Uncultured bacterial diversity in tropical maize (Zea mays L.) rhizosphere

Structure of maize (Zea mays L.) rhizosphere bacteria was evaluated to explore the feasibility of identifying novel rhizosphere bacteria using culture-independent method based on direct amplification and analysis of 16S rRNA gene (rRNA) sequences and especially to obtain a better understanding of bacterial community structure and diversity from maize. A total of 274 sequences were analyzed and assigned 48.00% Proteobacteria, 10.30% Actinobacteria, 9.90% Bacteroidetes, 6.60% Verrucomicrobia, 4.80% Acidobacteria, 1.80% Firmicutes, 1.50% Chloroflexi, 1.50% TM7, 1.10% Deinococcus-Thermus, 0.70% Planctomycetes, 0.70% Gemmatimonadetes and 0.40% Cyanobacteria. Economically important phyla Actinobacteria was second most dominant group after Proteobacteria, in our clone library. It would be interesting to hypothesize that root exudates from maize rhizosphere favors growth of Actinobacteria like microbes to eliminate pathogenic bacteria and decompose plant matter, for enhanced plant and soil health. An additional 12.8% of clone library (35 operational taxonomical units (OTUs) from 43 clones) with less than 94% similarity to any GenBank sequence could not be assigned to any known phylum and may represent unidentified bacterial lineages and suggests that a large amount of the rhizobacterial diversity remains to be characterized by culturing.     

Endophytic bacterial diversity in roots of Typha angustifolia L. in the constructed Beijing Cuihu Wetland (China)

We investigated the community structure of endophytic bacteria in narrowleaf cattail (Typha angustifolia L.) roots growing in the Beijing Cuihu Wetland, China, using the 16S rDNA library technique. In total, 184 individual sequences were used to assess the diversity of endophytic bacteria. Phylogenetic analysis revealed that 161 clones (87.5%) were affiliated with Proteobacteria, other clones grouped into Cytophaga/Flexibacter/Bacteroids (3.3%), Fusobacteria (3.8%), and nearly 5% were uncultured bacteria. In Proteobacteria, the beta and gamma subgroups were the most abundant, accounting for approximately 46% and 36.6% of all Proteobacteria, respectively. The dominant genera included Rhodoferax, Pelomonas, Uliginosibacterium, Pseudomonas, Aeromonas, Rhizobium, Sulfurospirillum, Ilyobacter and Bacteroides. While some of these endophytic bacteria are capable of fixing nitrogen and can therefore improve plant growth, other endophytes may play important biological roles by removing nitrogen, phosphorus and/or organic matter from the water body and thus have the potential to enhance the phytoremediation of eutrophic water bodies. These bacteria have the potential to degrade xenobiota such as methane, methanol, methylated amines, catechol, oxochlorate, urea, cyanide, and 2,4-dichlorophenol. Hence, the use of certain endophytic bacteria in the process of phytoremediation could be a powerful approach for the restoration of eutrophic systems.     

Abundance, diversity and antibiotics resistance pattern of Vibrio spp. in coral ecosystem of Kurusadai island

The abundance and species diversity of Vibrio associated with coral reef ecosystem of Kurusadai island, Tamil Nadu, India were evaluated. A total of twelve sampling locations including different live and dead coral surfaces, surrounding water and rock surface (negative control) were selected for the present study. Total viable and TCBS counts were found to be higher in dead coral as compared to that of live coral. Out of total 21 species of Vibrio isolated, 13 were identified up to species level based on biochemical tests and 16S rRNA gene sequence homology, while remaining 8 isolates did not show homology up to species level with any of the sequences available in the NCBI database. Moreover, these unidentified Vibrio spp. exhibited intra-species variation. This study indicated association of hitherto unknown Vibrio species with coral reef ecosystem of Kurusadai island. Assuming that only resistant bacteria can grow in the coral environment, susceptibility against a total of 20 antibiotics was evaluated. All the isolates exhibited resistance towards more than 6 antibiotics. Interestingly, none of the identified bacteria were previously reported to be of coral pathogen reflecting the healthy nature of the ecosystem. However, a continuous monitoring of the region will be prerequisite to envisage the role of these bacteria on the health status of the coral ecosystem.     

克雷伯菌16S rDNA和rpoB基因系统进化及序列特征分析

目的 比较分析克雷伯菌属的种之间16S rDNA和rpoB系统进化发育关系和序列进化特征.方法 选取经生化鉴定的克雷伯菌18株,提取菌株染色体作为模板,分别使用16S rDNA和rpoB通用引物扩增并测序16S rDNA和rpoB序列.与GenBank中目前已公布的8种克雷伯属菌株16S rDNA和rpoB序列各8条、除克雷伯菌外其他肠道菌株的16S rDNA和rpoB序列各9条一起,共计各35条16SrDNA和rpoB序列,在MEGA 4.0中建立进化树并进行分群分析,使用DNAStar/MegAlign程序比较分析8种克雷伯菌的种间16S rDNA和rpoB序列变异碱基位点,并做分歧度分析.结果 在所有受试的16SrDNA和rpoB的各35条序列中,16S rDNA和rpoB进化发育树都将克雷伯菌区分为3个群:分离的18株克雷伯菌中,15株肺炎亚种与GenBank中除产酸克雷伯菌和运动克雷伯菌外的其余6种克雷伯菌聚为一群(Ⅰ),其余3株克雷伯菌(FX246、FX280和FX286),经生化鉴定为产酸克雷伯菌,与GenBank中产酸克雷伯菌(DQ835530)聚为一群(Ⅱ);而GenBank中的运动克雷伯菌单独聚为一群(Ⅲ);进一步的分析,在rpoB进化发育树中,无沦足Ⅰ群和Ⅱ群,还足Ⅰ群内的两个亚群,rpoB进化发育树的结点处自引导值都明显高于16S rDNA进化发育树;而且rpoB对产酸克雷伯菌的分群优于16S rDNA.对克雷伯菌的序列分析发现,16S rDNA序列存在41个碱基变异位点,有4个易变区,rpoB序列存在63个碱基变异位点,有1个易变区;克雷伯菌16S rDNA和rpoB的相似性分别为95.9%～100%和90.2%～100%.进一步的克雷伯菌种间序列分歧值分析,rpoB分歧值(0～10.6)高于16S rDNA(0～4.0).结论 克雷伯菌rpoB比16S rDNA具有更高的分歧度,在克雷伯菌属内种的分子分类和鉴定中,rpoB比16S rDNA基因更具优越性.     

Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species

In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains.     

Universal DNA primers amplify bacterial DNA from human fetal membranes and link Fusobacterium nucleatum with prolonged preterm membrane rupture

A large number of bacterial species have been identified in fetal membranes after preterm labour (PTL) associated with intrauterine infection by microbiological culture. In this study, we have investigated a molecular and bioinformatic approach to organism identification which surmounts the need for specific and diverse microbiological culture conditions required by conventional methods. Samples of fetal membranes were taken from 37 preterm infants, and 6 normal term controls delivered by caesarean section, in which bacteria had been detected by in situ hybridization of 16S ribosomal RNA using a generic probe. Degenerate primers were designed to amplify bacterial 16S ribosomal DNA by PCR and used to amplify bacterial DNA from human fetal membranes. Amplicons were cloned, sequenced and bacteria were identified bioinformatically by comparison of sequences with known bacterial DNA genomes. In situ hybridization using an organism specific probe was then used to confirm the presence of the commonest identified organism in tissue samples. Bacterial DNA amplified from 15/43 samples, all from preterm deliveries, and the bioinformatic approach identified organisms in all cases. Multiple bacteria were identified including Mycoplasma hominis, Pasturella multocida, Pseudomonas PH1, Escherichia coli and Prevotella bivia. The commonest organism Fusobacterium nucleatum was found in 9/15 (60%) of samples. Ten of the 12 samples obtained after prolonged membrane rupture were positive for bacterial DNA, and 7 of these (70%) contained DNA from F. nucleatum. Bacteria from fetal membranes may be identified by molecular and bioinformatic methods. Further work is warranted to investigate the apparent linkage between F. nucleatum, fetal membrane rupture and preterm delivery.     

Comparative evaluation of prokaryotic 16S rDNA clone libraries and SSCP in groundwater samples

A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries.     

Phylogenetic diversity of bacterial communities in South China Sea mesoscale cyclonic eddy perturbations

Eddy pumping drives a set of biogeochemical processes by lifting deep waters into the euphotic zone. To address the potential effect of such physical processes upon the bacterial community, phylogenetic diversity was determined in two cold-core cyclonic eddies in the South China Sea. 16S rDNA terminal restriction fragment length polymorphism analysis of the microbial communities through the whole water column showed a wider depth range for the intermediate transition water mass at sites inside the eddies than for those outside. This water mass contained a relatively more complex community than the euphotic and deep-water zones. Stratification of prokaryotic populations between the surface and chlorophyll maximum layer of eddy-related sites versus homogeneity of communities in the euphotic zone of the reference site, revealed by statistical analysis of 16S rDNA libraries, is most likely a reflection of isopycnal displacement induced by differing water movement inside and outside eddies. Phylogenetic analysis revealed that eddy center sites were characterized by deep-water group Alteromonadales-affiliated clones, the psychrophilic genus Octadecabacter cluster and the nitrogen-fixing phototrophic Rhodospirillaceae cluster, while Paracoccus, an important functional group, abundantly existed at the reference site outside eddies. Our analysis revealed that bacterial community structure was significantly influenced by cyclonic eddy perturbations.     

Structural diversity and efficacy of culturable cellulose decomposing bacteria isolated from rice–pulse resource conservation practices

The diversity of cellulolytic bacteria from the rice–pulse system can be sourced for identification of efficient cellulose decomposing microbial strains. In the present study, the abundance, structural diversity, and cellulolytic potential of the culturable bacterial community were studied in 5‐year old rice–pulse system under different resource conservation technologies. Higher cellulose (68% more) and xylanase (35% more) activities were observed under zero tilled soil. The populations of cellulolytic bacteria were significantly higher (44%) in zero tillage (ZT) treatment than those of conventional practice. Results revealed that the cellulolytic bacterial diversity was found to be significantly higher under ZT practice, but the present population may not be sufficient for effective recycling of organic wastes in this system. Out of 290 bacterial isolates, 20 isolates had significantly higher cellulolytic activities, of which the top three superior isolates were received from ZT practice. The cellulolytic bacterial diversity based on 16S rDNA sequencing data revealed that the Firmicutes was the most dominant phyla and the Bacillus spp. were the common genus, the observation also showed that there were 17 different haplotypes were recorded among 20 isolates of cellulolytic bacteria. The present findings indicated that long‐term ZT in the rice–pulse system could be a unique source for efficient cellulose decomposing bacteria and further the efficient bacterial strains isolated from this system can be used as efficient bioinoculants for in situ as well as ex‐situ decomposition of rice straw particularly in conservation agriculture.     

Exploring the bacterial gut microbiota of supralittoral talitrid amphipods

Talitrid amphipods (sandhoppers and beach fleas) are typical of the supralittoral zone. They are known to thrive on stranded materials, including detrital marine angiosperms and macroalgae, as well as occasional dead animals. In this work, the gut microbiota of five species of talitrid amphipods (Talitrus saltator, Talorchestia ugolinii, Sardorchestia pelecaniformis, Orchestia montagui and Orchestia stephenseni) collected in Sardinia (Italy) has been investigated through: i) metabarcoding analysis of the amplified 16S rRNA V4 region; and ii) quantification of family 48 glycosyl hydrolase genes (GHF48), involved in cellulose degradation. Results indicate that, though talitrid gut biodiversity is not directly related to taxon or sampling locality, the animals' digestive tracts may host species-specific bacterial communities. In particular, gut microbiota of O. montagui, an inhabitant of Posidonia banquettes and macro-algae mat, showed the greatest differences in taxonomic composition and the highest proportion of GHF48 genes with respect to 16S rRNA genes. These results suggest that the different talitrid species may host species-specific bacterial communities whose function could partially reflect the different microhabitats and food preferences of their host.     

The interpersonal and intrapersonal diversity of human-associated microbiota in key body sites

The human body harbors 10 to 100 trillion microbes, mainly bacteria in our gut, which greatly outnumber our own human cells. This bacterial assemblage, referred to as the human microbiota, plays a fundamental role in our well-being. Deviations from healthy microbial compositions (dysbiosis) have been linked with important human diseases, including inflammation-linked disorders, such as allergies, obesity, and inflammatory bowel disease. Characterizing the temporal variations and community membership of the healthy human microbiome is critical to accurately identify the significant deviations from normality that could be associated with disease states. However, the diversity of the human microbiome varies between body sites, between patients, and over time. Environmental differences have also been shown to play a role in shaping the human microbiome in different cultures, requiring that the healthy human microbiome be characterized across life spans, ethnicities, nationalities, cultures, and geographic locales. In this article we summarize our knowledge on the microbial composition of the 5 best-characterized body sites (gut, skin, oral, airways, and vagina), focusing on interpersonal and intrapersonal variations and our current understanding of the sources of this variation.     

Culture-dependent and -independent molecular analysis of the bacterial community within uranium ore

The bacterial community structure within a uranium ore was investigated using culture-dependent and -independent clone library analysis and denaturing gradient gel electrophoresis of 16S rRNA genes. The major aerobic heterotrophic bacteria were isolated and identified, and their resistance to uranium and other heavy metals was characterized. Together with near neutral pH, moderate organic carbon content, elevated U and other heavy metals (V, Ni, Mn, Cu, etc.), the ore showed high microbial counts and phylotype richness. The bacterial community mainly consisted of uncultured Proteobacteria, with the predominance of γ - over β - and α -subdivisions, along with Actinobacteria and Firmicutes. A phylogenetic study revealed that nearly one-third of the community was affiliated to as yet uncultured and unidentified bacteria having a closer relationship to Pseudomonas. Lineages of Burkholderiaceae and Moraxellaceae were relatively more abundant in the total community, while genera affiliated to Xanthomonadaceae and Microbacteriaceae and Exiguobacterium were detected in the culturable fraction. More than 50% of the bacterial isolates affiliated to Stenotrophomonas, Microbacterium, Acinetobacter, Pseudomonas and Enterobacter showed resistance to uranium and other heavy metals. The study showed for the first time that uranium ore harbors major bacterial groups related to organisms having a wide range of environmentally significant functional attributes, and the most abundant members are possibly new groups/taxa. These findings provide new insights into U-ore geomicrobiology that could be useful in biohydrometallurgy and bioremediation applications.     

Temporal bacterial diversity and detection of putative methanotrophs in surface mats of Lonar crater lake

The phylogenetic diversity of bacterial communities in microbial mats of two different seasons from saline and hyperalkaline Lonar Lake was investigated using 16S rRNA gene library analysis. Arthrospira (Cyanobacteria) related clones (>80% of total clones) dominated libraries of both the seasons. Clear differences were found in both the seasons as the operational taxonomic units (OTUs) related to Fusibacter (LAI‐1 and LAI‐59) and Tindallia magadiensis (LAI‐27) found in post‐monsoon were not found in the pre‐monsoon library. Likewise, OTUs related to Planococcus rifietensis (LAII‐67), Bordetella hinzii (LAII‐2) and Methylobacterium variabile (LAII‐25) found in the pre‐monsoon were not found in post‐monsoon. The study was extended to identify methanotrophs in the surface mats. Libraries constructed with type I and type II methanotroph specific 16S rRNA gene primers showed the presence of clones (LAMI‐99 and LAMII‐2) closely related to Methylomicrobium buryaticum and Beijerinckiaceae family members. Denaturing gradient gel electrophoresis (DGGE) fingerprinting based on protein‐coding genes (pmoA and mxaF) further confirmed the detection of Methylomicrobium sp. Hence, we report here for the first time the detection of putative methanotrophs in surface mats of Lonar Lake. The finding of clones related to organisms with interesting functional attributes such as assimilation of C₁ compounds (LAII‐25, LAMI‐39, LAMI‐99 and LAMII‐2), non‐sulfur photosynthetic bacteria (LAMII‐43) and clones distantly affiliated to organisms of heavily polluted environments (LAI‐59 and LAMII‐52), is of significant note. These preliminary results would direct future studies on the functional dynamics of microbial mat associated food web chain in the extreme environment. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Microbial diversity analysis of former salterns in southern Taiwan by 16S rRNA-based methods

The microbiota diversity of the former salterns in southern Taiwan was investigated by denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). Soil samples from three salterns were analyzed using DGGE and 16S rRNA from 502 colonies representing 5 archaea and 18 bacteria taxonomic groups. Each representative taxonomic group was further identified, whereas 8.7% of clones were unclassified microorganisms. Chromohalobacter, Halomonas and Virgibacillus are dominant in the Biemen saltern, Chiguensis saltern and Szutsau saltern, respectively. During FISH analysis, several taxonomic-specific probes were used. The DAPI-stained-cell count in the Szutsao saltern had a higher number of microorganisms (4.58 x 10(7) cell/cm(3)) than the other salterns. Archaea occupied 2.7-6.6% whereas bacteria accounted for 37.2-52.9% of total microbial population at the three sites. Among these three sampling sites, the Szutsao saltern had the highest diversity in halophilic microbial composition, as indicated by DGGE and FISH.     

Nanopore 16S amplicon sequencing enables rapid detection of pathogen in knee periprosthetic joint infection

ObjectivesWe investigated whether nanopore 16S amplicon sequencing is capable of bacterial identification in patients with knee prosthetic joint infection (PJI), and we compared its efficacy with conventional culture studies.MethodsIn total, 36 patients who had clinical manifestation suspected of PJI were enrolled in this study. To begin, synovial fluids were aspirated from the affected knee using aseptic technique and tissues specimens were obtained during the surgery. Next, DNA was extracted from the synovial fluid or tissues, and 16S rDNA PCR was performed. In PCR positive cases, nanopore amplicon sequencing was then performed for up to 3 h. The results of amplicon sequencing were compared to those of conventional culture studies.ResultsOf the 36 patients enrolled, 22 were classified as true infections according to the MSIS criteria whereas 14 were considered uninfected. Among the 22 PJI cases, 19 cases were culture positive (CP-PJI) while three cases were culture negative (CN-PJI). In 14 of 19 (73.7 %) CP- PJI cases, 16S sequencing identified concordant bacteria with conventional culture studies with a significantly shorter turnaround time. In some cases, nanopore 16S sequencing was superior to culture studies in the species-level identification of pathogen and detection of polymicrobial infections. Altogether, in the majority of PJI candidate patients (32 of 36, 88.9 %), 16S sequencing achieved identical results to cultures studies with a significantly reduced turnaround time (100.9 ± 32.5 h vs. 10.8 ± 7.7 h, p < 0.001).ConclusionsNanopore 16S sequencing was found to be particularly useful for pathogen identification in knee PJI. Although the sensitivity was not superior to culture studies, the nanopore 16S sequencing was much faster, and species-level identification and detection of polymicrobial infections were superior to culture studies.     

Polymerase chain reaction, with sequencing, as a diagnostic tool in culture-negative bacterial meningitis

Objective: To evaluate the feasibility of using 16S rDNA universal primer PCR (followed by sequencing) and 65-kDa heat shock Mycobacterium tuberculosis protein gene PCR as a method to determine a bacterial etiology in culture-negative cerebrospinal fluid (CSF) samples.
Methods: One hundred and forty-nine CSF samples from 128 patients were processed. DNA was extracted from the CSF samples and amplified with the eubacterial 16S rDNA primers P11E and P13B, and with the 65-kDa heat shock protein gene mycobacterial primers. The amplicons were identified by sequencing and specific oligoprobe hybridization.
Results: Overall, a microbiological diagnosis was made in 11 of 125 ultimately culture-negative cases. The use of 65-kDa heat shock protein gene PCR was needed to improve the diagnosis of tuberculous meningitis; in four patients, prospectively studied, the outcome of antituberculous therapy could also be followed.
Conclusions: In culture-negative bacterial meningitis it is possible to improve the microbiological diagnosis by use of 16S rDNA amplification and sequencing, together with amplification of a more specific gene in mycobacteria.     

Identification of Bartonella species in rodents, shrews and cats in Denmark: detection of two B. henselae variants, one in cats and the other in the long-tailed field mouse

Small mammals and stray cats were trapped in two areas of North Zealand, Denmark, and their blood cultured for hemotrophic bacteria. Bacterial isolates were recovered in pure culture and subjected to 16S rDNA gene sequencing. Bartonella species were isolated from five mammalian species: B. grahamii from Microtus agrestis (field vole) and Apodemus flavicollis (yellow-necked field mouse); B. taylorii from M. agrestis, A. flavicollis and A. sylvaticus (long-tailed field mouse); B. tribocorum from A. flavicollis; B. vinsonii subsp. vinsonii from M. agrestis and A. sylvaticus; and B. birtlesii from Sorex vulgaris (common shrew). In addition, two variant types of B. henselae were identified: variant I was recovered from three specimens of A. sylvaticus, and B. henselae variant II from 11 cats; in each case this was the only B. henselae variant found. No Bartonella species was isolated from Clethrionomys glareolus (bank vole) or Micromys minutus (harvest mouse). These results suggest that B. henselae occurs in two animal reservoirs in this region, one of variant I in A. sylvaticus, which may be transmitted between mice by the tick Ixodes ricinus, and another of variant II in cats, which may be transmitted by the cat flea (Ctenocephalides felis). To our knowledge, this is the first report of the occurrence of B. henselae and B. tribocorum in Apodemus mice.