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.

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.

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.

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.

Metagenome tracking biogeographic agroecology: Phytobiota of tomatoes from Virginia, Maryland, North Carolina and California.

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.

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.

Distribution and Characterization of Salmonella enterica Isolates from Irrigation Ponds in the Southeastern United States.

Irrigation water has been implicated as a likely source of produce contamination by Salmonella enterica. Therefore, the distribution of S. enterica was surveyed monthly in irrigation ponds (n = 10) located within a prime agricultural region in southern Georgia and northern Florida. All ponds and 28.2% of all samples (n = 635) were positive for Salmonella, with an overall geometric mean concentration (0.26 most probable number [MPN]/liter) that was relatively low compared to prior reports for rivers in this region. Salmonella peaks were seasonal; the levels correlated with increased temperature and rainfall (P < 0.05). The numbers and occurrence were significantly higher in water (0.32 MPN/liter and 37% of samples) than in sediment (0.22 MPN/liter and 17% of samples) but did not vary with depth. Representative isolates (n = 185) from different ponds, sample types, and seasons were examined for resistance to 15 different antibiotics; most strains were resistant to streptomycin (98.9%), while 20% were multidrug resistant (MDR) for 2 to 6 antibiotics. DiversiLab repetitive extragenic palindromic-element sequence-based PCR (rep-PCR) revealed genetic diversity and showed 43 genotypes among 191 isolates, as defined by >95% similarity. The genotypes did not partition by pond, season, or sample type. Genetic similarity to known serotypes indicated Hadar, Montevideo, and Newport as the most prevalent. All ponds achieved the current safety standards for generic Escherichia coli in agricultural water, and regression modeling showed that the E. coli level was a significant predictor for the probability of Salmonella occurrence. However, persistent populations of Salmonella were widely distributed in irrigation ponds, and the associated risks for produce contamination and subsequent human exposure are unknown, supporting continued surveillance of this pathogen in agricultural settings.

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.

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.

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).

The presence of two cyclase thioesterases expands the conformational freedom of the cyclic Peptide occidiofungin.

Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by the Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and ß-hydroxyasparagine variants coordinates the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides.

Organically managed soils reduce internal colonization of tomato plants by Salmonella enterica serovar Typhimurium.

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 Salmonella Typhimurium strains (10(9) 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 Salmonella 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 Salmonella sp. was detected in plants grown from contaminated seed.

Occurrence and population density of Campylobacter jejuni in irrigation ponds on produce farms in the Suwannee River Watershed.

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.

Factors affecting the occurrence of Escherichia coli O157 contamination in irrigation ponds on produce farms in the Suwannee River Watershed.

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.

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.

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.

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.

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.

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.