Development and optimization of a novel immunomagnetic separation- bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth

Abstract

Salmonella is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of Salmonella in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of Salmonella enterica serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 10(4) CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of Salmonella serovar Enteritidis in broth cultures which can be applied to preenriched food samples.

FIG. 1

Flowchart of the IMS-bacteriophage protocol for detection of Salmonella serovar Enteritidis. Salmonella-positive and -negative scenarios are given. In step 1, Salmonella cells are captured and concentrated by IMS with anti-Salmonella Dynabeads. In step 2, phage are added to samples and incubated to allow attachment; exogenous phage are removed by washing the beads. In step 3 phage are amplified in the host and are released upon lysis; progeny phage are recovered and added to a healthy population of Salmonella serovar Enteritidis cells (SACs). In step 4, the assay end point is determined by measuring optical density (as shown) or fluorescence. Positive samples are identified based on decreased signals compared to the signals obtained for negative control samples.

FIG. 2

One-step growth curve for phage SJ2 in Salmonella serovar Enteritidis C721. The lag time is approximately 32 to 42 min with a burst size of about 245 PFU.

FIG. 3

Sensitivity of the IMS-bacteriophage assay as determined with five populations of Salmonella serovar Enteritidis. Values are expressed as percentages of the mean negative control value. The assay end point was determined by using optical density measurements obtained at 600 nm. The dark shaded bars show the results of assays performed with SACs from an overnight culture; the light shaded bars show the results of assays performed with freeze-dried SACs. The dashed line indicates the 70% cutoff value for positive tests. The error bars indicate 1 standard deviation.

FIG. 4

Sensitivity of the IMS-bacteriophage assay as determined with five populations of Salmonella serovar Enteritidis. Values are expressed as percentages of the mean negative control value. The assay end point was determined by using fluorescence measurements. The dashed line indicates the 70% cutoff value for positive tests. The error bars indicate 1 standard deviation.

FIG. 5

Relationship between plate counts and plate counts obtained following IMS for different dilutions of Salmonella serovar Enteritidis in broth. Two replicates from each 10-fold dilution (10³, 10⁴, 10⁵, 10⁶, 10⁷, and 10⁸ CFU/ml) were counted. The line is the fitted regression line (y = 0.89x + 0.39; r² = 0.99).