Multidrug Resistance among Gram Negative E. coli in Chicken Meat (Rafha-Saudi Arabia)

Aims: Multidrug resistant (MDR) bacteria pose a major public health issuer globally. The genes for antibiotic resistance are transferred vertically in the form of genomic DNA and horizontally in the form of plasmids or transposons. Antibiotic are extensively used in animal farming to treat and prevent animal diseases, and at sub-therapeutic doses, they are used to promote animal growth. This extensive use of antibiotics is causing an increase in resistance among bacteria. More frequent, chicken meat available at retail shops is reported to be contaminated with a variety of drug resistant bacteria including E. Coli. The aim of the present study was to investigate antibiotic resistance in Escherichia coli strains isolated from chicken meat available in the local shops of Rafha, Saudi Arabia. Place and Duration of Study: Department of basic health sciences, College of Pharmacy, Northern Border University, Rafha, Saudi Arabia, between February and October, 2019 Methodology: Eighty-six E. coli strains, isolated from chicken meat, were tested for their antibiotic resistance profile, using the disc diffusion method. Results: All the isolated E. coli strains were tested against 14 antibiotics. The maximum resistance was found against penicillin G (95%) followed by amoxicillin (85%), Cephalothin (81%), Erythromycin (72%), and Tetracycline (50%). Imipenem was the most effective agent of all with only 1% resistance followed by Cefepime with almost 6% resistance. A high percentage of the isolates (57%,) were multidrug resistant as they were non—susceptible to at least one antimicrobial in ≥3 antimicrobial classes including amoxicillin, erythromycin and tetracycline. Conclusion: The prevalence of MDR E. coli in retail chicken meat is very high and could pose a serious threat to public health. Original Research Article Ali and Chidrawar; JPRI, 33(58A): 38-48, 2021; Article no.JPRI.78567 39


INTRODUCTION
Antibiotic are used in the husbandry of livestock for many different purposes. They are used therapeutically for the treatment of various infectious diseases, for the metaphylactic treatment of a herd of animals when one of them gets disease, and for the prophylactic treatment against any anticipated disease. The antibiotics are also used at sub-therapeutic dose in animal food and water to promote animal growth and to improve efficiency of the food [1]. New FDA Veterinary Feed Directive has recommended stopping any such use [2]. This exercise has already been banned in Europe since 2006 [3].
The excessive misuse of antibiotics or antimicrobials kills the susceptible bacteria leaving behind only the resistant ones which increase in number overtime [4]. Bacteria acquire resistance through many ways. Some bacteria have natural resistance against some antibiotics; some acquire genetic mutations which make them resistant to some antibiotic while some others receive resistant genes present on plasmid from other bacteria [5]. Selective pressure of antibiotic overuse in human and animal has resulted in the propagation of antibiotic resistance bacteria which is linked with the attainment and spread of resistant genes among such bacteria. In this context, the probiotic bacterial strains are also considered to attain various antibiotic resistant genes and may transfer them to pathogenic bacterial strains [6].
Foods from animal origin, predominantly chicken and meat are found to be associated with foodborne diseases. Chicken may be contaminated with many different types of microorganisms including bacteria [7]. Many studies have reported the drug resistant E. coli in poultry [8], egg [9], milk [10] and raw meat [11]. Raw chicken is reported to possess highest percentages of drug resistant E. coli and Salmonella spp. [12]. Gram-negative bacteria especially Enterobacteriaceae organisms including E. coli and Salmonella are mostly reported to produce β-lactamase enzymes. These enzymes break βlactam ring of the β-lactam antibiotics. More than 1000 different types of β-lactamase enzymes have been reported so far [13]. Drug  E. coli is the most commonly found gramnegative pathogen in human colon and is the most common cause of UTIs. It also causes diarrhea and bacteremia [25]. Drug resistant E. coli strains can transfer antibacterial resistant genes not only to other E. coli strains but also to other common GIT bacteria [26]. Therefore, it is important to study multidrug resistance in E. coli found in food including chicken meat.
The aim of current study was to study the spectrum of drug resistance among E. coli isolated from chicken meat in Rafha, Saudi Arabia.

Sample Collection
The study was done at the Department of basic health sciences, College of Pharmacy, Northern Border University, Rafha city of Saudi Arabia, between February and October, 2019. Fresh and frozen chicken meat samples (sterile swabs) were collected in appropriate sterile containers from different chicken shops in Rafha and were immediately transported to the laboratory for bacteriological investigation.

Bacteriological Analysis
The samples were streaked onto MacConkey agar plates and incubated aerobically at 37 o C for 24 hours. The isolates which fermented lactose within 24 hours on MacConkey agar were further identified by PCR. For that purpose, isolated pink colonies were carefully picked and used for PCR identification of E. coli as well as disc diffusion method for antibiotic sensitivity testing. DNA (Fig. 1).

The Antibiotic Sensitivity Testing
The in vitro antimicrobial sensitivity testing for the E. coli isolates was carried out using the disc diffusion method. Multidrug resistance was defined as resistance to three or more classes of antibiotics. The antibiotic discs used in this study (Bioanalyse R 50 susceptibility discs) with their potency and standard zones of inhibition are given in Table 2. The results were analyzed using simple percentage prevalence values to find out the prevalence of drug resistant among isolated E. coli.

RESULTS
A total of 86 E. coli positive samples were included in the study. The E. coli strains isolated from all 86 chicken samples were tested against all the 14 antibiotics included in the study. The antibiotics used with potency and standard zone diameters for the interpretation of the results are given in Table 2. All the isolates were resistant to at least one or more antibiotics. The number of resistant, intermediate and susceptible isolates against various antibiotics found in this study are given in Table 3.

Frequency of Resistance
All isolates were resistant to more than one antibiotics. The number of isolates resistant to six different antibiotics was the maximum. Overall, equal or more than 8 isolates were resistant against 3 to 8 antibiotics. Number of isolates resistant to more than eight or less than three antibiotics was less than four (Fig. 3).

Multi drug Resistance
An isolate is defined as multidrug resistant if it is non-susceptible to at least one antimicrobial in ≥3 antimicrobial classes. A larger percentage of the isolates (57%,) were multidrug resistant as they were non-susceptible to amoxicillin, erythromycin and tetracycline. Among those resistant to these three classes, higher percentage was also resistant to Cotrimoxazole (68%), chloramphenicol (62%), ciprofloxacin (60%), and Gentamicin (56%) separately. A considerable number of the isolates were resistant to 5 or more different classes of antimicrobial agents (Table 4).

Tetracycline Susceptible Isolates
Since tetracycline is one of the most commonly used antibiotics used in chicken growth promotion, the data was further analyzed to see multidrug resistance against tetracycline susceptible isolates. Number of E. coli isolates susceptible to Tetracycline was 32 (37%). These isolates were also susceptible to Chloramphenicol (100%), Imipenem (100%),

DISCUSSION
Antibiotics are given to chicken and other animals at sub-therapeutic doses for growth promotion, which according to  Beta-lactam antibiotics, such as penicillins and cephalosporins are among the most commonly used antibiotics in chicken and most commonly prescribed antibiotics against bacterial infections in human [37,38]. As compared to Gram-positive bacteria, Gram-negatives usually have stronger intrinsic resistance against some antibiotics. This is because of the composition and complexity of their outer membrane and inner cell wall. The outer membrane of gram negative bacteria is made of lipopolysaccharides (LPS) and phospholipids. The inner cell wall is made of a single layer of peptidoglycans. Penicillin G is not able to penetrate the outer layer in gram negative bacteria [39]. In the current study, maximum resistance was observed against penicillin drugs especially Penicillin G (95%) which is quite understandable. The results are in accordance with other studies [40,41,42]. Resistance to Amoxicillin, Cephalothin (first generation cephalosporin) and erythromycin were also high, above 70% (85%, 81% and 72% respectively) and may be because of selective pressure due to over use of these antibiotics in poultry. These results aligned with the finding from other groups [43,44].
Interestingly, while studying AMR among Enterococcus species from food animal origin, Liu et al, 2013, [45] found similar resistance (72.8%) against erythromycin. Higher levels of resistance against amoxicillin and erythromycin among E. coli isolates were also reported in Bangladesh [46].
Imipenem and Cefepime were the most effective antimicrobials against the E. coli isolates found in this study. Similar results were reported in another study where zero resistance to Imipenem was observed [52]. On the contrary, amazingly higher level of resistance was reported against carbapenems including Imipenem (47.7%) and meropenem (41.9%) in Bangladesh. However, they could not give any clear explanation for these results as these antibiotics are not used in poultry farms in Bangladesh [53]. The same study also reported an unexpectedly high resistance against Cefepime (72.1%). Similarly, astonishingly higher resistance was reported against Cefepime (95.8%) in another study [54]. Close to the results reported by us, a study done in turkey reported zero resistance against Imipenem and Cefepime [55].
Two mechanisms may be responsible for Multidrug resistance in bacteria. First, bacteria may accumulate plasmids having multiple drug resistant genes. Second, they may have genes on their chromosome which code for the multidrug efflux pumps, removing the drug out of the cell. Whichever the mechanism be involved, the extensive unnecessary use of antimicrobials in poultry is the main cause of the development of multidrug resistance in bacteria especially E. coli. For multidrug resistant analysis, three antimicrobial classes amoxicillin, erythromycin and tetracycline were selected. Fifty-seven percent of the isolates were resistant to these three classes of antibiotics. Some of these were resistant to other classes of antibiotics as well. Some other studies have reported even much higher incidence of MDR in E. coli in chicken (94%) in India [56] and in Nepal (80.0%) [57]. In a combined study from China and Sudan, MDR was 80% and 54.4% respectively [58]. However, they defined multidrug-resistance as isolates showing resistance to two or more antimicrobial classes.

CONCLUSION
While all isolates in our study were resistant to two or more than two drugs, seventy-three isolates were resistant to 3-8 antibiotics, with some resistant to even more (9-12) antibiotics. These results indicate that chicken meat in the city is heavily contaminated with multidrug resistant E. coli which is due to the extensive use antibiotics in poultry farming. Authorities in the ministries of health and food must take notice of this and take necessary measures to reduce the extensive use of antibiotics in poultry.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.