Inactivation of Escherichia coli O157:H7 and Salmonella during washing of contaminated gloves in levulinic acid and sodium dodecyl sulfate solutions.

Field workers often wear gloves harvesting ready-to-eat produce; however, fields are not sterile environments and gloves may become contaminated numerous times during a working shift. This study explored the potential for inactivation of Escherichia coli O157:H7 and Salmonella when contaminated gloves were washed in levulinic acid (LV) and sodium dodecyl sulfate (SDS) solutions. Washing nitrile gloves with increasing concentrations of LV above 1.0% led to a decreased prevalence of glove contamination by Salmonella (P = 0.0000). A higher level of prevalence occurred for solid agar-cultured pathogens than liquid broth-cultured pathogens after nitrile gloves were washed in LV/SDS (P = 0.0000). Pathogens residing on latex gloves were more likely to be completely inactivated by washing in 0.5% LV/0.1% SDS solutions than nitrile or Canners gloves that exhibited inconsistent responses dependent on the pathogen strain. However, drying after washing nitrile gloves in 0.5% LV/0.1% SDS led to additional pathogen inactivation (P = 0.0394). Pathogen transfer from gloves to produce was implied as the pathogen prevalence on cantaloupe rind handled by LV/SDS-washed gloves was not statistically different from the prevalence on gloves (P = 0.7141). Hence, the risk of produce contamination may still exist but would be reduced by washing gloves in LV/SDS.

Ammonia Oxidizers in a Grazing Land with a History of Poultry Litter Application.

Poultry litter (PL) is widely applied on grazing lands in Georgia. However, it is not clear how its long-term use affects soil microorganisms and their function. We examined changes in activity and community structure of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a grazing land with a history of PL application and compared it to treatment with urea ammonium nitrate (UAN). Soil samples (0-15 cm) were collected in 2009 (after 15 yr of PL application) and in 2013 (after 2 yr of no application). The abundance and community composition of ammonia oxidizers (AO) were determined with molecular techniques that targeted Nitrification potential (NP) was used for measuring their activity. Abundance of AO was significantly higher in PL (7.41 and 7.10 log copies g soil for AOB and AOA, respectively) than in UAN plots (6.82 and 6.50 log copies g soil for AOB and AOA, respectively) in 2009. This is consistent with NP, which was higher in PL (0.78 mg NO -N kg h) than in UAN (0.50 mg NO-N kg h) plots in 2009. The abundance of AO and NP decreased in 2013. There was no treatment effect on the composition of AO. Correlation analysis suggested that AOB were functionally more important than AOA, indicating the need to target AOB for efficient management of N in PL-receiving soils. Overall, the difference in nitrification between PL and UAN was mainly caused by the change in AO abundance rather than composition, and AO were not negatively affected by an increase in PL-derived trace metal concentrations.

Modeling the Effects of Onsite Wastewater Treatment Systems on Nitrate Loads Using SWAT in an Urban Watershed of Metropolitan Atlanta.

Onsite wastewater treatment systems (OWTSs) can be a source of nitrogen (N) pollution in both surface and ground waters. In metropolitan Atlanta, GA, >26% of homes are on OWTSs. In a previous article, we used the Soil Water Assessment Tool to model the effect of OWTSs on stream flow in the Big Haynes Creek Watershed in metropolitan Atlanta. The objective of this study was to estimate the effect of OWTSs, including failing systems, on nitrate as N (NO-N) load in the same watershed. Big Haynes Creek has a drainage area of 44 km with mainly urban land use (67%), and most of the homes use OWTSs. A USGS gauge station where stream flow was measured daily and NO-N concentrations were measured monthly was used as the outlet. The model was simulated for 12 yr. Overall, the model showed satisfactory daily stream flow and NO-N loads with Nash-Sutcliffe coefficients of 0.62 and 0.58 for the calibration period and 0.67 and 0.33 for the validation period at the outlet of the Big Haynes Watershed. Onsite wastewater treatment systems caused an average increase in NO-N load of 23% at the watershed scale and 29% at the outlet of a subbasin with the highest density of OWTSs. Failing OWTSs were estimated to be 1% of the total systems and did not have a large impact on stream flow or NO-N load. The NO-N load was 74% of the total N load in the watershed, indicating the important effect of OWTSs on stream loads in this urban watershed.

Confirmation of the Impact of Onsite Wastewater Treatment Systems on Stream Base-Flow Nitrogen Concentrations in Urban Watersheds of Metropolitan Atlanta, GA.

Wastewater and lawn fertilizer potentially contribute to degraded water quality in urban watersheds. Previously we described a study from 2011 to 2012 in which we examined the effect of the density of onsite wastewater treatment systems (OWTS) on nitrogen concentrations in 24 small streams in metropolitan Atlanta. Our objective in this study was to confirm that the impact on water quality that we observed was due to OWTS and not lawn fertilizer. We sampled the same 24 streams again in 2013 and 2014, representing watersheds ranging in area from 0.18 to 8.8 km. We conducted regression analysis of the effect of OWTS and season, used dual-isotope analysis (nitrogen and oxygen in nitrate) to identify sources and determine the effect of denitrification and mixing, and conducted stream walks to identify areas where animals had access to the streams. Twelve streams were characterized as high-density (HD, more than 75 systems km) OWTS and 12 as low-density (LD, less than 75 systems km) OWTS. Water samples were collected three times a year under base-flow conditions, from November 2011 to July 2014, and analyzed for nitrate (NO-N), ammonium (NH-N), and total Kjeldahl nitrogen. Total nitrogen and NO-N concentrations increased linearly with increasing OWTS density above a threshold of about 75 OWTS km. Dual-isotope analysis of NO showed that stream NO originated predominantly from OWTS in HD watersheds and from a combination of animal waste and perhaps organic N in LD watersheds. Stream walks showed that livestock had access to some of the LD streams with high N concentrations. Our results confirm that HD OWTS can significantly degrade water quality at the watershed scale.

Prevalence and concentration of Salmonella and Campylobacter in the processing environment of small-scale pastured broiler farms.

A growing niche in the locally grown food movement is the small-scale production of broiler chickens using the pasture-raised poultry production model. Limited research exists that focuses on Salmonella and Campylobacter contamination in the environment associated with on-farm processing of pasture-raised broilers. The objective of this study was to establish data relative to Salmonella and Campylobacter prevalence and concentration in soil and mortality compost resulting from prior processing waste disposal in the small-scale, on-farm broiler processing environment. Salmonella and Campylobacter concentrations were determined in soil (n = 42), compost (n = 39), and processing wastewater (PWW; n = 46) samples from 4 small broiler farms using a 3-tube most probable number (MPN) method for Salmonella and direct plating method for Campylobacter. Salmonella prevalence and concentration (mean log10 MPN per sample weight or volume) in soil [60%, 0.97 (95% CI: 0.66 to 1.27)], compost [64%, 0.95 (95% CI: 0.66 to 1.24)], and wastewater [48%, 1.29 (95% CI: 0.87 to 1.71)] were not significantly different (P > 0.05). Although Campylobacter prevalence was not significantly different by sample type (64.3, 64.3, and 45.7% in soil, compost, and PWW, respectively), the concentration (mean log10 cfu) of this pathogen was significantly lower (P < 0.05) in wastewater [2.19 (95% CI: 0.36 to 3.03)] samples compared with soil [3.08 (95% CI: 2.23 to 3.94)], and compost [3.83 (95% CI: 2.71 to 4.95)]. These data provide insight into small-scale poultry production waste disposal practices and provides a record of data that may serve as a guide for future improvement of these practices. Further research is needed regarding the small-scale broiler production environment in relation to improving disposal of processing waste for optimum control of human pathogens.

Nutrient Sufficiency Ranges for Corn at the Early Growth Stage: Implications for Nutrient Management.

Growers rely on nutrient sufficiency ranges (NSRs) after plant tissue analysis to inform timely nutrient management decisions. The NSRs are typically established from survey studies across multiple locations, which could be confounded by several abiotic and biotic factors. We conducted field studies in 2020, 2021, and 2022 to validate the lower thresholds of the NSRs for corn (Zea mays) at the early growth stage as reported in the Southern Cooperative Series Bulletin #394. We induced various corn nutritional levels by making different nutrient application rates. If the NSRs are valid, samples within the same replication that satisfy the NSRs of all nutrients should have similar biomass accumulation. The results showed that the NSRs were not valid under the conditions tested. In total, 47.6% of the samples satisfied all the lower thresholds of the NSRs, and 25.4% of those samples had relative biomass <50%, with relative biomass even as low as 24.2% observed. Moreover, 9.6% of the total samples had P and Cu levels that failed to meet the lower threshold but still had relative biomass ≥75%. The findings highlight the sensitivity of corn to nutrient imbalance and the need to optimize nutrient diagnostic methods at the early growth stage.

Understanding the role of agricultural practices in the potential colonization and contamination by Escherichia coli in the rhizospheres of fresh produce.

To better protect consumers from exposure to produce contaminated with Escherichia coli, the potential transfer of E. coli from manure or irrigation water to plants must be better understood. We used E. coli strains expressing bioluminescence (E. coli O157:H7 lux) or multiantibiotic resistance (E. coli²(+)) in this study. These marked strains enabled us to visualize in situ rhizosphere colonization and metabolic activity and to track the occurrence and survival of E. coli in soil, rhizosphere, and phyllosphere. When radish and lettuce seeds were treated with E. coli O157:H7 lux and grown in an agar-based growth system, rapid bacterial colonization of the germinating seedlings and high levels of microbial activity were seen. Introduction of E. coli²(+) to soil via manure or via manure in irrigation water showed that E. coli could establish itself in the lettuce rhizosphere. Regardless of introduction method, 15 days subsequent to its establishment in the rhizosphere, E. coli²(+) was detected on the phyllosphere of lettuce at an average number of 2.5 log CFU/g. When E. coli²(+) was introduced 17 and 32 days postseeding to untreated soil (rather than the plant surface) via irrigation, it was detected at low levels (1.4 log CFU/g) on the lettuce phyllosphere 10 days later. While E. coli²(+) persisted in the bulk and rhizosphere soil throughout the study period (day 41), it was not detected on the external portions of the phyllosphere after 27 days. Overall, we find that E. coli is mobile in the plant system and responds to the rhizosphere like other bacteria.

Evaluating the impact of turf-care products on soil biological health.

Golf courses require extensive use of inputs to meet the needs of playability and aesthetics. The impact of these inputs on soil biological health is largely unknown. Two field trials were conducted at a golf course in Georgia to evaluate short-term effects of wetting agents (Cascade Plus and Duplex [C+D], Revolution [Rev]), plant growth regulators (PrimoMaxx [PM] and Cutless [CL]), and a product called PlantHelper (PH) on soil biological health by measuring microbial abundance and function. Quantitative polymerase chain reaction was used to measure microbial abundance, which included total bacteria, total fungi, and ammonia-oxidizing prokaryotes. Soil respiration and enzyme assays were used as additional indicators of soil health. In bentgrass putting green, total bacteria and ammonia-oxidizing bacteria decreased in abundance in response to the wetting agents and PH, indicating their sensitivity to the products. Whereas C+D stimulated urease activity, Rev and PH caused a short-lived but immediate increase in respiration, indicating that they acted as labile carbon sources. In a bermudagrass fairway, PM was the only product that caused an increase in total bacteria abundance. PrimoMaxx and CL caused a delayed increase in respiration, suggesting that they may have affected the microorganisms indirectly through their impact on root growth and exudate production later. Although CL caused a decrease in urease activity, none of the products significantly affected phosphatase activity. Overall, the products did not seem to have a lasting impact on soil biological health, although long-term studies are needed to confirm these observations.

Subcellular distribution and biotransformation of phenanthrene in pakchoi after inoculation with endophytic Pseudomonas sp. as probed using HRMS coupled with isotope-labeling.

Contamination of food-crops with polycyclic aromatic hydrocarbons (PAHs) poses a grave concern to food safety, especially when PAHs are internalized. We have demonstrated in our previous study that inoculation of crop with a phenanthrene-degrading endophytic Pseudomonas sp. Ph6-gfp could overcome this problem. Here, the subcellular distribution and biotransformation mechanism of phenanthrene in pakchoi (Brassica chinensis L.) seedlings with inoculation of Ph6-gfp were further investigated both in vitro and in vivo. The possible biotransformation products of phenanthrene were identified by high-resolution mass spectrometry (HRMS) coupled with 13C2-phenanthrene labeling. Results indicated that Ph6-gfp colonized pakchoi interior and reduced the content of phenanthrene in different cell compartments. Notably, the inoculation hindered the subcellular distribution of phenanthrene from intercellular space to subcellular fractions (i.e., cell wall, cell membrane, cell solution, and cell organelles), likely resulting from the interception and biodegradation of phenanthrene by the bacterium between the cell wall and intercellular space. Additionally, the conjugation reactions of phenanthrene-metabolites and endogenous plant compounds were enhanced as a result of the inoculation. We propose that endophytic degradation, plant metabolism, and conjugation reaction are the three possible biotransformation mechanisms that could account for the changes in phenanthrene inside the plant cell compartments. This is the first observation of endophytic bacteria (EB)-enhanced biotransformation and conjugation of phenanthrene in pakchoi at the subcellular level, which drive novel insights in regulating food-crop contamination with endophytes in PAH-contaminated matrices.

Inactivation of Escherichia coli O157:H7 and Salmonella deposited on gloves in a liquid state and subjected to drying conditions.

Gloves are worn by workers harvesting ready-to-eat produce as a deterrent for contaminating the produce with enteric pathogens that may reside on their hands. As fields are not sterile environments, the probability for gloves to become contaminated still exists and therefore it is critical to understand the conditions that affect the survival of pathogens on gloves. Both Escherichia coli O157:H7 and Salmonella deposited on glove surfaces in a liquid state survived longer when the pathogen had been suspended in lettuce sap than when suspended in water. Despite this protection, pathogens deposited on clean single-use gloves were more likely to survive during drying than pathogens deposited on dirty gloves (a film of lettuce sap had been applied to the surface prior to pathogen application and soil had been ground into the gloves). Survival of both E. coli O157:H7 and Salmonella was biphasic with the greatest losses occurring during the first hour of drying followed by much slower losses in the ensuing hours. Pathogens grown in rich media (tryptic soy broth) versus minimal media (M9) as well as those cultured on solid agar versus liquid broth were also more likely to be resistant to desiccation when deposited onto gloves. Although survival of E. coli O157:H7 on nitrile gloves was in general greater than it was on latex gloves, the relative survival of Salmonella on the two glove types was inconsistent. Due to these inconsistencies, no one glove type is considered better than another in reducing the risk for contamination with enteric pathogens. In addition, the extended survival of what are generally referred to as stress-resistant pathogens suggests that gloves either be changed frequently during the day or washed in a disinfectant to reduce the risk of glove contamination that could otherwise contaminate product handled with the contaminated gloves.

Isolating the impact of septic systems on fecal pollution in streams of suburban watersheds in Georgia, United States.

The presence of multiple sources of fecal pollution at the watershed level presents challenges to efforts aimed at identifying the influence of septic systems. In this study multiple approaches including targeted sampling and monitoring of host-specific Bacteroidales markers were used to identify the impact of septic systems on microbial water quality. Twenty four watersheds with septic density ranging from 8 to 373 septic units/km2 were monitored for water quality under baseflow conditions over a 3-year period. The levels of the human-associated HF183 marker, as well as total and ruminant Bacteroidales, were quantified using quantitative polymerase chain reaction. Human-associated Bacteroidales yield was significantly higher in high density watersheds compared to low density areas and was negatively correlated (r = -0.64) with the average distance of septic systems to streams in the spring season. The human marker was also positively correlated with the total Bacteroidales marker, suggesting that the human source input was a significant contributor to total fecal pollution in the study area. Multivariable regression analysis indicates that septic systems, along with forest cover, impervious area and specific conductance could explain up to 74% of the variation in human fecal pollution in the spring season. The results suggest septic system impact through contributions to groundwater recharge during baseflow or failing septic system input, especially in areas with >87 septic units/km2. This study supports the use of microbial source tracking approaches along with traditional fecal indicator bacteria monitoring and land use characterization in a tiered approach to isolate the influence of septic systems on water quality in mixed-use watersheds.

Ammonia-oxidizer communities in an agricultural soil treated with contrasting nitrogen sources.

The community of ammonia-oxidizing prokaryotes was examined in an agricultural soil treated for six seasons with contrasting nitrogen (N) sources. Molecular tools based on the genes encoding ammonia monooxygenase were used to characterize the ammonia oxidizer (AO) communities and their abundance. Soil DNA was extracted from soils sampled from silage corn plots that received no additional N (control), dairy waste compost, liquid dairy waste (LW), and ammonium sulfate (AS) treatments at approximately 100 and 200 kg available N ha(-1) over 6 years. The N treatment affected the quantity of AO based on estimates of amoA by real-time PCR. Ammonia oxidizing bacteria (AOB) were higher in soils from the AS200, AS100, and LW200 treatments (2.5 × 10(7), 2.5 × 10(7), and 2.1 × 10(7)copies g(-1) soil, respectively) than in the control (8.1 × 10(6) copies g(-1) soil) while the abundance of amoA encoding archaea [ammonia oxidizing archaea (AOA)] was not significantly affected by treatment (3.8 × 10(7) copies g(-1) soil, average). The ratio of AOA/AOB was higher in the control and compost treated soils, both treatments have the majority of their ammonium supplied through mineralization of organic nitrogen. Clone libraries of partial amoA sequences indicated AOB related to Nitrosospira multiformis and AOA related to uncultured Nitrososphaera similar to those described by soil fosmid 54d9 were prevalent. Profiles of the amoC-amoA intergenic region indicated that both Nitrosospira- and Nitrosomonas-type AOB were present in all soils examined. In contrast to the intergenic amoC-amoA profile results, Nitrosomonas-like clones were recovered only in the LW200 treated soil-DNA. The impact of 6 years of contrasting nitrogen sources applications caused changes in AO abundance while the community composition remained relatively stable for both AOB and AOA.

Degradation of multiwall carbon nanotubes by bacteria.

Understanding the environmental transformation of multiwall carbon nanotubes (MWCNTs) is important to their life cycle assessment and potential environmental impacts. We report that a bacterial community is capable of degrading (14)C-labeled MWCNTs into (14)CO2 in the presence of an external carbon source via co-metabolism. Multiple intermediate products were detected, and genotypic characterization revealed three possible microbial degraders: Burkholderia kururiensis, Delftia acidovorans, and Stenotrophomonas maltophilia. This result suggests that microbe/MWCNTs interaction may impact the long-term fate of MWCNTs.

Leaching of Mycobacterium avium Subsp paratuberculosis in Soil under In Vitro Conditions.

Mycobacterium avium subsp paratuberculosis (Map), the causative agent of Johne's disease, has a robust ability to survive in the environment. However, the ability of Map to migrate through soil to drainage tiles or ground water, leave the farm, and leak into local watersheds is inadequately documented. In order to assess the ability of Map to leach through soil, two laboratory experiments were conducted. In the first study, 8 columns (30 cm long each) of a sandy loam soil were treated with pure cultures of Map. Two soil moisture levels and two Map concentrations were used. The columns were leached with 500 mL of water once a week for three weeks, the leachate was collected, and detection analysis was conducted. In the second experiment, manure from Map negative cows (control) and Map high shedder cows (treatment) were deposited on 8 similar columns and the columns were leached with 500 mL of water once a week for four weeks. Map detection and numeration in leachate samples were done with RT-PCR and culture techniques, respectively. Using RT-PCR, Map could be detected in the leachates in both experiments for several weeks but could only be recovered using culture techniques in experiment one. Combined, these experiments indicate the potential for Map to move through soil as a result of rainfall or irrigation following application.

Nitrification exhibits Haldane kinetics in an agricultural soil treated with ammonium sulfate or dairy-waste compost.

An agricultural soil was treated with dairy-waste compost, ammonium-sulfate fertilizer or no added nitrogen (control) and planted to silage corn for 6 years. The kinetics of nitrification were determined in laboratory-shaken slurry assays with a range of substrate concentrations (0-20 mM NH(4)(+)) over a 24-h period for soils from the three treatments. Determined concentrations of substrate and product were fit to Michaelis-Menten and Haldane models. For all the treatments, the Haldane model was a better fit, suggesting that significant nitrification inhibition may occur in soils under high ammonium conditions similar to those found immediately after fertilization or waste applications. The maximum rate of nitrification (V(max)) was significantly higher for the fertilized and compost-treated soils (1.74 and 1.50 mmol N kg(-1) soil day(-1)) vs. control soil (0.98 mmol kg(-1) soil day(-1)). The K(m) and K(i) values were not significantly different, with average values of 0.02 and 27 mM NH(4)(+), respectively. Our results suggest that both N sources increased nitrifier community size, but did not shift the nitrifier community structure in ways that influenced enzyme affinity or sensitivity to ammonium. The K(m) values are comparable to those determined directly in other soils, but are substantially lower than those from most pure cultures of ammonia-oxidizing bacteria.