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Stimulated and unstimulated saliva samples have significantly different bacterial profiles.

ABSTRACT: Epidemiological studies use saliva on a regular basis as a non-invasive and easy-to-take sample, which is assumed to be a microbial representative of the oral cavity ecosystem. However, comparative studies between different kinds of saliva samples normally used in microbial studies are scarce. The aim of the current study was to compare oral microbiota composition between two different saliva samples collected simultaneously: non-stimulated saliva with paper points and stimulated saliva collected after chewing paraffin gum. DNA was extracted from saliva samples of ten individuals, then analyzed by 16S rRNA pyrosequencing to describe bacterial diversity. The results demonstrate significant differences between the microbiota of these two kinds of saliva. Stimulated saliva was found to contain an estimated number of species over three times higher than unstimulated saliva. In addition, bacterial composition at the class and genus level was radically different between both types of samples. When compared to other oral niches, both types of saliva showed some similarity to tongue and buccal mucosa, but they do not correlate at all with the bacterial composition described in supra- or sub-gingival dental plaque, questioning their use in etiological and epidemiological studies of oral diseases of microbial origin.

SUBMITTER: Gomar-Vercher S

PROVIDER: S-EPMC5983451 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Stimulated and unstimulated saliva samples have significantly different bacterial profiles.

Gomar-Vercher Sonia S Simón-Soro Aurea A Montiel-Company José María JM Almerich-Silla José Manuel JM Mira Alex A

PloS one 20180601 6

Epidemiological studies use saliva on a regular basis as a non-invasive and easy-to-take sample, which is assumed to be a microbial representative of the oral cavity ecosystem. However, comparative studies between different kinds of saliva samples normally used in microbial studies are scarce. The aim of the current study was to compare oral microbiota composition between two different saliva samples collected simultaneously: non-stimulated saliva with paper points and stimulated saliva collecte ...[more]

PMID: 29856779

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Project description:Obtaining complete, high-quality reference genomes is essential to the study of any organism. Recent advances in nanopore sequencing, as well as genome assembly and analysis methods, have made it possible to obtain complete bacterial genomes from metagenomic (i.e., multispecies) samples, including those from the human microbiome. In this study, methods are presented to obtain complete bacterial genomes from human saliva using complementary Oxford Nanopore (ONT) and Illumina sequencing. Applied to 3 human saliva samples, these methods resulted in 11 complete bacterial genomes: 3 Saccharibacteria clade G6 (also known as Ca. Nanogingivalaceae HMT-870), 1 Saccharibacteria clade G1 HMT-348, 2 Rothia mucilaginosa, 2 Actinomyces graevenitzii, 1 Mogibacterium diversum, 1 Lachnospiraceae HMT-096, and 1 Lancefieldella parvula; and one circular chromosome of Ruminococcaceae HMT-075 (which likely has at least 2 chromosomes). The 4 Saccharibacteria genomes, as well as the Actinomyces graeventizii genomes, represented the first complete genomes from their respective bacterial taxa. Aside from the complete genomes, the assemblies contained 147 contigs of over 500,000 bp each and thousands of smaller contigs, together representing a myriad of additional draft genomes including many which are likely nearly complete. The complete genomes enabled highly accurate pangenome analysis, which identified unique and missing features of each genome compared to its closest relatives with complete genomes available in public repositories. These features provide clues as to the lifestyle and ecological role of these bacteria within the human oral microbiota, which will be particularly useful in designing future studies of the taxa that have never been isolated or cultivated. IMPORTANCE Obtaining complete and accurate genomes is crucial to the study of any organism. Previously, obtaining complete genomes of bacteria, including those of the human microbiome, frequently required isolation of the organism, as well as low-throughput, manual sequencing methods to resolve repeat regions. Advancements in long-read sequencing technologies, including Oxford Nanopore (ONT), have made it possible to obtain complete, closed bacterial genomes from metagenomic samples. This study reports methods to obtain complete genomes from the human oral microbiome using complementary ONT and Illumina sequencing of saliva samples. Eleven complete genomes were obtained from 3 human saliva samples, with genomes of Saccharibacteria HMT-870, Saccharibacteria HMT-348, and Actinomyces graeventzii being the first complete genomes from their respective taxa. Obtaining complete bacterial genomes in a high-throughput manner will help illuminate the metabolic and ecological roles of important members of the human microbiota, particularly those that have remained recalcitrant to isolation and cultivation.

Project description:Understanding the molecular basis of how ticks adapt to feed on different animal hosts is central to understanding tick and tick-borne disease (TBD) epidemiology. There is evidence that ticks differentially express specific sets of genes when stimulated to start feeding. This study was initiated to investigate if ticks such as Ixodes scapularis and Amblyomma americanum that are adapted to feed on multiple hosts utilized the same sets of proteins to prepare for feeding. We exposed I. scapularis and A. americanum to feeding stimuli of different hosts (rabbit, human, and dog) by keeping unfed adult ticks enclosed in a perforated microfuge in close contact with host skin, but not allowing ticks to attach on host. Our data suggest that ticks of the same species differentially express tick saliva proteins (TSPs) when stimulated to start feeding on different hosts. SDS-PAGE and silver staining analysis revealed unique electrophoretic profiles in saliva of I. scapularis and A. americanum that were stimulated to feed on different hosts: rabbit, human, and dog. LC-MS/MS sequencing and pairwise analysis demonstrated that I. scapularis and A. americanum ticks expressed unique protein profiles in their saliva when stimulated to start feeding on different hosts: rabbit, dog, or human. Specifically, our data revealed TSPs that were unique to each treatment and those that were shared between treatments. Overall, we identified a total of 276 and 340 non-redundant I. scapularis and A. americanum TSPs, which we have classified into 28 functional classes including: secreted conserved proteins (unknown functions), proteinase inhibitors, lipocalins, extracellular matrix/cell adhesion, heme/iron metabolism, signal transduction and immunity-related proteins being the most predominant in saliva of unfed ticks. With exception of research on vaccines against Rhipicephalus microplus, which its natural host, cattle, research on vaccine against other ticks relies feeding ticks on laboratory animals. Data here suggest that relying on lab animal tick feeding data to select target antigens could result in prioritizing irrelevant anti-tick vaccine targets that are expressed when ticks feed on laboratory animals. This study provides the platform that could be utilized to identify relevant target anti-tick vaccine antigens, and will facilitate early stage tick feeding research.

Project description:BackgroundClinical and research interest in salivary peptidome and microbiota is ever-growing owing to its great value for diagnosis, risk assessment and prediction of prognosis in oral and systemic diseases. Saliva can be stimulated for the purpose of rapid collection, but currently there are no studies systematically addressing the similarities and differences of salivary peptidome and microbiota in different types of samples. The purpose of this study was to investigate the variations of salivary peptidome and microbial profiles in response to different stimulating conditions.MethodsUnstimulated saliva and three types of stimulated saliva samples (olfaction, gustation, and mastication stimulated saliva) were collected from 10 systematically and orally healthy donors. The peptidome profiles were detected by weak cation exchange magnetic beads and analyzed through matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS), while their microbial profiles were analyzed by 16S rDNA V3-V4 hypervariable region amplicon sequencing utilizing the Illumina MiSeq PE300 platform. The distance matrixes of salivary peptidome and microbial profiles were generated and the intra-individual distances were extracted, then the variations brought by different sampling conditions and repeated collections were compared.ResultsBy comparisons of the overall salivary peptidome and microbial profiles, olfactory stimulation led to minimal variations comparing with that of unstimulated saliva, but appreciable variations were observed between saliva samples collected with gustatory/masticatory stimulation and unstimulated saliva. The three types of stimulated saliva exhibited significantly different peptidome and microbial profiles.ConclusionsStimulated saliva collected in response to olfactory stimulation is an appropriate alternative to unstimulated saliva, whereas gustatory/masticatory stimulation introduced appreciable variations. It is suggested that only one type of stimulating method should be used throughout one peptidome/microbiome research, which provides comprehensive insight into the optimization of sampling methods for salivaomic studies in the future.

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Stimulated and unstimulated saliva samples have significantly different bacterial profiles.

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Stimulated and unstimulated saliva samples have significantly different bacterial profiles.

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