Molecular characterization of extended spectrum cephalosporin resistant Escherichia coli isolated from dogs

Article history: Received May 02, 2020 Accepted June 07, 2020 Available online June 23, 2021 Shepherd dogs have been implemented in the transmission and distribution of many threatening pathogens. The presence of extended-spectrum-cephalosporin resistant Escherichia coli (ESCR E. coli) in dog feces can constitute a significance risk to human health due to transmission of antibiotics resistance from dogs to humans, other animals and the surrounding environment. Therefore, in this study, phenotypic and molecular characterization of fecal ESCR E. coli were investigated in shepherd dogs accompanied sheepherders in urban areas. Sixty-seven fresh fecal samples were collected from shepherd dogs from different regions of Mosul city. Bacteriological examination of ESCR E. coli was done using MacConkey agar with cefotaxime followed by subsequent PCR confirmation of the CTX-M gene using specific primers and molecular characterization using specific primers directed to CTX-M-1, 2 and 9 groups. The results of bacterial examination showed successful confirmation of ESCR E. coli which has been isolated from fecal samples of shepherd dogs 58.2% (39/67). In addition, detection of CTX-M gene was confirmed in 53.7% (36/67) of E. coli isolates. Furthermore, molecular characterization of CTX-M gene revealed the presence of only one genotype belongs to CTX-M-1. However, both of CTXM-2 and CTX-M-9 genotypes were not detected in this study. This study concluded that shepherd dogs have an essential role in carrying and spreading of ESCR E. coli especially in urban regions.


Introduction
Dogs play an important role in human daily practices as they mainly kept as companion pets (1,2). In addition, they are used in many diverse activities such as animal grazing, livestock guarding, and thus it's in close contact with humans (3,4). One of the most common farming practices of sheep and goats in urban areas of Mosul city is keeping stray dogs as shepherd dogs along with these flocks to provide protection and control of the animal movement. Nevertheless, most of these dogs mainly did not receive any treatments or vaccinations which render them as potential risk for transmission of many pathogens and diseases (3)(4)(5)(6). Recent studies approved that many bacterial species have developed antimicrobial resistance especially in dogs (1,7,8) and this resistance might be transmitted to human (9). Spreading of beta-lactamase-producing Enterobacteriaceae is considered as one of the most important bacterial pathogens associated with high costs of health care and also unresponsiveness to treatment (7,10,11). Nevertheless, increased spreading of extended-spectrum β-lactamase (ESBL) bacteria was potentially related to the common pathogen for many infections. In addition, its treatment has become more difficult because of its resistance to antibiotics (12)(13)(14). Yet, the spreading of extended-spectrum cephalosporin resistance E. coli (ESCR E. coli) infections has complicated the scene and become a major challenge due to substantial genetic diversity, possession of virulence factors, large and wide hosts range, multifaceted potential and various pathogenic forms and the possibility of infection and spreading among multiple hosts within the environment (12)(13)(14)(15)(16). A plasmid-mediated CTX-M β-lactamase has become a dominant type of ESBLs, especially ESCR E. coli, rather other classical β-lactamases such as SHV-and TEMtypes (17)(18)(19). CTX-M type has been increasingly recorded in human and animal worldwide with most prevalent groups CTX-M-1, CTX-M-2 and CTX-M-9 (4,12,(17)(18)(19). Giving the fact, there is no information about spreading of ESCR E. coli in the shepherd dogs in Mosul city, in addition to lack of local studies which indicate the possibility of dogs as a transporter of ESCR E. coli. Therefore, this study aimed to detect and molecular characterize ESCR E. coli in shepherd dogs.

Sampling
A Total of 67 fresh fecal samples of apparently health shepherd dogs were collected in sterile containers from both sexes (male and female), ranging from 6 months to 3 years in age from different urban regions of Mosul city for the period from December 2019 to February 2020. The samples were transported immediately under cooling conditions to the Microbiology Laboratory, College of Veterinary Medicine, University of Mosul, for further microbiological examination.

Isolation and identification of ESCR E. coli
A loopful of the collected fecal sample was cultured on special MacConkey agar with cefotaxime (Foxime 500 mg, Tabuk, KSA) at 1 μg/ml final concentration according to Umeda et al. (15). After that, all the inoculated plates were put in the incubator at 37°C for 24 h. This medium inhibits all the bacteria that are sensitive to cefotaxime and allows only resistant ones to grow. All suspected fermented ESCR E. coli colonies were again subculture on MacConkey agar with cefotaxime for purification and subsequent identification using standard bacteriological methods (20).

Molecular characterization Extraction of DNA
Only ESCR E. coli positive colonies were subjected to DNA extraction using Bacteria DNA Preparation Cat No. PP-206S (Jena Bioscience, Germany). According to the manufacturer instructions, briefly, few fresh colonies on Brain Heart Infusion agar (Lab M, UK) were selected and suspended in an eppendorf microcentrifuge for cell Lysis. Then, followed by a step of precipitation of proteins. After that the supernatant was separated in a new sterile 1.5 ml eppendorf tube with 300 μl Isopropanol 99 %. This step was necessary for DNA precipitation. The tube was centrifuged and the supernatant was discarded followed by draining of the tube. The small DNA pellet was washed by adding washing buffer, inverted several times then centrifuged. The supernatant was eliminated and the tube was dried in air at room temperature. Hydration of DNA was done by adding 100 μl of Hydration Solution, followed by incubation at 65 °C for 1 h, and then the DNA was kept at -20 °C for subsequent testing.

Polymerase chain reaction (PCR) assay procedure
All the primers used in this study were detailed in ( Table  1). The molecular confirmation of E. coli was performed using species specific primers (E. coli: ECO223-F and ECO 455-R). While confirmation of the CTX-M gene was done by CTX-M-Uni-F and CTX-M-Uni-R. Subsequent characterization of CTX-M groups was done using 3 set of primers targeting G1, G2 and G9 groups of the CTX-M gene. Standard PCR reaction mixture was prepared for all protocols. Briefly, 25 µl containing 12.5 µl HS Prime Taq Premix (2X) (GeNet Bio, Korea), 1 µM of each primer 10 mmol (IDT, USA), 2.5 µl of DNA template final concentration (2 ng/µl) and 8 µl of PCR grade water. The PCR was done using thermal cycler (BioRad, T100, Bio-Rad, USA). The cycling conditions include 1 cycle of initial denaturation at 94 °C for 10 min, 35 cycles consisting of (initial denaturation at 94 °C for 30 sec, annealing at (55 °C for E. coli, 54 °C for CTX-M-Uni and 52 for CTX-M G1, G2, G3) for 30 sec, and extension at 72 °C for 45 sec). Finally, one cycle of final extension at 72 °C for 5 min. The reaction was set for cooling at 4 °C. Agarose gel electrophoresis was used to separate the PCR products using 1.2% agarose gel (Promega, USA) containing Prime safe Dye (GeNet Bio, Korea). The wells of agarose gel were loaded by 5 µl of each PCR product. The electrophoresis condition was set at 75 V, 300 mA for 50 min using Wide Mini-Sub Cell GT gel electrophoresis systems and basic power supply (Bio-Rad, USA) containing 1X TBE buffer (Bio-Rad, USA). A volume of 4 µl of DNA ladder, 100 bp (GeNet Bio, Korea) was used as molecular weight standard. Following electrophoresis, the gel was viewed using Gel doc Ez system (Bio-Rad-USA) to detect the specific bands.

Results
The bacterial isolation and identification showed successful recovery of ESCR E. coli from fecal samples 58.2% (39/67) using MacConkey agar with cefotaxime ( Figure 1). However, only 53.7% (36/67) isolates were confirmed E. coli by PCR producing expected product size of 232 bp (

Discussion
Globally, antibiotic resistance has become a serious threat and widely distributed in humans, animals and the environment (18,(24)(25)(26)(27). Nevertheless, dog feces are well known as a potential source of many zoonotic diseases and also spreading of different types of bacteria which are resistant to antibiotics (2,5,28,29). In this study we investigated a type of stray dogs that are used commonly as shepherd dogs in our community and they are almost with contact with the animals especially sheep and goats in addition to the grazers themselves. Our results showed that 53.7% (36/67) of the shepherd dogs were confirmed positive for ESCR E. coli and possess CTX-M gene using standard bacteriological culture and PCR as a molecular confirmation method. This result was in consistent with the previous findings obtained by Hong et al. (9), they reported that 49.2% (155/315) of dogs were positive for ESCR E. coli. Another recent study by Abbas et al. (30) (15) found that 22/151 (14.6%) of dogs were Cephalosporin-resistant Enterobacteriaceae with majority identifies as ESCR E. coli. This variation in the results may reflect the strict use of antibiotics in different countries. It is clear that the presence of ESCR Enterobacteriaceae especially ESCR E. coli in fecal samples of dogs is considered as a potential risk factor for spreading of antimicrobial resistance by these animals to other animal species, close contact humans and the environment (18,(32)(33)(34). This mainly occurred by contamination of the ground, pasture and water by feces of roaming shepherd dogs with ESCR type of bacteria. Among the ESBL genotypes of Enterobacteriaceae, the CTX-M type ESBL genes were frequently studied in both human and veterinary fields with almost dominant of ESCR E. coli (9,24,25,35). In this study the CTX-M-1 was the only detected genotype, while CTX-M-2 and CTX-M-9 were not confirmed. This was in agreement with Damborg et al. (29), they reported isolation of ESBL with predominant CTX-M-1 genotype confirmed by multilocus sequence typing (MLST). While Haenni et al.  (8,15). In addition, other genes also have been reported especially, those within ESBL such as SHV, CMY groups (37)(38)(39), TEM (39), or other types rather than ESBL such as AmpC β-lactamase (pAmpC) (28,33,37) and carbapenemase (25,40). Finally, the dramatic spreading of antibiotic resistance has a great impact on human in terms of one health concept though increasing the probability of fecal contamination by such kind of shepherd dogs, various animal species and also the environment worldwide. More studies have to be conducted to understand the relation between resistant bacteria isolates from different sources. Such information will surely help to mitigate and control the spreading of antibiotic resistance to a more dangerous level especially development of different pathogens, including multidrug-resistant once.

Conclusion
ESCR E. coli was successfully detected in shepherd dogs with common CTX-M G1 type. Shepherd dogs are considered as one of the most neglected sources of spreading and transmission of ESCR E. coli.