Isolation of multidrug-resistant Escherichia coli from turkeys in Dinajpur, Bangladesh, and their antibiogram profile.

OBJECTIVE: The study was carried out for molecular characterization and antibiotic resistance analysis of Escherichia coli isolated from different turkey farms in Dinajpur, Bangladesh.
MATERIALS AND METHODS: A total of 45 samples comprising feces (n = 23) and cloacal swabs (n = 22) were collected randomly from turkeys. The samples were subjected to isolation and identification of E. coli by cultural and biochemical characteristics, followed by polymerase chain reaction and sequencing. An antibiogram of the isolated E. coli isolates was carried out by following the Kirby-Bauer disk diffusion method.
RESULTS: Out of the 45 samples, 28 (62.21%) were positive for E. coli, of which 16 (35.55%) fecal samples were positive and 12 (26.66%) cloacal swabs were positive. The antibiotic sensitivity analysis revealed that all the E. coli isolates were 100% sensitive to levofloxacin, norfloxacin, neomycin, gentamicin, and nitrofurantoin. On the other hand, all the isolates were 100% resistant to amoxicillin, azithromycin, erythromycin, tetracycline, bacitracin, cephalexin, nalidixic acid, vancomycin, methicilin, piperacillin, pefloxacin, novobiocin, cefepime, trimethoprim, netilmicin, and aztreonam.
CONCLUSION: This study's results uncover the occurrence and antibiotic resistance pattern of E. coli in the study area's turkeys. Copyright: © Journal of Advanced Veterinary and Animal Research.

Introduction

Turkey (Meleagris gallopavo) production has gained special attention among Bangladesh’s people considering its progressive commercial value and comprehensive mandate for its products [1]. The reasons behind such consideration are that turkeys have less disease risk, elevated market value, lesser feed cost, and lower mortality than the rest of the poultry species [2]. In the last few years, the acceptance of turkeys in Bangladesh has increased through triggering alternative protein supplementation, income generation, and poverty alleviation. Furthermore, Bangladesh’s climate pattern supports the production system of different poultry species positively [3]. It is noticeable that poultry meat alone provides a significant amount of animal protein through meat production, accounting for 37% of total meat requirements in Bangladesh [4].

In a decade, globally, turkey meat production increased significantly, which is 5.6 million tons in 2012, whereas it was 5.1 million tons in 2003 [5]. Thus, turkey can be a useful alternative source of protein as the broiler meat has problems with higher disease incidence and lower taste. The drawbacks of broiler meat production tend to move farmers’ attention to turkey production in developing countries like Bangladesh. As an excellent forager, a turkey helps eradicate insects that are harmful to crops and vegetables [6]. This poultry species is more suitable to raise in any climatic condition if they are provided proper feeding and management, such as disease and predator protection [2]. Although there are many points in favor of turkey production over other poultry species, they still have some limitations that need to be addressed appropriately. It should be noted that farmers are not aware enough regarding feed efficiency, which needs particular attention because feed represents 70% of the total cost of the poultry production system [7].

Additionally, the farmers’ feeding system deserves special attention, as the turkey poults have higher protein requirements during their first 7 weeks [8]. On the other hand, farmers are not knowledgeable about turkey breeder ration, which was also observed in other parts of the world [9]. Only a few organisms are causing turkey diseases that need intensive observation to address commercially viable turkey production. Escherichia coli is the most abundant disease-producing strain in the domestic turkey production system among different microorganisms, leading to decreased egg and meat production and ultimately leading to the bird’s death [10]. E. coli infections and observable consequences include egg peritonitis, omphalitis, coligranuloma, swollen head syndrome, cellulitis, colisepticemia, and death of the birds [11]. Thus, E. coli affects the turkey production system through decreased feed efficiency, slow growth rate, and higher morbidity and mortality, inclining the turkey to other diseases [12]. Furthermore, the treatment of bacteria using different antibiotics gives the turkey antibiotic resistance, which further affects the turkey’s performance against diseases. Therefore, the present work was designed to isolate and characterize the avian E. coli in Bangladesh’s turkey population and formulate a control strategy against the isolates by selecting the best antibiotics [13].

Materials and Methods

Ethical approval

The Hajee Mohammad Danesh Science and Technology University’s ethical committee approved this study’s methodology [approval number: Hajee Mohammad Danesh Science and Technology University (HSTU)/IRT/3084].

Selection of study area

The present research work was carried out on different selected turkey farms in the Dinajpur district for 6 months from January 2019 to June 2019. Samples were collected and further research was conducted at the Department of Microbiology, Faculty of Veterinary and Animal Science, HSTU, Dinajpur-5200.

Collection of samples

A total of 45 samples were collected randomly from healthy and sick turkeys from different turkey farms (five from HSTU farm, eight from Ma Turkey farm, eight from Habib turkey farm, eight from Anowara turkey farm, eight from Liton turkey farm, and eight from Nazrul turkey farm) in Dinajpur district. Among the samples, 23 were fecal and 22 cloacal samples. According to age, 25 samples were collected from 4-week-old turkeys, and 20 samples were collected from 10-week-old turkeys.

Isolation of bacteria

In nutrient agar (NA) and nutrient broth media, the primary culture was conducted. Then, different bacteriological agar media such as MacConkey agar (MA), eosin methylene blue (EMB) agar, and tryptic soy agar were used for subculturing of suspected bacteria. The aseptic condition was maintained and incubated at 37°C for 24 h. For further investigation, the NA plates were maintained at 4°C. Pure cultures were obtained following the protocols published previously [14].

Identification of bacteria

To identify the bacterial flora, cultural, morphological, and biochemical characteristics were studied. Then, the morphology and staining characteristics of bacteria were evaluated by Gram stain [14]. Furthermore, biochemical tests, such as sugar fermentation, catalase, methyl red (MR), Voges–Proskauer (VP), Simmon’s citrate, triple sugar iron, and indole tests, were carried out using standard methods [15].

Genomic DNA extraction and purification

Genomic DNA isolation and purification from E. coli was initiated according to the protocol developed in our laboratory [16]. Briefly, genomic DNA was extracted using the chloroform-isoamyl alcohol method from E. coli isolates cultured in a sodium thioglycolate broth. Initially, cells were harvested by centrifugation in an IEC CL31R multispeed centrifuge (Thermo Scientific) at 2,400 g for 10 min. The pellets were then resuspended in 400 μl Tris-EDTA bu-er, containing 0.01 M Tris-HCl pH 7.4, 0.001 M EDTA with gentle agitation. After that, 10% SDS (Fisher Scientific), followed by proteinase K (Ambion), was supplemented to the suspension. The suspension was then incubated at 65°C for 1 h in a hybridization oven (Biometra OV2, Anachem, UK). While incubation was completed, 100 μl of 5M NaCl, followed by 100 μl CTAB/NaCl (prewarmed at 70°C), was added to the solutions. The solution was then gently inverted and was subjected to incubation for 20 min at 65°C and cooling at room temperature for 5 min. A ratio of 24:1 chloroform isoamyl alcohol (Sigma-Aldrich) was used in the solution and centrifuged at 1,300 g for 15 min. The supernatants were then collected in a new tube and treated with 5 μl RNase A (5 mg/ml in RNase A buffer containing 0.5M NaCl, 0.01M EDTA), incubated at 37°C for 30 min. Quantification and quality were examined using a nanodrop spectrophotometer, and the DNA was stored at –20°C for further use.

Polymerase chain reaction (PCR)

The reaction scale used for PCR was 25 ml consisting of 12.5 μl of 2× master mix [GENE Amp Fast PCR Master Mix (2×)], 2 μl template DNA, 0.2 μl Taq DNA polymerase, 0.5 μl forward primer, 0.5 μl reverse primer, and 9.3 μl molecular grade water. The samples were then subjected to initial denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 94°C for 1 min; annealing at 53°C for 1 min; extension at 72°C for 1 min, and a final extension at 72°C for 10 min [17].

Sequencing and phylogenetic analysis

PCR product of fecal origin E. coli was sequenced by the Genetic Analyzer 3,130 (Applied Biosystems) using the dideoxy chain termination method (Sanger and Coulson method) at the National Institute of Biotechnology (NIB), Savar, Dhaka. Sequencing was carried out using both forward and reverse directions. Obtained sequences were edited and analyzed by Molecular Evolutionary Genetics Analysis software [18]. A phylogenetic tree was made using the neighbor-joining method with 1,000-bootstrap replication [19,20]. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site [21]. The nucleotide sequence was submitted to GenBank under the accession number MW165527 (Feces/Turkey/2019)

Antibiogram study of isolated E. coli

Commonly used antibiotics were used to test the antibiotic sensitivity and resistance patterns of the isolated E. coli. Two lines, namely resistant and sensitive, were used to evaluate the sensitivity against antibiotic disks. Disk diffusion method or the Kirby–Bauer method [18] and guidelines of Clinical and Laboratory Standards Institute [20] were used to interpret the antimicrobial drug sensitivity on newly prepared and dried-up Mueller Hinton agar. A total of 20 antibiotics were used in this study. The antibiotics include amoxicillin (11 μg), aztreonam (11 μg), azithromycin (12 μg), bacitracin (5 μg), cefepime (7 μg), cephalexin (10 μg), erythromycin (10 μg), gentamicin (10 μg), kanamycin (14 μg), levofloxacin (25 μg), methicilin (5 μg), nalidixic acid (30 μg), neomycin (15 μg), netilmicin (9 μg), nitrofurantoin (22 μg), norfloxacin (20 μg), novobiocin (8 μg), pefloxacin (10 μg), tetracycline (30 μg), and trimethoprim (10 μg). The disks were purchased from Himedia, India.

Results

Isolation of E. coli

Out of the 45 samples (23 fecal and 22 cloacal swabs), 28 (62.21%) were positive for E. coli, of which 16 (35.55%) were positive for fecal samples and 12 (26.66%) were positive for cloacal swabs (Table 1). According to the age group, 17 (37.77%) samples were positive in 4-week-old turkeys and 11 (24.44%) were positive in 10-week-old turkeys. All the isolated organisms were positive in MA, EMB agar, MR, VP, Simmon’s citrate test, triple sugar iron test, motility indole urease test, sugar fermentation test, and catalase test.

Table 1.

Results of E. coli isolated from fecal and cloacal swabs of turkeys.

Name of farms	Samples collected (n = 45)		E. coli positive samples(28)		Occurrence rate (%)
	Fecal	Cloacal	Fecal	Cloacal	Overall
HSTU Farm	3	2	3	1	80.00%
Borogurgola	4	4	2	2	50.00%
Basherhat	4	4	2	1	37.50%
Farmerhat	4	4	4	2	75.00%
Bottoli	4	4	2	3	62.50%
Gopalganj	4	4	3	3	75.00%
Sub-total	23	22	16	12
Total	45		28		62.21

Antibiotic sensitivity test of E. coli

The antibiotic sensitivity pattern of E. coli revealed that all isolates were sensitive to levofloxacin, neomycin, gentamycin, and nitrofurantoin (Table 2). All E. coli were resistant to amoxicillin, azithromycin, erythromycin, tetracycline, bacitracin, cephalexin, nalidixic acid, vancomycin, methicilin, piperacillin, pefloxacin, novobiocin, cefepime, trimethoprim, netilmicin, norfloxacin, and aztreonam (Table 2). Two different categories, namely resistant and sensitive, were used to evaluate the results of sensitivity against antibiotic disks according to the guidelines of Clinical and Laboratory Standard Institute [22].

Table 2.

Resistant and sensitive percentage of isolated E. coli (n = 28).

Antimicrobial agents	Sensitive (%)	Resistant (%)
Amoxicillin (11 μg)	0 (0%)	28 (100%)
Azithromycin (12 μg)	0 (0%)	28 (100%)
Aztreonam (11 μg)	0 (0%)	28 (100%)
Bacitracin (5 μg)	0 (0.0%)	28 (100%)
Cefepime (7 μg)	0 (0%)	28 (100%)
Cephalexin (10 μg)	0 (0.0%)	28 (100%)
Erythromycin (10 μg)	0 (0.0%)	28 (100%)
Gentamicin (10 μg)	28 (100%)	0 (0.0%)
Kanamycin (14 μg)	0 (0%)	28 (100%)
Levofloxacin (25 μg)	28 (100%)	0 (0%)
Methicilin (5 μg)	0 (0%)	28 (100%)
Nalidixic acid (30 μg)	0 (0.0%)	28 (100%)
Neomycin (15 μg)	28 (100%)	0 (0%)
Netilmycin (9 μg)	0(0%)	28 (100%)
Nitrofurantoin (22 μg)	28 (0.0%)	0 (100%)
Norfloxacin (20 μg)	28 (100%)	0 (0.0%)
Novobiocin (8 μg)	0 (0%)	28 (100%)
Pefloxacin (10 μg)	0 (0%)	28 (100%)
Tetracycline (30 μg)	0 (0.0%)	28 (100%)
Trimethoprim (10 μg)	0 (0%)	28 (100%)

S = Sensitive, R = Resistant.

PCR, sequencing, and phylogenetic analysis

About 584-bp product from E. coli DNA was amplified with specific E1 and E2 primers [23] (Fig. 1). Sequencing of the PCR product was conducted at the NIB. Blast analysis revealed that our sequence had 100% homology with the sequences available at GenBank (Fig. 2). For example, bacteria with accession numbers MT649857 and MT649847 were isolated from a patient in China, CP054236 from urinary tract infection of a woman in Texas, USA, CP053281 from human, MT453873 from the superficial surgical incision of a patient from Iraq, MW116771 from the saliva of a cat from Bangladesh. These findings indicate the sequence of our E. coli isolated from turkeys clustered with the pathogenic E. coli. Hence, we assumed that the isolated E. coli of our study could be pathogenic.

Figure 1.

Amplification of DNA from E. coli. Lane M: Marker, Ladder is 50-bp, Lane 1–10: test samples.

Figure 2.

Phylogenetic analysis of the partial 16SrRNA gene of E. coli isolated from turkeys. The evolutionary history was inferred using the neighbor-joining method. The optimal tree with the sum of branch length = 2.46853609 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis involved 18 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGAX. Our sequence is marked in the tree with a black dot.

Discussion

The study was conducted for the isolation, identification, molecular characterization, and antibiotic sensitivity study of the E. coli isolated from turkey’s fecal and cloacal swabs. Among the 45 samples, 28 (62.22%) were positive for E. coli. E. coli isolated from fecal and cloacal swab samples collected from turkey farms were compared with the previous results [24-26]. The isolates of E. coli showing the results in biochemical tests, including MR, VP, and indole tests, were similar to the previous reports [27-29].

The colony characteristics of E. coli observed in NA and EMB agar were similar to previous findings [30]. In Gram stain, the isolated bacteria’s morphology exhibited Gram-negative, short-rods arranged in single or paired and motile, supported by an earlier study [31]. The E. coli isolates revealed a complete fermentation of five basic sugars by producing acid and gas supported by earlier research work [28,32]. About 80% of the samples were positive for E. coli from HSTU farms, 50% from Borogurgula turkey farms, 37.5% from Basherhat, 75% from Farmerhat turkey farms, 62.5% from Bottoli, and 75% from Gopalgonj. The incidence of E. coli. Isolated was from fecal and cloacal swab samples collected from turkey, as reported previously [24-26]. The different isolates of E. coli showed identical results in different biochemical tests, including MR, VP, and indole test, and a similar type of biochemical reaction was reported [27,29]. Molecular characterization revealed that the target E. coli DNA gene band found at 584-bp was similar to a previous study [23]. Antibiogram profile reports showed various degrees of antibiotic resistance in E. coli. strains isolated from poultry [33,34]. The antibiotic study revealed that all E. coli was 100% sensitive to nitrofurantoin, gentamicin, levofloxacin, norfloxacin, and neomycin. The isolates were 100% resistant to amoxicillin, azithromycin, erythromycin, tetracycline, bacitracin, cephalexin, nalidixic acid, vancomycin, methicilin, piperacillin, pefloxacin, novobiocin, cefepime, trimethoprim, netilmicin, and aztreonam. Several antibiotics were also used by Tawyabur et al. [35] and reported similar results. Moreover, Tawyabur et al. [35] studied the prevalence of multidrug-resistant E. coli in diseased and healthy turkeys. However, they did not find any significant difference in the antibiotics resistance pattern between these two groups.

Conclusion

In the context of this study, it may be concluded that both the fecal and cloacal samples of turkeys from different farms are reservoirs of E. coli. It also brings out the multidrug-resistant E. coli of the study area. Antibiotic drug resistance is a problem in treating turkey diseases associated with E. coli and a potential public health hazard to individuals that consume turkey meat.

List of abbreviations

MA = MacConkey Agar, NA = Nutrient agar, EMB = Eosin Methylene Blue, MR = Methyl Red, VP = Voges–Proskauer, HSTU = Hajee Mohammad Danesh Science and Technology University