A comparative study between overlay method and selective-differential media for recovery of stressed Enterobacter sakazakii cells from infant formula

This study compares the performance of different selective-differential media with the overlay method for recovery of stressed cells of Enterobacter sakazakii from infant formula milk (IFM). Five different selective-differential media were used in this study: OK medium, violet red bile agar (VRBA), Druggan–Forsythe–Iversen agar (DFI), Enterobacteriaceae enrichment (EE) agar, and fecal coliform agar (FCA). Tryptic soy agar supplemented with 0.1% sodium pyruvate (TSAP) was used as a control. The overlay method involved applying a thin layer (8 ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1×107 CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 °C for 10 min), a freeze–thaw cycle (−20 °C for 24 h, thawed at room temperature, frozen again at −20 °C, and thawed), acidic pH (pH 3.56 for 15 min), alkaline pH (pH 11.04 for 15 min), and desiccation (E. sakazakii was inoculated onto powdered IFM at a level of ca 1×106 CFU/g, held at 21 °C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30 d). No major differences were noticed between the control (TSAP) and the overlay methods. However, the overlay method recovered significantly higher numbers of stressed E. sakazakii cells compared to selective-differential media. Also, the selective-differential media exhibited some variability in terms of their capabilities to recover stressed cells of E. sakazakii. Among all the examined selective-differential media, DFI performed better for recovering stressed E. sakazakii cells. This study suggests that the overlay method may serve as a potential alternative to direct selective plating for best recovery of E. sakazakii from IFM.