Antimicrobial resistance and molecular characterization of pathogenic E . coli isolated from chickens

ARTICLE INFO Forty eight clinically diseased broiler chickens from different (n=18) farms from Beni Suef and El-Fayoum Governorates were subjected to euthanasia and post mortem examination. Lesions include airsaculitis, pericarditis and perihepatitis. Bacteriological examination showed that 22.9% of isolates were E. coli positive. Serogrouping of isolates revealed O125, O112, O91, O157, O115 and O25 % of each serogroups with an incidence rate of 18.2%, 9.1%, 9.1%, 9.1%, 9.1%, and 9.1%, respectively and four strains were untyped. Antimicrobial sensitivity tests against 17 antimicrobials showed that the most common resistance patterns were against penicillin, lincomycin, oxytetracycline, clindamycin, amoxycillin and erythromycin followed by nalidix acid and trimethoprim. On the other hand, the most potent antimicrobials were colistin sulphate, gentamycin, doxycycline and ceftriaxone followed by enrofloxacin, ciprofloxacin, norfloxacin, chloramphenicol and lastly ampicillin. PCR showed that all isolates had Blactam resistant gene (blaTEM) and tetracycline resistance gene A (tetA) but only 18 % have quinolones resistance gene A (qnrA). Article history:


Introduction
E. coli is a member of the family Enterobacteriaceae. It is a normal inhabitant in the digestive system of chickens. Stress factors like cold weather, dry dusty conditions, contaminated environment, over crowdedness, feed/water restriction, temperature extremes, poor ventilation or other stresses lead to E. coli infection associated with high mortality, reduced weight gain and consequently condemnation of birds at the slaughter, Kaul et al., (1992). E. coli strains were classified by Russo and Johnson, (2000) into three major groups: commensal strains, intestinal pathogenic strains, and extra-intestinal pathogenic E. coli (ExPEC) strains.
Some strains of E. coli produce a toxin called Shiga toxin which damages the lining of the intestine. The strains of E. coli that make this toxin are sometimes called Shiga toxinproducing E. coli (STEC). O 157 :H 7 E. coli strain can induce dangerous diseases in humans.
It causes abdominal cramps, vomiting, and bloody diarrhea. It is the leading cause of acute kidney failure in children. It can also cause lifethreatening manifestations such as adult kidney failure, bleeding, confusion, and seizures (Wasey and Salen, 2018). Although antimicrobials are considered a very important tool for the treatment of clinical disease and maintaining birds 'health and productivity, antimicrobial uses have been implicated as a risk factor in the dissemination and development of drug resistance (Gosh and LaPara, 2007). Increasing antimicrobial resistance is an important public health concern, and the emergence and spread of antimicrobial resistance is a complex problem driven by numerous interconnected factors. In-vitro antimicrobial susceptibility testing of veterinary pathogens can provide valuable guidance to the veterinarian in the choice of appropriate chemotherapy (Radwan et al., 2016). Moreover, it is very useful to detect the multidrug resistant isolates. Therefore, the appropriate antibiotic should better be selected on the basis of its sensitivity which could be detected by laboratory examination.
Since the introduction of antibiotics, there has been a tremendous increase in the resistance in diverse bacterial pathogens (Gold and Moellering, 1996). The resistance of E. coli species to antimicrobials is widespread and of concern to poultry veterinarians. This increasing resistance has received considerable national and international attention. Plasmids were the major vector in the dissemination of resistance genes through the bacterial population (Smalla et al., 2015;San Millan, 2018). There is a wide variety of E. coli being resistant to more than one antimicrobial agent, so we can use polymerase chain reaction (PCR) to detect antimicrobial resistance genes in E. coli isolates.
The aim of the present work was directed to study the antimicrobial resistance and molecular characterization of pathogenic E. coli isolated from chickens.

Chicken samples
A total of 48 clinical samples were collected aseptically from air sacs, liver and pericardium from chickens of different ages (2-5weeks) from flocks suspected to be infected with E. coli.

Bacteriological examination
MacConkey's broth, MacConkey bile salt lactose agar medium, eosine methylene blue, tryptone soya agar and semi-solid agar were used for bacteriological examination.

Microscopical examination
It was carried out using Gram's stain as described by Quinn et al., (2002) for morphological study.

Biochemical identification of the bacterial isolates
It was performed according to Quinn et al. (2002) including oxidase test, catalase test, indole production, methyl red test, Voges Proskauer test, citrate utilization test, sugar fermentation test, hydrogen sulphide (H 2 S) production on TSI medium and urea hydrolysis test.

Detection of antimicrobial resistant genes of E. coli by PCR
DNA Extraction was performed using QIA amp DNA mini kit according to the manufacturer's instruction. DNA was used for PCR (Sambrook et al., 1989) for detection of the target genes (blaTEM, tet A and qnrA). PCR products were separated by gel electrophoresis and visualized using U.V illuminator.

Prevalence of E. coli in the examined broiler chickens
Out of 48 samples taken from diseased broiler chickens from 18 different broiler farms from Beni-Suef and El-Fayoum Governorates, 11 E. coli isolates were recovered with a prevalence rate of 22.9%.

Antimicrobial sensitivity testing of E. coli isolated from broiler chickens
Results of in-vitro antimicrobial sensitivity testing of E. coli isolates are demonstrated in table (3).
MDR was detected in all E. coli isolates (100%). All isolates were MDR to at least 8 antimicrobials up to 16 antibacterial agents.

Antimicrobial resistance genes detection by PCR
PCR was applied on 11 isolates of MDR E. coli isolates to detect three antimicrobial resistant genes including β-lactam resistance gene (blaTEM), quinolones resistance gene A (qnrA) and tetracycline resistance gene A (tetA).
The results illustrated in the table (4) revealed that 100 % of the tested E. coli isolates harbored both blaTEM and tetA genes while only two isolates (18.2%) harbored qnrA gene.

Discussion
In this study the prevalence of E. coli in 18 different broiler farms from Beni Suef and Fayoum governorates during the year 2017 was about 22.9 %. Nearly similar incidence (23.5%) in El-Sharkia and (30%) in Alexandria were reported by Roshdy et al., (2012). Higher prevalences (91.8%) were detected by Derakhshanfar and Ghanbarpour, (2002); (82 %) by Jakaria et al., (2012) and (58%) by Akond et al., (2009). The differences in the prevalence rates of E. coli may be due to the difference in the season (Ashraf et al., 2015), difference in biosecurity or hygienic conditions from area to area and from farm to farm as well as the pathogenicity, and virulence of the strains and the immunological status of the flock.
In the present study, the percent of untyped E. coli was 36 % which to some extent agrees with that obtained by Allan et al., (1993) 39 % while differs from that obtained by Amer et al., (2018) 15 %.
Antimicrobials are usually used for treating infected animals and humans and also for prophylaxis and growth promotion of food producing animals. So inadequate selection and abuse of antimicrobials are the main causes of the emergence of resistance among various bacteria and this makes the treatment of bacterial infections more difficult (Aarestrup et al., 2008).
As commensal bacteria, E. coli is considered a reservoir of resistance genes for pathogenic bacteria; their resistance level is considered a good indicator for selection pressure by antibiotic use and for resistance problems to be expected in pathogens (Murray, 1992). Resistant E. coli strains can transfer antibiotic resistance determinants not only to other E. coli strains, but also to other bacteria within the gastrointestinal tract and to acquire resistance from other organisms (Österblad et al., 2000).
Resistance to ciprofloxacin was 81.8 % of tested isolates. This result goes hand with those obtained by Akond et al., (2009), 82 % and to some extent with that 79 % obtained by Yang et al., (2004). Lower resistance 67 % was reported by Salehi and Bonab, (2006); 30 % by Islam et al., (2008) and 19 %, as rarely used in poultry farming in Kenya, by Adelaide et al., (2008). On the other hand, high sensitivity to ciprofloxacin as 100 % was reported by Saidi et al., (2012) and 74.29 % by Sahoo et al., (2012). Also, Jakaria et al., (2012) found that E. coli isolates were highly sensitive to ciprofloxacin in his study.
In this study, Enrofloxacin, ciprofloxacin and norfloxacin gave the same results of resistance and sensitivity on the same isolates.
Resistance to fluoroquinolones was high which goes hand with the previous study of Rahimi, (2013), also (Li et al., 2005) reported that resistance to fluoroquinolones ranged from 57.1 % to 66.7 %.
All isolates tested for clindamycin were resistant, also (Amer et al., 2018) found that 80 % of isolates were resistant to clindamycin.
In this study, 45.5 % of isolates tested for ceftriaxone resistance were resistant. This result differs from that 2.4 % obtained by Tadeese et al., (2012). On the other hand, 91.43 % sensitivity was reported by Sahoo et al., (2012).
Colistin sulphate has the lowest rate of resistance (36.4 % of isolates were resistant). The highest rate of sensitivity was against this drug with 63.4 % sensitivity. This result goes hand with that found by Salehi and Bonab, (2006); Zakeri and Kashefi, (2012) both mentioned that resistance to colistin was low, also Messaï et al., (2013) found that 5.5 % of isolates were resistant to colistin. On the other hand Saberfar et al., (2008) found resistance in 99 % of isolates.
Meanwhile, 45.5 % of isolates were resistant to gentamycin. This result goes hand with the resistance of 46.6 % reported by Abd El Tawab et al., (2015) and to some extent with resistance of 50 % found by Islam et al., (2008) and 55 % obtained by Amer et al., (2018). Lower rate of resistance to gentamycin 39 % was published by Li et al., (2005); 14 % by Gregova et al., (2012); 12 % by Saberfar et al., (2008) and 5.5 % by Messaï et al., (2013). In this Study Gentamycin was the second potent drug after colistin against the E. coli isolates, There were many studies which discussed efficacy of Gentamycin on E. coli serotypes as sensitivity of 97.1 % found by Saidi et al., (2012); 85.72 % by Sahoo et al., (2012); 80 % by Akond et al., (2009) and also majority of isolates were sensitive to gentamycin as reported by Jakaria et al., (2012).
In this study, the most common resistance patterns were against penicillin, lincomycin, oxytetracycline, clindamycin, amoxycillin and erythromycin followed by nalidix acid and trimethoprim. On the other hand, the most potent antimicrobials were colistin sulphate, gentamycin, doxycycline and ceftriaxone followed by enrofloxacin, ciprofloxacin, norfloxacin, chloramphenicol and lastly ampicillin.
Multidrug resistance in E. coli has become a worrying issue that is increasingly observed in human and also in veterinary medicine worldwide (Poirel et al., 2018). In this study, there was multiple drug resistance to at least 8 antimicrobial up to 16 antimicrobials out of a total of 17 tested antimicrobials. No isolate was resistant to all antimicrobials used but there were 2 isolates which were intermediate sensitive to only one antimicrobial which either doxycycline or ceftriaxone. Such bacterial species has a great capacity to accumulate resistance genes, mostly through horizontal gene transfer) Poirel et al., 2018).
Efflux proteins have been the best studied of the Tet determinants and tetA, tetB, tetC, tetD, tetE, tetG, tetH, tetK, tetL, tetA(P) and otrB genes have been identified. All of these genes code for energy-dependent membrane-associated proteins that export tetracycline out of the cell. The export of tetracycline reduces the intercellular concentration of tetracycline and thus protects the bacterial ribosomes. Efflux genes are found in both Gram-positive and Gram-negative species. The Gram-positive genes tetK, tetL and tetA(P) and the Gram-negative genes tetA, tetC, tetD, tetE, tetG, tetH code for efflux proteins which confer resistance to tetracycline but not minocycline. In contrast, the Gram-negative tetB gene codes for an efflux protein which confers resistance to both tetracycline and minocycline (Roberts, 1996). Guay et al., (1994) suggested that the TetB protein's ability to confer minocycline resistance was not due to the TetB protein substrate specificity for minocycline, but rather TetB was a better pump when compared with other Tet efflux pumps. In the current study, all isolates have been found to carry the tetA(A)gene. β-lactam antibiotics are a class of antibiotics that contain a beta-lactam ring in their molecular structures. This includes penicillin derivativesbandncephalospor ins. Bacterianoftenndevelop resistance to βlactam antibiotics by synthesizing a βlactamase, an enzyme that attacks the β-lactam ring. β-lactam antibiotics are bactericidal, and act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls.
If the bacteria produce βlactamase or penicillinase enzyme, the enzyme will hydrolyze the β-lactam ring of the antibiotic and make the antibiotic ineffective. The genes encoding these enzymes may be inherently present on the bacterial chromosome or acquired via plasmid transfer (plasmid-mediated resistance), and β-lactamase gene expression may be induced by exposure to βlactams.
Β-lactamases from Gram-negative bacteria inactivate penicillins and cephalosporins by hydrolysis, and this is the predominant cause of resistance to these antibiotics. So far, more than 340 β-lactamases have been identified and on the basis of their amino acid sequences, substrate and inhibitor profiles. Gram-negative β-lactamases are divided into the four classes A to D. Class A enzymes, which include the plasmid-encoded broad-spectrum blaTEM and blaSHV families, and class C enzymes, which include the chromosomally encoded cephalosporinases, are the most frequently occurring enzymes (Monstein et al., (2007). In the current study, 100 % of isolates had blaTEM gene.
Since the plasmid-borne quinolone resistance gene qnr was reported in 1998, many additional qnr alleles have been discovered on plasmids or the bacterial chromosome. The plasmid-borne qnr genes currently comprise three families, qnrA, qnrB, and qnrS.
Quinolone resistance in Enterobacteriaceae results mostly from chromosomal mutations in genes coding for DNA gyrase (topoisomerase II), for efflux and outer membrane proteins, or for their regulatory elements (Hooper 2001). DNA gyrase and topoisomerase IV are responsible for decatenation of interlinked chromosomes in the bacterial cells; the key event in quinolone action is reversible trapping of gyrase-DNA and topoisomerase IV-DNA complexes. Complex formation with gyrase is followed by a rapid, reversible inhibition of DNA synthesis, cessation of growth. The 218-amino-acid protein qnrA, which belongs to the pentapeptide repeat family, protects DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones (Tran and Jacoby, 2002). qnrA confers resistance to nalidixic acid and increases MICs (Minimum inhibitory concentration) of fluoroquinolones up to 32-fold (Mammeri et al., 2005). In the current study, 18 % of isolates have qnrA (a plasmid-mediated quinolone resistance determinant).
The large problem associated with E. coli, is the acquisition of genes coding for extended-spectrum β-lactamases which conferring resistance to broad-spectrum cephalosporins, carbapenemases which conferring resistance to carbapenems, 16S rRNA methylases which conferring panresistance to aminoglycosides, plasmidmediated quinolone resistance (PMQR) genes which conferring resistance to fluoroquinolones, and mcr genes which conferring resistance to polymyxins. However, the spread of carbapenemase genes has been mainly recognized in the human sector but poorly recognized in animals (Poirel et al., 2018).
The past 20 years have witnessed major increases in the emergence and spread of multidrug-resistant bacteria and increasing resistance to newer compounds, such as fluoroquinolones and certain cephalosporins (Levy and Marshall, 2004).

Conclusion
Antimicrobial susceptibility testing showed that the most common resistance patterns were against penicillin, lincomycin, oxytetracycline, clindamycin, amoxicillin, and erythromycin followed by nalidixic acid and trimethoprim. On the other hand, the most potent antimicrobials were colistin sulphate, gentamycin, doxycycline and ceftriaxone followed by enrofloxacin, ciprofloxacin, norfloxacin, Chloramphenicol and lastly ampicillin.
Molecular examination for the presence of antimicrobial resistance genes showed that 100 % of isolates have both tet A (A) and blaTEM and 18 % of isolates have qnr.