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Helicobacter pylori infects the stomachs of one in two humans and can cause sequelae that include ulcers and cancer. Here we sequenced the genomes of 97 H. pylori isolates from 52 members of two families living in rural conditions in South Africa. From each of 45 individuals, two H. pylori strains were isolated from the antrum and corpus parts of the stomach, and comparisons of their genomes enabled us to study within-host evolution. In 5 of these 45 hosts, the two genomes were too distantly related to be derived from each other and therefore represented evidence of multiple infections. From the remaining 40 genome pairs, we estimated that the synonymous mutation rate was 1.38 × 10 ⁻⁵ per site per year, with a low effective population size within host probably reflecting population bottlenecks and immune selection. Some individuals showed very little evidence for recombination, whereas in others, recombination introduced up to 100-times more substitutions than mutation. These differences may reflect unequal opportunities for recombination depending on the presence or absence of multiple infections. Comparing the genomes carried by distinct individuals enabled us to establish probable transmission links. Transmission events were found significantly more frequently between close relatives, and between individuals living in the same house. We found, however, that a majority of individuals (27/52) were not linked by transmission to other individuals. Our results suggest that transmission does not always occur within families, and that coinfection with multiple strains is frequent and evolutionarily important despite a fast turnover of the infecting strains within-host.

Recombination plays a dominant role in the evolution of the bacterial pathogen Helicobacter pylori , but its dynamics remain incompletely understood. Here we use an in vitro transformation system combined with genome sequencing to study chromosomal integration patterns after natural transformation. A single transformation cycle results in up to 21 imports, and repeated transformations generate a maximum of 92 imports (8% sequence replacement). Import lengths show a bimodal distribution with averages of 28 and 1,645 bp. Reanalysis of paired H. pylori genomes from chronically infected people demonstrates the same bimodal import pattern in vivo . Restriction endonucleases (REases) of the recipient bacteria fail to inhibit integration of homeologous DNA, independently of methylation. In contrast, REases limit the import of heterologous DNA. We conclude that restriction-modification systems inhibit the genomic integration of novel sequences, while they pose no barrier to homeologous recombination, which reconciles the observed stability of the H. pylori gene content and its highly recombinational population structure. Uptake and integration of exogenous DNA into the bacterial genome play an important role in the evolution of the pathogen Helicobacter pylori . Here, the authors describe a bimodal pattern of chromosomal integration and show how restriction-modification systems limit the import of heterologous DNA.

Even though metagenomics have revolutionized the characterization of the human microbiome, detailed mechanistic studies are impracticable, as there is a dearth of robust culture collections. We now describe the development and use of a laser-assisted culturomics platform, incorporating the elements of a bioprinter, the culture conditions, the methods to characterize the microorganisms and a biobank. With laser-assisted bioprinting, the microorganisms can be rapidly and precisely transferred from clinical biofilms to highly organized arrays of microbial colonies, which are suitable for co-culturing and molecular analyses. The presented technique has propagated 99 of 100 microbial species and recovered 79% of abundant species from dental plaque in accordance with full 16S rRNA gene profiling of 691,199 sequences. Microscopy, spectroscopy and enzyme assays have been used to guide isolations. Processing of oral biofilms from four individuals has yielded 249 representative isolates, from 14 classes and 124 species in total. Functional profiling with bioprinting has indicated commensals which could potentially contribute to disease development. Isolates from peri-implantitis cover 85.4% of the transcriptionally active clinical biofilms at genus level. Taken together, this work provides the basis for generating on-demand culture collections and biofilms for research and clinical use. Representative microbial isolates and patient-specific biobanks are crucial for microbiome investigation and management. Here, authors develop laser-assisted microbial culturomics, combining high-throughput, precise bioprinting on diverse media with rapid, non-invasive analyses.

Smoking, a risk factor for periodontitis and peri-implantitis, is associated with shifts in the oral microbiome (OM) composition. Although smoking habits are almost always established before adulthood, data on effects of smoking on the OM in adolescents is rare. The aim of this study was to investigate the early impact of smoking on the OM composition in pupils. The adolescent cohort, aged 14–20, comprised 98 smokers and 98 non-smokers matched for several physiological co-variates. Buccal swabs were analysed for OM composition using high-throughput sequencing of the full-length 16 S rRNA gene targeting species-level resolution. Parameters of bacterial diversity and abundance of individual bacterial taxa were related to information on smoking. The microbiome dataset contained 733 species-level taxa. Streptococcus , Rothia , and Haemophilus dominated both groups, smokers and non-smokers. Smoking exerted a discernible influence on the overall microbial composition as measured by weighted UniFrac distances. The number of species-level bacterial taxa was significantly higher in individual smokers compared to non-smokers. Furthermore, several taxa, including known pathogens, exhibited significant differences in abundance between the two groups. The genera Veillonella , and Actinomyces , as well as and multiple Actinomyces species, Dialister invisus , Atopobium parvulum , Streptococcus mutans and Prevotella melaninogenica were significantly more abundant in smokers. Our findings indicated an early onset of smoking-related changes already in the oral microbiome of adolescents.

Background Peri-implant mucositis and peri-implantitis are highly prevalent biofilm-associated diseases affecting the tissues surrounding dental implants. As antibiotic treatment is ineffective to fully cure biofilm mediated infections, antimicrobial modifications of implants to reduce or prevent bacterial colonization are called for. Preclinical in vivo evaluation of the functionality of new or modified implant materials concerning bacterial colonization and peri-implant health is needed to allow progress in this research field. For this purpose reliable animal models are needed. Methods Custom made endosseous dental implants were installed in female Sprague Dawley rats following a newly established three-step implantation procedure. After healing of the bone and soft tissue, the animals were assigned to two groups. Group A received a continuous antibiotic treatment for 7 weeks, while group B was repeatedly orally inoculated with human-derived strains of Streptococcus oralis , Fusobacterium nucleatum and Porphyromonas gingivalis for six weeks, followed by 1 week without inoculation. At the end of the experiment, implantation sites were clinically assessed and biofilm colonization was quantified via confocal laser scanning microscopy. Biofilm samples were tested for presence of the administered bacteria via PCR analysis. Results The inner part of the custom made implant screw could be identified as a site of reliable biofilm formation in vivo . S. oralis and F. nucleatum were detectable only in the biofilm samples from group B animals. P. gingivalis was not detectable in samples from either group. Quantification of the biofilm volume on the implant material revealed no statistically significant differences between the treatment groups. Clinical inspection of implants in group B animals showed signs of mild to moderate peri-implant mucositis (4 out of 6) whereas the mucosa of group A animals appeared healthy (8/8). The difference in the mucosa health status between the treatment groups was statistically significant ( p  = 0.015). Conclusions We developed a new rodent model for the preclinical evaluation of dental implant materials with a special focus on the early biofilm colonization including human-derived oral bacteria. Reliable biofilm quantification on the implant surface and the symptoms of peri-implant mucositis of the bacterially inoculated animals will serve as a readout for experimental evaluation of biofilm-reducing modifications of implant materials.

Biofilm-associated peri-implant infections pose a major problem in modern medicine. The understanding of biofilm development is hampered by biofilm complexity and the lack of robust clinical models. This study comprehensively characterized the dynamics of early biofilm formation in the transmucosal passage of implant abutments in 12 patients. Biofilm structures and compositions were complex, diverse, subject-specific and dynamic. A total of 371 different bacterial species were detected. 100 phylogenetically diverse unnamed species and 35 taxonomically diverse disease-associated species comprised an average 4.3% and 3.1% of the community, respectively, but reached up to 12.7% and 21.7% in some samples. Oral taxa formed numerous positive associations and clusters and were characterized by a high potential for metabolic interactions. The subspecies diversity was highly patient-specific and species-dependent, with 1427 ASVs identified in total. The unprecedented depth of early biofilm characterization in this study will support the development of individualized preventive and early diagnostic strategies.

Many dinoflagellate species are notorious for the toxins they produce and ecological and human health consequences associated with harmful algal blooms (HABs). Dinoflagellates are particularly refractory to genomic analysis due to the enormous genome size, lack of knowledge about their DNA composition and structure, and peculiarities of gene regulation, such as spliced leader (SL) trans-splicing and mRNA transposition mechanisms. Alexandrium ostenfeldii is known to produce macrocyclic imine toxins, described as spirolides. We characterized the genome of A. ostenfeldii using a combination of transcriptomic data and random genomic clones for comparison with other dinoflagellates, particularly Alexandrium species. Examination of SL sequences revealed similar features as in other dinoflagellates, including Alexandrium species. SL sequences in decay indicate frequent retro-transposition of mRNA species. This probably contributes to overall genome complexity by generating additional gene copies. Sequencing of several thousand fosmid and bacterial artificial chromosome (BAC) ends yielded a wealth of simple repeats and tandemly repeated longer sequence stretches which we estimated to comprise more than half of the whole genome. Surprisingly, the repeats comprise a very limited set of 79-97 bp sequences; in part the genome is thus a relatively uniform sequence space interrupted by coding sequences. Our genomic sequence survey (GSS) represents the largest genomic data set of a dinoflagellate to date. Alexandrium ostenfeldii is a typical dinoflagellate with respect to its transcriptome and mRNA transposition but demonstrates Alexandrium-like stop codon usage. The large portion of repetitive sequences and the organization within the genome is in agreement with several other studies on dinoflagellates using different approaches. It remains to be determined whether this unusual composition is directly correlated to the exceptionally genome organization of dinoflagellates with a low amount of histones and histone-like proteins.

Endoprosthesis loosening is the major cause of arthroplasty failure. Currently, radiography, histo-pathological classification of the periprosthetic membrane, and microbiological examination are used retrospectively for diagnosis. Prospective options for early diagnosis are not available. The study presented here aimed to identify (prospective) molecular biomarkers of implant loosening in tissue samples from patients. Four patient cohorts (primary and revision arthroplasty due to aseptic loosening of hip or knee) were defined. Aseptic loosening of implants was assessed by the standardised approach of the Knee Society Total Knee Arthroplasty Roentgenographic Evaluation and Scoring System. Synovial fluid, bone marrow, and blood were collected from 96 patients. Mesenchymal stromal cells (BM-MSCs) were isolated from the bone marrow. Bulk RNA-sequencing of 28 samples (including a fifth patient cohort with knee arthrofibrosis as aseptic cause for revision surgery) showed that Glycoprotein Non-Metastatic Melanoma Protein B mRNA ( GPNMB ) was significantly upregulated in BM-MSCs derived from revision patients compared to patients with primary implantations. Elevated GPNMB mRNA levels were confirmed with qRT-PCR. Synovial fluid plasma study by ELISA revealed increased levels of GPNMB protein in patients undergoing revision surgery compared to patients undergoing primary arthroplasty. GPNMB concentration in synovial fluid plasma, which can be obtained non-invasively, could be a potential biomarker for early detection of implant loosening, ahead of current diagnostic procedures.

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10(-/-) mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10(-/-) mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.

Background The dinoflagellate Alexandrium minutum typically produces paralytic shellfish poisoning (PSP) toxins, which are known only from cyanobacteria and dinoflagellates. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive. Results We constructed and analysed an expressed sequence tag (EST) library of A. minutum , which contained 15,703 read sequences yielding a total of 4,320 unique expressed clusters. Of these clusters, 72% combined the forward-and reverse reads of at least one bacterial clone. This sequence resource was then used to construct an oligonucleotide microarray. We analysed the expression of all clusters in three different strains. While the cyanobacterial PSP toxin genes were not found among the A. minutum sequences, 192 genes were differentially expressed between toxic and non-toxic strains. Conclusions Based on this study and on the lack of identified PSP synthesis genes in the two existent Alexandrium tamarense EST libraries, we propose that the PSP toxin genes in dinoflagellates might be more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. As a starting point to identify possible PSP toxin-associated genes in dinoflagellates without relying on a priori sequence information, the sequences only present in mRNA pools of the toxic strain can be seen as putative candidates involved in toxin synthesis and regulation, or acclimation to intracellular PSP toxins.