RESUMO
Adaptive laboratory evolution (ALE) can be used to make bacteria less susceptible to oxidative stress. An alternative to large batch scale ALE cultures is to use microfluidic platforms, which are often more economical and more efficient. Microfluidic ALE platforms have shown promise, but many have suffered from subpar cell passaging mechanisms and poor spatial definition. A new approach is presented using a microfluidic Evolution on a Chip (EVoc) design which progressively drives microbial cells from areas of lower H2 O2 concentration to areas of higher concentration. Prolonged exposure, up to 72 h, revealed the survival of adaptive strains of Lacticaseibacillus rhamnosus GG, a beneficial probiotic often included in food products. After performing ALE on this microfluidic platform, the bacteria persisted under high H2 O2 concentrations in repeated trials. After two progressive exposures, the ability of L. rhamnosus to grow in the presence of H2 O2 increased from 1 mm H2 O2 after a lag time of 31 h to 1 mm after 21 h, 2 mm after 28 h, and 3 mm after 42 h. The adaptive strains have different morphology, and gene expression compared to wild type, and genome sequencing revealed a potentially meaningful single nucleotide mutation in the protein omega-amidase.
RESUMO
Enhancements in swabbing technology to increase sample collection efficacy would benefit the food industry. Specifically, these enhancements would assist the food industry in implementing the FDA Food Safety Modernization Act (FSMA) requirements by improving environmental monitoring effectiveness. A sonicating swab device, an example of an enhanced swabbing technology, was demonstrated previously to remove biofilm from stainless steel more efficiently than a standard cotton swab. Within this study, the performance of the sonicating swab was compared to that of the standard cotton swab for the recovery of Listeria monocytogenes from inoculated surfaces (plastic cutting board, wood cutting board, vinyl floor tile, and quarry clay floor tile). Additionally, we demonstrate the sonicating swab performance for collection of a microbiological sample from used commercial plastic cutting boards (noninoculated) in comparison to cotton swabs, foam swabs, and sponges. The sonicating swab captured significantly (P ≤ 0.05) more L. monocytogenes than the cotton swab for both the quarry tile and wood cutting board, while no significant differences were observed for the plastic cutting board or the vinyl floor tile. The sonicating swab consistently recovered significantly (P ≤ 0.05) more bacteria from the used cutting boards than did the standard cotton swab or the 3M Enviro swab, and it recovered significantly (P ≤ 0.05) more bacteria than the sponge swab for a majority of the time (4 of 6 trials). The results of this study indicate that swab technology can still be improved and that the sonicating swab is a viable technological enhancement which aids microbiological sample collection.IMPORTANCE Swabbing of surface areas for microbial contamination has been the standard for the detection and enumeration of microorganisms for many years. Inadequate surface sampling can result in foodborne illness outbreaks due to biotransfer of harmful microorganisms from food contact surfaces to foods. Swab material type, surface characteristics, and swabbing method used are a few of the factors associated with swabbing that can result in the variability of bacterial cell recovery for detection and enumeration. A previous study highlighted a sonicating swab prototype and its ability to recover cells from a stainless steel surface more efficiently and reliably than a standard swab method (T. A. Branck, M. J. Hurley, G. N. Prata, C. A. Crivello, and P. J. Marek, Appl Environ Microbiol 83:e00109-17, 2017, https://doi.org/10.1128/AEM.00109-17). This study expands upon the capabilities of the sonicating swab technology to recover cells from multiple surface types with increased performance over traditional swabbing methods as a tool to further assist in the prevention of foodborne illness outbreaks.
