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16s rRNA Sequencing with MR DNA

16S ribosomal  (rRNA) sequencing using next generation sequencing is a method used to identify and compare bacteria and archaea present within almost any type of sample. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study.




387. Inflamm Bowel Dis. 2011 Jan;17(1):179-84. doi: 10.1002/ibd.21339. Epub 2010 Sep



Disease phenotype and genotype are associated with shifts in

intestinal-associated microbiota in inflammatory bowel diseases.


Frank DN(1), Robertson CE, Hamm CM, Kpadeh Z, Zhang T, Chen H, Zhu W, Sartor RB,

Boedeker EC, Harpaz N, Pace NR, Li E.


Author information:

(1)Department of Molecular, Cellular and Developmental Biology, University of

Colorado, Boulder, Colorado 80045, USA.


BACKGROUND: Abnormal host-microbe interactions are implicated in the pathogenesis

of inflammatory bowel diseases. Previous 16S rRNA sequence analysis of intestinal

tissues demonstrated that a subset of Crohn's disease (CD) and ulcerative colitis

(UC) samples exhibited altered intestinal-associated microbial compositions

characterized by depletion of Bacteroidetes and Firmicutes (particularly

Clostridium taxa). We hypothesize that NOD2 and ATG16L1 risk alleles may be

associated with these alterations.

METHODS: To test this hypothesis, we genotyped 178 specimens collected from 35

CD, 35 UC, and 54 control patients for the three major NOD2 risk alleles (Leu

1007fs, R702W, and G908R) and the ATG16L1T300A risk allele, that had undergone

previous 16S rRNA sequence analysis. Our statistical models incorporated the

following independent variables: 1) disease phenotype (CD, UC, non-IBD control);

2) NOD2 composite genotype (NOD2(R) = at least one risk allele, NOD2(NR) = no

risk alleles); 3) ATG16L1T300A genotype (ATG16L1(R/R), ATG16L1(R/NR),

ATG16L1(NR/NR)); 4) patient age at time of surgery and all first-order

interactions. The dependent variable(s) were the relative frequencies of

bacterial taxa classified by applying the RDP 2.1 classifier to previously

reported 16S rRNA sequence data.

RESULTS: Disease phenotype, NOD2 composite genotype and ATG16L1 genotype were

significantly associated with shifts in microbial compositions by nonparametric

multivariate analysis of covariance (MANCOVA). Shifts in the relative frequencies

of Faecalibacterium and Escherichia taxa were significantly associated with

disease phenotype by nonparametric ANCOVA.

CONCLUSIONS: These results support the concept that disease phenotype and

genotype are associated with compositional changes in intestinal-associated



Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.


DOI: 10.1002/ibd.21339

PMCID: PMC3834564

PMID: 20839241  [PubMed - indexed for MEDLINE]



388. Indian J Med Microbiol. 2012 Oct-Dec;30(4):462-6. doi: 10.4103/0255-0857.103771.


16S rDNA-based metagenomic analysis of human oral plaque microbiota in patients

with atherosclerosis and healthy controls.


Ismail F(1), Baetzner C, Heuer W, Stumpp N, Eberhard J, Winkel A, Ismail I,

Haverich A, Stiesch M.


Author information:

(1)Department of Prosthetic Dentistry and Biomedical Material Sciences, Hannover

Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.


Erratum in

    Indian J Med Microbiol. 2014 Jul-Sep;32(3):359.

    Indian J Med Microbiol. 2014 Apr-Jun;32(2):178. Baetzner, C [added].


To address the question if an altered oral microbiota is associated with

atherosclerosis. Twenty patients suffering from atherosclerosis and 10 controls

were recruited. Clinical oral, medical and laboratory investigations were

performed. Oral bacteria were collected and 16S rDNA was sequenced following

Single strand conformation polymorphism.(SSCP) Probing pocket depths in patients

were significantly elevated. The oral microbiota of patients and controls were

dominated by Fusobacterium (16%/17%), Streptococcus (21%/14%), Prevotella

(10%/12%), Enterococcus (12%/12%), Porphyromonas (8%/7%), TM7 (0%/7%) and

Veillonella (6%/7%). Differences in diversity were not significant between

groups. The pathology of atherosclerosis may not be related to significant

qualitative changes of the oral microbiota.


DOI: 10.4103/0255-0857.103771

PMID: 23183474  [PubMed - indexed for MEDLINE]



389. ISME J. 2011 Apr;5(4):741-9. doi: 10.1038/ismej.2010.160. Epub 2010 Oct 21.


BIPES, a cost-effective high-throughput method for assessing microbial diversity.


Zhou HW(1), Li DF, Tam NF, Jiang XT, Zhang H, Sheng HF, Qin J, Liu X, Zou F.


Author information:

(1)Department of Environmental Health, School of Public Health and Tropical

Medicine, Southern Medical University, Guangzhou, Guangdong, China.


Pyrosequencing of 16S rRNA (16S) variable tags has become the most popular method

for assessing microbial diversity, but the method remains costly for the

evaluation of large numbers of environmental samples with high sequencing depths.

We developed a barcoded Illumina paired-end (PE) sequencing (BIPES) method that

sequences each 16S V6 tag from both ends on the Illumina HiSeq 2000, and the PE

reads are then overlapped to obtain the V6 tag. The average accuracy of Illumina

single-end (SE) reads was only 97.9%, which decreased from ∼99.9% at the start of

the read to less than 85% at the end of the read; nevertheless, overlapping of

the PE reads significantly increased the sequencing accuracy to 99.65% by

verifying the 3' end of each SE in which the sequencing quality was degraded.

After the removal of tags with two or more mismatches within the medial 40-70

bases of the reads and of tags with any primer errors, the overall base

sequencing accuracy of the BIPES reads was further increased to 99.93%. The BIPES

reads reflected the amounts of the various tags in the initial template, but long

tags and high GC tags were underestimated. The BIPES method yields 20-50 times

more 16S V6 tags than does pyrosequencing in a single-flow cell run, and each of

the BIPES reads costs less than 1/40 of a pyrosequencing read. As a laborsaving

and cost-effective method, BIPES can be routinely used to analyze the microbial

ecology of both environmental and human microbiomes.


DOI: 10.1038/ismej.2010.160

PMCID: PMC3105743

PMID: 20962877  [PubMed - indexed for MEDLINE]



390. Am J Clin Nutr. 2013 Jul;98(1):111-20. doi: 10.3945/ajcn.112.056689. Epub 2013

May 29.


Diet, microbiota, and microbial metabolites in colon cancer risk in rural

Africans and African Americans.


Ou J(1), Carbonero F, Zoetendal EG, DeLany JP, Wang M, Newton K, Gaskins HR,

O'Keefe SJ.


Author information:

(1)Department of Gastroenterology, Hepatology and Nutrition, School of Medicine,

University of Pittsburgh, Pittsburgh, PA 15213, USA.


BACKGROUND: Epidemiologic studies have suggested that most cases of sporadic

colon cancer can be attributed to diet. The recognition that colonic microbiota

have a major influence on colonic health suggests that they might mediate colonic


OBJECTIVE: To examine the hypothesis that the influence of diet on colon cancer

risk is mediated by the microbiota through their metabolites, we measured

differences in colonic microbes and their metabolites in African Americans with a

high risk and in rural native Africans with a low risk of colon cancer.

DESIGN: Fresh fecal samples were collected from 12 healthy African Americans aged

50-65 y and from 12 age- and sex-matched native Africans. Microbiomes were

analyzed with 16S ribosomal RNA gene pyrosequencing together with quantitative

polymerase chain reaction of the major fermentative, butyrate-producing, and bile

acid-deconjugating bacteria. Fecal short-chain fatty acids were measured by gas

chromatography and bile acids by liquid chromatography-mass spectrometry.

RESULTS: Microbial composition was fundamentally different, with a predominance

of Prevotella in native Africans (enterotype 2) and of Bacteroides in African

Americans (enterotype 1). Total bacteria and major butyrate-producing groups were

significantly more abundant in fecal samples from native Africans. Microbial

genes encoding for secondary bile acid production were more abundant in African

Americans, whereas those encoding for methanogenesis and hydrogen sulfide

production were higher in native Africans. Fecal secondary bile acid

concentrations were higher in African Americans, whereas short-chain fatty acids

were higher in native Africans.

CONCLUSION: Our results support the hypothesis that colon cancer risk is

influenced by the balance between microbial production of health-promoting

metabolites such as butyrate and potentially carcinogenic metabolites such as

secondary bile acids.


DOI: 10.3945/ajcn.112.056689

PMCID: PMC3683814

PMID: 23719549  [PubMed - indexed for MEDLINE]



391. Appl Environ Microbiol. 2009 Jan;75(2):381-6. doi: 10.1128/AEM.01731-08. Epub

2008 Nov 21.


Differential effects of Bifidobacterium pseudolongum strain Patronus and

metronidazole in the rat gut.


Vasquez N(1), Suau A, Magne F, Pochart P, Pélissier MA.


Author information:

(1)EA 3199, Laboratoire de Biologie, CNAM, 2 rue Conté, 75003 Paris, France.


In the luminal contents of metronidazole-treated rats, there was a dominant

Bifidobacterium species. A strain has been isolated, its 16S rRNA gene has been

sequenced, and the strain has been named Bifidobacterium pseudolongum strain

Patronus. In this study, using an experimental model of healthy rats, the effects

of metronidazole treatment and B. pseudolongum strain Patronus administration on

the luminal and mucosa-associated microbiota and on gut oxidation processes were

investigated. Metronidazole treatment and the daily gavage of rats with B.

pseudolongum strain Patronus increased the numbers of bifidobacteria in cecal

contents and in cecal mucosa-associated microbiota compared with those in control

rats. Metronidazole reduced the colonic oxidative damage to proteins. This is the

first evidence that B. pseudolongum strain Patronus exerts an effect on a

biomarker of oxidative damage by reducing the susceptibility to oxidation of

proteins in the colon and the small bowel. Antioxidant effects of metronidazole

could be linked to the bifidobacterial increase but also to other bacterial



DOI: 10.1128/AEM.01731-08

PMCID: PMC2620693

PMID: 19028910  [PubMed - indexed for MEDLINE]



392. J Clin Periodontol. 2012 May;39(5):425-33. doi: 10.1111/j.1600-051X.2012.01856.x.

Epub 2012 Mar 14.


Pyrosequencing reveals unique microbial signatures associated with healthy and

failing dental implants.


Kumar PS(1), Mason MR, Brooker MR, O'Brien K.


Author information:

(1)Division of Periodontology, College of Dentistry, The Ohio State University,

Columbus, OH, USA.


AIM: Although it is established that peri-implantitis is a bacterially induced

disease, little is known about the bacterial profile of peri-implant communities

in health and disease. The purpose of the present investigation was to examine

the microbial signatures of the peri-implant microbiome in health and disease.

MATERIALS AND METHODS: Subgingival and submucosal plaque samples were collected

from forty subjects with periodontitis, peri-implantitis, periodontal and

peri-implant health and analysed using 16S pyrosequencing.

RESULTS: Peri-implant biofilms demonstrated significantly lower diversity than

subgingival biofilms in both health and disease, however, several species,

including previously unsuspected and unknown organisms, were unique to this

niche. The predominant species in peri-implant communities belonged to the genera

Butyrivibrio, Campylobacter, Eubacterium, Prevotella, Selenomonas, Streptococcus,

Actinomyces, Leptotrichia, Propionibacterium, Peptococcus, Lactococcus and

Treponema. Peri-implant disease was associated with lower levels of Prevotella

and Leptotrichia and higher levels of Actinomyces, Peptococcus, Campylobacter,

non-mutans Streptococcus, Butyrivibrio and Streptococcus mutans than healthy

implants. These communities also demonstrated lower levels of Prevotella,

non-mutans Streptococcus, Lactobacillus, Selenomonas, Leptotrichia, Actinomyces

and higher levels of Peptococcus, Mycoplasma, Eubacterium, Campylobacter,

Butyrivibrio, S. mutans and Treponema when compared to periodontitis-associated


CONCLUSION: The peri-implant microbiome differs significantly from the

periodontal community in both health and disease. Peri-implantitis is a

microbially heterogeneous infection with predominantly gram-negative species, and

is less complex than periodontitis.


© 2012 John Wiley & Sons A/S.


DOI: 10.1111/j.1600-051X.2012.01856.x

PMCID: PMC3323747

PMID: 22417294  [PubMed - indexed for MEDLINE]



393. BMC Microbiol. 2015 Mar 14;15:65. doi: 10.1186/s12866-015-0398-4.


Composition of soil microbiome along elevation gradients in southwestern

highlands of Saudi Arabia.


Yasir M(1), Azhar EI(2,)(3), Khan I(4), Bibi F(5), Baabdullah R(6), Al-Zahrani

IA(7), Al-Ghamdi AK(8).


Author information:

(1)Special Infectious Agents Unit, King Fahd Medical Research Center, King

Abdulaziz University, Jeddah, Saudi Arabia.

(2)Special Infectious Agents Unit, King Fahd Medical Research Center, King

Abdulaziz University, Jeddah, Saudi Arabia. (3)Medical

Laboratory Technology Department, Faculty of Applied Medical Sciences, King

Abdulaziz University, Jeddah, Saudi Arabia. (4)Biochemistry

Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. (5)Special Infectious Agents Unit, King Fahd Medical

Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. (6)Special Infectious Agents Unit, King Fahd Medical

Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. (7)Medical Laboratory Technology Department, Faculty of

Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia. (8)Medical Laboratory Technology Department, Faculty of

Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.


BACKGROUND: Saudi Arabia is mostly barren except the southwestern highlands that

are susceptible to environmental changes, a hotspot for biodiversity, but poorly

studied for microbial diversity and composition. In this study,

454-pyrosequencing of 16S rRNA gene hypervariable region V6 was used to analyze

soil bacterial community along elevation gradients of the southwestern highlands.

RESULTS: In general, lower percentage of total soil organic matter (SOM) and

nitrogen were detected in the analyzed soil samples. Total 33 different phyla

were identified across the samples, including dominant phyla Proteobacteria,

Actinobacteria and Acidobacteria. Representative OTUs were grouped into 329 and

508 different taxa at family and genus level taxonomic classification,

respectively. The identified OTUs unique to each sample were very low

irrespective of the altitude. Jackknifed principal coordinates analysis (PCoA)

revealed, overall differences in the bacterial community were more related to the

quantity of specific OTUs than to their diversity among the studied samples.

CONCLUSIONS: Bacterial diversity and soil physicochemical properties did not show

consistent changes along the elevation gradients. The large number of OTUs shared

between the studied samples suggest the presence of a core soil bacterial

community in the southwestern highlands of Saudi Arabia.


DOI: 10.1186/s12866-015-0398-4

PMCID: PMC4374494

PMID: 25888310  [PubMed - indexed for MEDLINE]



394. Appl Environ Microbiol. 2004 May;70(5):2791-800.


Spatial distribution and stability of the eight microbial species of the altered

schaedler flora in the mouse gastrointestinal tract.


Sarma-Rupavtarm RB(1), Ge Z, Schauer DB, Fox JG, Polz MF.


Author information:

(1)Department of Civil and Environmental Engineering, Massachusetts Institute of

Technology, Cambridge, Massachusetts 02139, USA.


The overall complexity of the microbial communities in the gastrointestinal (GI)

tracts of mammals has hindered observations of dynamics and interactions of

individual bacterial populations. However, such information is crucial for

understanding the diverse disease-causing and protective roles that gut

microbiota play in their hosts. Here, we determine the spatial distribution,

interanimal variation, and persistence of bacteria in the most complex

defined-flora (gnotobiotic) model system to date, viz., mice colonized with the

eight strains of the altered Schaedler flora (ASF). Quantitative PCR protocols

based on the 16S rRNA sequence of each ASF strain were developed and optimized to

specifically detect as few as 10 copies of each target. Total numbers of the ASF

strains were determined in the different regions of the GI tracts of three C.B-17

SCID mice. Individual strain abundance was dependent on oxygen sensitivity, with

microaerotolerant Lactobacillus murinus ASF361 present at 10(5) to 10(7) cells/g

of tissue in the upper GI tract and obligate anaerobic ASF strains being

predominant in the cecal and colonic flora at 10(8) to 10(10) cells/g of tissue.

The variation between the three mice was small for most ASF strains, except for

Clostridium sp. strain ASF502 and Bacteroides sp. strain ASF519 in the cecum. A

comparison of the relative distribution of the ASF strains in feces and the colon

indicated large differences, suggesting that fecal bacterial levels may provide a

poor approximation of colonic bacterial levels. All ASF strains were detected by

PCR in the feces of C57BL/6 restricted flora mice, which had been maintained in

an isolator without sterile food, water, or bedding for several generations,

providing evidence for the stability of these strains in the face of potential

competition by bacteria introduced into the gut.



PMCID: PMC404395

PMID: 15128534  [PubMed - indexed for MEDLINE]



395. PLoS One. 2013 Nov 25;8(11):e80201. doi: 10.1371/journal.pone.0080201.

eCollection 2013.


Differential effects of antibiotic therapy on the structure and function of human

gut microbiota.


Pérez-Cobas AE(1), Artacho A, Knecht H, Ferrús ML, Friedrichs A, Ott SJ, Moya A,

Latorre A, Gosalbes MJ.


Author information:

(1)Unidad Mixta de Investigación en Genómica y Salud del Centro Superior de

Investigación en Salud Pública e Instituto Cavanilles de Biodiversidad y Biología

Evolutiva de la Universitat de València, Valencia, Spain ; CIBER en Epidemiología

y Salud Pública, Madrid, Spain.


The human intestinal microbiota performs many essential functions for the host.

Antimicrobial agents, such as antibiotics (AB), are also known to disturb

microbial community equilibrium, thereby having an impact on human physiology.

While an increasing number of studies investigate the effects of AB usage on

changes in human gut microbiota biodiversity, its functional effects are still

poorly understood. We performed a follow-up study to explore the effect of ABs

with different modes of action on human gut microbiota composition and function.

Four individuals were treated with different antibiotics and samples were taken

before, during and after the AB course for all of them. Changes in the total and

in the active (growing) microbiota as well as the functional changes were

addressed by 16S rRNA gene and metagenomic 454-based pyrosequencing approaches.

We have found that the class of antibiotic, particularly its antimicrobial effect

and mode of action, played an important role in modulating the gut microbiota

composition and function. Furthermore, analysis of the resistome suggested that

oscillatory dynamics are not only due to antibiotic-target resistance, but also

to fluctuations in the surviving bacterial community. Our results indicated that

the effect of AB on the human gut microbiota relates to the interaction of

several factors, principally the properties of the antimicrobial agent, and the

structure, functions and resistance genes of the microbial community.


DOI: 10.1371/journal.pone.0080201

PMCID: PMC3839934

PMID: 24282523  [PubMed - indexed for MEDLINE]



396. Lett Appl Microbiol. 2009 Apr;48(4):433-9. doi: 10.1111/j.1472-765X.2008.02547.x.

Epub 2009 Feb 2.


Novel 16S rRNA gene analyses reveal new in vitro effects of insoluble barley

fibres on the human faecal microbiota.


Rudi K(1), Zimonja M, Aasen IM, Knutsen SH, Sahlstrøm S.


Author information:

(1)Matforsk AS, Nofima Food, Osloveien, Norway.


AIMS: The aim of this work was to analyse the growth of human faecal microbiota

on barley dietary fibres (DF). It is generally accepted that insoluble DF are

health promoting, but the information is scarce about how these fibres affect the

gastrointestinal (GI) microbiota. A major reason for the limited knowledge is

that there are currently no proper tools to analyse the complete GI microbiota.

METHODS AND RESULTS: Here we present a novel 16S rRNA gene analytical approach

that enables the analyses of the complete microbiota, including the part that has

not yet been characterized. The basic principle of the method is use of 16S rRNA

gene signature sequences to determine both the phylogenetic relatedness and the

distribution of bacteria in the samples analysed. Using this approach, we

analysed the microbiota after in vitro fermentation of different barley fractions

with human faeces. Our main finding was that groups of actinobacteria were

selectively enriched by growth on the insoluble DF fractions.

CONCLUSIONS: Our novel analytical approaches revealed new enrichment patterns in

the taxa that respond to insoluble DF.

SIGNIFICANCE AND IMPACT OF THE STUDY: Our results may have major implications for

future understanding of insoluble DF health effects.


DOI: 10.1111/j.1472-765X.2008.02547.x

PMID: 19187495  [PubMed - indexed for MEDLINE]



397. Microbiology. 2008 May;154(Pt 5):1535-43. doi: 10.1099/mic.0.2007/014803-0.


The microbiome of the cloacal openings of the urogenital and anal tracts of the

tammar wallaby, Macropus eugenii.


Chhour KL(1), Hinds LA, Deane EM, Jacques NA.


Author information:

(1)Department of Biological Sciences, Division of Environmental and Life

Sciences, Macquarie University, NSW 2109, Australia.


The bacterial diversity of the openings of the urogenital and anal tracts of the

adult female tammar wallaby, Macropus eugenii, was determined in order to

ascertain whether the physical proximity of the openings of these tracts within

the cloaca affected the two populations of bacteria. Terminal restriction

fragment length polymorphism (T-RFLP) analyses of 42 wallabies identified 81

different terminal fragments, indicative of diverse and complex microbiomes at

these anatomical locations. Subsequent amplified rDNA restriction analysis

(ARDRA) identified 72 phylotypes from the urogenital tract and 50 from the anal

tract. Twenty-two of these phylotypes were common to both tracts. Phylogenetic

analysis of sequenced 16S rDNA showed that 83 % of the phylotypes were

unidentified species based on the premise that any sequence possessing <97 %

homology to a known bacterial species or phylotype was novel. Thus, despite the

close proximity of the openings of the urogenital and anal tracts within the

cloaca, the two sites retained a diverse range of distinct bacteria, with only a

small percentage of overlapping species.


DOI: 10.1099/mic.0.2007/014803-0

PMID: 18451062  [PubMed - indexed for MEDLINE]



398. World J Gastroenterol. 2013 Mar 14;19(10):1541-50. doi: 10.3748/wjg.v19.i10.1541.


Upper gastrointestinal microbiota and digestive diseases.


Wang ZK(1), Yang YS.


Author information:

(1)Department of Gastroenterology and Hepatology, Chinese PLA General Hospital,

Chinese PLA Medical Academy, Beijing 100853, China.


Metagenomics which combines the power of genomics, bioinformatics, and systems

biology, provide new access to the microbial world. Metagenomics permit the

genetic analysis of complex microbial populations without requiring prior

cultivation. Through the conceptual innovations in metagenomics and the

improvements in DNA high-throughput sequencing and bioinformatics analysis

technology, gastrointestinal microbiology has entered the metagenomics era and

become a hot topic worldwide. Human microbiome research is underway, however,

most studies in this area have focused on the composition and function of the

intestinal microbiota and the relationship between intestinal microbiota and

metabolic diseases (obesity, diabetes, metabolic syndrome, etc.) and intestinal

disorders [inflammatory bowel disease, colorectal cancer, irritable bowel

syndrome (IBS), etc.]. Few investigations on microbiota have been conducted

within the upper gastrointestinal tract (esophagus, stomach and duodenum). The

upper gastrointestinal microbiota is essential for several gastrointestinal

illnesses, including esophagitis, Barrett's esophagus, and esophageal carcinoma,

gastritis and gastric cancer, small intestinal bacterial overgrowth, IBS and

celiac disease. However, the constitution and diversity of the microbiota in

different sections of the upper gastrointestinal tract under health and various

disease states, as well as the function of microbiota in the pathogenesis of

various digestive diseases are still undefined. The current article provides an

overview of the recent findings regarding the relationship between upper

gastrointestinal microbiota and gastrointestinal diseases; and discusses the

study limitations and future directions of upper gastrointestinal microbiota



DOI: 10.3748/wjg.v19.i10.1541

PMCID: PMC3602471

PMID: 23539678  [PubMed - indexed for MEDLINE]



16s sequencing illumina or PGM low cost prices with MR DNA

MR DNA is a next generation sequencing provider with low cost 16s sequencing services.


Environ Sci Pollut Res Int. 2016 Jul;23(13):13245-54. doi: 10.1007/s11356-016-6474-y. Epub 2016 Mar 29.

Influence of zinc nanoparticles on survival of worms Eisenia fetida and taxonomic diversity of the gut microflora.

Yausheva Е1, Sizova Е2,3, Lebedev S2,3, Skalny A2, Miroshnikov S3, Plotnikov A4, Khlopko Y4, Gogoleva N5, Cherkasov S4.

Author information


The study was conducted to examine the effect of zinc nanoparticles on survival of worms Eisenia fetida and composition of the gut microflora. Analysis of the survival data has shown that the introduction of high doses of the nanoparticles causes death of worms in the second group with 35 % mortality rate and activates protective mechanisms realized as mucous film. DNA from the worm guts was extracted and 16S metagenomic sequencing was fulfilled using MiSeq (Illumina). Regarding the gut microflora of worms in the control group, high diversity of microorganisms (303 OTUs) was noted. Most of those belong to the taxa Firmicutes (51.9 % of the total high-quality united reads), Proteobacteria (24.1 % of the total), and Actinobacteria (13.3 % of the total), which were represented by numerous species of gen. Clostridium (C. saccharobutylicum, C. saccharoperbutylacetonicum, C. beijerinckii), gen. Pseudomonas (P. hydrogenovora, P. aeruginosa, and P. putida), gen. Bacillus (B. megaterium, B. silvestris), gen. Cellulomonas (B. megaterium, B. silvestris), and other numerically smaller genera. Adding of zinc nanoparticles to the substrate decreased the diversity of bacteria (78 OTUs) as well as percentage of bacteria belonging to the taxon Firmicutes (-41.6 %) and increased the proportion of Proteobacteria due to growth in abundance of gen. Verminephrobacter (+46 %) and gen. Ochrobactrum (+19.5 %).


16S metagenome; Eisenia fetida; High-throughput sequencing; Microcrystalline cellulose; Microflora; Nanoparticles; Taxa; Zinc

PMID: 27023811 DOI: 10.1007/s11356-016-6474-y

[PubMed - in process]

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Sci Rep. 2015 Jun 12;5:10044. doi: 10.1038/srep10044.

Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing.

Chao Y1, Mao Y2, Wang Z3, Zhang T2.

Author information


The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.


PMID: 26067561 PMCID: PMC4464384 DOI: 10.1038/srep10044

[PubMed - indexed for MEDLINE] Free PMC Article

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PLoS One. 2015 Jun 2;10(6):e0128711. doi: 10.1371/journal.pone.0128711. eCollection 2015.

Screening currency notes for microbial pathogens and antibiotic resistance genes using a shotgun metagenomic approach.

Jalali S1, Kohli S2, Latka C3, Bhatia S4, Vellarikal SK5, Sivasubbu S5, Scaria V1, Ramachandran S1.

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Fomites are a well-known source of microbial infections and previous studies have provided insights into the sojourning microbiome of fomites from various sources. Paper currency notes are one of the most commonly exchanged objects and its potential to transmit pathogenic organisms has been well recognized. Approaches to identify the microbiome associated with paper currency notes have been largely limited to culture dependent approaches. Subsequent studies portrayed the use of 16S ribosomal RNA based approaches which provided insights into the taxonomical distribution of the microbiome. However, recent techniques including shotgun sequencing provides resolution at gene level and enable estimation of their copy numbers in the metagenome. We investigated the microbiome of Indian paper currency notes using a shotgun metagenome sequencing approach. Metagenomic DNA isolated from samples of frequently circulated denominations of Indian currency notes were sequenced using Illumina Hiseq sequencer. Analysis of the data revealed presence of species belonging to both eukaryotic and prokaryotic genera. The taxonomic distribution at kingdom level revealed contigs mapping to eukaryota (70%), bacteria (9%), viruses and archae (~1%). We identified 78 pathogens including Staphylococcus aureus, Corynebacterium glutamicum, Enterococcus faecalis, and 75 cellulose degrading organisms including Acidothermus cellulolyticus, Cellulomonas flavigena and Ruminococcus albus. Additionally, 78 antibiotic resistance genes were identified and 18 of these were found in all the samples. Furthermore, six out of 78 pathogens harbored at least one of the 18 common antibiotic resistance genes. To the best of our knowledge, this is the first report of shotgun metagenome sequence dataset of paper currency notes, which can be useful for future applications including as bio-surveillance of exchangeable fomites for infectious agents.


PMID: 26035208 PMCID: PMC4452720 DOI: 10.1371/journal.pone.0128711

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Microb Ecol. 2015 Oct;70(3):701-9. doi: 10.1007/s00248-015-0611-x. Epub 2015 Apr 26.

Deciphering Cyanide-Degrading Potential of Bacterial Community Associated with the Coking Wastewater Treatment Plant with a Novel Draft Genome.

Wang Z1,2, Liu L1,3, Guo F1, Zhang T4.

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Biotreatment processes fed with coking wastewater often encounter insufficient removal of pollutants, such as ammonia, phenols, and polycyclic aromatic hydrocarbons (PAHs), especially for cyanides. However, only a limited number of bacterial species in pure cultures have been confirmed to metabolize cyanides, which hinders the improvement of these processes. In this study, a microbial community of activated sludge enriched in a coking wastewater treatment plant was analyzed using 454 pyrosequencing and Illumina sequencing to characterize the potential cyanide-degrading bacteria. According to the classification of these pyro-tags, targeting V3/V4 regions of 16S rRNA gene, half of them were assigned to the family Xanthomonadaceae, implying that Xanthomonadaceae bacteria are well-adapted to coking wastewater. A nearly complete draft genome of the dominant bacterium was reconstructed from metagenome of this community to explore cyanide metabolism based on analysis of the genome. The assembled 16S rRNA gene from this draft genome showed that this bacterium was a novel species of Thermomonas within Xanthomonadaceae, which was further verified by comparative genomics. The annotation using KEGG and Pfam identified genes related to cyanide metabolism, including genes responsible for the iron-harvesting system, cyanide-insensitive terminal oxidase, cyanide hydrolase/nitrilase, and thiosulfate:cyanide transferase. Phylogenetic analysis showed that these genes had homologs in previously identified genomes of bacteria within Xanthomonadaceae and even presented similar gene cassettes, thus implying an inherent cyanide-decomposing potential. The findings of this study expand our knowledge about the bacterial degradation of cyanide compounds and will be helpful in the remediation of cyanides contamination.



Activated sludge; Coking wastewater; Cyanides; Metagenome; Thermomonas

PMID: 25910603 DOI: 10.1007/s00248-015-0611-x

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Microb Cell Fact. 2015 Mar 14;14:33. doi: 10.1186/s12934-015-0218-4.

Dissecting microbial community structure and methane-producing pathways of a full-scale anaerobic reactor digesting activated sludge from wastewater treatment by metagenomic sequencing.

Guo J1,2, Peng Y3, Ni BJ4, Han X5,6, Fan L7, Yuan Z8.

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Anaerobic digestion has been widely applied to treat the waste activated sludge from biological wastewater treatment and produce methane for biofuel, which has been one of the most efficient solutions to both energy crisis and environmental pollution challenges. Anaerobic digestion sludge contains highly complex microbial communities, which play crucial roles in sludge treatment. However, traditional approaches based on 16S rRNA amplification or fluorescent in situ hybridization cannot completely reveal the whole microbial community structure due to the extremely high complexity of the involved communities. In this sense, the next-generation high-throughput sequencing provides a powerful tool for dissecting microbial community structure and methane-producing pathways in anaerobic digestion.


In this work, the metagenomic sequencing was used to characterize microbial community structure of the anaerobic digestion sludge from a full-scale municipal wastewater treatment plant. Over 3.0 gigabases of metagenomic sequence data were generated with the Illumina HiSeq 2000 platform. Taxonomic analysis by MG-RAST server indicated that overall bacteria were dominant (~93%) whereas a considerable abundance of archaea (~6%) were also detected in the anaerobic digestion sludge. The most abundant bacterial populations were found to be Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. Key microorganisms and related pathways involved in methanogenesis were further revealed. The dominant proliferation of Methanosaeta and Methanosarcina, together with the functional affiliation of enzymes-encoding genes (acetate kinase (AckA), phosphate acetyltransferase (PTA), and acetyl-CoA synthetase (ACSS)), suggested that the acetoclastic methanogenesis is the dominant methanogenesis pathway in the full-scale anaerobic digester.


In short, the metagenomic sequencing study of this work successfully dissected the detail microbial community structure and the dominated methane-producing pathways of a full-scale anaerobic digester. The knowledge garnered would facilitate to develop more efficient full-scale anaerobic digestion systems to achieve high-rate waste sludge treatment and methane production.


PMID: 25880314 PMCID: PMC4381419 DOI: 10.1186/s12934-015-0218-4

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Appl Biochem Biotechnol. 2015 Apr;175(7):3258-70. doi: 10.1007/s12010-015-1491-8. Epub 2015 Feb 10.

Metagenomic analysis of the sludge microbial community in a lab-scale denitrifying phosphorus removal reactor.

Lv XM1, Shao MF, Li J, Li CL.

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Denitrifying phosphorus removal is an attractive wastewater treatment process due to its reduced carbon source demand and sludge minimization potential. In the present study, the metagenome of denitrifying phosphorus removal sludge from a lab-scale anaerobic-anoxic SBR was generated by Illumina sequencing to study the microbial community. Compared with the aerobic phosphorus removal sludge, the denitrifying phosphorus removal sludge demonstrated quite similar microbial community profile and microbial diversity with sludge from Aalborg East EBPR WWTP. Proteobacteria was the most dominant phylum; within Proteobacteria, β-Proteobacteria was the most dominant class, followed by α-, γ-, δ-, and ε-Proteobacteria. The genes involved in phosphate metabolism and biofilm formation reflected the selective pressure of the phosphorus removal process. Moreover, ppk sequence from DPAO was outside the Accumulibacter clusters, which suggested different core phosphorus removal bacteria in denitrifying and aerobic phosphorus removal systems. In a summary, putative DPAO might be a novel genus that is closely related between Accumulibacter and Dechloromonas within Rhodocyclus. The microbial community and metabolic profiles achieved in this study will eventually help to improve the understanding of key microorganisms and the entire community in order to improve the phosphorus removal efficiency of EBPR processes.


PMID: 25820294 DOI: 10.1007/s12010-015-1491-8

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