Survival of Salmonella enterica serovar Newport in manure and manure-amended soils

Salmonella enterica serovar Newport has undergone a rapid epidemic spread in dairy cattle. This provides an efficient mechanism for pathogen amplification and dissemination into the environment through manure spreading on agricultural land. The objective of this study was to determine the survival characteristics of Salmonella serovar Newport in manure and manure-amended soils where the pathogen may be amplified. A multidrug-resistant (MDR) Salmonella serovar Newport strain and a drug-susceptible (DS) strain, both bovine isolates, were inoculated into dairy manure that was incubated under constant temperature and moisture conditions alone or after being mixed with sterilized or nonsterilized soil. Salmonella serovar Newport concentrations increased by up to 400% in the first 1 to 3 days following inoculation, and a trend of steady decline followed. With manure treatment, a sharp decline in cell concentration occurred after day 35, possibly due to microbial antagonism. For all treatments, decreases in Salmonella serovar Newport concentrations over time fit a first-order kinetic model. Log reduction time was 14 to 32 days for 1 log(10), 28 to 64 days for 2 log(10), and 42 to 96 days for 3 log(10) declines in the organisms' populations from initially inoculated concentrations. Most-probable-number monitoring data indicated that the organisms persisted for 184, 332, and 405 days in manure, manure-amended nonsterilized soil, and manure-amended sterilized soil, respectively. The MDR strain and the DS strain had similar survival patterns.     

Fate of Salmonella enterica Serovar Typhimurium on Carrots and Radishes Grown in Fields Treated with Contaminated Manure Composts or Irrigation Water

Three different types of compost, PM-5 (poultry manure compost), 338 (dairy cattle manure compost), and NVIRO-4 (alkaline-pH-stabilized dairy cattle manure compost), and irrigation water were inoculated with an avirulent strain of Salmonella enterica serovar Typhimurium at 10⁷ CFU g⁻¹ and 10⁵ CFU ml⁻¹, respectively, to determine the persistence of salmonellae in soils containing these composts, in irrigation water, and also on carrots and radishes grown in these contaminated soils. A split-plot block design plan was used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but with contaminated water applied) and five replicates for a total of 25 plots for each crop, with each plot measuring 1.8 × 4.6 m. Salmonellae persisted for an extended period of time, with the bacteria surviving in soil samples for 203 to 231 days, and were detected after seeds were sown for 84 and 203 days on radishes and carrots, respectively. Salmonella survival was greatest in soil amended with poultry compost and least in soil containing alkaline-pH-stabilized dairy cattle manure compost. Survival profiles of Salmonella on vegetables and soil samples contaminated by irrigation water were similar to those observed when contamination occurred through compost. Hence, both contaminated manure compost and irrigation water can play an important role in contaminating soil and root vegetables with salmonellae for several months.     

Identification of Specific Gene Sequences Conserved in Contemporary Epidemic Strains of Salmonella enterica

Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.     

In Situ Evaluation of Paenibacillus alvei in Reducing Carriage of Salmonella enterica Serovar Newport on Whole Tomato Plants

Recently, tomatoes have been implicated as a primary vehicle in food-borne outbreaks of Salmonella enterica serovar Newport and other Salmonella serovars. Long-term intervention measures to reduce Salmonella prevalence on tomatoes remain elusive for growing and postharvest environments. A naturally occurring bacterium identified by 16S rRNA gene sequencing as Paenibacillus alvei was isolated epiphytically from plants native to the Virginia Eastern Shore tomato-growing region. After initial antimicrobial activity screening against Salmonella and 10 other bacterial pathogens associated with the human food supply, strain TS-15 was further used to challenge an attenuated strain of S. Newport on inoculated fruits, leaves, and blossoms of tomato plants in an insect-screened high tunnel with a split-plot design. Survival of Salmonella after inoculation was measured for groups with and those without the antagonist at days 0, 1, 2, and 3 and either day 5 for blossoms or day 6 for fruits and leaves. Strain TS-15 exhibited broad-range antimicrobial activity against both major food-borne pathogens and major bacterial phytopathogens of tomato. After P. alvei strain TS-15 was applied onto the fruits, leaves, and blossoms of tomato plants, the concentration of S. Newport declined significantly (P ≤ 0.05) compared with controls. Astonishingly, >90% of the plants had no detectable levels of Salmonella by day 5 for blossoms. The naturally occurring antagonist strain TS-15 is highly effective in reducing the carriage of Salmonella Newport on whole tomato plants. The application of P. alvei strain TS-15 is a promising approach for reducing the risk of Salmonella contamination during tomato production.     

Differences in Growth of Salmonella enterica and Escherichia coli O157:H7 on Alfalfa Sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log₁₀ on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log₁₀. The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

Evaluation of the Potential Antimicrobial Resistance Transfer from a Multi-Drug Resistant Escherichia coli to Salmonella in Dairy Calves

Previous research conducted in our laboratory found a significant prevalence of multi-drug resistant (MDR) Salmonella and MDR Escherichia coli (MDR EC) in dairy calves and suggests that the MDR EC population may be an important reservoir for resistance elements that could potentially transfer to Salmonella. Therefore, the objective of the current research was to determine if resistance transfers from MDR EC to susceptible strains of inoculated Salmonella. The experiment utilized Holstein calves (approximately 3 weeks old) naturally colonized with MDR EC and fecal culture negative for Salmonella. Fecal samples were collected for culture of Salmonella and MDR EC throughout the experiment following experimental inoculation with the susceptible Salmonella strains. Results initially suggested that resistance did transfer from the MDR E. coli to the inoculated strains of Salmonella, with these stains demonstrating resistance to multiple antibiotics following in vivo exposure to MDR EC. However, serogrouping and serotyping results from a portion of the Salmonella isolates recovered from the calves post-challenge, identified two new strains of Salmonella; therefore transfer of resistance was not demonstrated under these experimental conditions.     

Escape of Steinernema feltiae from Alginate Capsules Containing Tomato Seeds

The entomogenous nematode Steinernema feltiae was encapsulated in an alginate matrix containing a tomato seed. When these capsules were placed on 0.8% agar for 7 days, the seed germinated and ca. 20% of the nematodes escaped from the capsules, whereas only 0.1% escaped from capsules without seeds. When capsules containing nematodes and a seed were planted into sterilized or nonsterilized soil, nematodes escaped to infect Galleria mellonella larvae. When seed in capsules containing ca. 274 nematodes per capsule were planted in nonsterilized soil, Galleria mortality was 90% 1 week later. Galleria mortality declined to 27%, 23%, and 0% in weeks 2, 4, and 8 postplant, respectively. In sterilized soil, Galleria mortality was 96% and did not differ significantly from the nonsterilized soil in week 1, but was significantly higher in sterilized soil over nonsterilized soil for week 2 (81%) and week 4 (51%). When capsules containing nematodes only were used, Galleria mortality was 71% in sterilized soil 1 week after planting and 58%, 33%, and 12% in weeks 2, 4, and 8 postplant, respectively. In nonsterilized soil, Galleria mortality was 8%, 30%, 21%, and 28% after 1, 2, 4, and 8 weeks, respectively, using only encapsulated nematodes. When the number of nematodes per capsule was increased to ca. 817, Galleria mortality was 92 % or higher in sterilized soil from week 1 to week 4.     

Steinernema feltiae (DD-136) and S. glaseri: Persistence in Soil and Bark Compost and Their Influence on Native Nematodes

Infective juveniles (J3) of the entomogenous nematodes Steinernema feltiae DD-136 (ca. 10,000 J3/100 ml) and S. glaseri (ca. 2,500 J3/100 ml) were incubated in steam-sterilized and nonsterilized sandy soil and bark compost for 8 weeks at 25 C. The nematodes were recovered by a two-step extraction procedure at 1-week intervals, and their infectivity to lepidopterous larvae (Spodoptera litura and Galleria mellonella) and their effect on the population and community of native nematodes in soil were determined. Survival of inoculated nematodes and mortality of insects were enhanced in sterilized media. Nonsterilized bark compost proved to be equally as suitable a medium as sterilized compost. In nonsterilized soil, the survival curve of S.feltiae declined more rapidly than that or S. glaseri which was less infective to insects despite its greater persistence even in nonsterilized soil. Soon after the addition of steinernematids to soil, the population of native nematodes showed a fluctuation with an increase in rhabditids and a decrease in other kinds of nematodes.     

Fate of Escherichia coli O157:H7 in Manure-Amended Soil

Escherichia coli O157:H7 cells survived for up to 77, >226, and 231 days in manure-amended autoclaved soil held at 5, 15, and 21°C, respectively. Pathogen populations declined more rapidly in manure-amended unautoclaved soil under the same conditions, likely due to antagonistic interactions with indigenous soil microorganisms. E. coli O157:H7 cells were inactivated more rapidly in both autoclaved and unautoclaved soils amended with manure at a ratio of 1 part manure to 10 parts soil at 15 and 21°C than in soil samples containing dilute amounts of manure. The manure-to-soil ratio, soil temperature, and indigenous microorganisms of the soil appear to be contributory factors to the pathogen's survival in manure-amended soil.     

Effects of Cattle Feeding Regimen and Soil Management Type on the Fate of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Manure, Manure-Amended Soil, and Lettuce

Survival of the green fluorescent protein-transformed human pathogens Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was studied in a laboratory-simulated lettuce production chain. Dairy cows were fed three different roughage types: high-digestible grass silage plus maize silage (6:4), low-digestible grass silage, and straw. Each was adjusted with supplemental concentrates to high and low crude protein levels. The pathogens were added to manure, which was subsequently mixed (after 56 and 28 days for E. coli O157:H7 and Salmonella serovar Typhimurium, respectively) with two pairs of organically and conventionally managed loamy and sandy soil. After another 14 days, iceberg lettuce seedlings were planted and then checked for pathogens after 21 days of growth. Survival data were fitted to a logistic decline function (exponential for E. coli O157:H7 in soil). Roughage type significantly influenced the rate of decline of E. coli O157:H7 in manure, with the fastest decline in manure from the pure straw diet and the slowest in manure from the diet of grass silage plus maize silage. Roughage type showed no effect on the rate of decline of Salmonella serovar Typhimurium, although decline was significantly faster in the manure derived from straw than in the manure from the diet of grass silage plus maize silage. The pH and fiber content of the manure were significant explanatory factors and were positively correlated with the rate of decline. With E. coli O157:H7 there was a trend of faster decline in organic than in conventional soils. No pathogens were detected in the edible lettuce parts. The results indicate that cattle diet and soil management are important factors with respect to the survival of human pathogens in the environment.     

Diversity and Antimicrobial Resistance of Salmonella enterica Isolates from Surface Water in Southeastern United States

A study of prevalence, diversity, and antimicrobial resistance of Salmonella enterica in surface water in the southeastern United States was conducted. A new scheme was developed for recovery of Salmonella from irrigation pond water and compared with the FDA''s Bacteriological Analytical Manual (8th ed., 2014) (BAM) method. Fifty-one isolates were recovered from 10 irrigation ponds in produce farms over a 2-year period; nine Salmonella serovars were identified by pulsed-field gel electrophoresis analysis, and the major serovar was Salmonella enterica serovar Newport (S. Newport, n = 29), followed by S. enterica serovar Enteritidis (n = 6), S. enterica serovar Muenchen (n = 4), S. enterica serovar Javiana (n = 3), S. enterica serovar Thompson (n = 2), and other serovars. It is noteworthy that the PulseNet patterns of some of the isolates were identical to those of the strains that were associated with the S. Thompson outbreaks in 2010, 2012, and 2013, S. Enteritidis outbreaks in 2011 and 2013, and an S. Javiana outbreak in 2012. Antimicrobial susceptibility testing confirmed 16 S. Newport isolates of the multidrug resistant-AmpC (MDR-AmpC) phenotype, which exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT), and to the 1st, 2nd, and 3rd generations of cephalosporins (cephalothin, amoxicillin-clavulanic acid, and ceftriaxone). Moreover, the S. Newport MDR-AmpC isolates had a PFGE pattern indistinguishable from the patterns of the isolates from clinical settings. These findings suggest that the irrigation water may be a potential source of contamination of Salmonella in fresh produce. The new Salmonella isolation scheme significantly increased recovery efficiency from 21.2 (36/170) to 29.4% (50/170) (P = 0.0002) and streamlined the turnaround time from 5 to 9 days with the BAM method to 4 days and thus may facilitate microbiological analysis of environmental water.     

Treatment with Ca(OH)2 for inactivation of Salmonella Typhimurium and Enterococcus faecalis in soil contaminated with infected horse manure

Aim: To investigate the inactivation of Salmonella enterica serovar Typhimurium and the faecal indicator Enterococcus faecalis in horse manure:soil mixtures by application of hydrated lime (Ca(OH)₂). Methods and Results: In laboratory incubations, the inhibitory effect of different concentrations of Ca(OH)₂, as well as different application techniques, was tested. Other variables were horse manure:soil ratio, incubation temperature (6 and 14°C) and soil type (sand/clay). Bacterial enumeration by the plate count method in samples taken at increasing intervals revealed that Ca(OH)₂ effectively reduced Salmonella Typhimurium numbers. However, to achieve a sufficient reduction, the Ca(OH)₂ had to be applied at a sufficient rate, and the amount required varied because of manure:soil ratio and incubation temperature. The results showed that a pH above 11 was needed and that a high pH had to be maintained for up to 7 days. An appropriate application technique for the Ca(OH)₂ was also important, so that a high pH was obtained throughout the whole material to be treated. In addition, a high manure:soil ratio in combination with a higher incubation temperature was found to rapidly neutralize the pH and to increase the risk of Salmonella re‐growth. Conclusions: Application of Ca(OH)₂ can be an efficient method for treating a Salmonella‐contaminated horse paddock. A high pH is a key factor in Salmonella inactivation, and thus, monitoring the pH during the treatment period is necessary. To avoid re‐growth excess manure should be removed for separate treatment elsewhere. Significance and Impact of the Study: Persistence of Salmonella in horse paddocks poses a risk of disease transmission to healthy animals and people who come into contact with these animals. An efficient method to de‐contaminate a Salmonella‐contaminated soil would be a valuable tool for animal welfare and for public health.     

Population Dynamics of Rhizobium leguminosarum Tn5 Mutants with Altered Cell Surface Properties Introduced into Sterile and Nonsterile Soils

The influence of cell surface properties on attachment to soil particles and on population dynamics of introduced bacteria was studied in sterilized and nonsterilized loamy sand and silt loam. Rhizobium leguminosarum RBL5523 and three Tn5 mutants (RBL5762, RBL5810, and RBL5811) with altered cell surface properties were used. Cellulose fibrils were not produced by RBL5762. Both RBL5810 and RBL5811 produced 80 to 90% less soluble exopolysaccharides and RBL5811 had, in addition, an altered lipopolysaccharide composition. In sterilized soil the total number of cells as well as the number of particle-associated cells of RBL5523 and RBL5810 were, in general, higher as compared with cell numbers of RBL5762 and RBL5811. Differences between strains in percentage of particle-associated cells in sterilized soil were only found at high inoculum densities, when populations increased little. In the nonsterilized silt loam, final population sizes, as well as numbers of particle-associated cells, of the parental strain (RBL5523) were higher than those of strains with altered cell surface properties after 56 and 112 days of incubation. But in general, differences in survival among the strains were not very marked. The importance of association with soil particles or aggregates for the survival of introduced cells was affirmed by the pronounced increase of the percentage of particle-associated cells during incubation in nonsterilized as well as sterilized soil. However, no clear relation among altered cell surface properties, particle association, and survival was found.     

Persistence of Mycobacterium avium subsp. paratuberculosis and Other Zoonotic Pathogens during Simulated Composting, Manure Packing, and Liquid Storage of Dairy Manure

Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis (Mycobacterium paratuberculosis). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated M. paratuberculosis, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55°C, manure packing at 25°C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with M. paratuberculosis at 10⁶ CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of M. paratuberculosis was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO₂, while no significant change in carbon content was observed in the liquid slurry. Escherichia coli, Salmonella, and Listeria were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55°C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days. M. paratuberculosis was detected by standard culture only on day 0 in all the treatments, but was undetectable in any treatment at 3 and 7 days. On days 14, 28, and 56, M. paratuberculosis was detected in the liquid storage treatment but remained undetectable in the compost and pack treatments. However, M. paratuberculosis DNA was detectable through day 56 in all treatments and up to day 175 in liquid storage treatments. Taken together, the results indicate that high-temperature composting is more effective than pack storage or liquid storage of manure in reducing these pathogens in dairy manure. Therefore, thermophilic composting is recommended for treatment of manures destined for pathogen-sensitive environments such as those for vegetable production, residential gardening, or application to rapidly draining fields.     

Use of Antibiotic Susceptibility Patterns and Pulsed-Field Gel Electrophoresis To Compare Historic and Contemporary Isolates of Multi-Drug-Resistant Salmonella enterica subsp. enterica Serovar Newport

Recently, multi-drug-resistant (MDR) Salmonella enterica subspecies enterica serovar Newport reemerged as a public and animal health problem. The antibiotic resistance of 198 isolates and the pulsed-field gel electrophoresis patterns (PFGE) of 139 isolates were determined. Serovar Newport isolates collected between 1988 and 2001 were included in the study. One hundred seventy-eight isolates were collected from the San Joaquin valley in California and came from dairy cattle clinical samples, human clinical samples, bulk tank milk samples, fecal samples from preweaned calves, and waterways. Twenty clinical isolates from humans from various regions of the United States were also included in the study. Resistance to 18 antibiotics was determined using a disk diffusion assay. PFGE patterns were determined using a single enzyme (XbaI). The PFGE and antibiogram patterns were described using cluster analysis. Although the antibiotic resistance patterns of historic (1988 to 1995) and contemporary (1999 to 2001) isolates were similar, the contemporary isolates differed from the historic isolates by being resistant to cephalosporins and florfenicol and in their general sensitivity to kanamycin and neomycin. With few exceptions, the contemporary isolates clustered together and were clearly separated from the historic isolates. One PFGE-antibiogram cluster combination was predominant for the recent isolates, which were taken from human samples from all parts of the United States, as well as in the isolates from California, indicating a rapid dissemination of this phenotypic strain. The data are consistent with the hypothesis that the reemergence of MDR serovar Newport is not simply an acquisition of further antibiotic resistance genes by the historic isolates but reflects a different genetic lineage.     

Salmonella enterica Serovar Typhimurium and Escherichia coli Contamination of Root and Leaf Vegetables Grown in Soils with Incorporated Bovine Manure

Bovine manure, with or without added Salmonella enterica serovar Typhimurium (three strains), was incorporated into silty clay loam (SCL) and loamy sand (LS) soil beds (53- by 114-cm surface area, 17.5 cm deep) and maintained in two controlled-environment chambers. The S. enterica serovar Typhimurium inoculum was 4 to 5 log CFU/g in manure-fertilized soil. The conditions in the two environmental chambers, each containing inoculated and uninoculated beds of manure-fertilized soil, simulated daily average Madison, Wis., weather conditions (hourly temperatures, rainfall, daylight, and humidity) for a 1 March or a 1 June manure application and subsequent vegetable growing seasons ending 9 August or 28 September, respectively. Core soil samples were taken biweekly from both inoculated and uninoculated soil beds in each chamber. Radishes, arugula, and carrots were planted in soil beds, thinned, and harvested. Soils, thinned vegetables, and harvested vegetables were analyzed for S. enterica serovar Typhimurium and Escherichia coli (indigenous in manure). After the 1 March manure application, S. enterica serovar Typhimurium was detected at low levels in both soils on 31 May, but not on vegetables planted 1 May and harvested 12 July from either soil. After the 1 June manure application, S. enterica serovar Typhimurium was detected in SCL soil on 7 September and on radishes and arugula planted in SCL soil on 15 August and harvested on 27 September. In LS soil, S. enterica serovar Typhimurium died at a similar rate (P ≥ 0.05) after the 1 June manure application and was less often detected on arugula and radishes harvested from this soil compared to the SCL soil. Pathogen levels on vegetables were decreased by washing. Manure application in cool (daily average maximum temperature of <10°C) spring conditions is recommended to ensure that harvested vegetables are not contaminated with S. enterica serovar Typhimurium. Manure application under warmer (daily average maximum temperature >20°C) summer conditions is not recommended when vegetable planting is done between the time of manure application and late summer. A late fall manure application will not increase the risk of contaminating vegetables planted the next spring, since further experiments showed that repeated freeze-thaw cycles were detrimental to the survival of S. enterica serovar Typhimurium and E. coli in manure-fertilized soil. The number of indigenous E. coli in soil was never significantly lower (P < 0.05) than that of S. enterica serovar Typhimurium, suggesting its usefulness as an indicator organism for evaluating the risk of vegetable contamination with manure-borne S. enterica serovar Typhimurium.     

Transport and Distribution of Salmonella enterica Serovar Typhimurium in Loamy and Sandy Soil Monoliths with Applied Liquid Manure

A leaching experiment, where liquid manure spiked with Salmonella enterica serovar Typhimurium (Tet⁺) DSM554 was applied to soil surfaces, was conducted on intact soil monoliths (60 cm in diameter and 100 cm long). A total of 6.5 × 10¹⁰ CFU was applied to each column. We found that Salmonella serovar Typhimurium could be transported to a 1-m depth in loamy soil at concentrations reaching 1.3 × 10⁵ CFU/ml of leachate. The test strain was found in concentrations ranging from 300 to 1.3⁵ cells/ml in loamy soil throughout the 27 days of the experiment, while concentrations below 20 cells/ml were sporadically detected in the leachates from sandy monoliths. Real-time PCR targeting invA DNA showed a clear correspondence between the total and culturable numbers of cells in the leachate, indicating that most cells leached were viable. On day 28, distribution of Salmonella serovar Typhimurium at five depths in the four monoliths was determined. The highest recovery rate, ranging from 1.5% to 3.8% of the total applied inoculum, was found in the top 0.2 m.The spreading of liquid manure on agricultural land is an economic and practical solution for improving soil quality. However, animal manure frequently contains zoonotic pathogenic bacteria such as certain Escherichia coli, Salmonella spp., and Campylobacter spp. (8, 9, 24). Salmonellosis is one of the most frequently reported food-borne diseases in Europe, accounting for 67.5% of reported food-borne outbreaks (32). Salmonella enterica serovar Typhimurium, which accounts for 23% of nonhuman isolates (from animal, food, feed, and environmental sources) and for 18% of human isolates, was the second-most-common serotype found worldwide from 2000 to 2004 (38). Human salmonellosis has been related to the consumption of water or foods contaminated with animal manure (9, 15), which frequently contains a variety of different enteric pathogenic microorganisms (27).Manure disposed of on agricultural land may create a risk for microbial contamination of surface water and groundwater. Field scale studies have reported manure bacteria in drainage water (5, 25, 36, 37). In addition to contamination originating from manure, pathogens in irrigation water may contaminate soil, water, crops, and, subsequently, animals and humans (7, 15). Disease outbreaks have been associated with water and food directly or indirectly contaminated with animal manure (9). Recent data from 1990 to 2004 showed that compared to beef, poultry, seafood, and eggs, fresh produce caused the second-highest number of food-borne disease outbreaks as well as the highest number of reported illnesses per outbreak (4).When microorganisms enter the soil environment, their survival and distribution is affected by various factors, including soil texture, pH, temperature, water saturation, and the intensity of rain events (17, 28). Preferential water movement in macropores is probably the primary route by which bacteria move down through the soil (1, 2, 23). Studies of bacterial transport have been made mostly on homogenized natural soils in laboratory soil columns (2, 18, 30, 33). The purpose of our study was to perform a large-scale experiment using intact 100-cm-deep soil monoliths to compare the leaching of Salmonella serovar Typhimurium in two structurally different soils: a loamy soil with high absorptive properties and macropores and a coarse sandy soil with less-absorptive properties and less preferential flow. The monoliths were initially exposed to normal precipitation events followed by intensive precipitation, where the leaching of Salmonella serovar Typhimurium cells was observed. After the leaching experiment, the concentrations of Salmonella serovar Typhimurium cells at five depths were determined. Furthermore, we validated bacterial enumerations based on cultures on agar plates with real-time PCR to determine if a proportion of the test strain entered a nonculturable state.     

Percolation and Survival of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Soil Amended with Contaminated Dairy Manure or Slurry

The effect of cattle manure and slurry application on percolation and survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was investigated for different soil depths after the addition of water. Four treatments were chosen for the first set of experiments: (i) addition of inoculated farmyard manure on the soil surface, (ii) mixing of inoculated farmyard manure with the top 10 cm of soil, (iii) addition of inoculated slurry on the soil surface, and (iv) injection of inoculated slurry into the top 10 cm of the soil. Homogeneity of water distribution in the soil profile was confirmed by a nondestructive nuclear magnetic resonance method. Survival data were fitted to a modified logistic model, and estimated survival times were compared. In the second set of experiments, pathogen-inoculated farmyard manure or slurry was applied to soil columns with 1-month-old lettuce plants. More pathogen cells percolated to greater depths after slurry than after manure application. Survival of E. coli O157:H7 was significantly longer in soil with slurry than in that with manure, while survival of Salmonella serovar Typhimurium was equally high with manure and slurry. The densities of the pathogens were not different in the rhizosphere compared to the bulk soil with manure, while the densities were higher by 0.88 ± 0.11 and 0.71 ± 0.23 log CFU per g (dry weight), respectively, in the rhizosphere than in bulk soil after slurry application. Our results suggest that surface application of manure may decrease the risk of contamination of groundwater and lettuce roots compared to injection of slurry.In the last 10 years food-borne disease outbreaks have increasingly been associated with the consumption of fresh vegetables and fruits contaminated with human pathogenic bacteria (3, 31). A significant number of the outbreaks were attributed to Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Bovine manure and slurry are the main environmental sources of these pathogens, with average concentrations between 10³ and 10⁴ CFU per g (dry weight) (gdw) of manure or slurry (24), but the density can be as high as 10⁷ CFU gdw⁻¹ of manure (10).Utilization of organic manures such as farmyard manure and slurry is the most economic and practical option for improving soil quality while providing as well an additional source of nutrients for growing plants. This is especially true for organic farms, where synthetic fertilizers cannot be used. Both organic and conventional soils can be fertilized with liquid slurry and/or farmyard manure. However, farmyard manure is more frequently used at organic farms.The survival of E. coli O157:H7 and Salmonella serovar Typhimurium is thought to be better in slurry than in farmyard manure (24; also A. V. Semenov, L. van Overbeek, N. Hidayah, A. J. Termorshuizen, and A. H. C. van Bruggen, submitted for publication) but is also dependent on the way manure or slurry is applied to agricultural fields (24). Survival of the pathogens may range from several days (turned composted manure) to more than a year (nonaerated manure) (9, 19). This broad difference in survival times is caused by various abiotic factors such as temperature (30, 38), presence of oxygen (A. V. Semenov, et al., submitted), and chemical composition (5) as well as by biological factors (e.g., microbial community composition) (5, 16, 30). The presence of plant roots is often neglected in controlled experiments although root exudates may support survival of human pathogens by providing a supply of easily available nutrients (18). Moreover, it has been shown that E. coli O157:H7 and Salmonella serovar Typhimurium may become associated with the surface of plants growing in soil amended with contaminated manure (15, 23) and may even be internalized by the plants (7, 18, 20, 32).When microorganisms are introduced on or in soil, their movement is mainly determined by the flow of percolating water (13). Water flow and the ultimate distribution of bacteria in soil are affected by soil texture, pH, temperature, and the structure of the root system in soil (17). Like other bacteria, E. coli O157:H7 and Salmonella serovar Typhimurium are able to move through the soil profile with water after rainfall or irrigation and can even reach the groundwater (2, 21). In field experiments, 20% of E. coli cells applied with contaminated slurry to the field were found in drain water (37). This water can contaminate plants when it is used for irrigation. Since E. coli O157:H7 can survive in well water up to 65 days (1), there is a high risk that private water supplies could be contaminated with enteric pathogens.Laboratory transport studies can mimic bacterial transport in field conditions only to a certain extent. The natural heterogeneity in field soil leads to the appearance of cracks and macropores through which water flow may occur while relatively homogeneous soil is commonly used in laboratory experiments. This may lead to underestimations of the movement of enteropathogens through the homogenized and possibly compacted soil. On the other hand, the presence of artificial boundaries (the so-called “wall effect”) and unexpected cracks may lead to overestimations of the movement of water and bacteria through the soil in mesocosms. The wall effect can be minimized by inserting sandpaper against the inner wall of soil columns while cracks can be minimized by careful packing of the soil. Nuclear magnetic resonance (NMR) can be used to check the homogeneity of water distribution in a soil column. NMR is a nondestructive and noninvasive spectroscopic method to measure static and dynamic water behavior in heterogeneous substrates (35). The data received from magnetic resonance images can give information about the spin density and spin relaxation values that reflect the interaction of water with the soil. These measurements have been proven to be highly correlated with water content in soils (35).While it was shown that water is the most important dispersal factor for percolation of bacteria in different types of soil (13, 36) as well as for percolation of enteropathogens under various management practices (11), the movement and distribution of E. coli O157:H7 and Salmonella serovar Typhimurium in soil after application of manure and slurry are still unclear. It is also not clear if and how survival of enteric pathogens is influenced by the depth of the soil where they end up after transport through the soil.The objectives of our study were the following: (i) to determine the extent of percolation of water and E. coli O157:H7 and Salmonella serovar Typhimurium from contaminated manure or slurry through a soil column, (ii) to determine the influence of application methods of manure and slurry on percolation and survival of these pathogens at different depths in a soil column, and (iii) to determine the influence of plant roots on percolation and survival of the pathogens, applied with manure or slurry, at different depths in bulk soil and the rhizosphere.