Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in human microbiome samples.

BACKGROUND: Sequencing variable regions of the 16S rRNA gene (≃300 bp) with Illumina technology is commonly used to study the composition of human microbiota. Unfortunately, short reads are unable to differentiate between highly similar species. Considering that species from the same genus can be associated with health or disease it is important to identify them at the lowest possible taxonomic rank. Third-generation sequencing platforms such as PacBio SMRT, increase read lengths allowing to sequence the whole gene with the maximum taxonomic resolution. Despite its potential, full length 16S rRNA gene sequencing is not widely used yet. The aim of the current study was to compare the sequencing output and taxonomic annotation performance of the two approaches (Illumina short read sequencing and PacBio long read sequencing of 16S rRNA gene) in different human microbiome samples. DNA from saliva, oral biofilms (subgingival plaque) and faeces of 9 volunteers was isolated. Regions V3-V4 and V1-V9 were amplified and sequenced by Illumina Miseq and by PacBio Sequel II sequencers, respectively. RESULTS: With both platforms, a similar percentage of reads was assigned to the genus level (94.79% and 95.06% respectively) but with PacBio a higher proportion of reads were further assigned to the species level (55.23% vs 74.14%). Regarding overall bacterial composition, samples clustered by niche and not by sequencing platform. In addition, all genera with > 0.1% abundance were detected in both platforms for all types of samples. Although some genera such as Streptococcus tended to be observed at higher frequency in PacBio than in Illumina (20.14% vs 14.12% in saliva, 10.63% vs 6.59% in subgingival plaque biofilm samples) none of the differences were statistically significant when correcting for multiple testing. CONCLUSIONS: The results presented in the current manuscript suggest that samples sequenced using Illumina and PacBio are mostly comparable. Considering that PacBio reads were assigned at the species level with higher accuracy than Illumina, our data support the use of PacBio technology for future microbiome studies, although a higher cost is currently required to obtain an equivalent number of reads per sample.

The multispecies microbial cluster of Fusobacterium, Parvimonas, Bacteroides and Faecalibacterium as a precision biomarker for colorectal cancer diagnosis.

The incidence of colorectal cancer (CRC) has increased worldwide, and early diagnosis is crucial to reduce mortality rates. Therefore, new noninvasive biomarkers for CRC are required. Recent studies have revealed an imbalance in the oral and gut microbiomes of patients with CRC, as well as impaired gut vascular barrier function. In the present study, the microbiomes of saliva, crevicular fluid, feces, and non-neoplastic and tumor intestinal tissue samples of 93 CRC patients and 30 healthy individuals without digestive disorders (non-CRC) were analyzed by 16S rRNA metabarcoding procedures. The data revealed that Parvimonas, Fusobacterium, and Bacteroides fragilis were significantly over-represented in stool samples of CRC patients, whereas Faecalibacterium and Blautia were significantly over-abundant in the non-CRC group. Moreover, the tumor samples were enriched in well-known periodontal anaerobes, including Fusobacterium, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Co-occurrence patterns of these oral microorganisms were observed in the subgingival pocket and in the tumor tissues of CRC patients, where they also correlated with other gut microbes, such as Hungatella. This study provides new evidence that oral pathobionts, normally located in subgingival pockets, can migrate to the colon and probably aggregate with aerobic bacteria, forming synergistic consortia. Furthermore, we suggest that the group composed of Fusobacterium, Parvimonas, Bacteroides, and Faecalibacterium could be used to design an excellent noninvasive fecal test for the early diagnosis of CRC. The combination of these four genera would significantly improve the reliability of a discriminatory test with respect to others that use a single species as a unique CRC biomarker.

Nitrate reduction capacity of the oral microbiota is impaired in periodontitis: potential implications for systemic nitric oxide availability.

The reduction of nitrate to nitrite by the oral microbiota has been proposed to be important for oral health and results in nitric oxide formation that can improve cardiometabolic conditions. Studies of bacterial composition in subgingival plaque suggest that nitrate-reducing bacteria are associated with periodontal health, but the impact of periodontitis on nitrate-reducing capacity (NRC) and, therefore, nitric oxide availability has not been evaluated. The current study aimed to evaluate how periodontitis affects the NRC of the oral microbiota. First, 16S rRNA sequencing data from five different countries were analyzed, revealing that nitrate-reducing bacteria were significantly lower in subgingival plaque of periodontitis patients compared with healthy individuals (P < 0.05 in all five datasets with n = 20-82 samples per dataset). Secondly, subgingival plaque, saliva, and plasma samples were obtained from 42 periodontitis patients before and after periodontal treatment. The oral NRC was determined in vitro by incubating saliva with 8 mmol/L nitrate (a concentration found in saliva after nitrate-rich vegetable intake) and compared with the NRC of 15 healthy individuals. Salivary NRC was found to be diminished in periodontal patients before treatment (P < 0.05) but recovered to healthy levels 90 days post-treatment. Additionally, the subgingival levels of nitrate-reducing bacteria increased after treatment and correlated negatively with periodontitis-associated bacteria (P < 0.01). No significant effect of periodontal treatment on the baseline saliva and plasma nitrate and nitrite levels was found, indicating that differences in the NRC may only be revealed after nitrate intake. Our results suggest that an impaired NRC in periodontitis could limit dietary nitrate-derived nitric oxide levels, and the effect on systemic health should be explored in future studies.

Dysimmunity in common variable immunodeficiency is associated with alterations in oral, respiratory, and intestinal microbiota.

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency characterized by decreased immunoglobulins and recurrent infections. Its aetiology remains unknown, and some patients present with severe non-infectious autoimmune or inflammatory complications with elevated associated morbimortality. Recently, intestinal dysbiosis has been proposed as a driver of immune dysregulation. In this study, we assessed the oral, respiratory, and gastrointestinal microbiota of 41 CVID patients (24 with dysimmune and 17 with infection complications) and 15 healthy volunteers using 16S rRNA gene sequencing to explore associations between microbiome profiles and CVID phenotypes. Profound differences in the composition of the microbiota in saliva, sputum, and stool were detected between dysimmune CVID patients and healthy individuals. Globally, respiratory species diversity and faecal bacterial richness were lower in CVID individuals with immune complications. Although a single species could not be identified as a robust predictor of dysimmunity, a combination of around 5-7 bacterial species in each type of sample could predict this severe phenotype with an accuracy of around 90% in the study population. Our study provides new insights into these previously unexplored but highly interrelated ecological niches among themselves and with patient profiles. Our data suggest that this disease-related systemic dysbiosis could be implicated in the immune dysregulation associated with severe cases of CVID.

Parvimonas micra can translocate from the subgingival sulcus of the human oral cavity to colorectal adenocarcinoma.

Oral and intestinal samples from a cohort of 93 colorectal cancer (CRC) patients and 30 healthy controls (non-CRC) were collected for microbiome analysis. Saliva (28 non-CRC and 94 CRC), feces (30 non-CRC and 97 CRC), subgingival fluid (20 CRC), and tumor tissue samples (20 CRC) were used for 16S metabarcoding and/or RNA sequencing (RNAseq) approaches. A differential analysis of the abundance, performed with the ANCOM-BC package, adjusting the P-values by the Holm-Bonferroni method, revealed that Parvimonas was significantly over-represented in feces from CRC patients (P-value < 0.001) compared to healthy controls. A total of 11 Parvimonas micra isolates were obtained from the oral cavity and adenocarcinoma of CRC patients. Genome analysis identified a pair of isolates from the same patient that shared 99.2% identity, demonstrating that P. micra can translocate from the subgingival cavity to the gut. The data suggest that P. micra could migrate in a synergistic consortium with other periodontal bacteria. Metatranscriptomics confirmed that oral bacteria were more active in tumor than in non-neoplastic tissues. We suggest that P. micra could be considered as a CRC biomarker detected in non-invasive samples such as feces.

Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis.

A few studies indicate that nitrate can reduce dysbiosis from a periodontitis point of view. However, these experiments were performed on samples from healthy individuals, and it is unknown if nitrate will be effective in periodontal patients, where the presence of nitrate-reducing bacteria is clearly reduced. The aim of this study was to test the effect of nitrate and a nitrate-reducing R. aeria (Ra9) on subgingival biofilms of patients with periodontitis. For this, subgingival plaque was incubated with 5 mM nitrate for 7 h (n = 20) or 50 mM nitrate for 12 h (n = 10), achieving a ~50% of nitrate reduction in each case. Additionally, Ra9 was combined with 5 mM nitrate (n = 11), increasing the nitrate reduced and nitrite produced (both p < 0.05). The addition of nitrate to periodontitis communities decreased biofilm mass (50 mM > 5 mM, both p < 0.05). Five millimolar nitrate, 50 mM nitrate and 5 mM nitrate + Ra9 led to 3, 28 and 20 significant changes in species abundance, respectively, which were mostly decreases in periodontitis-associated species. These changes led to a respective 15%, 63% (both p < 0.05) and 6% (not significant) decrease in the dysbiosis index. Using a 10-species biofilm model, decreases in periodontitis-associated species in the presence of nitrate were confirmed by qPCR (all p < 0.05). In conclusion, nitrate metabolism can reduce dysbiosis and biofilm growth of periodontitis communities. Five millimolar nitrate (which can be found in saliva after vegetable intake) was sufficient, while increasing this concentration to 50 mM (which could be achieved by topical applications such as a periodontal gel) increased the positive effects. Ra9 increased the nitrate metabolism of periodontitis communities and should be tested in vivo.

The bacteriology of diabetic foot ulcers and infections and incidence of Staphylococcus aureus Small Colony Variants.

Introduction. Uninfected diabetes-related foot ulcer (DFU) progression to diabetes-related foot infection (DFI) is a prevalent complication for patients with diabetes. DFI often progresses to osteomyelitis (DFI-OM). Active (growing) Staphylococcus aureus is the most common pathogen in these infections. There is relapse in 40-60 % of cases even when the initial treatment at the DFI stage apparently clears infection.Hypothesis. S. aureus adopts the quasi-dormant Small Colony Variant (SCV) state during DFU and consequently infection, and when present in DFI cases also permits survival in non-diseased tissues as a reservoir to cause relapse.Aim. The aim of this study was to investigate the bacterial factors that facilitate persistent infections.Methodology. People with diabetes were recruited from two tertiary hospitals. Clinical and bacterial data was taken from 153 patients with diabetes (51 from a control group with no ulcer or infection) and samples taken from 102 patients with foot complications to identify bacterial species and their variant colony types, and then compare the bacterial composition in those with uninfected DFU, DFI and those with DFI-OM, of whom samples were taken both from wounds (DFI-OM/W) and bone (DFI-OM/B). Intracellular, extracellular and proximal 'healthy' bone were examined.Results. S. aureus was identified as the most prevalent pathogen in diabetes-related foot pathologies (25 % of all samples). For patients where disease progressed from DFU to DFI-OM, S. aureus was isolated as a diversity of colony types, with increasing numbers of SCVs present. Intracellular (bone) SCVs were found, and even within uninfected bone SCVs were present. Wounds of 24 % of patients with uninfected DFU contained active S. aureus. All patients with a DFI with a wound but not bone infection had previously had S. aureus isolated from an infection (including amputation), representing a relapse.Conclusion. The presence of S. aureus SCVs in recalcitrant pathologies highlights their importance in persistent infections through the colonization of reservoirs, such as bone. The survival of these cells in intracellular bone is an important clinical finding supporting in vitro data. Also, there seems to be a link between the genetics of S. aureus found in deeper infections compared to those only found in DFU.

Novel bacterial proteolytic and metabolic activity associated with dental erosion-induced oral dysbiosis.

BACKGROUND: Dental erosion is a disease of the oral cavity where acids cause a loss of tooth enamel and is defined as having no bacterial involvement. The tooth surface is protected from acid attack by salivary proteins that make up the acquired enamel pellicle (AEP). Bacteria have been shown to readily degrade salivary proteins, and some of which are present in the AEP. This study aimed to explore the role of bacteria in dental erosion using a multi-omics approach by comparing saliva collected from participants with dental erosion and healthy controls. RESULTS: Salivary proteomics was assessed by liquid-chromatography mass spectrometry (LC-MS) and demonstrated two altered AEP proteins in erosion, prolactin inducible protein (PIP), and zinc-alpha-2 glycoprotein (ZAG). Immunoblotting further suggested that degradation of PIP and ZAG is associated with erosion. Salivary microbiome analysis was performed by sequencing the bacterial 16S rRNA gene (V1-V2 region, Illumina) and showed that participants with dental erosion had a significantly (p < 0.05) less diverse microbiome than healthy controls (observed and Shannon diversity). Sequencing of bacterial mRNA for gene expression (Illumina sequencing) demonstrated that genes over-expressed in saliva from erosion participants included H + proton transporter genes, and three protease genes (msrAB, vanY, and ppdC). Salivary metabolomics was assessed using nuclear magnetic resonance spectrometry (NMR). Metabolite concentrations correlated with gene expression, demonstrating that the dental erosion group had strong correlations between metabolites associated with protein degradation and amino acid fermentation. CONCLUSIONS: We conclude that microbial proteolysis of salivary proteins found in the protective acquired enamel pellicle strongly correlates with dental erosion, and we propose four novel microbial genes implicated in this process. Video Abstract.

Longitudinal changes in subgingival biofilm composition following periodontal treatment.

BACKGROUND: Current periodontal treatment involves instrumentation using hand and/or ultrasonic instruments, which are used either alone or in combination based on patient and clinician preference, with comparable clinical outcomes. This study sought to investigate early and later changes in the subgingival biofilm following periodontal treatment, to identify whether these changes were associated with treatment outcomes, and to investigate whether the biofilm responded differently to hand compared with ultrasonic instruments. METHODS: This was a secondary-outcome analysis of a randomized-controlled trial. Thirty-eight periodontitis patients received full-mouth subgingival instrumentation using hand (n = 20) or ultrasonic instrumentation (n = 18). Subgingival plaque was sampled at baseline and 1, 7, and 90 days following treatment. Bacterial DNA was analyzed using 16S rRNA sequencing. Periodontal clinical parameters were evaluated before and after treatment. RESULTS: Biofilm composition was comparable in both (hand and ultrasonics) treatment groups at all time points (all genera and species; p[adjusted] > 0.05). Large-scale changes were observed within groups across time points. At days 1 and 7, taxonomic diversity and dysbiosis were reduced, with an increase in health-associated genera including Streptococcus and Rothia equating to 30% to 40% of the relative abundance. When reassessed at day 90 a subset of samples reformed a microbiome more comparable with baseline, which was independent of instrumentation choice and residual disease. CONCLUSIONS: Hand and ultrasonic instruments induced comparable impacts on the subgingival plaque microbiome. There were marked early changes in the subgingival biofilm composition, although there was limited evidence that community shifts associated with treatment outcomes.

Subpopulations in Strains of Staphylococcus aureus Provide Antibiotic Tolerance.

The ability of Staphylococcus aureus to colonise different niches across the human body is linked to an adaptable metabolic capability, as well as its ability to persist within specific tissues despite adverse conditions. In many cases, as S. aureus proliferates within an anatomical niche, there is an associated pathology. The immune response, together with medical interventions such as antibiotics, often removes the S. aureus cells that are causing this disease. However, a common issue in S. aureus infections is a relapse of disease. Within infected tissue, S. aureus exists as a population of cells, and it adopts a diversity of cell types. In evolutionary biology, the concept of "bet-hedging" has established that even in positive conditions, there are members that arise within a population that would be present as non-beneficial, but if those conditions change, these traits could allow survival. For S. aureus, some of these cells within an infection have a reduced fitness, are not rapidly proliferating or are the cause of an active host response and disease, but these do remain even after the disease seems to have been cleared. This is true for persistence against immune responses but also as a continual presence in spite of antibiotic treatment. We propose that the constant arousal of suboptimal populations at any timepoint is a key strategy for S. aureus long-term infection and survival. Thus, understanding the molecular basis for this feature could be instrumental to combat persistent infections.

Metataxonomic and metaproteomic profiling of the oral microbiome in oral lichen planus - a pilot study.

Background: A growing body of evidence demonstrates a different bacterial composition in the oral cavity of patients with oral lichen planus (OLP). Patients and methods: Buccal swab samples were collected from affected and non-affected sites of six patients with reticular OLP and the healthy oral mucosa of six control subjects. 16S rRNA gene MiSeq sequencing and mass spectrometry-based proteomics were utilised to identify the metataxonomic and metaproteomic profiles of the oral microbiome in both groups. Results: From the metataxonomic analysis, the most abundant species in the three subgroups were Streptococcus oralis and Pseudomonas aeruginosa, accounting for up to 70% of the total population. Principal Coordinates Analysis showed differential clustering of samples from the healthy and OLP groups. ANCOM-BC compositional analysis revealed multiple species (including P. aeruginosa and several species of Veillonella, Prevotella, Streptococcus and Neisseria) significantly over-represented in the control group and several (including Granulicatella elegans, Gemella haemolysans and G. parahaemolysans) in patients with OLP. The metaproteomic data were generally congruent and revealed that several Gemella haemolysans-belonging peptidases and other proteins with inflammatory and virulence potential were present in OLP lesions. Conclusion: Our data suggest that several bacterial species are associated with OLP. Future studies with larger cohorts should be conducted to determine their role in the aetiology of OLP and evaluate their potential as disease biomarkers.

Personalized antibiotic selection in periodontal treatment improves clinical and microbiological outputs.

Introduction: Periodontitis is a biofilm-mediated disease that is usually treated by non-surgical biofilm elimination with or without antibiotics. Antibiotic treatment in periodontal patients is typically selected empirically or using qPCR or DNA hybridization methods. These approaches are directed towards establishing the levels of different periodontal pathogens in periodontal pockets to infer the antibiotic treatment. However, current methods are costly and do not consider the antibiotic susceptibility of the whole subgingival biofilm. Methods: In the current manuscript, we have developed a method to culture subgingival samples ex vivo in a fast, label-free impedance-based system where biofilm growth is monitored in real-time under exposure to different antibiotics, producing results in 4 hours. To test its efficacy, we performed a double-blind, randomized clinical trial where patients were treated with an antibiotic either selected by the hybridization method (n=32) or by the one with the best effect in the ex vivo growth system (n=32). Results: Antibiotic selection was different in over 80% of the cases. Clinical parameters such as periodontal pocket depth, attachment level, and bleeding upon probing improved in both groups. However, dental plaque was significantly reduced only in the group where antibiotics were selected according to the ex vivo growth. In addition, 16S rRNA sequencing showed a larger reduction in periodontal pathogens and a larger increase in health-associated bacteria in the ex vivo growth group. Discussion: The results of clinical and microbiological parameters, together with the reduced cost and low analysis time, support the use of the impedance system for improved individualized antibiotic selection.

Functional mgrA Influences Genetic Changes within a Staphylococcus aureus Cell Population over Time.

Prolonged survival in the host-bacteria microenvironment drives the selection of alternative cell types in Staphylococcus aureus, permitting quasi-dormant sub-populations to develop. These facilitate antibiotic tolerance, long-term growth, and relapse of infection. Small Colony Variants (SCV) are an important cell type associated with persistent infection but are difficult to study in vitro due to the instability of the phenotype and reversion to the normal cell type. We have previously reported that under conditions of growth in continuous culture over a prolonged culture time, SCVs dominated a heterogenous population of cell types and these SCVs harbored a mutation in the DNA binding domain of the gene for the transcription factor, mgrA. To investigate this specific cell type further, S. aureus WCH-SK2-ΔmgrA itself was assessed with continuous culture. Compared to the wild type, the mgrA mutant strain required fewer generations to select for SCVs. There was an increased rate of mutagenesis within the ΔmgrA strain compared to the wild type, which we postulate is the mechanism explaining the increased emergence of SCV selection. The mgrA derived SCVs had impeded metabolism, altered MIC to specific antibiotics and an increased biofilm formation compared to non-SCV strain. Whole genomic sequencing detected single nucleotide polymorphisms (SNP) in phosphoglucosamine mutase glmM and tyrosine recombinase xerC. In addition, several genomic rearrangements were detected which affected genes involved in important functions such as antibiotic and toxic metal resistance and pathogenicity. Thus, we propose a direct link between mgrA and the SCV phenotype. IMPORTANCE Within a bacterial population, a stochastically generated heterogeneity of phenotypes allows continual survival against current and future stressors. The generation of a sub-population of quasi-dormant Small Colony Variants (SCV) in Staphylococcus aureus is such a mechanism, allowing for persistent or relapse of infection despite initial intervention seemingly clearing the infection. The use of continuous culture under clinically relevant conditions has allowed us to introduce time to the growth system and selects SCV within the population. This study provides valuable insights into the generation of SCV which are not addressed in standard laboratory generated models and reveals new pathways for understanding persistent S. aureus infection which can potentially be targeted in future treatments of persistent S. aureus infection.

Functional changes in the oral microbiome after use of fluoride and arginine containing dentifrices: a metagenomic and metatranscriptomic study.

BACKGROUND: Tooth decay is one of the most prevalent diseases worldwide, and efficient tooth brushing with a fluoride-containing dentifrice is considered fundamental to caries prevention. Fluoride-containing dentifrices have been extensively studied in relation to enamel resistance to demineralization. Arginine (Arg) has also been proposed as a promising prebiotic to promote pH buffering through ammonia production. Here, we present the first metagenomic (DNA sequencing of the whole microbial community) and metatranscriptomic (RNAseq of the same community) analyses of human dental plaque to evaluate the effect of brushing with fluoride (Fl) and a Fl+Arg containing dentifrices on oral microbial composition and activity. Fifty-three patients were enrolled in a longitudinal clinical intervention study with two arms, including 26 caries-active and 27 caries-free adults. After a minimum 1-week washout period, dental plaque samples were collected at this post-washout baseline, 3 months after the use of a 1450-ppm fluoride dentifrice, and after 6 months of using a 1450-ppm fluoride with 1.5% arginine dentifrice. RESULTS: There was a shift in both the composition and activity of the plaque microbiome after 3 months of brushing with the fluoride-containing toothpaste compared to the samples collected at the 1-week post-washout period, both for caries-active and caries-free sites. Although several caries-associated bacteria were reduced, there was also an increase in several health- and periodontitis-associated bacteria. Over 400 genes changed proportion in the metagenome, and between 180 and 300 genes changed their expression level depending on whether caries-free or caries-active sites were analyzed. The metagenome and metatranscriptome also changed after the subjects brushed with the Fl+Arg dentifrice. There was a further decrease of both caries- and periodontitis-associated organisms. In both caries-free and caries-active sites, a decrease of genes from the arginine biosynthesis pathway was also observed, in addition to an increase in the expression of genes associated with the arginine deiminase pathway, which catabolizes arginine into ammonia, thereby buffering acidic pH. Bacterial richness and diversity were not affected by either of the two treatments in the two arms of the study. CONCLUSIONS: Our data demonstrate that long-term use of both assayed dentifrices changes the bacterial composition and functional profiles of human dental plaque towards a healthier microbial community, both in caries-free and caries-active sites. This observation was especially apparent for the Fl+Arg dentifrice. Thus, we conclude that the preventive benefits of tooth brushing go beyond the physical removal of dental plaque and that the active ingredients formulated within dentifrices have a positive effect not only on enamel chemistry but also on the metabolism of oral microbial populations. Video Abstract.

A Method of Transposon Insertion Sequencing in Comprehensively Identifying Vibrio vulnificus Genes Required for Growth in Human Serum.

One of the most powerful approaches to detect the loci that enable a pathogen to cause disease is the creation of a high-density transposon mutant library by transposon insertion sequencing (TIS) and the screening of the library using an adequate in vivo and/or ex vivo model of the disease. Here we describe the procedure for detection of the putative loci required for a septicemic pathogen to cause sepsis in humans by using TIS plus an ex vivo model of septicaemia: to grow the pathogen in fresh and inactivated human serum. We selected V. vulnificus because it is a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death in less than 24 h. To survive and proliferate in blood (or host serum), the pathogen requires mechanisms to overcome the innate immune defenses and metabolic limitations of this host niche. Initially, genes under-represented for insertions can be used to estimate the V. vulnificus essential gene set. Analysis of the relative abundance of insertion mutants in the library after exposure to serum would detect which genes are essential for the pathogen to overcome the diverse limitations imposed by serum.

Real-time monitoring of Pseudomonas aeruginosa biofilm growth dynamics and persister cells' eradication.

Biofilm formation and the appearance of persister cells with low metabolic rates are key factors affecting conventional treatment failure and antibiotic resistance. Using impedance-based measurements, crystal violet staining and traditional culture we have studied the biofilm growth dynamics of 13 Pseudomonas aeruginosa strains under the effect of seven conventional antibiotics. Real-time growth quantifications revealed that the exposure of established P. aeruginosa biofilms to certain concentrations of ciprofloxacin, ceftazidime and tobramycin induced the emergence of persister cells, that showed different morphology and pigmentation, as well increased antibiotic resistance. Whole-genome sequencing of wildtype and persister cells identified several SNPs, a genomic inversion and a genomic duplication in one of the strains. However, these mutations were not uniquely associated with persisters, suggesting that the persistent phenotype may be related to metabolic and transcriptional changes. Given that mannitol has been proposed to activate bacterial metabolism, the synergistic combination of mannitol and ciprofloxacin was evaluated on clinical 48 h P. aeruginosa biofilms. When administered at doses ≥320 mg/L, mannitol was capable of preventing persister cell formation by efficiently activating dormant bacteria and making them susceptible to the antibiotic. These results were confirmed using viable colony counting. As the tested ciprofloxacin-mannitol combination appeared to fully eradicate mature biofilms, we conclude that impedance-based biofilm diagnostics, which permits antibiotic susceptibility testing and the identification of persister cells, is of great potential for the clinical practice and could aid in establishing treatment breakpoints for emerging biofilm-related infections.

Bacterial Composition and Metabolomics of Dental Plaque From Adolescents.

Supragingival dental plaque samples were collected from 40 Swedish adolescents, including 20 with caries lesions (CAR) and 20 caries-free (CF). Fresh plaque samples were subjected to an ex vivo acid tolerance (AT) test where the proportion of bacteria resistant to an acid shock was evaluated through confocal microscopy and live/dead staining, and the metabolites produced were quantified by 1H Nuclear Magnetic Resonance (1H NMR). In addition, DNA was extracted and the 16S rRNA gene was sequenced by Illumina sequencing, in order to characterize bacterial composition in the same samples. There were no significant differences in AT scores between CAR and CF individuals. However, 7 out of the 10 individuals with highest AT scores belonged to the CAR group. Regarding bacterial composition, Abiotrophia, Prevotella and Veillonella were found at significantly higher levels in CAR individuals (p=0.0085, 0.026 and 0.04 respectively) and Rothia and Corynebacterium at significantly higher levels in CF individuals (p=0.026 and 0.003). The caries pathogen Streptococcus mutans was found at low frequencies and was absent in 60% of CAR individuals. Random-forest predictive models indicate that at least 4 bacterial species or 9 genera are needed to distinguish CAR from CF adolescents. The metabolomic profile obtained by NMR showed a significant clustering of organic acids with specific bacteria in CAR and/or high AT individuals, being Scardovia wiggsiae the species with strongest associations. A significant clustering of ethanol and isopropanol with health-associated bacteria such as Rothia or Corynebacterium was also found. Accordingly, several relationships involving these compounds like the Ethanol : Lactate or Succinate : Lactate ratios were significantly associated to acid tolerance and could be of predictive value for caries risk. We therefore propose that future caries risk studies would benefit from considering not only the use of multiple organisms as potential microbial biomarkers, but also their functional adaptation and metabolic output.

Draft Genome Sequences of Vibrio vulnificus Strains Recovered from Moribund Tilapia.

Potentially zoonotic Vibrio vulnificus strains were isolated from vibriosis outbreaks occurring on eastern Mediterranean tilapia farms between 2016 and 2019. In this work, the draft genome sequences of three representative isolates are presented.

Microbiology of molar-incisor hypomineralization lesions. A pilot study.

Objective: An insufficient mineralization (hypomineralization) in the teeth during the maturation stage of amelogenesis cause defects in 3-44% of children. Here, we describe for the first time the microbiota associated with these defects and compared it to healthy teeth within the same subjects. Methods: Supragingival dental plaque was sampled from healthy and affected teeth from 25 children with molar-incisor hypomineralization (MIH). Total DNA was extracted and the 16S rRNA gene was sequenced by Illumina sequencing in order to describe the bacterial composition. Results: We detected a higher bacterial diversity in MIH samples, suggesting better bacterial adhesion or higher number of niches in those surfaces. We found the genera Catonella, Fusobacterium, Campylobacter, Tannerella, Centipeda, Streptobacillus, Alloprevotella and Selenomonas associated with hypomineralized teeth, whereas Rothia and Lautropia were associated with healthy sites. Conclusion: The higher protein content of MIH-affected teeth could favour colonization by proteolytic microorganisms. The over-representation of bacteria associated with endodontic infections and periodontal pathologies suggests that, in addition to promote caries development, MIH could increase the risk of other oral diseases.