Cookson, A.L.; Cooley, W.A. and Woodward, M.J. (2002): The role of type 1 and curli fimbriae of Shiga toxin-producing Escherichia coli in adherence to abiotic surfaces. Int. J. Med. Microbiol. 292:195–205.

Costerton, W.; Lewandowski, Z.; Caldwell, E.; Korber, R. and Lappin-Scott, M. (1995): Microbial biofilms. In Annual Review of Microbiology, Vol. 49. Ornston, N., Ballows, A., and Greenberg, P. (eds). Palo Alto, CA: Annu Rev Inc, pp. 711–745.

Cruickshank, R.; Duguid, J.; Marmion, B. and Swain, R. (1975): Medical Microbiology "the practice of Medical Microbiology" 12^th Ed., Churchill Livingstone, Edinbrough, London and New-York.

Edwards, R. and Ewing, H. (1972): Identification of Enterobacteriacae. Minneapolis, Burgess Publishing Co., PP. 709.

Eftekharian, S.; Ghorbanpoor, M.; Seyfi Abad Shapouri, M.; Ghanbarpour, R.; Jafari, R. and Amani, A. (2016): Frequency of selected virulence-associated genes in intestinal and extra-intestinal Escherichia coli isolates from chicken.Iranian Journal of Veterinary Medicine, 10 (2):91-96.

EL-Sukhon, N.; Asad, M. and Al-Attar, M. (2002): Studies on the bacterial aetiology of airsacculities of broilers in Northern and Middle Jordan with special reference to E. coli. Avian Dis., 46(3): 605-612.

Filali, E.; Bell, G.; El Houadfi, M.; Huggins, B. and Cook, K., (1988): Antibiotic resistance of Escherichia coli strains isolated from chickens with colisepticaemia in Morocco. Comparative immunology, microbiology and infectious diseases, 11(2), pp. 121-124.

Finegold, M. and Martin, T. (1982): Diagnostic Microbiology. 6th ed., the C.V. Mosby Company, St. Louis, Toronto, London.

Ghanbarpour and Salehi (2010): Determination of Adhesin Encoding Genes in Escherichia coli Isolates from Omphalitis of Chicks. American Journal of Animal and Veterinary Sciences 5 (2): 91-96.

Ghanbarpour, R.; Sami, M.; Salehi, M. and Ouromiei, M. (2011): Phylogenetic background and virulence genes of Escherichia coli isolates from colisepticemic and healthy broiler chickens in Iran. Trop Anim Health Prod. 43:153-7.

Ghosh, P.; Mahanti, A.; Samanta, I.; Joardar, S.; Batab Yal, K., Dey, S.; Taraphder, S. and Isore, D. (2017): Occurrence of extended-spectrum cephalosporinase producing Escherichia coli in kuroiler birds. Vet. arhiv 87, 745- 757.

Hannan, A.; Saleem, S.; Chaudhary, S.; Barkaat, M. and Arshad, M. (2008): Anti-Bacterial Activity of Nigella Sativa against Clinical Isolates of Methicillin Resistant Staphylococcus aureus. J Ayub Med Coll Abbottabad; 20(3): 72-74

Idrees, M.; Shah, M.; Michael, S.; Qamar, R. and Bokhari, H. (2011): Antimicrobial Resistant Escherichia coli Strains Isolated from Food Animals in Pakistan. Pakistan J. Zool., vol. 43(2), pp. 303-310.

Jefferson, K. (2004): What drives bacteria to produce abiofilm? FEMS Microbiol. Lett. 236(2), pp.163–73.

Johnson, R.; Murray, C.; Gajewski, A.; Sullivan, M.; Snippes, P.; Kuskowski and A. Smith, E. (2003): Isolation and molecular characterization of nalidixic acid-resistant extraintestinal pathogenic Escherichia coli from retail chicken products. Antimicrob Agents Chemother. 47(7): 2161-8.

Kilic, A.; Ertafi, H.; Muz, A.; Ozbey, G. and Kalender, H. (2007): Detection of the eaeA Gene in Escherichia coli from Chickens by Polymerase Chain Reaction. Turk. J. Vet. Anim. Sci. 31(4): 215-218.

Kmeta, V. and Kmetovab, M. (2010): High Level of Quinolone Resistance in Escherichia coli from Healthy Chicken Broilers. Folia Microbiol. 55 (1), 79–82.

Kolar, M.; Bardon, J.; Sauer, P.; Kesselova, M.; Cekanova, D. and Hejnar, P. (2005): Fluoroquinolone-Resistant Escherichia coli and Proteus mirabilis in Poultry of Middle Moravia, Czech Republic. Acta Vet. Brno, 74: 249–253.

Kot, B.; Wicha, J.; Grużewska, A.; Piechota, M.; Wolska, K. and Obrębska, M. (2016): Virulence factors, biofilm-forming ability, and antimicrobial resistance of urinary Escherichia coli strains isolated from hospitalized patients. Turk J Med Sci, 46: 1908-1914.

Liu, Y.; C. Mckeever, C. and Malik, N. (2017): assessment of the Antimicrobial Activity of Olive Leaf Extract against Food borne Bacterial Pathogens. Front. Microbiol. 8:113.

Maram, M. Tawakol (2011): Studies on Salmonella on Chicken. M.V.Cs thesis, Bacteriology, Immunology and Mycology Department, Faculty of Vet. Med. Mansoura Univ.

Maram, M. Tawakol (2014): Molecular typing and detection of virulence and antibiotic resistant genes in E. coli isolated from chickens. Benha University, Faculty of Veterinary Medicine, pp.101.

Marhova, M.; Kostadinova, S. and Stoitsova, S. (2010): Biofilm-Forming Capabilities of Urinary Escherichia coli Isolates. Biotechnology & Biotechnological Equipment, 24:sup1, 589-593.

Miles,D.; Mclaughlin, W. and  Brown, P. (2006):Antimicrobial resistance of Escherichia coli isolates from broiler chickens and humans. Bmc vet res. Vol: (2) 2-7.

Momtaz, H.; Rahimi, E. and Moshkelani, S. (2012): Molecular detection of antimicrobial resistance genes in E. coli isolated from slaughtered commercial chickens in Iran. Veterinarni Medicina, 57 (4): 193–197.

Morad, R. (2013): Antibioresistance Profile of Avian pathogenic Escherichia coli Isolates Recovered from Broiler Chicken Farms with Colibacillosisin Kermanshah Province, Iran. Global Veterinaria 10 (4): 447-452.

Nataro, P. and Kaper, B. (1998): Diarrheagenic Escherichia coli. Clin. Microbiol. Rev., 11: 142-201.

Ngeleka, M.; Kwaga, P.; White, G.; Whittam, S.; Riddell, C.; Goodhope, R.; Potter, A. and Allan, B. (1996): Escherichia coli cellulitis in broiler chickens: Clonal relationships among strains and analysis of virulence-associated factor of isolates from diseased birds. Infect. Immun. 64: 3118-3126.  

Ogunleye, A.; Oyekunle, M. and Sonibare, A. (2008): Multidrug resistant Escherichia coli isolates of poultry origin in Abeokuta, South Western Nigeria. Veterinarski Arhiv78 (6), 501-509.

Otaki, Y. (1995): Poultry disease control programme in Japan. Asian Livestock, 20, 65-67.

Pacholewicz, E.; Liakopoulos, A.; Swart, A.; Gorte Maker, B.; Dierikx, C.; Havelaar, A. and Schmitt, H. (2015): Reduction of extended-spectrum-β lactamase- and AmpC-β-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses. Int. J. Food. Microbiol. 215, 57-63.

Pavlickova, S.; Klancnik, A.; Dolezalova, M.; Mozina, S. and Holko, I. (2017): Antibiotic resistance, virulence factors and biofilm formation ability in Escherichia coli strains isolated from chicken meat and wildlife in the Czech Republic. Journal of environmental science and health, part b, Vol (52).

Prigent-Combaret, C.; Brombacher, E.; Vidal, O.; Ambert, A.; Lejeune, P.; Landini, P. and Dorel, C. (2001): Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene. J. Bacteriol. 183:        7213–7223.

Quinn, J.; Markey, K.; Carter, E.; Donnelly, J. and Leonard, C. (2002): Veterinary Microbiology and Microbial Diseases. Black well scientific publications, Oxford, London.

Ramadan, H.; Awad, A. and Ateya, A. (2016): Detection of phenotypes, virulence genes and phylotypes of avian pathogenic and human diarrheagenic Escherichia coli in Egypt. J Infect Dev Ctries, 10(6): 584-591.

Robert, H.; John, K.; Pawin, P.; Keiko, H. and Christina, Z. (2002): Prevalence of Salmonella and E. coli, and Their Resistance to Antimicrobial Agents, In Farming Communities in Northern Thailand. Southeast Asian J Trop Med Public Health. Vol (33): 120-126.

Roberts, C. (2012): Antibiotic Discovery and Development. Acquired Tetracycline Resistance Genes. Springer, pp. 543-568.

Römling, U. (2002): Molecular biology of cellulose production in bacteria. Research in Microbiology 153: 205-212.

Ružauskas, M.; Šiugždinienė, R.; Krikštolaitis, R.; Virgailis, M. and Zienius, D. (2010): Prevalence and Antimicrobial Resistance of E. coli Isolated from Chicken Liver Sold in Retail Markets. Vet. Med. Zoot. T. 52 (74)   67-72.

Salehi, Z. and Bonab, F. (2006): Antibiotics Susceptibility Pattern of Escherichia coli Strains Isolated from Chickens with Colisepticemia in Tabriz Province, Iran. International Journal of Poultry Science 5 (7): 677-684

Schembri, A.; Givskov, M.  and Klemm, P. (2002): An attractive surface: gram-negative bacterial biofilms. Sci STKE, RE6.

Schembri, M.; Kjaergaard, K. and Klem, P. (2003): global gene expression in Escherichia coli biofilms. Mol microbial, (48): 253-267.

Sharada, R.; Ruban, S. and Thiyageeswaran, M. (2010): Isolation, Characterization and Antibiotic Resistance Pattern of Escherichia coli Isolated from Poultry. American-Eurasian Journal of Scientific Research 5(1): 18-22.

Srinivasan, P.; Balasubramaniam, G.; Murthy, T. and Balachandran, P. (2013): Bacteriological and pathological studies of egg peritonitis in commercial layer chicken in Namakkal area. Asian Pac J Trop Biomed, 3(12): 988-994.

Swayne, D.; Charman, E.; Glisson, J.; Jackwood, M.; Pearson, E. and Reed, M. (1998): Colibacillosis: A laboratory manual for the Isolation and identification of avian pathogens, fourth edition. Am. Assoc. Avian Pathol. 14-16.

Trkov, M.; Rupel, T.; Bertok, D.; Trontelj, S.; Avgustin, G. and Ambro, J. (2014): Molecular Characterization of Escherichia coli Strains Isolated from Different Food Sources. Food Technol. Biotechnol. 52 (2) 255–262.

Vandekerchove, D.; Vandemaele, F.; Adriaensen, C.; Zaleska, M.; Hernalsteens, P.; De Baets, L.; Butaye, P.; Van Immerseel, F.; Wattiau, P.; Laevens, H.; Mast, J.; Goddeeris, B. and Pasmans, F. (2005): Virulence-Associated Traits in Avian Escherichia coli: Comparison between Isolates from Colibacillosis-Affected and Clinically Healthy Layer Flocks. Veterinary Microbiology, 108, 75-87.

Wang, J.; Stanford, K.; Mcallister, T.; Johnson, R.; Chen, J.; Hou, H.; Zhang, G. and Niu, Y. (2016): Biofilm Formation, Virulence Gene Profiles, and Antimicrobial Resistance of Nine Serogroups of Non-O157 Shiga Toxin–Producing Escherichia coli. Foodborne Pathogens and Disease Vol. 13, No. 6

Yin, B.; Zhu, L.; Zhang, Y.; Dong, P.; Mao, Y.; Liang, R.; Niu, L. and Luo, X. (2018): The characterization of biofilm and detection of biofilm related genes in Salmonella isolated from beef processing plants. Foodborne pathogens and disease, Vol. 15, No. 10

Zibandeh, S.; Sharifiyazdi, H.; Asasi, K. and Abdi-Hachesoo, B. (2016): Investigation of tetracycline resistance genes in Escherichia coli isolates from broiler chickens during a rearing period in Iran.  Veterinarski Arhiv 86 (4), 565-572.