Expansion of Shiga Toxin-Producing Escherichia coli by Use of Bovine Antibiotic Growth Promoters

Abstract

Antibiotics are routinely used in food-producing animals to promote growth and prevent infectious diseases. We investigated the effects of bovine antibiotic growth promoters (bAGPs) on the propagation and spread of Shiga toxin (Stx)-encoding phages in Escherichia coli. Co-culture of E. coli O157:H7 and other E. coli isolated from cattle in the presence of sublethal concentrations of bAGPs significantly increased the emergence of non-O157, Stx-producing E. coli by triggering the SOS response system in E. coli O157:H7. The most substantial mediation of Stx phage transmission was induced by oxytetracyline and chlortetracycline, which are commonly used in agriculture. bAGPs may therefore contribute to the expansion of pathogenic Stx-producing E. coli.

Keywords: E. coli; STEC; Shiga toxin–producing Escherichia coli; Stx-encoding bacteriophages; antimicrobial resistance; bacteria; bovine antibiotic growth promoters.

Figure 1

Induction of Shiga toxin (Stx)2 phage propagation and SOS response by bovine antibiotic growth promoters (bAGPs). A) Stx2 phage induction in Escherichia coli O157:H7 EDL933 after 3 h exposure to subtherapeutic concentrations of bAGPs, including monensin (MON), tylosin (TYL), chlortetracycline (CTC), and oxytetracycline (OTC). Ciprofloxacin (CIP) was a control for phage induction. E. coli C600 was used as a phage-susceptible strain. B) Induction of stx2 expression by bAGPs. Fluorescence from an stx2::gfp transcriptional fusion indicates the level of Stx2 phage induction. C) Induction of recA transcription by bAGPs. The level of recA expression indicates the level of SOS response induction. The results show means and SDs of 3 independent experiments. Statistical significance was analyzed by using the Student t-test. **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2

Induction of Shiga toxin (Stx)2 by bovine antibiotic growth promoters (bAGPs) in Shiga toxin–producing Escherichia coli (STEC) strains from cattle. The levels of Stx2 phage induction were examined with 0.1 µg/mL monensin (MON), tylosin (TYL), chlortetracycline (CTC), and oxytetracycline (OTC). ONT, O antigen nontypable. The results show means and SDs of 3 independent experiments. Statistical significance was analyzed by using the Student t-test. **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3

Emergence of Shiga toxin (Stx)2–positive strains of Escherichia coli from cattle (isolates 1–6) by subtherapeutic bovine antibiotic growth promoters (bAGPs) at 0.1 µg/mL (A) and 0.01 µg/mL (B). Frequency of transfer of Stx phages to bovine E. coli isolates. The Stx pages originated from a detoxified derivative of E. coli O157:H7 EDL933, where stx2 was replaced with a kanamycin resistance cassette. The donor and recipient E. coli strains were co-cultured with or without monensin (MON), tylosin (TYL), chlortetracycline (CTC), and oxytetracycline (OTC). The transduction frequencies were calculated by dividing the number of transductants by the number of recipients. The results show means and SDs of 3 independent experiments. Statistical significance was analyzed by using the Student t-test in comparison with antibiotic-free cultures. **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4

Induction of Stx2 phages by treatment with high concentrations of chlortetracycline (CTC) and oxytetracycline (OTC). The phage titer was determined in Escherichia coli O157:H7 EDL933 by treatment with 1 to 8 µg/mL of CTC and OTC. The results show means and SDs of 3 independent experiments. Statistical significance was analyzed by using the Student t-test in comparison with antibiotic-free cultures. **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5

Transfer of Shiga toxin (Stx) phages by bovine antibiotic growth promoters (bAGPs) in Escherichia coli isolates from cattle. For the confirmation of Stx phage (Stx2Ф) transfer, E. coli O26 (stx2-negative, bovine isolate no. 1 in Figure 3) was used. A) PCR confirmation of the presence or absence of stx2, uspA, eaeA, and rfbE_O157 in EDL933 (Stx2Ф donor), O26 before transduction (Stx2Ф recipient), and O26 after transduction (transductant). B) Serotyping of E. coli O157 and O26. +, positive reaction; –, negative reaction. C) Sorbitol fermentation of donor, recipient, and transduced strains on sorbitol MacConkey agar plates. E. coli O157 does not ferment sorbitol, whereas non-O157 E. coli ferments sorbitol and produces pink colonies.

Figure 6

Induction of stx2 expression by treatment of oxytetracycline (OTC) and chlortetracycline (CTC) in combination with other antibiotics. Escherichia coli O157 harboring P_stx2::gfp was exposed to the following antibiotic combinations that are used in cattle for weight gain and feed efficiency: A) OTC/neomycin (NEO), and B) CTC/sulfamethazine (SMZ). The antibiotic combinations were prepared by mixing the indicated concentrations of each antibiotic. The results show means and SDs of a single representative experiment with triplicate samples. The experiment was repeated 3 times, and similar results were observed in all experiments. Statistical analysis was performed by using a Student t-test and GraphPad Prism 6 (http://www.graphpad.com/). NS, not significant; RFU, relative fluorescence units.