Whole microbial community viability is not quantitatively reflected by propidium monoazide sequencing approach.

BACKGROUND: High-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g., qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting "viable" communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark "PMA-seq" (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities. RESULTS: PMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis. However, in realistically complex communities (computer screens, computer mice, soil, and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability (even relative to variability in amplicon sequencing), with its performance largely affected by community properties such as initial biomass, sample types, and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the "PMA-responsive" microbes, we compared our results with previous PMA-based studies and found that PMA responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies. CONCLUSIONS: This study provides a comprehensive evaluation of PMA-seq exploring its quantitative potential in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities but provided only qualitative assessments in realistically complex community samples. Video abstract.

[Creation of a molecular genetic test system for early diagnosis and evaluation of the effectiveness of treatment of inflammatory periodontal diseases.]

Inflammatory periodontal diseases represent a serious dental and general medical problem due to the high prevalence among the adult population, the presence of clinical forms leading to the destruction of the dentition and tooth loss, insufficient treatment effectiveness and the frequency of relapse, including in connection with the formation of biofilms. A molecular genetic test system has been developed to evaluate the content of periodontopathogenic microorganisms Porphyromonas gingivalis, Treponema denticola, Streptococcus oralis, Streptococcus sanguis and Streptococcus sobrinus in the contents of periodontal pockets. The analytical characteristics of the test system were determined, and testing was carried out on clinical samples of patients with chronic generalized periodontitis of moderate severity. The constructed diagnostic kit allowed us to conduct a comparative analysis of the effectiveness of various types of treatment of inflammatory periodontal diseases based on quantitative data on the content of bacteria in the contents of periodontal pockets.

Rapid and Accurate Species Identification of Mitis Group Streptococci Using the MinION Nanopore Sequencer.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner "What's in My Pot?" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

Similar genomic patterns of clinical infective endocarditis and oral isolates of Streptococcus sanguinis and Streptococcus gordonii.

Streptococcus gordonii and Streptococcus sanguinis belong to the Mitis group streptococci, which mostly are commensals in the human oral cavity. Though they are oral commensals, they can escape their niche and cause infective endocarditis, a severe infection with high mortality. Several virulence factors important for the development of infective endocarditis have been described in these two species. However, the background for how the commensal bacteria, in some cases, become pathogenic is still not known. To gain a greater understanding of the mechanisms of the pathogenic potential, we performed a comparative analysis of 38 blood culture strains, S. sanguinis (n = 20) and S. gordonii (n = 18) from patients with verified infective endocarditis, along with 21 publicly available oral isolates from healthy individuals, S. sanguinis (n = 12) and S. gordonii (n = 9). Using whole genome sequencing data of the 59 streptococci genomes, functional profiles were constructed, using protein domain predictions based on the translated genes. These functional profiles were used for clustering, phylogenetics and machine learning. A clear separation could be made between the two species. No clear differences between oral isolates and clinical infective endocarditis isolates were found in any of the 675 translated core-genes. Additionally, random forest-based machine learning and clustering of the pan-genome data as well as amino acid variations in the core-genome could not separate the clinical and oral isolates. A total of 151 different virulence genes was identified in the 59 genomes. Among these homologs of genes important for adhesion and evasion of the immune system were found in all of the strains. Based on the functional profiles and virulence gene content of the genomes, we believe that all analysed strains had the ability to become pathogenic.

Biofilm formation on polyetheretherketone and titanium surfaces.

Objective: Polyetheretherketone (PEEK) is a polymer used in devices in orthopedic and dental rehabilitation. The aim of this in vitro study was to compare biofilm formation by a range of important oral bacterial species on PEEK, blasted PEEK, commercially pure titanium (cp-Ti), and titanium-6 aluminium-4 vanadium (Ti6Al4V). Material and methods: Coin-shaped samples were manufactured, and the surfaces were characterized using optical interferometry, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and contact angle measurements. Bacterial species of Streptococcus sanguinis, Streptococcus oralis, Enterococcus faecalis, and Streptococcus gordonii were cultured on the four material surfaces for varying amounts of time. Biofilms were quantified following staining with crystal violet. Results: Roughness and contact angle results showed blasted PEEK > PEEK > cp-Ti = Ti6Al4V. There was increased biofilm formation on blasted PEEK by S. sanguinis, S. oralis, and S. gordonii, whereas the bacterial adhesion was similar on PEEK, cp-Ti, and Ti6Al4V. The bacterial growth of E. faecalis was significantly higher on cp-Ti compared with the other three groups. Conclusion: The results, taking into consideration the biofilm formation, suggest that PEEK should perform as well as cp-Ti or TiAl6V4 when used as a dental restorative material.

High-resolution novel method for tracking bacteria in a multi-species biofilm.

The aim of this study is to establish a novel high resolution tracking ability of a specific bacterium in multispecies biofilm. A periodontal multispecies biofilm was constructed with Streptococcus sanguis, Actinomyces naeslundii, Porphyromonas gingivalis and Fusobacterium nucleatum. A single species was stained with fluorescein isothiocyanate (FITC). The mature biofilm was stained for viability (propidium iodide) and analysis was performed with flow cytometry. The sensitivity of the assay was compared with colony forming units (CFU) counts. A single cell suspension of P. gingivalis was grown in broth and biofilm to identify the location of these events on side scatter and forward scatter. The sensitivity of the assay was comparable to that of the CFU counts. The assay allows quantification of the ratio of a single bacterium within the biofilm, and its viable proportion. The described method is reproducible and of high resolution, and allows the examination of microbes' composition and viability within a biofilm structure.

The prevalence rate of periodontal pathogens and its association with oral squamous cell carcinoma.

Mounting evidence suggests a causal relationship between specific bacterial infections or microbial compositions and the development of certain malignant neoplasms. In this study, we performed research through 16S rRNA amplicon sequencing, qPCR and fluorescence in situ hybridization to certify the relationship between periodontal pathogens and oral squamous cell carcinoma (OSCC). Subgingival plaque, cancer and paracancerous tissues from 6 patients with OSCC were selected for mapping bacterial profiles by 16S rRNA amplicon sequencing. The research showed that periodontal pathogens were enriched in cancer and paracancerous tissues, while the bacterial profiles were similar between the cancer tissues and subgingival plaque. Furthermore, the relative abundance of Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus sanguinis was detected in 61 cancer tissues, paracancerous tissues and subgingival plaque samples and in 30 normal tissues by qPCR. The results revealed that P. gingivalis and F. nucleatum existed at higher levels in cancer tissue than in normal tissues and were correlated with subgingival plaques. P. gingivalis was detected using a special oligonucleotide probe in 60.7% of OSCC tissues, 32.8% of paracancerous tissues and 13.3% of normal tissues. Relevance analysis showed that P. gingivalis infection was positively associated with late clinical staging, low differentiation and lymph node metastasis in patients with OSCC, which was accompanied by deeper periodontal pockets, severe clinical attachment loss and loss of teeth. This study revealed that there might be a close relationship between oral microorganisms, particularly periodontal pathogens, and OSCC, which might enrich the pathogenesis of oral squamous carcinoma.

Genomic, Phenotypic, and Virulence Analysis of Streptococcus sanguinis Oral and Infective-Endocarditis Isolates.

Streptococcus sanguinis, an abundant and benign inhabitant of the oral cavity, is an important etiologic agent of infective endocarditis (IE), particularly in people with predisposing cardiac valvular damage. Although commonly isolated from patients with IE, little is known about the factors that make any particular S. sanguinis isolate more virulent than another or, indeed, whether significant differences in virulence exist among isolates. In this study, we compared the genomes of a collection of S. sanguinis strains comprised of both oral isolates and bloodstream isolates from patients diagnosed with IE. Oral and IE isolates could not be distinguished by phylogenetic analyses, and we did not succeed in identifying virulence genes unique to the IE strains. We then investigated the virulence of these strains in a rabbit model of IE using a variation of the Bar-seq (barcode sequencing) method wherein we pooled the strains and used Illumina sequencing to count unique barcodes that had been inserted into each isolate at a conserved intergenic region. After we determined that several of the genome sequences were misidentified in GenBank, our virulence results were used to inform our bioinformatic analyses, identifying genes that may explain the heterogeneity in virulence. We further characterized these strains by assaying for phenotypes potentially contributing to virulence. Neither strain competition via bacteriocin production nor biofilm formation showed any apparent relationship with virulence. Increased cell-associated manganese was, however, correlated with blood isolates. These results, combined with additional phenotypic assays, suggest that S. sanguinis virulence is highly variable and results from multiple genetic factors.

Salivary levels of Streptococcus mutans and Streptococcus sanguinis in early childhood caries: An in vivo study.

BACKGROUND: The oral microflora primarily involved in early childhood caries (ECC) is Streptococcus mutans while Streptococcus sanguinis is related to healthy caries-free mouth. The antagonism between the two at biological level is well recognized. AIM: This study aims to compare the pre- and posttreatment salivary levels of S. mutans and S. sanguinis in ECC. METHODS: This cross-sectional study was performed on 40 eligible participants. Clinical examination was done, and decayed teeth were recorded. Pre- and Posttreatment saliva sample was taken, processed and bacterial counts were compared. RESULTS: A statistically significant posttreatment reduction was found in S. mutans counts; however, a significant posttreatment elevation was seen in S. sanguinis counts (P < 0.001) using Wilcoxon signed-rank test. A significant weak positive correlation was found between S. mutans and decayed teeth in the mouth (r = 0.366 and P = 0.028) using Spearman's correlation. No significant gender association was found between pre- and posttreatment S. mutans and S. sanguinis counts (P = 0.908) and also between decayed teeth (P = 0.321) using Mann-Whitney U-test. CONCLUSION: Complete dental treatment procedure showed significant reduction and elevation in S. mutans and S. sanguinis counts, respectively. A positive association was observed between S. mutans and ECC while S. sanguinis were positively associated with posttreatment oral environment.

Patient with native valve infective endocarditis and concomitant bacterial myopericarditis.

A 39-year-old man with known mitral regurgitation (MR) presented with chest pain, nausea and dizziness. Troponin of 5801 ng/L and scooped ST segments indicated myopericarditis. Cardiac MRI demonstrated an epicardial late gadolinium enhancement pattern consistent with a significantly myocarditic syndrome. Initially afebrile, the patient reported fevers a week earlier when abroad where he received amoxicillin.The patient then began spiking temperatures and infective endocarditis (IE) was confirmed following blood cultures positive for Streptococcus sanguinis and Transoesophageal echocardiography (TOE) showing a vegetation on the anterior mitral valve leaflet. Patient underwent 6 weeks of intravenous benzylpenicillin and on resolution he was discharged to await valve surgery.A model is proposed where septic embolism from IE caused bacterial myopericarditis, triggering the initial presenting complaint. It is suggested that prior antibiotic therapy and paracetamol suppressed the systemic symptoms of IE.

Second look at Streptococcus sanguinis and the colon.

Although the link between Streptococcus bovis bacteraemia and colon cancer is well established, bacteraemia from other viridans group streptococci that commonly colonise colonic mucosa may also herald occult malignancy. We present a case of Streptococcus sanguinis bacteraemia in an elderly man with new anaemia that led to the detection and removal of a high-grade colon neoplasm. This case contributes to a growing body of literature contending that unexplained streptococcal bacteraemia merits a thorough workup that may include relatively invasive procedures such as endoscopy. Diagnostic colonoscopy provides an opportunity to prevent invasive malignancy that may outweigh bleeding and perforation risks in elderly patients.

Buckyballs conjugated with nucleic acid sequences identifies microorganisms in live cell assays.

BACKGROUND: Rapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system. METHODS: C60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy. RESULTS: The C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture. CONCLUSIONS: The technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.

Microbiomes of Site-Specific Dental Plaques from Children with Different Caries Status.

The oral microbiota associated with the initiation and progression of dental caries has yet to be fully characterized. The Human Oral Microbe Identification Using Next-Generation Sequencing (HOMINGS) approach was used to analyze the microbiomes of site-specific supragingival dental plaques from children with different caries status. Fifty-five children (2 to 7 years of age) were assessed at baseline and at 12 months and grouped as caries free (CF), caries active with enamel lesions (CAE), and caries active with dentin carious lesions (CA). Plaque samples from caries-free tooth surfaces (PF) and from enamel carious lesions (PE) and dentin carious lesions (PD) were collected. 16S community profiles were obtained by HOMINGS, and 408 bacterial species and 84 genus probes were assigned. Plaque bacterial communities showed temporal stability, as there was no significant difference in beta diversity values between the baseline and 12-month samples. Irrespective of collection time points, the microbiomes of healthy tooth surfaces differed substantially from those found during caries activity. All pairwise comparisons of beta diversity values between groups were significantly different (P < 0.05), except for comparisons between the CA-PF, CAE-PE, and CA-PE groups. Streptococcus genus probe 4 and Neisseria genus probe 2 were the most frequently detected taxa across the plaque groups, followed by Streptococcus sanguinis, which was highly abundant in CF-PF. Well-known acidogenic/aciduric species such as Streptococcus mutans, Scardovia wiggsiae, Parascardovia denticolens, and Lactobacillus salivarius were found almost exclusively in CA-PD. The microbiomes of supragingival dental plaque differ substantially among tooth surfaces and children of different caries activities. In support of the ecological nature of caries etiology, a steady transition in community species composition was observed with disease progression.

The MiiA motif is a common marker present in polytopic surface proteins of oral and urinary tract invasive bacteria.

Many surface virulence factors of bacterial pathogens show mosaicism and confounding phylogenetic origin. The Streptococcus gordonii platelet-binding GspB protein, the Streptococcus sanguinis SrpA adhesin and the Streptococcus pneumoniae DiiA protein, share an imperfect 27-residue motif. Given the disparate domain architectures of these proteins and its association to invasive disease, this motif was named MiiA from Multiarchitecture invasion-involved motif A. MiiA is predicted to adopt a beta-sheet folding, probably related to the Ig-like fold, with a symmetrical positioning of two conserved aspartic residues. A specific hidden Markov model profiling MiiA was built, which specifically detected the motif in proteins from 58 species, mainly in cell-wall proteins from Gram-positive bacteria. These proteins contained one to ten MiiA motifs, which were embedded within larger repeat units of 70-82 residues. MiiA motifs combined to other domains and elements such as coiled-coils and low-complexity regions. The species carrying MiiA-proteins included commensals from the urogenital tract and the oral cavity, which can cause opportunistic endocarditis and sepsis. Intra-protein MiiA repeats showed a complex mixture of orthologal, paralogal and inter-species relationships, suggestive of a multistep origin. Presence of these repeats in proteins involved in oligosaccharide recognition and lifestyle of species suggest a putative function for MiiA repeats in sugars binding, probably those present in receptors of epithelial and blood cells. MiiA modules appear to have been transferred horizontally between species co-habiting in the same niche to create their own MiiA-containing determinants. The present work provides a global study and a catalog of potential MiiA virulence factors that should be analyzed experimentally.

Bacterial invasion into radicular dentine-an in vitro study.

OBJECTIVES: We wanted to investigate differences in invasiveness into radicular dentinal tubules by monocultured and co-cultured bacteria frequently found in infected root canals. METHODS: Fifty-one human roots were incubated for 8 weeks with monocultured Streptococcus gordonii ATCC 10558, Streptococcus sanguinis ATCC 10556, and with five capnophiles/anaerobes as well as with capnophiles/anaerobes co-cultured with a streptococcal species. Thereafter, bacterial samples were cultured from the inner, middle, and outer third of the root dentine of longitudinally broken teeth (n = 5). In addition, scanning electron microscopy (SEM) images were obtained. RESULTS: Single gram-positive species were able to penetrate into the middle and outer third of the root dentine. Fusobacterium nucleatum ATCC 25586 was not found in any of the dentine specimens. Prevotella intermedia ATCC 25611 and Porphyromonas gingivalis ATCC 33277 were found in the inner and middle third. The bacterial load of streptococci was higher in all thirds in co-cultures compared to single infections. In co-cultures with streptococci, Actinomyces oris ATCC 43146 was found in the outer third in 9/10 samples, whereas P. intermedia ATCC 25611 was not detectable inside dentine. Co-culture with S. sanguinis ATCC 10556 enabled F. nucleatum ATCC 25586 to invade dentine; SEM images showed that F. nucleatum ATCC 25586 had a swollen shape. CONCLUSIONS: Invasiveness of bacteria into dentinal tubules is species-specific and may change depending on culturing as a single species or co-culturing with other bacteria. CLINICAL RELEVANCE: Oral streptococci may promote or inhibit invasion of capnophiles/anaerobes into radicular dentine.

Streptococcus sanguinis isolated from filler granuloma: Successful treatment with incision and drainage.

Filler granuloma is considered to be the result of delayed immune responses; growing evidence suggests that they may be secondary to biofilm formation. Dermal filler is technically a foreign body, and as the development of newer generations of dermal fillers lengthens their duration, it is possible that there is also an increased risk of biofilm formation. Here, we present a case report of a patient with Streptococcus sanguinis isolated from a filler granuloma, suggestive of biofilm formation. This case demonstrates the effective use of antibiotics after incision and drainage on antibiotic resistant biofilm.

Impedimetric antimicrobial peptide-based sensor for the early detection of periodontopathogenic bacteria.

Peri-implantitis, an inflammation caused by biofilm formation, constitutes a major cause of implant failure in dentistry. Thus, the detection of bacteria at the early steps of biofilm growth represents a powerful strategy to prevent implant-related infections. In this regard, antimicrobial peptides (AMPs) can be used as effective biological recognition elements to selectively detect the presence of bacteria. Thus, the aim of the present study was to combine the use of miniaturized and integrated impedimetric transducers and AMPs to obtain biosensors with high sensitivity to monitor bacterial colonization. Streptococcus sanguinis, which is one of the most prevalent strains in the onset of periodontal diseases, was used as a model of oral bacteria. To this end, a potent AMP derived from human lactoferrin was synthesized and covalently immobilized on interdigitated electrode arrays (IDEA). X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS) were employed to optimize and characterize the method of immobilization. Noteworthy, the interaction of Streptococcus sanguinis with AMP-coated sensors provoked significant changes in the impedance spectra, which were univocally associated with the presence of bacteria, proving the feasibility of our method. In this regard, the developed biosensor permits to detect the presence of bacteria at concentrations starting from 10(1) colony forming units (CFU)mL(-1) in KCl and from 10(2) CFUmL(-1) in artificial saliva. Moreover, the system was devoid of cytotoxicity for human fibroblasts. These results indicate that the proposed approach can be effective in the detection of initial stages of biofilm formation, and may be useful in the early prevention and treatment of peri-implantitis.

A New Experimental Design for Bacterial Microleakage Investigation at the Implant-Abutment Interface: An In Vitro Study.

PURPOSE: This study aimed to test bacterial microleakage at the implant-abutment interface (IAI) before and after dynamic loading using a new chewing simulation. MATERIALS AND METHODS: Fourteen implant systems (n = 5 samples of each) were divided into two groups: (1) systems with conical implant-abutment connections (IACs), and (2) systems with flat IACs. For collecting samples without abutment disconnection, channels (Ø = 0.3 mm) were drilled into implants perpendicularly to their axes, and stainless-steel cannulas were adhesively glued inside these channels to allow a sterilized rinsing solution to enter the implant interior and to exit with potential contaminants for testing. Implants were embedded in epoxy resin matrices, which were supported by titanium cylinders with lateral openings for inward and outward cannulas. Abutments were tightened and then provided with vertically adjustable, threaded titanium balls, which were cemented using composite cement. Specimens were immersed in a bacterial liquid and after a contact time of 15 minutes, the implant interior was rinsed prior to chewing simulation (0 N ≘ static seal testing). Specimens were exposed to a Frankfurt chewing simulator. Two hundred twenty force cycles per power level (110 in ± X-axis) were applied to simulate a daily masticatory load of 660 chewing cycles (equivalent to 1,200,000 cycles/5 years). The applied load was gradually increased from 0 N to a maximum load of 200 N in 25-N increments. The implant interior was rinsed to obtain samples before each new power level. All samples were tested using fluorescence microscopy; invading microorganisms could be counted and evaluated. RESULTS: No bacterial contamination was detected under static loading conditions in both groups. After loading, bacterial contamination was detected in one sample from one specimen in group 1 and in two samples from two specimens in group 2. CONCLUSION: Controlled dynamic loading applied in this study simulated a clinical situation and enabled time-dependent analysis regarding the bacterial seal of different implant systems. Conical IACs offer a better bacterial seal compared with flat IACs, which showed increased microleakage after dynamic loading. IAC design plays a crucial role in terms of bacterial colonization. Taking samples of the implant interior without abutment disconnection eliminates an error source.