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PubMed2006

Molecular characterization of subject-specific oral microflora during initial colonization of enamel.

The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.

Actinomyces↗

PubMed2006

Molecular techniques in wastewater: Understanding microbial communities, detecting pathogens, and real-time process control.

Traditionally, the detection of pathogens in water, wastewater, and other environmental samples is restricted by the ability to culture such organisms from complex environmental samples. During the last decade the use of molecular methods have supplied the means for examining microbial diversity and detecting specific organisms without the need for cultivation. The application of molecular techniques to the study of natural and engineered environmental systems has increased our insight into the vast diversity and interaction of microorganisms present in complex environments. In this paper, we will review the current and emerging molecular approaches for characterizing microbial community composition and structure in wastewater processes. Recent studies show that advances in microarray assays are increasing our capability of detecting hundreds and even thousands of DNA sequences simultaneously and rapidly. With the current progress in microfluidics and optoelectronics, the ability to automate a detection/identification system is now being realized. The status of such a system for wastewater monitoring is discussed.

DNA Fingerprinting↗

PubMed2006

Infection paradox: high abundance but low impact of freshwater benthic viruses.

The discovery of an abundant and diverse virus community in oceans and lakes has profoundly reshaped ideas about global carbon and nutrient fluxes, food web dynamics, and maintenance of microbial biodiversity. These roles are exerted through massive viral impact on the population dynamics of heterotrophic bacterioplankton and primary producers. We took advantage of a shallow wetland system with contrasting microhabitats in close proximity to demonstrate that in marked contrast to pelagic systems, viral infection, determined directly by transmission electron microscopy, and consequently mortality of prokaryotes were surprisingly low in benthic habitats in all seasons. This was true even though free viruses were abundant throughout the year and bacterial infection and mortality rates were high in surrounding water. The habitats in which we found this pattern include sediment, decomposing plant litter, and biofilms on aquatic vegetation. Overall, we detected viruses in only 4 of a total of approximately 15,000 bacterial cells inspected in these three habitats; for comparison, nearly 300 of approximately 5,000 cells suspended in the water column were infected. The strikingly low incidence of impact of phages in the benthos may have important implications, since a major portion of microbial biodiversity and global carbon and nutrient turnover are associated with surfaces. Therefore, if failure to infect benthic bacteria is a widespread phenomenon, then the global role of viruses in controlling microbial diversity, food web dynamics, and biogeochemical cycles would be greatly diminished compared to predictions based on data from planktonic environments.

Bacteria↗

PubMed1999

The search for unrecognized pathogens.

The distribution and diversity of microorganisms in the world are far greater than have been previously appreciated. Molecular, cultivation-independent methods have played a key role in this insight. To what extent do humans remain ignorant of microbial diversity within the human body and the settings in which microorganisms cause human disease? In addition to implicating microbial agents in nontraditional infectious diseases, the use of methods such as broad-range polymerase chain reaction, representational difference analysis, expression library screening, and host gene expression profiling may force a reassessment of the concepts of microbial disease causation.

Bacteria↗

PubMed2003

Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods.

DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley. DNA was also extracted from microbial enrichment cultures of sediment samples. 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons. Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons. Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82%. Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/ Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/ Aeromonas/Vibrio part of the gamma3 subdivision of the Proteobacteria.

Archaea↗

PubMed2006

Diversity and phylotype consistency of bacteria in the guts of three bee species (Apoidea) at an oilseed rape field.

The gut of insects may harbour one of the largest reservoirs of a yet unexplored microbial diversity. To understand how specific insects select for their own bacterial communities, the structural diversity and variability of bacteria found in the gut of different bee species was analysed. For three successive years, adults and larvae of Apis mellifera ssp. carnica (honey bee), and Bombus terrestris (bumble bee), as well as larvae of Osmia bicornis (red mason bee) were collected at a flowering oilseed rape field. Total DNA was extracted from gut material and the bacterial diversity was analysed, independent of cultivation, by genetic profiling with single-strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR)-amplified partial 16S rRNA genes. The SSCP profiles were specific for all bee species and for larvae and adults. Qualitative and quantitative differences were found in the bacterial community structure of larvae and adults of A. mellifera, but differences in B. terrestris were mainly quantitative. Sequencing of the PCR products revealed a dominance of Alpha-, Beta-, and Gammaproteobacteria, Bacteroidetes, and Firmicutes in all bee species. Single-strand conformation polymorphism profiles suggested a higher abundance and diversity of lactobacilli in adults of A. mellifera than in larvae. Further phylogenetic analyses indicated common bacterial phylotypes for all three bee species, e.g. those related to Simonsiella, Serratia, and Lactobacillus. Clades related to Delftia acidovorans, Pseudomonas aeruginosa or Lactobacillus intestinalis only contained sequences from larvae. Several of the bee-specific clusters also contained identical or highly similar sequences from bacteria detected in other A. mellifera subspecies from South Africa, suggesting the existence of cosmopolitan gut bacteria in bees.

Animals↗

PubMed2001

Phenotypic biomonitoring using multivariate flow cytometric analysis of multi-stained microorganisms.

A new method for monitoring phenotypic profiles of pure cultures and complex microbial communities was evaluated. The approach was to stain microorganisms with a battery of fluorescent dyes prior to flow cytometry analysis (FCM) and to analyse the data using multivariate methods, including principal component analysis and partial least squares. The FCM method was quantitatively evaluated using different mixtures of pure cultures as well as microbial communities. The results showed that the method could quantitatively and reproducibly resolve both populations and communities of microorganisms with 5% abundance in a diverse microbial background. The feasibility of monitoring complex microbial communities over time during the biodegradation of naphthalene using the FCM method was demonstrated. The biodegradation of naphthalene occurred to differing extents in microcosms representing three different types of aromatic-contaminated groundwater and a sample of bio-basin water. The FCM method distinguished each of these four microbial communities. The phenotypic profiles were compared with genotypic profiles generated by random-amplified polymorphic DNA analysis. The genotypic profiles of the microbial communities described only the microbial composition, and not their functional change, whereas the phenotypic profiles seemed to contain information on both the composition and the functional change of the microorganisms. Furthermore, event analysis of the FCM data showed that microbial communities with initially differing compositions could converge towards a similar composition if they had a capacity for high levels of degradation, whereas microbial communities with similar initial compositions could diverge if they differed in biodegrading ability.

Journal Article↗

PubMed2004

16 S rDNA primers and the unbiased assessment of thermophile diversity.

Our understanding of thermophile diversity is based predominantly on PCR studies of community DNA. "Universal" and domain-specific rRNA gene PCR primers have historically been used for the assessment of microbial diversity without adequate regard to the degree of specificity of primer pairs to different prokaryotic groups. In a reassessment of the published primers commonly used for "universal" and archaeal 16 S rDNA sequence amplification we note that substantial variations in specificity exist. An unconsidered choice of primers may therefore lead to significant bias in determination of microbial community composition. In particular, Archaea-specific primer sequences typically lack specificity for the Korarchaeota and Nanoarchaea and are often biased towards certain clades. New primer pairs specifically designed for "universal" archaeal 16 S rDNA sequence amplification, with homology to all four archaeal groups, have been designed. Here we present the application of these new primers for preparation of 16 S libraries from thermophile communities.

Base Sequence↗

PubMed2005

Effect of freezing and thawing on microbial activity and glyphosate degradation in two Norwegian soils.

Little research has been done on pesticide dissipation in cold climates and there is a need to focus on the influence of climate on pesticide degradation in soil. Glyphosate, N-(phosphonomethyl)glycine, is a herbicide frequently used for controlling perennial weeds through application after harvest and was used as a model compound for this study. The effect of freeze-thaw activity on the availability of glyphosate in soil, and consequently its mineralization by soil microorganisms, was studied through laboratory incubations of repacked soil cores treated with 14C-labelled glyphosate and subjected to different freeze-thaw treatments. Winter simulation regimes applied were constant thaw (+5 degrees C), constant freezing (-5 degrees C), unstable conditions with short fluctuations (24 h of -5 degrees C followed by 24 h of +5 degrees C), and long duration fluctuations (3 weeks of -5 degrees C followed by 3 weeks of +5 degrees C). Distribution of 14C-glyphosate was followed during the incubation through measurements of the mineralized fraction (14CO2), soil water fraction, KOH extractable fraction, and non-extractable fraction. Microbial parameters used to characterize the soils were estimates of size of microbial biomass, overall microbial activity and microbial diversity. The constant freezing treatment exhibited the lowest amount of glyphosate mineralization. The constant thawed treatment and the treatments with fluctuating temperature exhibited significantly increased mineralization. These results were in accordance with the observed concentration of glyphosate in soil water; the higher the activity, the lower the concentration. The amount of glyphosate extractable with KOH and the resulting non-extractable fraction, however, were not significantly affected by soil type or temperature regime. The glyphosate mineralization pattern was comparable with the overall microbial activity in the soils. Observed different levels of diversity might explain some of the difference in total glyphosate mineralization between soils.

Biodegradation, Environmental↗

PubMed2005

Changes in enzymes activity, substrate utilization pattern and diversity of soil microbial communities under cadmium pollution.

Heavy metal pollution has received increasing attention in recent years mainly because of the public awareness of environmental issues. In this study we have evaluated the effect of cadmium (Cd) on enzymes activity, substrate utilization pattern and diversity of microbial communities in soil spiked with 0, 20, 40, 60, 80, and 100 mg/kg Cd, during 60 d of incubation at 25 degrees C. Enzyme activities determined at 0, 15, 30, 45, and 60 d after heavy metal application (DAA) showed marked declines for various Cd treatments, and up to 60 DAA, 100 mg/kg Cd resulted in 50.1%, 47.4%, and 39.8% decreases in soil urease, acid phosphatase and dehydrogenase activities, respectively to control. At 60 DAA, substrate utilization pattern of soil microbial communities determined by inoculating Biolog ECO plates indicated that Cd addition had markedly inhibited the functional activity of soil microbial communities and multivariate analysis of sole carbon source utilization showed significantly different utilization patterns for 80 and 100 mg/kg Cd treatments. The structural diversity of soil microbial communities assessed by PCR-DGGE method at 60 DAA, illustrated that DGGE patterns in soil simplified with increasing Cd concentration, and clustering of DGGE profiles for various Cd treatments revealed that they had more than 50% difference with that of control.

Acid Phosphatase↗

PubMed2026

Comparative metagenomic assessment of Illumina-compatible library preparation methods, short-read lengths, and PacBio HiFi sequencing reveals differences in microbial and functional diversity recovery from a complex environmental sample.

UNLABELLED: Metagenomics enables comprehensive exploration of microbial communities but is influenced by library preparation and sequencing technologies, affecting recovery of microbial genomes and proteins. Here, we benchmarked six Illumina-compatible short-read library preparation conditions in triplicate at 2 × 150 bp and 2 × 250 bp read lengths alongside PacBio HiFi long-read sequencing using a composite environmental sample of marine mangrove sediment and terrestrial palm tree soil. Longer short reads (2 × 250 bp) combined with optimal library preparation approaches improved assembly quality, protein detection, and metagenome-assembled genome (MAG) recovery, achieving results approaching those of long-read sequencing. TruSeq libraries at 2 × 250 bp recovered more than sevenfold more unique proteins than the same kit at 2 × 150 bp (811,701 vs 110,108) using the same number of sequencing reads, while recovering a comparable number of high-quality MAGs to PacBio HiFi long-read sequencing (11 vs 18) and surpassing it in protein discovery by almost 10-fold (811,701 vs 87,745) at less than half of the sequencing cost. Furthermore, biosynthetic gene cluster analysis identified 46 biosynthetic gene clusters in TruSeq-250PE assemblies compared to 38 in PacBio HiFi, with several showing no close match in the MIBiG database. Although long reads yield more contiguity and complete genomes, longer short reads offer a cost-effective, scalable alternative for uncovering microbial and functional diversity. These findings provide critical guidance for metagenomic experimental design, demonstrating that strategic selection of library preparation chemistry and sequencing parameters can reveal more unknown microbial information in complex biomes without requiring additional sequencing depth. IMPORTANCE: Metagenomic outcomes are strongly influenced by library preparation and sequencing strategies, yet their combined effects in complex environmental samples remain poorly defined. Here, we provide the first direct comparison of Illumina NovaSeq short-read metagenomic sequencing at 2 × 150 bp and 2 × 250 bp across multiple library preparation kits, alongside PacBio HiFi long-read sequencing. We show that sequencing read length and library preparation critically shape assembly quality, protein recovery, and metagenome-assembled genome (MAG) reconstruction. These findings demonstrate that short-read sequencing at 2 × 250 bp, with appropriate library preparation, can match long-read technologies in MAG recovery while substantially surpassing them in protein discovery. With less than half of the sequencing price and a 3.5-fold reduction in cost per gigabase of usable data, this method facilitates more accessible large-scale metagenomic analysis within complex environmental systems.

Metagenomics↗

PubMed1998

The renaissance of microbiology.

Microbiology is finally occupying its true position as the pre-eminent field in life sciences. This is due to advances in molecular techniques that confirm the evolutionary significance of the biology of microbes. It is anticipated that the use of comparative genomics will provide information that will advance the understanding of mechanisms of pathogenesis and the importance of secondary metabolism in social microbiology. More emphasis on studies of microbial diversity will increase its value in both fundamental microbiology and its industrial applications.

Biological Evolution↗

PubMed2006

Investigating the heterogeneity of cell growth in microbial colonies by FTIR microspectroscopy.

Microorganisms rarely occur as individual cells in nature and are, instead, organized in complex multicellular communities such as colonies, fruiting bodies, or biofilms. Interest in the natural microbial life-style has increased during the last decade and a whole plethora of techniques has been used to gain insight into the development, structure and composition of diverse microbial communities. We have developed a technique for investigating the spatial heterogeneity of microbial growth in macro-colonies which essentially entails excision of the colonies with the underlying agar, freezing and subsequent cryotoming of the colonies, then FTIR microspectroscopic mapping of the cryosections. Colonies from Legionella, Bacillus, and Candida strains were chosen as model systems of multi-cellular communities to evaluate the technique. The results obtained indicate pronounced cell population heterogeneity even in relatively young colonies cultivated under laboratory conditions. Spectral data obtained from different positions within, e.g., a colony of Legionella bozemanii 120 h old indicated that levels of the storage material poly-beta-hydroxybutyric acid were significantly higher in cells at the surface of the colonies than in those growing at the bottom next to the agar surface. Similarly, in a 24-h-old macro-colony of Bacillus megaterium significantly more of the capsular compound polyglutamic acid was detected in upper layers than in deeper layers of the colony. Results demonstrate that FTIR microspectroscopy can be an useful tool for investigation of the spatial heterogeneity of cell growth within microbial macro-colonies. It is suggested that the method also can be adapted to the analysis of more complex multicellular communities, for example fruiting bodies, biofilms, or colonies growing under natural conditions.

Bacterial Physiological Phenomena↗

PubMed2000

Producer-decomposer co-dependency influences biodiversity effects.

Producers, such as plants and algae, acquire nutrients from inorganic sources that are supplied primarily by decomposers whereas decomposers, mostly fungi and bacteria, acquire carbon from organic sources that are supplied primarily by producers. This producer-decomposer co-dependency is important in governing ecosystem processes, which implies that the impacts of declining biodiversity on ecosystem functioning should be strongly influenced by this process. Here we show, by simultaneously manipulating producer (green algal) and decomposer (heterotrophic bacterial) diversity in freshwater microcosms, that algal biomass production varies considerably among microcosms (0.0-0.67 mg ml(-1)), but that neither algal nor bacterial diversity by itself can explain this variation. Instead, production is a joint function of both algal and bacterial diversity. Furthermore, the range in algal production in microscosms in which bacterial diversity was manipulated was nearly double (1.82 times) that of microcosms in which bacterial diversity was not manipulated. Measures of organic carbon use by bacteria in these microcosms indicate that carbon usage is the mechanism responsible for these results. Because both producer and microbial diversity respond to disturbance and habitat modification, the main causes of biodiversity loss, these results suggest that ecosystem response to changing biodiversity is likely to be more complex than other studies have shown.

Bacterial Physiological Phenomena↗

PubMed2003

Bacterial diversity and sulfur cycling in a mesophilic sulfide-rich spring.

An artesian sulfide- and sulfur-rich spring in southwestern Oklahoma is shown to sustain an extremely rich and diverse microbial community. Laboratory incubations and autoradiography studies indicated that active sulfur cycling is occurring in the abundant microbial mats at Zodletone spring. Anoxygenic phototrophic bacteria oxidize sulfide to sulfate, which is reduced by sulfate-reducing bacterial populations. The microbial community at Zodletone spring was analyzed by cloning and sequencing 16S rRNA genes. A large fraction (83%) of the microbial mat clones belong to sulfur- and sulfate-reducing lineages within delta-Proteobacteria, purple sulfur gamma-Proteobacteria, epsilon -Proteobacteria, Chloroflexi, and filamentous Cyanobacteria of the order Oscillatoria as well as a novel group within gamma-Proteobacteria. The 16S clone library constructed from hydrocarbon-exposed sediments at the source of the spring had a higher diversity than the mat clone library (Shannon-Weiner index of 3.84 compared to 2.95 for the mat), with a higher percentage of clones belonging to nonphototrophic lineages (e.g., Cytophaga, Spirochaetes, Planctomycetes, Firmicutes, and Verrucomicrobiae). Many of these clones were closely related to clones retrieved from hydrocarbon-contaminated environments and anaerobic hydrocarbon-degrading enrichments. In addition, 18 of the source clones did not cluster with any of the previously described microbial divisions. These 18 clones, together with previously published or database-deposited related sequences retrieved from a wide variety of environments, could be clustered into at least four novel candidate divisions. The sulfate-reducing community at Zodletone spring was characterized by cloning and sequencing a 1.9-kb fragment of the dissimilatory sulfite reductase (DSR) gene. DSR clones belonged to the Desulfococcus-Desulfosarcina-Desulfonema group, Desulfobacter group, and Desulfovibrio group as well as to a deeply branched group in the DSR tree with no representatives from cultures. Overall, this work expands the division-level diversity of the bacterial domain and highlights the complexity of microbial communities involved in sulfur cycling in mesophilic microbial mats.

Bacteria↗

PubMed2006

Microbial biogeography: putting microorganisms on the map.

We review the biogeography of microorganisms in light of the biogeography of macroorganisms. A large body of research supports the idea that free-living microbial taxa exhibit biogeographic patterns. Current evidence confirms that, as proposed by the Baas-Becking hypothesis, 'the environment selects' and is, in part, responsible for spatial variation in microbial diversity. However, recent studies also dispute the idea that 'everything is everywhere'. We also consider how the processes that generate and maintain biogeographic patterns in macroorganisms could operate in the microbial world.

Archaea↗

PubMed2006

Litter-forager termite mounds enhance the ectomycorrhizal symbiosis between Acacia holosericea A. Cunn. Ex G. Don and Scleroderma dictyosporum isolates.

The hypothesis of the present study was that the termite mounds of Macrotermes subhyalinus (MS) (a litter-forager termite) were inhabited by a specific microflora that could enhance with the ectomycorrhizal fungal development. We tested the effect of this feeding group mound material on (i) the ectomycorrhization symbiosis between Acacia holosericea (an Australian Acacia introduced in the sahelian areas) and two ectomycorrhizal fungal isolates of Scleroderma dictyosporum (IR408 and IR412) in greenhouse conditions, (ii) the functional diversity of soil microflora and (iii) the diversity of fluorescent pseudomonads. The results showed that the termite mound amendment significantly increased the ectomycorrhizal expansion. MS mound amendment and ectomycorrhizal inoculation induced strong modifications of the soil functional microbial diversity by promoting the multiplication of carboxylic acid catabolizing microorganisms. The phylogenetic analysis showed that fluorescent pseudomonads mostly belong to the Pseudomonads monteillii species. One of these, P. monteillii isolate KR9, increased the ectomycorrhizal development between S. dictyosporum IR412 and A. holosericea. The occurrence of MS termite mounds could be involved in the expansion of ectomycorrhizal symbiosis and could be implicated in nutrient flow and local diversity.

Acacia↗

PubMed2005

Bacterial diversity in the rhizosphere of Proteaceae species.

The Cape Floral Kingdom is an area of unique plant biodiversity in South Africa with exceptional concentrations of rare and endemic species and experiencing drastic habitat loss. Here we present the first molecular study of the microbial diversity associated with the rhizosphere soil of endemic plants of the Proteaceae family (Leucospermum truncatulum and Leucadendron xanthoconus). Genomic DNA was extracted from L. truncatulum rhizosphere soil, L. xanthoconus rhizosphere and non-rhizosphere soil and used as a template for the polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA gene (rDNA). Construction and sequencing of 16S rDNA libraries revealed a high level of biodiversity and led to the identification of several novel bacterial phylotypes. The bacterial community profiles were compared by 16S rDNA denaturing gradient gel electrophoresis (DGGE). Cluster analysis and biodiversity indices revealed that the rhizosphere soil samples were more similar to each other than to non-rhizosphere soil and the rhizosphere soil contained a bacterial diversity that was richer and more equitable compared with non-rhizosphere soil. A Chloroflexus and an Azospirillum genospecies were restricted to the L. xanthoconus rhizosphere soil and Stenotrophomonas genospecies was identified in all rhizosphere soil samples but was not present in the non-rhizosphere soil. Taxon-specific nested PCR and DGGE-identified differences between the Proteaceae plant rhizosphere soil with a Frankia genospecies restricted the L. truncatulum rhizosphere. Archaea-specific rDNA PCR, DGGE and DNA sequencing revealed that Crenarcheote genospecies were excluded from the plant rhizosphere soil and only present in non-rhizosphere soil.

Bacteria↗