Journal of Microbiology

Journal Article

Effect of dietary copper level on the gut microbiota and its correlation with serum inflammatory cytokines in Sprague-Dawley rats: Feng Zhang , Weijiang Zheng , Rong Guo , Wen Yao; J. Microbiol. 2017;55(9):694-702. Published online September 2, 2017; DOI: https://doi.org/10.1007/s12275-017-6627-9

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In China’s swine industry, copper is generally supplemented above the National Research Council (NRC) requirement (2012) because of its antimicrobial properties and the potential for growth promotion. Yet few are concerned about whether this excess supplementation is necessary. In this study, the 16S rRNA pyrosequencing was designed and used to investigate the effect of dietary copper level on the diversity of the fecal microbial community and the correlation of copper level with the serum level of inflammatory cytokines in Sprague-Dawley rat models. The results showed that the diet containing a high level of Cu (120 and 240 mg/kg) changed the microbial richness and diversity of rat feces associated with the increased copper content in the rat ileac and colonic digesta. Furthermore, a Pearson’s correlation analysis indicated that an accumulation of unabsorbed copper in the chyme was correlated with the microbial composition of the rat feces, which was linked with TNF-α in serum. The results suggest that dietary copper level may have a direct impact on circulating inflammatory cytokines in the serum, perhaps inducing an inflammatory response by altering the microbial composition of rat feces. Serum TNF-α could be the chief responder to excessive copper exposure.

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Research Support, Non-U.S. Gov'ts

Studies on seasonal dynamics of soil-higher fungal communities in Mongolian oak-dominant Gwangneung forest in Korea: Chang Sun Kim , Jong Woo Nam , Jong Won Jo , Sang-Yong Kim , Jae-Gu Han , Min Woo Hyun , Gi-Ho Sung , Sang-Kuk Han; J. Microbiol. 2016;54(1):14-22. Published online January 5, 2016; DOI: https://doi.org/10.1007/s12275-016-5521-1

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Abstract

We surveyed macrofungi biweekly at defined plots from April to December in 2014, in the Mongolian oak-dominant forest, Gwangneung Forest, Pochen-si, Korea, and analyzed a soilhigher fungal diversity during four seasons (represented by April, August, October, and December). Based on morphological observation of collected specimens, the collected macrofungi were classified into 2 phyla 3 classes 7 orders, 14 families, 21 genera, and 33 species (36 specimens). DNA-based community analyses indicated that soil-higher fungi were classified into 2 phyla, 18 classes, 49 orders, 101 families, and 155 genera (83,360 sequence reads), defined herein as 155 genus-level operational taxonomic units (GOTUs). In the present study, we evaluated and discussed the fungal diversity in seasonal dynamics and soil layers based on collected macrofungi and pyrosequencing data while considering environmental parameters (pH, exchangeable K, T-P, NH4+, NO3-, OM, WR, TOC, and T-N). Moreover, principal components analysis (PCA) showed distinct clusters of the GOTU assemblage associated with the seasons.

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Abstract

The concentration of major odor-causing compounds including phenols, indoles, short-chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) in response to the addition of powdered horse radish (PHR) and spent mushroom compost (SMC) was compared with control nontreated slurry (CNS) samples. A total of 97,465 rDNAs sequence reads were generated from three different samples (CNS, n = 2; PHR, n = 3; SMC, n = 3) using bar-coded pyrosequencing. The number of operational taxonomic units (OTUs) was lower in the PHR slurry compared with the other samples. A total of 11 phyla were observed in the slurry samples, while the phylogenetic analysis revealed that the slurry microbiome predominantly comprised members of the Bacteroidetes, Firmicutes, and Proteobacteria phyla. The rarefaction analysis showed the bacterial species richness varied among the treated samples. Overall, at the OTU level, 2,558 individual genera were classified, 276 genera were found among the three samples, and 1,832 additional genera were identified in the individual samples. A principal component analysis revealed the differences in microbial communities among the CNS, PHR, and SMC pig slurries. Correlation of the bacterial community structure with the Kyoto Encyclopedia of Genes and Genomes (KEGG) predicted pathways showed that the treatments altered the metabolic capabilities of the slurry microbiota. Overall, these results demonstrated that the PHR and SMC treatments significantly reduced the malodor compounds in pig slurry (P < 0.05).

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Abstract

The study of oceanic microbial communities is crucial for our understanding of the role of microbes in terms of biomass, diversity and ecosystem function. In this study, 16S rRNA gene tag pyrosequencing was used to investigate change in bacterial community structure between summer and winter water masses from Gosung Bay in the South Sea of Korea and Chuuk in Micronesia, located in the North and South Pacific Oceans, respectively. Summer and winter sampling from each water mass revealed highly diverse bacterial communities, containing ~900 Operational Taxonomic Units (OTUs). The microbial distribution and highly heterogeneous composition observed at both sampling sites were different from those of most macroorganisms. The bacterial communities in the seawater at both sites were most abundant in Proteobacteria during the summer in Gosung and in Bacterioidetes during the winter. The proportion of Cyanobacteria was higher in summer than in winter in Chuuk and similar in Gosung. Additionally, the microbial community during summer in Gosung was significantly different from other communities observed based on the unweighted UniFrac distance. These data suggest that in both oceanic areas sampled, the bacterial communities had distinct distribution patterns with spatially- and temporally-heterogeneous distributions.

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Abstract

This study examined the fecal bacterial diversity of 15-week-old pigs from three purebred lines: Duroc, Landrace, and Yorkshire. Taxon-dependent and -independent analyses were performed to evaluate differences in the fecal bacterial communities and to identify bacterial genera that can be used to discriminate breeds, following high-throughput pyrosequencing of 16S rRNA genes. Among the breeds evaluated, Landrace had the most diverse bacterial community composition. Prevotella, Blautia, Oscillibacter, and Clostridium were detected in all samples regardless of breed. On the other hand, Catenibacterium, Blautia, Dialister, and Sphaerochaeta were differentially detected among breeds, as demonstrated by the canonical loading plot. The discriminant analysis of principal components plot also showed clear separation of the three purebred pig lines, with a certain degree of similarity between Landrace and Yorkshire pigs and a distinct separation between Duroc pigs and the other two breeds. Other factors not related to breed, such as season or time of sampling and pen effects, may contribute to shaping the gut microbiota of pigs.

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Regional Effects on Chimera Formation in 454 Pyrosequenced Amplicons from a Mock Community: Sunguk Shin , Tae Kwon Lee , Jung Min Han , Joonhong Park; J. Microbiol. 2014;52(7):566-573. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3485-6

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Abstract

Chimeras are a frequent artifact in polymerase chain reaction and could be the underlying causes of erroneous taxonomic identifications, overestimated microbial diversity, and spurious sequences. However, little is known about the regional effects on chimera formation. Therefore, we investigated the chimera formation rates in different regions of phylogenetically important biomarker genes to test the regional effects on chimera formation. An empirical study of chimera formation rates was performed using the Roche GSFLXTM system with sequences of the V1/V2/V3 and V4/V5 regions of the 16S rRNA gene and sequences of the nifH gene from a mock microbial community. The chimera formation rates for the 16S V1/V2/V3 region, V4/V5 region, and nifH gene were 22.1–38.5%, 3.68–3.88%, and 0.31–0.98%, respectively. Some amplicons from the V1/V2/V3 regions were shorter than the typical length (~7–31%), reflecting incomplete extension. In the V1/V2/V3 and V4/V5 regions, conserved and hypervariable regions were identified. Chimeric hot spots were located in parts of conserved regions near the ends of the amplicons. The 16S V1/V2/V3 region had the highest chimera formation rate, likely because of long template lengths and incomplete extension. The amplicons of the nifH gene had the lowest frequency of chimera formation most likely because of variations in their wobble positions in triplet codons. Our results suggest that the main reasons for chimera formation are sequence similarity and premature termination of DNA extension near primer regions. Other housekeeping genes can be a good substitute for 16S rRNA genes inmolecularmicrobial studies to reduce the effects of chimera formation.

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Assessment of Microbial Diversity Bias Associated with Soil Heterogeneity and Sequencing Resolution in Pyrosequencing Analyses: Sokhee P. Jung , Hojeong Kang; J. Microbiol. 2014;52(7):574-580. Published online May 13, 2014; DOI: https://doi.org/10.1007/s12275-014-3636-9

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Abstract

It is important to estimate the true microbial diversities accurately for a comparative microbial diversity analysis among various ecological settings in ecological models. Despite drastically increasing amounts of 16S rRNA gene targeting pyrosequencing data, sampling and data interpretation for comparative analysis have not yet been standardized. For more accurate bacterial diversity analyses, the influences of soil heterogeneity and sequence resolution on bacterial diversity estimates were investigated using pyrosequencing data of oak and pine forest soils with focus on the bacterial 16S rRNA gene. Soil bacterial community sets were phylogenetically clustered into two separate groups by forest type. Rarefaction curves showed that bacterial communities sequenced from the DNA mixtures and the DNAs of the soil mixtures had midsize richness compared with other samples. Richness and diversity estimates were highly variable depending on the sequence read numbers. Bacterial richness estimates (ACE, Chao 1 and Jack) of the forest soils had positive linear relationships with the sequence read number. Bacterial diversity estimates NPShannon, Shannon and the inverse Simpson) of the forest soils were also positively correlated with the sequence read number. One-way ANOVA shows that sequence resolution significantly affected the α-diversity indices (P<0.05), but the soil heterogeneity did not (P>0.05). For an unbiased evaluation, richness and diversity estimates should be calculated and compared from subsets of the same size.

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Abstract

The bacteria resident in sponges collected from Chuuk Lagoon and Kosrae Island of Micronesia were investigated using the 16S rRNA gene PCR-tagged pyrosequencing method. These sponges were clustered into 5 groups based on their bacterial composition. Diversity indexes and cumulative rank abundance curves showed the different compositions of bacterial communities in the various groups of sponges. Reads related to the phylum Chloroflexi were observed predominantly (9.7–68.2%) in 9 sponges of 3 groups and unobserved in the other 2 groups. The Chloroflexi-containing group had similar bacterial patterns at the phylum and lower taxonomic levels, for example, significant proportions of Acidobacteria, Gemmatimonadetes, SBR1093, and PAUC34f were observed in most members of this group. The three groups in the Chloroflexi- containing group, however, showed some minor differences in the composition and diversity. The other two groups contained high proportions of Proteobacteria (>87%) or Bacteroidetes (>61%) and different composition and diversity compared to the Chloroflexi-containing group and each other. Four pairs of specimens with the same species showed similar bacterial profiles, but, the bacteria in sponges were highly specific at the individual level.

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Abstract

The majority of naturally occurring biofilms contain numerous microorganisms that have not yet been cultured. Additionally, there is little information available regarding the genetic structure and species diversity of these communities. Therefore, we characterised the species diversity, structure and metagenome of biofilms grown on stones and steel plates in the littoral zone of Lake Baikal (East Siberia, Russia) by applying three different approaches. First, light microscopy enabled identification of the species diversity of biofilm-forming cyanobacteria on different substrates with the dominance of Rivularia rufescens, Tolypothrix limbata, Chamaesiphon fuscus, Сh. subglobosus, and Heteroleibleinia pusilla. Additionally, scanning electron microscopy was used to show the spatial structure of biofilms. Finally, sequence analysis of 30,660 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to Cyanobacteria (8–46% sequences), Proteobacteria (14–43%), and Bacteroidetes (10– 41%). Rivularia sp., Pseudanabaena sp., and Chamaesiphon spp. were the dominant cyanobacterial phylotypes.

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Analysis of Bacterial Diversity in Sponges Collected off Chujado, an Island in Korea, Using Barcoded 454 Pyrosequencing: Analysis of a Distinctive Sponge Group Containing Chloroflexi: In-Hye Jeong , Kyoung-Ho Kim , Jin-Sook Park; J. Microbiol. 2013;51(5):570-577. Published online October 31, 2013; DOI: https://doi.org/10.1007/s12275-013-3426-9

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Abstract

The bacterial diversity of 14 sponges belonging to 5 different orders that were collected around Chuja Island, Korea was investigated using barcoded 454 pyrosequencing. The sponges contained many unidentified bacterial groups (e.g. more than half of the taxa at the family level) that were known only in environmental sequences and obtained from culture-independent methods. Five of the sponges were clustered into one notable group (CF group), which was distinguished from the other sponges in accordance with bacterial composition (the other sponges may be separated into more groups but clustering is not clear). The CF group contained high amounts of Chloroflexi (25.0–47.7%) and moderate amounts of Gemmatimonadetes (2.3–7.0%), AncK6 (0.6–2.2%), PAUC34f (0.8–6.0%), Acidobacteria (3.7–9.6%), and SBR1093 (1.8–5.6%) exclusively or almost exclusively to this group. Sponges in the CF group also showed higher diversity (e.g. Shannon index) than the other sponges and contained group-specific taxonomic lineages (e.g. class or family level) from group-specific phyla and even from the Proteobacteria and Actinobacteria, which were detected in all sponges at the phylum level. The CF group may be one of the most distinctive groups in sponges in terms of bacterial diversity.

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Bacterial Diversity in the Mountains of South-West China: Climate Dominates Over Soil Parameters: Dharmesh Singh , Lingling Shi , Jonathan M. Adams; J. Microbiol. 2013;51(4):439-447. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-2446-9

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Abstract: Certain patterns in soil bacterial diversity and community composition have become evident from metagenomics studies on a range of scales, from various parts of the world. For example, soil pH has generally been seen as dominating variation in bacterial diversity, above all other soil and climate parameters. It is important however to test the generality of these relationships by studying previously unsampled areas. We compared soil bacterial diversity and community composition under a wide range of climatic and edaphic conditions in mountainous Yunnan Province, SW China. Soil samples were taken from a range of primary forest types and altitudes, reflecting the great variation of forest environments in this region. From each soil sample, DNA was extracted and pyrosequenced for bacterial 16S rRNA gene identification. In contrast to other recent studies from other parts of the world, pH was a weaker predictor of bacterial community composition and diversity than exchangeable Ca2+ concentration, and also the more poorly defined >environmental parameter of elevation. Samples from within each forest type clustered strongly, showing the distinctive pattern of their microbial communities on a regional scale. It is clear that on a regional scale in a very heterogeneous environment, additional factors beyond pH can emerge as more important in determining bacterial diversity.

NOTE] Bacterial Diversity in Ornithogenic Soils Compared to Mineral Soils on King George Island, Antarctica: Ok-Sun Kim , Namyi Chae , Hyun Soo Lim , Ahnna Cho , Jeong Hoon Kim , Soon Gyu Hong , Jeongsu Oh; J. Microbiol. 2012;50(6):1081-1085. Published online December 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2655-7

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Abstract: In the Narębski Point area of King George Island of Antarctica, ornithogenic soils form on land under Chinstrap and Gentoo Penguin rookeries. The purpose of this study was to compare the bacterial community compositions in the gradient of contamination by penguin feces; mineral soil with no contamination, and soils with medium or high contamination. The discrimination between mineral soils and ornithogenic soils by characterization of physicochemical properties and bacterial communities was notable. Physicochemical analyses of soil properties showed enrichment of carbon and nitrogen in ornithogenic soils. Firmicutes were present abundantly in active ornithogenic soils, Bacteroidetes and Proteobacteria in a formerly active one, and several diverse phyla such as Proteobacteria, Actinobacteria, and Acidobacteria in mineral soils. Some predominant species belonging to the Firmicutes and Gammaproteobacteria may play an important role for the mineralization of nutrients in ornithogenic soils. Results of this study indicate that dominant species may play an important role in mineralization of nutrients in these ecosystems.

NOTE] Effects of PCR Cycle Number and DNA Polymerase Type on the 16S rRNA Gene Pyrosequencing Analysis of Bacterial Communities: Jae-Hyung Ahn , Byung-Yong Kim , Jaekyeong Song , Hang-Yeon Weon; J. Microbiol. 2012;50(6):1071-1074. Published online December 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2642-z

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67 Scopus

Abstract: The effects of PCR cycle number and DNA polymerase type on 16S rRNA gene pyrosequencing analysis were investigated using an artificially prepared bacterial community (mock community). The bacterial richness was overestimated at increased PCR cycle number mostly due to the occurence of chimeric sequences, and this was more serious with a DNA polymerase having proofreading activity than with Taq DNA polymerase. These results suggest that PCR cycle number must be kept as low as possible for accurate estimation of bacterial richness and that particular care must be taken when a DNA polymerase having proofreading activity is used.

PyroTrimmer: a Software with GUI for Pre-Processing 454 Amplicon Sequences: Jeongsu Oh , Byung Kwon Kim , Wan-Sup Cho , Soon Gyu Hong , Kyung Mo Kim; J. Microbiol. 2012;50(5):766-769. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2494-6

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21 Scopus

Abstract: The ultimate goal of metagenome research projects is to understand the ecological roles and physiological functions of the microbial communities in a given natural environment. The 454 pyrosequencing platform produces the longest reads among the most widely used next generation sequencing platforms. Since the relatively longer reads of the 454 platform provide more information for identification of microbial sequences, this platform is dedicated to microbial community and population studies. In order to accurately perform the downstream analysis of the 454 multiplex datasets, it is necessary to remove artificially designed sequences located at either ends of individual reads and to correct lowquality sequences. We have developed a program called Pyro-Trimmer that removes the barcodes, linkers, and primers, trims sequence regions with low quality scores, and filters out low-quality sequence reads. Although these functions have previously been implemented in other programs as well, PyroTrimmer has novelty in terms of the following features: i) more sensitive primer detection using Levenstein distance and global pairwise alignment, ii) the first stand-alone software with a graphic user interface, and iii) various options for trimming and filtering out the low-quality sequence reads. PyroTrimmer, written in JAVA, is compatible with multiple operating systems and can be downloaded free at http://pyrotrimmer.kobic.re.kr.

Characterization of the Bacterial and Archaeal Communities in Rice Field Soils Subjected to Long-Term Fertilization Practices: Jae-Hyung Ahn , Jaekyeong Song , Byung-Yong Kim , Myung-Sook Kim , Jae-Ho Joa , Hang-Yeon Weon; J. Microbiol. 2012;50(5):754-765. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2409-6

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115 Scopus

Abstract: The bacterial and archaeal communities in rice field soils subjected to different fertilization regimes for 57 years were investigated in two different seasons, a non-planted, drained season (April) and a rice-growing, flooded season (August), by performing soil dehydrogenase assay, real-time PCR assay and pyrosequencing analysis. All fertilization regimes increased the soil dehydrogenase activity while the abundances of bacteria and archaea increased in the plots receiving inorganic fertilizers plus compost and not in those receiving inorganic fertilizers only. Rice-growing and flooding decreased the soil dehydrogenase activity while they increased the bacterial diversity in rice field soils. The bacterial communities were dominated by Chloroflexi, Proteobacteria, and Actinobacteria and the archaeal communities by Crenarchaeota at the phylum level. In principal coordinates analysis based on the weighted Fast UniFrac metric, the bacterial and archaeal communities were separated primarily by season, and generally distributed along with soil pH, the variation of which had been caused by long-term fertilization. Variations in the relative abundance according to the season or soil pH were observed for many bacterial and archaeal groups. In conclusion, the microbial activity, prokaryotic abundance and diversity, and prokaryotic community structure in the rice field soils were changed by season and long-term fertilization.

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