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Eisenia fetida (Oligochaeta, Lumbricidae) activates fungal growth, triggering cellulose decomposition during vermicomposting.

Cellulose is the most abundant polymer in nature and constitutes a large pool of carbon for microorganisms, the main agents responsible for soil organic matter decomposition. Cellulolysis occurs as the result of the combined action of fungi and bacteria with different requirements. Earthworms influence decomposition indirectly by affecting microbial population structure and dynamics and also directly because the guts of some species possess cellulolytic activity. Here we assess whether the earthworm Eisenia fetida (Savigny 1826) digests cellulose directly (i.e., with its associated gut microbiota) and also whether the effects of E. fetida on microbial biomass and activity lead to a change in the equilibrium between fungi and bacteria. By enhancing fungal communities, E. fetida would presumably trigger more efficient cellulose decomposition. To evaluate the role of E. fetida in cellulose decomposition, we carried out an experiment in which pig slurry, a microbial-rich substrate, was treated in small-scale vermireactors with and without earthworms. The presence of earthworms in vermireactors significantly increased the rate of cellulose decomposition (0.43 and 0.26% cellulose loss day(-1), with and without earthworms, respectively). However, the direct contribution of E. fetida to degradation of cellulose was not significant, although its presence increased microbial biomass (C(mic)) and enzyme activity (cellulase and beta-glucosidase). Surprisingly, as fungi may be part of the diet of earthworms, the activity of E. fetida triggered fungal growth during vermicomposting. We suggest that this activation is a key step leading to more intense and efficient cellulolysis during vermicomposting of organic wastes.

Analysis of Variance↗

Analysis of different genetic traits and their association with biofilm formation in Staphylococcus epidermidis isolates from central venous catheter infections.

The aim of the present study was to characterize clinical isolates of Staphylococcus epidermidis, one of the bacterial species most often implicated in foreign-body-associated infections, for their ability to form biofilms and for the presence of mecA and IS256 element. Sixty-seven Staphylococcus epidermidis clinical isolates, obtained from implantable medical devices, were investigated. Overall, 70% of the strains were positive for ica operon genes, 85% possessed atlE, and 46% contained aap. In 89% of the population, the Congo red agar test confirmed the correlation between the presence of ica genes and slime expression. Almost all of the strains could be classified as biofilm producers by both the crystal violet assay and microscopy. The bacterial population studied showed a very high frequency of strains positive for mecA as well as for the IS256 element. Although well-structured biofilms have been previously observed only in those strains possessing genes belonging to the ica operon, this study demonstrates that strains lacking specific biofilm-formation determinants can be isolated from catheters and can form a biofilm in vitro. Hence, different and yet-to-be identified factors may work together in the formation and organization of complex staphylococcal microbial communities and sustain infections associated with implanted medical devices.

Anti-Bacterial Agents↗

Rhizosphere microbial activity during phytoremediation of diesel-contaminated soil.

To know microbial activity and diesel-removal efficiency influencing through plant roots, we examined the effect of the rhizosphere on phytoremediation of diesel-contaminated soils by alfalfa (Medicago sativa L.). Pots were treated with and without diesel and allowed to stabilize for 7 weeks, at which time four experimental/control groups were prepared: (1) planted diesel-contaminated soil, (2) unplanted diesel-contaminated soil, (3) planted uncontaminated soil, and (4) unplanted uncontaminated soil. Samples of rhizosphere and bulk soils were separately taken from all planted pots. After 7 weeks of alfalfa growth from seeds, the removal efficiencies in rhizosphere and bulk soil samples were 82.5% and 36.5 approximately 59.4%, respectively. The total microbial activity was highest in diesel-contaminated rhizosphere soils. Significantly more culturable soil bacteria and hydrocarbon-degraders were found in diesel-contaminated rhizosphere soil versus unplanted and uncontaminated bulk soil, with a greater increase seen in hydrocarbon-degraders (172-fold) versus general soil bacteria (14-fold). DGGE (Denaturing Gel Gradient Electrophoresis) analysis revealed that the bacterial community structure was most highly influenced by the combined presence of diesel contamination and plant roots (39.13% similarity compared to the control), but that diesel contamination alone had a higher influence (42.31% similarity compared to the control) than the rhizosphere (50.00% similarity compared to the control). Sequence analysis and BLAST searches revealed that all samples were dominated by members of alpha -, gamma -, delta - and epsilon -proteobacteria, and Chloroflexi. The rhizosphere samples additionally contained novel dominant members of alpha -proteobacteria and Cytophaga-Flexibacter-Bacteroides, while the diesel samples contained additional dominant alpha -proteobacteria and the rhizosphere plus diesel samples contained other epsilon -proteobacteria. Collectively, these findings indicate that the presence of plant roots (i.e., a rhizosphere) had a greater effect on bacterial activity in diesel contamination than did the absence of diesel contamination, whereas diesel contamination had a greater effect on bacterial community structure. These novel findings provide new insight into the mechanisms of phytoremediation.

Biodegradation, Environmental↗

Factors influencing the composition of bacterial communities found at abandoned copper-tailings dumps.

AIMS: To study the effect that copper residues exert on bacterial communities and the ability of bacteria to colonize different microhabitats in abandoned tailing dumps. METHODS AND RESULTS: We used the terminal-restriction fragment length polymorphism technique, a culture-independent molecular approach based on PCR amplification of ribosomal genes, to compare the structure of the bacterial communities from samples taken at two nearby located abandoned tailing dumps found in the Mediterranean-climate area of central Chile. Our results show that elevated available copper content in tailings has a strong effect on the bacterial community composition, but that other factors like pH and organic matter content also play an important role in the structure of these communities. We also found that the number of abundant bacteria in these samples was significantly lower than in soils not exposed to metal pollution. CONCLUSIONS: In addition to bioavailable copper, bacterial communities found in copper-tailings dumps are also affected by several other environmental factors. SIGNIFICANCE AND IMPACT OF THE STUDY: This first report on environmental factors influencing microbial communities in copper-tailings dumps will help to devise appropriate restoration procedures in this type of polluted habitat.

Bacteria↗

Indigenous microflora responses to introduction of cyanogenic strains of Pseudomonas fluorescens into soil.

The effects of cyanogenic Pseudomonas fluorescens strains introduced into soil on the kinetic of colony formation and bacterial community structure were investigated. About 7.8 x 10(8) and 1.2 x 10(9) cfu per g dry soil of TA1 and B2 were added to the soil portions, respectively. The parameters of colony formation by heterotrophic soil bacteria were determined. The bacterial community structure and phenotypic diversity were studied using concept of r/K strategies and echophysiological index, respectively. The physiological state of indigenous heterotrophic bacteria and gram-negative group did not change under the influence of the cyanogenic strains introduced. Phenotypic diversity of the soil bacteria also did not change significantly. However, some short-term shifts in community structure of indigenous heterotrophic bacteria were noticed. This study shows that the introduction of great numbers of cyanogenic P. fluorescens strains could be safely used as potential agents in biological control of soil-born pathogens.

Bacteria↗

Involvement of cross-genus phages in bacterial resistance to chlorine disinfection.

Chlorine disinfection resistance in pathogenic microorganisms poses severe environmental concerns and public health risks. While phages play critical roles in host adaptation to environmental stress, how poly-host phages contribute to bacterial resistance to chlorine disinfectants remains poorly understood. Here, we investigated shifts in the population dynamics, transcriptional profiles, and function potentials of cross-genus phage-bacterial communities under exposure to chlorine disinfectants in a continuously operated anaerobic-anoxic-oxic system over a 92-day period, using integrated metagenomic and metatranscriptomic approaches. In the presence and absence of chlorine disinfectants, the genomic abundance and diversity of phage and bacterial communities showed similar variation trends, and the community structures of both exhibited clear differences. A strong significant positive correlation was observed between phage and bacterial diversity under chlorine exposure (R&#x202f;=&#x202f;0.975, p&#x202f;=&#x202f;0.00,057), whereas no significant correlation was detected in the absence of chlorine disinfection (R&#x202f;=&#x202f;-0.314, p&#x202f;=&#x202f;0.613), suggesting that chlorine disinfectants may enhance phage-bacteria interactions. Host-associated phages exhibited high consistency with their corresponding putative hosts in terms of genomic abundance (M2&#x202f;=&#x202f;0.0945, p&#x202f;=&#x202f;0.001) and transcript abundance (M2&#x202f;=&#x202f;0.3668, p&#x202f;=&#x202f;0.001), and they were also significantly correlated with cross-genus phages in both genomic abundance (R&#x202f;=&#x202f;0.97, p&#x202f;<&#x202f;2.2e-16) and transcript abundance (R&#x202f;=&#x202f;0.83, p&#x202f;<&#x202f;2.2e-16), which collectively suggests the critical role of cross-genus phages in the resistance of microbial communities to chlorine disinfectants. Bipartite association network analysis shows that cross-genus phages carry highly homologous genes to their putative hosts and may be involved in the horizontal transfer of these genes among bacteria. These homologous genes are involved in DNA repair, redox balance regulation, environmental stress adaptation and efflux pump functions, suggesting a synergistic role between cross-genus phages and their putative hosts in chlorine resistance. Our findings reveal that cross-genus phages can contribute to the resistance of bacterial communities to chlorine disinfectants, providing the theoretical foundation for evaluating the role of poly-host phages in microbial communities.

Chlorine resistance↗

Feedback interactions between needle litter decomposition and rhizosphere activity.

The aim of our study was to identify interactions between the decomposition of aboveground litter and rhizosphere activity. The experimental approach combined the placement of labelled litter (delta13C=-37.9 per thousand ) with forest girdling in a 35-year-old Norway spruce stand, resulting in four different treatment combinations: GL (girdled, litter), GNL (girdled, no litter), NGL (not girdled, litter), and NGNL (not girdled, no litter). Monthly sampling of soil CO2 efflux and delta13C of soil respired CO2 between May and October 2002 allowed the partitioning of the flux into that derived from the labelled litter, and that derived from native soil organic matter and roots. The effect of forest girdling on soil CO2 efflux was detectable from June (girdling took place in April), and resulted in GNL fluxes to be about 50% of NGNL fluxes by late August. The presence of litter resulted in significantly increased fluxes for the first 2 months of the experiment, with significantly greater litter derived fluxes from non-girdled plots and a significant interaction between girdling and litter treatments over the same period. For NGL collars, the additional efflux was found to originate only in part from litter decomposition, but also from the decay of native soil organic matter. In GL collars, this priming effect was not significant, indicating an active role of the rhizosphere in soil priming. The results therefore indicate mutual positive feedbacks between litter decomposition and rhizosphere activity. Soil biological analysis (microbial and fungal biomass) of the organic layers indicated greatest activity below NGL collars, and we suppose that this increase indicates the mechanism of mutual positive feedback between rhizosphere activity and litter decomposition. However, elimination of fresh C input from both above- and belowground (GNL) also resulted in greater fungal abundance than for the NGNL treatment, indicating likely changes in fungal community structure (i.e. a shift from symbiotic to saprotrophic species abundance).

Biomass↗

Impact of iron salts on activated sludge and interaction with nitrite or nitrate.

Iron salts are often used in activated sludge treatment plants as coagulants or to improve reactor performance. Previous studies have indicated that iron itself has an impact on the activated sludge process. However, the interaction of iron with nitrite or nitrate present in the sludge has received little attention. In this research, the influence of addition of Fe(II) or Fe(III), alone or together with NO(2)(-) or NO(3)(-) on bench-scale activated sludge reactors was examined. Large differences were established between the distinct treatments, regarding reactor performance, sludge characteristics as well as microbial community. Ferric iron was more detrimental than ferrous iron. In some cases, nitrite was found to enhance inhibitory effects of the added iron, whereas nitrate had more a neutralizing effect. It was found that precipitation of phosphate by the iron was not responsible for the observed inhibition. Decrease in pH upon formation of iron hydroxides and the impairment of the floc structure could partially explain the toxicity of the iron dosages. The formation of toxic nitrogen oxides, such as nitric oxide, can also be of importance. The observed positive effect of nitrate on the floc activity is of interest and warrants further elucidation.

Biodegradation, Environmental↗

Study of nitrogen fixation in microbial communities of oil-contaminated marine sediment microcosms.

Aerobic microbial degradation of pollutant oil (petroleum) in aquatic environments is often severely limited by the availability of combined nitrogen. We therefore studied whether the microbial community enriched in marine sediment microcosms with an added oil layer and exposure to light harboured nitrogenase activity. The acetylene reduction (AR) assay indeed indicated active nitrogenase; however, similar activity was observed in oil-free control microcosms. In both microcosms, the AR rate was significantly reduced upon a dark shift, indicating that enriched cyanobacteria were the dominant diazotrophs. Analysis of structural dinitrogenase reductase genes (nifH) amplified from both microcosms indeed revealed NifH sequences related mostly to those of heterocystous cyanobacteria. NifH sequences typically affiliating with those of heterotrophic bacteria were more frequently retrieved from the oil-containing sediment. Expression analyses showed that mainly nifH genes similar to those of heterocystous cyanobacteria were expressed in the light. Upon a dark shift, nifH genes related to those of non-heterocystous cyanobacteria were expressed. Expression of nifH assignable to heterotrophs was apparently not significant. It is concluded that cyanobacteria are the main contributors of fixed nitrogen to oil-contaminated and pristine sediments if nitrogen is a limiting factor and if light is available. Hence, also the oil-degrading heterotrophic community may thus receive a significant part of combined nitrogen from cyanobacteria, even though oil vice versa apparently does not stimulate an additional nitrogen fixation in the enriched community.

Amino Acid Sequence↗

A graph-based approach for the visualisation and analysis of bacterial pangenomes.

BACKGROUND: The advent of low cost, high throughput DNA sequencing has led to the availability of thousands of complete genome sequences for a wide variety of bacterial species. Examining and interpreting genetic variation on this scale represents a significant challenge to existing methods of data analysis and visualisation. RESULTS: Starting with the output of standard pangenome analysis tools, we describe the generation and analysis of interactive, 3D network graphs to explore the structure of bacterial populations, the distribution of genes across a population, and the syntenic order in which those genes occur, in the new open-source network analysis platform, Graphia. Both the analysis and the visualisation are scalable to datasets of thousands of genome sequences. CONCLUSIONS: We anticipate that the approaches presented here will be of great utility to the microbial research community, allowing faster, more intuitive, and flexible interaction with pangenome datasets, thereby enhancing interpretation of these complex data.

Bacteria↗

Effects of large-scale poultry farms on aquatic microbial communities: a molecular investigation.

The effects of large-scale poultry production operations on water quality and human health are largely unknown. Poultry litter is frequently applied as fertilizer to agricultural lands adjacent to large poultry farms. Run-off from the land introduces a variety of stressors into the surface waters including nutrients, antimicrobials and pathogenic bacteria. The Delaware, Maryland and Virginia (Delmarva) Peninsula has the highest concentration of broiler chickens per farm acre in the United States and provides an ideal location for studying the effects of stressors from poultry farms. We investigated potential effects by characterizing shifts in the structure of aquatic bacterial communities. DNA was isolated from microorganisms in water samples from streams and rivers at varying distances from, or having different frequencies of, litter applications. Fingerprints of 16S rDNA amplicons from bacteria in water samples collected during late summer 2001 to late spring 2002 were produced by denaturing gradient gel electrophoresis (DGGE). A statistical analysis of multiple fingerprints from each sampling location demonstrated that each site harboured a bacterial community significantly different from the communities at other sites. Similarly, the bacterial communities from each sampling time differed significantly from communities at other sampling times. Most importantly, a competitive, library-based analysis showed time of sampling (month) had a greater effect on community structure than did location.

Agriculture↗

Symbiotic microorganisms, a key for ecological success and protection of plants.

Plant-associated microbial diversity encompasses symbionts, protecting their host against various aggressions. Mycorrhizal and rhizospheric microorganisms buffer effects of soil toxic compounds and soil-borne pathogens. Endophytic bacteria and fungi, some of which are vertically inherited through seeds, take part in plant protection by acting directly on aggressive factors (mainly pathogens and herbivores) or by enhancing plant responses. Plant protective microbial symbionts determine the ecological success of plants; they drastically modify plant communities and related trophic webs. This review suggests approaches to improve the inventory of diversity and functions of in situ plant-associated microorganisms.

Ecology↗

Effects of T4 lysozyme release from transgenic potato roots on bacterial rhizosphere communities are negligible relative to natural factors.

Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for Bacteria, Actinomycetales, or alpha- or beta-Proteobacteria. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.

Bacteria↗

Characterization of metal-resistant soil eubacteria by polymerase chain reaction--denaturing gradient gel electrophoresis with isolation of resistant strains.

Contamination of soils with heavy metal ions is a major problem on industrial and defense-related sites worldwide. The bioavailability and mobility of these contaminants is partially determined by the microbial biomass present at these sites. In this study, we have assessed the effect of the addition of a mixture of toxic metal salts on the prokaryotic community of microcosms consisting of sandy-loam soil using direct molecular analysis of the recoverable eubacterial 16S rDNA molecules by polymerase chain reaction--denaturing gradient gel electrophoresis (PCR-DGGE) and limited phospholipid fatty acid analysis (PLFA). Addition of toxic metals (nonradioactive surrogates of Sr, Co, Cs, Cd) resulted in rapid (ca. 1 week) changes in the DGGE profile of the indigenous eubacterial community when compared with pristine controls. These changes were stable over the course of the experiment (8 weeks). No changes in the eubacterial population of control microcosms were detected. The major changes in community structure in metal-contaminated microcosms consisted of the appearance of four novel bands not detected in controls. Sequence analysis of these bands suggested that two organisms related to the genus Acinetobacter and two related to the genus Burkholderia carried a selective advantage over other indigenous eubacteria under heavy metal induced stress. The Burkholderia spp. were then cultured and further characterized using lipid analysis.

Acinetobacter↗

Plasmids from Lactobacillus helveticus: distribution and diversity among natural isolates.

AIMS: To investigate the distribution and the level of diversity of extrachromosomal molecules in Lactobacillus helveticus strains in relation to their different ecological niches. METHODS AND RESULTS: The plasmid profile of 22 Lact. helveticus strains, isolated from five different Italian cheeses, was determined. Among the tested strains, there was a variable presence of plasmids: eight plasmid-free strains and the remaining with several plasmids that could be differentiated on the basis of number and molecular weight. The profiles showed between one and five plasmid bands, which size ranged between 2.3 and 31 kb. Four of these plasmids were further analyzed by restriction digestion and compared with the plasmids from Lact. helveticus ATCC 15009(T). Analyses and comparison of their primary structures and hybridization experiments revealed the presence of different DNA homology groups. CONCLUSIONS: This study indicates that within Lact. helveticus species, there is a high degree of variability in relation to the presence of plasmid molecules. Moreover, the structural diversity found among some of these plasmids allows to hypothesize the presence of different evolutionary lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on plasmid distribution and diversity should be considered as an essential component in a continuing effort to explore microbial diversity as well as to understand the real role of plasmids in the flow of genetic information in natural bacterial communities.

Cheese↗

Culture-independent molecular analysis of microbial constituents of the healthy human outer ear.

Molecular-phylogenetic sequence analyses have provided a new perspective on microbial communities by allowing the detection and identification of constituent microorganisms in the absence of cultivation. In this study we used broad-specificity amplification of ribosomal DNA (rDNA) genes to survey organisms present in the human outer ear canal. Samples were obtained from 24 individuals, including members of three extended families, in order to survey the resident microbiota and to examine microbial population structures in individuals related by familial or household associations. To examine the stability of the microbial populations, one individual was sampled four times and another twice over a 14-month period. We found that a distinct set of microbial types was present in the majority of the subjects sampled. The two most prevalent rDNA sequence types that were identified in multiple individuals corresponded closely to those of Alloiococcus otitis and Corynebacterium otitidis, commonly thought to be associated exclusively with infections of the middle ear. Our results suggest, therefore, that the outer ear canal may serve as a reservoir for normally commensal microbes that can contribute to pathogenesis upon introduction into the middle ear. Alternatively, culture analyses of diseases of the middle ear may have been confounded by these contaminating commensal organisms.

Bacteria↗

Changes in the microbiology of activated sludge upon the addition of iron salts with or without nitrite or nitrate.

Iron salts are frequently used in activated sludge treatment as coagulants or to improve reactor performance. However, the effect of the iron salts on the microbiology has received little attention. Furthermore, the interaction of iron ions with nitrite or nitrate present in the sludge has not been considered. In this research, the influence of ferrous and ferric iron additions, and their possible interactions with nitrite or nitrate at levels of a few mM, on the activated sludge microbiology of semi-continuous bench-scale reactors were examined. Conventional as well as molecular techniques have been used to investigate the microbial communities. Microscopic investigations showed that repeated addition of ferric iron at 2 mM gave rise to breaking of the sludge flocs, resulting in massive growth of sessile-protozoa and lowering of the numbers of nitrifiers. Ferrous iron at the same dosage resulted in large coherent flocs. Nitrite hereby seemed to enhance the detrimental effect of the ferric iron on the floc structure, whereas nitrate had a neutralizing character. 16S rRNA gene analysis showed a clear impact of the different iron respectively nitrogen treatments on the composition of bacterial communities. Cloning the sludge samples revealed the increased presence of denitrifying Feoxidizing bacteria in both the Fe(II) and Fe(III) reactors, suggesting a redox-state cycling between Fe(II) and Fe(III) in the mixed liquor.

Bacteria↗

Impact of biocontrol agents Pseudomonas fluorescens CHA0 and its genetically modified derivatives on the diversity of culturable fungi in the rhizosphere of mungbean.

AIMS: To assess whether Pseudomonas fluorescens strain CHA0 and its genetically modified derivatives, CHA0/pME3424 (antibiotic over-producer) and CHA89 (antibiotic-deficient) could have an impact on the fungal community structure and composition in the rhizosphere of mungbean. METHODS AND RESULTS: Under glasshouse conditions, mungbean was grown repeatedly in the same soil, which was inoculated with CHA0, CHA0/pME3424, CHA89 or was left untreated. Treatments were applied to soil at the start of each 36-day mungbean growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first, second and third cycles. The effects of CHA0 and CHA0/pME3424 did differ from the controls while CHA89 did not. Whereas all major fungal species were frequently isolated from both bacterized and nonbacterized rhizospheres, certain fungal species were exclusively promoted or specifically suppressed from Pseudomonas-treated soils. In general, fungal diversity and equitability tended to decrease with time while species richness slightly increased. Whilst a total of 29 fungal species were isolated from the mungbean rhizosphere, only eight species colonized the root tissues. CONCLUSIONS: Soil inoculation with Ps. fluorescens CHA0 or CHA0/pME3424 altered fungal community structure in mungbean rhizosphere but strain CHA89 failed to produce such effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens-mediated alteration in the composition and structure of fungal communities might have acute or lasting effects on ecosystem functioning. Furthermore, the study provides useful data pertinent to characterization of the fate of genetically modified inoculants (e.g. antibiotic-overproducing Pseudomonas strains) released into the environment.

Antibiosis↗