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Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

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We herein report an 81-year-old woman with no significant medical history who developed a fever, headache, and right eyelid swelling. Magnetic resonance imaging (MRI) showed eye proptosis, sphenoid opacity, enlarged cavernous sinus, and dilated right superior ophthalmic vein (SOV). Subsequent enhanced MRI revealed intraventricular debris and thrombosis in the right SOV and the left transverse and sigmoid sinuses. Blood cultures were positive for Aggregatibacter aphrophilus, as identified by mass spectrometry. The patient responded well to antibiotics, anticoagulants, and surgical drainage of sphenoid sinusitis. To our knowledge, this is the first case of A. aphirophilus sphenoid sinusitis causing orbital cellulitis, meningitis, and venous sinus thrombosis.

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To assess and compare the anti-microbial efficacy of 445 nm and 970 nm diode laser on mixed species biofilm of Aggregatibacter actinomycetemcomitans [A.a] and Porphyromonas gingivalis [P.g] cultured on machined pure titanium discs. A total of 65 machined pure titanium discs with no surface modifications with a 10-mm diameter and a 2-mm height were sterilized by autoclaving at 121 °C for 15 min and incubated with the commercially available bacterial strains ATCC(American Type Culture Collection- P.g 33277 and A.a 29522)mixture of Aggregatibacter actinomycetemcomitans(A.a) and Porphyromonas gingivalis(P.g).After a 2-week incubation period with the mixture of bacteria to develop a mixed species biofilm, the discs were divided into three groups: (1) no treatment (control), (2) 445 nm laser (test), (3) 970 nm laser (test). For each laser wavelength (445 and 970 nm), the discs were exposed to 1.0 W and 2.0 W in continuous wave mode for the times points of 15, 30, and 60 s. The antimicrobial efficacy was assessed by qPCR. A significant reduction in the levels of both species of bacteria was observed between control and the laser intervention groups. A higher efficacy for the 445 nm diode laser against Porphyromonas gingivalis and a similar efficacy against Aggregatibacter actinomycetemcomitans was observed as compared to the 970 nm group. 445 nm wavelength represents a potential and effective laser wavelength which can be used for the management of peri-implant infection. The present study findings also need to be further validated through clinical interventional trials.

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We report a case of a 16-year-old male with tricuspid valve infective endocarditis caused by Aggregatibacter aphrophilus and complicated by pulmonary septic embolisms. Multiple antimicrobial therapy was unsuccessful and surgical management was required. In this report, the authors highlight the importance of a high index of suspicion regarding the diagnosis of endocarditis and its possible complications.

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Antiseptics are widely used in oral healthcare to prevent or treat oral diseases, such as gingivitis and periodontitis. However, the incidence of bacteria being tolerant to standard antiseptics has sharply increased over the last few years. This stresses the urgency for surveillance against tolerant organisms, as well as the discovery of novel antimicrobials. Traditionally, susceptibility to antimicrobials is assessed by broth micro-dilution or disk diffusion assays, both of which are time-consuming, labor-intensive, and provide limited information on the mode of action of the antimicrobials. The abovementioned limitations highlight the need for the development of new methods to monitor and further understand antimicrobial susceptibility. In this study, we used real-time flow cytometry, combined with membrane permeability staining, as a quick and sensitive technology to study the quantitative and qualitative responses of two oral pathobionts to different concentrations of chlorhexidine (CHX), cetylpyridinium chloride (CPC), or triclosan. Apart from the real-time monitoring of cell damage, we further applied a phenotypic fingerprinting method to differentiate between the bacterial subpopulations that arose due to treatment. We quantified the pathobiont damage rate of different antiseptics at different concentrations within 15 minutes of exposure and identified the conditions under which the bacteria were most susceptible. Moreover, we detected species-specific and treatment-specific phenotypic subpopulations. This proves that real-time flow cytometry can provide information on the susceptibility of different microorganisms in a short time frame while differentiating between antiseptics and thus could be a valuable tool in the discovery of novel antimicrobial compound, while at the same time deciphering their mode of action. IMPORTANCE: With increasing evidence that microorganisms are becoming more tolerant to standard antimicrobials, faster and more accessible antimicrobial susceptibility testing methods are needed. However, traditional susceptibility assays are laborious and time-consuming. To overcome the abovementioned limitations, we introduce a novel approach to define antimicrobial susceptibility in a much shorter time frame with the use of real-time flow cytometry. Furthermore, phenotypic fingerprinting analysis can be applied on the data to study the way antiseptics affect the bacterial cell morphology over time and, thus, gain information on the mode of action of a certain compound.

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Aggregatibacter actinomycetemcomitans (A. actinomycetecomitans) is a Gram-negative bacterial species that is an essential component of the oral microbiota. Due to its aggregative properties, it plays a role in the pathogenesis of human diseases. The presence of the surface proteins Fim, Briae, and microvesicles enables the bacterium to adhere to the epithelial surface and the tooth's surface. The presence of leukotoxin A (LtxA), which plays an important role in the pathogenicity of the bacterium, has been associated with both periodontitis and the etiology of rheumatoid arthritis (RA). A. actinomycetecomitans is also associated with several other systemic diseases and complications, such as endocarditis and different abscesses. In addition to leukotoxin A, A. actinomycetecomitans possesses several different virulence factors, including bacteriocins, chemotaxis inhibitory factors, cytotoxic factors, Fc-binding proteins, immunosuppressive factors, lipopolysaccharide collagenase, fibroblast inhibitory factors, antibiotic resistance determinants, adhesins, invasive factors and factors that inhibit the function of polymorphonuclear leukocytes. The ability of A. actinomycetemcomitans lipopolysaccharide to induce macrophages to secrete the interleukins IL-1, IL-1ß, and tumor necrosis factor (TNF) is of considerable importance. The primary etiologic factor in the pathogenesis of periodontal disease is the oral biofilm colonized by anaerobic bacteria. Among these, A. actinomycetemcomitans occupies an important place as a facultative anaerobic bacterium. In addition, A. actinomycetemcomitans possesses many virulence factors that contribute to its potential to cause cancer. This article provides an overview of the virulence factors of A. actinomycetecomitans and its association with various systemic diseases, its oncogenic potential, and the treatment options for infections caused by A. actinomycetecomitans.

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Aggressive forms of periodontitis, especially in young patients, are often associated with an increased proportion of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans of the microbiota of the affected periodontal sites. One of the virulence factors of A. actinomycetemcomitans is a leukotoxin (LtxA) that induces a pro-inflammatory cell death process in leukocytes. A. actinomycetemcomitans exhibits a large genetic diversity and different genotypes vary in LtxA production capacity. The genotype JP2 is a heavy LtxA producer due to a 530-base pair deletion in the promoter for the toxin genes, and this trait has been associated with an increased pathogenic potential. The present study focused on the production and release of LtxA by different A. actinomycetemcomitans genotypes and serotypes under various growth conditions. Four different strains of this bacterium were cultured in two different culture broths, and the amount of LtxA bound to the bacterial surface or released into the broths was determined. The cultures were examined during the logarithmic and the early stationary phases of growth. The JP2 genotype exhibited the highest LtxA production among the strains tested, and production was not affected by the growth phase. The opposite was observed with the other strains. The composition of the culture broth had no effect on the growth pattern of the tested strains. However, the abundant release of LtxA from the bacterial surface into the culture broth was found in the presence of horse serum. Besides confirming the enhanced leucotoxicity of the JP2 genotype, the study provides new data on LtxA production in the logarithmic and stationary phases of growth and the effect of media composition on the release of the toxin from the bacterial membrane.

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Resumen El absceso cerebral es un proceso supurativo focal producido en la mayoría de los casos por agentes bacte rianos. Aggregatibacter aphrophilus es una bacteria gram negativa perteneciente al grupo HACEK, causante de endocarditis infecciosa, abscesos hepáticos, entre otras. Los abscesos cerebrales secundarios a este germen son infrecuentes y en la mayoría de los casos asociados a contactos con animales domésticos, pobre higiene dental o procedimientos odontológicos. El tratamiento consiste en drenaje del absceso (mayores de 2.5 cm) combinado con terapia antibiótica, idealmente betalactámicos. Se presenta el caso de un paciente varón de 64 años sin antecedentes de relevancia quien ingresó al servicio de emergencias por cuadro de cefalea, hemianopsias de una semana de evolución y posteriormente crisis tónico clónicas, en quien posteriormente en estudios imageno lógicos y cultivo de lesión cerebral se arribó al diagnóstico de absceso cerebral por A. aphrophilus. Este informe tiene como objetivo ilustrar al lector sobre la rareza de esta infección, debido a que A. aphrophilus forma parte normal de la flora orofaríngea y del tracto respiratorio, en los que rara vez ocasiona bacteriemias invasivas.

Abstract Brain abscess is a focal suppurative process pro duced in most cases by bacterial agents. Aggregati bacter aphrophilus is a gram-negative bacteria belong ing to the HACEK group, which causes infective endo carditis, liver abscesses, among others. Brain abscesses secondary to this germ are rare and, in most cases, it is associated with contact with pets, poor dental hygiene or dental procedures. Treatment consists of drainage of the abscess (greater than 2.5 cm) combined with antibiotic therapy, ideally beta-lactams. The case of a 64-year-old male patient with no relevant history is here presented. He was admitted to the emergency service due to headache, hemianopsia of a week's duration and later tonic-clonic seizures, in whom imaging studies and culture of a brain lesion subse quently revealed a brain abscess due to A. aphrophilus. This case aims to illustrate about the rarity of this infection, because A. aphrophilus is a normal part of the oropharyngeal flora and respiratory tract, in which it rarely causes invasive bacteremia.

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AIM: Evidence suggests that translocation of oral pathogens through the oral-gut axis may induce intestinal dysbiosis. This study aimed to evaluate the impact of a highly leukotoxic Aggregatibacter actinomycetemcomitans (Aa) strain on the gut microbiota, intestinal mucosal integrity and immune system in healthy mice. METHODS: Eight-week-old male C57BL6 mice were divided into control (n = 16) and JP2 groups (n = 19), which received intragastric gavage with PBS and with a suspension of Aa JP2 (HK921), respectively, twice a week for 4 weeks. Colonic lamina propria, fecal material, serum, gingival tissues, and mandibles were obtained for analyses of leukocyte populations, inflammatory mediators, mucosal integrity, alveolar bone loss, and gut microbiota. Differences between groups for these parameters were examined by non-parametric tests. RESULTS: The gut microbial richness and the number of colonic macrophages, neutrophils, and monocytes were significantly lower in Aa JP2-infected mice than in controls (p < .05). In contrast, infected animals showed higher abundance of Clostridiaceae, Lactobacillus taiwanensis, Helicobacter rodentium, higher levels of IL-6 expression in colonic tissues, and higher splenic MPO activity than controls (p < .05). No differences in tight junction expression, serum endotoxin levels, and colonic inflammatory cytokines were observed between groups. Infected animals presented also slightly more alveolar bone loss and gingival IL-6 levels than controls (p < .05). CONCLUSION: Based on this model, intragastric administration of Aa JP2 is associated with changes in the gut ecosystem of healthy hosts, characterized by less live/recruited myeloid cells, enrichment of the gut microbiota with pathobionts and decrease in commensals. Negligible levels of colonic pro-inflammatory cytokines, and no signs of mucosal barrier disruption were related to these changes.

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Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.

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Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is capable of intoxicating lymphocytes macrophages, mast cells and epithelial cells. Following Cdt binding to cholesterol, in the region of membrane lipid rafts, the CdtB and CdtC subunits are internalized and traffic to intracellular compartments. These events are dependent upon, cellugyrin, a critical component of synaptic like microvesicles (SLMVCg+). Target cells, such as Jurkat cells, rendered unable to express cellugyrin are resistant to Cdt-induced toxicity. Similar to Cdt, SARS-CoV-2 entry into host cells is initiated by binding to cell surface receptors, ACE-2, also associated with cholesterol-rich lipid rafts; this association leads to fusion and/or endocytosis of viral and host cell membranes and intracellular trafficking. The similarity in internalization pathways for both Cdt and SARS-CoV-2 led us to consider the possibility that cellugyrin was a critical component in both processes. Cellugyrin deficient Calu-3 cells (Calu-3Cg-) were prepared using Lentiviral particles containing shRNA; these cells were resistant to infection by VSV/SARS-CoV-2-spike pseudotype virus and partially resistant to VSV/VSV-G pseudotype virus. Synthetic peptides representing various regions of the cellugyrin protein were prepared and assessed for their ability to bind to Cdt subunits using surface plasmon resonance. Cdt was capable of binding to a region designated the middle outer loop (MOL) which corresponds to a region extending into the cytoplasmic surface of the SLMVCg+. SARS-CoV-2 spike proteins were assessed for their ability to bind to cellugyrin peptides; SARS-CoV-2 full length spike protein preferentially binds to a region within the SLMVCg+ lumen, designated intraluminal loop 1A. SARS-CoV-2-spike protein domain S1, which contains the receptor binding domains, binds to cellugyrin N-terminus which extends out from the cytoplasmic surface of SLMV. Binding specificity was further analyzed using cellugyrin scrambled peptide mutants. We propose that SLMVCg+ represent a component of a common pathway that facilitates pathogen and/or pathogen-derived toxins to gain host cell entry.

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The presence of periodontal pathogens is associated with an increased prevalence of rheumatoid arthritis (RA). The systemic antibody response to epitopes of these bacteria is often used as a proxy to study correlations between bacteria and RA. The primary aim of the present study is to examine the correlation between the presence of Aggregatibacter actinomycetemcomitans (Aa) in the oral cavity and serum antibodies against the leukotoxin (LtxA) produced by this bacterium. The salivary presence of Aa was analyzed with quantitative PCR and serum LtxA ab in a cell culture-based neutralization assay. The analyses were performed on samples from a well-characterized RA cohort (n = 189) and a reference population of blood donors (n = 101). Salivary Aa was present in 15% of the RA patients and 6% of the blood donors. LtxA ab were detected in 19% of RA-sera and in 16% of sera from blood donors. The correlation between salivary Aa and serum LtxA ab was surprisingly low (rho = 0.55 [95% CI: 0.40, 0.68]). The presence of salivary Aa showed no significant association with any of the RA-associated parameters documented in the cohort. A limitation of the present study is the relatively low number of individuals with detectable concentrations of Aa in saliva. Moreover, in the comparison of detectable Aa prevalence between RA patients and blood donors, we assumed that the two groups were equivalent in other Aa prognostic factors. These limitations must be taken into consideration when the result from the study is interpreted. We conclude that a systemic immune response to Aa LtxA does not fully reflect the prevalence of Aa in saliva. In addition, the association between RA-associated parameters and the presence of Aa was negligible in the present RA cohort.

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Microbes frequently experience nutrient deprivations in the natural environment and may enter dormancy. Aggregatibacter actinomycetemcomitans is known to establish long-term infections in humans. This study examined the dormancy-like phenotype of an A. actinomycetemcomitans strain D7S-1 and its isogenic smooth-colony mutant D7SS. A tissue culture medium RPMI-1640 was nutrient-deficient (ND) and unable to support A. actinomycetemcomitans growth. RPMI-1640 amended with bases was nutrient-limited (NL) and supported limited growth of A. actinomycetemcomitans less than the nutrient-enriched (NE) laboratory medium did. Strain D7S-1, after an initial 2-log reduction in viability, maintained viability from day 4 to day 15 in the NL medium. Strain D7SS, after 1-log reduction in viability, maintained viability from day 3 to day 5. In contrast, bacteria in the NE medium were either non-recoverable (D7S-1; >6-log reduction) or continued to lose viability (D7SS; 3-log reduction) on day 5 and beyond. Scanning and transmission electron microscopy showed that A. actinomycetemcomitans in the NL medium formed robust biofilms similar to those in the NE medium but with evidence of stress. A. actinomycetemcomitans in the ND medium revealed scant biofilms and extensive cellular damage. We concluded that A. actinomycetemcomitans grown in the NL medium exhibited a dormancy-like phenotype characterized by minimum growth, prolonged viability, and distinct cellular morphology.

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Introduction: Quorum-quenching enzyme Est816 hydrolyzes the lactone rings of N-acyl homoserine lactones, effectively blocking the biofilm formation and development of Gram-negative bacteria. However, its applications in the oral field is limited. This study aimed to evaluate the efficacy of enzyme Est816 in combination with antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in vitro and in vivo. Methods: The antimicrobial efficacy of enzyme Est816 in combination with minocycline, metronidazole, and amoxicillin was determined using the minimum inhibitory concentration test. The anti-biofilm effect of enzyme Est816 was assessed using scanning electron microscopy, live/dead bacterial staining, crystal violet staining, and real-time quantitative PCR. Biocompatibility of enzyme Est816 was assessed in human gingival fibroblasts (HGF) by staining. A rat model of periodontitis was established to evaluate the effect of enzyme Est816 combined with minocycline using micro-computed tomography and histological staining. Results: Compared to minocycline, metronidazole, and amoxicillin treatment alone, simultaneous treatment with enzyme Est816 increased the sensitivity of biofilm bacteria to antibiotics. Enzyme Est816 with minocycline exhibited the highest rate of biofilm clearance and high biocompatibility. Moreover, the combination of enzyme Est816 with antibiotics improved the antibiofilm effects of the antibiotics synergistically, reducing the expression of the virulence factor leukotoxin gene (ltxA) and fimbria-associated gene (rcpA). Likewise, the combination of enzyme Est816 with minocycline exhibited a remarkable inhibitory effect on bone resorption and inflammation damage in a rat model of periodontitis. Discussion: The combination of enzyme Est816 with antibiotics represents a prospective anti-biofilm strategy with the potential to treat periodontitis.

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The Gram-positive bacterium, Filifactor alocis is an oral pathogen, and approximately 50% of known strains encode a recently identified repeat-in-toxin (RTX) protein, FtxA. By assessing a longitudinal Ghanaian study population of adolescents (10-19 years of age; mean age 13.2 years), we recently discovered a possible correlation between deep periodontal pockets measured at the two-year follow-up, presence of the ftxA gene, and a high quantity of F. alocis. To further understand the contribution of F. alocis and FtxA in periodontal disease, we used qPCR in the present study to assess the carriage loads of F. alocis and the prevalence of its ftxA gene in subgingival plaque specimens, sampled at baseline from the Ghanaian cohort (n=500). Comparing these results with the recorded clinical attachment loss (CAL) longitudinal progression data from the two-year follow up, we concluded that carriers of ftxA-positive F. alocis typically exhibited higher loads of the bacterium. Moreover, high carriage loads of F. alocis and concomitant presence of the ftxA gene were two factors that were both associated with an enhanced prevalence of CAL progression. Interestingly, CAL progression appeared to be further promoted upon the simultaneous presence of F. alocis and the non-JP2 genotype of Aggregatibacter actinomycetemcomitans. Taken together, our present findings are consistent with the notion that F. alocis and its ftxA gene promotes CAL during periodontal disease.

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Brain abscess is a focal suppurative process produced in most cases by bacterial agents. Aggregatibacter aphrophilus is a gram-negative bacteria belonging to the HACEK group, which causes infective endocarditis, liver abscesses, among others. Brain abscesses secondary to this germ are rare and, in most cases, it is associated with contact with pets, poor dental hygiene or dental procedures. Treatment consists of drainage of the abscess (greater than 2.5 cm) combined with antibiotic therapy, ideally beta-lactams. The case of a 64-year-old male patient with no relevant history is here presented. He was admitted to the emergency service due to headache, hemianopsia of a week's duration and later tonic-clonic seizures, in whom imaging studies and culture of a brain lesion subsequently revealed a brain abscess due to A. aphrophilus. This case aims to illustrate about the rarity of this infection, because A. aphrophilus is a normal part of the oropharyngeal flora and respiratory tract, in which it rarely causes invasive bacteremia.

El absceso cerebral es un proceso supurativo focal producido en la mayoría de los casos por agentes bacterianos. Aggregatibacter aphrophilus es una bacteria gram negativa perteneciente al grupo HACEK, causante de endocarditis infecciosa, abscesos hepáticos, entre otras. Los abscesos cerebrales secundarios a este germen son infrecuentes y en la mayoría de los casos asociados a contactos con animales domésticos, pobre higiene dental o procedimientos odontológicos. El tratamiento consiste en drenaje del absceso (mayores de 2.5 cm) combinado con terapia antibiótica, idealmente betalactámicos. Se presenta el caso de un paciente varón de 64 años sin antecedentes de relevancia quien ingresó al servicio de emergencias por cuadro de cefalea, hemianopsias de una semana de evolución y posteriormente crisis tónico clónicas, en quien posteriormente en estudios imagenológicos y cultivo de lesión cerebral se arribó al diagnóstico de absceso cerebral por A. aphrophilus. Este informe tiene como objetivo ilustrar al lector sobre la rareza de esta infección, debido a que A. aphrophilus forma parte normal de la flora orofaríngea y del tracto respiratorio, en los que rara vez ocasiona bacteriemias invasivas.

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Aggregatibacter spp. is a rare cause for cardiac device infections. Due to limited data, the management of Aggregatibacter spp. device infections is not clearly defined but should always involve device removal and prolonged intravenous antibiotics.

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Peri-implantitis (PI), the most widespread condition in the oral cavity, affects patients globally; thus, advanced research in both in vitro and in vivo studies is required. This study aimed to develop peri-implantitis in the rat model by oral contamination with bacteria responsible for PI in humans. The study was carried out in three stages: the extraction of the maxillary first molar to reproduce the human edentation, the mounting of the implant, and finally, the contamination of the device by gavage with Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus oralis. The hematological examinations showed statistically significant increases for WBCs (white blood cells), Hb (hemoglobin), RBCs (red blood cells), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), and PLTs (platelets), but especially for the level of neutrophils and lymphocytes, and the systemic immunoinflammatory index completed the picture related to the inflammatory response triggered as a result of the activity of microorganisms pathogens on oral tissues. By examining the liver and kidney profile, we hypothesized that peri-implantitis is associated with systemic diseases, and the histopathological examination showed peri-implantitis lesions characterized by a marked inflammatory infiltrate with numerous neutrophils and lymphocytes. By corroborating all the results, we successfully developed a rat peri-implantitis model using a mixed bacterial infection through the oral gavage technique.

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The benefits of probiotics on dysbiotic microbiomes and inflammation are dependent on the tested strain, host factors, and the resident microbiome. There is limited knowledge on the effects of probiotics in A. actinomycetemcomitans-associated periodontitis. Thus, Lactobacillus acidophilus LA5 (LA5) was orally inoculated for 30 days in C57Bl/6 mice infected with A. actinomycetemcomitans JP2 (Aa) and S. gordonii (Sg). Alveolar bone loss, gingival gene expression, and oral and gut microbiomes were determined. LA5 controlled bone loss in Aa+Sg-infected mice, downregulated the expression of Il-1ß and upregulated Il-10 in gingival tissues, and altered the oral and gut microbiomes. LA5 increased the diversity of the oral microbiome of Aa+Sg infected mice, and Aa+Sg and Aa+Sg+LA5 oral or gut microbiomes clustered apart. LA5 induced shifts in Aa+Sg infected mice by increasing the abundance of Muribaculaceae and decreasing Bifidobacteriaceae in the oral cavity and increasing the abundance of Verrucomicrobiae and Eggerthellales in the gut. In conclusion, LA5 oral administration controls experimental Aa-associated periodontitis by altering inflammatory gene expression and the oral and gut microbiomes.

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Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with disease produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is released into the bacterial cytosol in a free form as well as in association with the surface of outer membrane vesicles (OMVs). We previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs: a highly abundant population of small (<100 nm) OMVs and a less abundant population of large (>300 nm) OMVs. Here, we have developed a protocol to isolate the OMVs produced during each specific phase of growth and used this to demonstrate that small OMVs are produced throughout growth and lack LtxA, while large OMVs are produced only during the exponential phase and are enriched with LtxA. Our results indicate that surface-associated DNA drives the selective sorting of LtxA into large OMVs. This study provides valuable insights into the observed heterogeneity of A. actinomycetemcomitans vesicles and emphasizes the importance of understanding these variations in the context of bacterial pathogenesis.

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