Antibiotic-resistant Escherichia coli and Salmonella spp. associated with dairy cattle and farm environment having public health significance

Abstract

Aim: The present study was carried out to determine load of total bacteria, Escherichia coli and Salmonella spp. in dairy farm and its environmental components. In addition, the antibiogram profile of the isolated bacteria having public health impact was also determined along with identification of virulence and resistance genes by polymerase chain reaction (PCR) under a one-health approach.

Materials and methods: A total of 240 samples of six types (cow dung - 15, milk - 10, milkers' hand wash - 10, soil - 10 water - 5, and vegetables - 10) were collected from four dairy farms. For enumeration, the samples were cultured onto plate count agar, eosin methylene blue, and xylose-lysine deoxycholate agar and the isolation and identification of the E. coli and Salmonella spp. were performed based on morphology, cultural, staining, and biochemical properties followed by PCR.The pathogenic strains of E. coli stx1, stx2, and rfbO157 were also identified through PCR. The isolates were subjected to antimicrobial susceptibility test against 12 commonly used antibiotics by disk diffusion method. Detection of antibiotic resistance genes ereA, tetA, tetB, and SHV were performed by PCR.

Results: The mean total bacterial count, E. coli and Salmonella spp. count in the samples ranged from 4.54±0.05 to 8.65±0.06, 3.62±0.07 to 7.04±0.48, and 2.52±0.08 to 5.87±0.05 log colony-forming unit/g or ml, respectively. Out of 240 samples, 180 (75%) isolates of E. coli and 136 (56.67%) isolates of Salmonella spp. were recovered through cultural and molecular tests. Among the 180 E. coli isolates, 47 (26.11%) were found positive for the presence of all the three virulent genes, of which stx1 was the most prevalent (13.33%). Only three isolates were identified as enterohemorrhagic E. coli. Antibiotic sensitivity test revealed that both E. coli and Salmonella spp. were found highly resistant to azithromycin, tetracycline, erythromycin, oxytetracycline, and ertapenem and susceptible to gentamycin, ciprofloxacin, and imipenem. Among the four antibiotic resistance genes, the most observable was tetA (80.51-84.74%) in E. coli and Salmonella spp. and SHV genes were the lowest one (22.06-25%).

Conclusion: Dairy farm and their environmental components carry antibiotic-resistant pathogenic E. coli and Salmonella spp. that are potential threat for human health which requires a one-health approach to combat the threat.

Keywords: Escherichia coli; Salmonella spp; antibiotic resistance genes; carbapenem resistance; dairy farm; one-health; virulence.

Figure-1

Polymerase chain reaction (PCR) amplification of 16S rRNA of Escherichia coli and invA gene of Salmonella spp. (a) PCR amplification of 16S rRNA of E. coli. Lane M: 100 bp DNA Marker, 1: Negative control, 2: Positive control, and 3-7: Representative E. coli isolates. (b) PCR amplification of invA gene of Salmonella spp. Lane M: 100 bp DNA Marker, 1: Negative control, 2-6: Representative Salmonella spp. isolates, and 7: Positive control.

Figure-2

Polymerase chain reaction (PCR) amplification of virulence genes of Escherichia coli. (a) PCR amplification of stx1gene of E. coli. Lane M: 100 bp DNA Marker, 1: Negative control, 2-6: Representative E. coli isolates, and 7: Positive control. (b) PCR amplification of stx2 gene of E. coli. Lane M: 100 bp DNA Marker, 1: Positive control, 2-5: Representative E. coli isolates, and 6: Negative control. (c) PCR amplification of rfbO157 gene of E. coli. Lane M: 100 bp DNA Marker, 1-5: Representative E. coli isolates, 6: Positive control, and 7: Negative control.

Figure-3

Distribution of stx1, stx2, and rfbO157 in isolated Escherichia coli.

Figure-4

Polymerase chain reaction (PCR) amplification of antibiotic resistance genes of Escherichia coli and Salmonella spp. (a) PCR amplification of ereA gene of erythromycin resistant E. coli and Salmonella spp. Lane M: 100 bp DNA Marker, 1: Negative control, 2-3: Representative E. coli isolates, 4-5: Representative Salmonella spp. isolates, 6: Positive control for E. coli, and 7: Positive control for Salmonella spp. (b) PCR amplification of tetA gene of tetracycline resistant E. coli and Salmonella spp. Lane M: 100 bp DNA Marker, 1: Negative control, 2-3: Representative E. coli isolates, 4-5: Representative Salmonella spp. isolates, 6: Positive control for E. coli, and 7: Positive control for Salmonella spp. (c) PCR amplification of tetB gene of tetracycline resistant E. coli and Salmonella spp. Lane M: 100 bp DNA Marker, 1: Negative control, 2-3: Representative E. coli isolates, 4-5: Representative Salmonella spp. isolates, 6: Positive control for E. coli. and 7: Positive control for Salmonella spp. (d) PCR amplification of SHV gene of ertapenem, imipenem and meropenem resistant E. coli and Salmonella spp. Lane M: 100 bp DNA Marker, 1: Negative control,2: Positive control for E. coli, 3: Positive control for Salmonella spp., 4-5: Representative E. coli isolates, and 6-7: Representative Salmonella spp. isolates.