Enhanced inactivation of Salmonella and Pseudomonas biofilms on stainless steel by use of T-128, a fresh-produce washing aid, in chlorinated wash solutions.

The effect of the washing aid T-128 (generally recognized as safe [GRAS] formulation, composed mainly of phosphoric acid and propylene glycol) on inactivation of Salmonella and Pseudomonas populations in biofilms on stainless steel was evaluated under conditions of increasing organic matter loads in chlorinated wash solutions dominated by hypochlorous acid. Biofilms were formed statically on stainless steel coupons suspended in 2% lettuce extract after inoculation with Salmonella enterica serovar Thompson or Newport or with Pseudomonas fluorescens. Coupons with biofilms were washed in chlorine solutions (0, 0.5, 1, 2, 5, 10, or 20 mg/liter at pH 6.5, 5.0 and 2.9), with or without T-128, and with increasing loads of organic matter (0, 0.25, 0.5, 0.75, or 1.0% lettuce extract). Cell populations on coupons were dispersed using intermittent, pulsed ultrasonication and vortexing and enumerated by colony counts on XLT-4 or Pseudomonas agars. Cell responses to fluorescent viability staining of biofilm treatment washing solutions were examined using confocal laser scanning microscopy. Results showed that 0.1% T-128 (without chlorine) reduced P. fluorescens biofilm populations by 2.5 log(10) units but did not reduce Salmonella populations. For both Salmonella and Pseudomonas, the sanitizing effect of free chlorine (1.0 to 5.0 mg/liter) was enhanced (P < 0.05) when it was combined with T-128. Application of T-128 decreased the free chlorine depletion rate caused by increasing organic matter in wash waters and significantly (P < 0.05) augmented inactivation of bacteria in biofilms compared to treatments without T-128. Image analysis of surfaces stained with SYTO and propidium iodide corroborate the cultural assay results showing that T-128 can aid in reducing pathogen viability in biofilms and thus can aid in sanitizing stainless steel contact surfaces during processing of fresh-cut produce.

Fabrication of biomimetically patterned surfaces and their application to probing plant-bacteria interactions.

We have developed a two-step replica molding method for rapid fabrication of biomimetically patterned plant surfaces (BPS) using polydimethylsiloxane (PDMS-BPS) and agarose (AGAR-BPS). Beyond providing multiple identical specimens that faithfully reproduce leaf surface microstructure, this approach also offers unique chemical, physical, and biological features. PDMS-BPS provide good structural durability for SEM examination, have surface wettability comparable to plant surfaces for coating development, and allow for real-time monitoring of biosynthesis through incorporation into microfluidic devices. AGAR-BPS are compatible with bacterial growth, recovery, and quantification, and enable investigation of the effects of surface topography on spatially varying survival and inactivation of Escherichia coli cells during biocide treatment. Further development and application of these biomimetically patterned surfaces to study (and possibly modify) other aspects of plant-bacteria interactions can provide insight into controlling pathogen contamination in a wide range of applications.

Native microflora in fresh-cut produce processing plants and their potentials for biofilm formation.

Representative food contact and nonfood contact surfaces in two mid-sized, fresh-cut processing facilities were sampled for microbiological analyses after routine daily sanitization. Mesophilic and psychrotrophic bacteria on the sampled surfaces were isolated by plating on nonselective bacterial media. Alternatively, bacteria were isolated after an incubation period that allowed the formation of heterogeneous biofilms on stainless steel beads. Of over 1,000 tested isolates, most were capable of forming biofilms, with approximately 30 % being strong or moderate biofilm formers. Selected isolates (117) were subjected to species identification by using the Biolog Gen III microbial identification system. They distributed among 23 genera, which included soil bacteria, plant-related bacteria, coliforms, and opportunistic plant- or human-pathogenic bacteria. The most commonly identified bacteria species were Pseudomonas fluorescens, Rahnella aquatilis, and Ralstonia insidiosa. The high prevalence of R. insidiosa, a strong biofilm former, and P. fluorescens, a moderate biofilm former, suggests that they were established residents in the sampled plants. These results suggest that native microflora capable of forming biofilms are widely distributed in fresh-produce processing environments.