Evaluating the efficacy of peroxyacetic acid in preventing Salmonella cross-contamination on tomatoes in a model flume system.

The use of flume tanks for tomato processing has been identified as a potential source of cross-contamination, which could result in foodborne illness. This study's objective was to assess the efficacy of peroxyacetic acid (PAA) at a concentration of ≤80 mg/L in preventing Salmonella enterica cross-contamination under various organic loads in a benchtop model tomato flume tank. The stability of 80 mg/L PAA at different chemical oxygen demand (COD) levels was also tested. Tomatoes were spot inoculated with a five-serovar rifampin-resistant (rif+) Salmonella cocktail (106 or 108 colony forming unit (CFU)/tomato). Inoculated (n = 3) and uninoculated (n = 9) tomatoes were introduced into the flume system containing 0-80 mg/L PAA and 0 or 300 mg/L COD. After washing for 30, 60, or 120 s, uninoculated tomatoes were sampled and analyzed for cross-contamination. All experiments were conducted in triplicate. Increasing the organic load (measured as COD) affected the stability of PAA in water with significantly faster dissociation when exposed to 300 mg/L COD. The concentration of PAA, inoculum level, COD levels, and time intervals were all significant factors that affected cross-contamination. Cross-contamination occurred at the high inoculum level (108 CFU/tomato) even when 80 mg/L PAA was present in the model flume tank, regardless of the organic load level. When the tomatoes were contaminated at a level of 106 CFU/tomato, concentrations as low as 5 mg/L of PAA were effective in preventing cross-contamination at 0 mg/L COD; however, 100 % tomatoes (9/9) were positive when the organic load increased to 300 mg/L COD. When the PAA concentration was increased to 10 mg/L, it effectively prevented cross-contamination in the tank, regardless of the presence of organic load. These results suggest that using PAA at concentrations below the maximum limit remains effective in limiting bacterial cross-contamination and offers a more environment-friendly option for tomato packinghouse operators.

Influence of Free Chlorine and Contact Time on the Reduction of Salmonella Cross-Contamination of Tomatoes in a Model Flume System.

ABSTRACT: The process of washing tomatoes in dump (flume) tanks has been identified as a potential source of cross-contamination. This study's objective was to assess the potential for Salmonella enterica cross-contamination at various inoculation levels in the presence of free chlorine (HOCl) and organic matter. Uninoculated tomatoes were introduced into a laboratory-based model flume containing tomatoes inoculated with a cocktail of five rifampin-resistant S. enterica serovars at 104, 106, or 108 CFU per tomato in water containing 0 or 25 mg/L HOCl and 0 or 300 mg/L chemical oxygen demand (COD). Uninoculated tomatoes exposed to the inoculated tomatoes were removed from the water after 5, 30, 60, and 120 s and placed in bags containing tryptic soy broth supplemented with rifampin and 0.1% sodium thiosulfate. Following incubation, enrichment cultures were plated on tryptic soy agar supplemented with rifampin and xylose lysine deoxycholate agar to determine the presence of Salmonella. HOCl and pH were measured before and after each trial. The HOCl in water containing 300 mg/L COD significantly declined (P ≤ 0.05) by the end of each 120-s trial, most likely due to the increased demand for the oxidant. Higher inoculum levels and lower HOCl concentrations were significant factors (P ≤ 0.05) that contributed to increased cross-contamination. At 25 mg/L HOCl, no Salmonella was recovered under all conditions from uninoculated tomatoes exposed to tomatoes inoculated at 104 CFU per tomato. When the inoculum was increased to 106 and 108 CFU per tomato, cross-contamination was observed, independent of COD levels. The results from this study indicate that the currently required sanitizer concentration (e.g., 100 or 150 mg/L) for flume water may be higher than necessary and warrants reevaluation.

Determining Bacterial Load and Water Quality Parameters of Chlorinated Tomato Flume Tanks in Florida Packinghouses.

ABSTRACT: Monitoring and maintenance of water quality in dump tanks or flume systems is crucial to maintaining proper sanitizer levels to prevent pathogen cross-contamination during postharvest washing of tomatoes, but there is limited information on how organic matter influences sanitizer efficacy in the water. The main objective of this study was to monitor water quality in flume tanks and evaluate the efficacy of postharvest washing of tomatoes in commercial packinghouses. Flume tank water samples (n = 3) were collected on an hourly basis from three packinghouses in Florida and analyzed for pH, total dissolved solids (TDS), free chlorine, chemical oxygen demand (COD), oxidation-reduction potential, and turbidity. Additionally, three flume-water samples were collected and tested for total aerobic plate count (APC), total coliforms (TC), and Escherichia coli. Fresh tomatoes (n = 3), both before and after washing, were collected and analyzed for the same bacterial counts. Turbidity, COD, and TDS levels in flume water increased over time in all packinghouses. Correlations observed include COD and turbidity (r = 0.631), turbidity and TDS (r = 0.810), and oxidation-reduction potential and chlorine (r = 0.660). APC for water samples had an average range of 0.0 to 4.7 log CFU/mL and TC average range of 0.0 to 4.7 log CFU/mL. All water samples were negative for E. coli. The average APC for pre- and postflume tomatoes from the three packinghouses was 6.0 log CFU per tomato and ranged from 2.2 to 7.4 log CFU per tomato. The average TC count was <1.5 and 7.0 log CFU per tomato for pre- and postwash tomatoes, respectively. There was no significant effect (P > 0.05) of postharvest washing on the microbiological qualities of tomatoes. Water quality in flume tanks deteriorated over time in all packinghouses during a typical operational day of 4 to 8 h.

Prevalence, Concentration, and Antimicrobial Resistance Profiles of Salmonella Isolated from Florida Poultry Litter.

ABSTRACT: For over a decade, Salmonella contamination has increasingly led to outbreaks of foodborne illness associated with fresh produce. The use of untreated animal manures, or biological soil amendments of animal origin, to amend agricultural soils holds a risk of contamination from foodborne pathogens, such as Salmonella. This study was conducted to determine the prevalence, concentration, serotypes, and antimicrobial resistance profiles of Salmonella in poultry litter from Florida farms. Litter pH, total Kjeldahl nitrogen, total ammonia nitrogen, total phosphorus (P2O5), total potassium (K2O), moisture content, total solids, total ash, organic matter, and aerobic plate count (APC) were also measured. Litter samples (n = 54) were collected from 18 broiler farms across three seasons (spring, summer, and winter). Salmonella concentrations were enumerated using a most-probable-number (MPN) method, and antimicrobial susceptibility testing was performed. The prevalence of Salmonella in litter samples was 61.1%, with a geometric mean of 0.21 ± 20.7 MPN/g. Across all seasons, Salmonella concentrations were not influenced by the chemical, physical, or microbial properties measured. Recovered Salmonella isolates (n = 290) were grouped into serogroups O:4 (43.1%), O:7 (26.9%), O:8 (11.0%), O:1,3,10,19 (7.9%), and O:9,46 (7.2%). Serotyping Salmonella isolates (n = 47) resulted in 12 serotypes, with the most common being Typhimurium (27.7%), Kentucky (17.0%), Enteritidis (14.9%), and Mbandaka (14.9%). Antimicrobial resistance to tetracycline (29.8%), sulfisoxazole (23.4%), and streptomycin (14.9%) was observed. No isolates were resistant to more than two antimicrobial agents. This study provides valuable information for future risk assessments for the use of poultry litter as an untreated biological soil amendment of animal origin.

Survival of Escherichia coli O157 in autoclaved and natural sandy soil mesocosms.

While the soil microbiome may influence pathogen survival, determining the major contributors that reduce pathogen survival is inconclusive. This research was performed to determine the survival of E. coli O157 in autoclaved and natural (unautoclaved) sandy soils. Soils were inoculated with three different E. coli O157 strains (stx1+/stx2+, stx1-/stx2-, and stx1-/stx2+), and enumerated until extinction at 30°C. There was a significant difference in the survival of E. coli O157 based on soil treatment (autoclaved versus natural) at 30°C on days 1 (P = 0.00022), 3, (P = 2.53e-14), 7 (P = 5.59e-16), 14 (P = 1.072e-12), 30 (P = 7.18e-9), and 56 (P = 0.00029), with greater survival in autoclaved soils. The time to extinction (two consecutive negative enrichments) for all three strains was 169 and 84 days for autoclaved and natural soils, respectively. A separate E. coli O157 trial supplemented with 16S rRNA gene sequencing of the soil microbiome was performed at 15°C and 30°C on days 0, 7, 14, and 28 for each soil treatment. Greater species richness (Chao1, P = 2.2e-16) and diversity (Shannon, P = 2.2e-16) was observed in natural soils in comparison with autoclaved soils. Weighted UniFrac (beta-diversity) showed a clear distinction between soil treatments (P = 0.001). The greatest reduction of E. coli O157 was observed in natural soils at 30°C, and several bacterial taxa positively correlated (relative abundance) with time (day 0 to 28) in these soils (P < 0.05), suggesting that the presence of those bacteria might cause the reduction of E. coli O157. Taken together, a clear distinction in E. coli O157 survival, was observed between autoclaved and natural soils along with corresponding differences in microbial diversity in soil treatments. This research provides further insights into the bacterial taxa that may influence E. coli O157 in soils.

Prevalence and concentration of stx+ E. coli and E. coli O157 in bovine manure from Florida farms.

Fresh produce outbreaks due to Shiga toxin-producing Escherichia coli (STEC) continue to occur in the United States (US). Manure-amended soils can pose a public health risk when used for growing raw agricultural commodities. Knowing the prevalence and concentration of STEC in untreated biological soil amendments of animal origin (BSAAO) is important to help guide the most appropriate pre-harvest interval(s) following application to limit risks from these soil amendments. Bovine manure samples were collected from 12 farms in Florida, including samples from piles, lagoons, barns, and screened solids. Two methods were used to detect stx1/2 and rfbE genes in samples. A prevalence rate of 9% for stx1 and/or stx2 and 19% for rfbE was observed from the 518 bovine manure samples evaluated. A most probable number (MPN) assay was performed on stx+ samples when applicable. The geometric mean for stx+ samples (n = 20) was 3.37 MPN g-1 (0.53 log MPN g-1) with a maximum value of 6,800 MPN g-1 (3.83 log MPN g-1). This research was part of a larger nationwide geographical study on the prevalence and concentration of STEC in bovine manure to help guide regulations on feasible pre-harvest intervals for the application of untreated BSAAO.

Survival of Salmonella and Escherichia coli in Two Different Soil Types at Various Moisture Levels and Temperatures.

With the increased consumption of fresh produce, a proportional increase in numbers of produce-related foodborne illness has been observed. An estimate of foodborne illness during 1998 to 2008 attributed ∼46% of the incidences to produce. Any foodborne illness associated with produce can have devastating consequences to the industry. The most recent data from the Centers for Disease Control and Prevention implicate leafy vegetables, vine-stalk vegetables, root vegetables, and sprouts as the most common cause of produce-related foodborne outbreaks. Excess rainfall or flooding, mainly by altering levels of soil moisture and oxygen content, affects the microbial community in soil. The goal of this research was to determine the survivability of a three-serovar Escherichia coli and a five-serovar Salmonella enterica cocktail in microcosms prepared with Candler sand (CS) and Orangeburg sandy loam (OSL) soils. Microcosms were prepared with low, medium, and high volumetric water contents and were incubated at 20 and 30°C. Serotyping was used to determine which E. coli or Salmonella serovar(s) from each cocktail persisted. Microcosm inoculation levels were ∼7.0 log CFU/g. Sampling for CS and OSL microcosms incubated at 20°C ended on day 364 and 357, respectively. The reduction of Salmonella and E. coli to below the limit of detection (extinction) in CS microcosms (incubated at 30°C at all volumetric water content [VWC] levels) was reached on day 168 and 56, respectively. Extinction of Salmonella and E. coli in OSL microcosms (incubated at 30°C at all VWCs) was reached on day 168 and 224, respectively. Of the Salmonella and E. coli serovars analyzed, Salmonella Javiana persisted the longest in both soil types, whereas E. coli O104:H4 and E. coli O145 persisted the longest in CS and OSL microcosms, respectively. Results from the current study suggest that soil type and temperature influenced pathogen persistence in CS and OSL soils more than moisture level and pathogen type.

Determination of Optimum Sanitizer Levels for Prevention of Salmonella Cross-Contamination of Mature Round Tomatoes in a Laboratory Model Flume System.

Salmonella has been reported to be involved in several foodborne illness outbreaks, many of which resulted from consumption of raw tomatoes. This research aimed to optimize and evaluate the concentration of free chlorine (hypochlorous acid [HOCl]) used as a sanitizer to prevent cross-contamination of tomatoes inoculated with a cocktail of five rifampin-resistant Salmonella enterica serovars in a laboratory-based model flume system. Organic load, prepared using sterilized Scotts Premium Topsoil, was added in different quantities to the flume wash water to simulate real-world packinghouse conditions. In a typical packinghouse operation utilizing a recirculating flume system, the organic matter washed from tomato surfaces accumulates over time. In this study, different concentrations (0, 25, 50, 75, and 100 ppm) of HOCl were used as sanitizers under three organic load conditions (0, 650, and 1,000 mg/L chemical oxygen demand). Results showed that 100 ppm of HOCl was necessary to prevent Salmonella cross-contamination of uninoculated tomatoes in the model flume system in the presence of organic loading. Also, when treated with 100 ppm of HOCl, Salmonella levels were reduced by >4.5 log CFU per tomato from inoculated tomatoes in the presence of organic load. At 75 ppm of HOCl, Salmonella cross-contamination was prevented, but only in the absence of organic loading. In studies in which plate counts were negative, whole tomato enrichment studies were performed. No cross-contamination of uninoculated tomatoes was recorded when 100 ppm of HOCl was used, even in the presence of high organic load (1,000 mg/L chemical oxygen demand). Although sanitizer application reduces contamination on tomato surfaces, the primary function of sanitizers in the wash water is to prevent cross-contamination.

Persistence of Indigenous Escherichia coli in Raw Bovine Manure-Amended Soil.

The Food Safety Modernization Act attempts to reduce produce-related foodborne illness by using preventive rather than reactive measures. The goal of this research was to determine the persistence of manure-borne generic Escherichia coli under laboratory and field conditions. The population density of naturally occurring E. coli was ∼7.2 and 5.4 log CFU/10 g in pre- and postscreened manures, respectively. Postscreened (i.e. after the liquid manure has passed through a screen) manure was applied at light, medium, and heavy rates to fields in Live Oak and Citra, FL, during the fall and summer. Laboratory microcosms of the manure-amended soils (comparable to the field's heavy application rate of manure) from both locations were maintained at 20 and 30°C. Persistence of E. coli, moisture content, and pH were monitored until E. coli became unrecoverable. The longest E. coli persistence seen in field trails was during the summer and fall trials from Citra (heavy application) that terminated on day 112 and day 280, respectively. The rate of E. coli decay ranged from 0.02 to 0.04 log CFU per day across all manure application rates, seasons, and locations. In the microcosm studies, the E. coli became extinct on day 210 in the 30°C, whereas they became unrecoverable on day 420 in the 20°C microcosms. The relatively prolonged persistence of E. coli in the microcosms suggests that survival under laboratory conditions does not mimic real-world survival rates and may not be adequate for predicting E. coli persistence in the field. The persistence data also suggest that the risk from E. coli associated with new contamination events, such as wild life intrusion, runoff, or other vectors, may be greater than the risk associated with the long-term survival of manure-borne E. coli, although more work is needed to confirm this hypothesis.

Sulfate and chromate increased each other's uptake and translocation in As-hyperaccumulator Pteris vittata.

We investigated the effects of chromate (CrVI) and sulfate on their uptake and translocation in As-hyperaccumulator Pteris vittata. Plants were exposed to 1) 0.1 mM CrVI and 0, 0.25, 1.25 or 2.5 mM sulfate or 2) 0.25 mM sulfate and 0, 0.5, 2.5 or 5.0 mM CrVI for 1 d in hydroponics. P. vittata accumulated 26 and 1261 mg kg(-1) Cr in the fronds and roots at CrVI0.1, and 2197 and 1589 mg kg(-1) S in the fronds and roots at S0.25. Increasing sulfate concentrations increased Cr root concentrations by 16-66% and helped CrVI reduction to CrIII whereas increasing CrVI concentrations increased frond sulfate concentrations by 3-27%. Increasing sulfate concentrations enhanced TBARS concentrations in the biomass, indicating oxidative stress caused lipid peroxidation in plant cell membranes. However, addition of 0.25-2.5 mM sulfate alleviated CrVI's toxic effects and decreased TBARS from 23.5 to 9.46-12.3 µmol g(-1) FW. Though CrVI was supplied, 78-96% of CrIII was in the biomass, indicating efficient CrVI reduction to CrIII by P. vittata. The data indicated the amazing ability of P. vittata in Cr uptake at 289 mg kg(-1) h(-1) with little translocation to the fronds. These results indicated that P. vittata had potential in Cr phytoremediation in contaminated sites but further studies are needed to evaluate this potential. The facts that CrVI and sulfate helped each other in uptake by P. vittata suggest that CrVI was not competing with sulfate uptake in P. vittata. However, the mechanisms of how sulfate and CrVI enhance each other's accumulation in P. vittata need further investigation.

Cell hydration as a biomarker for estimation of biological effects of nonionizing radiation on cells and organisms.

"Changes in cell hydration" have been hypothesized as an input signal for intracellular metabolic cascade responsible for biological effects of nonionizing radiation (NIR). To test this hypothesis a comparative study on the impacts of different temperature and NIR (infrasound frequency mechanical vibration (MV), static magnetic field (SMF), extremely low frequency electromagnetic field (ELF EMF), and microwave (MW)) pretreated water on the hydration of barley seeds in its dormant and germination periods was performed. In dormant state temperature sensitivity (Q 10) of seed hydration in distilled water (DW) was less than 2, and it was nonsensitive to NIR treated DW, whereas during the germination period (48-72 hours) seeds hydration exhibited temperature sensitivity Q 10 > 2 and higher sensitivity to NIR treated DW. Obtained data allow us to suggest that the metabolic driving of intracellular water dynamics accompanied by hydrogen bonding and breaking is more sensitive to NIR-induced water structure changes in seed bathing aqua medium than the simple thermodynamic processes such as osmotic gradient driven water absorption by seeds in dormant state. Therefore, cell hydration is suggested to be a universal and extrasensitive biomarker for detection of biological effects of NIR on cells and organisms.

Detoxification of toxic heavy metals by marine bacteria highly resistant to mercury.

Pollution in industrial areas is a serious environmental concern, and interest in bacterial resistance to heavy metals is of practical significance. Mercury (Hg), Cadmium (Cd), and lead (Pb) are known to cause damage to living organisms, including human beings. Several marine bacteria highly resistant to mercury (BHRM) capable of growing at 25 ppm (mg L(-1)) or higher concentrations of mercury were tested during this study to evaluate their potential to detoxify Cd and Pb. Results indicate their potential of detoxification not only of Hg, but also Cd and Pb. Through biochemical and 16S rRNA gene sequence analyses, these bacteria were identified to belong to Alcaligenes faecalis (seven isolates), Bacillus pumilus (three isolates), Bacillus sp. (one isolate), Pseudomonas aeruginosa (one isolate), and Brevibacterium iodinium (one isolate). The mechanisms of heavy metal detoxification were through volatilization (for Hg), putative entrapment in the extracellular polymeric substance (for Hg, Cd and Pb) as revealed by the scanning electron microscopy and energy dispersive x-ray spectroscopy, and/or precipitation as sulfide (for Pb). These bacteria removed more than 70% of Cd and 98% of Pb within 72 and 96 h, respectively, from growth medium that had initial metal concentrations of 100 ppm. Their detoxification efficiency for Hg, Cd and Pb indicates good potential for application in bioremediation of toxic heavy metals.

Bioremediation of toxic substances by mercury resistant marine bacteria.

Bioremediation of toxic substances includes microbe-mediated enzymatic transformation of toxicants to non-toxic, often assimilable, forms. Mercury-resistant marine bacteria are found to be very promising in dealing with mercury, and a host of other highly toxic heavy metals and xenobiotics. In the present studies we have shown that the Pseudomonas aeruginosa CH07 (NRRL B-30604) has been able to degrade a variety of PCB congeners including a complete degradation of CB-126 and CB-181. The culture was able to remove over 70% Cd from growth medium when supplemented with 100 ppm Cd. The same bacterium rapidly biotransformed/removed toxic mercury from wastewater in a bioreactor system.

Tolerance to various toxicants by marine bacteria highly resistant to mercury.

Bacteria highly resistant to mercury isolated from seawater and sediment samples were tested for growth in the presence of different heavy metals, pesticides, phenol, formaldehyde, formic acid, and trichloroethane to investigate their potential for growth in the presence of a variety of toxic xenobiotics. We hypothesized that bacteria resistant to high concentrations of mercury would have potential capacities to tolerate or possibly degrade a variety of toxic materials and thus would be important in environmental pollution bioremediation. The mercury-resistant bacteria were found to belong to Pseudomonas, Proteus, Xanthomonas, Alteromonas, Aeromonas, and Enterobacteriaceae. All these environmental bacterial strains tolerant to mercury used in this study were capable of growth at a far higher concentration (50 ppm) of mercury than previously reported. Likewise, their ability to grow in the presence of toxic xenobiotics, either singly or in combination, was superior to that of bacteria incapable of growth in media containing 5 ppm mercury. Plasmid-curing assays done in this study ascertained that resistance to mercury antibiotics, and toxic xenobiotics is mediated by chromosomally borne genes and/or transposable elements rather than by plasmids.