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Bacterial community dynamics in the human gut microbiome can have profound effects on human health and disease, yet experimental limitations make it extremely difficult to study this environment with sufficient ecological validity. To work toward physiological relevance, this study ostensibly aimed to assess the morphological changes in Bacillus subtilis colonies in response to pH transience. However, before observing colony responses at pH transition points, static hard substrate experiments in acidic conditions revealed a novel colony growth phenotype. The bulk of the work that follows this observation aims to characterize and explain the novel ‘microcolony’ phenotype; this phenotype is characterized by an asymmetrical formation of cell clusters within the inoculation drop initially, which then converge to form a dense, cohesive colony with minimal radial expansion. Experiments revealed a lack of swarming motility and increased chaining in acidic conditions – with no evidence to support a pH sensation and chemotaxis element to the phenotype. Our results suggest that apparent increased cell-cell adhesion under acidic conditions is a result of both pH-driven matrix protein aggregation and a pH-driven defect in cell division machinery. While both theories are strongly supported by our findings and the existing literature, we recommend further experiments to assess the contributions of each of these factors in determining colony morphology.

Induction of acquired immunological tolerance is the ultimate goal in transplantation. So far the acceptance of a mismatched graft is achieved through immunosuppression that requires long-term treatment and a variety of methods have been explored to prevent rejection and achieve transplant tolerance in mouse models. There are several factors that contribute to acquired tolerance. Recent studies have focused on the inhibition of costimulatory molecules and TLRs in Dendritic Cells (DCs), as a key to the mechanisms underlying the barrier to tolerance induction. Dendritic cells are the sentinels of the immune system. Immature Dendritic cells, which are characterized by low MHC Class II expression and weak T cell stimulation ability, reside in all organs of the body sampling the environment for antigens to bring back to the lymph nodes for T and B cell tolerization or activation, depending on the presence of danger signals. One of these danger signals is LPS from gram-negative Bacteria that can induce DC maturation by triggering TLR4, a surface PRR that is also stimulated by endogenous danger signals, like HMGB1, released during inflammation and tissue damage. Mature DCs highly express MHC II and costimulatory molecules and are potent T cell stimulators. However, LPS has multiple effects on DCs. Indeed, unpublished results from our lab also show that LPS induces DC cell death in vitro and in vivo. It has also been reported that DCs treated with LPS during their development remained in an immature state and they induced alloantigen-specific anergy of CD4+ T cells in vitro. The effects of the simultaneous exposure of DCs to LPS and endogenous danger signals requires further investigation. Therefore, we developed a mouse skin transplant model to determine the effects of LPS and endogenous danger signals, released during engraftment, on DC functions and the ability to induce rejection vs tolerance in transplantation. We used the spontaneous model of skin rejection of a single minor histocompatibility mismatch, the male-specific H-Y antigen. We performed skin grafts from the tail or ear of female or male C57BL/6 mice onto syngeneic female recipients. We administered 4 treatments of PBS 0.5ml or LPS 0.5ml at 25ug/mouse every other day starting from day 0. We observed that control mice transplanted with male skin completely rejected the graft between 24-34 days, while mice transplanted with male skin and treated with LPS did not show rejection of the graft until an average of 64 days and 50% of did not rejected at all. When we administered a different DC stimulator, the TLR9 ligand CpG, we found on the contrary that it induced acceleration of the graft rejection. To understand the mechanism underlying these results, we studied the DCs in vivo. Upon organ transplantation, DCs migrate out of the graft in the first 3 days. Studying the phenotype of the DCs migrating out of the skin graft, we found a sharp decrease of DCs in the skin graft as early as 48 hours post transplant and the loss of DCs was more severe with treatments of LPS. The analysis of the DCs in the epidermal sheets of the graft showed that mice treated with LPS treatment had strongly decreased numbers of DCs compared to mice injected with either PBS or with CpGs. Moreover, we analyzed the DCs from the graft-draining Lymph Nodes (Brachial and Inguinal), and from Spleen. We found again decreased numbers of DCs in both the Spleens and Lymph Nodes of grafted mice treated with LPS compared to mice injected with either PBS or CpGs. Based on these findings, we hypothesize that one of the mechanisms in which LPS prolongs graft survival is that it decreases the number of DCs leaving the graft to stimulate the immune response. LPS is either killing the DCs or holding them outside of the Lymph Nodes, not allowing for antigen presentation during the first week after transplantation when most of the DAMPS from the surgery and ischemia are released. / Microbiology and Immunology

Objectives: Successful treatment of severe human periodontitis lesions has been shown to be highly dependent upon adequate suppression or eradication of key bacteria, most often species classified as red and orange complex periodontal pathogens, in the subgingival microbiome of diseased periodontal sites. Multiple clinical studies have reported superior therapeutic outcomes when conventional mechanical root debridement of severe periodontitis lesions is supplemented with professional periodontal pocket delivery of a povidone-iodine antiseptic solution, which offers antimicrobial effects against red and orange complex periodontal pathogens. However, many questions involving the clinical application of povidone-iodine in periodontal therapy remain unresolved, such as which concentration of povidone-iodine is preferred, and how long of a contact time period is needed between povidone-iodine and targeted bacteria in periodontal pockets. Previous in vitro studies on the effects of povidone-iodine on periodontal bacterial pathogens employed 5-minute or longer contact times and most often tested laboratory stock strains of microorganisms, even though inflamed periodontal pockets undergo a rapid washout of introduced fluids, and bacterial stock collections frequently develop altered properties and decreased virulence in comparison to freshly-recovered wild-type clinical isolates. To address some of these issues, the objective of this study was to further explore the in vitro effects of povidone-iodine on periodontal bacterial pathogens by employing a subgingival biofilm species eradication assay to test the antimicrobial effects of a 60- second in vitro exposure of 5% and 10% povidone-iodine on freshly-isolated red and orange complex periodontal pathogens from severe human periodontitis lesions. Methods: Paper point subgingival biofilm samples from 22 adults with severe periodontitis that were to be discarded after microbiological analysis at the Oral Microbiology Testing Service Laboratory at Temple University School of Dentistry were secondarily employed in this study. Dilution aliquots from each subgingival specimen were mixed with either 10% or 5% povidone-iodine for a 60-second in vitro contact time period, and then neutralized with 3% sodium thiosulfate. The mixtures were then inoculated onto enriched Brucella blood agar plates, and incubated anaerobically for 7 days at 37°C. Bacterial species growing subsequent to the 60-second povidone-iodine contact time were considered to be resistant to that concentration of povidone-iodine. Total viable counts in povidone-iodine exposed subgingival specimens were quantitated, and established phenotypic criteria employed to identify the following red and orange complex periodontal pathogens: Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, Campylobacter rectus, Fusobacterium nucleatum group species, and Streptococcus constellatus. Other cultivable isolates recovered from povidone-iodine exposed subgingival specimens were identified using matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry and Bruker MALDI Biotyper analytic software. Subgingival sample dilution aliquots not exposed to povidone-iodine were similarly processed as controls for comparison with povidone-iodine exposed specimens, and were additionally inoculated onto enriched Brucella blood agar plates supplemented with either metronidazole at 16 mg/L, doxycycline at 4 mg/L, amoxicillin at 8 mg/L, or clindamycin at 4 mg/L, which represent recognized non-susceptible drug breakpoint concentrations for each of the antibiotics, followed by anaerobic incubation for 7 days at 37°C. In vitro antibiotic resistance was noted when any of the evaluated red and orange complex periodontal pathogens displayed growth on one or more of the antibiotic- supplemented enriched Brucella blood agar plates. Paired t-tests compared mean total subgingival viable counts, and mean total subgingival proportions of the evaluated anaerobic red and orange complex periodontal pathogens per patient, between subgingival biofilm samples exposed and not exposed in vitro to 10% or 5% povidone-iodine, with a P-value of < 0.05 required for statistical significance. Comparisons were also made between antibiotic-resistance and susceptible strains of the evaluated red and orange complex periodontal pathogens to assess their in vitro sensitivity to 10% and 5% povidone-iodine. Results: Subgingival specimens exposed in vitro for 60-seconds to either 10% or 5% povidone-iodine yielded significantly lower total subgingival viable counts per patient than those not exposed to povidone-iodine (P < 0.001, paired t-test), with no statistically significant differences found between the two povidone-iodine concentrations (P = 0.125, paired t-test). All evaluated red and orange complex periodontal pathogens were suppressed below detection levels after 10% povidone-iodine in vitro exposure, except for one F. nucleatum strain in one patient specimen. As a result, 21 (95.5%) of the patient subgingival biofilm samples were culture-negative for red and orange complex periodontal pathogens after 10% povidone-iodine in vitro exposure. A similar suppression of red and orange complex periodontal pathogens was found following 5% povidone iodine in vitro exposure, where only one patient sample yielded P. intermedia/nigrescens, and two other samples F. nucleatum. This resulted in 19 (86.4%) of patient subgingival biofilm samples being devoid of red and orange complex periodontal pathogens after 5% povidone-iodine in vitro exposure. Total subgingival proportions of red and orange complex periodontal pathogens averaged 0.5% per patient in subgingival specimens exposed in vitro to 10% povidone-iodine, and 0.7% per patient in those exposed to 5% povidone-iodine, which were both significantly lower than 14.8% mean proportions detected in subgingival biofilms not exposed to povidone-iodine (P < 0.0001, paired t-test). No statistically significant differences were found between 10% and 5% povidone-iodine relative to suppression of total red and orange complex periodontal pathogen proportions (P = 0.743, paired t-test). Both povidone-iodine concentrations were similarly active against red and orange complex periodontal pathogens which were resistant in vitro to breakpoint levels of doxycycline, amoxicillin, or clindamycin, as compared to antibiotic-susceptible clinical isolates. Various Streptococcus species, particularly Streptococcus oralis, were the most frequently recovered microorganisms in subgingival biofilm specimens after 60-second in vitro exposure to povidone-iodine, indicative of their in vitro resistance to both 10% and 5% povidone-iodine. Conclusions: Povidone-iodine exerted marked antimicrobial effects against both antibiotic-resistant and susceptible fresh clinical isolates of red and orange complex periodontal pathogens, and total viable counts, in subgingival biofilm specimens from severe periodontitis patients after only a brief (60-second) in vitro contact time, with no statistically significant differences found between 10% and 5% concentrations of povidone-iodine. The profound suppression of red and orange complex periodontal pathogens, and concurrent predominance of periodontal health-associated Streptococcus species, that occurs in subgingival biofilm specimens following in vitro exposure to povidone-iodine, further supports the clinical use of povidone-iodine in periodontal therapy as an adjunct to mechanical root debridement in altering pathogenic subgingival microbial populations towards one compatible with periodontal health. / Oral Biology

Objectives: Prevotella intermedia and Prevotella nigrescens are two genetically distinct, gram-negative, anaerobic rods associated with the subgingival microbiome of human periodontitis. The two species are frequently isolated from subgingival dental plaque biofilms in chronic periodontitis patients clinically experiencing progressive destructive disease activity. In anaerobically-incubated liquid or solid culture media, P. intermedia and P. nigrescens exhibit nearly identical phenotypic properties, with regard to their colony morphology features and biochemical properties, which differ from other subgingival Prevotella and non-Prevotella microbial species. As a result, rapid differentiation and identification of P. intermedia/nigrescens group organisms from other bacterial species in anaerobically-cultivated subgingival dental plaque biofilms has been based upon examination of culture isolates for a dark-pigmented colony appearance, presence of brick-red autofluorescence of colonies to long-wave ultraviolet light exposure, and biochemical testing demonstrating a lack of colony lactose fermentation. However, the accuracy of this phenotypic-based identification scheme for periodontal P. intermedia/nigrescens group species has yet to be validated with a broad-based reference method that encompasses testing for a wide array of microbial species. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and associated analytic software, is recently approved for clinical microbiology diagnostic use in the United States by the Food and Drug Administration, and is capable of definitively identifying 4,613 different microbial species based on mass spectra of their bacterial proteins. To date, no performance evaluation has been carried out comparing the phenotypic-based identification scheme for periodontal P. intermedia/ nigrescens group species with definitive MALDI-TOF mass spectrometry identification of the organisms. As a result, the purpose of this study was to assess with MALDI-TOF mass spectrometry the accuracy of the rapid phenotypic-based periodontal P. intermedia/ nigrescens group species identification scheme widely utilized since 1986 by clinical periodontal microbiology laboratories and periodontal microbiology culture-based research studies. Methods: 84 fresh subgingival cultivable isolates from 23 chronic periodontitis patients were presumptively identified on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. intermedia/nigrescens group species based on their dark-pigmented colony morphology, presence of brick-red autofluorescence under long-wave ultraviolet light, and a negative MUG fluorescence test for lactose fermentation activity. Each of the putative P. intermedia/nigrescens clinical isolates were subjected to MALDI-TOF mass spectrometry analysis using a bench top mass spectrometer, Bruker FlexControl 3.0 software, and MALDI Biotyper 3.1 software (Bruker Daltonics, Billerica, MA, USA), which contains mass spectra for P. intermedia and P. nigrescens in its reference library of bacterial protein profiles. A MALDI Biotyper log score of equal to or larger than 1.7 was required for reliable taxonomic classification of the clinical isolates, with scores of equal to or larger than 2.0 representing more definitive species identification. Results: A total of 60 (71.4%) of the putative P. intermedia/nigrescens clinical isolates were reliably identified with MALDI-TOF mass spectrometry as either P. intermedia (25 isolates, with eight isolates exhibiting MALDI Biotyper log scores of equal to or larger than 2.0), or P. nigrescens (35 isolates, with nine isolates exhibiting MALDI Biotyper log scores of equal to or larger than 2.0). Among the 24 putative P. intermedia/nigrescens clinical isolates generating MALDI Biotyper log scores < 1.7, indicating a less reliable species identification, only P. intermedia (14 isolates) or P. nigrescens (10 isolates) were listed by the analytic software as the first choice among the most likely bacterial species. No other bacterial species other than P. intermedia or P. nigrescens were identified by MALDI-TOF mass spectrometry for any of the 84 tested putative P. intermedia/ nigrescens clinical isolates. Conclusions: These findings document, for the first time with MALDI-TOF mass spectrometry, the relative accuracy of a rapid phenotypic-based periodontal P. intermedia/nigrescens identification scheme based on dark-pigmented colony morphology, presence of brick-red long-wave ultraviolet light autofluorescence, and a negative MUG test for lactose fermentation activity. 100% of the 84 presumptive P. intermedia/nigrescens clinical isolates tested were identified with MALDI-TOF mass spectrometry, with varying levels of reliability, as being only either P. intermedia or P. nigrescens. These findings provide validation for the continued use of this rapid phenotypic identification scheme for periodontal P. intermedia/nigrescens. / Oral Biology

Objectives: Silver diamine fluoride is approved by the United States Food and Drug Administration for intraoral human treatment of tooth hypersensitivity, and it has also been employed world-wide as an emerging method to arrest tooth decay. A 38% silver diamine fluoride formulation, comprised of 25% silver, 5% fluoride, and 8% ammonia as a solvent, is commercially available in the United States. One of the main mechanisms underlying the dental caries arrest potential of silver diamine fluoride is the silver component, which exerts pronounced antimicrobial activity against cariogenic bacteria. Interestingly, studies initiated in the late 1990s demonstrated marked susceptibility of periodontal bacterial pathogens to silver nitrate. However, efforts to develop silver-based, slow-release biodegradable wafers for subgingival placement into periodontal pockets were not commercially successful. At present, no commercial products are available which employ silver ions to combat periodontal bacterial pathogens in periodontal disease treatment. It is not known whether the 38% silver diamine fluoride product commercially available in the United States possesses antimicrobial activity against periodontal bacterial pathogens, and potentially, have application in periodontal therapeutic regimens. As a result, the objective of this study was to test the in vitro antimicrobial effects of silver diamine fluoride on freshly-isolated red and orange complex periodontal pathogens from severe human periodontitis lesions. Methods: Paper point subgingival biofilm samples from 24 adults with severe periodontitis that were to be discarded after microbiological analysis at the Temple University School of Dentistry Oral Microbiology Testing Service Laboratory were secondarily employed in this study. Dilution aliquots from each subgingival specimen were mixed with either 38% or 19% silver diamine fluoride, inoculated onto enriched Brucella blood agar plates, and incubated anaerobically for 7 days at 37°C. Bacterial species growing subsequent to the silver diamine fluoride exposure were considered to be resistant to that concentration of silver diamine fluoride. Total viable counts in silver diamine fluoride-exposed subgingival specimens were quantitated, and established phenotypic criteria employed to identify the following red and orange complex periodontal pathogens: Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, Campylobacter rectus, Fusobacterium nucleatum group species, and Streptococcus constellatus. Other cultivable isolates recovered from silver diamine fluoride-exposed subgingival specimens were identified using matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry and Bruker MALDI Biotyper analytic software. Subgingival sample dilution aliquots not exposed to silver diamine fluoride were similarly processed as controls for comparison with silver diamine fluoride-exposed specimens Paired t-tests compared mean total subgingival viable counts, and mean total subgingival proportions of the evaluated anaerobic red and orange complex periodontal pathogens per patient, between subgingival biofilm samples exposed and not exposed in vitro to 38% or 19% silver diamine fluoride, with a P-value of < 0.05 required for statistical significance. Results: Subgingival specimens exposed in vitro to either 38% or 19% silver diamine fluoride yielded significantly lower total subgingival viable counts per patient than those not exposed to silver diamine fluoride (P < 0.001, paired t-test), with no statistically significant differences found between 38% and 19% silver diamine fluoride exposures (P = 0.370, paired t-test). All evaluated red and orange complex periodontal pathogens were suppressed below detection levels in 21 (87.5%) of subgingival samples after in vitro exposure to 38% silver diamine fluoride. Three other patient specimens treated with 38% silver diamine fluoride each had persistence of P. micra. Similarly, 21 (87.5%) of subgingival specimens also were culture-negative for red and orange complex periodontal pathogens after 19% silver diamine fluoride exposure, with two other patient samples showing persistence of P. micra, and a third sample persistence of S. constellatus. Total subgingival proportions of red and orange complex periodontal pathogens averaged 0.6% per patient in subgingival specimens exposed in vitro to 38% silver diamine fluoride, and 0.5% per patient in those exposed to 19% silver diamine fluoride, which were both significantly lower than 25.9% mean proportions detected in subgingival biofilms not exposed to silver diamine fluoride (P < 0.0001, paired t-test). No statistically significant differences were found between 38% and 19% silver diamine fluoride relative to suppression of total red and orange complex periodontal pathogen proportions (P = 0.345, paired t-test). Various Streptococcus species, particularly Streptococcus oralis, were the most frequently recovered microorganisms in subgingival biofilm specimens after exposure to both 38% and 19% silver diamine fluoride, indicative of their in vitro resistance to silver diamine fluoride. Conclusions: Silver diamine fluoride demonstrated substantial antimicrobial activity against fresh clinical isolates of red and orange complex periodontal pathogens, and total viable counts, in subgingival biofilm specimens from severe periodontitis patients, with no statistically significant differences found between silver diamine fluoride concentrations of 38% and 19%. The dramatic in vitro suppression of red and orange complex periodontal pathogens in subgingival biofilm specimens by silver diamine fluoride, along with its selection of silver diamine fluoride-resistant species of Streptococcus that are associated with periodontal health, suggests a new therapeutic use for silver diamine fluoride in the management of human periodontal infections. / Oral Biology

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