There are some microbes that stick like glue to whatever they touch; if they are foodborne pathogens, this makes surface disinfection and food decontamination extremely difficult. However, even within the same species this stickiness can vary. For example, strains of Listeria monocytogenes show a great deal of variation here, which inspired Tiong et al. (2016) to use tandem mass spectrometric (MS/MS) proteomic analysis to investigate the cell surface proteins that might be responsible1.
Many potential foodborne pathogens secrete biofilms to ensure contact with both food matrices and abiotic surfaces, such as processing equipment. In this way, the microbes manipulate the local environment to assure favorable conditions for multiplication and survival. As covered previously on Examining Food, biofilms and other surface adhesion factors make it extremely difficult to manage these pathogens and there is a strong likelihood of their persistence in the food processing environment. Moreover, strongly adherent microbes are more likely to show increased invasion and infectiveness since these biofilm adhesion factors also act as virulence factors and avoid the host immune response.
Tiong et al. took two strains of L. monocytogenes with well-characterized differences in adherence, and examined the surface proteins secreted by them during attachment to an abiotic surface. The two strains (CW35 – low adherence; 99-38, high adherence) came from primary isolates of contaminated food products—frankfurter and raw ground beef respectively. The team confirmed adherence properties using microplate adherence assays in addition to standard strain serotyping and morphological characterization.
Once characterized, the researchers used two methods to examine the proteomes of each strain, growing the bacteria in both standard suspension as a planktonic culture and as adherent or sessile in the presence of glass bead culture surfaces. They extracted the proteins using a modified UB-Ghost approach for gathering surface proteomes. This involved establishing microbial growth on the glass bead surfaces then washing off cells and purifying the recovered material to remove cytoplasmic elements. Following this, the team harvested surface proteomes in a buffered urea solution. They separated proteins by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) before proteomic analysis of tryptic digests using a hybrid LTQ-Orbitrap XL mass spectrometer in conjunction with nano-liquid chromatography (2D-nanoLC-MSMS). Tiong et al. searched the spectral data against the L. monocytogenes EGD-e database, in addition to using other bioinformatics tools for functional characterization.
From the results, the researchers found that 99-38 showed 30x greater adherence than the CW35 strain. Following proteomic analysis, they identified 640 proteins overall. Of these, 107 came from attached cells, with 21 of these unique to the strongly adherent cells. Further quantitative analysis showed that 22 proteins showed differential expression in the strongly adherent strain, with 11 showing a 5-fold increase and 11 increased by 10-fold.
The team then examined the data for protein localization and functional characteristics, finding that 503 and 93 proteins identified as non-envelope associated for planktonic and sessile cells respectively. They identified 124 proteins as belonging to the microbial envelope. According to analysis, many of these proteins identified as bifunctional and could be classified as ‘moonlighting proteins’, which take on different roles depending on the cell’s environment. Further examination showed that 141 proteins identified during analysis associated with the cell surface.
Noting that the attachment surface for these studies was abiotic, Tiong et al. suggest that further work on biological matrices such as food surfaces might yield differing surface proteomes. However, the team is confident that the results shown demonstrate the variety of surface adhesion factors at work in colonization of food preparation surfaces. More extensive evaluation could yield valuable results for food safety initiatives in determining sanitation procedures and also in detecting virulence in the presence of foodborne pathogen contamination.
Learn more about Listeria monocytogenes testing in our food and beverage community
1. Tiong, H.K. et al. (2016) “Comparison of Surface Proteomes of Adherence Variants of Listeria Monocytogenes Using LC-MS/MS for Identification of Potential Surface Adhesins“, Pathogens 5 (2) doi:10.3390/pathogens5020040