Concentration of Bacterial Pathogens Using Apolipoprotein H

Introduction: Concentration of bacteria from food or environmental samples prior to detection could reduce or even eliminate the need for cultural enrichment.  A broadly reactive ligand with the ability to concentrate a variety of microbes from relevant sample matrices could facilitate this type of sample preparation. The human plasma protein Apolipoprotein H (ApoH) has been shown to have a high affinity for a number of Gram- and Gram+ bacteria. 

Purpose: To investigate the utility of ApoH conjugated to magnetic beads for the capture and concentration of select foodborne bacterial pathogens. 

Methods: Overnight cultures of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serovar Enteritidis, and Staphylococcus aureus were serially diluted in proprietary binding buffer to concentrations of 10³, 10⁵ and 10⁷CFU/100 µl. Suspensions were supplemented with 10μl of ApoH conjugated magnetic beads (ApoH Technologies, Villeneuve St Georges, France) and incubated for 60 min at 4°C with rotation. The beads were captured by magnet and washed twice.  Both bead and supernatants suspensions were retained for cultural enumeration of bacteria.  An aliquot of the beads was also subjected to DNA extraction followed by detection of each pathogen using a SYBR green qPCR method targeting the 16S rDNA gene. 

Results: Based on loss to supernatant, the ApoH beads showed high capture efficiency (73.4-100%) for all four pathogens tested and at all three concentrations (10³, 10⁵ and 10⁷ CFU/100 µl.) In most cases, there were no statistically significant differences in capture efficiencies when comparing pathogens or initial cell concentration (P > 0.05, n = 3).  The SYBR green qPCR results were more variable but in general, assay detection limits after ApoH capture and qPCR were approximately one log CFU higher compared to input cell numbers. 

Significance: ApoH conjugated magnetic beads show promise for concentration of bacterial pathogens in preparation for detection using cultural methods or qPCR.