The composition of environmental microbiota in three tree fruit packing facilities changed over seasons and contained taxa indicative of L. monocytogenes contamination.

BACKGROUND: Listeria monocytogenes can survive in cold and wet environments, such as tree fruit packing facilities and it has been implicated in outbreaks and recalls of tree fruit products. However, little is known about microbiota that co-occurs with L. monocytogenes and its stability over seasons in tree fruit packing environments. In this 2-year longitudinal study, we aimed to characterize spatial and seasonal changes in microbiota composition and identify taxa indicative of L. monocytogenes contamination in wet processing areas of three tree fruit packing facilities (F1, F2, F3). METHODS: A total of 189 samples were collected during two apple packing seasons from floors under the washing, drying, and waxing areas. The presence of L. monocytogenes was determined using a standard culturing method, and environmental microbiota was characterized using amplicon sequencing. PERMANOVA was used to compare microbiota composition among facilities over two seasons, and abundance-occupancy analysis was used to identify shared and temporal core microbiota. Differential abundance analysis and random forest were applied to detect taxa indicative of L. monocytogenes contamination. Lastly, three L. monocytogenes-positive samples were sequenced using shotgun metagenomics with Nanopore MinION, as a proof-of-concept for direct detection of L. monocytogenes' DNA in environmental samples. RESULTS: The occurrence of L. monocytogenes significantly increased from 28% in year 1 to 46% in year 2 in F1, and from 41% in year 1 to 92% in year 2 in F3, while all samples collected from F2 were L. monocytogenes-positive in both years. Samples collected from three facilities had a significantly different microbiota composition in both years, but the composition of each facility changed over years. A subset of bacterial taxa including Pseudomonas, Stenotrophomonas, and Microbacterium, and fungal taxa, including Yarrowia, Kurtzmaniella, Cystobasidium, Paraphoma, and Cutaneotrichosporon, were identified as potential indicators of L. monocytogenes within the monitored environments. Lastly, the DNA of L. monocytogenes was detected through direct Nanopore sequencing of metagenomic DNA extracted from environmental samples. CONCLUSIONS: This study demonstrated that a cross-sectional sampling strategy may not accurately reflect the representative microbiota of food processing facilities. Our findings also suggest that specific microorganisms are indicative of L. monocytogenes, warranting further investigation of their role in the survival and persistence of L. monocytogenes. Video Abstract.

Effect of processing on the anti-inflammatory efficacy of cocoa in a high fat diet-induced mouse model of obesity.

Obesity causes inflammation which may lead to development of co-morbidities like cardiovascular diseases. Cocoa is a popular food ingredient that has been shown to mitigate obesity and inflammation in preclinical models. Cocoa typically undergoes fermentation and roasting prior to consumption, which can affect the polyphenol content in cocoa. The aim of this study was to compare the effect of fermentation and roasting protocols on the ability of cocoa to mitigate obesity, gut barrier dysfunction, and chronic inflammation in high fat (HF)-fed, obese C57BL/6J mice. We found that treatment of mice with 80 mg/g dietary cocoa powder for 8 weeks reduced rate of body weight gain in both male and female mice (46-57%), regardless of fermentation and roasting protocol. Colonic length was increased (11-24%) and gut permeability was reduced (48-79%) by cocoa supplementation. Analysis of the cecal microbiome showed that cocoa, regardless of fermentation and roasting protocol, reduced the ratio of Firmicutes to Bacteroidetes. Multivariate statistical analysis of markers of inflammation and body weight data showed sex differences in the effect of both the HF diet as well as cocoa supplementation. Based on this data there was strong protective efficacy from cocoa supplementation especially for the more processed cocoa samples. Overall, this study shows that anti-obesity and anti-inflammatory efficacy of cocoa is resilient to changes in polyphenol content and composition induced by fermentation or roasting. Further, this study shows that although cocoa has beneficial effects in both males and females, there are significant sex differences.

Ability of Two Strains of Lactic Acid Bacteria To Inhibit Listeria monocytogenes by Spot Inoculation and in an Environmental Microbiome Context.

We evaluated the ability of two strains of lactic acid bacteria (LAB) to inhibit L. monocytogenes using spot inoculation and environmental microbiome attached-biomass assays. LAB strains (PS01155 and PS01156) were tested for antilisterial activity toward 22 phylogenetically distinct L. monocytogenes strains isolated from three fruit packing environments (F1, F2, and F3). LAB strains were tested by spot inoculation onto L. monocytogenes lawns (108 and 107 CFU/mL) and incubated at 15, 20, 25, or 30°C for 3 days. The same LAB strains were also cocultured at 15°C for 3, 5, and 15 days in polypropylene conical tubes with L. monocytogenes and environmental microbiome suspensions collected from F1, F2, and F3. In the spot inoculation assay, PS01156 was significantly more inhibitory toward less concentrated L. monocytogenes lawns than more concentrated lawns at all the tested temperatures, while PS01155 was significantly more inhibitory toward less concentrated lawns only at 15 and 25°C. Furthermore, inhibition of L. monocytogenes by PS01156 was significantly greater at 15°C than higher temperatures, whereas the temperature did not have an effect on the inhibitory activity of PS01155. In the assay using attached environmental microbiome biomass, L. monocytogenes concentration was significantly reduced by PS01156, but not PS01155, when cocultured with microbiomes from F1 and F3 and incubated for 3 days at 15°C. Attached biomass microbiota composition was significantly affected by incubation time but not by LAB strain. This study demonstrates that LAB strains that may exhibit inhibitory properties toward L. monocytogenes in a spot inoculation assay may not maintain antilisterial activity within a complex microbiome. IMPORTANCE Listeria monocytogenes has previously been associated with outbreaks of foodborne illness linked to consumption of fresh produce. In addition to conventional cleaning and sanitizing, lactic acid bacteria (LAB) have been studied for biocontrol of L. monocytogenes in food processing environments that are challenging to clean and sanitize. We evaluated whether two specific LAB strains, PS01155 and PS01156, can inhibit the growth of L. monocytogenes strains in a spot inoculation and in an attached-biomass assay, in which they were cocultured with environmental microbiomes collected from tree fruit packing facilities. LAB strains PS01155 and PS01156 inhibited L. monocytogenes in a spot inoculation assay, but the antilisterial activity was lower or not detected when they were grown with environmental microbiota. These results highlight the importance of conducting biocontrol challenge tests in the context of the complex environmental microbiomes present in food processing facilities to assess their potential for application in the food industry.