Native Microbial Aggregates On Alfalfa

Interpretive Summary: In nature, bacteria are often present as large grouping of cells attached to each other and to the solid surface. These groupings are called biofilms. Bacteria in biofilms are much more resistant to killing by antimicrobial compounds. As biofilms on plant surfaces are poorly described and sprouts have been responsible for numerous outbreaks of foodborne illness, in this study we examined the surface of alfalfa, clover and mung bean sprouts for biofilms using a relatively new technique called confocal scanning laser microscopy (CSLM). Unlike other types of microscopy, CSLM allows for the study of biofilms in their natural state without the need fixation steps that can introduce artifacts and also allows for the determination of biofilm thickness. The study indicated that biofilms were abundant on sprout surfaces. Over 150 of the biofilms were examined for area and thickness. The area of the biofilms ranged from 32 to 7283 µm2. The depth of the biofilms ranged from 1.7 to 12.6 µm. This study clearly indicated that biofilms on sprout surfaces were not simply monolayers of microbial cells. As complex biofilms on sprouts may harbor pathogenic bacteria, interventions aimed at ensuring the microbial safety of sprouts and reducing spoilage need to be effective against plant and human pathogens present in biofilms.

Technical Abstract: In nature, bacteria are often organized in biofilms. Biofilms on inert surfaces have been studied in depth using confocal scanning laser microscopy (CSLM) with a variety of fluorescent probes. In contrast, there have been few studies of biofilms on living surfaces including plants. CSLM in combination with the LIVE/DEAD® BacLightTM Viability Kit (Molecular Probes, Inc.) was used in a survey of biofilms on alfalfa, clover and mung bean sprouts purchased from retail outlets. Biofilm area and thickness were quantified by use of a 20X dry lens due to its large free working distance and field of view. Values for measured thickness (z- axis) were corrected based on point spread functions of fluorescent latex spheres. Biofilms were readily observed and consisted primarily of live bacteria. Average areas for biofilms for the sprout samples were 201 um2 to 470 um2 (range of 32 to 7283 um2). The average depth of the observed biofilms was from 2.0 to 6.3 um (range of 1.7 to 12.6 um) indicating only a single to a few layers of cells. Bacteria in fully hydrated biofilms as imaged by CSLM appeared to be less densely packed when compared to biofilms imaged previously in our laboratory with traditional scanning electron microscopy techniques. Despite a lack of considerable depth, biofilms on plant surfaces may harbor plant and human pathogens making their eradication more problematic.