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An improved direct plate method for the enumeration of stressed Escherichia coli O157:H7 from food
J McCarthy1, R Holbrook, P J Stephens
1Unilever Research, Bedford, UK. jan.mccarthy@unilever.com
Abstract:
The use of sorbitol MacConkey agar (SMAC) performed poorly in supporting growth of stressed Escherichia coli O157:H7 cells. Up to a 3-log difference was observed between counts on SMAC and tryptone soy agar (TSA). It is critical in the risk assessment of certain foods to be able to enumerate stressed and healthy E. coli O157:H7 in a background of potentially healthy competing bacteria. Investigations carried out to overcome the inhibitory effect of SMAC included the reduction of the selective agent concentration, inclusion of a recovery stage in broth prior to plating out, addition of recovery agents, and delayed exposure to the selective agent. The only successful approach was delayed exposure to the selective agent. This was achieved by resuscitating the stressed cells on a membrane placed on the surface of a TSA plate and, after a defined time period sufficient for full resuscitation, transferring the membrane to the surface of a SMAC plate. The choice of membrane material was critical for maintaining the positive sorbitol color change used to identify wild-type E. coli. Track-etched polycarbonate membranes allowed the typical color reactions to be visualized, whereas cellulose acetate did not. The method was validated with E. coli O157:H7 cells stressed by low pH and high salt conditions, whereby all cells that would previously be undetectable on direct inoculation of SMAC were countable.
Insights
Stressed Escherichia coli O157:H7 enumeration on sorbitol MacConkey agar (SMAC) was improved using a delayed selective agent exposure method. This technique, involving resuscitation on tryptone soy agar (TSA) before SMAC plating, successfully recovered previously undetectable stressed cells.
Area of Science:
- Food Microbiology
- Bacteriology
- Diagnostic Microbiology
Background:
- Stressed Escherichia coli O157:H7 is difficult to enumerate using standard sorbitol MacConkey agar (SMAC).
- Accurate enumeration of stressed E. coli O157:H7 is critical for food safety risk assessments.
- Traditional SMAC media exhibit poor recovery rates for stressed cells, showing up to a 3-log difference compared to tryptone soy agar (TSA).
Purpose of the Study:
- To develop a reliable method for enumerating stressed Escherichia coli O157:H7.
- To overcome the inhibitory effects of SMAC on stressed bacterial cells.
- To improve the sensitivity of E. coli O157:H7 detection in food matrices.
Main Methods:
- Investigated methods to overcome SMAC inhibition, including reduced selective agent concentration, broth recovery, recovery agents, and delayed selective agent exposure.
- Developed a two-stage plating technique: resuscitation of stressed cells on TSA, followed by transfer to SMAC using track-etched polycarbonate membranes.
- Validated the method using E. coli O157:H7 cells subjected to low pH and high salt stress.
Main Results:
- Delayed exposure to the selective agent on SMAC was the only successful approach to improve recovery.
- Track-etched polycarbonate membranes were critical for maintaining the sorbitol fermentation color change necessary for E. coli identification.
- The developed method enabled the enumeration of previously undetectable stressed E. coli O157:H7 cells.
Conclusions:
- A novel resuscitation method using delayed exposure to SMAC significantly enhances the recovery of stressed Escherichia coli O157:H7.
- This improved enumeration technique is vital for accurate food safety evaluations.
- The choice of membrane material is crucial for the efficacy of the delayed plating method on SMAC.