Comparison of a novel strategy for the detection and isolation of Salmonella in shell eggs with the FDA bacteriological analytical manual method

Guodong Zhang, Eve Thau, Eric W. Brown, and Thomas S. Hammack
Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD 20740

The reference FDA Bacteriological Analytical Manual (BAM) method (2013 version) for the detection of Salmonella in eggs requires two weeks to complete. The objective of this project was to improve the BAM method for the detection and isolation of Salmonella in whole shell eggs. A novel protocol, using 1000 g liquid eggs for direct preenrichment with 2 L tryptic soy broth (TSB) followed by enrichment using Rappaport-Vassiliadis and Tetrathionate broths, was compared with the standard BAM method, which requires 96 h room temperature incubation of whole shell egg samples followed by preenrichment in TSB supplemented with FeSO₄. Four Salmonella ser. Enteritidis (4 phage types) and one Salmonella ser. Heidelberg isolates were used in the study. Bulk inoculated pooled liquid eggs, weighing 52 or 56 kg (approximately 1100 eggs) were used in each trial. Twenty 1000 g test portions were withdrawn from the pooled eggs for both the alternative and the reference methods. Test portions were inoculated with Salmonella at 1 to 5 CFU/1000 g eggs. Two replicates were performed for each isolate. In the eight trials conducted with Salmonella ser. Enteritidis, the alternative method was significantly (p<0.05) more productive than the reference method in 3 trials, and significantly (p<0.05) less productive than the reference method in 1 trial. There were no significant (p<0.05) differences between the two methods for the other 4 trials. For Salmonella ser. Heidelberg, combined data from two trials showed the alternative method was significantly (p<0.05) more efficient than the bam method. We have concluded that the alternative method, described herein, has the potential to replace the current bam culture method for detection and isolation of Salmonella from shell eggs based on the following factors: (1) the alternative method is 4 days shorter than the reference method; (2) it uses regular TSB instead of the more complicated TSB supplemented with FeSO₄; (3) it was equivalent or superior to the reference method in 9 out of 10 trials for the detection of Salmonella in shell eggs, (4) it could dectect Salmonella as low as 1 cell in 1000 g eggs.

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TABLE 1. Comparison of new and reference FDA BAM culture methods for the detection of Salmonella ser. Enteritidis in shell eggs¹

¹ All uninoculated controls were negative. Second trial for phage type 8 and both trials for phage type 14b had 5 uninoculated controls. The rest had 3 uninoculated controls.
² MPN, Most Probable Number.
³ Fisher's exact test was conducted using SAS 9.3_M 1.

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TABLE 2. Comparison of new and reference FDA BAM culture methods for the detection of Salmonella ser. Heidelberg in shell eggs¹

¹ Each trial had 3 uninoculated controls. All uninoculated controls were negative.
² MPN, Most Probable Number.
³ Fisher's exact test was conducted using SAS 9.3_M 1.