Detection of Antibiotic Resistance Genes in Escherichia coli Isolated from Chicken Eggs

Total 150 chicken eggs were randomly collected from farms, households, and markets of Rajshahi and Naogaon districts. Isolation and identification of E. coli was done by using cultural and staining characteristics, and biochemical properties. Antibiotic sensitivity assay was also performed for the isolated E. coli by using disc diffusion method. Characterization and detection of antibiotic resistance genes in E. coli isolates were done through PCR analysis. Prevalence of E. coli was 30.00% in chicken eggs in Rajshahi and Naogaon districts. Results of antibiotic sensitivity patterns of the isolated E. coli showed 57.78%, 33.33%, 31.11%, 28.89%, 26.67%, 17.78%, 15.56%, 13.33%, 13.33%, and 11.11% resistance to ciprofloxacin, doxycycline, oxytetracycline, ceftriaxone, amoxycillin, erythromycin, levofloxacin, enrofloxacin, gentamycin, and neomycin, respectively. The isolates also showed 94.44%, 82.22%, 80.00%, 77.78%, 66.67%, 60.00%, 55.56%, 48.89%, 46.67%, and 33.33% sensitive to neomycin, enrofloxacin, erythromycin, levofloxacin, gentamycin, ceftriaxone, oxytetracycline, doxycycline, amoxicillin, and ciprofloxacin, respectively. All 45 phenotypic identified isolates were confirmed as E. coli by PCR analysis. Genotypic identified isolates of E. coli were further characterized by PCR to detect antibiotic resistance genes. Out of 45 E. coli isolates 37 (82.22%) showed tetracycline resistance (tetA ) gene, 32 (71.11%) showed tetracycline resistance (tetB ) gene, and interestingly all 45 (100%) isolates showed quinolone group resistance ( gyrA ) gene by PCR analysis.


Introduction
Poultry industry is a promising sector for poverty elevation in Bangladesh.Poultry is a significant agricultural industry that began in the 1980s (Hoque et al., 1997).The poultry business in Bangladesh predominantly produces chickens, though duck, pigeon, quail, geese, turkey, and guinea fowl are also available.Bangladesh is now producing 15.52 billion eggs versus a current yearly need of 17.13 billion.In Bangladesh, the poultry sector provides 36% of total protein intake through egg and meat consumption.Chicken eggs and meat are the cheapest source of animal protein in Bangladesh and it is accepted by all religious, economic, social, and demographic groups (Simon 2009).Chicken eggs are one of the most important foods of animal origin which contain many nutrients.High-nutritional-value eggs containing omega-3 fatty acids, which are essential for human vision and brain function (Maki et al., 2003).Eggs are also an important source of minerals like phosphorus and iron, and a good source of vitamins like A, D, E, K, and B1, B2, B12, choline, and selenium.
*Corresponding author e-mail: mozaffor@ru.ac.bdDespite their nutritional value, chicken eggs can cause health problems when contaminated with pathogenic bacteria.Eggs can be contaminated with pathogenic bacteria such as Escherichia coli, Salmonella sp., Proteus sp., Listeria sp., Staphylococcus sp., Streptococcus sp., Bacillus sp.(Lee et al., 2016) owing to infected ovaries, and contact with contaminated cages, soil, or water (Svobodova and Tu-mova 2015).Although eggs possess several antibacterial proteins, the level of egg contamination is influenced by several factors such as microbial number in the environment and storage conditions (Scott and Silversides 2000).When the shell cracks, the egg's contents could become contaminated (Neira et al., 2017).It has already been proven that eating contaminated eggs can result in foodborne illnesses (Osimani et al., 2016;Chousalkar and Gole 2018).
Antibiotics are widely used to treat infectious diseases or to promote chicken growth.The usage of antibiotics had facilitated their efficient production and also improved chicken health and well-being by lowering disease incidences.But unfortunately, the unauthorized use of these antibiotics or inappropriate withdrawal period of time before the slaughtering of chickens could lead to the contamination of edible tissues with antibiotics with potential adverse effects on human health (Donoghue 2003).The use of antibiotics to treat bacterial infections without susceptibility testing may lead to the emergence of antibiotic resistance.Antibiotics in poultry are poorly absorbed in the gut and usually excreted without metabolism.Antibiotic resistance continues to be a major issue in human and animal health.Pathogens isolated from food are increasingly showing resistance (Akoachere et al., 2009).Food contamination with antibiotic resistant bacteria can therefore be a major threat to public health, as the antibiotic resistance genes can be transferred to other bacteria (Van et al., 2007;Adesiji et al., 2011).Furthermore, the transfer of these resistant bacteria to humans has public health significance by increasing the number of food-borne illnesses and treatment failure (Adesiji et al., 2011).The indiscriminate use and higher doses of antibiotics are common which eventually accumulates antibiotic residues in the edible tissues of poultry (Kabir et al., 2004;Nisha 2008;Goetting et al., 2011).All antibiotic residues produce potential threats of direct toxicity in humans with possible development of resistant strains due to low and continuous exposure to antibiotics (Nisha 2008).In this regard finding out alternatives of antibiotics are crucially important for the production of safe poultry products.

The Study Area and Sources of Sample
A baseline survey was conducted at farms and households in Rajshahi and Naogaon districts.The baseline survey of 50 farms (commercial layer chicken) and 50 households (indigenous chicken) was conducted randomly in Rajshahi and Naogaon districts with semi-structures questionnaires.After collection of data from these farms and households, total 150 egg samples were collected randomly from selected farms, households and markets, and brought to the Microbiology Laboratory, Department of Veterinary and Animal Sciences, University of Rajshahi for microbiological analysis.

Isolation and Identification of E. coli
After enrichment in nutrient broth or on nutrient agar, Grams staining was performed on a smear of bacterial culture on a sterile slide for morphological study.The culture was also streaked onto EMB agar, MacConkey agar, SS agar, and brilliant green agar which were incubated at 37ºC for 18-24 hours aerobically for the isolation of bacteria.Following that, microorganisms were identified using a variety of biochemical tests using standard methods as described by Merchant and Packer (1967) and Cheesbrough (1985).

Antibiogram assay of Isolated E. coli
The disc diffusion method (Bauer and Kirby 1966) was used to test the sensitivity patterns of the isolated E. coli.In brief, isolated E. coli were cultured into the nutrient broth.Then 200 µl of broth culture was taken by micropipette and placed onto a Mueller Hinton agar (MHA) plate and spread evenly over the entire surface with a sterile glass rod spreader.The predetermined battery of antibiotic discs was dispensed onto the surface keeping a distance of about 1 cm apart.The plate was placed in an incubator at 37 0 C and after 16-18 hours of incubation and examined.The susceptibility test of the E. coli isolates was performed against ten commonly used antibiotic discs including; gentamycin, oxytetracycline, amoxicillin, ciprofloxacin, ceftriaxone, doxycycline, erythromycin, enrofloxacin, levofloxacin, and neomycin (Table 1).Susceptibility zones were measured and interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI 2022).

Genotypic Identification of E. coli Isolates
Extraction and purification of genomic DNA All broth cultures of E. coli were incubated at 37°C for 24 hours.Then 1 ml of cultured broth was taken in eppendrof tubes and centrifuged at 10,000 rpm for 5 minutes.The supernatant was removed, and the pellet was resuspended in 1 ml of sterile distilled water.The eppendrof tubes were vortexed and centrifuged again.The supernatant was removed, and the pellet was resuspended in 200 μl of deionized distilled water.The eppendorf tubes were transferred in boiling water bath and boiled for 15 minutes.Immediately after boiling, the eppendorf tubes were placed into ice and kept 10 minutes for cold shock.The tubes were then centrifuged at 10,000 rpm for 10 minutes.Finally, the supernatant was collected as genomic DNAs.The DNAs were analysed by agarose gel electrophoresis on 1% agarose and visualised using the UV transilluminator.DNA concentrations and purity were determined spectrophotometrically.
Confirmation of E. coli isolates by PCR Primers were F: GACCTCGGTTTAGTTCACAGA and R: CACACGCTGACG CTGACCA, amplicon size 585 bp for E. coli detection was used from the published article.Each PCR assay was carried out with a 25 μl mixture containing 6.5 μl nuclease free water, 12.5 μl Taq master mix (1X), 1.0 μl of each primer (20 nmol/μl) and 3 μl of the DNA template.The PCR assay was done in a thermocycler using the following cycle: after an initial denaturation of five minutes at 95 o C, the reaction mixture was subjected to 35 amplification cycles of one minute at 94 o C, one minute at 55 o C, one minute at 72 o C and final extension of ten minutes at 72 o C. The PCR products were separated by electrophoresis on 1.5 % agarose gel and visualized using UV transilluminator.A 100 bp DNA molecular marker was used to determine the size of the amplicons.

Detection of antibiotic resistance genes in E. coli isolates by PCR
Primers for antibiotic resistance genes such as gentamicin (aac3-IV), tetracycline (tetA, tetB and tetC), betalactams (blaTEM, blaSHV and blaCMY), erythromycin (ereA) and Quinolone (gyrA) were used from the published article (Table 2).Each PCR assay was carried out with a 25 μl mixture containing 6.5 μl nuclease free water, 12.5 μl Taq master mix (1X), 1.0 μl of each primer (20 nmol/μl) and 3 μl of the DNA template.Then the PCR assay was done in a thermocycler using the following cycle: after an initial denaturation of five minutes at 95 o C, the reaction mixture were subjected to 35 amplification cycles of one minute at 94 o C, one minute at 55 o C, one minutes at 72 o C and final extension of ten minutes at 72 o C. The PCR products were separated by electrophoresis on 1.5 % agarose gel and visualized using UV transilluminatior.A 100 bp DNA molecular marker was used to determine the size of the amplicons.

Isolation and Identification of E. coli
All egg samples were analyzed by different staining, cultural and biochemical methods to determine the prevalence of E. coli.On nutrient agar, the development of round, clear, moist, smooth, low convex, and glistening colonies indicated the growth of E. coli (Fig. 1).On MacConkey agar the E. coli isolates produced lactose fermenting bright pink color colonies and on EMB agar produced greenish-black colonies with metallic sheen.The morphology of the isolated E. coli showed a Gram-negative, small rod shaped appearance, arranged in single or paired (Fig. 2).In sugar fermentation tests, all the isolates fermented dextrose, sucrose, lactose, maltose, and mannitol with acid and gas production (Fig. 3).Similarly, all the isolates showed positive to MR test, indole test, catalase test, and TSI agar slant reaction but negative to Simmon's citrate reaction and VP test.The results of cultural, staining and biochemical tests of isolated E. coli are similar to the findings of Freeman (1985), Buxton andFraser (1977), andMerchant andPacker (1967).

Prevalence of E. coli in Chicken eggs
The overall prevalence of E. coli was 30.00% in chicken eggs in Rajshahi and Naogaon districts of Bangladesh.The prevalence of E. coli was 36.00% in commercial layer chicken eggs, 24.00% in indigenous chicken eggs, and 30.00% in market eggs (Table 3).These findings are in agreement with Islam et al. (2018) who reported 34.64% of chicken eggs were contaminated with E. coli.Similarly, Akond et al. (2009) reported that 42% of egg surface was polluted with E. coli in poultry and poultry farms environments in Bangladesh.4).These results are in agreement with the findings of Bantawa et al. (2019) who stated that E. coli exhibited the highest resistance to amoxycillin (100%), followed by tetracycline (93%), nalidixic acid (25%), and cefotaxime (19%) isolated from meat samples collected from eastern Nepal.

Genotypic Confirmation of E. coli Isolates
Phenotypic identified E. coli isolates were confirmed by PCR experiment.The 585 bp amplicon of phenotypic identified E. coli was amplified by PCR with the genus specific primers.All 45 isolates (100%) were confirmed as E. coli by PCR analysis (Fig. 4).
Legends: μg = Microgram, mm = millimetre.S = Sensitive, I = Intermediate sensitive, R = Resistant, ≥ = Greater than or equal to, ≤ = Less than or equal to.Table 2. Primers list used for the detection of antibiotic resistance genes in E. coli Gentamicin aac

Fig. 1 .
Fig. 1.Growth of E. coli on nutrient agar.Growth of E. coli on nutrient agar indicated by the development of smooth, circular, white to grayish white colony.

Fig. 3 .
Fig. 3. Sugar fermentation test for the isolated E. coli.E. coli isolates fermented dextrose, lactose, sucrose, maltose and mannitol with the production of acid and gas.

Figure 4 .
Figure 4. Agarose gel profile of PCR products of E. coli.Lane 1 indicated marker and Lane 2-4 indicated PCR products.

Figure 5 .
Figure 5. Antibiotic sensitivity and resistant patterns (zone of inhibitions) of isolated E. coli on Mueller Hinton agar.

Figure 6 .
Figure 6.Antibiotic sensitivity and resistant patterns of isolated E. coli

Table 1 .
Antibiotics discs with their concentration

Table 3 .
The prevalence of E. coli in chicken eggs in Rajshahi and Naogaon districts

Table 4 .
Momtaz et al. (2012)ty and resistant patterns of isolated E. coliGenotypic identified isolates of E. coli were further characterized by PCR to detect antibiotic resistance genes.The 577 bp amplicon of tetracycline resistance gene (tetA) was amplified by PCR with the gene specific primers.Out of 45 isolates 37 (82.22%)showedtetracyclineresistance gene (tetA) by PCR analysis.The 634 bp amplicon of tetracycline resistance gene (tetB) was amplified by PCR with the gene specific primers.Out of 45 isolates 35 (77.77%) showed tetracycline resistance gene (tetB) by PCR analysis.Similarly, the 577 bp amplicon of quinolone group resistance gene (gyrA) was amplified by PCR with the gene specific primers.Interestingly, all 45 (100%) isolates of E. coli showed quinolone group resistance gene (gyrA) by PCR analysis.However, we could not detect gentamycin resistance gene (aac(3)-II), tetracycline resistance gene (tetC), β-lactamases resistance genes (blaTEM, blaSHV and blaCMY), and erythromycin resistance gene (ereA) in E. coli.Similarly,Momtaz et al. (2012)detected tetracycline resistance (tetA and tetB) genes, erythromycin resistance gene (eraA) and sulfonamide resistance gene (sul1) in E. coli that was isolated in Iran from chicken meat.