Comparison of Three Air Sampling Methods for the Quantification of Salmonella, Shiga-toxigenic Escherichia coli (STEC), Coliforms, and Generic E. coli from Bioaerosols of Cattle and Poultry Farms.

Recent fresh produce outbreaks potentially associated with bioaerosol contamination from animal operations in adjacent land highlighted the need for further study to better understand the associated risk. The purpose of this research was to evaluate three sampling methods for quantifying target bacterial bioaerosols from animal operations. A dairy cattle and poultry farm located in Georgia, U.S. were visited six times each. Air was collected for 10 min using: 2-stage Andersen impactor with and without mineral oil overlay and impingement samplers. Sampling devices were run concurrently at 0.1, 1, and 2 m heights (n = 36). Andersen samplers were loaded with CHROMagar™ Salmonella, CHROMagar™ STEC, or Brilliance™ coliforms/E. coli. The impingement sampler contained buffered peptone water (20 mL) which was vacuum filtered through a 0.45 µm filter and placed onto the respective media. Plates were incubated at 37 ℃ for 48 h. PCR confirmation followed targeting ttr for Salmonella and stx1, stx2, and eae genes for STEC. No significant differences were found among methods to quantify coliforms and E. coli. Salmonella and STEC bioaerosols were not detected by any of the methods (Limit of detection: 0.55 log CFU/m3). E. coli bioaerosols were significantly greater in the poultry (2.76-5.00 log CFU/m3) than in the cattle farm (0.55-2.82 log CFU/m3) (p < 0.05), and similarly distributed at both stages in the Andersen sampler (stage 1:>7 µm; stage 2: 0.65-7 µm particle size). Sampling day did not have a significant effect on the recovery of coliforms/E. coli bioaerosols in the poultry farm when samples were taken at the broiler house exhaust fan (p > 0.05). A greater and constant emission of coliforms and E. coli bioaerosols from the poultry farm warrants further investigation. These data will help inform bioaerosol sampling techniques which can be used for the quantification of bacterial foodborne pathogens and indicator organisms for future research.

Longitudinal Survey of Aeromonas hydrophila and Foodborne Pathogens in a Commercial Aquaponics System.

Aquaponic production of fresh produce is a sustainable agricultural method becoming widely adopted, though few studies have investigated potential food safety hazards within commercial systems. A longitudinal study was conducted to isolate and quantify several foodborne pathogens from a commercial, aquaponic farm, and to elucidate their distribution throughout. The survey was conducted over 2 years on a controlled-environment farm containing Nile tilapia (Oreochromis niloticus) and lettuce (Lactuca sativa). Samples (N = 1,047) were collected bimonthly from three identical, independent systems, and included lettuce leaves, roots, fingerlings (7-126 d old), feces from mature fish (>126 d old), water, and sponge swabs collected from the tank interior surface. Most probable number of generic Escherichia coli were determined using IDEXX Colilert Quanti-Tray. Enumeration and enrichment were used to detect Shiga toxin-producing E. coli (STEC), Salmonella enterica, Listeria monocytogenes, Aeromonas spp., Aeromonas hydrophilia, and Pseudomonas aeruginosa. Generic E. coli, STEC, L. monocytogenes, and S. enterica were not detected in collected samples. P. aeruginosa was isolated from water (7/351; 1.99%), swabs (3/351; 0.85%), feces (2/108; 1.85%), and lettuce leaves (2/99; 2.02%). A. hydrophila was isolated from all sample types (623/1047; 59.50%). The incidence of A. hydrophila in water (X2 = 23.234, p < 0.001) and sponge samples (X2 = 21.352, p < 0.001) increased over time.

Transfer of generic Escherichia coli and attenuated Salmonella enterica Typhimurium from the soil to the surface of in-shell pecans during harvest.

During harvest pecan nuts are at risk of contamination with foodborne pathogens from extended contact with the ground. The objective of this study was to determine the potential transfer of Escherichia coli and Salmonella from the ground to in-shell pecans during the harvesting process. Plots (2 m2) were sprayed with 1 L of a rifampicin (rif) resistant strain of either E. coli TVS 353 or an attenuated Salmonella Typhimurium inoculum at a low (∼4 log CFU/ml), mid (∼6 log CFU/ml) or high (∼8 log CFU/ml) concentrations. The following day, nuts were mechanically harvested and samples from each plot were collected at 1 min, 4 h, and 24 h. Samples were enumerated for Salmonella and E. coli on tryptic soy agar supplemented with rif. The Salmonella levels in the soil from the inoculated plots were 2.0 ± 0.3, 4.1 ± 0.1, and 6.4 ± 0.2 log CFU/g for the low, mid, and high inocula, respectively. The E. coli levels in the soil from the inoculated plots were 1.5 ± 0.4, 3.7 ± 0.3, and 5.8 ± 0.1 log CFU/g for the low, mid, and high inocula, respectively. There was a significant difference in the average daily rainfall among the three trials. Trial 3 received 23.8 ± 9.2 cm, while trials 1 and 2 received much less (0.1 ± 0.1 0.0 ± 0.0 cm, respectively). Inoculation concentration and trial were significant (P<0.05) factors that influenced the transfer of E. coli and Salmonella to pecans. For the high inoculum treatment, bacterial transfer to pecans ranged from 0.7 ± 0.3 to 4.1 ± 0.2 for E. coli and 1.3 ± 0.7 to 4.3 ± 0.4 log CFU/g for Salmonella. For the medium inoculum treatment, transfer ranged from <0.3 to 1.5 ± 0.1 for E. coli and <0.3 to 1.9 ± 0.2 log CFU/g for Salmonella. For the low treatment, transfer ranged from <0.3 to 0.4 ± 0.2 and <0.3 to 0.5 ± 0.1 log CFU/g for E. coli and Salmonella, respectively. These results show the need for implementing agricultural practices that prevent potential transfer of foodborne pathogens onto the surface of in-shell pecans during harvest.

Relationship of the Poultry Microbiome to Pathogen Colonization, Farm Management, Poultry Production, and Foodborne Illness Risk Assessment.

Despite the continuous progress in food science and technology, the global burden of foodborne illnesses remains substantial, with pathogens in food causing millions of infections each year. Traditional microbiological culture methods are inadequate in detecting the full spectrum of these microorganisms, highlighting the need for more comprehensive detection strategies. This review paper aims to elucidate the relationship between foodborne pathogen colonization and the composition of the poultry microbiome, and how this knowledge can be used for improved food safety. Our review highlights that the relationship between pathogen colonization varies across different sections of the poultry microbiome. Further, our review suggests that the microbiome profile of poultry litter, farm soil, and farm dust may serve as potential indicators of the farm environment's food safety issues. We also agree that the microbiome of processed chicken samples may reveal potential pathogen contamination and food quality issues. In addition, utilizing predictive modeling techniques on the collected microbiome data, we suggest establishing correlations between particular taxonomic groups and the colonization of pathogens, thus providing insights into food safety, and offering a comprehensive overview of the microbial community. In conclusion, this review underscores the potential of microbiome analysis as a powerful tool in food safety, pathogen detection, and risk assessment.

Influence of antibiotic-resistance and exudate on peroxyacetic acid tolerance in O157 and non-O157 Shiga toxin producing E. coli.

Shiga toxin producing Escherichia coli (STEC) continues to cause foodborne outbreaks associated with beef and beef products despite consistent use of antimicrobial interventions. In this study, the influence of antibiotic resistance (ABR) in E. coli O157:H7 H1730, O157:H7 43,895, O121:H19 and O26:H11 on tolerance to peroxyacetic acid (PAA) was evaluated. Further, bactericidal concentrations of PAA in the presence of nutrient rich media (Tryptic Soy Broth, TSB and beef exudates) and nutrient deficient media (Sterile Deionized Water, SDW and Phosphate Buffered Saline, PBS) were evaluated for all bacterial strains. Antibiotic resistance to ampicillin (amp C), or ampicillin and streptomycin (amp P strep C) was generated in each bacterial strain through incremental exposure to the antibiotics or by plasmid transformation (n = 12 total strains). The mean bactericidal concentrations of PAA were higher (p ≤ 0.05) in nutrient rich media (205.55 ± 31.11 in beef exudate and 195.83 ± 25.00 ppm in TSB) than in nutrient deficient media (57.91 ± 11.97 ppm in SDW and 56.66 ± 9.56 ppm in PBS). Strain O157: H7 ampP strepC was the most tolerant to PAA (p ≤ 0.05). At 200 ppm in nutrient rich media and 60 ppm in nutrient deficient media, all bacterial strains declined in population to below the limit of detection. Analysis of the beef exudates indicated the presence of diverse amino acids that have been associated with acid tolerance. The results from this study indicate that beef exudates could contribute to acid tolerance and suggest that some STEC bacterial strains with certain ABR profiles might be more tolerant to PAA.

Using E. coli population to predict foodborne pathogens in pastured poultry farms.

Escherichia coli shows the potential of indicating foodborne pathogens. The purpose of this study is to investigate the association between E. coli and foodborne pathogens such as Campylobacter, Salmonella, and Listeria in pastured poultry farms, as well as in related processing facilities. Five different sample types: (i) feces, (ii) soil, (iii) whole carcass rinse during processing (WCR-P), (iv) whole carcass rinse of final product after chilling and storage (WCR-F), and (v) ceca were measured for E. coli populations. A logistic regression model for pathogen presence was developed for each sample type. The E. coli population significantly increased the predicted probability of Salmonella presence for soil and WCR-P samples (p = 0.0011 and p = 0.0157 respectively). For Campylobacter, the initial prevalence in feces and ceca were high and a decreasing trend of detecting Campylobacter was observed as E. coli concentration increased. In soil and WCR-P models, the probability of the presence of Campylobacter significantly increased as E. coli population increased. These models provide a practical and effective way of evaluating the relationship between E. coli and foodborne pathogens and enable prediction of foodborne pathogen presence based on E. coli prevalence within the pastured poultry farm-to-fork continuum.

Inactivation of Escherichia coli in apple cider using atmospheric cold plasma.

Atmospheric cold plasma (ACP) is a promising non-thermal technology that has the potential to inactivate microorganisms in foods. In this work, the inactivation of E. coli K12, acid-adapted E. coli K12, and E. coli O157:H7 in apple cider by ACP was investigated using feed gases as simulated air (SA) (80 % N2 + 20 % O2) and a mixture of 90 % N2 + 10 % O2 with various processing times (0 to 180 s). We obtained the reduced the populations of both acid-adapted and non-adapted E. coli K12 by 5 log CFU/mL within 120 s, and E. coli O157:H7 within 90 s. Additionally, no significant changes in the °Brix, pH, temperature, or titratable acidity (TA) of apple cider were observed after exposure to ACP. However, processing times longer than 120 s resulted in significant changes in the pH values. The highest concentration of ozone and hydrogen peroxide reached to 0.22 ± 0.1 mg/L for CG in 180 s and 0.07 ± 0.01 mg/L for SA in 150 s, respectively. Both acid-adapted and non-acid adapted E. coli K12 was found to be more resistant to ACP processing than E. coli O157:H7 after the 90 s, so it could serve as a surrogate for E. coli O157:H7. When we compared the effect of the gas type on inactivation, non-selective media, the results showed no significant differences between the gas types, while selective media demonstrated significant differences. In optical absorption spectroscopy measurements of plasma species, primarily ozone peaks were observed. Furthermore, the optical absorption spectroscopy also revealed that the inactivation of the bacteria could be attributed to some plasma species with wavelengths between 190 and 308 nm. The findings provided a perspective on the use of ACP as a method for decontaminating fruit juices as a non-thermal processing.

Sunlight Parameters Influence the Survival and Decline of Salmonella and Escherichia coli in Water.

ABSTRACT: The effect of variations in temperature, UV radiation, and sunlight intensity on Escherichia coli, E. coli O157:H7, Salmonella Newport, and antibiotic resistant (ABR) variants of E. coli O157:H7 and Salmonella Newport exposed to sunlight was evaluated. Bacterial strains suspended in sterile deionized water at a concentration of 8 log CFU/mL were exposed to sunlight on three different days for 180 min; control treatments were stored in the dark. The mean temperature of 30.08 and 26.57°C on day 1 and day 3, respectively, was significantly different (P < 0.05). The UV intensity was significantly different on all 3 days, and sunlight intensity significantly differed on day 3 (P < 0.05). Bacterial population decline positively correlated with temperature, sunlight, and UV intensity. Differences in bacterial population declines differed among species, ABR profile, and day of exposure (P < 0.05). On day 1 and day 2, the populations of E. coli dropped below the limit of detection (1 log CFU/mL), whereas the percentage of live cells was 67 and 6.6%, respectively. The artificial neural network model developed to predict bacterial survival under different environmental conditions suggested that Salmonella cells were more resistant than E. coli cells. The ABR strains had significantly higher numbers of viable cells after sunlight exposure (P < 0.05). Sunlight-exposed cells resuscitated in tryptic soy broth varied in maximum population density and maximum specific growth rate based on bacterial species and presence of ABR. Morphological changes such as viable but nonculturable state transition and filament formation were detected in subpopulations of sunlight-exposed bacteria. Daily fluctuations in UV and sunlight intensity can result in significant variations in bacterial decline and recovery.

Predictive model for growth of Clostridium perfringens during cooling of cooked pork supplemented with sodium chloride and sodium pyrophosphate.

A dynamic model was developed to predict growth of Clostridium perfringens in cooked ground pork supplemented with salt (0-3% wt/wt) and sodium pyrophosphate (0-0.3% wt/wt) under varying temperatures. C. perfringens (NCTC 8238, NCTC 8239, and NCTC 10240) spores were heat shocked, cooled, and inoculated into ground pork. Isothermal bacterial growth was quantified with variable salt and phosphate concentrations at temperatures ranging from 15 to 51 °C. The primary Baranyi model was fitted to all C. perfringens growth profiles and gave a satisfactory fit (R2 ≥ 0.85). A quadratic polynomial secondary model was developed (P < 0.0001) to predict the maximum specific growth rate as a function of temperature, salt, and phosphate concentrations (R2 = 0.93). A dynamic model was developed and validated using growth data retrieved from 7 published studies. Thirty three out of 44 predictions were within the acceptable prediction zone (-0.5 ≤ prediction error ≤ 1.0). The developed predictive model can be used to minimize the risk of C. perfringens in pork products supplemented with additives during cooling.

Using farm management practices to predict Campylobacter prevalence in pastured poultry farms.

Contamination of poultry products by Campylobacter is often associated with farm management practices and processing plant practices. A longitudinal study was conducted on 11 pastured poultry farms in southeastern United States from 2014 to 2017. In this study, farm practices and processing variables were used as predictors for a random forest (RF) model to predict Campylobacter prevalence in pastured poultry farms and processing environments. Individual RF models were constructed for fecal, soil and whole carcass rinse after processing (WCR-P) samples. The performance of models was evaluated by the area under curve (AUC) from the receiver operating characteristics curve. The AUC values were 0.902, 0.894, and 0.864 for fecal, soil, and WCR-P models, respectively. Relative importance plots were generated to predict the most important variable in each RF model. Animal source of feces was identified as the most important variable in fecal model and the soy content of the brood feed was the most important variable for soil model. For WCR-P model, the average flock age showed the strongest impact on RF model. These RF models can help pastured poultry growers with food safety control strategies to reduce Campylobacter prevalence in pastured poultry farms.

Postharvest Reduction of Salmonella enterica on Tomatoes Using a Pelargonic Acid Emulsion.

A novel produce wash consisting of pelargonic acid (PEL) emulsions was tested on tomatoes contaminated with a five-serovar Salmonella enterica cocktail. Ability to reduce contamination on the inoculated tomato surface, as well as mitigation of subsequent cross-contamination to uninoculated tomatoes washed in re-used/spent wash water were examined. Sanitizer efficacy was also examined over 1 and 7 d storage time (8 °C, recommended for red ripe tomatoes) and in the presence of 0.5% (w/v) organic load. PEL performed statistically the same (p ≤ 0.05) at both 30 mM and 50 mM concentrations and resulted in greater than 1, 5 and 6 log CFU/g Salmonella reductions at 0 h, 1 d and 7 d, respectively, when compared to a water-only or no rinse (NR) treatment. This was also a significantly greater reduction than was observed due to chlorine (sodium hypochlorite) and peroxyacetic acid (PAA) at all time points (p ≤ 0.01). Organic load had no impact on sanitizer efficacy for all examined treatments. Finally, PEL had a deleterious impact on tomato texture. At 1 d, ca. 5 N and 7 N were required to achieve tomato skin penetration and compression, respectively, compared to >9 N and 15 N required by all other treatments (p ≤ 0.05). While PEL sanitizers effectively reduced inoculated Salmonella and subsequent transfer to uninoculated tomatoes, reformulation may be necessary to prevent deleterious quality impacts on produce.

Contamination of spinach at germination: A route to persistence and environmental reintroduction by Salmonella.

The effects of using contaminated seed and water on the persistence and internalization of Salmonella Newport in organic spinach cultivars- Lazio, Space, Emilia and Waitiki were studied. Seeds were contaminated by either immersing in a suspension of Salmonella and then sprouted or were sprouted in Salmonella contaminated water in the dark at 25 °C. After 5 days, germinated sprouts were analyzed for S. Newport population and internalization. Germinated sprouts were potted in soil and grown in a plant incubator for 4 weeks. Leaves, stems and roots were sampled for Salmonella population by plating on CHROMagar™. Plants surface-sterilized with chlorine were analyzed for internalized pathogen. Potting soil and water runoff were sampled for Salmonella after 4 weeks of plant growth. Contaminated seeds and irrigation water had S. Newport populations of 7.64±0.43 log CFU/g and 7.12±0.04 log CFU/ml, respectively. Sprouts germinated using contaminated water or seeds had S. Newport populations of 8.09±0.04 and 8.08±0.03 log CFU/g, respectively and had a Salmonella population that was significantly higher than other spinach tissues (P<0.05). Populations of S. Newport in leaves, stem and roots of spinach plants were as follows: contaminated seed- 2.82±1.69, 1.69±0.86, and 4.41±0.62 log CFU/ml; contaminated water- 3.56±0.90, 3.04±0.31, and 4.03±0.42 log CFU/ml of macerated tissue suspension, respectively. Internalization was observed in plants developing from contaminated seeds and in sprouts germinated using contaminated water. S. Newport populations of 2.82±0.70 log CFU/g and 1.76±0.46 log CFU/ml were recovered from soil and water runoff, respectively. The results indicate that contamination of spinach during germination can result in persistence, internalization and environmental reintroduction of Salmonella.

Microbial degradation, spectral analysis and toxicological assessment of malachite green by Streptomyces chrestomyceticus S20.

Malachite green (MG), a triphenylmethane dye is extensively used for coloring silk, aquaculture and textile industries, it has also has been reported toxic to life forms. This study aimed to investigate the biodegradation potential of MG by actinobacteria. The potent actinobacterial strain S20 used in this study was isolated from forest soil (Sabarimala, Kerala, India) and identified as Streptomyces chrestomyceticus based on phenotype and molecular features. Strain S20 degraded MG up to 59.65 ± 0.68% was studied in MSM medium and MG (300 mg l-1) and degradation was increased (90-99%) by additions of 1% glucose and yeast extract into the medium at pH 7. The treated metabolites from MG by S20 characterized by FT-IR and GC-MS. The results showed MG has been degraded into nontoxic compounds evaluated by (1) phytotoxic assay on Vigna radiata, (2) microbial toxicity on Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Streptococcus sp. and Escherichia coli, (3) cytotoxicity assay in a human cell line (MCF 7). The toxicity studies demonstrated that the byproducts from MG degradation by S. chrestomyceticus S20 were no toxic to plants and microbes and less toxic to human cells as compared to the parent MG. Perhaps this is the first work reported on biodegradation of MG by S. chrestomyceticus which could be a potential candidate for the removal of MG from various environments.

Modified Coring Tool Designs Reduce Iceberg Lettuce Cross-Contamination.

Contaminated coring tools may transfer bacteria to iceberg lettuce. The efficiency of coring tool design modifications in reducing bacterial transfer to lettuce heads was evaluated under simulated field operations. The standard coring tool consists of a stainless steel cylindrical tube welded to a tab that is inserted into a plastic handle. Design modifications included removal of the welded portion, incorporation of a shorter front straight bottom edge, or an angled bottom edge toward the front. In the first study, coring tools of four different designs were inoculated by dipping in a tryptic soy broth (TSB) suspension that contained 8.85 Log CFU/mL of Escherichia coli K-12 and then were used to core 100 lettuce heads, consecutively. Use of the standard tool resulted in 91% ± 9% positive lettuce heads. Removing the welded surface from the standard tool resulted in the highest reduction of E. coli transfer (44% ± 11.9% positive lettuce heads, P < 0.05), whereas incorporation of a short front straight edge with no welding resulted in 65.6% ± 5.6% of the cored lettuce heads being positive for E. coli. Removal of the welded surface resulted in a 40% decrease in E. coli contamination among the last 20 cored lettuce heads (81 to 100), which indicates that coring tool design modifications resulted in reduced cross-contamination. In the second study, the transfer of Salmonella to coring tools after their immersion in rinsing solutions was evaluated using imaging. The tools were dip inoculated for 2 min in water, water with lettuce extract, or TSB containing 7 Log CFU/mL bioluminescent Salmonella Newport; they were then imaged to observe spatial distribution of bacteria. There was greater retention and spatial distribution of Salmonella on the surface of tools immersed in water containing lettuce extract than in TSB and water. The results of the second study indicate that rinsing solutions that contain lettuce particulate and organic load could facilitate cross-contamination of Salmonella Newport to tool surfaces.

Airborne soil particulates as vehicles for Salmonella contamination of tomatoes.

The presence of dust is ubiquitous in the produce growing environment and its deposition on edible crops could occur. The potential of wind-distributed soil particulate to serve as a vehicle for S. Newport transfer to tomato blossoms and consequently, to fruits, was explored. Blossoms were challenged with previously autoclaved soil containing S. Newport (9.39log CFU/g) by brushing and airborne transfer. One hundred percent of blossoms brushed with S. Newport-contaminated soil tested positive for presence of the pathogen one week after contact (P<0.0001). Compressed air was used to simulate wind currents and direct soil particulates towards blossoms. Airborne soil particulates resulted in contamination of 29% of the blossoms with S. Newport one week after contact. Biophotonic imaging of blossoms post-contact with bioluminescent S. Newport-contaminated airborne soil particulates revealed transfer of the pathogen on petal, stamen and pedicel structures. Both fruits and calyxes that developed from blossoms contaminated with airborne soil particulates were positive for presence of S. Newport in both fruit (66.6%) and calyx (77.7%). Presence of S. Newport in surface-sterilized fruit and calyx tissue tested indicated internalization of the pathogen. These results show that airborne soil particulates could serve as a vehicle for Salmonella. Hence, Salmonella contaminated dust and soil particulate dispersion could contribute to pathogen contamination of fruit, indicating an omnipresent yet relatively unexplored contamination route.