Effects of Media on Recovery of Escherichia coliO157:H7 and Pseudomonas fluorescens from Spinach

Abstract

AbstractEscherichia coli O157:H7 is an important foodborne pathogen implicated in contamination of leafy greens and can cause hemorrhagic colitis in humans. Efficient recovery and enumeration of E. coli O157:H7 (Ec) and Pseudomonas fluorescens from produce are crucial for efficacy of biocontrol of Ec. Biocontrol refers to the suppression, reduction or control of one organism with another. P. fluorescens (Pf) is used for biocontrol research due to its colonization potential and suppressive activity against many microbes. We evaluated the sensitivity and selectivity of Restaino and Frampton (R & F) E. coli O157:H7 chromogenic medium (RFCM) and the effect of storage time and temperature on recovery of Ec and Pf from cultures and spinach. Ec and Pf were inoculated at the same level. Microbial counts differed between media and Ec was identified on RFCM based on color (blue‐black precipitate), while Pf had distinct white/translucent colonies that fluoresce under ultraviolet light. Bacterial populations from mixed cultures plated on media and recovered ranged from 3.6–8.7 and 4.2–7.7 log cfu/mL for Ec and Pf, respectively. Microbial counts on spinach ranged from 6.0–8.2 and 6.5–8.6 log cfu/g for the above bacteria. Bacterial recovery increased with storage time of 0–48 h, implying a possible increase of contact time for bacterial attachment. The RFCM was best suited for simultaneous recovery of Ec and Pf based on differentiation of colonies on media and can be an effective tool for biocontrol research.Practical ApplicationsEfficient recovery and accurate enumeration of Escherichia coli O157:H7, the human pathogen and Pseudomonas fluorescens, the biocontrol agent, from leafy greens are crucial for assessment of biocontrol efficacy. P. fluorescens has been used for biocontrol of some foodborne and plant pathogenic microbes from leafy greens, phyllosphere and rhizosphere of agronomic crops based on its competitive and antagonistic ability. Other than the generation of antibiotic resistant strains (foodborne pathogens) or use of green fluorescent‐labeled mutants, there are limited options available for simultaneous recovery and enumeration of biocontrol and pathogenic bacteria. This research provides information on the recovery and differentiation of E. coli O157:H7 and P. fluorescens from media and inoculated spinach. The potential implication is a methodology for evaluation of the effectiveness of a biocontrol agent, with applications to other leafy greens. Accurate recovery and enumeration of biocontrol and pathogenic bacteria would promote optimal determination of biocontrol efficacy, a component of postharvest intervention strategy. This study validates the use of selective medium with specific applications for this type of research.