Differential microbiota shift on whole romaine lettuce subjected to source or forward processing and on fresh-cut products during cold storage.

Romaine lettuce in the U.S. is primarily grown in California or Arizona and either processed near the growing regions (source processing) or transported long distance for processing in facilities serving distant markets (forward processing). Recurring outbreaks of Escherichia coli O157:H7 implicating romaine lettuce in recent years, which sometimes exhibited patterns of case clustering in Northeast and Midwest, have raised industry concerns over the potential impact of forward processing on romaine lettuce food safety and quality. In this study, freshly harvested romaine lettuce from a commercial field destined for both forward and source processing channels was tracked from farm to processing facility in two separate trials. Whole-head romaine lettuce and packaged fresh-cut products were collected from both forward and source facilities for microbiological and product quality analyses. High-throughput amplicon sequencing targeting16S rRNA gene was performed to describe shifts in lettuce microbiota. Total aerobic bacteria and coliform counts on whole-head lettuce and on fresh-cut lettuce at different storage times were significantly (p < 0.05) higher for those from the forward processing facility than those from the source processing facility. Microbiota on whole-head lettuce and on fresh-cut lettuce showed differential shifting after lettuce being subjected to source or forward processing, and after product storage. Consistent with the length of pre-processing delays between harvest and processing, the lettuce quality scores of source-processed romaine lettuce, especially at late stages of 2-week storage, was significantly higher than of forward-processed product (p < 0.05).

Microbial Composition and Diversity of High-demand Street-vended Foods in Ecuador.

Developing countries such as Ecuador carry a heavy food safety burden but reports on the microbiological quality of their foods are scarce. In this investigation, the microbial diversity of 10 high-risk and mass-consumption street-vended foods including bolones, encebollado, food dressings, ceviche, chopped fruits, fruit juices, fruit salads, cheese, raw chicken, and ground beef in Quito, Guayaquil, and Cuenca, three major population centers in Ecuador, were evaluated using 16S rRNA gene High Throughput Sequencing. In total, 1,840 amplicon sequence variants (ASVs) were classified into 23 phyla, 253 families, 645 genera, and 829 species. In the tested food samples, Proteobacteria and Firmicutes were the most abundant phyla accounting for 97.41% of relative abundance (RA). At genus level, 10 dominant genera were identified: Acinetobacter (12.61% RA), Lactococcus (12.08% RA), Vibrio (8.23% RA), Weissella (7.43% RA), Aeromonas (6.18% RA), Photobacterium (6.32% RA), Pseudomonas (3.92% RA), Leuconostoc (3.51% RA), Klebsiella (3.49% RA), and Cupriavidus (2.86% RA). The highest microbial diversity indices were found in raw chicken, encebollados, fruit salads, and fruit juices from Guayaquil and Cuenca. From sampled foods, 29 species were classified as food spoilage bacteria and 24 as opportunistic pathogenic bacteria. Two groups associated with human diseases were identified, including 11 enteric species and 26 species of fecal bacteria. The occurrence of recognized and opportunistic pathogenic bacteria, as well as enteric and fecal microorganisms, in the street-vended foods indicated extensive risks for the consumers' health. This study demonstrated the application of culture-independent amplicon sequencing in providing a more comprehensive view of microbial safety for street-vended food, which could be a useful tool to facilitate the control of foodborne diseases.

Cultivar was more influential than bacterial strain and other experimental factors in recovery of Escherichia coli O157:H7 populations from inoculated live Romaine lettuce plants.

The varied choice of bacterial strain, plant cultivar, and method used to inoculate, retrieve, and enumerate Escherichia coli O157:H7 from live plants could affect comparability among studies evaluating lettuce-enterobacterial interactions. Cultivar, bacterial strain, incubation time, leaf side inoculated, and sample processing method were assessed for their influence in recovering and quantifying E. coli O157:H7 from live Romaine lettuce. Cultivar exerted the strongest effect on E. coli O157:H7 counts, which held up even when cultivar was considered in interactions with other factors. Recovery from the popularly grown green Romaine "Rio Bravo" was higher than from the red variety "Outredgeous." Other modulating variables were incubation time, strain, and leaf side inoculated. Sample processing method was not significant. Incubation for 24 hours post-lettuce inoculation yielded greater counts than 48 hours, but was affected by lettuce cultivar, bacterial strain, and leaf side inoculated. Higher counts obtained for strain EDL933 compared to a lettuce outbreak strain 2705C emphasized the importance of selecting relevant strains for the system being studied. Inoculating the abaxial side of leaves gave higher counts than adaxial surface inoculation, although this factor interacted with strain and incubation period. Our findings highlight the importance of studying interactions between appropriate bacterial strains and plant cultivars for more relevant research results, and of standardizing inoculation and incubation procedures. The strong effect of cultivar exerted on the E. coli O157:H7-lettuce association supports the need to start reporting cultivar information for illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk.IMPORTANCEThe contamination of Romaine lettuce with Escherichia coli O157:H7 has been linked to multiple foodborne disease outbreaks, but variability in the methods used to evaluate E. coli O157:H7 association with live lettuce plants complicates the comparability of different studies. In this study, various experimental variables and sample processing methods for recovering and quantifying E. coli O157:H7 from live Romaine lettuce were assessed. Cultivar was found to exert the strongest influence on E. coli O157:H7 retrieval from lettuce. Other modulating factors were bacterial incubation time on plants, strain, and leaf side inoculated, while sample processing method had no impact. Our findings highlight the importance of selecting relevant cultivars and strains, and of standardizing inoculation and incubation procedures, in these types of assessments. Moreover, results support the need to start reporting cultivars implicated in foodborne illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk.

Effects of Fumigation on the Reduction of Salmonella enterica in Soil.

Due to the phaseout of methyl bromide (MeBr), there is a need for broad-spectrum soil fumigation alternatives for pest management. Little is known about the impact of fumigation alternatives on foodborne pathogens, such as Salmonella, in agricultural soils. This study investigated the effect of MeBr alternative fumigants on Salmonella reduction in soil. Sandy loam soil was collected from a conventional farmed vegetable field and inoculated with either Salmonella Newport J1892 or Typhimurium ATCC 14028 (5.9 ± 0.3 log10 colony-forming unit [CFU]/g). Each of the four fumigants labeled for pest management (1,3-dichloropropene, chloropicrin, dimethyl disulfide, and metam sodium) was applied at labeled maximum application field levels to soil in pots and stored for a 2-week period. Sterile water was used as a control. Following the 2-week period, Salmonella concentrations in soil samples were enumerated at 1, 7, 14, and 21 days postfumigation. The mean concentration of Salmonella Newport was significantly higher than that of Salmonella Typhimurium 1 day after fumigation (p = 0.015). Fumigation using 1,3-dichloropropene or dimethyl disulfide significantly reduced Salmonella Newport and Salmonella Typhimurium concentrations, compared with the sterile water control. The rate of Salmonella reduction in soil treated with dimethyl disulfide was higher (0.17 ± 0.02 log10 CFU/g/day), compared with soil treated with the other fumigants (0.10-0.12 log10 CFU/g/day). Due to the reduction of Salmonella, alternative fumigation treatments may mitigate potential Salmonella contamination in soil within farm environments.

Listeria monocytogenes loss of cultivability on carrot is associated with the formation of mesosome-like structures.

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.

Pre-harvest biocontrol of Listeria and Escherichia coli O157 on lettuce and spinach by lactic acid bacteria.

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.

Growth and inactivation of Listeria monocytogenes in sterile extracts of fruits and vegetables: Impact of the intrinsic factors pH, sugar and organic acid content.

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.

Use of a silver-based sanitizer to accelerate Escherichia coli die-off on fresh-cut lettuce and maintain produce quality during cold storage: Laboratory and pilot-plant scale tests.

Outbreaks and product recalls involving romaine and iceberg lettuce are frequently reported in the United States. Novel technologies are needed to inactivate pathogens without compromising product quality and shelf life. In this study, the effects of a process aid composed of silver dihydrogen citrate, glycerin, and lactic acid (SGL) on Escherichia coli and Listeria monocytogenes concentrations on lettuce immediately after washing and during cold storage were evaluated. Sensory and quality attributes of fresh-cut iceberg lettuce were also evaluated. Laboratory results indicated that application of SGL solution for 30 s as a first step in the washing process resulted in a 3.15 log reduction in E. coli O157:H7 immediately after washing. For E. coli O157:H7 a significant difference between SGL treatment and all other treatments was maintained until day 7. On day zero, SGL led to a 2.94 log reduction of L. monocytogenes. However, there was no significant difference between treatments with or without SGL regardless of storage time. Pilot-plant results showed that samples receiving SGL spray followed by chlorinated flume wash exhibited a greater reduction (1.48 log) in nonpathogenic E. coli populations at the end of shelf life than other treatments (p < 0.05). Additional pilot plant tests were conducted to investigate the hypothesis that SGL residues could continue to impact microbial survival on the final washed lettuce. Results show that pathogens introduced subsequent to flume washing of lettuce pretreated with SGL solution were not affected by antimicrobial residues. The final quality and shelf life of flume washed lettuce were also unaffected by pretreatment with SGL. In conclusion, the results of this study demonstrate that this new technology has the potential to accelerate E. coli die-off on fresh-cut lettuce during cold storage and improve product safety, while not affecting quality throughout the shelf life of the finished products.

Dynamics of Listeria monocytogenes and the microbiome on fresh-cut cantaloupe and romaine lettuce during storage at refrigerated and abusive temperatures.

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.

Microbiological Quality of High-Demand Food from Three Major Cities in Ecuador.

ABSTRACT: Bacterial foodborne diseases are among the most important public health issues worldwide, but in Ecuador, reports on the microbiological quality of food are scarce. In this cross-sectional study, 450 samples of high-demand Ecuadorian food, including bolon, encebollado, sauces, ceviche, fruit, fruit juice, fruit salad, cheese, raw chicken, and ground beef, were collected from popular street markets in the cities of Guayaquil, Quito, and Cuenca. Populations of total aerobic mesophilic bacteria, total coliforms, fecal coliforms, Escherichia coli, Salmonella enterica, and Listeria monocytogenes were examined on composited samples by plate count following the local regulations (Norma Tecnica Ecuatoriana, Instituto Ecuatoriano de Normalización) for each kind of food. The individual and interaction effects of the city and food type on the levels of each bacterial group were assessed by two-way analysis of variance. Selected colonies from each culture were identified using Biolog OmniLog ID and sequencing of the V3 to V4 region on the 16S rRNA gene. Average total aerobic mesophilic bacteria, total coliform, fecal coliform, and E. coli levels were 5.10 ± 0.12, 2.50 ± 0.16, 1.09 ± 0.12, and 0.83 ± 0.12 log CFU/g or mL, respectively, with significant variations among the cities. The prevalence of Salmonella in chicken and sauces and L. monocytogenes in cheese and fruit salad was greater than 20%. Opportunistic pathogens including Klebsiella pneumoniae, Staphylococcus sciuri, and Enterococcus spp. were frequently identified in the samples from all three cities. High prevalence of spoilage microorganisms such as Bacillus amyloliquefaciens and biocontrol bacteria such as Lactococcus lactis was also observed. This is the first report on the microbiological quality of food from Ecuador.

Salmonella inactivation and sponge/microfiber mediated cross-contamination during papaya wash with chlorine or peracetic acid as sanitizer.

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.

Factors Impacting Chemical and Microbiological Quality of Wash Water during Simulated Dump Tank Wash of Grape Tomatoes.

ABSTRACT: Fresh and fresh-cut tomatoes are high in phytonutrients. However, illness outbreaks associated with contaminated tomatoes have significantly impacted public health and the economic well-being of the tomato industry. Scientific information is needed to develop an effective, practical food safety standard to reduce pathogen contamination. The aim of this study was to assess factors impacting the deterioration of the quality of tomato wash water and the proliferation of indigenous microorganisms during a simulated dump tank washing process. Freshly harvested grape tomatoes were sorted into four groups: prime, defective, underripe, and nontomato debris. Tomatoes with leaf or stem harvest debris, combined or separate, were washed in tap water with or without free chlorine. Water samples were analyzed for total dissolved solids, turbidity, chemical oxygen demand, and chlorine demand. Microbial populations in water and on tomatoes as impacted by chlorine concentration and water filtration (300 µm) were also quantified. Field debris and defective tomatoes were the major contributors to microbial populations in wash water. Field debris, although accounting for <1% of the total weight of harvested material, contributed 37.84% of total dissolved solids, 46.15% of turbidity, 48.77% of chemical oxygen demand, and 50.55% of chlorine demand in the wash water. Water quality deterioration was proportional to the cumulative quantity of tomatoes and debris washed, and free chlorine at ≥5 mg/L significantly reduced the Enterobacteriaceae, aerobic mesophilic bacteria, and yeast and mold populations. These results highlight the importance of minimizing field debris and defective fruits in harvested grape tomatoes to reduce the microbial load and prevent deterioration of wash water quality. This information will be useful for the development of data-driven harvesting and packinghouse food safety practices for grape tomatoes.

Survival of Salmonella enterica and shifts in the culturable mesophilic aerobic bacterial community as impacted by tomato wash water particulate size and chlorine treatment.

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.

Genome Sequences of Brevundimonas naejangsanensis Strain FS1091 and Bacillus amyloliquefaciens Strain FS1092, Isolated from a Fresh-Cut-Produce-Processing Plant.

The complete genome sequences of Brevundimonas naejangsanensis strain FS1091 and Bacillus amyloliquefaciens strain FS1092, which were isolated from a commercial fresh-cut-produce-processing facility, were determined. Both FS1091 and FS1092 have one circular chromosome of approximately 3.15 and 4.24 Mb, respectively.

Salmonella inactivation and cross-contamination on cherry and grape tomatoes under simulated wash conditions.

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.

Differential Effects of Growth Medium Salinity on Biofilm Formation of Two Salmonella enterica Strains.

ABSTRACT: Salmonella enterica is a prominent foodborne pathogen, including diverse serotypes that are prolific biofilm formers. Its ability to form biofilm can be affected by multiple environmental factors. In this study, the effect of salinity on biofilm formation by S. enterica was evaluated by using two recently isolated strains of Salmonella serotypes Enteritidis and Newport. Although supplementing the growth medium with a low concentration (0.5 to 2%) of sodium chloride (NaCl) slightly enhanced biofilm formation for the strain S. enterica serovar Enteritidis 110, it sharply reduced or abolished biofilm formation by the strain S. enterica serovar Newport 193. This differential effect of salinity on S. enterica strains of different serotypes was poorly correlated with inhibition of planktonic growth but strongly correlated with cell motility. Examining genes known to affect biofilm formation showed that the expression of adrA, csgD, and fliC, which encode proteins required for surface adhesion and cell motility, was significantly downregulated with salinity increase in Salmonella Newport 193 but not in Salmonella Enteritidis 110. Therefore, it is plausible that the differential effect of salinity on biofilm formation by Salmonella Enteritidis 110 and Salmonella Newport 193 resulted from the differential regulation to genes required for cell adherence and motility.

Microbiome convergence following sanitizer treatment and identification of sanitizer resistant species from spinach and lettuce rinse water.

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.

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.

Quantitative Proteomic Analysis of Staphylococcus aureus Treated With Punicalagin, a Natural Antibiotic From Pomegranate That Disrupts Iron Homeostasis and Induces SOS.

Staphylococcus aureus, a bacterial, food-borne pathogen of humans, can contaminate raw fruits and vegetables. While physical and chemical methods are available to control S. aureus, scientists are searching for inhibitory phytochemicals from plants. One promising compound from pomegranate is punicalagin, a natural antibiotic. To get a broader understanding of the inhibitory effect of punicalagin on S. aureus growth, high-throughput mass spectrometry and quantitative isobaric labeling was used to investigate the proteome of S. aureus after exposure to a sublethal dose of punicalagin. Nearly half of the proteins encoded by the small genome were interrogated, and nearly half of those exhibited significant changes in accumulation. Punicalagin treatment altered the accumulation of proteins and enzymes needed for iron acquisition, and it altered amounts of enzymes for glycolysis, citric acid cycling, protein biosynthesis, and purine and pyrimidine biosynthesis. Punicalagin treatment also induced an SOS cellular response to damaged DNA. Transcriptional comparison of marker genes shows that the punicalagin-induced iron starvation and SOS responses resembles those produced by EDTA and ciprofloxacin. These results show that punicalagin adversely alters bacterial growth by disrupting iron homeostasis and that it induces SOS, possibly through DNA biosynthesis inhibition.

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.