Enhanced propagation of Granulicatella adiacens from human oral microbiota by hyaluronan.

Host determinants for formation/composition of human oral microbiota remain to be clarified, although microorganisms entering the mouth cannot necessarily colonize the oral environment. Here we show that human oral-abundant bacteria degraded host glycosaminoglycans (GAGs) in saliva and gingiva, and certain bacteria significantly grew on hyaluronan (HA), a kind of GAGs. Microbial communities from teeth or gingiva of healthy donors assimilated HA. Metagenomic analysis of human oral microbiota under different carbon sources revealed HA-driven Granulicatella growth. HA-degrading bacterial strains independently isolated from teeth and gingiva were identified as Granulicatella adiacens producing extracellular 130 kDa polysaccharide lyase as a HA-degrading enzyme encoded in a peculiar GAG genetic cluster containing genes for isomerase KduI and dehydrogenase DhuD. These findings demonstrated that GAGs are one of the host determinants for formation/composition of oral microbiota not only for colonization but also for the adaptation to the host niche. Especially, HA enhanced the G. adiacens propagation.

Molecular and Structural Parallels between Gluten Pathogenic Peptides and Bacterial-Derived Proteins by Bioinformatics Analysis.

Gluten-related disorders (GRDs) are a group of diseases that involve the activation of the immune system triggered by the ingestion of gluten, with a worldwide prevalence of 5%. Among them, Celiac disease (CeD) is a T-cell-mediated autoimmune disease causing a plethora of symptoms from diarrhea and malabsorption to lymphoma. Even though GRDs have been intensively studied, the environmental triggers promoting the diverse reactions to gluten proteins in susceptible individuals remain elusive. It has been proposed that pathogens could act as disease-causing environmental triggers of CeD by molecular mimicry mechanisms. Additionally, it could also be possible that unrecognized molecular, structural, and physical parallels between gluten and pathogens have a relevant role. Herein, we report sequence, structural and physical similarities of the two most relevant gluten peptides, the 33-mer and p31-43 gliadin peptides, with bacterial pathogens using bioinformatics going beyond the molecular mimicry hypothesis. First, a stringent BLASTp search using the two gliadin peptides identified high sequence similarity regions within pathogen-derived proteins, e.g., extracellular proteins from Streptococcus pneumoniae and Granulicatella sp. Second, molecular dynamics calculations of an updated α-2-gliadin model revealed close spatial localization and solvent-exposure of the 33-mer and p31-43 peptide, which was compared with the pathogen-related proteins by homology models and localization predictors. We found putative functions of the identified pathogen-derived sequence by identifying T-cell epitopes and SH3/WW-binding domains. Finally, shape and size parallels between the pathogens and the superstructures of gliadin peptides gave rise to novel hypotheses about activation of innate immunity and dysbiosis. Based on our structural findings and the similarities with the bacterial pathogens, evidence emerges that these pathologically relevant gluten-derived peptides could behave as non-replicating pathogens opening new research questions in the interface of innate immunity, microbiome, and food research.

The unusual isolation of carnobacteria in eyes of healthy salmonids in high-mountain lakes.

Carnobacteria are common bacteria in cold and temperate environments; they are also reported during fish mortality events. In a previous study, carnobacteria were isolated from the eyes of healthy wild salmonids from a high-mountain lake. To better understand these findings, salmonids were captured from three high-mountain lakes (Lower and Upper Balma Lake, Rouen Lake; northwest Italy) during August 2019 and subjected to bacteriological and histological examination. Although all were healthy, 8.7% (Lower Balma Lake), 24% (Upper Balma Lake), and 32.6% (Rouen Lake) were positive for carnobacteria colonization of the eyes. A Trojan-horse effect was hypothesized to explain carnobacteria isolation in the eye. This immune-escaping macrophage-mediated mechanism has been identified in other Gram-positive bacteria. Biochemical, molecular, and phylogenetic analysis were carried out on isolated bacteria (Carnobacterium maltaromaticum and C. divergens). Based on previous references for carnobacteria isolated from fish, C. maltaromaticum strains were tested for the pisA precursor gene of the bacteriocin piscicolin 126. Carnobacterium maltaromaticum strains were found to display genotypic heterogeneity and a low percentage of pisA positive amplification. Features of geomorphology, geographic isolation, and microbiota common to the three lakes are thought to be possibly related to our findings. Moreover, terrestrial insects collected from the lake shoreline and the stomach contents were screened for the presence of carnobacteria. The salmonids in these high-mountain environments feed mainly on terrestrial insects, which are considered possible vectors for carnobacteria that might catabolize the exoskeleton chitin. All insects tested negative for carnobacteria, but as a small number of samples were analyzed, their role as possible vectors of infection cannot be excluded. Further studies are needed to corroborate our research hypothesis.

Proteomics of extracellular vesicles produced by Granulicatella adiacens, which causes infective endocarditis.

When oral bacteria accidentally enter the bloodstream due to transient tissue damage during dental procedures, they have the potential to attach to the endocardium or an equivalent surface of an indwelling prosthesis and cause infection. Many bacterial species produce extracellular vesicles (EVs) as part of normal physiology, but also use it as a virulence strategy. In this study, it was hypothesized that Granulicatella adiacens produce EVs that possibly help it in virulence. Therefore, the objectives were to isolate and characterize EVs produced by G. adiacens and to investigate its immune-stimulatory effects. The reference strain G. adiacens CCUG 27809 was cultured on chocolate blood agar for 2 days. From subsequent broth culture, the EVs were isolated using differential centrifugation and filtration protocol and then observed using scanning electron microscopy. Proteins in the vesicle preparation were identified by nano LC-ESI-MS/MS. The EVs proteome was analyzed and characterized using different bioinformatics tools. The immune-stimulatory effect of the EVs was studied via ELISA quantification of IL-8, IL-1ß and CCL5, major proinflammatory cytokines, produced from stimulated human PBMCs. It was revealed that G. adiacens produced EVs, ranging in diameter from 30 to 250 nm. Overall, G. adiacens EVs contained 112 proteins. The proteome consists of several ribosomal proteins, DNA associated proteins, binding proteins, and metabolic enzymes. It was also shown that these EVs carry putative virulence factors including moonlighting proteins. These EVs were able to induce the production of IL-8, IL-1ß and CCL5 from human PBMCs. Further functional characterization of the G. adiacens EVs may provide new insights into virulence mechanisms of this important but less studied oral bacterial species.

Genome-guided analysis allows the identification of novel physiological traits in Trichococcus species.

BACKGROUND: The genus Trichococcus currently contains nine species: T. flocculiformis, T. pasteurii, T. palustris, T. collinsii, T. patagoniensis, T. ilyis, T. paludicola, T. alkaliphilus, and T. shcherbakoviae. In general, Trichococcus species can degrade a wide range of carbohydrates. However, only T. pasteurii and a non-characterized strain of Trichococcus, strain ES5, have the capacity of converting glycerol to mainly 1,3-propanediol. Comparative genomic analysis of Trichococcus species provides the opportunity to further explore the physiological potential and uncover novel properties of this genus. RESULTS: In this study, a genotype-phenotype comparative analysis of Trichococcus strains was performed. The genome of Trichococcus strain ES5 was sequenced and included in the comparison with the other nine type strains. Genes encoding functions related to e.g. the utilization of different carbon sources (glycerol, arabinan and alginate), antibiotic resistance, tolerance to low temperature and osmoregulation could be identified in all the sequences analysed. T. pasteurii and Trichococcus strain ES5 contain a operon with genes encoding necessary enzymes for 1,3-PDO production from glycerol. All the analysed genomes comprise genes encoding for cold shock domains, but only five of the Trichococcus species can grow at 0 °C. Protein domains associated to osmoregulation mechanisms are encoded in the genomes of all Trichococcus species, except in T. palustris, which had a lower resistance to salinity than the other nine studied Trichococcus strains. CONCLUSIONS: Genome analysis and comparison of ten Trichococcus strains allowed the identification of physiological traits related to substrate utilization and environmental stress resistance (e.g. to cold and salinity). Some substrates were used by single species, e.g. alginate by T. collinsii and arabinan by T. alkaliphilus. Strain ES5 may represent a subspecies of Trichococcus flocculiformis and contrary to the type strain (DSM 2094T), is able to grow on glycerol with the production of 1,3-propanediol.

Microbiome response to controlled shifts in ammonium and LCFA levels in co-digestion systems.

Anaerobic co-digestion using protein-rich and lipid-rich co-substrates is limited by the accumulation of ammonia and long chain fatty acids (LCFAs), which are important inhibitors of the anaerobic microorganisms. This work aimed to study the microbial community dynamics during gradual and abrupt increase in ammonium and LCFAs concentrations by applying several molecular techniques, as well as during gradual decrease. For this purpose, two anaerobic reactors co-digesting three agro-industrial wastes underwent abrupt and gradual changes of ammonium and LCFAs concentrations. Both variations provoked volatile fatty acids (VFAs) accumulation, mainly acetic acid up to 4.5gL(-1). High ammonium levels were correlated to an increase in Pseudomonadaceae, Carnobacteriaceae and Clostridiadaceae families and to a drop in Syntrophomonadaceae. However, high LCFA levels provoked an increase in the Anaerobaculaceae and Peptococcaceae families. Both perturbations resulted in greater variations in the archaeal domain, going from Methanosaeta dominance in steady state to hydrogenotrophic pathway during the disturbance periods. During the abrupt changes, Bacteria domain experienced a minimal change, which indicates the adaptation bacterial populations to high ammonium and LCFAs levels. Species belonging to Porphyromonadaceae and Tissierellaceae families linked to VFAs consumption rose their presence during the recovery period. This study identifies a subset of microbial communities linked to high ammonia and LCFA concentrations, useful for optimizing the high-rate co-digestion processes dealing with lipid and protein-rich co-substrates.

[Bioconversion of cellulose to methane by a consortium consisting of four microbial strains].

Cellulose was usually degraded by microbial communities in natural habitats. Construction of a simple cellulolytic consortium is necessary to understand the underlying interaction within microorganisms involved in cellulose conversion. A screening approach was developed to obtain a simple microbial community with the ability of cellulose degradation to methane. This technique was based on the method of enrichment culture accompanying with denaturing gel gradient electrophoresis (DGGE) fingerprint detection technology and roll-tube method. Moreover, a four-strain mixed culture capable of degrading cellulose to methane was isolated from Zoige alpine wetland of the Tibetan Plateau. The results showed that the microbial consortia consisted of three functional groups: the cellulolytic bacterium Clostridium glycolicum, the non-celluloytic bacteria group of Trichococcus flocculiformis and Parabacteroides merdae, and the methanogenic bacterium Methanobacterium subterraneum. This four-strain co-culture can convert cellulose to methane. In the future, the isolated cellulolytic consortia could provide a platform for controlling metabolic pathways and genetic modification involved in methane production from cellulose.

1,3-Propanediol production from glycerol by a newly isolated Trichococcus strain.

A coccal bacterium (strain ES5) was isolated from methanogenic bioreactor sludge with glycerol as the sole energy and carbon source. Strain ES5 fermented glycerol to 1,3-propanediol as main product, and lactate, acetate and formate as minor products. The strain was phylogenetically closely related to Trichococcus flocculiformis; the rRNA gene sequence similarity was 99%. However, strain ES5 does not show the typical growth in chains of T. flocculiformis. Moreover, T. flocculiformis does not ferment glycerol. Strain ES5 used a variety of sugars for growth. With these substrates, lactate, acetate and formate were the main products, while 1,3-propanediol was not formed. The optimum growth temperature of strain ES5 ranges from 30-37°C, but like several other Trichoccoccus strains, strain ES5 is able to grow at low temperature (< 10°C). Therefore, strain ES5 may be an appropriate catalyst for the biotechnological production of 1,3-propanediol from glycerol at low ambient temperature.

Impact of anaerobic and oligotrophic conditions on survival of Alloiococcus otitidis, implicated as a cause of otitis media.

The survival of Alloiococcus otitidis (NCFB2890) with different nutritional supplements, including brain-heart infusion broth (BHI), phosphate-buffered saline (PBS), distilled water (DW), and middle ear effusion (MEE), as well as various atmospheres (aerobic, microaerobic, anaerobic), was compared using cultures, LIVE/DEAD staining, and transmission electron microscopy. The bacterial morphological traits and viability were maintained in BHI and MEE under aerobic conditions but were rapidly lost in PBS and DW. In contrast, anaerobic conditions did not support viability at all. Thus, the bacteria critically required an aerobic atmosphere for its survival as well as the appropriate nutrients, implying that culture of this pathogen from clinical specimens would become more difficult through oxygen depletion depending on a slight change in the middle ear atmosphere.