Cascading effects of composts and cover crops on soil chemistry, bacterial communities and the survival of foodborne pathogens.

AIMS: Recent foodborne disease outbreaks have caused farmers to re-evaluate their practices. In particular, concern that soil amendments could introduce foodborne pathogens onto farms and promote their survival in soils has led farmers to reduce or eliminate the application of animal-based composts. However, organic amendments (such as composts and cover crops) could bolster food safety by increasing soil microbial diversity and activity, which can act as competitors or antagonists and reduce pathogen survival. METHODS AND RESULTS: Leveraging a study of a 27-year experiment comparing organic and conventional soil management, we evaluate the impacts of composted poultry litter and cover crops on soil chemistry, bacterial communities and survival of Salmonella enterica and Listeria monocytogenes. We found that bacterial community composition strongly affected pathogen survival in soils. Specifically, organic soils managed with cover crops and composts hosted more macronutrients and bacterial communities that were better able to suppress Salmonella and Listeria. For example, after incubating soils for 10 days at 20°C, soils without composts retained fourfold to fivefold more Salmonella compared to compost-amended soils. However, treatment effects dissipated as bacterial communities converged over the growing season. CONCLUSIONS: Our results suggest that composts and cover crops may be used to build healthy soils without increasing foodborne pathogen survival. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work suggests that animal-based composts do not promote pathogen survival and may even promote bacterial communities that suppress pathogens. Critically, proper composting techniques are known to reduce pathogen populations in biological soil amendments of animal origin, which can reduce the risks of introducing pathogens to farm fields in soil amendments. Thus, animal-based composts and cover crops may be a safe alternative to conventional fertilizers, both because of the known benefits of composts for soil health and because it may be possible to apply amendments in such a way that food-safety risks are mitigated rather than exacerbated.

Bacterial population dynamics after foliar fertilization of almond leaves.

AIMS: To describe the effects of foliar fertilizer application on the bacterial populations of almond tree leaves. METHODS AND RESULTS: We applied a commercially available foliar fertilizer or a water control onto the leaves of almond trees and collected leaves after 1, 7, 14 and 56 days and examined their bacterial populations by 16S rRNA gene sequence analysis. After 1 day, we observed significant differences in 3 of the 4 predominant bacterial phyla, and 5 of the 13 predominant bacterial families. After 7 days, we observed significant differences in all of the predominant phyla, and 8 of the 13 predominant families. After 14 days, the number of significant differences decreased, and after 56 days only 2 of the 13 predominant families differed significantly. CONCLUSIONS: Foliar fertilization significantly altered the bacterial population structure of almond leaves as compared to the water control. While most of the observed perturbation was transient, significant differences remained after 56 days. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the effects of foliar fertilization on the bacterial populations of almond leaves and provides new insights as to how this process alters the leaf bacterial population structure.

Bacterial populations on the surfaces of organic and conventionally grown almond drupes.

AIMS: To compare the bacterial populations on organically and conventionally grown almond drupes before and after hull split. METHODS AND RESULTS: We constructed 16S rRNA gene libraries, containing approx. 3000 sequences each, from the bacteria from organically and conventionally grown drupes before and after hull split. We observed that before hull split both conventionally and organically grown drupes were colonized by relatively few types of bacteria that were mostly common phyllosphere-associated Proteobacteria. However, the organically grown drupes contained significantly more Alphaproteobacteria and the conventionally grown drupes contained significantly more Gammaproteobacteria. The conventionally grown drupes also contained significantly more sequences associated with the phylum Actinobacteria. After hull split, we observed a significant increase in bacterial diversity, with many newly appearing sequences that were not normally associated with the phyllosphere. CONCLUSIONS: Organic and conventional growing methodologies influence the types of bacteria on almond drupes and hull split results in a burst of microbial diversification. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of organic produce is increasing due to consumer preferences, but it was unknown how this methodology affects the bacterial populations on almond drupes. This is the first study to compare the bacterial populations of organically and conventionally grown almond drupes.

Bacterial population structure and dynamics during the development of almond drupes.

AIMS: To describe the bacterial populations and their dynamics during the development of almond drupes. METHODS AND RESULTS: We examined 16S rRNA gene libraries derived from the bacterial populations on almond drupes at three stages of development: (i) when the drupes were full sized, but before embryo development, (ii) when the drupe hulls first began to split and (iii) when the drupes were fully mature, but before harvesting. Our data revealed that the immature drupes were colonized by relatively few types of bacteria, belonging mostly to common phyllosphere-associated bacteria within the genera Pseudomonas, Pantoea, Methylobacterium and Sphingomonas. However, after the hulls first began to split, the level of bacterial diversity increased and continued to do so until the drupes were fully mature. At the last sampling period, we observed several sequences belonging to bacteria that are not usually associated with the phyllosphere, including some identical to Salmonella enterica. CONCLUSIONS: The bacterial populations on almond drupes before hull split were composed of relatively few types, most of which were commonly associated with the phyllosphere. However, after hull split, the level of microbial diversity increased, which was mostly due to increased levels of bacteria that are not normally associated with the phyllosphere, including Salm. enterica. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the bacterial populations associated with almond drupes and their dynamics during development. Of specific significance is the observation that Salm. enterica was present on the drupes just prior to harvesting, which may represent a critical control point.

Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa) and subsequent exposure to air.

AIMS: To describe, at high resolution, the bacterial population dynamics and chemical transformations during the ensiling of alfalfa and subsequent exposure to air. METHODS AND RESULTS: Samples of alfalfa, ensiled alfalfa and silage exposed to air were collected and their bacterial population structures compared using 16S rRNA gene libraries containing approximately 1900 sequences each. Cultural and chemical analyses were also performed to complement the 16S gene sequence data. Sequence analysis revealed significant differences (P < 0·05) in the bacterial populations at each time point. The alfalfa-derived library contained mostly sequences associated with the Gammaproteobacteria (including the genera: Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly sequences associated with the lactic acid bacteria (LAB) (including the genera: Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even greater percentages of LAB, especially among the genus Lactobacillus, and a significant drop in bacterial diversity. CONCLUSIONS: In-depth 16S rRNA gene sequence analysis revealed significant bacterial population structure changes during ensiling and again during exposure to air. SIGNIFICANCE AND IMPACT OF THE STUDY: This in-depth description of the bacterial population dynamics that occurred during ensiling and simulated feed out expands our knowledge of these processes.

Effects of sodium bisulfate on the bacterial population structure of dairy cow waste.

AIMS: To determine the effects of sodium bisulfate (SBS) on the bacterial populations in cattle waste. METHODS AND RESULTS: We applied SBS at 0, 60, 70 or 100 kg week(-1) to cattle waste as it accumulated on the floors of four cattle pens, housing eight cattle each. We observed significant pH decreases in all of the treated wastes on day one; however, the 60 kg week(-1) treatment returned to control levels by day four, while the others remained significantly lower. Heterotrophic plate counts of the waste revealed that all treatments reduced the bacterial populations in the wastes on day one; however, all returned to control levels by day four. The 16S rRNA gene libraries derived from the wastes revealed significant reductions in sequences associated with the phyla Bacteroidetes and Firmicutes and increases in the Proteobacteria, Actinobacteria and Spirochaetes on day one, but resembled the control by day seven. Sequences associated with Escherichia coli increased significantly after SBS application, but became undetectable by day seven. CONCLUSIONS: SBS application significantly alters the bacterial population structure of waste during the first few days of application, but the populations return to almost normal after 7 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of SBS to animal waste can reduce emissions; however, biosecurity precautions must be rigorously maintained during the initial application to ensure that pathogenic E. coli is not released into the environment.

Acidification of calf bedding reduces fly development and bacterial abundance.

Environmental stressors, such as high fly density, can affect calf well-being. Sodium bisulfate (SBS) is an acidifier that reduces the pH of flooring and bedding, creating a medium that neither bacteria nor immature flies (also known as larvae or maggots) can thrive in. Two experiments were conducted to investigate the application of SBS to a mixture of rice hull calf bedding and calf slurry (BED) to reduce house fly (Musca domestica L.) larval density and the abundance of bacteria. In experiment 1, dish pans containing 1L of BED and 3,000 house fly eggs were treated with SBS at concentrations of 0, 8.9, 17.7, and 26.5g of SBS/0.05m(2) of BED (CON, LOW, MED, and HIGH, respectively), with each SBS concentration applied to 4 individual pans (16 pans total). Reapplication of the same SBS concentrations in each pan occurred 3 times/wk throughout the 23-d trial. Larval house fly survival was significantly reduced in all pans with SBS relative to CON pans, with lowest survival rates in the MED and HIGH pans (99% and 100% reduction, respectively). The mean pH for each treatment was inversely related to the SBS concentration. In experiment 2, pans containing 1L of BED and 3,000 house fly eggs were treated with either 0g of SBS (CON), 8.9g of SBS/0.05m(2) of BED with reapplication of the acidifier 3 times/wk (SB3x), or 8.9g of SBS/0.05m(2) of BED applied only once at 48h before the end of the 8 d-trial (SB48). Larval house fly survival and bacterial concentrations were reduced (90% larval reduction and 68% bacterial reduction) in the SB3x treatment relative to the CON. Mean pH was also reduced in SB3x pans relative to CON or SB48 pans. Overall, acidification of calf BED using the acidifier SBS resulted in a reduction of bacteria and house fly larval survival. This form of fly control might be expected to reduce adult fly production and, therefore, fly-related stress in calves.

Induction of purple sulfur bacterial growth in dairy wastewater lagoons by circulation.

AIMS: To determine whether circulation of dairy wastewater induces the growth of phototrophic purple sulfur bacteria (PSB). METHODS AND RESULTS: Two dairy wastewater lagoons that were similar in size, geographic location, number and type of cattle loading the lagoons were chosen. The only obvious visual difference between them was that one was stagnant and the water was brown in colour (Farm 1), and the other was circulated and the water was red in colour because of the presence of PSB that contained carotenoid pigments (Farm 2). Both wastewaters were sampled monthly for 3 months and assayed for PSB and extractable carotenoid pigments (ECP). After this point, circulators were placed in the wastewater lagoon on Farm 1, and samples were taken monthly for 9 months and assayed for PSB and ECP. Before the installation of circulators, no PSB-like 16S rRNA sequences or ECP were observed in the wastewater from Farm 1; however, both were observed in the wastewater from Farm 2. After the installation of circulators, statistically greater levels of PSB and extractable carotenoid pigments were observed in the wastewater from Farm 1. CONCLUSIONS: Circulation enhances the growth of PSB in dairy wastewater. SIGNIFICANCE AND IMPACT OF THIS STUDY: Because PSB utilize H(2)S and volatile organic acids (VOA) as an electron source for photosynthesis, and VOA and alcohols as a carbon source for growth, the increase in these bacteria should reduce H(2)S, volatile organic compounds and alcohol emissions from the lagoons, enhancing the air quality in dairy farming areas.

Comparison of bacterial populations and chemical composition of dairy wastewater held in circulated and stagnant lagoons.

AIMS: This study compared the chemical, physical and bacterial composition of circulated and stagnant dairy wastewaters. METHODS AND RESULTS: Samples taken from circulated and stagnant wastewater lagoons, over a 1-year period, were analysed for 10 chemical (total N, NH3, NO3, NO2, Na, Ca, HCO3, Fe, P and K) and six physical (biological oxygen demand, chemical oxygen demand, dissolved solids, electrical conductivity, pH and sodium absorption ratio) parameters and were found to be similar. The 16S rDNA genes from the samples were amplified, cloned and BLAST analysed. In total, 996 stagnant and 1052 circulated wastewater derived sequences were obtained, comprising 294 and 362 operational taxonomic units (OTUs) from the circulated and stagnant wastewaters respectively. Coverage estimates of the OTUs identified were 72.1% for the stagnant, and 63.6% for the circulated wastewater libraries. The greatest difference between the two wastewaters was a c. sixfold greater number of sequences representative of the family Chromatiaceae in the circulated wastewater derived library and a c. fivefold greater number of sequences representative of the phylum Chloroflexi in the stagnant wastewater derived library. CONCLUSIONS: Circulation of dairy wastewater does not affect any of the chemical or physical parameters tested; however, circulation does alter the bacterial community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence that circulation of dairy wastewater promotes the growth of bacteria within the family Chromatiaceae and that stagnant systems promote the growth of the phylum Chloroflexi.

Differential gene expression in mononuclear phagocytes infected with pathogenic and non-pathogenic mycobacteria.

The pathogenic mycobacteria are an insidious group of bacterial pathogens that cause the deaths of millions of people every year. One of the reasons these pathogens are so successful is that they are able to invade and replicate within host macrophages, one of the first lines of defence against intruding pathogens. In contrast, non-pathogenic mycobacteria, such as Mycobacterium smegmatis are killed rapidly by macrophages. In order to understand better the series of events that allow pathogenic mycobacteria to survive and replicate within macrophages, while the non-pathogenic mycobacteria are killed rapidly, we inoculated the human monocytic cell line U937 with pathogenic (M. tuberculosis and M. avium) and non-pathogenic (M. smegmatis) mycobacteria and monitored the expression of over 3500 genes at 4, 12 and 24 h post-inoculation using a commercially available gene array system. We observed multiple differences in the gene expression patterns of monocytes infected with pathogenic and non-pathogenic mycobacteria including genes involved in cytokine, lymphokine and chemokine production, adhesion, apoptosis, signal transduction, transcription, protein cleavage, actin polymerization and growth. We also observed differences in gene expression profiles in monocytes infected with M. tuberculosis or M. avium, indicating that there are differences in the host pathogen interactions of mononuclear phagocytes infected with different pathogenic mycobacterial species. These results increase the understanding of the mechanisms used by pathogenic mycobacteria to cause disease, the host response to these organisms, and provide new insights for antimycobacterial intervention strategies.

Phenotypic and genomic analyses of the Mycobacterium avium complex reveal differences in gastrointestinal invasion and genomic composition.

Mycobacterium avium and Mycobacterium intracellulare are closely related organisms and comprise the Mycobacterium avium complex. These organisms share many common characteristics, including the ability to cause life-threatening respiratory infections in people with underlying lung pathology or immunological defects and occasionally in those with no known predisposing conditions. However, the ability to invade the mucosa of the gastrointestinal tract and cause disseminated disease in AIDS patients has not been epidemiologically linked to M. intracellulare and appears to be unique to M. avium. We compared the abilities of M. avium and M. intracellulare to tolerate the acidic conditions of the stomach, to resist the membrane-disrupting activity of cationic peptides, and to invade intestinal epithelial cells in vitro and in vivo. We observed that M. avium and M. intracellulare were both tolerant to the acidic conditions encountered in the stomach and resistant to cationic peptides. However, when strains of M. avium and M. intracellulare were examined for their ability to enter cultured human intestinal cells or mouse intestinal mucosa, we observed that M. avium could invade more efficiently than M. intracellulare. To elucidate the basis of this pathogenic difference and identify genes involved in the invasion of the intestinal mucosa, we performed chromosomal DNA subtractive hybridization using M. avium and M. intracellulare chromosomal DNAs. In all, 21 genes that were present in M. avium but absent in M. intracellulare were identified, including some that may be associated with the ability of M. avium to invade the intestinal mucosa.

Spatial-Temporal and Quantitative Analysis of Growth and EPS I Production by Ralstonia solanacearum in Resistant and Susceptible Tomato Cultivars.

ABSTRACT One susceptible and two resistant cultivars of tomato were tested for differences in infection by Ralstonia solanacearum and for the subsequent multiplication, colonization, and production of the wilt-inducing virulence factor, exopolysaccharide I (EPS I). Bacterial ingress into the taproot was fastest in the susceptible cv. Marion, followed by the resistant cvs. L285 (fivefold slower) and Hawaii 7996 (15-fold slower). Once inside the taproot, R. solanacearum colonized, to some extent, almost all regions of the resistant and susceptible plants. However, colonization occurred sooner in the susceptible than in the resistant cultivars, as measured by viablecell counts of bacteria in the midstems. Rates of multiplication and maximum bacterial cell densities were also greater in the susceptible than in the resistant cultivars. Growth experiments utilizing xylem fluid from infected and uninfected plants indicated that neither antimicrobial activities nor reduced levels of growth-supporting nutrients in the xylem fluids were responsible for the reduced bacterial multiplication in the resistant cultivars. Quantification of EPS I in the infected plants, using an enzyme-linked immunosorbent assay, revealed that the bacterial populations in the susceptible cultivar produced greater amounts of EPS I per plant than those in the resistant cultivars. Immunofluorescence microscopy using antibodies against either EPS I or R. solanacearum cells revealed that bacteria and EPS I were distributed throughout the vascular bundles and intercellular spaces of the pith in the susceptible cultivar, whereas in the resistant cultivars, bacteria and EPS I were restricted to the vascular tissues.

Bacterial oxidation of mercury metal vapor, Hg(0).

We used metalloregulated luciferase reporter fusions and spectroscopic quantification of soluble Hg(II) to determine that the hydroperoxidase-catalase, KatG, of Escherichia coli can oxidize monatomic elemental mercury vapor, Hg(0), to the water-soluble, ionic form, Hg(II). A strain with a mutation in katG and a strain overproducing KatG were used to demonstrate that the amount of Hg(II) formed is proportional to the catalase activity. Hg(0) oxidation was much decreased in stationary-phase cells of a strain lacking KatG, suggesting that the monofunctional hydroperoxidase KatE is less effective at this reaction. Unexpectedly, Hg(0) oxidation also occurred in a strain lacking both KatE and KatG, suggesting that activities other than hydroperoxidases may carry out this reaction. Two typical soil bacteria, Bacillus and Streptomyces, also oxidize Hg(0) to Hg(II). These observations establish for the first time that bacteria can contribute, as do mammals and plants, to the oxidative phase of the global Hg cycle.

Role of Extracellular Polysaccharide and Endoglucanase in Root Invasion and Colonization of Tomato Plants by Ralstonia solanacearum.

ABSTRACT Ralstonia solanacearum is a soilborne plant pathogen that normally invades hosts through their roots and then systemically colonizes aerial tissues. Previous research using wounded stem infection found that the major factor in causing wilt symptoms was the high-molecular-mass acidic extracellular polysaccharide (EPS I), but the beta-1,4-endoglucanase (EG) also contributes to virulence. We investigated the importance of EPS I and EG for invasion and colonization of tomato by infesting soil of 4-week-old potted plants with either a wild-type derivative or genetically well-defined mutants lacking EPS I, EG, or EPS I and EG. Bacteria of all strains were recovered from surface-disinfested roots and hypocotyls as soon as 4 h after inoculation; that bacteria were present internally was confirmed using immunofluorescence microscopy. However, the EPS-minus mutants did not colonize stems as rapidly as the wild type and the EG-minus mutant. Inoculations of wounded petioles also showed that, even though the mutants multiplied as well as the wild type in planta, EPS-minus strains did not spread as well throughout the plant stem. We conclude that poor colonization of stems by EPS-minus strains after petiole inoculation or soil infestation is due to reduced bacterial movement within plant stem tissues.