Abasiofiok M. Ibekwe

Changes in developing plant microbial community structure as affected by contaminated water

The effects of sand and clay soils and water contaminated by Escherichia coli O157:H7 on the development of rhizosphere and phyllosphere microbial communities were analyzed to determine the influence of plant age on microbial community structure and composition. Community bacterial nucleic acids were extracted from lettuce rhizosphere and phyllosphere samples at different stages of plant development after the soils were irrigated with water contaminated with E. coli O157:H7 at planting and 15 days after planting. PCR was used to amplify 16S ribosomal RNA (rRNA) for total bacterial community composition and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominant bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere and phyllosphere, such as Pseudomonas, Acidobacterium, Bacillus and Agrobacterium. The PCR-DGGE patterns observed for rhizosphere samples were more complex than those obtained from the bulk soil and the phyllosphere. The Shannon index of diversity (H) was used to determine the complexity of the DGGE bands from the phyllosphere, rhizosphere and the bulk soils at different growth stages. A higher diversity was observed in the clay soil than sandy soil during the first week. Few changes in diversity were observed after the first week. The results show that microbial community development in lettuce may take about 7-12 days and this may be the most likely period for maximum pathogen contamination in plants.

Distinct Soil Bacterial Communities Revealed under a Diversely Managed Agroecosystem

Land-use change and management practices are normally enacted to manipulate environments to improve conditions that relate to production, remediation, and accommodation. However, their effect on the soil microbial community and their subsequent influence on soil function is still difficult to quantify. Recent applications of molecular techniques to soil biology, especially the use of 16S rRNA, are helping to bridge this gap. In this study, the influence of three land-use systems within a demonstration farm were evaluated with a view to further understand how these practices may impact observed soil bacterial communities. Replicate soil samples collected from the three land-use systems (grazed pine forest, cultivated crop, and grazed pasture) on a single soil type. High throughput 16S rRNA gene pyrosequencing was used to generate sequence datasets. The different land use systems showed distinction in the structure of their bacterial communities with respect to the differences detected in cluster analysis as well as diversity indices. Specific taxa, particularly Actinobacteria, Acidobacteria, and classes of Proteobacteria, showed significant shifts across the land-use strata. Families belonging to these taxa broke with notions of copio- and oligotrphy at the class level, as many of the less abundant groups of families of Actinobacteria showed a propensity for soil environments with reduced carbon/nutrient availability. Orders Actinomycetales and Solirubrobacterales showed their highest abundance in the heavily disturbed cultivated system despite the lowest soil organic carbon (SOC) values across the site. Selected soil properties ([SOC], total nitrogen [TN], soil texture, phosphodiesterase [PD], alkaline phosphatase [APA], acid phosphatase [ACP] activity, and pH) also differed significantly across land-use regimes, with SOM, PD, and pH showing variation consistent with shifts in community structure and composition. These results suggest that use of pyrosequencing along with traditional analysis of soil physiochemical properties may provide insight into the ecology of descending taxonomic groups in bacterial communities.

Persistence of Escherichia coli O157:H7 in major leafy green producing soils.

Persistence of Escherichia coli O157:H7 in 32 (16 organically managed and 16 conventionally managed) soils from California (CA) and Arizona (AZ) was investigated. Results showed that the longest survival (ttd, time needed to reach detection limit, 100 CFU g(-1) dry soil) of E. coli O157:H7 was observed in the soils from Salinas Valley, CA and in organically managed soils from AZ. Detrended correspondence analysis revealed that the survival profiles in organically managed soils in Yuma, AZ were different from the ones in conventionally managed soils from the same site. Principal component analysis and stepwise regression analysis showed that E. coli O157:H7 survival in soils was negatively correlated with salinity (EC) (P < 0.001), while positively correlated with assimilable organic carbon (AOC) and total nitrogen (TN) (P < 0.01). Pearson correlation analysis revealed that a greater ttd was associated with a larger δ (time needed for first decimal reduction in E. coli population). EC was negatively correlated and TN was positively correlated (P < 0.05) with δ, suggesting that EC and TN likely have a direct impact on ttd. On the other hand, AOC showed a close correlation with p (the shape parameter) that was not directly related to ttd, indicating that AOC might have an indirect effect in the overall survival of E. coli O157:H7 in soils. Our data showed that AOC and EC significantly affected the survival of E. coli O157:H7 in leafy green producing soils and the development of good agricultural practices (manure/composting/irrigation water source management) in the preharvest environment must be followed to minimize foodborne bacterial contamination on fresh produce.

Bacterial community composition in low‐flowing river water with different sources of pollutants

Pollution of water resources is a major risk to human health and water quality throughout the world. The purpose of this study was to determine the influence of pollutant sources from agricultural activities, urban runoffs, and runoffs from wastewater treatment plants (WWTPs) on bacterial communities in a low-flowing river. Bacterial community structure was monitored using terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene clone library. The results were analyzed using nonmetric multidimensional scaling (NMDS) and UniFrac, coupled with principal coordinate analysis (PCoA) to compare diversity, abundance, community structure, and specific functional groups of bacteria in surface water affected by nonpoint sources. From all the sampling points, Bacteria were numerically dominated by three phyla – the Proteobacteria, Bacteroidetes, and Cyanobacteria – accounting for the majority of taxa detected. Overall results, using the b diversity measures UniFrac, coupled with PCoA, showed that bacterial contamination of the low-flowing river was not significantly different between agricultural activities and urban runoff.

Bacterial community dynamics in surface flow constructed wetlands for the treatment of swine waste.

Constructed wetlands are generally used for the removal of waste from contaminated water. In the swine production system, wastes are traditionally flushed into an anaerobic lagoon which is then sprayed on agricultural fields. However, continuous spraying of lagoon wastewater on fields can lead to high N and P accumulations in soil or lead to runoff which may contaminate surface or ground water with pathogens and nutrients. In this study, continuous marsh constructed wetland was used for the removal of contaminants from swine waste. Using pyrosequencing, we assessed bacterial composition within the wetland using principal coordinate analysis (PCoA) which showed that bacterial composition from manure influent and lagoon water were significantly different (P=0.001) from the storage pond to the final effluent. Canonical correspondence analysis (CCA) showed that different bacterial populations were significantly impacted by ammonium--NH4 (P=0.035), phosphate--PO4(3-) (P=0.010), chemical oxygen demand--COD (P=0.0165), total solids--TS (P=0.030), and dissolved solids--DS (P=0.030) removal, with 54% of the removal rate explained by NH4+PO4(3-) according to a partial CCA. Our results showed that different bacterial groups were responsible for the composition of different wetland nutrients and decomposition process. This may be the major reason why most wetlands are very efficient in waste decomposition.

Influence of fumigants on soil microbial diversity and survival of E. coli O157:H7

The aim of this study was to assess the effects of soil fumigation with methyl bromide (MeBr; CH3Br) and methyl iodide (MeI, iodomethane; CH3I) on the microbial community structure and diversity in two soils and determine the effects of microbial diversity on the survival of Escherichia coli O157:H7 from contaminated irrigation water. Polymerase chain reaction (PCR) was used to amplify 16S rRNA from total bacterial community composition and the products were subjected to denaturing gradient gel electrophoresis (DGGE). The Shannon-Weaver index of diversity (H′) was used to determine the effects of both fumigants on soil microbial diversity. The effect was more severe in sandy soil than in clay soil at the normal application rate of MeBr and MeI. Our results showed that MeBr and MeI have about the same effects on soil microbial diversity. The two fumigants had greater impact on microbial diversity in sandy soil than in clay soil and this resulted in higher survival of E. coli O157:H7 in sandy soil than in clay soil during the 50 days that the study was conducted. MeBr has been used as soil fumigant for >40 years with no serious detrimental effects on agricultural production and our research also suggests that the use of MeI may also produce no long-term detrimental effects on agricultural production since both fumigants had about the same effects on soil microbial communities. Therefore, soil systems with reduced microbial diversity may offer greater opportunities for the survival of pathogenic bacteria such as E. coli O157:H7.

Topological data analysis of Escherichia coli O157:H7 and non-O157 survival in soils

Shiga toxin-producing E. coli O157:H7 and non-O157 have been implicated in many foodborne illnesses caused by the consumption of contaminated fresh produce. However, data on their persistence in soils are limited due to the complexity in datasets generated from different environmental variables and bacterial taxa. There is a continuing need to distinguish the various environmental variables and different bacterial groups to understand the relationships among these factors and the pathogen survival. Using an approach called Topological Data Analysis (TDA); we reconstructed the relationship structure of E. coli O157 and non-O157 survival in 32 soils (16 organic and 16 conventionally managed soils) from California (CA) and Arizona (AZ) with a multi-resolution output. In our study, we took a community approach based on total soil microbiome to study community level survival and examining the network of the community as a whole and the relationship between its topology and biological processes. TDA produces a geometric representation of complex data sets. Network analysis showed that Shiga toxin negative strain E. coli O157:H7 4554 survived significantly longer in comparison to E. coli O157:H7 EDL 933, while the survival time of E. coli O157:NM was comparable to that of E. coli O157:H7 EDL 933 in all of the tested soils. Two non-O157 strains, E. coli O26:H11 and E. coli O103:H2 survived much longer than E. coli O91:H21 and the three strains of E. coli O157. We show that there are complex interactions between E. coli strain survival, microbial community structures, and soil parameters.

Potential pathogens, antimicrobial patterns and genotypic diversity of Escherichia coli isolates in constructed wetlands treating swine wastewater

Escherichia coli populations originating from swine houses through constructed wetlands were analyzed for potential pathogens, antimicrobial susceptibility patterns, and genotypic diversity. Escherichia coli isolates (n = 493) were screened for the presence of the following virulence genes: stx1, stx2 and eae (Shiga toxin-producing E. coli [STEC]), heat-labile enterotoxin (LT) genes and heat stable toxin STa and STb (enterotoxigenic E. coli (ETEC), cytotoxin necrotizing factors 1 and 2 (cnf1 and cnf2 [necrotoxigenic E. coli- NTEC]), as well as O and H antigens, and the presence of the antibiotic resistance genes blaTEM, blaSHV, blaCMY-2, tet A, tet B, tet C, mph(A), aadA, StrA/B, sul1, sul2 and sul3. The commensal strains were further screened for 16 antimicrobials and characterized by BOX AIR-1 PCR for unique genotypes. The highest antibiotic resistance prevalence was for tetracycline, followed by erythromycin, ampicillin, streptomycin, sulfisoxazole and kanamycin. Our data showed that most of the isolates had high distribution of single or multidrug-resistant (MDR) genotypes. Therefore, the occurrence of MDR E. coli in the wetland is a matter of great concern due to possible transfer of resistance genes from nonpathogenic to pathogenic strains or vice versa in the environment.

Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations

Microbial diversity of agricultural soils has been well documented, but information on leafy green producing soils is limited. In this study, we investigated microbial diversity and community structures in 32 (16 organic, 16 conventionally managed soils) from California (CA) and Arizona (AZ) using pyrosequencing, and identified factors affecting bacterial composition. Results of detrended correspondence analysis (DCA) and dissimilarity analysis showed that bacterial community structures of conventionally managed soils were similar to that of organically managed soils; while the bacterial community structures in soils from Salinas, California were different (P<0.05) from those in soils from Yuma, Arizona and Imperial Valley, California. Canonical correspondence analysis (CCA) and artificial neural network (ANN) analysis of bacterial community structures and soil variables showed that electrical conductivity (EC), clay content, water-holding capacity (WHC), pH, total nitrogen (TN), and organic carbon (OC) significantly (P<0.05) correlated with microbial communities. CCA based variation partitioning analysis (VPA) showed that soil physical properties (clay, EC, and WHC), soil chemical variables (pH, TN, and OC) and sampling location explained 16.3%, 12.5%, and 50.9%, respectively, of total variations in bacterial community structure, leaving 13% of the total variation unexplained. Our current study showed that bacterial community composition and diversity in major fresh produce growing soils from California and Arizona is a function of soil physiochemical characteristics and geographic distances of sampling sites.

Influence of soil fumigation by methyl bromide and methyl iodide on rhizosphere and phyllosphere microbial community structure

Rhizosphere and phyllosphere microbial communities were evaluated on roots and leaves of growth chamber-grown lettuce (Lactuca sativa (L.) cv. Green Forest) plants by culture-dependent and -independent methods after soil fumigation. Denaturing gradient gel electrophoresis (DGGE) with 16S rRNA primers followed by cloning and sequencing was used to identify major rRNA bands from the rhizosphere and phyllosphere. Three weeks after fumigation, there were no differences (P = 0.16) in rhizosphere microbial communities between the fumigated treatments and the control. The same effect was observed during week seven after fumigation (P=0.49). Also, no significant differences (P=0.49) were found in the phyllosphere microbial communities between the fumigated treatments and the control during the growth period of the plant. A majority of the bands in the rhizosphere were related to known bacterial sequences with a 96 to 100 % sequence similarity. Some of the derived sequences were related to Pseudomonas syringae pv. tomato DC300 and Bradyrhizobium japonicum USDA 110. A total of 23 isolates were identified from leaf surface by both culture-dependent and independent methods, and only Photorhabdus luminescens was found on leaf surface using both techniques. All the Biolog isolates from phyllosphere were from the Proteobacteria phylum compared to the culture-independent bands from the leaves that were from different bacterial phyla. Based on our data, methyl bromide (MeBr) and methyl iodide (MeI) did not have any significant negative effects on rhizosphere and phyllosphere microbial communities throughout the growing period of lettuce.