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

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

Mutations in cdsA and pgsA Correlate with Daptomycin Resistance in Streptococcus mitis and S. oralis.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

Streptococcus Oralis meningitis from right sphenoid Meningoencephalocele and cerebrospinal fluid leak.

BACKGROUND: Streptococcus oralis belongs to the Streptococcus mitis group and is part of the normal flora of the nasal and oropharynx (Koneman et al., The Gram-positive cocci part II: streptococci, enterococci and the 'Streptococcus-like' bacteria. Color atlas and textbook of diagnostic microbiology, 1997). Streptococcus oralis is implicated in meningitis in patients with decreased immune function or from surgical manipulation of the central nervous system. We report a unique case of meningitis by Streptococcus oralis in a 58-year-old patient with cerebral spinal fluid leak due to right sphenoid meningoencephalocele. CASE PRESENTATION: A 58-year-old female presented in the emergency department due to altered mental status, fevers, and nuchal rigidity. Blood cultures were positive for Streptococcus oralis. Magnetic resonance stereotactic imaging of head with intravenous gadolinium showed debris in lateral ventricle occipital horn and dural thickening/enhancement consistent with meningitis. There was also a right sphenoidal roof defect, and meningoencephalocele with cerebrospinal fluid leak as a result. The patient was treated with ceftriaxone and had endoscopic endonasal repair of defect. She had complete neurologic recovery 3 months later. CONCLUSIONS: Cerebrospinal fluid leak puts patients at increased risk for meningitis. Our case is unique in highlighting Streptococcus oralis as the organism implicated in meningitis due to cerebrospinal fluid leak.

A Descriptive Study of Chlorhexidine as a Disinfectant in Cleft Palate Surgery.

OBJECTIVES: Chlorhexidine is seen as the golden standard of disinfectants. It is widely used to clean surgical sites; however, many studies indicate resistance of pathogens to chlorhexidine. One study indicated that pathogenic microorganisms were isolated from the soft palate cleft region in 57% of patients with facial clefts. The objectives of our study were to determine (1) if chlorhexidine application is effective in removing pathogens from the surgical site in these patients, and (2) if any pathogens are isolated, determine if they are resistant to other antimicrobials. DESIGN: A descriptive observational study. SETTINGS: A private practice that specializes in facial cleft surgery, with a country-wide patient base. All procedures were executed by one oral and maxillofacial surgeon. PARTICIPANTS: All patients (N=50) who presented for primary repair of the soft palate cleft were included in the study. INCLUSION CRITERIA: written consent from parent(s), and patient cleared as systemically healthy by a pediatric physician. EXCLUSION CRITERIA: patient(s) with systemic infections (eg, flu) and/or any local infections (eg, tonsillitis). There were 25 males and 25 females with an average age of 7 months and 16 days included in the study. METHODS: Swabs were taken from the surgical site of all 50 patients with cleft soft palate and were sent for culture, identification and antimicrobial sensitivity. The swabs were taken before disinfecting the site as well as after 2 minutes of disinfecting the surgical site with chlorhexidine. Results were compared against each other. RESULTS: Positive cultures with 28 different pathogenic microorganisms that were identified in 47 patients before cleaning the surgical site with the chlorhexidine. The most dominant pathogens were K. pneumonia (n=22), H. influenza (n=18) and S. aureus (n=10). Of the pathogens found, 13 (46%) were still present on the swabs taken after disinfecting with chlorhexidine. K. pneumonia (n= 13), H. influenza (n=11) and S. aureus (n=9) were still the most prevalent pathogens. CONCLUSIONS: This study demonstrated that 61 of the total of 113 pathogens isolated (54%), survived after 2 minutes of disinfecting the surgical and surrounding area with chlorhexidine, thus intensifying the chances of post-operative infection.

Streptococcus mitis and S. oralis Lack a Requirement for CdsA, the Enzyme Required for Synthesis of Major Membrane Phospholipids in Bacteria.

Synthesis and integrity of the cytoplasmic membrane are fundamental to cellular life. Experimental evolution studies have hinted at unique physiology in the Gram-positive bacteria Streptococcus mitis and S. oralis These organisms commonly cause bacteremia and infectious endocarditis (IE) but are rarely investigated in mechanistic studies of physiology and evolution. Unlike in other Gram-positive pathogens, high-level (MIC ≥ 256 µg/ml) daptomycin resistance rapidly emerges in S. mitis and S. oralis after a single drug exposure. In this study, we found that inactivating mutations in cdsA are associated with high-level daptomycin resistance in S. mitis and S. oralis IE isolates. This is surprising given that cdsA is an essential gene for life in commonly studied model organisms. CdsA is the enzyme responsible for the synthesis of CDP-diacylglycerol, a key intermediate for the biosynthesis of all major phospholipids in prokaryotes and most anionic phospholipids in eukaryotes. Lipidomic analysis by liquid chromatography-mass spectrometry (LC-MS) showed that daptomycin-resistant strains have an accumulation of phosphatidic acid and completely lack phosphatidylglycerol and cardiolipin, two major anionic phospholipids in wild-type strains, confirming the loss of function of CdsA in the daptomycin-resistant strains. To our knowledge, these daptomycin-resistant streptococci represent the first model organisms whose viability is CdsA independent. The distinct membrane compositions resulting from the inactivation of cdsA not only provide novel insights into the mechanisms of daptomycin resistance but also offer unique opportunities to study the physiological functions of major anionic phospholipids in bacteria.

[Reconstruction of Anterior Mitral Leaflet Using Fresh Autologous Pericardial Patch for Active Infective Endocarditis;Report of a Case].

Several reports have described that the prognosis of patients with mitral valve regurgitation due to active infective endocarditis (IE) is better after mitral valve plasty (MVP) than replacement (MVR). However, extensive destruction of valve tissue might cause difficulties with MVP. We repaired a widely-affected anterior mitral leaflet (AML) using an autologous pericardial patch. A 44-year-old woman with mitral regurgitation presented with prolonged fever and backache. We made a diagnosis of active IE accompanied by mitral valve regurgitation. We performed MVP, widely resected the infected areas of the AML, and reconstructed the defective area using the pericardial patch. She was discharged after four weeks of antibiotic therapy, when she was free of recurrence. The pericardial patch facilitated MVP and was effective for treating mitral valve regurgitation due to active IE.

Arthritis-induced alveolar bone loss is associated with changes in the composition of oral microbiota.

Rheumatoid arthritis (RA) and periodontitis (PD) are chronic inflammatory disorders that cause bone loss. PD tends to be more prevalent and severe in RA patients. Previous experimental studies demonstrated that RA triggers alveolar bone loss similarly to PD. The aim of this study was to investigate if arthritis-induced alveolar bone loss is associated with modification in the oral microbiota. Checkerboard DNA-DNA hybridization was employed to analyze forty oral bacterial species in 3 groups of C57BL/6 mice: control (n = 12; without any challenge); Y4 (n = 8; received oral inoculation of Aggregatibacter Actinomycetemcomitans strain FDC Y4) and AIA group (n = 12; chronic antigen-induced arthritis). The results showed that AIA and Y4 group exhibited similar patterns of bone loss. The AIA group exhibited higher counts of most bacterial species analyzed with predominance of Gram-negative species similarly to infection-induced PD. Prevotella nigrescens and Treponema denticola were detected only in the Y4 group whereas Campylobacter showae, Streptococcus mitis and Streptococcus oralis were only found in the AIA group. Counts of Parvimonas micra, Selenomonas Noxia and Veillonella parvula were greater in the AIA group whereas Actinomyces viscosus and Neisseira mucosa were in large proportion in Y4 group. In conclusion, AIA is associated with changes in the composition of the oral microbiota, which might account for the alveolar bone loss observed in AIA mice.

Streptococcal co-infection augments Candida pathogenicity by amplifying the mucosal inflammatory response.

Mitis-group streptococci are ubiquitous oral commensals that can promote polybacterial biofilm virulence. Using a novel murine oral mucosal co-infection model we sought to determine for the first time whether these organisms promote the virulence of C. albicans mucosal biofilms in oropharyngeal infection and explored mechanisms of pathogenic synergy. We found that Streptococcus oralis colonization of the oral and gastrointestinal tract was augmented in the presence of C. albicans. S. oralis and C. albicans co-infection significantly augmented the frequency and size of oral thrush lesions. Importantly, S. oralis promoted deep organ dissemination of C. albicans. Whole mouse genome tongue microarray analysis showed that when compared with animals infected with one organism, the doubly infected animals had genes in the major categories of neutrophilic response/chemotaxis/inflammation significantly upregulated, indicative of an exaggerated inflammatory response. This response was dependent on TLR2 signalling since oral lesions, transcription of pro-inflammatory genes and neutrophil infiltration, were attenuated in TLR2(-/-) animals. Furthermore, S. oralis activated neutrophils in a TLR2-dependent manner in vitro. In summary, this study identifies a previously unrecognized pathogenic synergy between oral commensal bacteriaand C. albicans. This is the first report of the ability of mucosal commensal bacteria to modify the virulence of an opportunistic fungal pathogen.

An in vivo comparison of internal bacterial colonization in two dental implant systems: identification of a pathogenic reservoir.

OBJECTIVES: The aim of this study was to compare internal bacterial colonization in two implant systems, one screw root form (SRF) with an external hexagon connection and one plateau root form (PRF) with a Morse taper internal connection. MATERIALS AND METHODS: Thirty-two implants; 12 SRF and 20 PRF, were sampled in 15 patients. All implants had been in function for at least 6 months prior to sampling. The implant restoration was removed and 10 µl of sterile saline was introduced into the implant well via a sterile glass syringe. The saline was drawn back up and transferred to the laboratory for microbiological analysis. The number of aerobic and anaerobic colony forming units per millilitre was determined and the dominant micro-organism in each sample was identified by 16s rRNA gene amplicon sequencing. RESULTS: There was a significant difference between bleeding on probing around the SRF implants (3%) and the PRF implants (28%) (p = 0.0496). Bacterial colonization was identified at 11 SRF and 19 PRF implants. The numbers of anaerobic bacteria recovered from PRF implants was significantly higher than that from SRF implants (p = 0.0002). Streptococcus species and Enterococcus faecalis were found to dominate. CONCLUSIONS: This in vivo study demonstrated bacterial colonization in both types of implant systems, irrespective of the type of connection. Significantly greater anaerobic counts were found in the Morse taper internal connection implants.

Interproximal biofilm removal by intervallic use of a sonic toothbrush compared to an oral irrigation system.

BACKGROUND: The purpose of this in-vitro study was to investigate the potential of biofilm removal in interproximal tooth regions using intervallic cleaning with an oral irrigator or a sonic toothbrush. METHODS: Three-species biofilms (Streptococcus mutans (OMZ 918), Streptococcus oralis SK 248 (OMZ 60), Actinomyces naeslundii (OMZ 745)) were grown on hydroxyapatite discs for 3 days in culture media. Every 24 h, specimens were incubated for 15 min in resazurin solution (i.e., culture medium and 10 % v/v alamarBlue®) to measure the metabolic activity with a fluorescence spectrophotometer in relative fluorescence units (rfu) at baseline. Then, specimens were fixed in interproximal holding devices and underwent treatment with an oral irrigator (WF; Waterpik® Sensonic WP-100E), an active sonic toothbrush (WPa), or an inactive sonic toothbrush (WPi; Waterpik® Sensonic SR-3000E) for 10 s (n = 18/group). Untreated biofilms served as controls (CO). After treatment, bacterial activity was re-measured, and specimens were re-grown in fresh medium for 24 h until next cleaning procedure. Altogether, cleaning was repeated in intervals of three treatment days (d1, d2, d3). After d3, SEM images were taken (n = 8) and CFU was measured (n = 3). Metabolic activity was analyzed for each disc separately, rfu values were averaged for d1 to compare initial biofilm stability, and ratios of baseline and post-treatment values were compared. Results were analyzed using ANOVA with the post-hoc Scheffé test, or Kruskal-Wallis with post-hoc Mann-Whitney test. RESULTS: Median baseline rfu-values of d1 resulted in 7821.8 rfu (interquartile range = 5114.5). Highest reduction in metabolic activity was recorded significantly for the oral irrigator used for 10 s (residual activity per day d1: WF 17.9 %, WPa 58.8 %, WPi 82.5 %, CO 89.6 %; d2: WF 36.8 %, WPa 85.2 %, WPi 82.5 %, CO 90.0 %; d3: WF 17.2.%, WPa 79.6 %, WPi 96.3 %, CO 116.3 %). SEM images of untreated specimens (CO) and specimens treated with the sonic toothbrush (WPa and WPi) showed huge amounts of biofilm, while oral irrigator-treated specimens (WF) revealed barely any bacteria. CFU data confirmed the graduations between the groups. CONCLUSIONS: Cleaning of interproximal regions achieved better success with an oral irrigator as compared to the use of a sonic toothbrush. (350/350 words).

Histopathology of streptococcus mitis/oralis endophthalmitis after intravitreal injection with bevacizumab: a report of 7 patients.

PURPOSE: To report the histopathologic findings of a series of patients from an outbreak of Streptococcal endophthalmitis after intravitreal injection of bevacizumab prepared by a single compounding pharmacy. DESIGN: Case series. PARTICIPANTS: Seven surgical specimens (5 enucleated globes and 2 evisceration specimens) from 7 patients with endophthalmitis after intravitreal injection of bevacizumab. METHODS: Retrospective case series, including clinical data and histopathologic specimens examined by light microscopy. MAIN OUTCOME MEASURES: Review of clinical data included baseline visual acuity, clinical intervention, and time elapsed from injection to loss of globe. Histopathologic specimens were reviewed for pathologic changes at all tissue levels. RESULTS: Seven of 12 total patients (4 women, 3 men; mean age, 77.7 years) from an outbreak of Streptococcus mitis/oralis endophthalmitis after bevacizumab injection ultimately sustained loss of the affected globe, with an average of 139.1 days elapsed between injection and globe loss. Mean time from injection to presentation was 2.86 days (range, 1-6), and all patients were initially treated with vitreous tap and injection. Although histologic review of surgical specimens disclosed a wide range of pathologic tissue changes, recurring patterns of tissue damage were evident. All 5 enucleated globes displayed retinal detachment, fibrous proliferation with cyclitic membrane formation, rubeosis iridis, and secondary angle closure. All 7 specimens displayed persistent choroidal inflammation, in 1 case 208 days after injection. Six of 7 specimens had foci of retinal necrosis. Although vitreous cultures were positive in all cases, no organisms were identified by light microscopy in any of the 7 specimens. CONCLUSIONS: S. mitis/oralis endophthalmitis is a devastating complication of intravitreal injection with bevacizumab with a high rate of globe loss (7 of 12 patients, 58.3%) and a wide variety of severe pathologic tissue changes. Although no organisms were identified in the examined tissues, persistent inflammation was present in all cases, and fibrous proliferation resulted in cyclitic membrane formation and retinal detachment in all enucleated globes. These findings suggest that potential globe-salvaging interventions must address a pattern of changes involving persistent, chronic inflammation and fibrovascular proliferation as key components.

Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model.

BACKGROUND AND OBJECTIVES: Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. MATERIAL AND METHODS: PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 µm). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. RESULTS: When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10 . P. gingivalis showed a vitality reduction in the biofilm of 3 log10 , while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction. CONCLUSION: These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms.

Streptococcus mitis/oralis endophthalmitis management without phakic intraocular lens removal in patient with iris-fixated phakic intraocular lens implantation.

BACKGROUND: To report a case of Streptococcus mitis/oralis endophthalmitis management which had developed after complicated iris-fixated phakic intraocular (pIOL) lens implantation. CASE PRESENTATION: A 23-year-old-woman received pIOL implantation followed secondary intraocular intervention to lower intraocular pressure. The patient presented with severe pain and decreased visual acuity and was managed with intravitreal and intracameral antibiotic injection with topical applications of fortified antibiotics. Culture of aqueous humor was positive for S. mitis/oralis, which was sensitive to the empiric antibiotic regimen. Clinical features started to improve 5 days after treatment and the pIOL was left in place. The uncorrected distant visual acuity and endothelial cell count were 20/32 and 3143 cells/mm2 four weeks after treatment, respectively. CONCLUSION: S. mitis/oralis endophthalmitis after pIOL implantation could be managed with appropriate antibiotic administration without pIOL removal if accompanied by rapid clinical improvement after the initial intensive management in the absence of vitreous involvement.

Bacteremia after piezocision.

INTRODUCTION: The aim of this study was to investigate the presence of transient bacteremia after a piezocision procedure. METHODS: The sample consisted of 30 subjects (24 women, 6 men; mean age, 19.6 ± 0.7 years; range, 18.1-22.4 years) with the American Society of Anesthesiologists' physical status I. All patients had Class I skeletal and dental relationships and had fixed orthodontic treatment with the Damon system. The piezocision surgery was performed 1 week after the placement of the orthodontic appliances in all patients. Two 20-mL venous blood samples were collected before and 30 to 60 seconds after the first microincision using an aseptic technique. The samples were inoculated into BACTEC Plus aerobic and anaerobic blood culture bottles and were assessed in the BACTEC blood culture analyzer (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md). The results were analyzed statistically using the McNemar test, with P <0.05 indicating statistical significance. RESULTS: No significant difference between the preoperative and postoperative samples was determined with respect to transient bacteremia (P = 0.250). No bacteremia was detected in the pretreatment samples, although Gemella sanguinis, Streptococcus pluranimalium, and Streptococcus mitis/oralis were detected in 3 postoperative blood samples. CONCLUSIONS: The piezocision procedure might be related to transitory bacteremia. Hence, orthodontists should consider the possibility of bacterial endocarditis in at-risk patients when piezocision is part of the treatment plan.

Characterization of recombinant fluoroquinolone-resistant pneumococcus-like isolates.

Fourteen fluoroquinolone-resistant streptococcal isolates with recombinant DNA topoisomerase genes, preliminarily identified as pneumococci, were further characterized using phenotypic and genotypic approaches. Phenotypic tests classified them as atypical pneumococci. Phylogenetic relationships were analyzed by using the sequences of seven housekeeping alleles from these isolates and from isolates of Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. Four isolates grouped with S. pneumoniae, seven grouped with S. pseudopneumoniae, and three grouped with S. mitis. These results generally agreed with those obtained with an optochin susceptibility test and with the organization of the atp operon chromosomal region, encoding the F(o)F(1) H(+)-ATPase (the target of optochin). All seven isolates grouping with S. pseudopneumoniae share the same spr1368-atpC-atpA gene order; all four grouping with S. pneumoniae share the spr1368-IS1239-atpC-atpA order, and two out of the three grouping with S. mitis share the spr1284-atpC-atpA order. In addition, evidence for recombination within the seven housekeeping alleles of the S. pseudopneumoniae population was provided by several methods: the index of association (0.4598, P < 0.001), the pairwise homoplasy index, and the split-decomposition method. This study confirms the existence of pneumococci among the alpha-hemolytic streptococci with DNA topoisomerase genes showing a mosaic structure and reveals a close relationship between atypical pneumococci and S. pseudopneumoniae.

Simultaneous detection of Streptococcus pneumoniae, S. mitis, and S. oralis by a novel multiplex PCR assay targeting the gyrB gene.

A multiplex PCR (mPCR) protocol was developed for simultaneous detection of the gyrB gene in Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis, and the specificity was evaluated using 141 coccus strains. Genomic DNAs purified from S. pneumoniae, S. mitis, and S. oralis strains were efficiently detected with size differences, whereas no PCR products were amplified from any of the reference strains tested. A pilot study of 47 human oral swab specimens was conducted in parallel, and the mPCR assay identified S. pneumoniae in 1 sample, S. mitis in 8 samples, and S. oralis in 2 samples, providing a powerful means for characterization at the level of species compared with traditional culture analysis. Our results suggest that the mPCR protocol presented here is a sensitive and promising tool for the rapid detection and discrimination of S. pneumoniae, S. mitis, and S. oralis from clinical specimens.

Streptococcus endophthalmitis outbreak after intravitreal injection of bevacizumab: one-year outcomes and investigative results.

PURPOSE: To report the 1-year clinical outcomes of an outbreak of Streptococcus endophthalmitis after intravitreal injection of bevacizumab, including visual acuity outcomes, microbiological testing, and compound pharmacy investigations by the Food and Drug Administration (FDA). DESIGN: Retrospective consecutive case series. PARTICIPANTS: Twelve eyes of 12 patients who developed endophthalmitis after receiving intravitreal bevacizumab prepared by a single compounding pharmacy. METHODS: Medical records of patients were reviewed; phenotypic and DNA analyses were performed on microbes cultured from patients and from unused syringes. An inspection report by the FDA based on site visits to the pharmacy that prepared the bevacizumab syringes was summarized. MAIN OUTCOME MEASURES: Visual acuity, interventions received, time to intervention, microbiological consistency, and FDA inspection findings. RESULTS: Between July 5 and 8, 2011, 12 patients developed endophthalmitis after intravitreal bevacizumab from syringes prepared by a single compounding pharmacy. All patients received initial vitreous tap and injection, and 8 patients (67%) subsequently underwent pars plana vitrectomy (PPV). After 12 months follow-up, outcomes have been poor. Seven patients (58%) required evisceration or enucleation, and only 1 patient regained pre-injection visual acuity. Molecular testing using real-time polymerase chain reaction, partial sequencing of the groEL gene, and multilocus sequencing of 7 housekeeping genes confirmed the presence of a common strain of Streptococcus mitis/oralis in vitreous specimens and 7 unused syringes prepared by the compounding pharmacy at the same time. An FDA investigation of the compounding pharmacy noted deviations from standard sterile technique, inconsistent documentation, and inadequate testing of equipment required for safe preparation of medications. CONCLUSIONS: In this outbreak of endophthalmitis, outcomes have been generally poor, and PPV did not improve visual results at 1-year follow-up. Molecular testing confirmed a common strain of S. mitis/oralis. Contamination seems to have occurred at the compounding pharmacy, where numerous problems in sterile technique were noted by public health investigators.

Validation of a quantitative real-time PCR assay and comparison with fluorescence microscopy and selective agar plate counting for species-specific quantification of an in vitro subgingival biofilm model.

BACKGROUND AND OBJECTIVE: Subgingival biofilms are the prime etiological factor of periodontal disease. Owing to their complex polymicrobial nature, quantification of individual bacterial species within the biofilm for research and diagnostic purposes can be methodologically challenging. The aims of this study were to establish a quantitative real-time PCR (qPCR) assay to quantify the bacteria used in our 10-species in vitro 'subgingival' biofilm model and to compare the quantitative outcome with fluorescence microscopy and colony-forming unit (CFU) counts on selective agar plates. MATERIAL AND METHODS: The 10 species included in the in vitro biofilm were Streptococcus oralis, Streptococcus anginosus, Veillonella dispar, Fusobacterium nucleatum, Treponema denticola, Tannerella forsythia, Actinomyces oris, Campylobacter rectus, Porphyromonas gingivalis and Prevotella intermedia. The numbers of each species were quantified at two time points using qPCR, microscopy counting following fluorescence in-situ hybridization (FISH) or immunofluorescence staining, and counting of CFUs after growth on selective agar plates. RESULTS: All 10 species were successfully quantified using qPCR and FISH or immunofluorescence, and the eight species culturable on selective agar plates were also quantified by counting the numbers of CFUs after growth on selective agar. In early biofilm cultures, all methods showed a significant correlation, although the absolute numbers differed between methods. In late biofilm cultures, measurements obtained using qPCR and FISH or immunofluorescence, but not by CFU counts, maintained significant correlation. CFU counts yielded lower values than did measurements made using the other two methods. CONCLUSION: Quantitative PCR and epifluorescence microscopy can be easily combined with each other to determine species-specific bacterial numbers within biofilms. However, conventional bacterial cultures cannot be as efficiently combined using these molecular detection methods. This may be crucial in designing and selecting appropriate clinical diagnostic methods for subgingival biofilm samples.

Early microbial succession in redeveloping dental biofilms in periodontal health and disease.

BACKGROUND AND OBJECTIVE: The development of dental biofilms after professional plaque removal is very rapid. However, it is not clear whether most bacterial species return at similar rates in periodontally healthy and periodontitis subjects or if there are differences in bacterial recolonization between supragingival and subgingival biofilms in periodontal health and disease. MATERIAL AND METHODS: Supragingival and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from seven teeth in randomly selected quadrants after 1, 2, 4 and 7 d of no oral hygiene and analyzed using checkerboard DNA-DNA hybridization. The percentage of DNA probe counts were averaged within subjects at each time-point. Ecological succession was determined using a modified moving-window analysis. RESULTS: Succession in supragingival biofilms from subjects with periodontitis and from healthy individuals was similar. At 1 d, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1-4 d. At 4-7 d, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival plaque redevelopment. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 d, by Capnocytophaga sputigena and P. nigrescens. No significant increase in the proportions of periodontal pathogens was observed in any of the clinical groups or locations. CONCLUSION: There is a defined order in bacterial species succession in early supragingival and subgingival biofilm redevelopment after professional cleaning.