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Efficacy of the thin agar layer method for the recovery of stressed Cronobacter spp. (Enterobacter sakazakii)
Tareq M Osaili1, Anas A Al-Nabulsi, Reyad R Shaker
1Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, P.O. Box (3030), Irbid, 22110, Jordan. tosaili@just.edu.jo
Insights
The thin agar layer (TAL) method using Druggan-Forsythe-Iversen (DFI)+TSA effectively recovers stressed Cronobacter spp. This method improves detection of these opportunistic pathogens, even after exposure to various environmental challenges.
Area of Science:
- Microbiology
- Food Safety
- Pathogen Detection
Background:
- Cronobacter spp. (Enterobacter sakazakii) are opportunistic pathogens, posing risks to all age groups, especially infants.
- Sublethal stress can injure Cronobacter cells, hindering detection by standard selective media without extended recovery.
- Effective recovery methods are crucial for identifying stressed Cronobacter in food safety contexts.
Purpose of the Study:
- To evaluate the efficacy of the thin agar layer (TAL) method for recovering stressed Cronobacter spp.
- To compare the recovery rates of Cronobacter cells exposed to various stresses using different agar media.
- To determine if TAL enhances the detection of sublethally injured Cronobacter cells.
Main Methods:
- Cronobacter sakazakii and C. muytjensii strains were subjected to multiple stress conditions (starvation, heat, cold, acid, alkaline, chlorine, ethanol) and desiccation.
- Cell recovery was assessed on nonselective tryptone soy agar (TSA), selective violet red bile glucose agar (VRBGA), selective Druggan-Forsythe-Iversen (DFI) agar, and TAL combinations (VRBGA+TSA, DFI+TSA).
- Comparative analysis of recovery rates and differentiation in mixed cultures was performed.
Main Results:
- No significant difference in recovery of stressed, desiccated Cronobacter spp. was observed between TSA, DFI+TSA, and VRBGA+TSA media.
- Violet red bile glucose agar (VRBGA) alone showed significantly lower recovery rates for stressed cells.
- The DFI+TSA combination demonstrated superior differentiation of Cronobacter spp. in mixed bacterial cultures compared to VRBGA+TSA.
Conclusions:
- The thin agar layer (TAL) method, specifically DFI+TSA, is an effective approach for direct recovery of stressed Cronobacter spp.
- This method shows promise for improved detection of Cronobacter, even when cells are sublethally injured by various stresses.
- The findings suggest adopting the TAL method DFI+TSA for enhanced Cronobacter spp. isolation in food safety surveillance.
Abstract:
Cronobacter spp. (Enterobacter sakazakii) are emerging opportunistic pathogens for all age groups, and are of particular concern when it comes to infants. Prior to contaminating food, the organism may be exposed to a variety of stresses, leading to a generation of sublethally injured cells that may not be detected by selective media unless a protracted recovery period is included in the isolation procedure. This study evaluated the efficacy of the thin agar layer (TAL) method for the recovery of Cronobacter cells that had been exposed to various stress conditions. Five strains of C. sakazakii and C. muytjensii were exposed to starvation, heat, cold, acid, alkaline, chlorine, or ethanol, with or without further exposure to desiccation stress. The recovery of the stressed cells was determined on tryptone soy agar (TSA; nonselective control medium), violet red bile glucose agar (VRBGA; selective agar), Druggan-Forsythe-Iversen (DFI; selective agar), and TAL media (viz., VRBGA overlaid with TSA, and DFI overlaid with TSA). Regardless of stress type, there were no significant differences among the recoveries of stressed desiccated Cronobacter spp. cultures on TSA, DFI+TSA, and VRBGA+TSA, but there was significantly less recovery on VRBGA. The recovery of prestressed desiccated Cronobacter spp. on DFI+TSA was similar to that on TSA, whereas the recovery on VRBGA+TSA was lower. DFI+TSA performed better than VRBGA+TSA did in differentiating Cronobacter spp. within mixed bacterial cultures. The results of this study suggest the use of the TAL method DFI+TSA as an improved method for the direct recovery of stressed Cronobacter spp.
