Culture-independent Molecular Techniques Research Articles

DdPCR enables rapid detection of bloodstream infections in patients on home parenteral nutrition: A prospective cohort study

IntroductionChronic intestinal failure patients (CIF) require a central venous access device (CVAD) to administer parenteral nutrition. Most serious complication related to a CVAD is a central line-associated bloodstream infection (CLABSI). The golden standard to diagnose a CLABSI are blood cultures, however, they may require 1–5 days before getting a result. Droplet digital polymerase chain reaction (ddPCR) for the detection of pathogen 16S/28S rRNA is a novel culture-independent molecular technique that has been developed to enhance and expedite infection diagnostics within two and a half hours. In this study, we prospectively compared ddPCR with blood cultures to detect pathogens in whole blood. MethodsWe included adult CIF patients with a clinical suspicion of CLABSI in this prospective single-blinded clinical study. Blood cultures were routinely collected and subsequently two central samples from the CVAD and two peripheral samples from a peripheral venous access point. Primary outcome was the sensitivity and specificity of ddPCR. ResultsIn total, 75 patients with 126 suspected CLABSI episodes were included, with 80 blood samples from the CVAD and 114 from peripheral veins. The central ddPCR samples showed a sensitivity of 91% (95%CI 77–98), and specificity of 96% (95%CI 85–99). Peripheral ddPCR samples had a sensitivity of 63% (95%CI 46–77) and specificity of 99% (95%CI 93–100). ConclusionddPCR showed a high sensitivity and specificity relative to blood cultures and enables rapid pathogen detection and characterization. Clinical studies should explore if integrated ddPCR and blood culture outcomes enables a more rapid pathogen guided CLABSI treatment and enhancing patient outcomes.

A retrospective study of the detection of sepsis pathogens comparing blood culture and culture-independent digital PCR

Fast and precise identification of microorganisms in the early diagnosis of sepsis is crucial for enhancing patient outcomes. Digital PCR (dPCR) is a highly sensitive approach for absolute quantification that can be utilized as a culture-independent molecular technique for diagnosing sepsis pathogens. We performed a retrospective investigation on 69 ICU patients suspected of sepsis. Our findings showed that a multiplex dPCR diagnostic kit outperformed blood culture in detecting the 15 most frequent bacteria that cause sepsis. Ninety-two bacterial strains were identified using dPCR at concentrations varying from 34 copies/mL to 105,800 copies/mL. The detection rate of dPCR was much greater than that of BC, with 27.53% (19/69) versus 73.91% (51/69). The sensitivity of dPCR was 63.2%. Our research indicated that dPCR outperforms blood culture in the early detection of sepsis-causing microorganisms. The diagnostic kit can detect a greater variety of pathogens with quantitative data, including polymicrobial infections, and has a quicker processing time. DPCR is a valuable technique that could aid in the proper management of sepsis.

Identifying Active Rather than Total Methanotrophs Inhabiting Surface Soil Is Essential for the Microbial Prospection of Gas Reservoirs.

Methane-oxidizing bacteria (MOB) have long been recognized as an important bioindicator for oil and gas exploration. However, due to their physiological and ecological diversity, the distribution of MOB in different habitats varies widely, making it challenging to authentically reflect the abundance of active MOB in the soil above oil and gas reservoirs using conventional methods. Here, we selected the Puguang gas field of the Sichuan Basin in Southwest China as a model system to study the ecological characteristics of methanotrophs using culture-independent molecular techniques. Initially, by comparing the abundance of the pmoA genes determined by quantitative PCR (qPCR), no significant difference was found between gas well and non-gas well soils, indicating that the abundance of total MOB may not necessarily reflect the distribution of the underlying gas reservoirs. 13C-DNA stable isotope probing (DNA-SIP) in combination with high-throughput sequencing (HTS) furthermore revealed that type II methanotrophic Methylocystis was the absolutely predominant active MOB in the non-gas-field soils, whereas the niche vacated by Methylocystis was gradually filled with type I RPC-2 (rice paddy cluster-2) and Methylosarcina in the surface soils of gas reservoirs after geoscale acclimation to trace- and continuous-methane supply. The sum of the relative abundance of RPC-2 and Methylosarcina was then used as specific biotic index (BI) in the Puguang gas field. A microbial anomaly distribution map based on the BI values showed that the anomalous zones were highly consistent with geological and geophysical data, and known drilling results. Therefore, the active but not total methanotrophs successfully reflected the microseepage intensity of the underlying active hydrocarbon system, and can be used as an essential quantitative index to determine the existence and distribution of reservoirs. Our results suggest that molecular microbial techniques are powerful tools for oil and gas prospecting.

Detection Of Viable but Non Culturable (VBNC) Bacteria Using of Propidium Monoazide PCR: A Review

Microorganisms in their natural habitats are subjected to a broad range of stressors. In order to survive and retain their vitality under those demanding conditions, they enter a state known as viable but nonculturable (VBNC). Traditional laboratory techniques cannot identify microbes in the VBNC state, but they can be brought back to life in the right circumstances. As a result, VBNC pathogens seriously jeopardize public health and food safety. More than 100 microbe species have been found to have entered the VBNC state to date, owing to a variety of chemical and physical processes. Since the VBNC syndrome was first discovered forty years ago, new methods for inducing, detecting, resuscitating, and understanding its molecular pathways have been created. To determine their effect on public health, live bacterial foodborne pathogens must be found and measured. Due to the existence of viable but nonculturable cells (VBNC), which can retain metabolic and pathogenic activity, traditional growth-based approaches have the potential to significantly underestimate the true numbers. The similar issue arises when using different culture-independent molecular techniques, such PCR, which can detect DNA from dead cells in addition to VBNC bacteria. Viability dyes and PCR have been used to selectively identify and quantify viable foodborne microorganisms, overcoming this drawback.

Functional diversity of nanohaloarchaea within xylan-degrading consortia.

Extremely halophilic representatives of the phylum Candidatus Nanohaloarchaeota (members of the DPANN superphyla) are obligately associated with extremely halophilic archaea of the phylum Halobacteriota (according to the GTDB taxonomy). Using culture-independent molecular techniques, their presence in various hypersaline ecosystems around the world has been confirmed over the past decade. However, the vast majority of nanohaloarchaea remain uncultivated, and thus their metabolic capabilities and ecophysiology are currently poorly understood. Using the (meta)genomic, transcriptomic, and DNA methylome platforms, the metabolism and functional prediction of the ecophysiology of two novel extremely halophilic symbiotic nanohaloarchaea (Ca. Nanohalococcus occultus and Ca. Nanohalovita haloferacivicina) stably cultivated in the laboratory as members of a xylose-degrading binary culture with a haloarchaeal host, Haloferax lucentense, was determined. Like all known DPANN superphylum nanoorganisms, these new sugar-fermenting nanohaloarchaea lack many fundamental biosynthetic repertoires, making them exclusively dependent on their respective host for survival. In addition, given the cultivability of the new nanohaloarchaea, we managed to discover many unique features in these new organisms that have never been observed in nano-sized archaea both within the phylum Ca. Nanohaloarchaeota and the entire superphylum DPANN. This includes the analysis of the expression of organism-specific non-coding regulatory (nc)RNAs (with an elucidation of their 2D-secondary structures) as well as profiling of DNA methylation. While some ncRNA molecules have been predicted with high confidence as RNAs of an archaeal signal recognition particle involved in delaying protein translation, others resemble the structure of ribosome-associated ncRNAs, although none belong to any known family. Moreover, the new nanohaloarchaea have very complex cellular defense mechanisms. In addition to the defense mechanism provided by the type II restriction-modification system, consisting of Dcm-like DNA methyltransferase and Mrr restriction endonuclease, Ca. Nanohalococcus encodes an active type I-D CRISPR/Cas system, containing 77 spacers divided into two loci. Despite their diminutive genomes and as part of their host interaction mechanism, the genomes of new nanohaloarchaea do encode giant surface proteins, and one of them (9,409 amino acids long) is the largest protein of any sequenced nanohaloarchaea and the largest protein ever discovered in cultivated archaea.

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

Non-cyanobacterial diazotrophs: Global diversity, distribution, ecophysiology, and activity in marine waters.

Biological dinitrogen (N2) fixation supplies nitrogen to the oceans, supporting primary productivity, and is carried out by some bacteria and archaea referred to as diazotrophs. Cyanobacteria are conventionally considered to be the major contributors to marine N2 fixation, but non-cyanobacterial diazotrophs (NCDs) have been shown to be distributed throughout ocean ecosystems. However, the biogeochemical significance of marine NCDs has not been demonstrated. This review synthesizes multiple datasets, drawing from cultivation-independent molecular techniques and data from extensive oceanic expeditions, to provide a comprehensive view into the diversity, biogeography, ecophysiology, and activity of marine NCDs. A NCD nifH gene catalog was compiled containing sequences from both PCR-based and PCR-free methods, identifying taxa for future studies. NCD abundances from a novel database of NCD nifH-based abundances were colocalized with environmental data, unveiling distinct distributions and environmental drivers of individual taxa. Mechanisms that NCDs may use to fuel and regulate N2 fixation in response to oxygen and fixed nitrogen availability are discussed, based on a metabolic analysis of recently available Tara Oceans expedition data. The integration of multiple datasets provides a new perspective that enhances understanding of the biology, ecology, and biogeography of marine NCDs and provides tools and directions for future research.

Is Stomach a Sterile Environment

The stomach is considered a sterile organ for a long time due to anatomical and physiological features, till the discovery of Helicobacter pylori in 1982, which demolished the conception of sterile stomach. The pathogenicity of Helicobacter pylori is enhanced by several virulence factors. Initially, with the aid of culture-based techniques which were later followed by advanced culture-independent molecular techniques, whereby the complexity and biodiversity of gastric microbiota were revealed. Commensals, as well as pathogenic microbes have developed mechanisms to ensure successful colonization in the gastric environment. A number of published literature suggests the correlation of these bacteria with gastric diseases including gastric cancer and peptic ulcer disease as well as the beneficial relationships like probiotics. This review summarizes current information on the correlation of complexity and diversity of gastric microbiota and host in health and disease.

Mycobacterial diversity in soil samples from King George Island, Antarctica

Mycobacteria are ubiquitous microorganisms and may be found in many different environments, either associated or not to diseases. Culture-independent molecular techniques brought new perspectives for research regarding soil microorganisms, which is relevant to microbiologists and industry. Therefore, new research is necessary to broaden our knowledge about ecology and physiology of mycobacteria in extreme environments. The aim of this study was to verify the presence of mycobacteria in Antarctica soil samples and to standardize molecular and culture-dependent techniques to their isolation. Soil DNA was extracted and submitted to denaturing gradient gel electrophoresis (DGGE), rrs mycobacterial gene amplification and partial sequencing. Thereby, 10 colonies morphologically similar to mycobacteria were isolated from 10 different samples, resulting in 15 DNA sequences where Mycobacterium neglectum and Mycobacterium kumamotonense were identified, suggesting that Mycobacterium terrae complex representatives are ubiquitous in the region. This study proposes a simple method for mycobacteria cultivation from soil samples and shows that ornithogenic soils can be the source of organic matter and low pH, modulating the mycobacterial Antarctica diversity. Further research aiming to explore mycobacterial diversity in soil are needed to increase knowledge and better explore their evolution, their relation with animal and human species and their potential biotechnological use.

An Overview of Adenoid Microbiome Using 16S rRNA Gene Sequencing-Based Metagenomic Analysis.

Background and Objectives: the upper respiratory tract harbors the highest bacterial density in the whole respiratory system. Adenoids, which are located in the nasopharynx, are a major site of bacterial colonies in the upper airways. Our goal was to use culture-independent molecular techniques to identify the breadth of bacterial diversity in the adenoid vegetations of children suffering from chronic rhinosinusitis and obstructive sleep apnea. Materials and methods: in total, 21 adenoid samples were investigated using amplification and sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene. Results: among the most common bacterial species found were Veillonella atypica, Fusobactrium nucelatum, Shaalia odontolytica, and Moraxella catarrhalis. Veillonella atypica and Fusbacterium nucelatum dominated the microbiome in all 21 samples, attributing to more than 60% of all detected genetic material. Conclusions: since both Veillonella atypica and Fusobacterium nucleatum are, predominantly, oral cavity and dental microorganisms, our findings may suggest oral microbiome migration deeper into the oropharynx and nasopharynx where these bacteria colonize adenoid vegetations.

Phylogenetic diversity of Actinobacteria from Momela soda lakes, Arusha National Park, Tanzania

The Momela soda lakes consist of seven small, hypersaline, alkaline lakes, situated in the East African rift valley at Arusha National Park, Tanzania. The lakes are fed by separate underground water sources with slightly varying mineral contents resulting in colour variation and supporting different kinds of prokaryotic and eukaryotic species. In this study, the diversity of Actinobacteria in surface water and sediments of five Lakes were investigated using culture-dependent and culture-independent molecular techniques. A total of 34 out of 112, and 13 out of 85, representatives of Actinobacteria isolates and clones, respectively, were selected for gene sequencing using the CD-HIT program. Analysis of their 16S rRNA gene sequences displayed the presence of species affiliated to 15 different genera, namely Mycobacterium, Rhodococcus, Microbacterium, Isoptericola, Dietzia, Leucobacter, Jonesia, Nesterenkonia, Micrococcus, Streptomyces, Hoyosella, Norcadiopsis, Cellulomonas, Bogoriella, and Agromyces. The results showed 5 and 12 putative new Actinobacteria isolates and clones, respectively. This is the first report of isolation of bacteria from the genus Mycobacterium from a soda lake globally, as well as the genera Hoyosella, Isoptericola, Jonesia, Micrococcus, Leucobacter and Agromyces from a soda lake in East Africa. Because Actinobacteria are known as a source of biotechnologically important compounds, the species revealed set a platform to search for novel bioactive compounds.

Sediment-associated microbial community profiling: sample pre-processing through sequential membrane filtration for 16S rRNA amplicon sequencing

BackgroundSequential membrane filtration as a pre-processing step for capturing sediment-associated microorganisms could provide good quality and integrity DNA that can be preserved and kept at ambient temperatures before community profiling through culture-independent molecular techniques. However, the effects of sample pre-processing via filtration on DNA-based profiling of sediment-associated microbial community diversity and composition are poorly understood. Specifically, the influences of pre-processing on the quality and quantity of extracted DNA, high-throughput DNA sequencing reads, and detected microbial taxa need further evaluation.ResultsWe assessed the impact of pre-processing freshwater sediment samples by sequential membrane filtration (from 10, 5 to 0.22 μm pore size) for 16S rRNA-based community profiling of sediment-associated microorganisms. Specifically, we examined if there would be method-driven differences between non- and pre-processed sediment samples regarding the quality and quantity of extracted DNA, PCR amplicon, resulting high-throughput sequencing reads, microbial diversity, and community composition. We found no significant difference in the qualities and quantities of extracted DNA and PCR amplicons, and the read abundance after bioinformatics processing (i.e., denoising and chimeric-read filtering steps) between the two methods. Although the non- and pre-processed sediment samples had more unique than shared amplicon sequence variants (ASVs), we report that their shared ASVs accounted for 74% of both methods’ absolute read abundance. More so, at the genus level, the final collection filter identified most of the genera (95% of the reads) captured from the non-processed samples, with a total of 51 false-negative (2%) and 59 false-positive genera (3%). We demonstrate that while there were differences in shared and unique taxa, both methods revealed comparable microbial diversity and community composition.ConclusionsOur observations highlight the feasibility of pre-processing sediment samples for community analysis and the need to further assess sampling strategies to help conceptualize appropriate study designs for sediment-associated microbial community profiling.

Bacterial population monitoring during alcoholic fermentation of mezcal in Durango by DGGE

Mezcal is the second most important alcoholic beverage produced in Mexico, and the microorganisms present during the fermentation process are primarily responsible for its organoleptic characteristics. Among these, the bacterial populations have been less studied, mainly due to the difficulty of recovering them. The aim was to apply a culture-independent molecular technique to detect the bacterial populations involved in spontaneous mezcal fermentation in Durango State. Samples were obtained from several stages in the mezcal production line. Denaturing gradient gel electrophoresis (DGGE) analysis was performed using DNA extracted from isolated microorganisms and metagenomes obtained from samples directly taken from the fermentation process. This study indicated that non-isolated bacteria were significant in the fermentation process and indicated that the lactic acid and acetic acid bacterial populations present in the monitored process are similar to those present in other regions during fermentation, although they differ in some species that may play essential roles in the development of the typical organoleptic properties of mezcal produced in the State of Durango. Furthermore, this study indicates that culture-independent analysis by DGGE can reveal the bacterial diversity during the mezcal fermentation process without the use of complex isolation procedures and can be a useful tool for the analysis, monitoring and control of mezcal fermentation processes.

The Lung Microbiome during Health and Disease

Healthy human lungs have traditionally been considered to be a sterile organ. However, culture-independent molecular techniques have reported that large numbers of microbes coexist in the lung and airways. The lungs harbor diverse microbial composition that are undetected by previous approaches. Many studies have found significant differences in microbial composition between during health and respiratory disease. The lung microbiome is likely to not only influence susceptibility or causes of diseases but be affected by disease activities or responses to treatment. Although lung microbiome research has some limitations from study design to reporting, it can add further dimensionality to host-microbe interactions. Moreover, there is a possibility that extending understanding to the lung microbiome with new multiple omics approaches would be useful for developing both diagnostic and prognostic biomarkers for respiratory diseases in clinical settings.

Impact of non-antibiotic drugs on the human intestinal microbiome

Introduction The gut microbiota is composed of trillions of microbial cells and viruses that interact with hosts. The composition of the gut microbiota is influenced by several factors including age, diet, diseases or medications. The impact of drugs on the microbiota is not limited to antibiotics and many non-antibiotic molecules significantly alter the composition of the intestinal microbiota. Areas covered This review focuses on the impact of four of the most widely prescribed non-antibiotic drugs in the world: Proton-pump inhibitors, metformin, statins and non-steroidal anti-inflammatory. We conducted a systematic review by searching online databases including Medline, Web of science and Scopus for indexed articles published in English until February 2021. We included studies assessing the intestinal microbiome alterations associated with proton pump inhibitors (PPIs), metformin, statins and nonsteroidal anti-inflammatory drugs (NSAIDs). Only studies using culture-independent molecular techniques were included. Expert opinion The taxonomical signature associated with non-antibiotic drugs are not yet fully described, especially in the field of metabolomic. The identification of taxonomic profiles associated a specific molecule provides information on its mechanism of action through interaction with the intestinal microbiota. Many side effects could be related to the dysbiosis induced by these molecules.

Bacterial community analysis on Sclerotium-suppressive soil.

Difficulties in controlling the soil-borne plant pathogenic fungus Sclerotium rolfsii favoured the analysis of its suppressive soil for better understanding. In the present study, culture-independent molecular technique was used to analyse the bacterial communities of suppressive soil and conducive soil. Hence, metagenomic DNAs from both kinds of soils were directly extracted and their sequence polymorphism was analysed by targeting hypervariable domains, V4 + V5, of the 16S rRNA gene. The results of 16S rRNA gene-driven bacterial community diversity analysis along with soil physicochemical and biological properties clearly discriminated S. rolfsii suppressive soil from conducive soil. The dominant phylogenetic group of suppressive soil is Actinobacteria followed by Proteobacteria. The other groups include Acidobacteria, Firmicutes and Cyanobacteria. In contrast, conducive soil had very few Actinobacterial sequences and was dominated by Gamma- and Betaproteobacteria. Based on the relative proportion of different bacterial communities, their diversity and species richness were observed more in suppressive soil than in conducive soil. The present study identifies the dominant bacterial community which shares S. rolfsii suppressiveness.

Oral Lactobacillus Species in Systemic Sclerosis.

In systemic sclerosis (SSc), the gastrointestinal tract (GIT) plays a central role in the patient’s quality of life. The microbiome populates the GIT, where a relationship between the Lactobacillus and gastrointestinal motility has been suggested. In this study, the analysis of oral Lactobacillus species in SSc patients and healthy subjects using culture-independent molecular techniques, together with a review of the literature on microbiota and lactobacilli in SSc, has been carried out. Twenty-nine SSc female patients (mean age 62) and twenty-three female healthy subjects (HS, mean age 57.6) were enrolled and underwent tongue and gum swab sampling. Quantitative PCR was conducted in triplicate using Lactobacillus specific primers rpoB1, rpoB1o and rpoB2 for the RNA-polymerase β subunit gene. Our data show significantly (p = 0.0211) lower LactobacillusspprpoB sequences on the tongue of patients with SSc compared to HS. The mean value of the amount of Lactobacillus ssprpoB gene on the gumsofSSc patients was minor compared to HS. A significant difference between tongue and gums (p = 0.0421) was found in HS but not in SSc patients. In conclusion, our results show a lower presence of Lactobacillus in the oral cavity of SSc patients. This strengthens the hypothesis that Lactobacillus may have both a protective and therapeutic role in SSc patients.

Interactions of Bacteriophages and Bacteria at the Airway Mucosa: New Insights Into the Pathophysiology of Asthma.

The airway epithelium is the primary site where inhaled and resident microbiota interacts between themselves and the host, potentially playing an important role on allergic asthma development and pathophysiology. With the advent of culture independent molecular techniques and high throughput technologies, the complex composition and diversity of bacterial communities of the airways has been well-documented and the notion of the lungs' sterility definitively rejected. Recent studies indicate that the microbial composition of the asthmatic airways across the spectrum of disease severity, differ significantly compared with healthy individuals. In parallel, a growing body of evidence suggests that bacterial viruses (bacteriophages or simply phages), regulating bacterial populations, are present in almost every niche of the human body and can also interact directly with the eukaryotic cells. The triptych of airway epithelial cells, bacterial symbionts and resident phages should be considered as a functional and interdependent unit with direct implications on the respiratory and overall homeostasis. While the role of epithelial cells in asthma pathophysiology is well-established, the tripartite interactions between epithelial cells, bacteria and phages should be scrutinized, both to better understand asthma as a system disorder and to explore potential interventions.

A Longitudinal Study of the Human Oropharynx Microbiota Over Time Reveals a Common Core and Significant Variations With Self-Reported Disease.

Our understanding of human microbial communities, in particular in regard to diseases is advancing, yet the basic understanding of the microbiome in healthy subjects over time remains limited. The oropharynx is a key target for colonization by several important human pathogens. To understand how the oropharyngeal microbiome might limit infections, and how intercurrent infections might be associated with its composition, we characterized the oropharyngeal microbiome of 18 healthy adults, sampled weekly over a 40-weeks using culture-independent molecular techniques. We detected nine phyla, 202 genera and 1438 assignments on OTU level, dominated by Firmicutes, Bacteroidetes, and Proteobacteria on phylum level. Individual microbiomes of participants were characterized by levels of high alpha diversity (mean = 204.55 OTUs, sd = 35.64), evenness (19.83, sd = 9.74) and high temporal stability (mean Pearson’s correlation between samples of 0.52, sd = 0.060), with greater differences in microbiome community composition between than within individuals. Significant changes in community composition were associated with disease states, suggesting that it is possible to detect specific changes in OTU abundance and community composition during illness. We defined the common core microbiota by varying occurrence and abundance thresholds showing that individual core microbiomes share a substantial number of OTUs across participants, chiefly Streptococci and Veillonella. Our results provide insights into the microbial communities that characterize the healthy human oropharynx, community structure and variability, and provide new approaches to define individual and shared cores. The wider implications of this result include the potential for modeling the general dynamics of oropharynx microbiota both in health and in response to antimicrobial treatments or probiotics.

Impact of the Post-Transplant Period and Lifestyle Diseases on Human Gut Microbiota in Kidney Graft Recipients.

Gaining long-term graft function and patient life quality remain critical challenges following kidney transplantation. Advances in immunology, gnotobiotics, and culture-independent molecular techniques have provided growing insights into the complex relationship of the microbiome and the host. However, little is known about the over time-shift of the gut microbiota in the context of kidney transplantation and its impact on both graft and health stability. Here we aimed to characterize the structure of gut microbiota within stable kidney graft recipients. We enrolled forty kidney transplant patients after at least three months of transplantation and compared them to eighteen healthy controls. The overall microbial community structure of the kidney transplanted group was clearly different from control subjects. We found lower relative abundances of Actinobacteria, Bacteroidetes, and Verrucomicrobia within the patient group and a higher abundance of Proteobacteria compared to the control group. Both richness and Shannon diversity indexes were significantly lower in the kidney graft recipients than in healthy controls. Post-graft period was positively correlated with the relative abundance of the Proteobacteria phylum, especially Escherichia.Shigella genus. Interestingly, only Parabacteroides was found to significantly differentiate patients that were not suffering from lifestyle diseases and those who suffer from post-graft complications. Furthermore, network analysis showed that the occurrence of lifestyle diseases was significantly linked with a higher number of negative interactions of Sutterella and Succinivibrio genera within patients. This study characterizes gut microbiome fluctuation in stable kidney transplant patients after a long post-allograft period. Analysis of fecal microbiota could be useful for nephrologists as a new clinical tool that can improve kidney allograft monitoring and outcomes.