Uncovering Bacterial Diversity with MRDNA 16s, 18s, ITS Amplicon Assays

141. Eur J Dent. 2015 Jan-Mar;9(1):127-32. doi: 10.4103/1305-7456.149661.


16S rRNA gene-based metagenomic analysis identifies a novel bacterial

co-prevalence pattern in dental caries.


Jagathrakshakan SN(1), Sethumadhava RJ(1), Mehta DT(2), Ramanathan A(2).


Author information:

(1)Department of Prosthodontia, Sree Balaji Dental College and Hospital, Bharath

University, Narayanapuram, Pallikaranai, Chennai, Tamil Nadu, India.

(2)Department of Human Genetics Laboratory, Central Research Facility, Sree

Balaji Medical and Dental College and Hospital, Bharath University,

Narayanapuram, Pallikaranai, Chennai, Tamil Nadu, India.


OBJECTIVE: To identify the prevalence of acidogenic and nonacidogenic bacteria in

patients with polycaries lesions, and to ascertain caries specific bacterial

prevalence in relation to noncaries controls.

MATERIALS AND METHODS: Total genomic DNA extracted from saliva of three adults

and four children from the same family were subjected to 16S rRNA gene sequencing

analysis on a next generation sequencer, the PGS-Ion Torrent. Those bacterial

genera with read counts > 1000 were considered as significant in each of the

subject and used to associate the occurrence with caries.

RESULTS AND CONCLUSION: Sequencing analysis indicated a higher prevalence of

Streptococcus, Rothia, Granulicatella, Gemella, Actinomyces, Selenomonas,

Haemophilus and Veillonella in the caries group relative to controls. While

higher prevalence of Streptococcus, Rothia and Granulicatella were observed in

all caries samples, the prevalence of others was observable in 29-57% of samples.

Interestingly, Rothia and Selenomonas, which are known to occur within anaerobic

environments of dentinal caries and subgingival plaque biofilms, were seen in the

saliva of these caries patients. Taken together, the study has identified for the

first time a unique co-prevalence pattern of bacteria in caries patients that may

be explored as distinct caries specific bacterial signature to predict

cariogenesis in high-risk primary and mixed dentition age groups.


DOI: 10.4103/1305-7456.149661

PMCID: PMC4319289

PMID: 25713496  [PubMed]



142. J Clin Microbiol. 2014 Oct;52(10):3583-9. doi: 10.1128/JCM.01459-14. Epub 2014

Jul 23.


Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of

prosthetic joint infection: a prospective multicenter cross-sectional study.


Bémer P(1), Plouzeau C(2), Tande D(3), Léger J(4), Giraudeau B(4), Valentin

AS(5), Jolivet-Gougeon A(6), Vincent P(6), Corvec S(7), Gibaud S(7), Juvin ME(7),

Héry-Arnaud G(3), Lemarié C(8), Kempf M(8), Bret L(9), Quentin R(5), Coffre C(4),

de Pinieux G(10), Bernard L(11), Burucoa C(2); Centre de Référence des Infections

Ostéo-articulaires du Grand Ouest (CRIOGO) Study Team.


Collaborators: Cottin J, Rousselet M, Bizot P, Abgueguen P, Quentin-Roue I,

Gérard R, Stindel E, Ansart S, Boisson R, Guilloux A, Crémet L, Moreau A,

Touchais S, Gouin F, Boutoille D, Asseray N, Guigon A, Guinard J, Michenet P,

Razanabola F, Mille C, Cognée A, Milin S, Gayet L, Roblot F, Moal G, Guinard J,

Stock N, Polard J, Arvieux C, Rosset P, Gras G.


Author information:

(1)CHU Nantes, Laboratoire de Bactériologie, Nantes, France

pascale.bemer@chu-nantes.fr. (2)CHU Poitiers, Laboratoire de Bactériologie,

Poitiers, France. (3)CHU Brest, Laboratoire de Bactériologie, Brest, France.

(4)INSERM, CIC 1415, Tours, France. (5)CHU Tours, Laboratoire de Bactériologie,

Tours, France. (6)CHU Rennes, Laboratoire de Bactériologie, Rennes, France.

(7)CHU Nantes, Laboratoire de Bactériologie, Nantes, France. (8)CHU Angers,

Angers, France. (9)CHU Orléans, Laboratoire de Bactériologie, Orléans, France.

(10)CHU Tours, Laboratoire d'Anatomo-Pathologie, Tours, France. (11)CHU Tours,

Service des Maladies Infectieuses, Tours, France.


There is no standard method for the diagnosis of prosthetic joint infection

(PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis

remains to be defined. We performed a prospective multicenter study to assess the

contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all

patients suspected to have PJIs and a few uninfected patients undergoing primary

arthroplasty (control group) were included. Five perioperative samples per

patient were collected for culture and 16S rRNA gene PCR sequencing and one for

histological examination. Three multicenter quality control assays were performed

with both DNA extracts and crushed samples. The diagnosis of PJI was based on

clinical, bacteriological, and histological criteria, according to Infectious

Diseases Society of America guidelines. A molecular diagnosis was modeled on the

bacteriological criterion (≥ 1 positive sample for strict pathogens and ≥ 2 for

commensal skin flora). Molecular data were analyzed according to the diagnosis of

PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35

control cases were included. PJI was confirmed in 215/264 suspected cases, 192

(89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases

[85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n =

16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The

molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases

with bacteriological PJI documentation and 8 treated cases without

bacteriological documentation) and in 2/49 cases without confirmed PJI

(sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a

lack of sensitivity in the diagnosis of PJI on a multicenter routine basis.


Copyright © 2014, American Society for Microbiology. All Rights Reserved.


DOI: 10.1128/JCM.01459-14

PMCID: PMC4187742

PMID: 25056331  [PubMed - indexed for MEDLINE]



143. PLoS One. 2013 Apr 8;8(4):e60811. doi: 10.1371/journal.pone.0060811. Print 2013.


Species identification and profiling of complex microbial communities using

shotgun Illumina sequencing of 16S rRNA amplicon sequences.


Ong SH(1), Kukkillaya VU, Wilm A, Lay C, Ho EX, Low L, Hibberd ML, Nagarajan N.


Author information:

(1)Genome Institute of Singapore, Genome #02-01, Singapore, Singapore.


The high throughput and cost-effectiveness afforded by short-read sequencing

technologies, in principle, enable researchers to perform 16S rRNA profiling of

complex microbial communities at unprecedented depth and resolution. Existing

Illumina sequencing protocols are, however, limited by the fraction of the 16S

rRNA gene that is interrogated and therefore limit the resolution and quality of

the profiling. To address this, we present the design of a novel protocol for

shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify

more than 90% of sequences in the Greengenes database and with the ability to

distinguish nearly twice as many species-level OTUs compared to existing

protocols. Using several in silico and experimental datasets, we demonstrate that

despite the presence of multiple variable and conserved regions, the resulting

shotgun sequences can be used to accurately quantify the constituents of complex

microbial communities. The reconstruction of a significant fraction of the 16S

rRNA gene also enabled high precision (>90%) in species-level identification

thereby opening up potential application of this approach for clinical microbial



DOI: 10.1371/journal.pone.0060811

PMCID: PMC3620293

PMID: 23579286  [PubMed - indexed for MEDLINE]



144. Acta Biochim Pol. 2016;63(2):315-9. doi: 10.18388/abp.2015_1145. Epub 2016 Feb



Metagenomic 16s rRNA investigation of microbial communities in the Black Sea

estuaries in South-West of Ukraine.


Bobrova O(1), Kristoffersen JB(2), Oulas A(2), Ivanytsia V(1).


Author information:

(1)Department of Microbiology, Virology and Biotechnology, Odessa National I. I.

Mechnikov University, Odessa, Ukraine. (2)Institute of Marine Biology,

Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Heraklion,



The Black Sea estuaries represent interfaces of the sea and river environments.

Microorganisms that inhabit estuarine water play an integral role in all

biochemical processes that occur there and form unique ecosystems. There are many

estuaries located in the Southern-Western part of Ukraine and some of them are

already separated from the sea. The aim of this research was to determine the

composition of microbial communities in the Khadzhibey, Dniester and Sukhyi

estuaries by metagenomic 16S rDNA analysis. This study is the first complex

analysis of estuarine microbiota based on isolation of total DNA from a biome

that was further subjected to sequencing. DNA was extracted from water samples

and sequenced on the Illumina Miseq platform using primers to the V4 variable

region of the 16S rRNA gene. Computer analysis of the obtained raw sequences was

done with QIIME (Quantitative Insights Into Microbial Ecology) software. As the

outcome, 57970 nucleotide sequences were retrieved. Bioinformatic analysis of

bacterial community in the studied samples demonstrated a high taxonomic

diversity of Prokaryotes at above genus level. It was shown that majority of 16S

rDNA bacterial sequences detected in the estuarine samples belonged to phyla

Cyanobacteria, Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia,

Planctomycetes. The Khadhzibey estuary was dominated by the Proteobacteria

phylum, while Dniester and Sukhyi estuaries were characterized by dominance of

Cyanobacteria. The differences in bacterial populations between the Khadzhibey,

Dniester and Sukhyi estuaries were demonstrated through the Beta-diversity

analysis. It showed that the Khadzhibey estuary's microbial community

significantly varies from the Sukhyi and Dniester estuaries. The majority of

identified bacterial species is known as typical inhabitants of marine

environments, however, for 2.5% of microbial population members in the studied

estuaries no relatives were determined.


DOI: 10.18388/abp.2015_1145

PMID: 26929931  [PubMed - in process]


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