Effect of two shampoo formulations on the prokaryotic and eukaryotic microbiota composition of the human scalp.

OBJECTIVE: The human scalp is characterized by a moderately diverse microbial community, comprising prokaryotic (bacteria) and eukaryotic (fungi) members. Although the details are far from being fully understood, the human scalp microbiota is implicated in several scalp disorders, in particular dandruff formation. Hence, the protection of an intact and diverse scalp microbiota can be regarded as a quality criterion for hair and scalp care formulations. In this study, we investigated the influence of two commercially available, non-antimicrobial shampoo formulations on the structure of the scalp microbiota. METHODS: Scalp microbiota samples, obtained by swab sampling from two cohorts of probands (n = 25, each), were analysed before and after daily use of two different shampoo formulations for 2 weeks, respectively. A polyphasic approach was used, comprising quantitative cultivation of bacteria and fungi on selective media as well as sequencing of PCR-amplified 16S rRNA and 18S rRNA genes, respectively. RESULTS: All analyses revealed a microbiota composition typical for the human scalp. While in particular fungal germ numbers increased significantly during the treatments, overall bacterial and fungal community composition was not affected, based on alpha- and beta-diversity measures. However, we observed an increase in structural bacterial diversity with the age of the probands. CONCLUSIONS: Over an application period of 2 weeks, the investigated shampoo induced quantitative but no qualitative changes in the scalp microbial community structure of the investigated probands, suggesting no adverse but rather preserving or even stimulating effects of the underlying formulations on the scalp microbiota. Further investigation will have to clarify if this is also true for longer application periods and if the formulations might affect community functionality, for example microbial gene expression, rather than community composition.

OBJECTIF: Le cuir chevelu humain se caractérise par une communauté microbienne modérément diversifiée, comprenant des membres procaryotes (bactéries) et eucaryotes (champignons). Bien que l'on soit loin de comprendre totalement les détails, le microbiote du cuir chevelu humain est impliqué dans différents troubles du cuir chevelu, en particulier la formation de pellicules. La protection du microbiote du cuir chevelu intact et diversifié peut être considérée comme un critère de qualité pour les formulations de soins pour les cheveux et le cuir chevelu. Dans cette étude, nous avons examiné l'influence de deux formulations de shampooing non antimicrobien disponibles dans le commerce sur la structure du microbiote du cuir chevelu. MÉTHODES: Des échantillons de microbiote du cuir chevelu, obtenus par écouvillonnage dans deux cohortes de proposants (n = 25 dans chaque cohorte), ont été analysés respectivement avant et après l'utilisation quotidienne de deux formulations de shampooing pendant deux semaines. Une approche en plusieurs phases a été utilisée, dont une culture quantitative de bactéries et de champignons sur des milieux sélectifs et un séquençage respectivement des gènes de l'ARN ribosomique 16S et de l'ARN ribosomique 18S amplifiés par PCR. RÉSULTATS: Toutes les analyses ont révélé une composition du microbiote typique pour le cuir chevelu humain. Bien que le nombre de germes fongiques en particulier ait augmenté significativement pendant les traitements, la composition globale des communautés bactériennes et fongiques n'a pas été affectée, d'après les mesures de diversité alpha et bêta. Cependant, nous avons observé une augmentation de la diversité bactérienne structurelle avec l'âge des proposants. CONCLUSIONS: Sur une période d'utilisation de deux semaines, le shampooing étudié a induit des modifications quantitatives, mais pas qualitatives, de la structure des communautés microbiennes du cuir chevelu des proposants étudiés, ce qui suggère qu'il n'y a pas d'effets indésirables, mais qu'il y a des effets de préservation, voire de stimulation, des formulations sous-jacentes sur le microbiote du cuir chevelu. Des recherches supplémentaires devront clarifier si cela s'avère également pour des périodes d'utilisation plus longues et si les formulations peuvent affecter la fonctionnalité des communautés, par exemple, l'expression des gènes microbiens, plutôt que la composition des communautés.

Gut microbiota dysbiosis in Parkinson disease: A systematic review and pooled analysis.

BACKGROUND AND PURPOSE: The role of the gut microbiome in the pathogenesis of Parkinson disease (PD) is under intense investigation, and the results presented are still very heterogeneous. These discrepancies arise not only from the highly heterogeneous pathology of PD, but also from widely varying methodologies at all stages of the workflow, from sampling to final statistical analysis. The aim of the present work is to harmonize the workflow across studies to reduce the methodological heterogeneity and to perform a pooled analysis to account for other sources of heterogeneity. METHODS: We performed a systematic review to identify studies comparing the gut microbiota of PD patients to healthy controls. A workflow was designed to harmonize processing across all studies from bioinformatics processing to final statistical analysis using a Bayesian random-effects meta-analysis based on individual patient-level data. RESULTS: The results show that harmonizing workflows minimizes differences between statistical methods and reveals only a small set of taxa being associated with the pathogenesis of PD. Increased shares of the genera Akkermansia and Bifidobacterium and decreased shares of the genera Roseburia and Faecalibacterium were most characteristic for PD-associated microbiota. CONCLUSIONS: Our study summarizes evidence that reduced levels of butyrate-producing taxa in combination with possible degradation of the mucus layer by Akkermansia may promote intestinal inflammation and reduced permeability of the gut mucosal layer. This may allow potentially pathogenic metabolites to transit and enter the enteric nervous system.

RNA-based stable isotope probing provides no indication for rapid α-synuclein assimilation by murine gut bacteria.

In Parkinson's disease (PD), α-synuclein is a key protein in the process of neurodegeneration. Besides motor symptoms, most PD patients additionally suffer from gastrointestinal tract (GIT) dysfunctions, even several years before the onset of motor disabilities. Studies have reported a dysbiosis of gut bacteria in PD patients compared to healthy controls and have suggested that the enteric nervous system (ENS) can be involved in the development of the disease. As α-synuclein was found to be secreted by neurons of the ENS, we used RNA-based stable isotope probing (RNA-SIP) to identify gut bacteria that might be able to assimilate this protein. The gut contents of 24 mice were pooled and incubated with isotopically labelled (13C) and unlabelled (12C) α-synuclein. After incubation for 0, 4 and 24 h, RNA was extracted from the incubations and separated by density gradient centrifugation. However, RNA quantification of density-resolved fractions revealed no incorporation of the 13C isotope into the extracted RNA, suggesting that α-synuclein was not assimilated by the murine gut bacteria. Potential reasons and consequences for follow-up-studies are discussed.

Minority report: small-scale metagenomic analysis of the non-bacterial kitchen sponge microbiota.

Kitchen sponges are particularly well known to harbor a high number and diversity of bacteria, including pathogens. Viruses, archaea, and eukaryotes in kitchen sponges, however, have not been examined in detail so far. To increase knowledge on the non-bacterial kitchen sponge microbiota and its potential hygienic relevance, we investigated five used kitchen sponges by means of metagenomic shot-gun sequencing. Viral particles were sought to be enriched by a filter step during DNA extraction from the sponges. Data analysis revealed that ~ 2% of the sequences could be assigned to non-bacterial taxa. Each sponge harbored different virus (phage) species, while the present archaea were predominantly affiliated with halophilic taxa. Among the eukaryotic taxa, besides harmless algae, or amoebas, mainly DNA from food-left-overs was found. The presented work offers new insights into the complex microbiota of used kitchen sponges and contributes to a better understanding of their hygienic relevance.

Do it yourself! - Initial experiences with self-synthesized CsTFA for RNA-SIP analyses.

Cesium trifluoroacetate (CsTFA) is a gradient medium for isopycnic centrifugation in RNA-based Stable Isotope Probing (RNA-SIP), an important means to link the structure and function of microbial communities. We report a protocol to easily synthesize CsTFA from cesium carbonate (Cs2CO3) and trifluoroacetic acid (TFA) and show that self-synthesized CsTFA performs similarly to commercial CsTFA in the separation of isotopically labelled and unlabelled bacterial RNA.

Association between Parkinson's disease and the faecal eukaryotic microbiota.

Parkinson's disease (PD) is one of the most common neurodegenerative disease, and is so far not considered curable. PD patients suffer from several motor and non-motor symptoms, including gastrointestinal dysfunctions and alterations of the enteric nervous system. Constipation and additional intestinal affections can precede the classical motor symptoms by several years. Recently, we reported effects of PD and related medications on the faecal bacterial community of 34 German PD patients and 25 age-matched controls. Here, we used the same collective and analysed the V6 and V7 hypervariable region of PCR-amplified, eukaryotic 18S rRNA genes using an Illumina MiSeq platform. In all, 53% (18) of the PD samples and 72% (18) of the control samples yielded sufficient amplicons for downstream community analyses. The PD samples showed a significantly lower alpha and a different beta eukaryotic diversity than the controls. Most strikingly, we observed a significantly higher relative abundance of sequence affiliated with the Geotrichum genus in the PD samples (39.7%), when compared to the control samples (0.05%). In addition, we observed lower relative abundances of sequences affiliated with Aspergillus/Penicillium, Charophyta/Linum, unidentified Opisthokonta and three genera of minor abundant zooflagellates in the PD samples. Our data add knowledge to the small body of data about the eukaryotic microbiota of PD patients and suggest a potential association of certain gut eukaryotes and PD.

Site-specific molecular analysis of the bacteriota on worn spectacles.

Regularly touched surfaces are usually contaminated with microorganisms and might be considered as fomites. The same applies for spectacles, but only little is known about their microbial colonization. Previous cultivation-based analyses from our group revealed a bacterial load strongly dominated by staphylococci. To better account for aerotolerant anaerobes, slow growing and yet-uncultivated bacteria, we performed an optimized 16S rRNA gene sequencing approach targeting the V1-V3 region. 30 spectacles were swab-sampled at three sites, each (nosepads, glasses and earclips). We detected 5232 OTUs affiliated with 19 bacterial phyla and 665 genera. Actinobacteria (64%), Proteobacteria (22%), Firmicutes (7%) and Bacteroidetes (5%) were relatively most abundant. At genus level, 13 genera accounted for 84% of the total sequences of all spectacles, having a prevalence of more than 1% relative abundance. Propionibacterium (57%), Corynebacterium (5%), Staphylococcus (4%), Pseudomonas, Sphingomonas and Lawsonella (3%, each) were the dominant genera. Interestingly, bacterial diversity on the glasses was significantly higher compared to nosepads and earclips. Our study represents the first cultivation-independent study of the bacteriota of worn spectacles. Dominated by bacteria of mostly human skin and epithelia origin and clearly including potential pathogens, spectacles may play a role as fomites, especially in clinical environments.

Towards safer stable isotope probing - effect of formamide on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation.

RNA-based stable isotope probing (RNA-SIP) is used in molecular microbial ecology to link the identity of microorganisms in a complex community with the assimilation of a distinct substrate. The technique is highly dependent on a reliable separation of isotopic-labeled RNA from unlabeled RNA by isopycnic density gradient ultracentrifugation. Here we show that 13C-labeled and unlabeled Escherichia coli RNA can be sufficiently separated by isopycnic ultracentrifugation even in the absence of formamide. However, a slightly lower starting density is needed to obtain a distribution pattern similar to that obtained when formamide was used. Hence, the commonly used addition of formamide to the centrifugation solution might not be needed to separate 13C-labeled RNA from unlabeled RNA, but this must be verified for more complex environmental mixtures of RNA. Clearly, an omission of formamide would increase the safety of RNA-SIP analyses.

A single Thaumarchaeon drives nitrification in deep oligotrophic Lake Constance.

Ammonia released during organic matter mineralization is converted during nitrification to nitrate. We followed spatiotemporal dynamics of the nitrifying microbial community in deep oligotrophic Lake Constance. Depth-dependent decrease of total ammonium (0.01-0.84 µM) indicated the hypolimnion as the major place of nitrification with 15 N-isotope dilution measurements indicating a threefold daily turnover of hypolimnetic total ammonium. This was mirrored by a strong increase of ammonia-oxidizing Thaumarchaeota towards the hypolimnion (13%-21% of bacterioplankton) throughout spring to autumn as revealed by amplicon sequencing and quantitative polymerase chain reaction. Ammonia-oxidizing bacteria were typically two orders of magnitude less abundant and completely ammonia-oxidizing (comammox) bacteria were not detected. Both, 16S rRNA gene and amoA (encoding ammonia monooxygenase subunit B) analyses identified only one major species-level operational taxonomic unit (OTU) of Thaumarchaeota (99% of all ammonia oxidizers in the hypolimnion), which was affiliated to Nitrosopumilus spp. The relative abundance distribution of the single Thaumarchaeon strongly correlated to an equally abundant Chloroflexi clade CL500-11 OTU and a Nitrospira OTU that was one order of magnitude less abundant. The latter dominated among recognized nitrite oxidizers. This extremely low diversity of nitrifiers shows how vulnerable the ecosystem process of nitrification may be in Lake Constance as Central Europe's third largest lake.

Effect of Parkinson's disease and related medications on the composition of the fecal bacterial microbiota.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders. PD patients suffer from gastrointestinal dysfunctions and alterations of the autonomous nervous system, especially its part in the gut wall, i.e., the enteric nervous system (ENS). Such alterations and functional gastrointestinal deficits often occur years before the classical clinical symptoms of PD appear. Until now, only little is known about PD-associated changes in gut microbiota composition and their potential implication in PD development. In order to increase knowledge in this field, fecal samples of 34 PD patients and 25 healthy, age-matched control persons were investigated. Here, the V4 and V5 hypervariable region of bacterial 16S rRNA genes was PCR-amplified and sequenced using an Ion Torrent PGM platform. Within the PD group, we observed a relative decrease in bacterial taxa which are linked to health-promoting, anti-inflammatory, neuroprotective or other beneficial effects on the epithelial barrier, such as Faecalibacterium and Fusicatenibacter. Both taxa were lowered in PD patients with elevated levels of the fecal inflammation marker calprotectin. In addition, we observed an increase in shares of the Clostridiales family XI and their affiliated members in these samples. Finally, we found that the relative abundances of the bacterial genera Peptoniphilus, Finegoldia, Faecalibacterium Fusicatenibacter, Anaerococcus, Bifidobacterium, Enterococcus, and Ruminococcus were significantly influenced by medication with L-dopa and entacapone, respectively. Our data confirm previously reported effects of COMT inhibitors on the fecal microbiota of PD patients and suggest a possible effect of L-dopa medication on the relative abundance of several bacterial genera.

Effect of endometriosis on the fecal bacteriota composition of mice during the acute phase of lesion formation.

Accumulating evidence indicates that there is an interaction between the gut microbiota and endometriotic lesions. The new formation of these lesions is associated with stem cell recruitment, angiogenesis and inflammation, which may affect the composition of the gut microbiota. To test this hypothesis, we herein induced endometriotic lesions by transplantation of uterine tissue fragments from green fluorescent protein (GFP)+ donor mice into the peritoneal cavity of GFP- C57BL/6 wild-type mice. Sham-transplanted animals served as controls. Fecal pellets of the animals were collected 3 days before as well as 7 and 21 days after the induction of endometriosis to analyze the composition of the gut microbiota by means of 16S ribosomal RNA gene sequencing. The transplantation of uterine tissue fragments resulted in the establishment of endometriotic lesions in all analyzed mice. These lesions exhibited a typical histomorphology with endometrial glands surrounded by a vascularized stroma. Due to their bright GFP signal, they could be easily differentiated from the surrounding GFP- host tissue. Bacterial 16S rRNA genes were successfully PCR-amplified from the DNA extracts of all obtained mice fecal samples. However, no significant effect of endometriosis induction on the composition of the bacterial microbiota was detected with our experimental setup. Our findings allow careful speculation that endometriosis in mice does not induce pronounced dysbiosis during the acute phase of lesion formation.

Influence of Sampling Site and other Environmental Factors on the Bacterial Community Composition of Domestic Washing Machines.

Modern, mainly sustainability-driven trends, such as low-temperature washing or bleach-free liquid detergents, facilitate microbial survival of the laundry processes. Favourable growth conditions like humidity, warmth and sufficient nutrients also contribute to microbial colonization of washing machines. Such colonization might lead to negatively perceived staining, corrosion of washing machine parts and surfaces, as well as machine and laundry malodour. In this study, we characterized the bacterial community of 13 domestic washing machines at four different sampling sites (detergent drawer, door seal, sump and fibres collected from the washing solution) using 16S rRNA gene pyrosequencing and statistically analysed associations with environmental and user-dependent factors. Across 50 investigated samples, the bacterial community turned out to be significantly site-dependent with the highest alpha diversity found inside the detergent drawer, followed by sump, textile fibres isolated from the washing solution, and door seal. Surprisingly, out of all other investigated factors only the monthly number of wash cycles at temperatures ≥ 60 °C showed a significant influence on the community structure. A higher number of hot wash cycles per month increased microbial diversity, especially inside the detergent drawer. Potential reasons and the hygienic relevance of this finding need to be assessed in future studies.

RNA-Based Stable Isotope Probing (RNA-SIP) in the Gut Environment.

The RNA-SIP technology allows for linking the structure and function of complex microbial communities, that is, the identification of microbial key players involved in distinct degradation and assimilation processes under in situ conditions. Being dependent on RNA, this technique is particularly suited for environments with high numbers of very active, that is, significantly RNA-expressing microorganisms, such as intestinal tract samples. We use RNA-SIP for the identification of bacteria involved in the degradation and assimilation of prebiotic carbohydrates in order to better understand the functionality of these medically and economically important nutrients in human and animal intestinal environments.

RNA-based stable isotope probing (RNA-SIP) to unravel intestinal host-microbe interactions.

The RNA-SIP technology, introduced into molecular microbial ecology in 2002, is an elegant technique to link the structure and function of complex microbial communities, i.e. to identify microbial key-players involved in distinct degradation and assimilation processes under in-situ conditions. Due to its dependence of microbial RNA, this technique is particularly suited for environments with high numbers of very active, i.e. significantly RNA-expressing, bacteria. So far, it was mainly used in environmental studies using microbiotas from soil or water habitats. Here we outline and summarize our application of RNA-SIP for the identification of bacteria involved in the degradation and assimilation of prebiotic carbohydrates in intestinal samples of human and animal origin. Following an isotope label from a prebiotic substrate into the RNA of distinct bacterial taxa will help to better understand the functionality of these medically and economically important nutrients in an intestinal environment.

In Vivo Assessment of Resistant Starch Degradation by the Caecal Microbiota of Mice Using RNA-Based Stable Isotope Probing-A Proof-of-Principle Study.

Resistant starch (RS) is the digestion resistant fraction of complex polysaccharide starch. By reaching the large bowel, RS can function as a prebiotic carbohydrate, i.e., it can shape the structure and activity of bowel bacterial communities towards a profile that confers health benefits. However, knowledge about the fate of RS in complex intestinal communities and the microbial members involved in its degradation is limited. In this study, 16S ribosomal RNA (rRNA)-based stable isotope probing (RNA-SIP) was used to identify mouse bowel bacteria involved in the assimilation of RS or its derivatives directly in their natural gut habitat. Stable-isotope [U13C]-labeled native potato starch was administrated to mice, and caecal contents were collected before 0 h and 2 h and 4 h after administration. 'Heavy', isotope-labeled [13C]RNA species, presumably derived from bacteria that have metabolized the labeled starch, were separated from 'light', unlabeled [12C]RNA species by fractionation of isolated total RNA in isopycnic-density gradients. Inspection of different density gradients showed a continuous increase in 'heavy' 16S rRNA in caecal samples over the course of the experiment. Sequencing analyses of unlabeled and labeled 16S amplicons particularly suggested a group of unclassified Clostridiales, Dorea, and a few other taxa (Bacteroides, Turicibacter) to be most actively involved in starch assimilation in vivo. In addition, metabolic product analyses revealed that the predominant 13C-labeled short chain fatty acid (SCFA) in caecal contents produced from the [U13C] starch was butyrate. For the first time, this study provides insights into the metabolic transformation of RS by intestinal bacterial communities directly within a gut ecosystem, which will finally help to better understand its prebiotic potential and possible applications in human health.