Isolation and Characterization of Multidrug-Resistant Escherichia coli and Salmonella spp. from Healthy and Diseased Turkeys

Diseases caused by Escherichia coli (E. coli) and Salmonella spp. can negatively impact turkey farming. The aim of this study was to isolate and characterize multidrug-resistant (MDR) E. coli and Salmonella spp. in healthy and diseased turkeys. A total of 30 fecal samples from healthy turkeys and 25 intestinal samples from diseased turkeys that died of enteritis were collected. Bacterial isolation and identification were based on biochemical properties and polymerase chain reaction (PCR). Antibiogram profiles were determined by disk diffusion. The tetracycline-resistance gene tetA was detected by PCR. All samples were positive for E. coli. Only 11 samples (11/30; 36.67%) were positive for Salmonella spp. from healthy turkeys, whereas 16 (16/25; 64%) samples were positive for Salmonella spp. from diseased turkeys. E. coli isolated from diseased turkeys showed higher resistance to levofloxacin, gentamicin, chloramphenicol, ciprofloxacin, streptomycin, and tetracycline. Salmonella spp. isolated from healthy turkeys exhibited higher resistance to gentamicin, chloramphenicol, ciprofloxacin, streptomycin, imipenem, and meropenem. All E. coli and Salmonella spp. from both healthy and diseased turkeys were resistant to erythromycin. Salmonella spp. from both healthy and diseased turkeys were resistant to tetracycline. Multidrug resistance was observed in both E. coli and Salmonella spp. from diseased turkeys. Finally, the tetA gene was detected in 93.1% of the E. coli isolates and in 92.59% of the Salmonella spp. isolates. To the best of our knowledge, this is the first study to isolate and characterize tetA-gene-containing MDR E. coli and Salmonella spp. from healthy and diseased turkeys in Bangladesh. Both microorganisms are of zoonotic significance and represent a significant public health challenge.


Introduction
Turkey (Meleagris gallopavo) farming is a profitable business in many countries. In Bangladesh, turkey farming generates a higher profit than broiler and layer farming due to lower feeding cost, higher market price, and high demand from consumers. In addition, turkey is generally more adaptable under different weather conditions and less prone to disease than other poultry birds [1,2].

Detection of tetA Gene
Of the 29 E. coli isolates phenotypically resistant to tetracycline, tetA was detected in 27 (27/29; 93.1%). In the case of Salmonella spp., tetA was detected in 25 of the 27 isolates (25/27; 92.59%). The prevalence of tetA was similar in healthy and diseased turkeys for both E. coli and Salmonella spp. (Figure 1).
E. coli isolated from healthy turkeys showed eight resistance patterns, while E. coli isolated from diseased turkeys showed ten resistance patterns. Salmonella isolated from healthy and diseased turkeys showed four and seven resistance patterns, respectively ( Table 2). Among the antibiogram types, pattern E-MEM-CIP showed the highest prevalence in E. coli (14 isolates). On the other hand, the E-CIP-TE pattern showed the highest prevalence in Salmonella (five isolates) ( Table 2).

Discussion
In this study, we report the detection of MDR E. coli and Salmonella spp. from healthy and diseased turkeys. This is significant to human health due to the zoonotic nature of these pathogens. Moreover, most E. coli and Salmonella spp. isolates were found to be MDR, which makes it difficult to treat the infected turkeys [37][38][39][40][41][42]. Antibiograms can guide the choice of therapies for colibacillosis and salmonellosis in turkeys. The incorrect choice of antibiotics is not only associated with the development of AMR but can also have significant negative economic impacts.
Whereas all samples were positive for E. coli, only 49.09% (27/55) of the samples were positive for Salmonella spp., which were significantly more prevalent in diseased than in healthy turkeys. The isolation and characterization of E. coli and Salmonella spp. from turkeys revealed the presence of the tetA gene. The gut microflora of poultry typically includes E. coli and Salmonella spp. [43]. Detection of Salmonella spp. in diseased turkeys that died of enteritis suggests that Salmonella was the causative factor of enteritis. Previously, Kar et al. [8] reported the detection of E. coli and Salmonella spp. from cloacal swabs of turkeys but did not use any molecular techniques, such as the PCR technology used in this study. PCR is a robust and rapid detection method with increased sensitivity and specificity for detecting Salmonella in food, environmental, and clinical samples [44]. The invA gene has been the target for many PCR protocols, as it is found in almost all known serovars of Salmonella [45]. This gene encodes an inner membrane protein necessary for invasion of epithelial cells by Salmonella [46]. We were able to observe higher rates of E. coli and Salmonella spp. compared to the study of Kar et al. [8], which may be attributed to the highly sensitive nature of the molecular techniques used in this study.
The detection of E. coli and Salmonella spp. from fecal materials and intestinal contents of healthy turkeys indicates intestinal colonization [47]. The findings also indicate that fecal materials may be a source of transmission of E. coli and Salmonella spp. to other birds. The detection of the virulence gene invA in the isolated Salmonella spp. indicates the potential pathogenic nature of these isolates. It is also possible for these pathogens to be introduced into the food chain causing food-borne diseases [48].
Antibiotic resistance is a major public health problem. The misuse and abuse of antimicrobial agents contributed to the emergence and dissemination of antibiotic-resistant pathogens in animals and humans [49]. Location-specific information on antibiotic resistance patterns in different geographical areas is important for the successful treatment of outbreaks and infections. The isolated E. coli and Salmonella spp. were found to be resistant to levofloxacin, erythromycin, ciprofloxacin, meropenem, and tetracycline. This antibiotic resistance profile can be due to the frequent use of antibiotics in poultry for therapeutic and growth promotion purposes [32,33]. The presence of antibiotic-resistant E. coli and Salmonella spp. in fecal materials of healthy turkeys indicates the role of these birds as spreaders of resistant microorganisms in farm environments.
Several studies detected the tetA gene in E. coli and Salmonella spp. from dairy farms, boiler farms, house flies, and aquatic environments [31,33,[50][51][52]. However, there were no studies on the detection of the tetA gene in E. coli and Salmonella from turkeys. Among the isolates phenotypically resistant to tetracycline, 93.1% of the E. coli isolates and 92.59% of Salmonella spp. isolates were positive for the tetA gene. The tetA has been shown to be the most common genetic component in tetracycline-resistant E. coli and Salmonella spp. [9,[53][54][55]. Generally remaining in mobile genetic components (integrons, transposons, and plasmids), tetA can be easily transferred to different bacteria.
Resistance to carbapenems (imipenem and meropenem) may be due to the transmission of bacteria from human sources, especially that carbapenems are not approved for use in livestock [56]. Future detailed studies at the genetic level are needed to test this hypothesis. According to the WHO, carbapenem-resistant E. coli and Salmonella spp. are considered to be among the most critical pathogens [57]. The detection of carbapenem-resistant E. coli and Salmonella spp. in turkeys has to be treated as an urgent public health problem.
Antibiotic treatment failures in poultry has been highly attributed to the MDR nature of the pathogens [58]. In the present study, the majority of the isolated E. coli (48/55; 87.27%) and Salmonella spp. (24/27; 88.89%) were MDR. More MDR E. coli and Salmonella spp. were retrieved from diseased turkeys than from healthy turkeys. The higher MDR in diseased turkeys may have been caused by the selection pressure resulting from the excessive use of several classes of antibiotics. However, the differences were statistically insignificant as in Table 2 (p = 0.112 and p = 0.056 for E. coli and Salmonella spp., respectively). The statistical insignificance indicates that the bacteria were MDR regardless of whether the source was healthy or diseased turkeys. To avoid the development of MDR, the use of antibiotics should be more strategic and selective.

Ethics Statement
No ethical permission was required for the study. During sample collection, verbal permission was taken from farm owners.

Study Design
A pilot survey was conducted prior to the start of the current study to identify the different turkey farming areas in Bangladesh, disease outbreaks in these farms, and antibiotic treatment regimens. Based on the survey results, seven antibiotics were selected. In addition, two carbapenem antibiotics were included based on reports that indicated that E. coli could be resistant to carbapenems in poultry [31,50,59]. Guided by bird mortality rates and antibiotic use reports from the survey, five farms from two districts were selected for sample collection. The birds were categorized into healthy and diseased birds. Six healthy and five diseased bird samples were randomly collected from each farm resulting in a total of 55 samples from the five farms. Freshly dropped feces from healthy birds and intestinal contents from diseased birds that had avian colibacillosis and/or Salmonellosis were collected for analysis.

Study Areas and Collection of Samples
The study was conducted in two districts of Bangladesh namely Mymensingh (24. Freshly dropped fecal samples (n = 30) were aseptically collected using sterile cotton buds from healthy turkeys. During the postmortem examination, 5 g of intestinal contents (n = 25) was collected from each turkey that died of enteritis and had lesions of avian colibacillosis and/or salmonellosis.
Immediately after collection, samples were transferred to sterile zip-lock bags. Samples were transported to the laboratory maintaining cold chain. Collected samples were transferred into sterile test tubes containing freshly prepared nutrient broth (5 mL) and were incubated aerobically at 37 • C overnight for the growth of bacteria.

Isolation of E. coli and Salmonella spp.
Isolation of E. coli and Salmonella spp. was based on culture on Eosin Methylene Blue (EMB) and Xylose Lysine Deoxycholate (XLD) agar (HiMedia, India) plates, respectively. Initially, freshly grown broth cultures were streaked on EMB and XLD agar media using sterile inoculating loops. This was followed by aerobic incubation of the inoculated agar plates at 37 • C overnight to obtain pure colonies. Single green-colored metallic-sheen colonies on EMB agar media and black-centered colonies on XLD agar media represented the growth of E. coli and Salmonella spp., respectively. For further confirmation, selected colonies were subjected to morphological study by Gram staining and biochemical tests such as the methyl red test, sugar fermentation test, Voges-Proskauer test, motility test, urease test, and indole test [22,31].

Study Areas and Collection of Samples
The study was conducted in two districts of Bangladesh namely Mymensingh (24.7539° N, 90.4073° E) and Tangail (24.2513° N, 89.9167° E) during the period from June 2018 to November 2019. The study areas are represented in Figure 2. Freshly dropped fecal samples (n = 30) were aseptically collected using sterile cotton buds from healthy turkeys. During the postmortem examination, 5 g of intestinal contents (n = 25) was collected from each turkey that died of enteritis and had lesions of avian colibacillosis and/or salmonellosis.
Immediately after collection, samples were transferred to sterile zip-lock bags. Samples were transported to the laboratory maintaining cold chain. Collected samples were transferred into sterile test tubes containing freshly prepared nutrient broth (5 mL) and were incubated aerobically at 37 °C overnight for the growth of bacteria.

Isolation of E. coli and Salmonella spp.
Isolation of E. coli and Salmonella spp. was based on culture on Eosin Methylene Blue (EMB) and Xylose Lysine Deoxycholate (XLD) agar (HiMedia, India) plates, respectively. Initially, freshly grown broth cultures were streaked on EMB and XLD agar media using sterile inoculating loops. This was followed by aerobic incubation of the inoculated agar plates at 37 °C overnight to obtain pure colonies. Single green-colored metallic-sheen colonies on EMB agar media and black-centered

Molecular Detection of E. coli and Salmonella spp.
Isolation of E. coli and Salmonella spp. were confirmed by polymerase chain reaction (PCR) targeting E. coli 16S rRNA gene and Salmonella genus specific invA genes respectively (Table 3). tetA F: GGTTCACTCGAACGACGTCA R: CTGTCCGACAAGTTGCATGA 577 57 [62] For PCR, genomic DNA of E. coli and Salmonella spp. was extracted by the boiling method as described by Sobur et al. [50]. Briefly, a pure colony collected from freshly grown culture was initially taken into an Eppendorf tube containing molecular-grade water (100 µL) followed by mixing gently through vortexing. Subsequently, the mixture was boiled for 10 min, cooled for 10 min, and centrifuged for 10 min at 1400 rpm. Finally, the supernatant was collected as the source for the genomic DNA for PCR and stored at −20 • C until further use.
PCR tests were carried out in a final volume of 25 µL with 12.5 µL of the master mix (2X) (Promega, Madison, WI, USA), 4 µL of genomic DNA (50 ng/µL), 1 µL of each primer, and 6.5 µL of nuclease-free water. After amplification, PCR products were subjected to gel electrophoresis in 1.5% agarose, followed by staining and visualizing by 0.25% ethidium bromide solution and ultraviolet trans-illuminator (Biometra, Göttingen, Germany). A DNA ladder (100 bp; Promega, Madison, WI, USA) was used to assess the sizes of PCR amplicons.

Molecular Detection of Tetracycline Resistance tetA Gene
E. coli and Salmonella isolates resistant to tetracycline were screened by PCR for the detection of the tetracycline-resistance tetA gene using the primer and protocol described by Randall et al. [62].

Statistical Analysis
Chi-square tests were performed using the SPSS software (IBM SPSS version 25.0, IBM, Chicago, IL, USA). p-values less than 0.05 (p < 0.05) were considered to be statistically significant.

Conclusions
The isolation and characterization of tetA-gene-containing-MDR E. coli and Salmonella spp. from turkeys are concerning. The potential ability of these MDR bacteria to enter into the food chain can expose humans to serious health risks. Bacterial surveillance programs should be implemented in order to control the emergence of bacterial resistance in turkey farms in Bangladesh and elsewhere in the world. This should be a concerted effort that is best carried out via bacterial surveillance networks across different countries. Additionally, holistic and multi-sectoral approaches, such as the one health approach, need to be implemented [66]. Guided by top health professionals and scientists, these strategies can provide effective solutions to the complex, multifaceted global challenge of AMR.