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Outbreaks of Salmonellosis have been traced to contaminated tomato. The produce production environment poses a risk for Salmonella contamination; however, little is known about the effects of pest management practices on Salmonella during production. The study objective was to evaluate pesticide application on the inactivation of Salmonella on tomato leaves. Thirty greenhouse‐grown tomato plants were inoculated with S. enterica serovars Newport or Typhimurium. Inoculation was performed by dipping tomato leaves in an 8‐log CFU/mL Salmonella suspension with 0.025% (vol/vol) Silwet L‐77 surfactant for 30 s, for a starting concentration of 6–7 log CFU/mL. Plants were treated with one of four pesticides, each with a different mode of action [acibenzolar‐S‐methyl, copper‐hydroxide, peroxyacetic acid (PAA), and streptomycin]. Pesticides were applied at manufacturers' labeled rate for plant disease management with water as a control treatment. Salmonella was enumerated at 0.125 (3 h), 2, 6, and 9 days post‐inoculation (dpi), and counts log‐transformed. Growth of Salmonella was not observed. At 2 dpi, PAA and streptomycin significantly reduced surface Salmonella concentrations of inoculated tomato leaves (0.7 and 0.6‐log CFU/g, respectively; p ≤ 0.05), while significant Salmonella log reduction occurred in the ground tomato leaves after copper hydroxide treatment (0.8‐log CFU/g; p ≤ 0.05), compared to the control. No significant differences in Salmonella populations on tomato leaf surface and in ground leaves were observed from 2 to 9 dpi, regardless of pesticide application. These findings suggest single in‐field pesticide applications may not be an effective mitigation strategy in limiting potential Salmonella contamination. Future research, including multiple in‐field pesticide applications, or pesticide use in combination with other mitigation strategies, may offer intriguing management practices to limit possible preharvest contamination. [ABSTRACT FROM AUTHOR]

Outbreaks of foodborne illnesses linked to fresh fruits and vegetables have been key drivers behind a wide breadth of research aiming to fill data gaps in our understanding of the total ecology of agricultural water sources such as ponds and wells and the relationship of this ecology to foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. Both S. enterica and L. monocytogenes can persist in irrigation water and have been linked to produce contamination events. Data describing the abundance of these organisms in specific agricultural water sources are valuable to guide water treatment measures. Here, we profiled the culture independent water microbiota of four farm ponds and wells correlated with microbiological recovery of S. enterica (prevalence: pond, 19.4%; well, 3.3%), L. monocytogenes (pond, 27.1%; well, 4.2%) and fecal indicator testing. Correlation between abiotic factors, including water parameters (temperature, pH, conductivity, dissolved oxygen percentage, oxidation reduction potential, and turbidity) and weather (temperature and rainfall), and foodborne pathogens were also evaluated. Although abiotic factors did not correlate with recovery of S. enterica or L. monocytogenes (p > 0.05), fecal indicators were positively correlated with incidence of S. enterica in well water. Bacterial taxa such as Sphingomonadaceae and Hymenobacter were positively correlated with the prevalence and population of S. enterica , and recovery of L. monocytogenes was positively correlated with the abundance of Rhizobacter and Comamonadaceae (p < 0.03). These data will support evolving mitigation strategies to reduce the risk of produce contamination by foodborne pathogens through irrigation. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus (Vp) is a common marine halophilic food‐borne pathogen, mainly found in seafood and food with a high salt content. Gastrointestinal reactions such as diarrhea, headache, vomiting, nausea, and abdominal cramps may occur after eating food infected with Vp. This study aimed to screen for high‐affinity aptamers that specifically recognize Vp. A high‐affinity modified aptamer screening kit was used to rapidly screen aptamers of the food‐borne Vp. The first round of screening involved release of target aptamers from the microspheres. The "false‐positive" aptamers were eliminated after specific binding to and elution of Vp in the second round. The second round of screening of the aptamers involved polymerase chain reaction (PCR), and the abundance of a sequence was determined using next‐generation sequencing. Nine high‐affinity aptamer sequences were obtained, and the first eight modified aptamer sequences were derived using a cloud‐based intelligent software of the American AM Biotech Co. Escherichia coli (E. coli) was used as a control, and aptamer ID 12 with the highest affinity for Vp was selected using real‐time PCR. According to the principle of color change caused by nano‐gold condensing under salt induction, Salmonella, Listeria monocytogenes (L. monocytogenes), and E. coli were used as counter‐screening bacteria, and the aptamer ID12 was combined with nano‐gold. The results showed that aptamer ID12 has strong specificity for Vp. Based on these findings, this study developed a simple, innovative, and rapid method for screening Vp aptamers. [ABSTRACT FROM AUTHOR]

The Eastern Shore of Virginia (ESV) is a major agricultural region in Virginia and in the past has been linked to some tomato-associated outbreaks of salmonellosis. In this study, water samples were collected weekly from irrigation ponds and wells in four representative vegetable farms (Farms A–D, each farm paired with one pond and one well) and a creek as well. In addition, water samples from two sites in the Chesapeake Bay on the ESV were collected monthly. Poultry litter was sampled monthly from three commercial broiler farms. Soil samples were collected monthly after fertilization with poultry litter from 10 farms in 2014 and another 14 farms in 2015. A most probable number method was used to detect Salmonella enterica presence and concentration in collected samples. Presumptive Salmonella colonies were confirmed by the cross-streaking method. Molecular serotyping was carried out to determine the Salmonella serovars. The average prevalence of Salmonella in pond, well, creek, and bay water samples was 19.3, 3.3, 24.2, and 29.2%, respectively. There were significant spatial and temporal differences for Salmonella incidence in various water sources. The prevalence of S. enterica in four tested ponds from farms A, B, C, and D were 16, 12, 22, and 27%, respectively. While the prevalence of S. enterica in irrigation wells was significantly lower, some well water samples tested positive during the study. Salmonella Newport was found to be the predominant serovar isolated from water samples. All poultry houses of the three tested broiler farms were Salmonella -positive at certain sampling points during the study with prevalence ranging from 14.3 to 35.4%. Salmonella was found to be able to survive up to 4 months in poultry litter amended soils from the tested farms in 2014, and up to 6 months in 2015. This research examined the dynamics of S. enterica in relationship to water source, poultry litter, and amended soil in a major agricultural area, and provides useful information for food safety risk assessments. [ABSTRACT FROM AUTHOR]

A study was conducted to examine the complete covalent structures of two purified closely related antifungal compounds, which were observed by the experiments of TOCSY, NOESY, ROESY, [super 13]C HSQC 2D NMR, and ESI-MS and GC. Findings suggest that the antifungal compound interrupts normal membrane morphology.

Abstract 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. Highlights • Zone 3 microbiome in a fresh produce facility was determined by 16S rDNA survey. • The effect of routine sanitation on microbiome was evaluated. • Microbiome from surfaces on production areas was more affected by sanitation than on peripheral surfaces. • A core residential microbiota in the facility was identified. [ABSTRACT FROM AUTHOR]

Between 2000 and 2010 the Eastern Shore of Virginia was implicated in four Salmonella outbreaks associated with tomato. Therefore, a multi-year study (2012–2015) was performed to investigate presumptive factors associated with the contamination of Salmonella within tomato fields at Virginia Tech's Eastern Shore Agricultural Research and Extension Center. Factors including irrigation water sources (pond and well), type of soil amendment: fresh poultry litter (PL), PL ash, and a conventional fertilizer (triple superphosphate – TSP), and production practices: staked with plastic mulch (SP), staked without plastic mulch (SW), and non-staked without plastic mulch (NW), were evaluated by split-plot or complete-block design. All field experiments relied on naturally occurring Salmonella contamination, except one follow up experiment (worst-case scenario) which examined the potential for contamination in tomato fruits when Salmonella was applied through drip irrigation. Samples were collected from pond and well water; PL, PL ash, and TSP; and the rhizosphere, leaves, and fruits of tomato plants. Salmonella was quantified using a most probable number method and contamination ratios were calculated for each treatment. Salmonella serovar was determined by molecular serotyping. Salmonella populations varied significantly by year; however, similar trends were evident each year. Findings showed use of untreated pond water and raw PL amendment increased the likelihood of Salmonella detection in tomato plots. Salmonella Newport and Typhimurium were the most frequently detected serovars in pond water and PL amendment samples, respectively. Interestingly, while these factors increased the likelihood of Salmonella detection in tomato plots (rhizosphere and leaves), all tomato fruits sampled (n = 4800) from these plots were Salmonella negative. Contamination of tomato fruits was extremely low (< 1%) even when tomato plots were artificially inoculated with an attenuated Salmonella Newport strain (104 CFU/mL). Furthermore, Salmonella was not detected in tomato plots irrigated using well water and amended with PL ash or TSP. Production practices also influenced the likelihood of Salmonella detection in tomato plots. Salmonella detection was higher in tomato leaf samples for NW plots, compared to SP and SW plots. This study provides evidence that attention to agricultural inputs and production practices may help reduce the likelihood of Salmonella contamination in tomato fields. [ABSTRACT FROM AUTHOR]

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 ± 0.61 log CFU/g, were reduced by 1.33 ± 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. [ABSTRACT FROM AUTHOR]

Ammonia-oxidizing archaea (AOA) have more importance in ammonia oxidation than ammonia-oxidizing bacteria (AOB) in acidic red soils. The aim of this study was to investigate if the abundance and composition of AOA could be altered by long-term application of organic manure in an acidic red soil. The abundance and composition of AOA were evaluated by polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) targeting archaealamoAgenes after long-term (24-year) application of mineral fertilizer and/or organic manure. The treatments were: non-fertilized control, mineral nitrogen (N) fertilizer only, mineral N, phosphorus (P) and potassium (K) fertilizer only, organic manure only, and organic manure plus mineral NPK fertilizer. The abundance of archaealamoAgenes was significantly increased after the long-term application of organic manures, either with or without mineral NPK fertilizer. So were the Shannon and Richness diversity indices of AOA deduced from the DGGE patterns. Phylogenetic analyses showed that most of the AOA sequences from various fertilization treatments were affiliated with group 1.1b thaumarchaea and only one with the group 1.1a-associated thaumarchaea. Nitrification potential was significantly increased after the long-term application of organic manures in comparison with the non-fertilized control. Our results strengthened the importance of organic manure in promoting the growth of AOA and thus nitrification potential in the acidic red soils. [ABSTRACT FROM AUTHOR]

Abstract: The 2013 Produce Safety Rules in Food Safety Modernization Act (FSMA) require regular testing for generic Escherichia coli in agricultural water intended for pre-harvest contact with the edible portion of fresh produce. However, the use of fecal contamination indicators frequently does not correctly reflect distribution of foodborne pathogens such as Salmonella enterica, and ensuring food safety may require direct detection and enumeration of pathogens in agricultural settings. Herein we report the evaluation of different cost-effective methods for quantification, isolation, and confirmation of Salmonella in irrigation pond water and sediment samples. A most probably number (MPN) dual enrichment culture method was used in combination with differential and selective agars, XLT4 and CHROMagar™ Salmonella plus (CSP). The necessity for PCR confirmation was evaluated, and methods were compared by cost and performance measures (i.e., sensitivity, specificity, positive predictive value, and negative predictive value). Statistical analyses showed that using XLT4 as the initial selective agar to isolate Salmonella colonies improved recovery compared to CSP agar; however, PCR confirmation was required to avoid false positive results on either agar. Therefore, a novel cross-streaking method utilizing CHROMagar™ agar for individual colony confirmation of Salmonella presence/absence on XLT4 was developed. This method classifies the colony as positive if typical Salmonella appearance is observed on both agars. Statistical analysis showed that this method was as effective as PCR for species confirmation of pure individual strains isolated from enrichment cultures (sensitivity=0.99, specificity=1.00, relative to PCR). This method offers a cost-effective alternative to PCR that would increase the capacity and sensitivity of Salmonella evaluation. [Copyright &y& Elsevier]

Listeria monocytogenes (Lm) outbreaks and recalls associated with fresh produce in recent years have heightened concerns and demands from industry and consumers to more effectively mitigate the contamination risk of this foodborne pathogen on fresh produce. In this study, the growth of Lm and indigenous bacteria on fresh-cut cantaloupe and romaine lettuce held at refrigerated (4 °C) and abusive (10–24 °C) temperatures was determined by both culture dependent and independent methods. Composition and dynamics of bacterial communities on Lm inoculated and non-inoculated samples were analyzed by 16S rRNA high-throughput sequencing. Fresh-cut cantaloupe provided favorable growth conditions for Lm proliferation (1.7 and >6 log increase at refrigerated and abusive temperatures, respectively) to overtake indigenous bacteria. The Lm population also increased on fresh-cut lettuce, but the growth rate was lower than that of the total mesophilic bacteria, resulting in 0.4 and >2 log increase at refrigerated and abusive temperatures. Microbial diversity of fresh-cut cantaloupe was significantly lower than that of fresh-cut romaine lettuce. The Shannon index of microbial communities on cantaloupe declined after storage, but it was not significantly changed on lettuce samples. Shifts in the bacterial microbiome on cantaloupe were mainly affected by Lm inoculation, while both inoculation and storage temperature played significant roles on lettuce bacterial communities. Multiple indigenous bacteria, including Leuconostoc and Weissella spp., were negatively correlated to Lm abundance on romaine lettuce, and were determined by bioassay as potential anti-listerial species. Data derived from this study contribute to better understanding of the relationship between Lm and indigenous microbiota on fresh-cut produce during storage. • Assessed microbiome shifts on fresh-cut cantaloupe and romaine lettuce during storage. • Determined Listeria monocytogenes interactions with fresh-cut cantaloupe and romaine lettuce bacteria • Observed L. monocytogenes growth as impacted by the complexity of fresh-cut produce microbiome • Isolated and identified multiple L. monocytogenes antagonist strains. [ABSTRACT FROM AUTHOR]

• A water-soluble corn zein formulation impregnated with nisin was used as fruit coating. • L. monocytogenes survival on coated nectarine and apple was significantly reduced. • Coated nectarines and apples had improved firmness and fresh weight retention. A corn zein based water-soluble coating formulation impregnated with nisin at 1 g L−1 (CoZWN), or without nisin (CoZW), was evaluated for performance against Listeria monocytogenes as edible coating on nectarines and apples. In comparison to uncoated (NC) control and coating with conventional petroleum-based wax (Wax), CoZWN coated nectarine showed higher reduction of L. monocytogenes population after storage for 2 d at 2.2 °C in simulation of refrigerated transportation, followed by additional 2 d at 22 °C in simulation of retail display. L. monocytogenes populations were reduced by 1.3 or 1.1 log in comparison to uncoated controls when coating was applied before or after L. monocytogenes inoculation, respectively. The performance of this nisin-impregnated corn zein formulation was further validated as a post-contamination coating using Gala apples stored at room temperature (22 °C) for up to 10 d in simulation of retail display. Gala apples coated with CoZWN accelerated the decline of L. monocytogenes populations during the early stage of simulated storage, and outperformed Wax through the entire period against L. monocytogenes. CoZWN did not significantly impact the survival and growth of molds and yeasts on nectarines but performed comparably to Wax against molds and yeasts on Gala apples. Data presented in this study indicated that nisin-impregnated corn zein coating is a promising strategy for mitigating L. monocytogenes contamination on fresh produce. It also demonstrated that this water-soluble coating could be impregnated with other natural antimicrobials to target a wide range of foodborne pathogens. [ABSTRACT FROM AUTHOR]

Campylobacter spp., especially Campylobacter jejuni, are common causal agents of gastroenteritis globally. Poultry, contaminated water, and fresh produce are considered to be the main sources for infection by this pathogen. In this study, occurrence and population density of C. jejuni from vegetable irrigation ponds in the Suwannee River watershed were investigated and the relationship to environmental factors was analyzed. Two water samples were collected from each of 10 ponds every month from January 2011 to February 2012. Campylobacter jejuni was detected by quantitative real-time PCR. Nine of the 10 ponds were positive for C. jejuni some of the time with an overall prevalence of 19.3%. The highest counts were obtained in spring 2011. Oxidation-reduction potential and total nitrogen concentration were positively correlated ( P < 0.05) with mean population and occurrence of C. jejuni, while temperature and dissolved oxygen percent saturation (DO%) were negatively correlated with mean population ( P < 0.05). Presence of this pathogen was related to bacterial community composition. No correlations were found between C. jejuni and fecal indicators. Increasing DO% of irrigation water and limiting nitrogen pollution in the ponds are suggested to reduce the contamination risk of C. jejuni in a major fruit and vegetable growing area. [ABSTRACT FROM AUTHOR]

A two-phase experiment was conducted twice to investigate the effects of soil management on movement of Salmonella enterica Typhimurium in tomato plants. In the first phase, individual leaflets of 84 tomato plants grown in conventional or organic soils were dip inoculated two to four times before fruiting with either of two Sahnonella Typhimurium strains (1010) CFU/ml; 0.025% [vol/vol] Silwet L-77). Inoculated and adjacent leaflets were tested for Salmonella spp. densities for 30 days after each inoculation. Endophytic bacterial communities were characterized by polymerase chain reaction denaturing gradient gel electrophoresis before and after inoculation. Fruit and seed were examined for Salmonella spp. incidence. In phase 2, extracted seed were planted in conventional soil, and contamination of leaves and fruit of the second generation was checked. More Salmonella spp. survived in inoculated leaves on plants grown in conventional than in organic soil. The soil management effect on Sahnonella spp. survival was confirmed for tomato plants grown in two additional pairs of soils. Endophytic bacterial diversities of tomato plants grown in conventional soils were significantly lower than those in organic soils. All contaminated fruit (1%) were from tomato plants grown in conventional soil. Approximately 5% of the seed from infested fruit were internally contaminated. No Sahnonella sp. was detected in plants grown from contaminated seed. [ABSTRACT FROM AUTHOR]

Outbreaks of enteritis caused by Escherichia coli O157 associated with fresh produce have resulted in questions about the safety of irrigation water; however, associated risks have not been systematically evaluated. In this study, the occurrence and distribution of the human pathogen E. coli O157 from vegetable irrigation ponds within the Suwannee River Watershed in Georgia were investigated, and the relationship to environmental factors was analyzed. Surface and subsurface water samples were collected monthly from 10 vegetable irrigation ponds from March 2011 to February 2012. Escherichia coli O157 was isolated from enriched filtrates on CHROMagar and sorbitol MacConkey agar media and confirmed by an agglutination test. Presence of virulence genes stx1, stx2 , and eae was tested by polymerase chain reaction. In addition, 27 environmental variables of the sampled ponds were measured. Denaturing gradient gel electrophoresis was conducted for the analysis of bacterial communities in the water samples. Biserial correlation coefficients were calculated to evaluate the log10 colony-forming unit per millilitre correlations between the environmental factors and the occurrence of E. coli O157. Stepwise and canonical discriminant analyses were used to determine the factors that were associated with the presence and absence of E. coli O157 in water samples. All 10 ponds were positive for E. coli O157 some of the time, mainly in summer and fall of 2011. The temporal distribution of this bacterium differed among the 10 ponds. Temperature, rainfall, populations of fecal coliform, and culturable bacteria were positively correlated with the occurrence of E. coli O157 ( P < 0.05), while the total nitrogen concentration, oxidation-reduction potential, and dissolved oxygen concentration were negatively correlated with the occurrence of this pathogen ( P < 0.05). Temperature and rainfall were the most important factors contributing to the discrimination between samples with and without E. coli O157, followed by bacterial diversity and culturable bacteria population density. Bacterial numbers and diversity, including fecal coliforms and E. coli O157, increased after rainfall (and possibly runoff from pond margins) in periods with relatively high temperatures, suggesting that prevention of runoff may be important to minimize the risk of enteric pathogens in irrigation ponds. [ABSTRACT FROM AUTHOR]

Internal contamination of Salmonella in plants is attracting increasing attention for food safety reasons. In this study, three different tomato cultivars "Florida Lanai", "Crown Jewel", "Ailsa Craig" and the transgenic line Sp5 of "Ailsa Craig" were inoculated with 1 ml GFP-labeled Salmonella Typhimurium through guttation droplets at concentrations of 109 or 107 CFU/ ml. Survival of Salmonella on/in tomato leaves was detected by both direct plating and enrichment methods. Salmonella cells survived best on/in the inoculated leaves of cultivar "Ailsa Craig" and decreased fastest on/in "Florida Lanai" leaves. Increased guttation in the abscisic acid over-expressing Sp5 plants may have facilitated the entrance of Salmonella into leaves and the colonization on the surface of tomato leaves. Internalization of Salmonella Typhimurium in tomato leaves through guttation drop inoculation was confirmed by confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can enter tomato leaves through hydathodes and move into the vascular system, which may result in the internal translocation of the bacteria inside plants. [ABSTRACT FROM AUTHOR]

Abstract: Salmonella enterica strains with rdar (red dry and rough) and saw (smooth and white) morphotypes have previously been associated with tomato outbreaks but the dispersal mechanisms of these morphotypes are still poorly understood. In this study, Salmonella adhesion was distinguished from attachment by comparing different contact periods. Initial adhesion of rdar and saw morphotypes of Salmonella was compared in relation to tomato plants with different leaf trichome densities. Trichome densities were increased or reduced by treatment with jasmonic or salicylic acid, respectively. The overall effect of Salmonella morphotype and trichome density on splash dispersal was assessed in a rain simulator and correlated to cell hydrophobicity and initial adhesion. The presence of the rdar morphotype increased initial adhesion at high trichome densities but not at low trichome densities. Attachment of the rdar strain occurred after 30s contact time regardless of trichome density. Splash dispersal was slightly further for the saw morphotype than the rdar morphotype of S. enterica at all trichome densities. Salmonella cells of both morphotypes survived significantly better on the surface of high trichome density leaflets. [Copyright &y& Elsevier]

Abstract: Outbreaks of Salmonella enterica have been associated with tomatoes and traced back to production areas but the spread of Salmonella in agricultural fields is still poorly understood. Post-rain Salmonella transfer from a point source to the air and then to tomato plants was evaluated. GFP-labeled kanamycin-resistant S. enterica serovar Typhimurium (108 CFU/mL) with and without expression of the rdar morphotype (rough colonies; cells with fimbriae and cellulose) was used as the point source in the center of a rain simulator. Rain intensities of 60 and 110mm/h were applied for 5, 10, 20, and 30min. Petri dishes with lactose broth and tomato plants with fruit (50–80cm high) were placed in the simulator after the rain had ceased. Salmonella recovery from air was maximum (300CFU/plate) after a rain episode of 60mm/h for 10min at distances of at least 85.5cm above the source and when the rdar morphotype strain was used. Small scale experiments showed that the smooth-colony strain without fimbriae precipitated from the air in significantly higher numbers than the rdar strain. Transfer of aerial Salmonella with the rdar morphotype to tomato fruits on plants followed a beta distribution (2.5950, 4.7393) within the generalized range from 0 to 30min of rain. Results show for the first time that Salmonella may transfer from rain to the air and contaminate tomato fruits at levels that could possibly be infectious to humans. [Copyright &y& Elsevier]

A striking feature of Burkholderia contaminans strain MS14 is the production of a glycolipopeptide named occidiofungin. Occidiofungin has a broad range of antifungal activities against plant and animal pathogens. In this study, a complete covalent structure characterization and identification of the whole genomic DNA region for the occidiofungin gene (ocf) cluster are described. Discovery of the presence of 2,4-diaminobutyric acid and 3-chloro-β-hydroxytyrosine and elucidation of the structure of a novel C18 fatty amino acid residue have been achieved. In addition, seven additional putative open reading frames (the genes from ocfI to ocfN [ocfI-N] and ORF16) were identified. Transcription of all the putative genes ocfI-N identified in the region except ORF16 was regulated by both ambR1 and ambR2. Elucidation of the structure and the ocf gene cluster provides insight into the biosynthesis of occidiofungin and promotes future aims at understanding the biosynthetic machinery. This work provides new avenues for optimizing the production and synthesis of structural analogs of occidiofungin. [ABSTRACT FROM AUTHOR]

Burkholderia contaminans strain MS14 has a broad range of antifungal activities to plant and human pathogens. In previous studies, a 22.7-kb genomic fragment harboring six genes was shown to be involved in the production of an antifungal oligopeptide in B. contaminans strain MS14. In this study, another LuxR-type regulatory gene, named ambR1, was identified downstream of the ambR2 gene, and three new ORFs were found upstream of ORF6 of the 22.7-kb fragment. Site-directed mutagenesis revealed that ambR1 was required for expression of the antifungal activity against the indicator fungus Geotrichum candidum. Transcription of all the putative genes (ORFs 2–9) identified in the region except ORF1 was regulated by both ambR1 and ambR2. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44( ambR1∷ nptII). Two of the three ORFs identified upstream from the ORF6 were predicted to encode two nonribosomal peptide synthetases (ORF7 and ORF9), and an insertion mutation in ORF9 resulted in the loss of antifungal activity against G. candidum. These results suggest that ambR1 is the key regulatory gene controlling the production of the antifungal activity of strain MS14. [ABSTRACT FROM AUTHOR]

Abstract: Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. When compared with the wild type strain, antifungal activities of mutants MS14MT13 and MS14MT18 against Geotrichum candidum were eliminated, and the activity of mutant MS14MT15 was reduced by approximately 80%. Sequence analysis of a 22.7-kb DNA segment revealed the presence of six open-reading frames (ORFs), including the genes disrupted in the mutants. The wild type level of antifungal activity was observed for mutant MS14MT15 (ambR2::Tn5) complemented in trans with the intact ambR2 gene, which encodes a LuxR type regulator. AmbR2 positively regulates the transcription of the other five ORFs identified, which including two nonribosomal peptide synthetase genes required for biosynthesis of antifungal compound. The compound consists of amino acid residues having a molecular weight of 1200.6Da. These results show that strain MS14 possesses a novel set of genes dedicated to the biosynthesis of an antifungal oligopeptide. [Copyright &y& Elsevier]

Fresh-cut leafy greens are washed in water with antimicrobials such as free chlorine to mitigate risk of pathogen cross-contamination. Validation of the process in a commercial setting poses challenges, including the identification of effective surrogates that can be used directly on the processing line during commercial operation. The objective of this study was to evaluate the use of an abiotic, DNA-based indicator as a potential surrogate for Escherichia coli O157:H7 during the washing of spinach. E. coli O157:H7 and DBAS (DNA barcode abiotic surrogate) were inoculated onto spinach leaves and washed in water with free chlorine. E. coli O157:H7 and DBAS populations were enumerated by selective plate counting or qPCR, respectively, and by laser scanning confocal microscopy. DBAS displayed high sensitivity to free chlorine at concentrations ≥12.5 mg/L, similar to concentrations commonly used in leafy green washing. Linear regression analysis indicated that inactivation of E. coli O157:H7 and DBAS had comparable inactivation responses (R 2 = 0.76). Microscopy analysis also showed similar rates of decontamination between the two analytes from the surface of spinach. These results show that DBAS is a promising abiotic surrogate for onsite validation of antimicrobial processes targeting foodborne pathogens such as E. coli O157:H7 on fresh produce. • A GRAS substance with embedded DNA was tested as abiotic surrogate for foodborne pathogen. • This DNA-based abiotic surrogate (DBAS) showed comparable adhesion on spinach as an E. coli O157:H7 strain. • DBAS and E. coli O157:H7 strain showed comparable inactivation when treated in chlorinated water. [ABSTRACT FROM AUTHOR]

Imported papayas from Mexico have been implicated in multiple salmonellosis outbreaks in the United States in recent years. While postharvest washing is a critical process to remove latex, dirt, and microbes, it also has the potential of causing cross-contamination by foodborne pathogens, with sponge or other fibrous rubbing tools often questioned as potential harboring or transmitting risk. In this study, Salmonella inactivation and cross-contamination via sponges and microfiber wash mitts during simulated papaya washing and cleaning were investigated. Seven washing treatments (wash without sanitizer; wash at free chlorine 25, 50, and 100 mg/L, and at peracetic acid 20, 40, and 80 mg/L), along with unwashed control, were evaluated, using Salmonella strains with unique antibiotic markers differentially inoculated on papaya rind (serovars Typhimurium, Heidelberg, and Derby) and on wash sponge or microfiber (serovars Typhimurium, Newport, and Braenderup). Salmonella survival and transfer on papaya and on sponge/microfiber, and in wash water were detected using selective plating or enrichment. The washing and cleaning process reduced Salmonella on inoculated papayas by 1.69–2.66 and 0.69–1.74 log for sponge and microfiber cleaning, respectively, with the reduction poorly correlated to sanitizer concentration. Salmonella on inoculated sponge or microfiber was under detection limit (1.00 log CFU/cm2) by plate count, but remained recoverable by selective enrichment. Transference of Salmonella from inoculated papaya to sponge/microfiber, and vice versa , could be detected sporadically by selective enrichment. Sponge/microfiber mediated Salmonella cross-contamination from inoculated to uninoculated papayas was frequently detectable by selective enrichment, but rendered undetectable by wetting sponge/microfiber in sanitizing wash water (FC 25–100 mg/L or PAA 20–80 mg/L) between washing different papaya fruits. Therefore, maintaining adequate sanitizer levels and frequently wetting sponge/microfiber in sanitizing wash water can effectively mitigate risks of Salmonella cross-contamination associated with postharvest washing, especially with regard to the use of sponge or microfiber wash mitts. • Salmonella inactivation and cross-contamination during papaya washing were evaluated. • Papaya washing at 25, 50, and 100 mg/L FC, and 20, 40, and 80 mg/L PAA was tested. • Washing with above sanitizers reduced Salmonella on papayas (0.69–2.66 log) and sponge/microfiber (>5 log). • Sponge/microfiber mediated cross-contamination was detectable by selective enrichment. • Cross-contamination was reduced to undetectable with wetting sponge/microfiber in sanitizing wash water between wiping papayas. [ABSTRACT FROM AUTHOR]

Chemical sanitizers are commonly used for fresh produce washing to reduce potential presence of foodborne pathogens and spoilage bacteria, and to prevent cross-contamination. In this study, we evaluated the susceptibility of foodborne pathogens, including Shiga-toxigenic Escherichia coli , Salmonella enterica , and Listeria monocytogenes, to free chlorine (FC) and peracetic acid (PAA) in spinach and romaine lettuce rinsates as simulated leafy green wash water. All inoculated pathogens exhibited notably enhanced tolerance (over 2 log of increased recovery) to FC, but not to PAA, when treated in fresh produce rinsates in comparison to that in sterile water. Both FC and PAA effectively inactivated the inoculated foodborne pathogens, rendering them undetectable after 30 s exposure to 10 mg/L FC or 30 mg/L PAA, by selective plating or non-selective enrichment. However, potentially viable cells for all the inoculated foodborne pathogens were detected by PMA-qPCR, especially in wash water samples treated with PAA at concentrations up to 50 mg/L. While laser confocal microscopy after differential staining suggested significant presence of viable but non-culturable (VBNC) populations in PAA treated wash water samples, resuscitation in vitro of the potential VBNC cells was not achieved. This study provides scientific data to better understand the risks of potential VBNC foodborne pathogens during fresh produce washing. • Pathogen sanitizer susceptibility in produce wash water assessed. • Culture-dependent and -independent detections showing great discrepancies. • Potentially sanitizer induced VBNC explored. • Resuscitation of potential VBNC populations no achieved in vitro. [ABSTRACT FROM AUTHOR]

Washing in chlorinated water is widely practiced for commercial fresh produce processing. While known as an effective tool for mitigating food safety risks, chlorine washing could also represent an opportunity for spreading microbial contaminations under sub-optimal operating conditions. This study evaluated Salmonella inactivation and cross-contamination in a simulated washing process of cherry and grape tomatoes. Commercially harvested tomatoes and the associated inedible plant matter (debris) were differentially inoculated with kanamycin resistant (KanR) or rifampin resistant (RifR) Salmonella strains, and washed together with uninoculated tomatoes in simulated packinghouse dump tank (flume) wash water. Washing in chlorinated water resulted in significantly higher Salmonella reduction on tomatoes than on debris, achieving 2–3 log reduction on tomatoes and about 1 log reduction on debris. Cross-contamination by Salmonella on tomatoes was significantly reduced in the presence of 25–150 mg/L free chlorine, although sporadic cross-contamination on tomatoes was detected when tomatoes and debris were inoculated at high population density. The majority of the sporadic cross-contaminations originated from Salmonella inoculated on debris. These findings suggested that debris could be a potentially significant source of contamination during commercial tomato washing. • Salmonella inactivation and cross-contamination during simulated tomato flume washing were examined. • Chlorine inactivation of Salmonella on debris was less efficient than on tomato. • Sporadic cross-contamination was observed at all free chlorine levels tested, primarily from debris. • Debris may be a risk for harboring and disseminating foodborne pathogens in wash water. [ABSTRACT FROM AUTHOR]

Fresh produce, as a known or suspected source of multiple foodborne outbreaks, harbors large populations of diverse microorganisms, which are partially released into wash water during processing. However, the dynamics of bacterial communities in wash water during produce processing is poorly understood. In this study, we investigated the effect of chlorine (FC) and peracetic acid (PAA) on the microbiome dynamics in spinach and romaine lettuce rinse water. Treatments with increasing concentrations of sanitizers resulted in convergence of distinct microbiomes. The resultant sanitizer resistant microbiome showed dominant presence by Bacillus sp., Arthrobacter psychrolactophilus , Cupriavidus sp., and Ralstonia sp. Most of the FC and PAA resistant bacteria isolated from spinach and lettuce rinse water after sanitation were gram positive spore forming species including Bacillus , Paenibacillus , and Brevibacillus spp., while several PAA resistant Pseudomonas spp. were also isolated from lettuce rinse water. Inoculation of foodborne pathogens altered the microbiome shift in spinach rinse water under PAA treatment, but not in lettuce rinse water or FC treated samples. These inoculated foodborne pathogens were not isolated among the sanitizer resistant strains. • Effect of chlorine and PAA treatments on produce wash water microbiomes was evaluated. • Bacteria resistant to chlorine and PAA in spinach and lettuce rinse water were identified. • Sanitizer treatment of different wash water resulted in microbiome convergence. • Inoculation with foodborne pathogens did not significantly change microbiome dynamics. [ABSTRACT FROM AUTHOR]

Raw carrot is known to have antimicrobial activity against Listeria monocytogenes , but the mechanism of action has not been fully elucidated. In this study, we examined carrot antilisterial activity against several strains of Listeria species (including L. grayi , L. innocua , L. seeligeri , and L. welshimeri) and L. monocytogenes. A representative strain of L. monocytogenes was subsequently used for further characterizing carrot antilisterial activity. Exposure to fresh-cut carrot for 15 min resulted in a similar loss of cultivability, ranging from 2.5 to 4.7 log units, across all Listeria strains evaluated. L. monocytogenes recovered from the fresh-cut surface of different raw carrots was 1.6 to 4.1 log lower than levels obtained from paired boiled carrot samples with abolished antilisterial activity. L. monocytogenes levels recovered from fresh-cut carrot were 2.8 to 3.1 log lower when enumerated by culture-dependent methods than by the culture-independent method of PMAxx-qPCR, a qPCR assay that is performed using DNA pre-treated to selectively sequester DNA from cells with injured membranes. These results suggested that L. monocytogenes loss of cultivability on fresh-cut carrot was not associated with a loss of L. monocytogenes cell membrane integrity and putative cell viability. Transmission electron microscopy imaging revealed that L. monocytogenes rapidly formed mesosome-like structures upon exposure to carrot fresh-cut surface but not upon exposure to boiled carrot surface, suggesting there may be an association between the formation of these mesosome-like structures and a loss of cultivability in L. monocytogenes. However, further research is necessary to conclude the causality of this association. • The antilisterial activity of fresh-cut carrot against Listeria was examined. • Carrot antlisterial activity resulted in loss of cultivability in all Listeria species tested. • Carrot antilisterial activity did not damage L. monocytogenes cell membranes. • L. monocytogenes forms mesosome-like structures on fresh-cut carrot surface. [ABSTRACT FROM AUTHOR]

Recent outbreaks linked to contaminated leafy greens underline the need for identifying effective natural approaches to improve produce safety at pre-harvest level. Lactic acid bacteria (LAB) have been evaluated as biocontrol agents in food products. In this study, the efficacy of a cocktail of LAB including Lactococcus lactis , Lactiplantibacillus plantarum , Lactobacillus johnsonii , and Lactobacillus acidophilus as pre-harvest biocontrol agents against Listeria and Escherichia coli O157 on lettuce and spinach was investigated. Bacterial pathogens L. monocytogenes and E. coli O157:H7 and the non-pathogenic surrogates L. innocua and E. coli O157:H12 were used to spray-inoculate cultivars of lettuce and spinach grown in growth chamber and in field, respectively. Inoculated plants were spray-treated with water or a cocktail of LAB. On day 0, 3, and 5 post-inoculation, four samples from each group were collected and bacterial populations were determined by serial dilution and spiral plating on selective agars. LAB treatment exhibited an immediate antimicrobial efficacy against L. monocytogenes and E. coli O157:H7 on "Green Star" lettuce by ~2 and ~ 1 log reductions under growth chamber conditions, respectively (P < 0.05). The effect of LAB against E. coli O157:H7 on "New Red Fire" lettuce remained effective during the 5-day period in growth chamber (P < 0.05). Treatment of LAB delivered an effective bactericidal effect against E. coli O157:H12 immediately after application on the field-grown lettuce plants (P < 0.05). Approximately 1 log L. innocua reduction was observed on "Matador" and "Palco" spinach on day 5 (P < 0.05). Results of this study support that LAB could potentially be applied as biocontrol agents for controlling Listeria and E. coli O157 contamination on leafy greens at the pre-harvest level. • LAB inactivated Listeria and E. coli O157 on spinach and lettuce. • Leafy green cultivars affected the effectiveness of LAB against foodborne pathogens. • LAB could potentially be used for improving leafy green safety at farm. [ABSTRACT FROM AUTHOR]

Intrinsic characteristics of fresh produce, such as pH, water activity, acid content and nutrient availability are critical factors in determining the survival and growth of Listeria monocytogenes (Lm). In this study, sterile fresh produce juice was used to analyze Lm growth potential among 14 different commodities and to identify physicochemical characteristics in those juices that affect Lm growth. Significant growth of Lm was observed in juices with pH ≥5.6 and low acidity (0.04–0.07 % titratable acidity (TA)) (cantaloupe, carrot, celery, green pepper, parsley, and romaine lettuce), slight reduction of Lm was observed in juices with pH 4.1 (tomato) and pH 3.9 (mango), and no Lm counts were recovered from juices with pH ≤3.8 and high acidity (0.28–1.17 % TA) (apple, blueberry, grape, peach, and pineapple). Although these acidic fruit juices possessed a high sugar content, the pH and acidity of produce juice seemed to be the primary determinants for Lm growth. The neutralization of acidic juices (i.e., Fuji and Gala apple, blueberry, grape, mango, pineapple, peach, and tomato) enabled Lm growth at 37 °C in all juices except for Gala apple and peach. Strong decline in Lm populations in Gala apple, grape and peach juices might be linked to sensitivity to organic acids, such as malic acid. Furthermore, Lm populations significantly decreased in pH-neutral (7.6) cauliflower juice, suggesting that potential antilisterial substances may play a role in Lm decline in cauliflower juice. • L. monocytogenes growth potential was assessed in sterile juices from a broad range of fresh produce. • Fresh produce pH and acidity are the main determinants of L. monocytogenes growth. • pH neutralization of acidic juices enabled L. monocytogenes growth at 37 °C. [ABSTRACT FROM AUTHOR]

Abstract Describing baseline microbiota associated with agricultural commodities in the field is an important step towards improving our understanding of a wide range of important objectives from plant pathology and horticultural sustainability, to food safety. Environmental pressures on plants (wind, dust, drought, water, temperature) vary by geography and characterizing the impact of these variable pressures on phyllosphere microbiota will contribute to improved stewardship of fresh produce for both plant and human health. A higher resolution understanding of the incidence of human pathogens on food plants and co-occurring phytobiota using metagenomic approaches (metagenome tracking) may contribute to improved source attribution and risk assessment in cases where human pathogens become introduced to agro-ecologies. Between 1990 and 2007, as many as 1990 culture-confirmed Salmonella illnesses were linked to tomatoes from as many as 12 multistate outbreaks (Bell et al., 2012; Bell et al., 2015; Bennett et al., 2014; CDC, 2004; CDC, 2007; Greene et al., 2005a; Gruszynski et al., 2014). When possible, source attribution for these incidents revealed a biogeographic trend, most events were associated with eastern growing regions. To improve our understanding of potential biogeographically linked trends in contamination of tomatoes by Salmonella , we profiled microbiota from the surfaces of tomatoes from Virginia, Maryland, North Carolina and California. Bacterial profiles from California tomatoes were completely different than those of Maryland, Virginia and North Carolina (which were highly similar to each other). A statistically significant enrichment of Firmicutes taxa was observed in California phytobiota compared to the three eastern states. Rhizobiaceae, Sphingobacteriaceae and Xanthobacteraceae were the most abundant bacterial families associated with tomatoes grown in eastern states. These baseline metagenomic profiles of phyllosphere microbiota may contribute to improved understanding of how certain ecologies provide supportive resources for human pathogens on plants and how components of certain agro-ecologies may play a role in the introduction of human pathogens to plants. Highlights • At the time measured, bacterial profiles from the phyllosphere of California tomatoes were completely different than those of Maryland, Virginia and North Carolina (which were highly similar to each other). • As environmental pressures on plants (wind, dust, drought, water, temperature) vary by geography, it appears that risk factors for introduction of human pathogens to plants may also vary. • Describing baseline microbiota associated with agricultural commodities in the field is an important step towards improving our understanding of a wide range of important objectives from plant pathology and horticultural sustainability, to food safety. [ABSTRACT FROM AUTHOR]

Particulates of harvest debris are common in tomato packinghouse dump tanks, but their role in food safety is unclear. In this study we investigated the survival of Salmonella enterica and the shifts in relative abundance of culturable mesophilic aerobic bacteria (cMAB) as impacted by particulate size and interaction with chlorine treatment. Particulates suspended in grape tomato wash water spanned a wide size range, but the largest contribution came from particles of 3–20 μm. Filtration of wash water through 330 μm, applied after 100 mg/L free chlorine (FC) wash, reduced surviving cMAB by 98%. The combination of filtration (at 330 μm or smaller pore sizes) and chlorinated wash also altered the cMAB community, with the survivors shifting toward Gram-positive and spore producers (in both lab-simulated and industrial conditions). When tomatoes and harvest debris inoculated with differentially tagged Salmonella were washed in 100 mg/L FC for 1 min followed by filtration, only cells originating from harvest debris survived, with 85 and 93% of the surviving cells associated with particulates larger than 330 and 63 μm, respectively. This suggests that particulates suspended in wash water can protect Salmonella cells from chlorine action, and serve as a vector for cross-contamination. • Debris particulates 3–20 μm in size in grape tomato wash water predominate. • Filtering (≤330 μm) after washing (100 mg/L FC) reduces Salmonella survival 98%. • Gram-positives, spore-formers dominate survivor microbiome of filtered wash water. • Particulates in tomato wash water protect Salmonella from chlorine disinfection. [ABSTRACT FROM AUTHOR]