Study on antibiotic resistance pathern of E. coli in diarrheic calves of Tabriz dairy farms

Introduction: Diarrhea is a very important issue in calves, especially in the first days after birth, due to mortality and economic losses. Identifying the infectious and non-infectious agents responsible for diarrhea is crucial and can help to understand how to control this disease. The most important bacterial agents causing diarrhea in calves are Escherichia coli (E. coli), Salmonella spp. and Clostridium perfringens type C and D. Among infectious agents, pathogenic strains of E. coli have been known as the most important causes of diarrhea in calves. Enteropathogenic E. coli (EPEC is the main pathogenic agents in developing countries. These strains do not produce toxins and their virulence is represented in the form of lesions in the villi of the intestinal epithelial cells Therefore, phenotypic and genotypic studies on E. coli strains that cause diarrhea in calves can be helpful for understanding their prevalence control methods.
Material and method: In this study, rectal swabs were collected from 1 to 30-day-old calves with diarrhea during 2021. Samples were collected from the rectum using sterile swabs and put into tubes containing sterile saline normal solution. The samples were immediately transferred in an ice box to the bacteriology laboratory.The antibiotic-resistant pattern of the isolates was determined by the Kirby-Bauer disk diffusion susceptibility test. The Müller-Hinton agar was used to performing the antibiotic susceptibility. Then, standard microbial suspension (106CFU/ml) was prepared for the test. Bacterial-impregnated swabs were streaked on the surface of the Müller-Hinton agar. Then, antibiotic discs including nalidixic acid (30μg), ciprofloxacin (5μg), penicillin (10IU), imipenem (10μg), sulfamethoxazole-trimethoprim (23.75/1.25 µg), gentamicin (10μg), tetracycline (30μg), ceftriaxone (30μg), colistin (10μg), and ofloxacin (5 µg) were placed on the bacterial culture. Finally, the plates were incubated at 35 °C, and the results were evaluated after 18 hours. To the extraction of DNA from isolated bacteria,
The boiling method was used for DNA extraction. The isolates were cultured on nutrient agar. After incubation at 37 ° C for 24 h, fresh colonies were used for DNA extraction. Three to five colonies of each isolate were completely dissolved in 200 µL of sterile distilled water. The micro tubes were placed in a bain-marie (100 ° C) for 10-15 min. Then, the tubes were centrifuged at 14,000 rpm for 10 min. The supernatant was transferred to another sterile tube and used for PCR. The prevalence of the pathogenic genes (eae and bfp) for enteropathogenic E. coli (EPEC) strains and verotoxigenic E. coli (VTEC) (stx1 and stx2) strains was investigated using PCR. The primers designed in this study were also used in previous studies. The PCR reaction was performed using the thermal program to amplify the target genes (eae and bfp) (stx1 and stx2). PCR products were subjected to electrophoresis using 1% (w/v) agarose gel for 45 minutes. The gel was stained with Safe Red (Sinagen, Iran). Ultraviolet transillumination (UV Tech, Canada) was used for the visualization of DNA.

Results and discussion: Out of 105 samples, 81 isolates (85.05%) of E. coli were identified. In the present study, no bacterial agent was isolated from 24 cases (25.2% of samples) of the calves with diarrhea. This indicates that other factors such as viruses, parasites, and even nutritional factors also contribute to the occurrence of diarrhea. According to the symptoms of acute diarrhea (lethargy, muscle weakness, severe dehydration, and ataxia) and chronic diarrhea (mild symptoms and watery diarrhea) in calves, in this study, the prevalence of diarrhea, based on severe and mild symptoms, was 72.8% and 27.2%, respectively. In this research, the prevalence of diarrhea in calves was investigated between two age ranges. It was found that the prevalence rate of diarrhea in the age range of 1-7 days and 7-30 days was 63.6% and 36.4%, respectively. Also, 63% of the calves with diarrhea did not consume colostrum and or had incomplete colostrum consumption, while 37% of calves with diarrhea had a history of colostrum consumption. The difference in the prevalence of E. coli can be related to various reasons, such as the immunization of pregnant cows before giving birth, compliance with hygienic principles in cattle breeding, as well as feeding colostrum to newborn calves in sufficient quantities and at the right time. As it was determined in this study, the highest rate of prevalence of diarrhea (60.5%) was observed in traditional cattle farms, followed by semi-industrial cattle farms (30.9%) and industrial cattle farms (8.6%). One of the most important reasons for the low rate of infection in industrial and semi-industrial farms is the observance of hygiene principles in calving and the regular cleaning of the bedding, and the regular observance of feeding times (especially with colostrum). According to these results, it seems that the prevalence of diarrhea is more common at younger ages (56.6% in the age group of 1-7 days compared to 43.6% in the age group of 7-30 days). Besides the decrease in the levels of maternal antibodies in the calf's serum, the prevalence of diarrhea in the 2-4th weeks can also be related to the change in the diet, because at this age in Iran, farmers usually start feeding the calves with flour, chopped fodder, and other substitutes of milkThe resistance of the isolates was observed for penicillin (56.84%), tetracycline (38.3%), sulfamethoxazole-trimethoprim (38.3%), gentamicin (29.6%), ceftazidime (29.2%), colistin (25.9%), imipenem (23.5%), ceftriaxone (22.21%), nalidixic acid (22.2%), ciprofloxacin (14.8%) and ofloxacin (9.9%) were detected in the isolates. Furtheremore, 16% of the isolates had the eae gene and 24.7% had both the stx1 and stx2 genes. In the present study, the infection rate of calves with diarrhea with VTEC strains was higher than EPEC strains. According to the results of the present work, the presence of antibiotic resistance in E. coli isolates indicates the high importance of this bacterium in terms of epidemiology and public health.