Evaluation of enterohemorrhagic Escherichia coli (EHEC) growth media against six EHEC strains

Authors: ADAMS, DANIEL - Creatv Micro Tech, Inc; ZHU, PEIXUAN - Creatv Micro Tech, Inc; Shelton, Daniel; STEFANSSON, STEINGRIMUR - Creatv Micro Tech, Inc; LI, SHUHONG - Creatv Micro Tech, Inc; AMSTUTZ, PLATTE - Creatv Micro Tech, Inc; TANG, CHA-MEI - Creatv Micro Tech, Inc

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/27/2009
Publication Date: 4/27/2009
Citation: Adams, D., Zhu, P., Shelton, D.R., Stefansson, S., Li, S., Amstutz, P., Tang, C. 2009. Evaluation of enterohemorrhagic Escherichia coli (EHEC) growth media against six EHEC strains. Meeting Abstract.

Interpretive Summary:

Technical Abstract: Introduction: Food safety specialists continue to be challenged by the need to determine the presence of pathogenic bacteria in foods. This is especially true for E. coli O157:H7 and emerging EHEC strains. Such EHECs survive well in meat and poultry, and although stressed, can remain viable in dry or low nutrient conditions on fresh produce. All test methods continue to depend on enrichment to resuscitate the stressed viable cells and to provide an adequate number of cells for sensitivity and statistical validity of the assay. There is a need for an enrichment medium that provides rapid recovery from a variety of stress conditions for a broad range of EHEC strains and a rapid growth rate. In our study we compared 24 enrichment media that are either commercially available, or commonly used, media preparations. We tested the media to determine the time required to resuscitate stressed cells and their growth rates after resuscitation (doubling time). We tested the media against six EHEC strains that had been subjected to three different stress conditions. Our goal was to find one or more medium that could be used with a rapid immunoassay or PCR test. We found that the resuscitation and doubling times vary greatly, which impacts the choice of an optimal growth medium. Materials and Methods: Six EHEC strains were used in this study: four O157:H7, one O26, and one O111. All strains were conditioned by culturing in 10 mL of minimal lactose broth for 18 hours at 37 deg C under static conditions. Cell concentration was then measured and standardized. Control experiments were conducted by placing 200 microliters of each medium into microplate wells and spiking each with 2 microliters of the conditioned E. coli strain culture. The plates were incubated at 42 deg C, with OD readings at 620 nm taken at time 0 and every hour for eight hours. The cells were subjected to stress conditions of low pH, desiccation, and nutrient depletion. Low pH experiments were conducted by adjusting the cell culture pH to 3.0 for 30 min before adding 2 microliters to microplate wells containing the respective medium. Desiccation experiments were conducted by placing 2 microliters cell culture in the wells, incubating at 37 deg C for 30 minutes, and then adding media. Nutrient Depletion experiments were conducted by replacing the culture media with de-ionized water, incubating at 37 deg C for 24 hours, and then adding 2 microliters to the wells. Results and Discussions: Doubling times were calculated based on the time required to increase OD from 0.010 (10x6 cfu/ml) to 0.100 (10x7 cfu/ml). The time to resuscitate was determined as the incremental time required for stressed cells to reach OD 0.100, as compared to the time required to reach OD 0.100 for the unstressed controls. Conclusions: Twelve of the media had doubling times of 18-30 min for all six strains under the control conditions. • Effect of Stress on doubling time. Eight of the media demonstrated the same doubling times (18-30 min) for resuscitated cells as under control conditions: LB broth, RapidChek for E. coli O157 (SDI), Medium B (proprietary reagent, Creatv MicroTech), "Medium C" (name withheld by manufacturer), NZCYM, TSB, SOC and R&F E.coli O157:H7 Enrichment Broth (R&F Laboratories). • Resuscitation time. - Low pH. Low pH had relatively little effect on the resuscitation times of the 24 media. - Desiccation. The fastest resuscitation was provided by LB broth, Medium B, NZCYM, Universal Pre-Enrichment broth, and Nutrient broth. - Nutrient Depletion. The fastest resuscitation was provided by LB broth, RapidChek for E. coli O157, Nutrient broth, Medium B, Medium C, NZCYM, TSB and SOC. - Strain variation. Of the media already mentioned above, the following provided the most consistent and rapid doubling times for all stains tested: LB broth, RapidChek for E. coli O157, Medium B, Medi