Anaerobic soil disinfestation, amendment-type, and irrigation regimen influence Salmonella survival and die-off in agricultural soils.

AIMS: This study investigated Salmonella concentrations following combinations of horticultural practices including anaerobic soil disinfestation (ASD), soil amendment type and irrigation regimen. METHODS AND RESULTS: Sandy-loam soil was inoculated with a five-serovar Salmonella cocktail (5.5 ± 0.2 log CFU per gram) and subjected to one of six treatments: (i) no soil amendment, ASD (ASD control), (ii) no soil amendment, no-ASD (non-ASD control) and (iii-vi) soil amended with pelletized poultry litter, rye, rapeseed or hairy vetch with ASD. The effect of irrigation regimen was determined by collecting samples 3 and 7 days after irrigation. Twenty-five-gram soil samples were collected pre-ASD, post-soil saturation (i.e. ASD-process), and at 14 time-points post-ASD, and Salmonella levels enumerated. Log-linear models examined the effect of amendment type and irrigation regimen on Salmonella die-off during and post-ASD. During ASD, Salmonella concentrations significantly decreased in all treatments (range: -0.2 to -2.7 log CFU per gram), albeit the smallest decrease (-0.2 log CFU per gram observed in the pelletized poultry litter) was of negligible magnitude. Salmonella die-off rates varied by amendment with an average post-ASD rate of -0.05 log CFU per gram day (CI = -0.05, -0.04). Salmonella concentrations remained highest over the 42 days post-ASD in pelletized poultry litter, followed by rapeseed, and hairy vetch treatments. Findings suggested ASD was not able to eliminate Salmonella in soil, and certain soil amendments facilitated enhanced Salmonella survival. Salmonella serovar distribution differed by treatment with pelletized poultry litter supporting S. Newport survival, compared with other serovars. Irrigation appeared to assist Salmonella survival with concentrations being 0.14 log CFU per gram (CI = 0.05, 0.23) greater 3 days, compared with 7 days post-irrigation. CONCLUSIONS: ASD does not eliminate Salmonella in soil, and may in fact, depending on the soil amendment used, facilitate Salmonella survival. SIGNIFICANCE AND IMPACT OF THE STUDY: Synergistic and antagonistic effects on food safety hazards of implementing horticultural practices should be considered.

Strain-Level Identification of Bacterial Tomato Pathogens Directly from Metagenomic Sequences.

Routine strain-level identification of plant pathogens directly from symptomatic tissue could significantly improve plant disease control and prevention. Here we tested the Oxford Nanopore Technologies (ONT) MinION sequencer for metagenomic sequencing of tomato plants either artificially inoculated with a known strain of the bacterial speck pathogen Pseudomonas syringae pv. tomato or collected in the field and showing bacterial spot symptoms caused by one of four Xanthomonas species. After species-level identification via ONT's WIMP software and the third-party tools Sourmash and MetaMaps, we used Sourmash and MetaMaps with a custom database of representative genomes of bacterial tomato pathogens to attempt strain-level identification. In parallel, each metagenome was assembled and the longest contigs were used as query with the genome-based microbial identification Web service LINbase. Both the read-based and assembly-based approaches correctly identified P. syringae pv. tomato strain T1 in the artificially inoculated samples. The pathogen strain in most field samples was identified as a member of Xanthomonas perforans group 2. This result was confirmed by whole genome sequencing of colonies isolated from one of the samples. Although in our case metagenome-based pathogen identification at the strain level was achieved, caution still must be exercised in interpreting strain-level results because of the challenges inherent to assigning reads to specific strains and the error rate of nanopore sequencing.

Impact of routine sanitation on the microbiomes in a fresh produce processing facility.

Indigenous bacterial populations in fresh-cut produce processing facilities can have a profound effect on the survival and proliferation of inadvertently contaminating foodborne pathogens. In this study, environmental samples were collected from a variety of Zone 3 sites in a processing plant before and after daily routine sanitation. Viable mesophilic aerobic bacteria population was evaluated using both culturing method and quantitative real-time PCR (qPCR) after propidium monoazide treatment. Zone 3 surface microbiota were analyzed using 16S rRNA gene amplicon sequencing with the Qiime2 bioinformatic pipeline. Over 8000 bacterial species across 4 major phyla were identified in Zone 3 microbiomes in the processing facility. Overall, effective bacterial reduction was observed at the sampling sites on the production floor, while sanitation effect on peripheral surfaces was less evident. Effective sanitation resulted in both quantitative and qualitive shifts of Zone 3 microbiota. Several species were highly abundant at multiple sample sites for both winter and summer samplings. Based on the spatial and temporal distribution of the most abundant species, a Zone 3 core microbiome in the processing facility was tentatively described to included Cupriavidus sp., Pseudomonas sp., Ralstonia sp., Arthrobacter psychrolactophilus, Pseudomonas veronii, Stenotrophomonas sp., and an unknown species of the family Enterobacteriaceae.

Shifts in spinach microbial communities after chlorine washing and storage at compliant and abusive temperatures.

Fresh produce, like spinach, harbors diverse bacterial populations, including spoilage and potentially pathogenic bacteria. This study examined the effects of produce washing in chlorinated water and subsequent storage on the microbiota of spinach. Baby spinach leaves from a commercial fresh-cut produce processor were assessed before and after washing in chlorinated water, and then after one week's storage at 4, 10, and 15 °C. Microbial communities on spinach were analyzed by non-selective plating, qPCR, and 16S rDNA amplicon sequencing. Bacterial populations on spinach, averaging 6.12 ±â€¯0.61 log CFU/g, were reduced by 1.33 ±â€¯0.57 log after washing. However, populations increased by 1.77-3.24 log after storage, with larger increases occurring at higher temperature (15 > 10 > 4 °C). The predominant phylum identified on unwashed spinach leaves was Proteobacteria; dominant genera were Pseudomonas and Sphingomonas. Bacterial communities shifted significantly after chlorine washing and storage. Several Proteobacteria species, such as Stenotrophomonas sp. and Erwinia sp., were relatively tolerant of chlorine treatment, while species of Flavobacterium and Pedobacter (phylum Bacteroidetes) grew rapidly during storage, especially at abusive temperatures. Cupriavidus sp. and Ralstonia sp. showed significant increases after washing. After storage, microbial communities on spinach appeared to shift back toward the pre-washing distributions.