Terminal Restriction Fragment Polymorphism Research Articles

The impact of Beauveria species bioinocula on the soil microbial community structure in organic strawberry plantations.

The multifunctionality of microorganisms, including entomopathogenic fungi, represents a feature that could be exploited to support the development, marketing, and application of microbial-based products for plant protection. However, it is likely that this feature could affect the composition and dynamics of the resident soil microorganisms, possibly over a longer period. Therefore, the methodology utilized to evaluate such impact is critical for a reliable assessment. The present study was performed to evaluate the impact of strains of Beauveria brongniartii and Beauveria bassiana on soil bacterial and fungal communities using an approach based on the terminal restriction fragment polymorphism (T-RFLP) analysis. Soil samples in the vicinity of the root system were collected during a 3-year period, before and after the bioinocula application, in two organic strawberry plantations. Specific primers were used for the amplification of the bacterial 16S rRNA gene and the fungal ITS region of the ribosome. Data of the profile analysis from T-RFLP analysis were used to compare the operational taxonomic unit (OTU) occurrence and intensity in the inoculated soil with the uninoculated control. With regard to the impact on the bacterial community, both Beauveria species were not fully consistently affecting their composition across the seasons and fields tested. Nevertheless, some common patterns were pointed out in each field and, sometimes, also among them when considering the time elapsed from the bioinoculum application. The impact was even more inconsistent when analyzing the fungal community. It is thus concluded that the application of the bioinocula induced only a transient and limited effect on the soil microbial community, even though some changes in the structure dynamic and frequency of soil bacterial and fungal OTUs emerged.

Effects of Short-Term Soil Tillage Management on Activity and Community Structure of Denitrifiers under Double- Cropping Rice Field.

Soil physical and chemical characteristics, soil potential denitrification rates (PDR), community composition and nirK-, nirS- and nosZ-encoding denitrifiers were studied by using MiSeq sequencing, quantitative polymerase chain reaction (qPCR), and terminal restriction fragment polymorphism (T-RFLP) technologies base on short-term (5-year) tillage field experiment. The experiment included four tillage treatments: conventional tillage with crop residue incorporation (CT), rotary tillage with crop residue incorporation (RT), no-tillage with crop residue retention (NT), and rotary tillage with crop residue removed as control (RTO). The results indicated that soil organic carbon, total nitrogen and NH4+-N contents were increased with CT, RT and NT treatments. Compared with RTO treatment, the copies number of nirK, nirS and nosZ in paddy soil with CT, RT and NT treatments were significantly increased. The principal coordinate analysis indicated that tillage management and crop residue returning management were the most and the second important factors for the change of denitrifying bacteria community, respectively. Meanwhile, this study indicated that activity and community composition of denitrifiers with CT, RT and NT treatments were increased, compared with RTO treatment. This result showed that nirK, nirS and nosZ-type denitrifiers communities in crop residue applied soil had higher species diversity compared with crop residue removed soil, and denitrifying bacteria community composition were dominated by Gammaproteobacteria, Deltaproteobacteria, and Betaproteobacteria. Therefore, it is a beneficial practice to increase soil PDR level, abundance and community composition of nitrogen-functional soil microorganism by combined application of tillage with crop residue management.

Root Endophytes of Coffee (Coffea arabica): Variation Across Climatic Gradients and Relationships with Functional Traits

The root microbiome of Central American coffee trees was studied from four different sites experiencing different annual temperatures and precipitation levels, sampling from plots grown conventionally and under agroforestry management (with shade trees). Total community DNA was separately extracted from roots from four trees sampled from each site/management pair and analyzed using terminal restriction fragment polymorphism analysis and also next generation sequencing (Illumina) of fungal and bacterial ribosomal amplicons. Community profiles were analyzed for site and management effects and correlations to environmental parameters and tree leaf and root economic traits. Communities of both bacteria and fungi varied with site locations, but were not impacted by management system type. They also both varied strongly with environmental parameters. Fungal communities also showed significant variation that could be attributed to plant leaf and root traits. Pooled DNA samples from each site/management regime were used to generate amplicons for next generation sequencing to determine the dominant members of the coffee root microbiome at these locations. Core bacterial genera included Pantoea, Enterobacter, and Burkholderia, while fungal core communities were dominated by members of Cladosporium, Penicillium, Exidiopsis, Trechispora, and Mycena. The potential ecological function of these microbial associates is discussed.

Responses of archaeal, bacterial, and functional microbial communities to growth season and nitrogen fertilization in rice fields

Fertilization provides excess N to soil microorganisms, thus possibly affecting soil microbial abundance, activity, and community composition during rice cultivation. The abundance and diversity of archaeal, bacterial, and functional microbial communities in rice soils upon different N fertilization regimes (no fertilizer, urea, and controlled-release fertilizer) were investigated by sampling four seasonal growth stages (seedling, tillering, vegetative, maturing) under field conditions. The abundance of bacteria was significantly affected by fertilization and seasonal time, while that of the archaea was not significantly affected. Analysis of terminal restriction fragment polymorphism (T-RFLP) of 16S rRNA genes showed no effect of N fertilization on the archaeal and bacterial community composition, but changes with plant growth time. This result was confirmed by the patterns of pyrosequencing of bacterial 16S rRNA genes. The function of the methanogenic microbial community was assayed at maturing plant growth stage by determining CH4 production rates and stable isotope fractionation in the absence and presence of methyl fluoride, an inhibitor of acetoclastic methanogenesis. N fertilization had a pronounced effect on the CH4 production rate but not on the pathway of CH4 formation. Additionally, the abundance of functional microbial communities related to CH4 and N2O emissions was measured by qPCR of functional genes. Similarly to the taxonomic composition, rice growth season showed a significant effect on the abundance of the functional microbial communities represented by the mcrA, pmoA, nirK, nirS, and nosZ genes, while N addition had usually no significant effect. A similar result was also obtained by correlation analysis between CH4 and N2O emission rates and abundances of the functional microbial gene copies. In summary, rice growth time had pronounced effects on abundance, composition, and function of microbial communities in the rice soil, while the effect of N fertilization was negligible on the level of both specific functional genes and taxonomic 16S rRNA genes.

Effects of Rhizosphere and Long-Term Fertilization Practices on the Activity and Community Structure of Denitrifiers Under Double-Cropping Rice Field

ABSTRACTThe soil physicochemical properties, soil denitrification rates (PDR), denitrifiers via nitrite reductases (nirK and nirS) and nitrous oxide reductase (nosZ), abundance and community composition of denitrifiers in both the rhizosphere and bulk soil from a long-term (32 year) fertilizer field experiment conducted during late rice season were investigated by using the MiSeq sequencing, quantitative PCR, terminal restriction fragment polymorphism (T-RFLP). The experiment including four treatments: without fertilizer input (CK), chemical fertilizer alone (MF), rice straw residue and chemical fertilizer (RF), and organic manure and chemical fertilizer (OM). The results showed that the application of rice straw residue and organic manure increased soil organic carbon (C), total nitrogen (N), and NH4+-N contents. The nirK, nirS, and nosZ copy numbers with OM and RF treatments were significant higher than that of the MF and CK treatments in the rhizosphere and bulk soil (p < 0.05). The principal coordinate analysis (PCoA) analysis showed that the different parts of root zone are the most important factors for the variation of denitrifying bacteria community, and the different fertilization treatments is the second important factors for the variation of denitrifying bacteria community. The MiSeq sequencing result showed that nirK, nirS and nosZ-type denitrifiers communities within bulk soil had lower species diversity compared with rhizosphere soil, and were dominated by Rhizobiales, Rhodobacterales, Burkholderiales, and Pseudomonadales. As a result, the application of fertilization practices had significant effects on soil N and PDR levels, and affected the abundance and community composition of N-functional microbes.

Effects of different fertilizer application methods on the community of nitrifiers and denitrifiers in a paddy soil

Nitrifiers and denitrifiers are the key drivers of N cycling in paddy soil. Little is known about the effects of different fertilization methods, especially side bar fertilization, on the community of nitrifiers and denitrifiers in paddy soils. Here we assess the relationships between soil physicochemical properties, denitrification and nitrification activities, and the underlying microbial communities in a surface layer of paddy field soil treated with different fertilization methods. Soil was unfertilized (control), treated with conventional chemical fertilizer (CF), CF plus pig manure (MC), or slow-release fertilizer (SR), or by slow-release side bar fertilization (SB). Soil was sampled after one season of early and late rice growth. We determined soil physicochemical properties, potential nitrification rates (PNR), and denitrification enzyme activities (DEA). Ammonia-oxidizing archaeal (AOA) and bacterial (AOB) communities were assessed via their ammonia monooxygenase (amoA) genes, and denitrifiers via nitrite reductases (nirK and nirS) and nitrous oxide reductase (nosZ). Quantitative PCR was used to assess gene abundance, terminal restriction fragment polymorphism (T-RFLP) to investigate fertilization effects on microbial communities, and clone library sequencing and phylogenetic analysis to explore the taxonomic diversity of the nitrifiers and denitrifiers. Fertilization significantly increased the amount of NH4 +-N in the soil of SB and MC treatments, whereas MC lowered the NO3 −-N level. DEA was higher for MC and CF than the other treatments. The PNR in MC-treated soil was significantly lower than that in CF-treated soil. There were no significant differences in AOA and nirS copy numbers; however, nirK and nosZ copy numbers were higher for MC compared with CF. The number of AOA terminal restriction fragments (TRFs) increased significantly with N addition and reached the highest level for SB, whereas the number of AOB TRFs did not change significantly between treatments. Similarly, the number of nirK TRFs increased under fertilization, with the highest number obtained for SR; however, no significant change was observed for nirS and nosZ TRFs across different treatments, except for their relative abundance. All AOA amoA genotypes were in archaeal group 1.1b, whereas 95% AOB were in Nitrosospira cluster 3a. More than 40% of nirS OTUs were affiliated with Herbaspirillum, a key N-cycle player in this paddy soil. The SB and MC treatments had significant effects on soil N, DEA, and PNR levels, and affected the community of N-functional microbes. SB in combination with pig manure would contribute to the improvement of paddy soil fertility.

Effects of Short and Long-Term Hydrological Regimes on the Composition and Function of Denitrifying Bacteria in Experimental Wetlands

We investigated effects of short- and long-term hydrologic fluctuations on denitrification in constructed wetland basins. N2 and N2O emission rates, their ratio and a number of physicochemical variables were compared between pulsed (with annual drawdown-flooding cycles) and static (constantly flooded) wetland basins. The abundance and structure of general and denitrifying bacterial communities were compared based on quantitative PCR and terminal restriction fragment polymorphism analyses of 16S rRNA and nosZ genes, respectively. These variables significantly differed among sampling months, but not between the two long-term hydrologic treatments. The same set of variables were measured and compared among samples from different sites of one pulsed basin. The single basin experiment revealed temporal shifts in measured variables, which coincided with short-term hydrological changes. Bank sites, which had larger changes in abiotic conditions, showed greater variability in denitrifying activities than basin sites. Significant difference in N2 and N2O emission rates, 16S rRNA and nosZ gene abundance and structural composition was also identified between basin and bank sites. Our results suggest that activity and community structure of denitrifying bacteria in wetland soils are insensitive to moderate hydrology change, but can be significantly impacted by large hydrological fluctuation at both short-term and long-term scales.

Gastric microbiota in the functional dyspepsia patients treated with probiotic yogurt

ObjectiveTo investigate the structure of the gastric microbiota in functional dyspepsia (FD) and its role in the pathophysiology.DesignWe compared the basic physiological properties of the gastric fluid (GF) and the...

Change of methane production pathway with sediment depth in a lake on the Tibetan plateau

Methane production is the terminal step in the degradation of organic matter in most anoxic lake sediments. It has been suggested that CH4 production in lake sediments is dominated by hydrogenotrophic methanogenesis, especially in deeper layers. We investigated the vertical sediment profile from the surface to 50cm depth in the sediment of Bangong Co, an oligotrophic high altitude lake on the Tibetan plateau. We measured CH4 production, stable carbon isotopefractionation and the archaeal community structure. We found that the methane production rates, the acetate concentrations and the numbers of bacteria and archaea strongly decreased with sediment depth. The enrichment factor (ε) for hydrogenotrophic methanogenesis also decreased with depth, while δ13C of acetate stayed fairly constant. The contribution of hydrogenotrophic methanogenesis to total CH4 production increased with depth from ~36% to 100%. Analysis of terminal restriction fragment polymorphism (T-RFLP) of archaeal 16S rRNA genes showed that the relative abundance of aceticlastic (Methanosaetaceae) methanogens also decreased with depth disappearing completely at 50cm depth. Our study firstly showed that the methanogenic pathway and the methanogenic archaeal community systematically changed with sediment depth in a high altitude lake, probably controlled by the availability of easily degradable organic matter.

Population Dynamics and Community Composition of Ammonia Oxidizers in Salt Marshes after the Deepwater Horizon Oil Spill.

The recent oil spill in the Gulf of Mexico had significant effects on microbial communities in the Gulf, but impacts on nitrifying communities in adjacent salt marshes have not been investigated. We studied persistent effects of oil on ammonia-oxidizing archaeal (AOA) and bacterial (AOB) communities and their relationship to nitrification rates and soil properties in Louisiana marshes impacted by the Deepwater Horizon oil spill. Soils were collected at oiled and unoiled sites from Louisiana coastal marshes in July 2012, 2 years after the spill, and analyzed for community differences based on ammonia monooxygenase genes (amoA). Terminal Restriction Fragment Polymorphism and DNA sequence analyses revealed significantly different AOA and AOB communities between the three regions, but few differences were found between oiled and unoiled sites. Community composition of nitrifiers was best explained by differences in soil moisture and nitrogen content. Despite the lack of significant oil effects on overall community composition, we identified differences in correlations of individual populations with potential nitrification rates between oiled and unoiled sites that help explain previously published correlation patterns. Our results suggest that exposure to oil, even 2 years post-spill, led to subtle changes in population dynamics. How, or if, these changes may impact ecosystem function in the marshes, however, remains uncertain.

Site preparation and fertilization of wet forests alter soil bacterial and fungal abundance, community profiles and CO2 fluxes

Nitrogen (N) fertilization and mechanical site preparation are used to increase productivity of forests and prevent paludification in wet climates. However, these practices may also alter soil microbial community structure and rates of decomposition and respiration. The effects of mounding, drainage and fertilization were investigated at a mounded hybrid spruce site and a drained coastal western redcedar – western hemlock site in British Columbia (B.C.), Canada. CO2 efflux was measured at four dates over a one-year period using static closed chambers. Total bacterial and fungal ribosomal marker abundance and structure were assessed using quantitative PCR and terminal-restriction fragment polymorphism (T-RFLP), respectively. Mounding reduced soil water content of planting sites, but also removed much of the forest floor layer from the rooting zone and led to a significant decrease in CO2 efflux rates, as well as bacterial and fungal abundance and diversity. Drainage increased CO2 fluxes and led to significant differences in T-RFLP profiles in drained and undrained samples. Fertilization had variable effects on the microbial community and CO2 fluxes at the two sites. Site preparation and fertilization can improve regeneration of wet forests that would otherwise be subject to paludification, but can lead to shifts in carbon fluxes and microbial community structure. Major disturbances to soil structure and composition (e.g., following mounding) can alter the microbial community structure in a way that suppresses microbial diversity and function for up to one year following treatment.

Impact of long-term fertilizer treatment on the microeukaryotic community structure of a rice field soil

Microeukaryotes play many different functional roles in soil ecosystems, but the environmental factors that shape their community composition are poorly understood. Here we studied the impact of chemical and organic fertilizers on the microeukaryotic soil communities of rice field plots under long-term (82 years) fertilizer management. Terminal restriction fragment polymorphism (T-RFLP) and clone library analyses of 18S rRNA genes and their transcripts obtained from soils subjected to five different fertilizer treatments showed that fertilizer management strongly influenced microeukaryotic community composition in the soils and that its impact was greater than that of water management or seasonality. Among the different chemical fertilizers, the effects of phosphorus fertilizer on community composition were more pronounced than those of either nitrogen or potassium. Soils without phosphorus fertilizer were characterized by a high relative abundance of diatoms and algivorous nematodes whereas in phosphorus-fertilized soils green algae predominated. Different rates of organic fertilizer (rice straw compost) application had distinctive effects on the DNA-based microeukaryotic community, especially its algal composition. Thus, protistan grazers were the largest group in the potentially active community from soils with a higher amount of organic fertilizer application. Our findings provide evidence that long-term fertilization shapes the microeukaryotic community composition of rice field soils. The results imply that fertilizer management of rice field soils is an important determinant of the structure and function of the microbial food web.

English

Following an increased interest in management practices designed to reduce posed phytotoxicity during phytoremediation experiments, crude oil polluted soil and variants from conditioners-aided phytoremediation experiment were subjected to terminal restriction fragment polymorphism (t-RFLP) to evalute the biodiversity of bacterial microflora of polluted soil and amendments conditions. Genetic fingerprinting showed that hydrocarbons stress led to depletion of the genetic resources of soil microflora and to a radical change in its qualitative composition. The amended stressed soils not only has a greater number of species present, but the individuals in the community are distributed more equitably among these species. Non-uniform marginal regain of community was clear with applied conditioner. Positive associations, however were observed with conditioner and phyto-assisted clean-up attempts. Keywords: Crude oil pollution, soil conditioners, microbial diversity, phytoremediation, environment African Journal of Biotechnology , Vol 13(44) 4220-4225

Effect of Disinfectant, Water Age, and Pipe Materials on Bacterial and Eukaryotic Community Structure in Drinking Water Biofilm

Availability of safe, pathogen-free drinking water is vital to public health; however, it is impossible to deliver sterile drinking water to consumers. Recent microbiome research is bringing new understanding to the true extent and diversity of microbes that inhabit water distribution systems. The purpose of this study was to determine how water chemistry in main distribution lines shape the microbiome in drinking water biofilms and to explore potential associations between opportunistic pathogens and indigenous drinking water microbes. Effects of disinfectant (chloramines, chlorine), water age (2.3 days, 5.7 days), and pipe material (cement, iron, PVC) were compared in parallel triplicate simulated water distribution systems. Pyrosequencing was employed to characterize bacteria and terminal restriction fragment polymorphism was used to profile both bacteria and eukaryotes inhabiting pipe biofilms. Disinfectant and water age were both observed to be strong factors in shaping bacterial and eukaryotic community structures. Pipe material only influenced the bacterial community structure (ANOSIM test, P < 0.05). Interactive effects of disinfectant, pipe material, and water age on both bacteria and eukaryotes were noted. Disinfectant concentration had the strongest effect on bacteria, while dissolved oxygen appeared to be a major driver for eukaryotes (BEST test). Several correlations of similarity metrics among populations of bacteria, eukaryotes, and opportunistic pathogens, as well as one significant association between mycobacterial and proteobacterial operational taxonomic units, provides insight into means by which manipulating the microbiome may lead to new avenues for limiting the growth of opportunistic pathogens (e.g., Legionella) or other nuisance organisms (e.g., nitrifiers).

The Choice of PCR Primers Has Great Impact on Assessments of Bacterial Community Diversity and Dynamics in a Wastewater Treatment Plant

Assessments of bacterial community diversity and dynamics are fundamental for the understanding of microbial ecology as well as biotechnological applications. We show that the choice of PCR primers has great impact on the results of analyses of diversity and dynamics using gene libraries and DNA fingerprinting. Two universal primer pairs targeting the 16S rRNA gene, 27F&1492R and 63F&M1387R, were compared and evaluated by analyzing the bacterial community in the activated sludge of a large-scale wastewater treatment plant. The two primer pairs targeted distinct parts of the bacterial community, none encompassing the other, both with similar richness. Had only one primer pair been used, very different conclusions had been drawn regarding dominant phylogenetic and putative functional groups. With 27F&1492R, Betaproteobacteria would have been determined to be the dominating taxa while 63F&M1387R would have described Alphaproteobacteria as the most common taxa. Microscopy and fluorescence in situ hybridization analysis showed that both Alphaproteobacteria and Betaproteobacteria were abundant in the activated sludge, confirming that the two primer pairs target two different fractions of the bacterial community. Furthermore, terminal restriction fragment polymorphism analyses of a series of four activated sludge samples showed that the two primer pairs would have resulted in different conclusions about community stability and the factors contributing to changes in community composition. In conclusion, different PCR primer pairs, although considered universal, target different ranges of bacteria and will thus show the diversity and dynamics of different fractions of the bacterial community in the analyzed sample. We also show that while a database search can serve as an indicator of how universal a primer pair is, an experimental assessment is necessary to evaluate the suitability for a specific environmental sample.

Evaluation of molecular community analysis methods for discerning fecal sources and human waste

Molecular microbial community analyses provide information on thousands of microorganisms simultaneously, and integrate biotic and abiotic perturbations caused by fecal contamination entering water bodies. A few studies have explored community methods as emerging approaches for microbial source tracking (MST), however, an evaluation of the current state of this approach is lacking. Here, we utilized three types of community-based methods with 64 blind, single- or dual-source, challenge samples generated from 12 sources, including: humans (feces), sewage, septage, dogs, pigs, deer, horses, cows, chickens, gulls, pigeons, and geese. Each source was a composite from multiple donors from four representative geographical regions in California. Methods evaluated included terminal restriction fragment polymorphism (TRFLP), phylogenetic microarray (PhyloChip), and next generation (Illumina) sequencing. These methods correctly identified dominant (or sole) sources in over 90% of the challenge samples, and exhibited excellent specificity regardless of source, rarely detecting a source that was not present in the challenge sample. Sensitivity, however, varied with source and community analysis method. All three methods distinguished septage from human feces and sewage, and identified deer and horse with 100% sensitivity and 100% specificity. Method performance improved if the composition of blind dual-source reference samples were defined by DNA contribution of each single source within the mixture, instead of by Enterococcus colony forming units. Data analysis approach also influenced method performance, indicating the need to standardize data interpretation. Overall, results of this study indicate that community analysis methods hold great promise as they may be used to identify any source, and they are particularly useful for sources that currently do not have, and may never have, a source-specific single marker gene.

Acidobacteriadominate the active bacterial communities of Arctic tundra with widely divergent winter-time snow accumulation and soil temperatures

The timing and extent of snow cover is a major controller of soil temperature and hence winter-time microbial activity and plant diversity in Arctic tundra ecosystems. To understand how snow dynamics shape the bacterial communities, we analyzed the bacterial community composition of windswept and snow-accumulating shrub-dominated tundra heaths of northern Finland using DNA- and RNA-based 16S rRNA gene community fingerprinting (terminal restriction fragment polymorphism) and clone library analysis. Members of the Acidobacteria and Proteobacteria dominated the bacterial communities of both windswept and snow-accumulating habitats with the most abundant phylotypes corresponding to subdivision (SD) 1 and 2 Acidobacteria in both the DNA- and RNA-derived community profiles. However, different phylotypes within Acidobacteria were found to dominate at different sampling dates and in the DNA- vs. RNA-based community profiles. The results suggest that different species within SD1 and SD2 Acidobacteria respond to environmental conditions differently and highlight the wide functional diversity of these organisms even within the SD level. The acidic tundra soils dominated by ericoid shrubs appear to select for diverse stress-tolerant Acidobacteria that are able to compete in the nutrient poor, phenolic-rich soils. Overall, these communities seem stable and relatively insensitive to the predicted changes in the winter-time snow cover.

Terminal-Restriction Fragment Length Polymorphism (T-RFLP) Analysis for Changes in the Gut Microbiota Profiles of Indomethacin- and Rebamipide-Treated Mice

Background/Aims: We investigated the effects of indomethacin and rebamipide on the gut microbiota profiles using terminal restriction fragment polymorphism (T-RFLP) analysis. Materials and Methods: Female C57BL/6J mice were given indomethacin (10 mg/kg, s.c.) once a day and 2.5 mg rebamipide orally 3 times a day. After 7 days, they were sacrificed, and luminal contents were obtained from the ileum and cecum. The gut microbiota communities were analyzed by T-RFLP analysis with BslI digestion. Results: T-RFLP analyses showed that rebamipide and indomethacin had no significant effects on the gut microbiota profiles in the ileum and cecum. In contrast, the combination of rebamipide + indomethacin induced a significant change in the gut microbiota. The changes in the microbiota composition induced by the combination of rebamipide + indomethacin were characterized by the increase in the orders Bifidobacteriales and Lactobacillales, the genera Bacteroides and Prevotella and the family Clostridiaceae. The diversity of the gut microbiota community generated by the combination of rebamipide + indomethacin was significantly higher than those induced by either rebamipide or indomethacin alone. Conclusion: The combination of rebamipide + indomethacin induces remarkable changes in the gut microbiota composition and diversity. The clinical activity of rebamipide on nonsteroidal anti-inflammatory drug-induced intestinal injury may be exerted through a modulation of the gut microbiota.

Molecular Survey of the Occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and Amoeba Hosts in Two Chloraminated Drinking Water Distribution Systems

The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems.

Culture-Based Assessment of Microbial Communities in Soil Suppressive to Potato Common Scab.

A field in East Lansing, MI, showed a decline of potato common scab compared with an adjacent potato field. To confirm that the decline was due to biological factors, the soil was assayed. In the greenhouse, putative common-scab-suppressive soil (SS) was either treated with various temperatures or mixed with autoclaved SS at various ratios. Pathogenic Streptomyces scabies was incorporated into the treated soil at 106 CFU/cm3 of soil, followed by planting of either potato or radish. Disease severity was negatively correlated with the percentage of SS in the mixture and positively correlated with temperature above 60°C. The soil was screened for four groups of potential antagonists (general bacteria, streptomycetes, fluorescent pseudomonads, and bacilli) pairing in culture with S. scabies. The frequency of antagonistic bacteria in SS was higher than common-scab-conducive soil (CS) in all four groups but only pseudomonads and streptomycetes were significantly higher. The population of pathogenic Streptomyces spp. in the rhizosphere of CS was significantly higher than SS. Dilution plating of CS and SS samples showed no clear trends or differences in populations of total fungi, total bacteria, streptomycetes, fluorescent pseudomonads, and bacilli but terminal restriction fragment polymorphism analysis revealed two distinct microbial communities were present in SS and CS.