Enhanced recovery and molecular techniques for determining bacterial colonists and viable pathogens on the complex phylloplane matrix

Abstract

The rise in demand for fresh fruits and vegetables has seen an increase in the risk of gastrointestinal disease, by pathogens such as E. coli O157:H7 and Salmonella enterica, since such produce is eaten uncooked. Although most produce is washed in chlorinated water, this processing may not be sufficient and can result in the formation of hazardous compounds. Classical cell culture techniques as well as novel episcopic differential contrast and epifluorescence microscopy (EDIC/EF) combined with the BacLightTM kit (to distinguish live and dead bacteria), the DAPI assay (to distinguish bacterial colonists and inorganic debris) and GFP-Salmonella strains were combined for the first time to study the complex leaf surface (phylloplane). EDIC/EF microscopy was shown to be advantageous compared to other methodologies; as well as being able to visualize GFP-labelled Salmonella inoculated onto the phylloplane it was possible to observe the naturally residing microflora on this difficult matrix. The viable pathogens and microflora were shown to colonize by four strategies: they form clusters on the phylloplane; or single cells integrate into pre-existing aggregates of biofilm microcolonies; they become entrapped in niches such as stomata; or they actively swim into the stomata and become subsurface (confirmed using laser scanning confocal microscopy, LSCM). The clusters were sometimes surrounded by slime, suggesting the formation of biofilm on the phylloplane. The effects of treatments to the phylloplane were not directly comparable, due to large biological variations in each field of view; smaller treated sample areas should allow for qualitative and quantitative comparisons. The Stomacher is at present used worldwide for the mechanical release of microorganisms from various matrices; here it was compared to the Pulsifier, which was shown to be more efficient in terms of cell recovery and causing less damage to the watercress phylloplane. Surface attachment was investigated by use of the Pulsifier release principle and refinements in its protocol were made. Pulsifier recovery techniques showed the inefficiencies of potential disinfectants in killing attached microorganisms, since they were not susceptible to attack until released into aqueous suspension. It is these ‘protected’ cells that then subsequently go on to produce foodborne illnesses. Further study showed the molecular signalling molecule nitric oxide (NO), to be an important physiological release agent, for enhanced recovery of coliforms, but not Salmonella, from the phylloplane. Chemical methods of decontamination such as the use of ozone were shown to be efficient at reducing the numbers of viable cells, particularly when combined with pulsification mechanical release of cells into aqueous suspension, resulting in between 1- and 2-log reductions. However, this procedure is not ideal, due to chemical damage to the phylloplane and problems in maintaining constant ozone concentrations, both in the laboratory and at the factory. It was shown that chlorine levels could be reduced to 20 ppm compared to the industry standard of 90-120 ppm, this producing similar log reductions of between 1- and 2-log. The Pulsifier and NO were shown in combination to provide effective mechanical and physiological detachment strategies, releasing almost 4-log cells. It was found that 20 or 500 nM of NO, produced a 3-log dispersion of bacterial cells, including biofilm aggregates off the surface of watercress leaves. These studies demonstrate the importance of microbial physiology in the attachment of microorganisms on fresh produce phylloplanes and suggest that disinfection procedures are unnecessary for sanitation.

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ORCID for Charles W. Keevil: orcid.org/0000-0003-1917-7706

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