Identification of Virulence Factors of Escherichia coli Isolates from Fecal Samples of Calves in Southern Brazil

Abstract

Background: Escherichia coli (E. coli) is an enteropathogen that commonly causes diarrhea in calves. However, not all E. coli isolates are pathogenic. The aim of this study was to identify E. coli virulence factors derived from fecal samples collected in the southern region of Rio Grande do Sul state (RS) from calves with and without diarrhea, as well as investigate the antimicrobial susceptibility of E. coli isolates from calves with diarrhea.

Materials, Methods & Results: Forty stool samples were collected in 12 farms, each one from calves having one day to six months of age, with and without diarrhea. The total DNA of from these isolates was extracted and a PCR using primers specific for the virulence factors Stx1, Eae, F41, F5 and STa was conducted. The susceptibility testing used the disk diffusion method and the susceptibility profile was evaluated against the following antimicrobials: ampicillin, penicillin, chloramphenicol, enrofloxacin, gentamicin, trimethoprim, sulfonamide, tetracycline and streptomycin. From all calves, 15 (15/40, 37.5%) had diarrheal stools and 25 (25/40, 62.5%) had normal or semi-liquid stools. Twelve (12/40; 30%) E. coli isolates showed at least one virulence factor. These factors were found in four isolates (4/15; 26.6%) from diarrheal stools and eight isolates (8/25; 28.5%) from normal stool. The Stx1 factor was identified in five isolates (5/40; 12.5%), and the Eae and the Sta factors in one (1/40; 0.2%) and in atypical associations between Stx1 and Eae and also between Eae and F41 in two isolates (2/40; 0.5%). Also, the Eae and Sta factors were identified in one isolate (1/40; 0.2%). The susceptibility test showed resistance to penicillin and tetracycline in 93% and 80% of the tested isolates, respectively.

Discussion: The identification of virulence factors is necessary because E. coli is an enterobacterium present in calves gastrointestinal tract, to prove its pathogenicity. The virulence factor most commonly found in E. coli isolates derived from feces of calves with and without diarrhea in the southern region of the RS was the Stx1 (Shiga toxin-producing E. coli STEC). It is likely that the highest occurrence of E. coli isolates positive for the Stx1 virulence factor was due to the fact that cattle were the main reservoirs of this type of bacteria. The occurrence of enterohemorrhagic E. coli (EHEC) in animals of nine and 34 days of life, respectively, is highlighted. Studies have shown that contamination of animal foods with EHEC can cause enteric disorders, hemorrhagic colitis, and uremic hemolytic syndrome (UHS) in humans. Although in the present study the identification of the Stx2 factor was not performed, authors describe that the presence of the genes encoding Stx2 and Eae is determinant in the occurrence of UHS. In the susceptibility test, it was observed that E. coli isolates from diarrheal stools showed resistance to antimicrobials penicillin (10 mg) and tetracycline (30 mg) [93% and 80%, respectively], ampicillin (10 mg) [47%], streptomycin (10 mg) [47%], trimethoprim (5 mg) [47%] and sulfonamide (300 mg) [53%]. Although the percentage of antimicrobial resistance varies among studies, it is believed that the indiscriminate use of antimicrobial therapies as a common practice among rural properties contributes to bacterial resistance to these drugs. The sensitivity profile to antimicrobials showed that the analyzed Escherichia coli isolates are resistant to the antimicrobials commonly used for diarrhea treatment in the southern region of the Rio Grande do Sul state, Southern Brazil.