Bacterial Small Subunit rRNA Genes Research Articles

Limited effects of azithromycin on the oropharyngeal microbiome in children with CF and early pseudomonas infection

BackgroundTobramycin inhalation solution (TIS) and chronic azithromycin (AZ) have known clinical benefits for children with CF, likely due to antimicrobial and anti-inflammatory activity. The effects of chronic AZ in combination with TIS on the airway microbiome have not been extensively investigated. Oropharyngeal swab samples were collected in the OPTIMIZE multicenter, randomized, placebo-controlled trial examining the addition of AZ to TIS in 198 children with CF and early P. aeruginosa infection. Bacterial small subunit rRNA gene community profiles were determined. The effects of TIS and AZ were assessed on oropharyngeal microbial diversity and composition to uncover whether effects on the bacterial community may be a mechanism of action related to the observed changes in clinical outcomes.ResultsSubstantial changes in bacterial communities (total bacterial load, diversity and relative abundance of specific taxa) were observed by week 3 of TIS treatment for both the AZ and placebo groups. On average, these shifts were due to changes in non-traditional CF taxa that were not sustained at the later study visits (weeks 13 and 26). Bacterial community measures did not differ between the AZ and placebo groups.ConclusionsThis study provides further evidence that the mechanism for AZ’s effect on clinical outcomes is not due solely to action on airway microbial composition.

Development of LNA oligonucleotide–PCR clamping technique in investigating the community structures of plant-associated bacteria

Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.

Nitrospira‐dominated biofilm within a thermal artesian spring: a case for nitrification‐driven primary production in a geothermal setting

Water chemistry, energetic modeling, and molecular analyses were combined to investigate the microbial ecology of a biofilm growing in a thermal artesian spring within Hot Springs National Park, AR. This unique fresh water spring has a low dissolved chemical load and is isolated from both light and direct terrestrial carbon input - resulting in an oligotrophic ecosystem limited for fixed carbon and electron donors. Evaluation of energy yields of lithotrophic reactions putatively linked to autotrophy identified the aerobic oxidation of methane, hydrogen, sulfide, ammonia, and nitrite as the most exergonic. Small subunit (SSU) rRNA gene libraries from biofilm revealed a low-diversity microbial assemblage populated by bacteria and archaea at a gene copy ratio of 45:1. Members of the bacterial family 'Nitrospiraceae', known for their autotrophic nitrite oxidation, dominated the bacterial SSU rRNA gene library (approximately 45%). Members of the Thaumarchaeota ThAOA/HWCGIII (>96%) and Thaumarchaeota Group I.1b (2.5%), which both contain confirmed autotrophic ammonia oxidizers, dominated the archaeal SSU rRNA library. Archaea appear to dominate among the ammonia oxidizers, as only ammonia monooxygenase subunit A (amoA) genes belonging to members of the Thaumarchaeota were detected. The geochemical, phylogenetic, and genetic data support a model that describes a novel thermophilic biofilm built largely by an autotrophic nitrifying microbial assemblage. This is also the first observation of 'Nitrospiraceae' as the dominant organisms within a geothermal environment.

A PCR-based toolbox for the culture-independent quantification of total bacterial abundances in plant environments

A major obstacle in the culture-independent estimation of the abundance of bacteria associated with plants is contamination with plant organelles, which precludes the use of universal rRNA bacterial primers in quantitative PCR applications. We present here a PCR-based method that allows a priori determination of the degree of chloroplast and mitochondrial contamination in DNA samples from plant environments. It is based on differential digestibility of chloroplast, mitochondrial and bacterial small subunit rRNA gene amplicons with the restriction enzymes AfeI and BbvCI. Using this method, we demonstrated for field-grown lettuce plants that even a gentle washing protocol, designed to recover the microbial community and its metagenome from the leaf surface, resulted in substantial contamination with chloroplast DNA. This finding cautions against the use of universal primer pairs that do not exclude chloroplast DNA from amplification, because they risk overestimation of bacterial population sizes. In contrast, contamination with mitochondrial 18S rRNA was minor in the lettuce phyllosphere. These findings were confirmed by real-time PCR using primer sets specific for small subunit rRNA genes from bacteria, chloroplasts, and mitochondria. Based on these results, we propose two primer pairs (534f/783r and mito1345f/mito1430r) which between them offer an indirect means of faithfully estimating bacterial abundances on plants, by deduction of the mito1345f/mito1430r-based mitochondrial count from that obtained with 534f/783r, which amplifies both bacterial and mitochondrial DNA but excludes chloroplast. In this manner, we estimated the number of total bacteria on most leaves of field-grown lettuce to be between 10 5 and 10 6 g − 1 of leaf, which was 1–3 orders of magnitudes higher than the number of colony-forming units that were retrieved from the same leaf surfaces on agar plates.

Bar-Coded Pyrosequencing Reveals Shared Bacterial Community Properties along the Temperature Gradients of Two Alkaline Hot Springs in Yellowstone National Park

An understanding of how communities are organized is a fundamental goal of ecology but one which has historically been elusive for microbial systems. We used a bar-coded pyrosequencing approach targeting the V3 region of the bacterial small-subunit rRNA gene to address the factors that structure communities along the thermal gradients of two alkaline hot springs in the Lower Geyser Basin of Yellowstone National Park. The filtered data set included a total of nearly 34,000 sequences from 39 environmental samples. Each was assigned to one of 391 operational taxonomic units (OTUs) identified by their unique V3 sequence signatures. Although the two hot springs differed in their OTU compositions, community resemblance and diversity changed with strikingly similar dynamics along the two outflow channels. Two lines of evidence suggest that these community properties are controlled primarily by environmental temperature. First, community resemblance decayed exponentially with increasing differences in temperature between samples but was only weakly correlated with physical distance. Second, diversity decreased with increasing temperature at the same rate along both gradients but was uncorrelated with other measured environmental variables. This study also provides novel insights into the nature of the ecological interactions among important taxa in these communities. A strong negative association was observed between cyanobacteria and the Chloroflexi, which together accounted for approximately 70% of the sequences sampled. This pattern contradicts the longstanding hypothesis that coadapted lineages of these bacteria maintain tightly cooccurring distributions along these gradients as a result of a producer-consumer relationship. We propose that they instead compete for some limiting resource(s).

Culture-Independent Analysis of Indomethacin-Induced Alterations in the Rat Gastrointestinal Microbiota

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed for a variety of inflammatory conditions; however, the benefits of this class of drugs are accompanied by deleterious side effects, most commonly gastric irritation and ulceration. NSAID-induced ulceration is thought to be exacerbated by intestinal microbiota, but previous studies have not identified specific microbes that contribute to these adverse effects. In this study, we conducted a culture-independent analysis of approximately 1,400 bacterial small-subunit rRNA genes associated with the small intestines and mesenteric lymph nodes of rats treated with the NSAID indomethacin. This is the first molecular analysis of the microbiota of the rat small intestine. A comparison of clone libraries and species-specific quantitative PCR results from rats treated with indomethacin and untreated rats revealed that organisms closely related to Enterococcus faecalis were heavily enriched in the small intestine and mesenteric lymph nodes of the treated rats. These data suggest that treatment of NSAID-induced ulceration may be facilitated by addressing the microbiological imbalances.

Microbial community in a geothermal aquifer associated with the subsurface of the Great Artesian Basin, Australia

To investigate the biomass and phylogenetic diversity of the microbial community inhabiting the deep aquifer of the Great Artesian Basin (GAB), geothermal groundwater gushing out from the aquifer was sampled and analyzed. Microbial cells in the groundwater were stained with acridine orange and directly counted by epifluorescence microscopy. Microbial cells were present at a density of 10(8)-10(9) cells per liter of groundwater. Archaeal and bacterial small-subunit rRNA genes (rDNAs) were amplified by PCR with Archaea- and Bacteria-specific primer sets, and clone libraries were constructed separately. A total of 59 clones were analyzed in archaeal and bacterial 16S rDNA libraries, respectively. The archaeal 16S rDNA clones were divided into nine operated taxonomic units (OTUs) by restriction fragment length polymorphism. These OTUs were closely related to the methanogenic genera Methanospirillum and Methanosaeta, the heterotrophic genus Thermoplasma, or miscellaneous crenarchaeota group. More than one-half of the archaeal clones (59% of total 59 clones) were placed beside phylogenetic clusters of methanogens. The majority of the methanogen-related clones (83%) was closely related to a group of hydrogenotrophic methanogens (genus Methanospirillum). The bacterial OTUs branched into seven phylogenetic clusters related to hydrogen-oxidizing thermophiles in the genera Hydrogenobacter and Hydrogenophilus, a sulfate-reducing thermophile in the genus Thermodesulfovibrio, chemoheterotropic bacteria in the genera Thermus and Aquaspirillum, or the candidate division OP10. Clones closely related to the thermophilic hydrogen-oxidizers in the genera Hydrogenobacter and Hydrogenophilus were dominant in the bacterial clone library (37% of a total of 59 clones). The dominancy of hydrogen-users strongly suggested that H(2) plays an important role as a primary substrate in the microbial ecosystem of this deep geothermal aquifer.

The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis.

The Ribosomal Database Project (RDP-II) provides the research community with aligned and annotated rRNA gene sequences, along with analysis services and a phylogenetically consistent taxonomic framework for these data. Updated monthly, these services are made available through the RDP-II website (http://rdp.cme.msu.edu/). RDP-II release 9.21 (August 2004) contains 101 632 bacterial small subunit rRNA gene sequences in aligned and annotated format. High-throughput tools for initial taxonomic placement, identification of related sequences, probe and primer testing, data navigation and subalignment download are provided. The RDP-II email address for questions or comments is rdpstaff@msu.edu.

Serial analysis of ribosomal sequence tags (SARST): a high-throughput method for profiling complex microbial communities.

Two decades of culture-independent studies have confirmed that microbial communities represent the most complex and concentrated pool of phylogenetic diversity on the planet. There remains a need for innovative molecular tools that can further our knowledge of microbial diversity and its functional implications. We present the method and application of serial analysis of ribosomal sequence tags (SARST) as a novel tool for elucidating complex microbial communities, such as those found in soils and sediments. Serial analysis of ribosomal sequence tags uses a series of enzymatic reactions to amplify and ligate ribosomal sequence tags (RSTs) from bacterial small subunit rRNA gene (SSU rDNA) V1-regions into concatemers that are cloned and sequenced. This approach offers a significant increase in throughput over traditional SSU rDNA clone libraries, as up to 20 RSTs are obtained from each sequencing reaction. To test SARST and measure the bias associated with this approach, RST libraries were prepared from a defined mixture of pure cultures and from duplicate arctic soil DNA samples. The actual RST distribution reflected the theoretical composition of the original defined mixture. Data from duplicate soil libraries (1345 and 1217 RSTs, with 525 and 505 unique RSTs, respectively) indicated that replication provides a strongly correlated RST profile (r(2) = 0.80) and division-level distribution of RSTs (r(2) = 0.99). Using sequence data from abundant soil RSTs, we designed specific primers that successfully amplified a larger portion of the SSU rDNA for further phylogenetic analysis. These results suggest that SARST is a powerful approach for reproducible high-throughput profiling of microbial diversity amenable to medical, industrial or environmental microbiology applications.

Fidelity of select restriction endonucleases in determining microbial diversity by terminal-restriction fragment length polymorphism.

An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected < or =70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).

Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds.

Five brightly red-pigmented, motile, rod-shaped, extremely halophilic bacteria were isolated from saltern crystallizer ponds in Alicante (two strains) and Mallorca (three strains), Spain. They grew optimally at salt concentrations between 20 and 30% and did not grow below 15% salts. Thus, these isolates are among the most halophilic organisms known within the domain Bacteria. The temperature optimum was 37-47 degrees C. A single, yet to be identified pigment was present, with an absorption maximum at 482 nm and a shoulder at 506-510 nm. The G+C content of the DNA was 66.3-67.7 mol% and, together, they formed a homogeneous genomic group with DNA-DNA similarities above 70%. The 16S rRNA gene sequences were almost identical to sequences recovered earlier from the saltern biomass by amplification of bacterial small-subunit rRNA genes from DNA extracted from the environment. This phylotype, earlier described as 'Candidatus Salinibacter', was shown by fluorescence in situ hybridization to contribute between 5 and 25% of the prokaryote community of the saltern crystallizers. We have therefore succeeded in isolating a bacterium from the natural environment that, although being a major component of the community, was previously known by its phylotype only. Isolation of the organism now allows formal description of a novel genus and species, for which we propose the name Salinibacter ruber gen. nov., sp. nov. The type strain is strain M31T (= DSM 13855T = CECT 5946T).

Effect of protistan bacterivory on coastal bacterioplankton diversity

Four protist exclusion experiments were conducted to test the hypothesis that marine bacterivorous protists selectively feed on different bacterioplankton genotypes and affect the taxonomc diversity of coastal marine bacterioplankton communities. In these experiments, the changes in bacterial community composition of seawater samples from which protists were removed by filtration were followed and compared to those of untreated control water samples. Bacterioplankton community structure was inferred from the relative abundance of bacterial small subunit rRNA genes (SSU rDNAs) by a recently developed technique (length heterogeneity analysis by PCR (LH-PCR; Suzuki et al. 1998). The results of the experiments show that the community structure did not dran~atically change up to a 24 h incubation period in any of the treatments. However there were significant dfferences in filtered water samples and controls between 24 and 48 h of incubation. In the absence of bacterivores some SSU rDNAs that were rare in the original water samples dominated the bacterioplankton SSU rDNA pool after 48 h of incubation. Protists appeared to be capable of controlling bacterioplankton taxonomic diversity under these manipulated conditions. The results also agree with the hypothesis that aquatic bactenoplankton communiries are composed oi s111dii ceub ~ i l d l e x a p e p ~ ~ ; a ; i ~ ~ ~ , CIII; uc 111 0 J;u:& 6: low physiological turnover.

A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature.

An assessment of 10 tetrameric restriction enzymes (TREs) was conducted by using a computer-simulated restriction fragment length polymorphism (RFLP) analysis for over 100 proximally and distally related bacterial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logistic simplicity, commercial availability, and economy. However, the rationale for selecting the type and number of TREs has not been systematically evaluated. Our objective was to identify the optimal combination of TREs for RFLP screening of cloned SSU rRNA genes from undefined bacterial clone libraries. After computer-simulated TRE digestion, the resultant fragments were categorized on the basis of the frequency of different restriction fragment size classes. Three groups of distribution patterns for the TREs were determined and further examined via graphical exploratory data analysis. The RFLP size-frequency distribution data for each group of enzymes were then used to infer phylogenetic relationships via the neighbor-joining method. The resulting bootstrap values and the correct placement of node bifurcations were used as additional criteria to evaluate the efficacy of the selected TREs. These RFLP data were compared with known phylogenetic relationships based on SSU rRNA sequence analysis as defined by the Ribosomal Database Project. A heuristic approach testing random combinations of TREs showed that three or more TRE combinations detected > 99% of the operational taxonomic units (OTUs) within the model data set. OTUs that remained undetected after three TRE treatments had a median sequence similarity of 96.1%. Of the 10 restriction enzymes examined, HhaI, RsaI, and BstUI (group 3) were the most efficacious at detecting and differentiating bacterial SSU rRNA genes on the basis of their ability to correctly classify OTUs. Group 3 TREs are therefore recommended for screening in studies using bacterial SSU rRNA genes as descriptors of in situ microbial diversity.