Comparative persistence of Salmonella and Escherichia coli O157:H7 in loam or sandy loam soil amended with bovine or swine manure.

The fate of Salmonella and Escherichia coli O157:H7 in swine or dairy manure amended into sandy loam or loam soil under field conditions was studied. Soil was amended with manure inoculated with a Salmonella or E. coli O157:H7 cocktail, then transferred to 0.22 µm pore size membrane walled vials. The vials were then placed on the surface or at 15 cm depth in the test plots. Pathogen numbers, soil moisture, rainfall, and temperature were measured throughout the three trials (20-47 weeks duration) representing spring or fall application. Survival curves were characterized by having an initial rapid decline in pathogen numbers followed by a slower inactivation phase with an occasional increase in culturable cells. The CT99.9 values (time to reach a 3 log CFU reduction) varied from 2 to 120 days, with the most rapid decrease being observed on the surface of sandy loam soil. The persistence of pathogens is primarily governed by variations in moisture and temperature, although season of application along with manure and soil type also contribute. To generate more accurate predictive pathogen models, there is a need for laboratory-based trials to mirror the dynamic variation in temperature and soil moisture encountered within the natural environment.

Dairy Manure Total Solid Levels Impact CH Flux and Abundance of Methanogenic Archaeal Communities.

Stored liquid dairy manures are methane (CH) emission hotspots because of the large amount of slurry volatile solids (VS) converted into CH by methanogens under anaerobic conditions. Our research has indicated that a reduction of total solids (TS) of slurries before storage can reduce CH emissions. In the current study, methanogen abundance was characterized in tanks with different CH emissions. Using mesoscale slurry storage facilities equipped for continuous gaseous emission monitoring, we stored dairy slurries having TS from 9.5 to 0.3% for up to 6 mo. Samples were taken after Day 30 and Day 120 of the storage (20 May-16 Nov. 2010) from the upper and bottom layers of the slurries. Methanogenic communities were studied by targeting the gene encoding the α subunit methyl-coenzyme M reductase (), which catalyzes the final step of methanogenesis. Interestingly, mean abundances of methanogens increased by ∼8 and 23% at the top and bottom sections, respectively, as slurry TS decreased from 9.5 to 0.3%. Cumulative CH emissions, however, decreased by ∼70% as slurry TS decreased from 9.5 to 0.3%. Nevertheless, compared with Day 30 of storage, mean abundances of methanogens were relatively higher at Day 120 (up to 19%), consistent with an increase in the cumulative CH emissions. Polymerase chain reaction denaturing gel electrophoresis analysis indicated a low methanogen diversity, with most bands sequenced closely related to the genus (>95% amino acid sequence similarity), the hydrogenotrophic methanogens. Results suggest that available carbon substrate and not methanogen abundance may be limiting cumulative CH emissions at reduced TS levels of dairy slurries.

The Extent of Manure Removal from Storages and Its Impact on Gaseous Emissions.

Manure remaining in storage due to incomplete removal is a source of microbial inoculum that may affect methane (CH), nitrous oxide (NO), and ammonia (NH) emissions during subsequent storage. Manure removal was studied by loading fresh manure into outdoor concrete tanks (10.6 m) that contained previously stored manure (inoculum) at six levels (0, 5, 10, 15, 20, and 25%, with 0% representing an empty tank). Emissions were continuously measured for 6-mo storage periods (warm and cold seasons) using flow-through chambers. Fluxes during the warm season (average manure temperature at 80 cm depth, = 17°C) were 25 times higher for CH, 20 times higher for NO, and 2.9 times higher for NH compared with the cold season ( = 4°C). Cumulative CH emissions increased linearly with the level of added inoculum in the cold season ( = 0.98). A similar linear increase was observed in the warm season from 0 to 20% inoculum ( = 0.91), after which a decrease in emissions was observed at 25%. Reducing inoculum from 15 to 5% reduced CH emissions by 26% in the warm season and 45% in the cold season. There was no clear effect of inoculum on NO and NH emissions, suggesting that complete manure storage emptying does not alter their emissions.

Relationships between dairy slurry total solids, gas emissions, and surface crusts.

Livestock slurry storages are sources of methane (CH4), nitrous oxide (NO2), and ammonia (NH3) emissions. Total solids (TS) content is an indicator of substrate availability for CH4 and N2O production and NH3 emissions and is related to crust formation, which can affect these gas emissions. The effect of TS on these emissions from pilot-scale slurry storages was quantified from 20 May through 16 Nov. 2010 in Nova Scotia, Canada. Emissions from six dairy slurries with TS ranging from 0.3 to 9.5% were continuously measured using flow-through steady-state chambers. Methane emissions modeled using the USEPA methodology were compared with measured data focusing on emissions when empty storages were filled, and retention times were >30 d with undegraded volatile solids (VS) remaining in the system considered available for CH4 production (VS carry-over). Surface crusts formed on all the slurries. Only the slurries with TS of 3.2 and 5.8% were covered completely for ∼3 mo. Nitrous oxide contributed <5% of total greenhouse gas emissions for all TS levels. Ammonia and CH4 emissions increased linearly with TS despite variable crusting, suggesting substrate availability for gas production was more important than crust formation in regulating emissions over long-term storage. Modeled CH4 emissions were substantially higher than measured data in the first month, and accounting for this could improve overall model performance. Carried-over VS were a CH4 source in months 2 through 6. The results of this study suggest that substrate availability regulates emissions over long-term storage and that modifying the USEPA model to better describe carbon cycling is warranted.

Bacterial contamination of tile drainage water and shallow groundwater under different application methods of liquid swine manure.

A 2 year field experiment evaluated liquid manure application methods on the movement of manure-borne pathogens (Salmonella sp.) and indicator bacteria (Escherichia coli and Clostridium perfringens) to subsurface water. A combination of application methods including surface application, pre-application tillage, and post-application incorporation were applied in a randomized complete block design on an instrumented field site in spring 2007 and 2008. Tile and shallow groundwater were sampled immediately after manure application and after rainfall events. Bacterial enumeration from water samples showed that the surface-applied manure resulted in the highest concentration of E. coli in tile drainage water. Pre-tillage significantly (p < 0.05) reduced the movement of manure-based E. coli and C. perfringens to tile water and to shallow groundwater within 3 days after manure application (DAM) in 2008 and within 10 DAM in 2007. Pre-tillage also decreased the occurrence of Salmonella sp. in tile water samples. Indicator bacteria and pathogens reached nondetectable levels within 50 DAM. The results suggest that tillage before application of liquid swine manure can minimize the movement of bacteria to tile and groundwater, but is effective only for the drainage events immediately after manure application or initial rainfall-associated drainage flows. Furthermore, the study highlights the strong association between bacterial concentrations in subsurface waters and rainfall timing and volume after manure application.

Evaluation of Petrifilm EC method for enumeration of E. coli from soil.

AIMS: To evaluate the suitability of commercially available Petrifilm EC plates for enumeration of Escherichia coli from soil. METHODS AND RESULTS: A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 10(2), 10(3), 10(5) CFU g(-1) of soil. The efficiency of recovery on Petrifilm EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m-FC basal medium supplemented with 3-bromo-4-chloro-5-indoyl-beta-D-glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) broth. Petrifilm EC and m-FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0.05) were observed between Petrifilm EC, m-FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure-applied field soil samples showed a significant difference (P < 0.05) between the Petrifilm EC method and the m-FC method in enumerating E. coli possibly as a result of false positives on m-FC. CONCLUSION: The Petrifilm EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g(-1) soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The commercially available Petrifilm EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests.

Microbial population profiles of the microflora associated with pre- and postharvest tomatoes contaminated with Salmonella typhimurium or Salmonella montevideo.

AIMS: To determine the microflora profiles of pre- and postharvest tomatoes contaminated with Salmonella montevideo or S. typhimurium DT104. METHODS AND RESULTS: Salmonella montevideo or S. typhimurium was inoculated onto the flowers of tomato plants with the microflora of the subsequent fruit examined using a combination of Source Carbon Utilization and 16S rDNA-PCR profiling. From 16S rDNA profiles it was evident that tomatoes derived from Salmonella inoculated plants harboured a different microbial population compared to nontreated controls. The same result was observed for tomatoes inoculated at postharvest and subsequently stored for 14 days at 15 degrees C. From sequencing analysis it was found that tomatoes derived from Salmonella inoculated plants but testing negative for the enteric pathogen, frequently harboured Enterobacter and Bacillus spp. In contrast, both bacterial types were not found associated with tomatoes testing positive for Salmonella. CONCLUSIONS: Salmonella introduced onto tomatoes at pre- or postharvest alters the composition of the microbial community. The presence of Enterobacter and Bacillus spp negatively affects the persistence of Salmonella on preharvest tomatoes. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella appears to modify rather than become integrated into the microbial communities associated with tomatoes. Yet, the presence of antagonistic bacteria appears to reduce the persistence of the enteric pathogen.

Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase.

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.