Food Safety Interventions: Adhesin-Specific Nanoparticles for Removal of Foodborne Pathogens

Tzuen-Rong Jeremy Tzeng

Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA

E-mail: tzuenr@clemson.edu

ABSTRACT

Many pathogenic bacteria use carbohydrate-binding proteins (adhesins) to adhere to specific host cell receptors (carbohydrate receptors) to initiate infection. The application of synthetic carbohydrate receptor analogs, which compete with host for bacterial adhesins, could serve as an anti-bacterial-adhesion strategy. Polymeric nanoparticles with polystyrene core and polyethylene glycol tethers covalently functionalized with carbohydrate molecules were synthesized and characterized. Specifically, multivalent D-mannose biofunctionalized nanoparticles bound strongly with Escherichia coli ORN178 expressing FimH adhesin resulted in significant nanoparticle-mediated bacterial aggregations.

Applications utilizing carbohydrate biofunctionalized nanoparticles have potentials as alternatives to antibiotics for the removal and control of foodborne pathogens. Their potential toxicities as well as applications in biosensor development are also discussed.

Introduction

Pathogens establish infection after adhering to host tissue through multivalent carbohydrate-lectin interactions. Inhibition of bacterial cell adhesion to intestinal epithelium is an effective anti-enteropathogen strategy. Aggregated bacteria cells bind poorly to host epithelium cells due to peristaltic pressure and produce reduced colony forming units on media. Numerous publications have established the scientific basis for the syntheses and the application of different carbohydrate and multivalent glycoconjugate analogs for the purpose of anti-adhesion strategy.

Figure 1. Schematic diagram (a) and the TEM image (b) of the synthetic mannose bioactive nanoparticles (previous work, Qu et al. 2005a).

Nanoparticles have found uses in biology and medicine for applications such as biological labeling and drug delivery. In our previous work (Luo et al. 2005, Qu et al. 2005a) we reported the synthesis of carbohydrate biofunctionalized nanoparticles (Figure 1). We have also shown that carbohydrate-functionalized nanoparticles are able to bind to pathogenic bacteria and mediate bacterial cell aggregation (Qu et al. 2005a,b). This was demonstrated by an EM study, which showed that the mixing of nanoparticles with bacterial cells in solution led to the formation of cell aggregates (Figure 2), thus indicating a high level of binding affinity between the bacteria and the NPs (Qu et al. 2005a).

Materials and Methods

Aggregation of E. coli mediated by D-mannose nanoparticles

Early stationary culture of E. coli ORN178 (fim⁺) that expresses wild-type type 1 pili and ORN208 (fim^-) that expresses abnormal type 1 pili were harvested and washed twice with phosphate buffered saline. The bacterial suspension was then mixed with an aqueous suspension of the nanoparticles and centrifuged. The supernatant containing free nanoparticles was removed, and the pellet was washed with PBS, centrifuged, and then re-suspended in PBS. Cells were then fixed in cacodylate buffered glutaraldehyde for transmission electron microscope imaging (TEM). Agglutination of E. coli ORN178 cells by D-mannose biofunctionalized nanoparticles is shown in Figure 2.

Figure 2. Lower (a) and higher (b) magnification of TEM images of E. coli aggregation mediated by synthetic mannose bioactive nanoparticles (previous work, Qu et al. 2005a).

In vitro and in vivo biocompatibility of mannosylated polystyrene nanoparticles

The applications of nanoparticles necessitate contact with animal or human tissues, potentially via inhalation, oral ingestion, or exposure to the eyes and skin routes. In vitro and in vivo exposure sensitivity studies were conducted to investigate cellular and tissue-level responses for each of these routes of exposure. MTS, trypan blue, and live/dead assays were conducted to determine the response of human lung, colon, and dermal fibroblasts and rat lung macrophages to nanoparticle exposure over a range of concentrations. Ocular and skin sensitivity studies were conducted with New Zealand albino rabbits, and oral ingestion and inhalation studies were conducted with Sprague Dawley rats using approved protocols (Shyamprasad et al. 2006).

Results and Discussion

The agglutination mediated by D-mannose biofunctionalized nanoparticles is shown to be adhesin specific as the same experimental procedures done with E. coli ORN208, a variant of E. coli ORN178 deficient in mannose-specific adhesin (Qu et al. 2005a), resulted in no agglutination. Bare nanoparticles core with PEG tethers lacking the surface D-mannose functional groups did not cause agglutination of E. coli ORN178 or ORN208.

Polystyrene-PEG-Mannose nanoparticles did not exhibit consistent significant levels of toxicity towards human lung and dermal fibroblasts and rat lung macrophages. Very low, but significant levels of toxicity towards human colon fibroblasts were observed. The ocular studies revealed no signs of discomfort or acute inflammation. The dermal studies showed no signs of edema or erythema. No inflammatory response was evident in blood or tissue samples of the lungs, gastrointestinal tract, liver, kidney, and spleen that were collected in the inhalation and oral ingestion studies. The results from these studies provide evidence that these nanoparticles may be safe for use as antibacterial agents.

Conclusion

Multivalent nanoparticles fabricated to display specific receptor molecules could potentially be used to physically remove targeted pathogens from hosts. The same adhesin-receptor specificities could be used to fabricate biosensors for the detection of foodborne pathogens.

Although minimum cytotoxicity have been observed with polystyrene nanoparticles, the cytotoxicity of nanomaterials in their intended application formats, e.g. sizes, valencies, liquid, or powder form, as well as the environmental impacts of the released nanomaterials must be evaluated.

Acknowledgements

Luo, P. G.; Tzeng, T.-R.; Qu, L.; Lin, Y.; Caldwell, E.; Latour, R. A.; Stutzenberger, F.; Sun, Y.-P. “Quantitative Analysis of Bacterial Aggregation Mediated by Bioactive Nanoparticles.” J. Biomed. Nanotech. 2005, 1, 291-296.

Qu L, Luo P G, Taylor S, Lin Y, Huang W, Tzeng T-RJ, Stutzenberger F, Latour RA, Sun Y-P. 2005a Visualizing adhesion-induced agglutination of Escherichia coli with mannosylated nanoparticles.” J. Nanosci. Nanotech. 5, 319-322.

Qu L, Gu L, Li H, Taylor S, Elkin T, Luo PG, Tzeng T-RJ, Jiang X, Latour R A, Williams A, Sun Y-P 2005b. Galactosylated polymeric nanoparticles: synthesis and adhesion interactions with Escherichia coli. J. Biomed. Nanotech. 1 61-67.

Shyamprasad M, Qu L, Lin Y, Sun Y-P, Tzeng T-R, Stutzenberger, F, Latour R. 2006. In vitro and in vivo biocompatibility of mannosylated polystyrene nanoparticles. J. Biomed. Nanotech. 2, 1-10.