Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients.

BACKGROUND: Stenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains. RESULTS: All S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness. CONCLUSIONS: The main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

Role of excessive inflammatory response to Stenotrophomonas maltophilia lung infection in DBA/2 mice and implications for cystic fibrosis.

Stenotrophomonas maltophilia is a pathogen that causes infections mainly in immunocompromised patients. Despite increased S. maltophilia isolation from respiratory specimens of patients with cystic fibrosis (CF), the real contribution of the microorganism to CF pathogenesis still needs to be clarified. The aim of the present study was to evaluate the pathogenic role of S. maltophilia in CF patients by using a model of acute respiratory infection in DBA/2 mice following a single exposure to aerosolized bacteria. The pulmonary bacterial load was stable until day 3 and then decreased significantly from day 3 through day 14, when the bacterial load became undetectable in all infected mice. Infection disseminated in most mice, although at a very low level. Severe effects (swollen lungs, large atelectasis, pleural adhesion, and hemorrhages) of lung pathology were observed on days 3, 7, and 14. The clearance of S. maltophilia observed in DBA/2 mouse lungs was clearly associated with an early and intense bronchial and alveolar inflammatory response, which is mediated primarily by neutrophils. Significantly higher levels of interleukin-1beta (IL-1beta), IL-6, IL-12, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), GROalpha/KC, MCP-1/JE, MCP-5, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-2, and TARC were observed in infected mice on day 1 with respect to controls. Excessive pulmonary infection and inflammation caused systemic effects, manifested by weight loss, and finally caused a high mortality rate. Taken together, our results show that S. maltophilia is not just a bystander in CF patients but has the potential to contribute to the inflammatory process that compromises respiratory function.

Clinical, microbiologic, and biochemical effects of subgingival administration of a Xanthan-based chlorhexidine gel in the treatment of periodontitis: a randomized multicenter trial.

BACKGROUND: The use of locally delivered antibacterials containing chlorhexidine (CHX) was proposed to improve the effectiveness of non-surgical periodontal treatment. The present multicenter randomized study investigated the effects of a xanthan-based chlorhexidine (Xan-CHX) gel used as an adjunct to scaling and root planing (SRP) in the treatment of chronic periodontitis. METHODS: Ninety-eight systemically healthy subjects with moderate to advanced periodontitis were recruited in four centers (59 females and 39 males; aged 24 to 58 years). For each subject, two experimental sites located in two symmetric quadrants were chosen with probing depths (PD) >or=5 mm and positive for bleeding on probing (BOP). These two sites were randomized at the split-mouth level with one receiving a single SRP treatment and the other receiving a single SRP + Xan-CHX gel treatment. Supragingival plaque, modified gingival index, PD, clinical attachment level (CAL), and BOP were evaluated at baseline (prior to any treatment) and after 3 and 6 months. At the same times, subgingival microbiologic samples and gingival crevicular fluid (GCF) were collected for the analysis of total bacterial counts (TBCs), including the identification of eight putative periodontopathogens, and alkaline phosphatase (ALP) activity, respectively. RESULTS: The Xan-CHX treatment group showed greater improvements compared to the SRP group for PD and CAL at 3 and 6 months (P <0.001). The differences in PD reduction between the treatments were 0.87 and 0.83 mm at 3 and 6 months, respectively (P <0.001); for CAL, these were 0.94 and 0.90 mm, respectively (P <0.001). Similar behavior was seen when the subgroup of pockets >or=7 mm was considered. The percentage of sites positive for BOP was similar between the treatments at each time point. For the comparisons between the treatment groups, no differences were seen in the TBCs and GCF ALP activity at baseline and 6 months; in contrast, slightly, but significantly, lower scores were recorded for the Xan-CHX treatment group at 3 months (P = 0.018 and P = 0.045, respectively). Moreover, greater reductions in the percentages of sites positive for the eight putative periodontopathic bacteria were generally seen for the Xan-CHX treatment group compared to SRP alone. CONCLUSIONS: The adjunctive use of Xan-CHX gel promoted greater PD reductions and CAL gains compared to SRP alone. These results were concomitant with better microbiologic and biochemical outcomes when Xan-CHX gel use was added to SRP, particularly up to 3 months after treatment.

Comparison of culture methods and multiplex PCR for the detection of periodontopathogenic bacteria in biofilm associated with severe forms of periodontitis.

Conventional culture methods and Multiplex PCR, both of which we have been used for a long time in our clinical microbiology laboratory, were compared for their ability to detect a selected panel of periodontopathic bacteria: Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia. Tests were performed in a single subgingival sample taken from a periodontal diseased site with a probing depth equal to or greater than 6mm. The results were compared site-by-site, taking into account the quality and the presence or absence of pathogens. 529 samples of subgingival plaque were analysed and the prevalence of the six species monitored varied in relation to the species itself and the method of detection. The most represented species is F. nucleatum, with a percentage of positive variability between 44.9% PCR and 46.5% culture test. Generally, the lowest prevalence was determined by culture test, with the exception of E. corrodens and F. nucleatum, which, unlike other bacteria, have been seen in higher percentages in culture with respect to PCR. For both methods, there was a good degree of accuracy in the determination of A. actinomycetemcomitans, C. rectus, E. corrodens, and P. gingivalis. It becomes weak for F. nucleatum and P. intermedia. Both culture and PCR techniques introduced many methodological problems when applied in oral microbiology, but the ideal technique for accurate detection of pathogens in subgingival plaque samples has yet to be developed.

Graft versus host disease-related Hafnia alvei colonization and probable infection.

We describe the case of a graft versus host disease (GvHD) patient, in whom Hafnia alvei was cultured as a single organism, and at high bacterial counts from stool samples, from the onset of the disease until its resolution. This case is a further example of the contentious role of this species in causing human intestinal disease. Furthermore, it focuses on enteric damage by GvHD as a risk factor for acquiring H. alvei colonization, and probably infection.

Femoral prosthesis infection by Rhodotorula mucilaginosa.

This case report is a case history of a femoral prosthesis infection caused by Rhodotorula mucilaginosa in a human immunodeficiency virus patient. Though the pathogenicity of this organism for bone tissue has been previously reported, this is the first reported case of an orthopedic prosthesis infection by this species of the genus Rhodotorula.

Factors associated with adherence to and biofilm formation on polystyrene by Stenotrophomonas maltophilia: the role of cell surface hydrophobicity and motility.

We tested 40 clinical Stenotrophomonas maltophilia strains to investigate the possible correlation between adherence to and formation of biofilm on polystyrene, and cell surface properties such as hydrophobicity and motility. Most of the strains were able to adhere and form biofilm, although striking differences were observed. Eleven (27.5%) of the strains were hydrophobic, with hydrophobicity greatly increasing as S. maltophilia attached to the substratum. A positive correlation was observed between hydrophobicity and levels of both adhesion and biofilm formation. Most of the isolates showed swimming and twitching motility. A highly significant negative correlation was observed between swimming motility and level of hydrophobicity. Hydrophobicity is thus a significant determinant of adhesion and biofilm formation on polystyrene surfaces in S. maltophilia.

Internal decontamination of dental implants: an in vivo randomized microbiologic 6-month trial on the effects of a chlorhexidine gel.

BACKGROUND: Microbial penetration inside an implant's internal cavity results in a bacterial reservoir that has been associated with an area of inflamed connective tissue facing the fixture-abutment junction. The aim of the present clinical trial was to evaluate the effectiveness of a 1% chlorhexidine gel on the internal bacterial contamination of implants with screw-retained abutments. METHODS: Thirty subjects (age range: 27.3 to 54.2 years) underwent single implant restoration. Three months after prosthodontic restoration, the modified sulcus bleeding index, modified plaque index, full-mouth plaque score, and full-mouth bleeding score were recorded. Microbiologic samples were also collected from the internal part of each fixture. Subjects were then divided into two equal groups: control and test groups (CG and TG, respectively). The CG had the abutment screwed and the crown cemented without any further intervention. Conversely, the TG had the internal part of the fixture filled with a 1% chlorhexidine gel before the abutment placement and screw tightening. Six months later, microbiologic and clinical procedures were repeated in both groups. Total bacterial count and multiplex polymerase chain analysis were performed to detect specific pathogens. RESULTS: Clinical parameters remained stable throughout the study. From baseline to the 6-month examination, the total bacterial counts underwent a significant reduction in the TG (P<0.05). Detection of the single pathogen species did not show any significant differences. However, periopathogens were detected more frequently in the CG. CONCLUSION: The application of a 1% chlorhexidine gel seemed to be an effective method to reduce bacterial colonization of the implant cavity over a 6-month period.

Two cases of vaginitis caused by itraconazole-resistant Saccharomyces cerevisiae and a review of recently published studies.

Genitourinary infections caused by non-Candida yeasts are uncommon, and especially due to Saccharomyces cerevisiae. We describe the cases of two adult females with vulvovaginal infections caused by itraconazole-resistant S. cerevisiae who made a full recovery after oral fluconazole therapy. We also provide a concise review of recently published studies on this topic.

Clinical and microbiologic effects of subgingival controlled-release delivery of chlorhexidine chip in the treatment of periodontitis: a multicenter study.

BACKGROUND: The main therapeutic approach for periodontal diseases is mechanical treatment of root surfaces via scaling and root planing (SRP). Multicenter clinical trials have demonstrated that the adjunctive use of a chlorhexidine (CHX) chip is effective in improving clinical results compared to SRP alone. However, some recent studies failed to confirm these clinical results, and conflicting results were reported regarding the effects of the CHX chip on subgingival microflora. The aim of this study was to provide further data on the clinical and microbiologic effects of CHX chips when used as an adjunct to SRP. METHODS: A total of 116 systemically healthy individuals with moderate to advanced periodontitis, aged 33 to 65 years, were recruited from the Departments of Periodontology of four Italian universities. For each subject, two experimental sites were chosen that had probing depths (PD) > or =5 mm and bleeding on probing (BOP) and were located in two symmetric quadrants. These two sites were randomized at the split-mouth level, with one receiving SRP treatment alone and the other receiving treatment with SRP plus one CHX chip (SRP + CHX). PD, relative attachment level (RAL), and BOP were evaluated at baseline, prior to any treatment, and after 3 and 6 months. Supragingival plaque and the modified gingival index were evaluated at baseline and after 15 days and 1, 3, and 6 months. Subgingival microbiologic samples were harvested at baseline and after 15 days and 1, 3, and 6 months, cultured for total bacterial counts (TBCs), and investigated by polymerase chain reaction analysis for the identification of eight putative periodontopathogens. RESULTS: When all of the pockets were considered, the PD and RAL were significantly less at 3 and 6 months compared to the baseline scores (P <0.01) for both treatments. Moreover, the PD was reduced in the SRP + CHX treatment group compared to the SRP treatment group at 3 and 6 months, whereas the RAL was similar for both treatments at 3 months and was reduced in the SRP + CHX treatment group at 6 months. The differences in PD reductions between the treatments were 0.30 and 0.55 mm at 3 and 6 months, respectively (P <0.01); for the RAL gain, the differences were 0.28 and 0.64 mm, respectively (P <0.001). The TBCs decreased significantly with both treatments. A similar, although less evident, pattern was noted when only the pockets with an initial PD > or =7 mm were considered. The percentage of sites positive for BOP was similar between the treatments at each time point. At 15 days and 1 month, the TBC for the SRP + CHX treatment group was significantly lower than for the SRP treatment group (P <0.01 and P <0.05, respectively). Over time, both treatments generally reduced the percentages of sites positive for the eight putative periodontopathic bacteria, although greater reductions were seen often for the SRP + CHX treatment group. CONCLUSIONS: The adjunctive use of the CHX chip resulted in a significant PD reduction and a clinical attachment gain compared to SRP alone. These results were concomitant with a significant benefit of SRP + CHX treatment on the subgingival microbiota.

Diagnosis in periodontology: a further aid through microbiological tests.

Most of the current knowledge of the complex microbiology of oral biofilms, which initiates and maintains periodontal lesions, has been facilitated by the introduction of molecular techniques. Several studies exalt the high sensitivity and specificity of molecular tests in the detection and quantification of periodontal pathogens. Although they have large a diffusion, the old method of bacterial culture remains nowadays the gold standard when determining the utility of a new microbial test. Moreover, cultures have the important advantage of allowing an antibiotic sensitivity test and this is much more important during the treatment of patients with aggressive periodontitis.

Use of diode laser 980 nm as adjunctive therapy in the treatment of chronic periodontitis. A randomized controlled clinical trial.

The primary goal of periodontal therapy is the removal of supra and subgingival bacterial deposits by mechanical debridement consisting in scaling and root-planing (SRP) using manual or power-driven instruments. The complete removal of bacteria and their toxins from periodontal pockets is not always achieved with conventional mechanical treatment. The use of lasers as an adjunctive therapy for periodontal disease may improve tissue healing by bactericidal and detoxification effects. The aim of this study was to compare the effectiveness of Diode laser used as adjunctive therapy of SRP to that of SRP alone for non surgical periodontal treatment in patients with chronic periodontitis. Nineteen pairs of teeth with untreated chronic periodontitis were selected in 13 patients and randomly treated by SRP alone (control group) or by SRP + laser irradiation (test group). Clinical measurements (PPD, CAL, BOP, GI, PI) were performed before treatment at baseline (T0) and at T1 (after 4 weeks), T2 (8 weeks), T3 (12 weeks), T4 (6 months). Subgingival plaque samples were taken at baseline and after treatment and examined for 8 periopathogens bacteria using PCR technique. The present study showed that the additional treatment with diode laser may lead to a slightly improvement of clinical parameters, whereas no significant differences between test and control group in reduction of periodontopathogens were found.

Biofilm formation by the emerging fungal pathogen Trichosporon asahii: development, architecture, and antifungal resistance.

Trichosporon asahii is the most common cause of fatal disseminated trichosporonosis, frequently associated with indwelling medical devices. Despite the use of antifungal drugs to treat trichosporonosis, infection is often persistent and is associated with high mortality. This drove our interest in evaluating the capability of T. asahii to form a biofilm on biomaterial-representative polystyrene surfaces through the development and optimization of a reproducible T. asahii-associated biofilm model. Time course analyses of viable counts and a formazan salt reduction assay, as well as microscopy studies, revealed that biofilm formation by T. asahii occurred in an organized fashion through four distinct developmental phases: initial adherence of yeast cells (0 to 2 h), germination and microcolony formation (2 to 4 h), filamentation (4 to 6 h), and proliferation and maturation (24 to 72 h). Scanning electron microscopy and confocal scanning laser microscopy revealed that mature T. asahii biofilms (72-h) displayed a complex, heterogeneous three-dimensional structure, consisting of a dense network of metabolically active yeast cells and hyphal elements completely embedded within exopolymeric material. Antifungal susceptibility testing demonstrated a remarkable rise in the MICs of sessile T. asahii cells against clinically used amphotericin B, caspofungin, voriconazole, and fluconazole compared to their planktonic counterparts. In particular, T. asahii biofilms were up to 16,000 times more resistant to voriconazole, the most active agent against planktonic cells (MIC, 0.06 microg/ml). Our results suggest that the ability of T. asahii to form a biofilm may be a major factor in determining persistence of the infection in spite of in vitro susceptibility of clinical isolates.

Effectiveness of ultrasonic instruments in the therapy of severe periodontitis: a comparative clinical-microbiological assessment with curettes.

Patients with deep periodontal pockets were treated with either Vector System (TG) or manual instruments (CG). Clinical assessments by supragingival plaque (PL+), gingival index (GI), bleeding on probing (BOP), probing depth (PD), clinical attachment level (CAL) and subgingival plaque collection for microbiological analysis were made prior to and after treatment. Multiplex Polymerase Chain Reaction was used to determine the presence of Actinobacillus actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythensis and Treponema denticola. GI, PD, CAL and the number of BOP+ sites underwent a significant reduction over time in both groups. When compared to baseline, the pair-wise analyses showed significantly lower PD and CAL at 6 months in the CG and significant reductions in the GI, PD, CAL and a number of BOP+ sites at 3 and 6 months in the TG. For microbiological results, significant reductions were seen for C. rectus and R. gingivalis in the CG and for T. forsythensis, E. corrodens and T. denticola in the TG. The total bacterial count underwent a reduction in both groups. Both ultrasonic and manual debridement are equally effective in non-surgical periodontal therapy of severe periodontitis in terms of clinical and microbiological effects.

In vitro pharmacodynamic characteristics of amphotericin B, caspofungin, fluconazole, and voriconazole against bloodstream isolates of infrequent Candida species from patients with hematologic malignancies.

Time-kill and postantifungal effect (PAFE) of amphotericin B, caspofungin, fluconazole, and voriconazole were determined against clinical isolates of Candida guilliermondii, Candida kefyr, and Candida lusitaniae. Azoles displayed fungistatic activity and no measurable PAFE, regardless of the concentration tested. Amphotericin B and caspofungin demonstrated concentration-dependent fungicidal activity, although amphotericin B only produced a significant dose-dependent PAFE against all isolates tested.

Clinical and microbiological effects of different restorative materials on the periodontal tissues adjacent to subgingival class V restorations.

OBJECTIVES: The relationship between subgingival dental restorations and periodontal health has been thoroughly investigated for many years. However, longitudinal data on the subgingival microflora features after the placement of well-finished subgingival restorations are still lacking. Therefore, this study compares the short-term clinical and microbiological features occurring in the gingiva after the completion of different subgingival restorations. MATERIAL AND METHODS: Sixteen systemically healthy subjects, 10 males and six females (ages: 31.7-45.8 years; mean age 39.3+/-5.1 years), who were non-smokers and were positive for the presence of three cervical abrasion/erosion defects to be restored in three different adjacent teeth were enrolled in this study. The cervical abrasion/erosion defects were each restored by using one of three different materials: amalgam, glass ionomer cement, or composite resin. Immediately before class V cavity preparations and restorations (baseline), clinical monitoring and subgingival plaque sampling were performed in the mid-buccal aspect of each experimental restored tooth and in one adjacent sound, non-treated, control tooth. These procedures were repeated every 4 months over the following 1 year. RESULTS: Throughout the study, the clinical parameters recorded did not change significantly in any of the experimental groups, and no differences were detected among them at each clinical session. Over this time, no significant changes in the composition of the subgingival microflora were observed in amalgam, glass ionomer cement, and control groups. Conversely, in the composite resin group, there was a significant increase in the total bacterial counts, and a significant (p<0.05) decrease in Gram-positive, aerobic bacteria, which was associated with a significant (p<0.05) increase in the Gram-negative, anaerobic microbiota. CONCLUSIONS: Over a 1-year observation period, amalgam, glass ionomer cement, and composite resin subgingival restorations do not significantly affect the clinical parameters recorded. However, composite resin restorations may have some negative effects on the quantity and quality of subgingival plaque.