Detection of bacteria in dental samples using the Periodic acid-Schiff (PAS) histological stain.

The bacterial cell wall mainly consists of glycoproteins and polysaccharides, which could be detected in dental tissue with specific stain protocols. The present study aimed to investigate bacteria stainability in dental histological samples of human teeth by a histochemical method. Eight extracted teeth, because severely decayed, were decalcified, dehydrated, paraffin-embedded, and serially sectioned at 4 µm thickness each. The serial sections were then stained with Periodic acid-Schiff (PAS). Furthermore, SEM analysis was performed on the same slide of one previously histologically investigated tooth to acquire more details on the structures stained by the PAS method obtained from the histological procedures. Afterward, some American Type Culture Collection (ATCC) strains, smeared on glass slides, were stained following the staining method used in histological samples. Stained rod and cocci forms by PAS stain, observed under light microscopy, were predominantly detected inside dentinal tubules and root canal space of histologically examined specimens, suggesting their bacterial origin. Additional SEM analysis on the identical histological stained slide showed the precise nature of these forms (bacteria) and supplemental information regarding their vitality status. In addition, ATCC smeared strain samples showed variable PAS stainability of microorganisms investigated. Due to its properties, the PAS histochemical stain could be a valid and helpful aid for non- or weakly stainable microorganisms in infected tissues to be associated with other methods of investigation.

Host Environment Shapes S. aureus Social Behavior as Revealed by Microscopy Pattern Formation and Dynamic Aggregation Analysis.

Understanding how bacteria adapt their social behavior to environmental changes is of crucial importance from both biological and clinical perspectives. Staphylococcus aureus is among the most common infecting agents in orthopedics, but its recalcitrance to the immune system and to antimicrobial treatments in the physiological microenvironment are still poorly understood. By means of optical and confocal microscopy, image pattern analysis, and mathematical modeling, we show that planktonic biofilm-like aggregates and sessile biofilm lifestyles are two co-existing and interacting phases of the same environmentally adaptive developmental process and that they exhibit substantial differences when S. aureus is grown in physiological fluids instead of common lab media. Physicochemical properties of the physiological microenvironment are proposed to be the key determinants of these differences. Besides providing a new tool for biofilm phenotypic analysis, our results suggest new insights into the social behavior of S. aureus in physiological conditions and highlight the inadequacy of commonly used lab media for both biological and clinical studies of bacterial development.

A spacer infection by Candida albicans secondary to a Staphylococcus capitis prosthetic joint infection: a case report.

BACKGROUND: Prosthetic joint infection (PJI) is one of the most feared complications following total arthroplasty surgeries. Gram-positive bacteria are the most common microorganisms implicated in PJIs, while infections mediated by fungi only account for 1% of cases. When dealing with PJIs, a two-stage revision arthroplasty is widely used. Briefly, a spacer is introduced until re-implantation of the definitive prosthesis to provide skeleton stabilization while delivering antibiotics in the site of the infection. Sometimes, antimicrobial therapy may fail, but the isolation of a second microorganism from the spacer is uncommon and even less frequent that of a yeast. CASE PRESENTATION: Here is described a case of a 75-year-old woman who underwent two-stage revision surgery of the left hip prosthesis secondary to a Staphylococcus capitis infection, whose spacer was found to be infected by Candida albicans at a later time. Briefly, the patient underwent revision surgery of the hip prosthesis for a suspected PJI. After the debridement of the infected tissue, an antibiotic-loaded spacer was implanted. The microbiological analysis of the periprosthetic tissues and the implant depicted a S. capitis infection that was treated according to the antimicrobial susceptibility profile of the clinical isolate. Three months later, the patient was admitted to the emergency room due to local inflammatory signs. Synovial fluid was sent to the laboratory for culture. No evidence of S. capitis was detected, however, a yeast was identified as Candida albicans. Fifteen days later, the patient was hospitalized for the removal of the infected spacer. Microbiological cultures confirmed the results of the synovial fluid analysis. According to the susceptibility profile, the patient was treated with fluconazole (400 mg/day) for 6 months. Seven months later, the patient underwent second-stage surgery. The microbiological tests on the spacer were all negative. After 12 months of follow-up, the patient has fully recovered and no radiological signs of infection have been detected. CONCLUSIONS: Given the exceptionality of this complication, it is important to report these events to better understand the clinical outcomes after the selected therapeutic options to prevent and forestall the development of either bacterial or fungal spacer infections.

Superior Osteo-Inductive and Osteo-Conductive Properties of Trabecular Titanium vs. PEEK Scaffolds on Human Mesenchymal Stem Cells: A Proof of Concept for the Use of Fusion Cages.

Fusion cages composed of titanium and its alloys are emerging as valuable alternative to standard polyetheretherketone (PEEK) ones routinely used in cervical and lumbar spine surgery. Aim of this study was to evaluate osteo-inductive and osteo-conductive ability of an innovative trabecular titanium (T-Ti) scaffold on human mesenchymal stem cells (hMSCs), in both absence and presence of biochemical osteogenic stimuli. Same abilities were assessed on PEEK and standard 2D plastic surface, the latter meant as gold-standard for in vitro differentiation studies. hMSCs adhered and colonized both T-Ti and PEEK scaffolds. In absence of osteogenic factors, T-Ti triggered osteogenic induction of MSCs, as demonstrated by alkaline phosphatase activity and calcium deposition increments, while PEEK and standard 2D did not. Addition of osteogenic stimuli reinforced osteogenic differentiation of hMSCs cultured on T-Ti in a significantly higher manner with respect to standard 2D plastic culture surfaces, whereas PEEK almost completely abolished the process. T-Ti driven differentiation towards osteoblasts was confirmed by gene and marker expression analyses, even in absence of osteogenic stimuli. These results clearly indicate superior in vitro osteo-inductive and osteo-conductive capacity of T-Ti compared to PEEK, and make ground for further studies supporting the use of T-Ti cages to improve bone fusion.

Bacterial adhesion on spinal implants: An in vitro study of "hot spots".

Few studies evaluated bacterial colonization of spinal implants from a "topographic" point of view. This lack of knowledge could hinder the development of more effective strategies in the prevention and treatment of postoperative spinal infections. The aim of this in vitro study was the analysis of the adhesion pattern of sessile cells on conventional spinal implants, to identify "hot spots" on implants where bacterial adhesion could be favored. Clinically relevant Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa isolates were grown on commercially available end product spinal implants. To identify sessile cells attached to implant surfaces, confocal laser scan microscopy was used. Different areas from the spinal instrumentations (both Ti and CoCr) were selected for biofilm quantification. Bacterial biofilm was markedly increased in the cut of the rods, both Ti and CoCr, as the uneven surface deriving from the cut might foster cell adhesion. Though not statistically significant, a difference was observed between the rod and the area of the notch, possibly as a consequence of the smoothening effect deriving from the bending of the rod. Finally, the amount of biofilm produced on cobalt-chromium surfaces was always more significant than that formed on titanium surfaces. This study highlights how bacterial adhesion through biofilm formation is favored on the surfaces of higher irregularity and that staphylococci are able to increase sessile biomass on CoCr surfaces. These preliminary results show how surface modifications on the implants may play a key role in bacterial adhesion, opening an exciting field for future research.

In Vitro Evaluation of Gentamicin or Vancomycin Containing Bone Graft Substitute in the Prevention of Orthopedic Implant-Related Infections.

Antibiotic-loaded bone graft substitutes are attractive clinical options and have been used for years either for prophylaxis or therapy for periprosthetic and fracture-related infections. Calcium sulfate and hydroxyapatite can be combined in an injectable and moldable bone graft substitute that provides dead space management with local release of high concentrations of antibiotics in a one-stage approach. With the aim to test preventive strategies against bone infections, a commercial hydroxyapatite/calcium sulfate bone graft substitute containing either gentamicin or vancomycin was tested against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, harboring different resistance determinants. The prevention of bacterial colonization and biofilm development by selected microorganisms was investigated along with the capability of the eluted antibiotics to select for antibiotic resistance. The addition of antibiotics drastically affected the ability of the selected strains to adhere to the tested compound. Furthermore, both the antibiotics eluted by the bone graft substitutes were able to negatively impair the biofilm maturation of all the staphylococcal strains. As expected, P. aeruginosa was significantly affected only by the gentamicin containing bone graft substitutes. Finally, the prolonged exposure to antibiotic-containing sulfate/hydroxyapatite discs did not lead to any stable or transient adaptations in either of the tested bacterial strains. No signs of the development of antibiotic resistance were found, which confirms the safety of this strategy for the prevention of infection in orthopedic surgery.

Evaluation of Cytotoxicity and Antibacterial Activity of a New Class of Silver Citrate-Based Compounds as Endodontic Irrigants.

In the present study, the cytotoxicity and the antimicrobial activity of two silver citrate-based irrigant solutions were investigated. Cytotoxicity of various concentrations (0.25%, 0.5%, 1%, 2.5%, 5%) of both solutions (BioAKT and BioAKT Endo) was assessed on L-929 mouse fibroblasts using the MTT assay. For the quantitative analysis of components, an infrared (I.R.) spectroscopy was performed. The minimum inhibitory and minimal bactericidal concentrations (M.I.C. and M.B.C., respectively) were ascertained on Enterococcus faecalis strain ATCC 4083. For biofilm susceptibility after treatment with the irrigating agent, a minimum biofilm eradication concentration (M.B.E.C.) and confocal laser scanning microscope (C.L.S.M.) assays were performed. Quantification of E. faecalis cell biomass and percentage of live and dead cells in the biomass was appraised. Normality of data was analyzed using the D'Agostino & Pearson's test and the Shapiro-Wilk test. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's test. Both silver citrate solutions showed mouse fibroblasts viability >70% when diluted to 0.25% and 0.5%. Conversely, at higher concentrations, they were extremely cytotoxic. F.T.-IR spectroscopy measurements of both liquids showed the same spectra, indicating similar chemical characteristics. No substantial contrast in antimicrobial activity was observed among the two silver citrate solutions by using broth microdilution methods, biofilm susceptibility (MBEC-HTP device), and biomass screening using confocal laser scanning microscopy (C.L.S.M.) technique. Both solutions, used as root canal irrigants, exhibited significant antimicrobial activity and low cytocompatibility at dilutions greater than 0.5%.

Phenotypic Modulation of Biofilm Formation in a Staphylococcus epidermidis Orthopedic Clinical Isolate Grown Under Different Mechanical Stimuli: Contribution From a Combined Proteomic Study.

One of the major causes of prosthetic joint failure is infection. Recently, coagulase negative Staphylococcus epidermidis has been identified as an emergent, nosocomial pathogen involved in subclinical prosthetic joint infections (PJIs). The diagnosis of PJIs mediated by S. epidermidis is usually complex and difficult due to the absence of acute clinical signs derived from the host immune system response. Therefore, analysis of protein patterns in biofilm-producing S. epidermidis allows for the examination of the molecular basis of biofilm formation. Thus, in the present study, the proteome of a clinical isolate S. epidermidis was analyzed when cultured in its planktonic or sessile form to examine protein expression changes depending on culture conditions. After 24 h of culture, sessile bacteria exhibited increased gene expression for ribosomal activity and for production of proteins related to the initial attachment phase, involved in the capsular polysaccharide/adhesin, surface associated proteins and peptidoglycan biosynthesis. Likewise, planktonic S. epidermidis was able to aggregate after 24 h, synthesizing the accumulation associate protein and cell-wall molecules through the activation of the YycFG and ArlRS, two component regulatory pathways. Prolonged culture under vigorous agitation generated a stressful growing environment triggering aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. Further studies will be essential to support these findings in order to further delineate the complex mechanisms of biofilm formation of S. epidermidis and they could provide the groundwork for the development of new drugs against biofilm-related infections, as well as the identification of novel biomarkers of subclinical or chronic infections mediated by these emerging, low virulence pathogens.

Identification and Characterization of Planktonic Biofilm-Like Aggregates in Infected Synovial Fluids From Joint Infections.

Recent in vitro studies reported the exceptional ability of some bacterial species to form biofilm-like aggregates in human and animal synovial fluids (SF), but evidences from infected clinical samples are still lacking. In this study, we investigated whether this bacterial phenotype was present in infected SFs collected from joint infections and if it was maintained in in vitro settings. SFs sent for culture to the Laboratory of Microbiology of our institute were directly analyzed by means of confocal laser scanning microscopy (CLSM), and the infective agents were isolated for further in vitro tests. Moreover, sterile SF was collected from patients who did not receive previous antibiotic therapy to investigate the formation of bacterial aggregates, together with biofilm and matrix production on a titanium surface. Finally, antibiotic susceptibility studies were performed by using bovine SF. Four Staphylococcus aureus, one Staphylococcus lugdunensis, and one Prevotella bivia strain were identified in the infected SFs. The CLSM analysis showed that all staphylococci were present as a mixture of single cells and bacterial clumps surrounded by an exopolymeric substance, which comprised SF-derived fibrin, while all P. bivia cells appeared separated. Despite that, differences in the ability to aggregate between S. aureus and S. lugdunensis were observed in clinical SFs. These different phenotypes were further confirmed by in vitro growth, even though the application of such ex vivo approach lead all staphylococci to form exceptionally large microbial aggregates, which are several folds bigger than those observed in clinical samples. Planktonic aggregates challenged for antibiotic susceptibility revealed a sharp increase of recalcitrance to the treatments. Although this is still at a preliminary stage, the present work confirmed the ability of staphylococci to form free-floating biofilm-like aggregates in infected SF from patients with joint infections. Furthermore, the obtained results pointed out that future in vitro research on joint infections will benefit from the use of human- or animal-derived SF. Even though this approach should be carefully validated in further studies comprising a larger microbial population, these findings pose new challenges in the treatment of infected native and prosthetic joints and for the approach to new investigations.

Ability of adhesion and biofilm formation of pathogens of periprosthetic joint infections on titanium-niobium nitride (TiNbN) ceramic coatings.

BACKGROUND: Orthopedic metal implants are notoriously associated with release of metallic ions able to cause biological adverse reactions which might lead to implant loosening and failure. To limit any possible adverse reactions, ceramic coatings for orthopedic metal implants have been introduced. However, information regarding the interaction of these coatings with microbes responsible for periprosthetic joint infections (PJIs) is lacking. Hence, the aim of the present in vitro study is to assess the microbial affinity to a titanium-niobium nitride (TiNbN) coating. METHODS: Adhesion and biofilm formation of clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Cutibacterium acnes were assessed on TiNbN-coated titanium discs in comparison with uncoated titanium and cobalt-chrome alloys discs, with either smooth or rough surfaces. Bacterial adhesion was performed by counting adhered bacteria in the first hours of incubation, and the biofilm formation was performed by means of a spectrophotometric assay and by confocal laser scan microscopy after 72 hours of incubation. RESULTS: Overall, Staphylococcus aureus and Staphylococcus epidermidis, among the most common bacteria responsible for PJIs, displayed a significantly decreased attachment in the first hours of contact and, when cultured in presence of TiNbN coating, in comparison with CoCrMo. Biofilm formation of the four tested strains was comparable on all alloys. CONCLUSIONS: Although the onset of a PJI is more complex than in an in vitro scenario, these findings suggest that TiNbN-coated orthopedic implants do not increase PJIs risk while ameliorating tribological and surface properties could represent a valid choice to limit possible complications such as metal hypersensitivity.

Antimicrobial effectiveness of etidronate powder (Dual Rinse® HEDP) and two EDTA preparations against Enterococcus faecalis: a preliminary laboratory study.

This study aimed to evaluate the antimicrobial and antibiofilm activity of two chelating agents: ethylenediaminetetraacetic acid (EDTA) combined or not with detergents, and etidronic acid combined with sterile saline. The bacterial inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined on Enterococcus faecalis ATCC 4083 strain. Antimicrobial tests were performed on a biofilm model after treatment with the chelating agents at different times (1, 3, and 5 min) using a biofilm eradication concentration (MBEC) and confocal laser scanning microscope (CLSM) assays. Quantification of cell biomass and percentage of live and dead cells in the biomass was assessed for each group. The normality of the distributions for each variable was assessed using the D'Agostino and Pearson's omnibus normality test. The comparison of bacterial viability among groups and between any two groups was performed using the non-parametric Kruskal-Wallis one-way analysis of variance and the Dunn's test, respectively. No significant between-group difference was observed regarding biomass reduction. On the other hand, EDTA combined with detergents displayed a substantial increase of the dead bacteria ranging between 35 and 43%; whereas, the number of cells killed in the control group and in the other treated groups always ranged between 1 and 6%, at all experimental times. The addition of detergents to EDTA can improve its anti-biofilm activity by reducing EPS production and enhancing the killing of sessile bacterial cells. Clinical relevance EDTA presents a relevant antimicrobial activity when combined with surface-active agents.

Proteomic Analysis Reveals a Biofilm-Like Behavior of Planktonic Aggregates of Staphylococcus epidermidis Grown Under Environmental Pressure/Stress.

Prosthetic joint replacement failure has a huge impact on quality of life and hospitalization costs. A leading cause of prosthetic joint infection is bacteria-forming biofilm on the surface of orthopedic devices. Staphylococcus epidermidis is an emergent, low-virulence pathogen implicated in chronic infections, barely indistinguishable from aseptic loosening when embedded in a mature matrix. The literature on the behavior of quiescent S. epidermidis in mature biofilms is scarce. To fill this gap, we performed comparative analysis of the whole proteomic profiles of two methicillin-resistant S. epidermidis strains growing in planktonic and in sessile form to investigate the molecular mechanisms underlying biofilm stability. After 72-h culture of biofilm-forming S. epidermidis, overexpression of proteins involved in the synthesis of nucleoside triphosphate and polysaccharides was observed, whereas planktonic bacteria expressed proteins linked to stress and anaerobic growth. Cytological analysis was performed to determine why planktonic bacteria unexpectedly expressed proteins typical of sessile culture. Images evidenced that prolonged culture under vigorous agitation can create a stressful growing environment that triggers microorganism aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. The choice of a unique late time point provided an important clue for future investigations into the biofilm-like behavior of planktonic cells. Our preliminary results may inform comparative proteomic strategies in the study of mature bacterial biofilm. Finally, there is an increasing number of studies on the aggregation of free-floating bacteria embedded in an extracellular matrix, prompting the need to gain further insight into this mode of bacterial growth.

Chlorquinaldol, a topical agent for skin and wound infections: anti-biofilm activity and biofilm-related antimicrobial cross-resistance.

PURPOSE: Persistence of skin and wound infections is nowadays accepted being linked to bacterial biofilms, which are highly recalcitrant to treatments and contribute to maintain a constant inflammation state and prevent a correct healing. Topical antimicrobials are the most common first-line self-medications; however, treatment failure is not uncommon and emerging resistance to antibiotics is alarming. Chlorquinaldol is an antimicrobial with a wide spectrum of activity and desirable characteristics for topical application. Aim of this study was to evaluate the efficacy of chlorquinaldol to prevent or eradicate S. aureus and P. aeruginosa biofilms, in comparison to classic topical antibiotics like gentamicin and fusidic acid. METHODS: Minimum inhibitory concentrations (MIC) were assessed for each strain and subinhibitory concentrations (½ and » MIC) were used in the biofilm assay. Antimicrobial assays were performed during biofilm formation or were applied on mature biofilms and were evaluated by means of crystal violet assay and confocal laser scan microscopy. RESULTS: Chlorquinaldol and gentamicin were the most effective antimicrobials in both eradicating and preventing pathogens biofilm; however, resistance to methicillin and impermeability to carbapenems impaired chlorquinaldol effect. In addition, similarly to other hydroxyquinolines, aspecific metal chelation is here proposed as chlorquinaldol mode of action. CONCLUSION: Relying on an acceptable antibiofilm and a wide spectrum of activity, an aspecific mode of action and consequent absence of resistance development, chlorquinaldol proved to be a good antimicrobial for topical use.

Effects of a cream containing 5% hyaluronic acid mixed with a bacterial-wall-derived glycoprotein, glycyrretinic acid, piroctone olamine and climbazole on signs, symptoms and skin bacterial microbiota in subjects with seborrheic dermatitis of the face.

Objective: A new cream formulation containing hyaluronic acid 5%, complexed with a mix of a bacterial-wall-derived glycoprotein and peptide glycan complex (EDS), has been recently developed. We evaluated in a prospective, assessor-blinded, 6-week study the efficacy and tolerability of EDS in the treatment of facial seborrheic dermatitis (SD) and the effects on skin microbiota. Subjects and methods: Seventy-five subjects (mean age 46; 60 men) with moderate-severe SD of the face were enrolled. EDS cream was applied twice daily. The primary outcome was the evolution of the Investigator Global Assessment (IGA) score, evaluating erythema, scale/flaking, grade of seborrhea and itch. Superficial skin bacterial microbiome at baseline and after treatment was assessed, using the 16S rRNA gene methodology, in affected and non-affected face areas. Local tolerability was evaluated checking self-reported side effects at each visit. Results: Baseline IGA scores (mean±SD) was 10±3. The use of EDS reduced IGA score significantly by 70% at week 3 and by 88% at week 6. An increase in the abundance of Cutibacterium acnes genera associated with a significant drop of Staphylococcus genera presence was detected in affected areas. The ratio of relative abundance of genera Cutibacterium/Staphylococcus increased significantly after treatment in affected areas. The product was very well tolerated. Conclusion: Treatment with EDS applied twice daily for 6 consecutive weeks was associated with a reduction of the signs and symptoms of SD. Furthermore, after EDS cream treatment, a reequilibrating effect on facial skin microbiota was observed. The product was very well tolerated.

Is Implant Coating With Tyrosol- and Antibiotic-loaded Hydrogel Effective in Reducing Cutibacterium (Propionibacterium) acnes Biofilm Formation? A Preliminary In Vitro Study.

BACKGROUND: Studies have suggested that Cutibacterium acnes (formerly known as Propionibacterium) is the most frequently isolated pathogen after shoulder arthroplasty. To address the burden of periprosthetic joint infections associated with this pathogen, new prevention methods are needed. Tyrosol has a promising record of effectiveness in the field of biofilm-associated infections; however, to our knowledge, it has not been tested against C. acnes thus far. QUESTIONS/PURPOSES: In this in vitro study, we asked: (1) Is tyrosol effective in inhibiting and eradicating C. acnes planktonic growth? (2) Is there synergy between tyrosol and rifampicin? (3) Is supplementation of hydrogel with tyrosol at the minimum inhibitory and subinhibitory concentrations efficacious in reducing free-floating C. acnes growth? (4) Is implant hydrogel coating (either alone or combined with tyrosol, rifampicin, or vancomycin) beneficial in reducing C. acnes biofilm formation? (5) Is the administration of soluble tyrosol an effective measure against C. acnes biofilm formation? METHODS: We assessed C. acnes planktonic growth and eradication by inspecting visually the results of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. We also evaluated macroscopically the presence of synergy among tyrosol and rifampicin by means of the MIC checkerboard testing. Thereafter, we addressed colorimetrically the efficacy of tyrosol-loaded Defensive Antibacterial Coating (DAC®) hydrogel against the C. acnes free-floating form by means of the XTT cell proliferation reduction assay. Then, we explored photometrically the effect of hydrogel and soluble tyrosol at reducing C. acnes biofilm formation on titanium alloy disks that simulated orthopaedic implants by using the minimum biofilm inhibition concentration assay. In particular, 16 disks were sequentially allocated to each of the following testing conditions: (1) hydrogel alone; (2) tyrosol-loaded hydrogel; (3) rifampicin-supplemented hydrogel; (4) vancomycin-loaded hydrogel; and (5) soluble tyrosol. Subsequently, implants were sonicated and cell viability was evaluated in terms of the XTT assay. RESULTS: Tyrosol was effective in inhibiting C. acnes planktonic (free-floating) growth demonstrating MIC values of 63 mM (9 mg/mL) and MBC values of 250 mM (35 mg/mL). Concerning synergy assessment, the checkerboard testing revealed additivity among tyrosol and rifampicin with a fractional inhibitory concentration index of 0.56. In addition, a hydrogel coating with tyrosol at the MIC showed no difference in the inhibition of free-floating C. Acnes form over control (median absorbance [MA] for tyrosol-supplemented hydrogel versus control groups were 0.21 [interquartile range {IQR}, 0.19-0.24] versus 0.26 [IQR, 0.23-0.31], p = 0.066). Furthermore, loaded hydrogel with tyrosol at 597 mg/mL (1 M) was no more effective than control in reducing C. acnes biofilm formation (MAs for tyrosol versus control were 0.12 [IQR, 0.11-0.13] versus 0.14 [IQR, 0.12-0.16], respectively; p = 0.076). This was also the case when we considered hydrogel in conjunction with vancomycin and rifampicin (MAs for vancomycin at 2% and 5% and rifampicin at 1% versus biofilm control were 0.139 [IQR, 0.133-0.143] and 0.141 [IQR, 0.133-0.143] and 0.135 [IQR, 0.128-0.146] versus 0.142 [IQR, 0.136-0.144], correspondingly). In contrast, soluble tyrosol at 597 mg/mL (1 M) inhibited biofilm formation compared to control (MAs for tyrosol and control groups were 0.11 [IQR, 0.09-0.13] versus 0.13 [IQR, 0.12-0.14], p = 0.007). CONCLUSIONS: Although the implant coating with hydrogel (either pure or supplemented with antimicrobial agents) did not diminish C. acnes biofilm development in vitro, soluble tyrosol at 597 mg/mL (1 M) exceeded the meaningful biofilm inhibition threshold of 80%. CLINICAL RELEVANCE: The results of the current preclinical investigation did not support the use of a fast, bioresorbable hydrogel as a coating method against C. acnes biofilms. Instead, direct local administration of soluble tyrosol at high concentrations should be further tested in future animal studies.

Putative Microbial Population Shifts Attributable to Nasal Administration of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a.

Changes in bacterial composition of nasal microbiota may alter the host's susceptibility to several infectious and allergic diseases such as chronic rhinosinusitis and allergic rhinitis. The aim of this study was to evaluate the effects of 1-week administration of a probiotic product, composed by a combination of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a, on the nostril microbiota. Differences in the nasal microbiota composition were investigated by using a next-generation sequencing approach. A strong and significant decrease in Staphylococcus aureus abundance was detected immediately after the bacterial administration. Moreover, comparing the microbial networks of nostril microbiota before and 1 month after the end of treatment, we detected an increase in the total number of both bacterial nodes and microbial correlations, with particular regard to the beneficial ones. Furthermore, a less abundance of microbial genera commonly associated to potential harmful bacteria has been observed. These results suggest a potential ability of S. salivarius 24SMBc and S. oralis 89a to regulate and reorganize the nasal microbiota composition, possibly favoring those microorganisms that may be able to limit the overgrowth of potential pathogens.

Salivary calculi microbiota: new insights into microbial networks and pathogens reservoir.

Sialolithiasis represents the most common disorders of salivary glands in middle-aged patients. It has been hypothesized that the retrograde migration of bacteria from the oral cavity to gland ducts may facilitate the formation of stones. Thus, in the present study, a microbiome characterization of salivary calculi was performed to evaluate the abundance and the potential correlations between microorganisms constituting the salivary calculi microbiota. Our data supported the presence of a core microbiota of sialoliths constituted principally by Streptococcus spp., Fusobacterium spp. and Eikenella spp., along with the presence of important pathogens commonly involved in infective sialoadenitis.

Probiotics Streptococcus salivarius 24SMB and Streptococcus oralis 89a interfere with biofilm formation of pathogens of the upper respiratory tract.

BACKGROUND: Infections of the ears, paranasal sinuses, nose and throat are very common and represent a serious issue for the healthcare system. Bacterial biofilms have been linked to upper respiratory tract infections and antibiotic resistance, raising serious concerns regarding the therapeutic management of such infections. In this context, novel strategies able to fight biofilms may be therapeutically beneficial and offer a valid alternative to conventional antimicrobials. Biofilms consist of mixed microbial communities, which interact with other species in the surroundings and communicate through signaling molecules. These interactions may result in antagonistic effects, which can be exploited in the fight against infections in a sort of "bacteria therapy". Streptococcus salivarius and Streptococcus oralis are α-hemolytic streptococci isolated from the human pharynx of healthy individuals. Several studies on otitis-prone children demonstrated that their intranasal administration is safe and well tolerated and is able to reduce the risk of acute otitis media. The aim of this research is to assess S. salivarius 24SMB and S. oralis 89a for the ability to interfere with biofilm of typical upper respiratory tract pathogens. METHODS: To investigate if soluble substances secreted by the two streptococci could inhibit biofilm development of the selected pathogenic strains, co-cultures were performed with the use of transwell inserts. Mixed-species biofilms were also produced, in order to evaluate if the inhibition of biofilm formation might require direct contact. Biofilm production was investigated by means of a spectrophotometric assay and by confocal laser scanning microscopy. RESULTS: We observed that S. salivarius 24SMB and S. oralis 89a are able to inhibit the biofilm formation capacity of selected pathogens and even to disperse their pre-formed biofilms. Diffusible molecules secreted by the two streptococci and lowered pH of the medium revealed to be implied in the mechanisms of anti-biofilm activity. CONCLUSIONS: S. salivarius 24SMB and S. oralis 89a possess desirable characteristics as probiotic for the treatment and prevention of infections of the upper airways. However, the nature of the inhibition appear to be multifactorial and additional studies are required to get further insights.

BAG-S53P4 as bone graft extender and antimicrobial activity against gentamicin- and vancomycin-resistant bacteria.

AIM: To evaluate the suitability of bioactive glass (BAG)-S53P4 as a bone-graft extender for large bony defect filling in bone and joint infection. MATERIALS & METHODS: Antimicrobial activity of BAG-S53P4 against clinically relevant strains isolated from bone and joint infections was evaluated by means of time-kill curves in presence of bone graft. Furthermore, the susceptibility to BAG of strains resistant to vancomycin and gentamicin was assessed. RESULTS: Though attenuated, BAG maintains a good in vitro antimicrobial activity in presence of human body fluids and tissues contained in bone graft, with the exception of Enterococcus faecalis. CONCLUSION: BAG-S53P4 is a suitable bone substitute that can be used as an extender with autologous bone graft to promote better fusion and healing.

In vitro comparison between α-tocopheryl acetate and α-tocopheryl phosphate against bacteria responsible of prosthetic and joint infections.

Biofilm-related infections represent a recurrent problem in the orthopaedic setting. In recent years, great interest was directed towards the identification of novel molecules capable to interfere with pathogens adhesion and biofilm formation on implant surfaces. In this study, two stable forms of α-tocopherol, the hydrophobic acetate ester and the water-soluble phosphate ester, were tested in vitro as coating for titanium prosthesis. Antimicrobial activity against microorganisms responsible of prosthetic and joints infections was assessed by broth microdilution method. In addition, α-tocopherol esters were evaluated for both their ability to hamper bacterial adhesion to and biofilm formation on sandblasted titanium surfaces. Results showed that only α-tocopheryl phosphate displayed antimicrobial activity against the tested strains. Both esters were able to significantly interfere with bacterial adhesion and to prevent biofilm formation, especially by Staphylococcus aureus and Staphylococcus epidermidis. The activity of α-tocopheryl phosphate was greater than that of α-tocopheryl acetate. Alterations at membrane levels have been reported in literature and may be likely responsible for the interference on bacterial adhesion and biofilm formation shown by α-tocopherol esters. Although further studies are needed to better investigate the mechanisms of action and the spectrum of activity of α-tocopherol esters, these characteristics together with the positive effect on wound healing and immune response, make these molecules promising candidate for coating in order to prevent implant-associated infections.