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PubMed2005

Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

Bacteria↗

PubMed2005

Identifying causes for N2O accumulation in a lab-scale sequencing batch reactor performing simultaneous nitrification, denitrification and phosphorus removal.

The recently described process of simultaneous nitrification, denitrification and phosphorus removal (SNDPR) has a great potential to save capital and operating costs for wastewater treatment plants. However, the presence of glycogen-accumulating organisms (GAOs) and the accumulation of nitrous oxide (N(2)O) can severely compromise the advantages of this process. In this study, these two issues were investigated using a lab-scale sequencing batch reactor performing SNDPR over a 5-month period. The reactor was highly enriched in polyphosphate-accumulating organisms (PAOs) and GAOs representing around 70% of the total microbial community. PAOs were the dominant population at all times and their abundance increased, while GAOs population decreased over the study period. Anoxic batch tests demonstrated that GAOs rather than denitrifying PAOs were responsible for denitrification. N(2)O accumulated from denitrification and more than half of the nitrogen supplied in a reactor cycle was released into the atmosphere as N(2)O. After mixing SNDPR sludge with other denitrifying sludge, N(2)O present in the bulk liquid was reduced immediately if external carbon was added. We therefore suggest that the N(2)O accumulation observed in the SNDPR reactor is an artefact of the low microbial diversity facilitated by the use of synthetic wastewater with only a single carbon source.

Bacteria, Aerobic↗

PubMed2006

Estimation of intracellular phosphorus content of phosphorus-accumulating organisms at different P:COD feeding ratios.

The intracellular phosphorus content of phosphorus-accumulating organisms (PAO) was determined based on a stoichiometric equation and phosphorus balance for an enhanced biological phosphorus removal system fed with different P:COD ratios. The data indicated that a higher P:COD feeding ratio could significantly promote the growth of PAO. As the P:COD feeding ratio increased from 0.02:1 to 0.04:1 and 0.16:1, the phosphorus in the sludge increased considerably from 0.053 to 0.084 and 0.205 mg P (mg VSS(aerobic))(-1), respectively, indicating a dynamic condition in the microbial population. From the calculations, the mass fractions of the PAO, glycogen-accumulating organisms, and ordinary heterotrophs changed from 0.10-0.15, 0.83-0.88, and 0.02 at 0.02:1 to 0.19-0.28, 0.70-0.79, and 0.02 at 0.04:1 and to 0.478-0.71, 0.26-0.50, 0.03 at 0.16:1 P:COD feeding ratios, respectively. Despite the variation in microbial diversity, the calculated phosphorus contents of the PAO at all P:COD feeding ratios were consistent between 0.241 and 0.378 mg P (mg VSS(PAO))(-1). The initial specific phosphorus release and uptake rates were 84.7-167.9 mg P (g VSS(PAO))(-1)h(-1) and 52.8-90.0 mgP (g VSS(total))(-1)h(-1), respectively.

Aerobiosis↗

PubMed2026

Agricultural sprinkler irrigation systems as environmental reservoirs and airborne dissemination sources of Legionella pneumophila.

Sprinkler irrigation systems are critical for modern agriculture but represent largely unrecognized aquatic environments capable of sustaining opportunistic human pathogens. Among them, Legionella pneumophila is of particular concern due to its ability to colonize engineered water systems, persist under fluctuating environmental conditions, and be transmitted through aerosols. In this study, we conducted a comprehensive microbiological and genomic investigation of irrigation ponds and ditches in a rural area of north-east Spain where two zones were sampled. Metagenomic profiling revealed highly diverse microbial communities encompassing more than 20,000 species, including 21 airborne-transmissible bacterial pathogens of clinical relevance. Notably, L. pneumophila was detected in both zones, with a relative abundance of up to 4.6 %. Culture-based isolation confirmed the presence of L. pneumophila serogroup 1, Pontiac group, Benidorm subgroup, sequence type 15. Phylogenetic analysis demonstrated a close relationship between this environmental strain and clinical isolates obtained during a Legionnaires' disease outbreak occurred in 2015, which had remained without a confirmed environmental source. Meteorological data from the exposure period revealed wind conditions favouring long-distance aerosol dispersion from irrigated fields toward residential areas. Our findings provide evidence that irrigation infrastructures can act as environmental reservoirs and dissemination routes of L. pneumophila among other airborne pathogens. These results underscore the need to incorporate agricultural irrigation systems into routine environmental surveillance, outbreak investigations, and public health risk assessments.

Legionella pneumophila↗

PubMed2026

Boosting domestic wastewater treatment with quorum signal-augmented heterotrophic nitrification-aerobic denitrification bacterial-algal aerobic granular sludge.

The aerobic bacterial-algal granular sludge (ABGS) enhanced with heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria, as a novel symbiotic technology, exhibits fluctuating treatment efficiency and unstable performance primarily due to the unstable symbiotic relationship. This study proposes an innovative approach to strengthening the bacteria-algae symbiosis by introducing exogenous signaling molecules. Concurrently, high-throughput, correlation analysis of environmental factors and metagenomic sequencing techniques are employed to elucidate the enhancement mechanisms of the signaling molecules. The results demonstrate that signaling molecule enhancement boosted total nitrogen (TN) removal efficiency by 24.51 % in the bacteria-algae symbiotic system (X1). Scanning electron microscopy (SEM) characterization revealed that the addition of signaling molecules resulted in more compact aerobic granular sludge (AGS) and markedly improved stability. High-throughput sequencing showed signaling molecules enriched denitrifying bacteria (Hydrogenophaga, Pseudoxanthomonas, Thauera, Zoogloea) and organic-degrading Desulfomicrobium, optimizing microbial diversity and enhancing nitrogen/organic removal. Correlation analysis of environmental factors indicate that the addition of C8-HSL facilitates the enrichment and functional activation of specific genera. Metagenomic analysis revealed that signaling molecules enhanced the system's denitrification performance by modulating gene expression and associated metabolic pathways. Quantitative polymerase chain reaction (qPCR) analysis further confirmed that the signaling molecules upregulated the expression of the napA, nirK, and nirS genes. An increased abundance of the napA gene facilitated aerobic denitrification (NO₃⁻-N→NO₂⁻-N), while upregulated abundance of the nirK and nirS genes accelerated nitrite reduction (NO₂⁻-N→N₂). This study aims to provide theoretical and practical foundations for implementing advanced bacteria-algae symbiotic technologies.

Denitrification↗

PubMed2003

Review and re-analysis of domain-specific 16S primers.

The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the "universal" primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective "universal" Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.

Archaea↗

PubMed2004

PCR-DGGE fingerprinting: novel strategies for detection of microbes in food.

Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting was recently introduced into food microbiology. This paper describes the technique and reports on the state-of-the-art application of this technique to food and food-related ecosystems. Applications of PCR-DGGE in several fields of food microbiology are reviewed: the identification of microorganisms isolated from food, the evaluation of microbial diversity during food fermentation, and microbiological and commercial food quality assessment. Potentials and limitations of this culture-independent approach in food microbiology are indicated and future perspectives are discussed.

Bacteria↗

PubMed2004

A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis.

Denaturant gradient gel electrophoresis (DGGE) is a widely used method for mutation analysis and for studies of microbial diversity. Particular combinations of target gene fragments and primers may give rise to erroneous DGGE profiles. We report on a very straightforward means to eliminate the artifactual 'double bands' that can be encountered in several applications.

Artifacts↗

PubMed2005

Culture independent PCR: an alternative enzyme discovery strategy.

Degenerate primers were designed for use in a culture-independent PCR screening of DNA from composite fungal communities, inhabiting residues of corn stovers and leaves. According to similarity searches and alignments amplified clone sequences affiliated with glycosyl hydrolase family 7 and glycosyl hydrolase family 45 though significant sequence divergence was observed. Glycosyl hydrolases from families 7 and 45 play a crucial role in biomass conversion to fuel ethanol. Research in this renewable energy source has two objectives: (i) To contribute to development of a renewable alternative to world's limited crude fossil oil reserves and (ii) to reduce air pollution. Amplification with 18S rDNA-specific primers revealed species within the ascomycetous orders Sordariales and Hypocreales as well as basidiomycetous order Agaricales to be present in these communities. Our study documents the value of culture-independent PCR in microbial diversity studies and could add to development of a new enzyme screening technology.

Amino Acid Sequence↗

PubMed2005

An improved expression plasmid for affinity purification of Staphylococcus aureus gyrase A subunit.

Of the bacterial topoisomerases, the gyrase A subunit (GyrA) of Staphylococcus aureus is particularly difficult to purify because of its tendency to form inclusion bodies. Previous attempts at purification yielded low concentrations of protein with reduced specific activity. To overcome this problem, we modified the commercially available plasmid expression vector, pBAD/Thio-TOPO, via the addition of DNA sequences encoding a hexahistidine tag upstream and a cleavage site for tobacco etch virus protease downstream of the gene encoding thioredoxin. The resulting expression system consisting of the modified plasmid, pSAGA7, and the recommended host strain, Escherichia coli TOP 10, facilitated high level expression of soluble GyrA and its affinity purification to over 95% homogeneity. Purified GyrA had high biological activity as evidenced by a specific activity of 4.3x10(5)U/mg. The pSAGA7/TOP10 expression system also facilitated the expression and purification of a subunit of S. aureus topoisomerase IV, ParE, and a recently discovered protein unrelated to topoisomerases, QnrB, two "hard to purify" proteins. We conclude that pSAGA7 might be useful for high-level soluble expression and purification of diverse microbial proteins.

Chromatography, Affinity↗

PubMed2006

Phylogenetic diversity of culturable bacteria from alpine permafrost in the Tianshan Mountains, northwestern China.

Microbes have been discovered in permafrost sediments for nearly a century. However, microbiological analyses of alpine permafrost are very scarce. This study is a first attempt to describe the phylogenetic diversity of a culturable bacterial community isolated from alpine permafrost in the Tianshan Mountains in northwestern China. Aerobic 2.5-6.0x10(5) CFU/gdw (CFU per 1 gram of dry weight) on modified PYGV medium were recovered from alpine permafrost samples at 4 degrees C; among these, 91 bacterial isolates with different morphotypes were characterized by phenotypic properties, such as morphology, colony pigmentation, Gram staining, endospore formation and temperature range of growth. The isolates were further categorized based on amplified rDNA restriction analysis (ARDRA), and 51 representative isolates possessing distinct ARDRA patterns selected for subsequent 16S rDNA sequencing and phylogenetic analysis. The phylogenetic trees placed the 51 isolates in four major groups: the high-G+C Gram-positives, the low-G+C Gram-positives, Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum. The most abundant and diverse isolates were members of Gram-positive bacteria, particularly the Arthrobacter as a dominant group in alpine permafrost culturable populations. Results of the Jukes-Cantor evolutionary distance matrix suggested that the vast majority of the isolates were different strains of known species, and three may represent new species within the genus Chryseobacterium of the CFB phylum. From this study, it is proposed that alpine permafrost sediments in the Tianshan Mountains provide a specific ecological niche for prolonging survival of diverse microbial lineages.

Arthrobacter↗

PubMed2004

Determination of biodegradable dissolved organic carbon in waters: comparison of batch methods.

An effect of different types of bacterial inocula upon the final biodegradable dissolved organic carbon (BDOC) result was investigated in samples of both low and high BDOC concentrations. Stream water and leaf leachate samples were incubated either with free, suspended bacteria or with bacteria attached to the stream sediment particles or attached to artificial substrata. The time course of dissolved organic carbon (DOC) decomposition was observed using absorbance analysis of DOC. BDOC determination by means of commonly used suspended bacteria as the inoculum made for an underestimation of BDOC between 5% and 25%, compared with attached bacterial community (biofilm). The reason for these findings could be the higher microbial diversity, higher metabolic activity of attached bacteria and abiotic adsorption of organic molecules to inorganic support and biofilm matrix surfaces. Adsorbed DOC is easily hydrolyzed and utilized by biofilm bacteria.

Alnus↗

PubMed2004

Unravelling rhizosphere-microbial interactions: opportunities and limitations.

The rhizosphere is a biologically active zone of the soil around plant roots that contains soil-borne microbes including bacteria and fungi. Plant-microbe interactions in the rhizosphere can be beneficial to the plant, the microbes or to neither of them. One of the major difficulties that plant biologists and microbiologists face when studying these interactions is that many groups of microbes that inhabit this zone are not cultivable in the laboratory. Recent developments in molecular biology methods are shedding some light on rhizospheric microbial diversity. This review discusses recent findings and future challenges in the study of plant-microbe interactions in the rhizosphere.

Bacteria↗

PubMed2006

Spatial scaling of microbial biodiversity.

A central goal in ecology is to understand the spatial scaling of biodiversity. Patterns in the spatial distribution of organisms provide important clues about the underlying mechanisms that structure ecological communities and are central to setting conservation priorities. Although microorganisms comprise much of Earth's biodiversity, little is known about their biodiversity scaling relationships relative to that for plants and animals. Here, we discuss current knowledge of microbial diversity at local and global scales. We focus on three spatial patterns: the distance-decay relationship (how community composition changes with geographic distance), the taxa-area relationship, and the local:global taxa richness ratio. Recent empirical analyses of these patterns for microorganisms suggest that there are biodiversity scaling rules common to all forms of life.

Animals↗

PubMed2006

Multidimensional modelling to investigate interspecies hydrogen transfer in anaerobic biofilms.

Anaerobic digestion is a multistep process, mediated by a functionally and phylogenetically diverse microbial population. One of the crucial steps is oxidation of organic acids, with electron transfer via hydrogen or formate from acetogenic bacteria to methanogens. This syntrophic microbiological process is strongly restricted by a thermodynamic limitation on the allowable hydrogen or formate concentration. In order to study this process in more detail, we developed an individual-based biofilm model which enables to describe the processes at a microbial resolution. The biochemical model is the ADM1, implemented in a multidimensional domain. With this model, we evaluated three important issues for the syntrophic relationship: (i) Is there a fundamental difference in using hydrogen or formate as electron carrier? (ii) Does a thermodynamic-based inhibition function produced substantially different results from an empirical function? and; (iii) Does the physical co-location of acetogens and methanogens follow directly from a general model. Hydrogen or formate as electron carrier had no substantial impact on model results. Standard inhibition functions or thermodynamic inhibition function gave similar results at larger substrate field grid sizes (> 10 microm), but at smaller grid sizes, the thermodynamic-based function reduced the number of cells with long interspecies distances (> 2.5 microm). Therefore, a very fine grid resolution is needed to reflect differences between the thermodynamic function, and a more generic inhibition form. The co-location of syntrophic bacteria was well predicted without a need to assume a microbiological based mechanism (e.g., through chemotaxis) of biofilm formation.

Bacteria, Anaerobic↗

PubMed1998

Natural bioburden levels detected on flexible gastrointestinal endoscopes after clinical use and manual cleaning.

BACKGROUND: Colonoscopes present a special bacterial decontamination challenge because the colon has a large and diverse microbial population. METHODS: Bioburden of colonoscope insertion tube surfaces and suction channels were determined after use and after manual cleaning. RESULTS: After use bioburden in suction channels averaged 7.0 x 10(9) colony-forming units (cfu). Cleaning reduced this level to 1.3 x 10(5). Cleaning of tube surfaces reduced the after-use bioburden from a level of 5.1 x 10(5) to 2.2 x 10(4) cfu. Gram-negative rods accounted for approximately 99% of the bioburden within the suction channel after use and after cleaning. After use flora were predominantly Escherichia coli and Bacteroides. The flora shifted to waterborne Pseudomonas organisms, and other members of the family Enterobacteriaceae after cleaning. Gram-positive bacteria were the primary isolates from the device surfaces both after use (56%) and after cleaning (47%). Because gram-positive cocci and diphtheroids are a part of the normal microbiota of the skin, these bacteria may have been introduced by the hospital environment or by handling. CONCLUSIONS: After the cleaning of in-use colonoscopes, fewer than 10(6) vegetative bacteria could be recovered. This value is several logs lower than some previous estimates. This finding may be useful in the formulation of sterilization and disinfection cycles. Microflora from the colonoscopes indicated that the cleaning process introduced waterborne and enteric microorganisms, which highlights the importance of sanitation in the device reprocessing area.

Bacteria, Aerobic↗

PubMed1993

Heat shock (stress) proteins and autoimmunity in rheumatic diseases.

The rheumatic diseases (RDs) are characterized by acute and chronic inflammation, and autoimmunity plays a major role in their pathogenesis. RDs are for the most part of unknown etiology, but recent evidence indicates that heat shock or stress proteins (HSPs) may have an important role in the etiology/pathogenesis of RDs. HSPs are produced by prokaryotic and eukaryotic cells and are grouped according to molecular weight. Phylogenetically, HSPs are very old and are remarkably conserved molecules in evolution from bacteria to humans. HSPs are induced by a variety of cellular stresses in addition to heat; cognates are expressed constitutively and are essential in a number of normal functions. Some HSPs serve as molecular chaperones, the latter defined as proteins that mediate folding of other polypeptides and either promote their assembly into oligomeric structures or disassemble the final product. Conservation of structure and function of many HSPs may provide a link between immunity to infection and the autoimmune features of RDs. Evidence is reviewed from clinical and laboratory observations that diverse microbial agents, including viruses, bacteria, and parasites, may have putative roles in the development and pathogenesis of some RDs. HSPs also are discussed in relation to the major histocompatibility complex, HLA antigens, and disease associations and how they may alter the balance between tolerance and autoimmunity. Studies are reviewed that are supportive or nonsupportive of the concept of microbial infection associated with autoimmunity; individuals first react to microbial immunizations or infections with enhanced cellular/humoral responses to the agent's HSPs. With the enhanced immune response, cross-reactivity may occur with an HSP of the stressed host because of structural similarities to the microbial HSP. If all of these events occur, the host's homologous HSP or stressed cells now become true autoantigen(s). This sequence has implications for the etiology of immune-mediated RDs, the concept of epitope sharing, and the accompanying autoimmunity. A recurring theme emphasized in some reports to understand better the role of HSPs in autoimmunity is the need to select patients with early-onset disease. A minor subpopulation of T lymphocytes express a CD3-associated T-cell receptor (TCR) heterodimer composed of gamma and delta polypeptide chains. The gamma delta + T cells have several unique features. When analyzed by the polymerase chain reaction, lymphocytes with TCR-gamma delta appear to reflect the polyclonal expansion of preexisting gamma delta clones. They are found in peripheral lymphoid tissue in very low percentage (< 5%) but may represent the majority of T cells within epithelial tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed2003

Molecular typing of Lactobacillus delbrueckii of dairy origin by PCR-RFLP of protein-coding genes.

Thirty-five strains of Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus isolated from dairy products were typed by restriction fragment length polymorphism (RFLP) of protein-coding genes. The strains were analysed by RFLP of PCR amplified, infragenic fragments of the following housekeeping genes: beta-galactosidase, lactose permease, and proline dipeptidase. Sequencing of the variable regions of the 16S rDNA was then performed on a reduced number of strains. PCR-RFLP analysis evidenced wide strain heterogeneity. Strains were grouped into genotypes according to both subspecies assignment and infra-species genetic polymorphism. This polymorphism was related to the presence of microbial groups within subspecies populations. The low infra-species sequence polymorphism detected in the variable region of the 16S rRNA gene did not enable to group the strains with the same sensitivity reached by PCR-RFLP of protein-coding genes. PCR-RFLP of protein-coding genes applied to L. delbrueckii seems a promising tool to evaluate microbial diversity within bacterial subpopulations. Differences among bacterial subpopulations based upon molecular heterogeneity in protein-coding genes would enable to better understand the role of strains from different ecological niches.

Bacterial Typing Techniques↗