Evaluation of Carvacrol for the Control of Escherichia coli O157 on Cattle Hide and Carcass Cuts

Bacterial strains and control reagents

Four toxigenic E. coli O157 strains (380-94, T6H11, J21, and 13C1T3) were obtained from the Teagasc Food Research Centre, Ashtown culture collection. All strains were made resistant to nalidixic acid (50 μg/mL) and streptomycin sulphate (1000 μg/mL), as outlined by Park (1978) to aid in the recovery of the inoculated strains.

Preparation of inoculum and carvacrol

All bacterial cultures were inoculated into 10 mL of tryptone soya broth (TSB; Merck, Whitehouse Station, NJ) and incubated overnight at 37°C. The overnight E. coli O157 cultures were centrifuged at 8000×g for 10 min and washed in 10 mL of phosphate-buffered saline (PBS) prior to use. A cocktail of E. coli O157 strains were prepared in PBS (total volume 20 mL) to achieve a final concentration of 4–5 log₁₀ CFU/cm² or 5–6 log₁₀ CFU/cm² to hide and carcass surfaces, respectively.

A stock solution of carvacrol (purchased as a liquid from Sigma-Aldrich, St. Louis, MO) in ethanol (v/v; ratio of 90:10) was prepared to make an emulsification to aid in application, and diluted in sterile water (50 mL) to achieve a final concentration of 10, 20, or 30 mg/mL carvacrol.

Hide and carcass samples

Cattle hide (approximately 2×2 m) and carcass pieces (20×20 cm; from pre-chilled carcasses) were obtained either during in-house processing at Teagasc Food Research Centre, Ashtown or from a local abattoir (from the slaughter line), and transported immediately to the laboratory for processing. The conditions of the hides obtained were dry and moderately dirty. The carcass cuts were obtained from the brisket area of the carcass. The hide or carcass cuts did not undergo any pre-treatments or washes at the abattoir prior to collection. The large sections of hide were cut into smaller 30×30 cm pieces, and one piece was used for each treatment. Each hide and carcass piece was pegged onto a stainless steel board, and smaller sections on each hide (20×20 cm) and carcass (15×15 cm) piece were marked with a permanent marker. The prepared inoculum of E. coli O157 was gently poured onto the marked areas and spread evenly using a sterile plastic plate spreader. Hide and carcass pieces were then left to dry for 1 h in a laminar flow hood to allow for bacterial attachment. The volume of inoculum that was applied dried substantially on both hide and carcass pieces prior to treatment. Inoculated hide and carcass pieces were treated with either a 50-mL wash containing 10, 20, and 30 mg/mL carvacrol or with 50 mL of sterile distilled water only. Treatments were applied to marked areas using a hand-held pressure spray bottle and left in contact for 10 min. A piece of hide and carcass inoculated with the marked E. coli O157 cocktail but not treated with any washes (no wash) was used as a control. A hide and carcass piece not inoculated or treated was tested for the presence of enteric bacteria that possess the same antibiotic-resistant profile as the marked E. coli O157 strains applied in the study. The hide and carcass pieces were held at room temperature throughout the experiment. The assays for all treatments were performed in triplicate (n=3) with separate hide and carcass pieces and inoculum cocktails.

Sampling and enumeration of E. coli O157

The swab samples for microbiological analysis were performed on the hide and carcass portions in accordance with Regulation (EC) 2073/2005. The swab was then placed into sterile stomacher bags (Seward Laboratory, London, UK) and diluted in 90 mL of modified TSB containing streptomycin sulfate (1000 μg/mL) and nalidixic acid (50 μg/mL; mTSB-nas) and stomached for 2 min. The swab samples were performed in duplicate using different areas of each treated sample. The enumeration of E. coli O157 was immediately performed as outlined by McGee et al. (2004), where all stomached swab and excision samples were serially diluted in MRD and 0.1-mL aliquots were spread plated in duplicate onto tryptic soya agar (TSA; Oxoid, Basingstoke Hampshire, UK) and incubated at 37°C for 2 h. Plates were then overlaid with sorbitol MacConkey agar containing streptomycin sulphate (1,000 μg/mL) and nalidixic acid (50 μg/mL; SMAC-nas) and incubated at 37°C for 48 h.

Statistical analysis

All counts for all experiments were log₁₀ transformed, and one-way analysis of means and comparisons of means (Tukey's method) were performed on all data sets using Minitab 12.1 (Minitab Inc., Minneapolis, MN).