In Vitro Investigation of the Antimicrobial Effect of Three Bisphosphonates Against Different Bacterial Strains.

PURPOSE: Since the first descriptions of medication-related osteonecrosis of the jaw (MRONJ) in 2003, the pathogenesis has remained unanswered. Recent histomorphometric studies have found several microorganisms, including Actinomyces, Bacillus, Fusobacterium, Staphylococcus, Streptococcus, Selenomonas, Treponema, and Candida albicans in necrotic bone. Polymerase chain reaction studies have recently confirmed the occurrence of 48 genera. Only a few studies have examined the antimicrobial effect of bisphosphonates (BPs). The influence of bacterial growth on the etiology remains unclear. The aim of the present study was the in vitro investigation of the antimicrobial effect of 3 BPs against different bacterial strains. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 48 strains from 40 species were determined in microdilution assays against pamidronic, ibandronic, and zoledronic acid. RESULTS: Growth of gram-positive oral microbiota, which account for most microorganisms in MRONJ, was present for 2 of 22 species; 6 of 26 gram-negative species and 9 of 13 anaerobes were inhibited. The MIC values were compared with the BP bone concentrations from previous reports. Of the 48 strains, 9 had an MIC or MBC less than the bone concentrations. CONCLUSIONS: The results of the present study have demonstrated that BPs have an inhibitory effect on selected bacterial species and might inhibit the growth of some relevant pathogens in osteonecrosis. However, most of the species tested were unaffected at the concentration levels assumed present in the human jawbone. The clinical relevance of these in vitro data will better be clarified with reliable data on the BP concentrations in the human jawbone. The present study has provided a first approach toward the assessment of the interaction of oral bacteria and BPs.

Comparison of antibacterial-coated and non-coated suture material in intraoral surgery by isolation of adherent bacteria.

OBJECTIVES: In general surgery the incidence of postoperative wound infections is reported to be lower using triclosan-coated sutures. In intraoral surgery, sutures are faced with different bacterial species and the question arises whether the antibacterial-coated suture material has the same positive effects. MATERIALS AND METHODS: Triclosan-coated and uncoated suture materials were applied in 17 patients undergoing wisdom tooth extraction. Postoperatively, sutures were removed and adherent bacteria were isolated, colony-forming units (cfu) were counted, and species identified. RESULTS: Oral bacteria were found in high numbers (cfu>10(7)) on both Vicryl and the triclosan-coated Vicryl Plus. The total number of bacteria isolated from Vicryl Plus was 37% higher than for Vicryl, mainly due to increased numbers of anaerobes. The number of bacterial strains identified was higher for Vicryl ( n=203) than for Vicryl Plus (n=198), but the number of pathogens was higher on Vicryl Plus (n=100) than on Vicryl (n=97). Fewer Gram-positive strains were found on Vicryl Plus (n=95) than on Vicryl (n=107) and, conversely, more Gram-negative strains on Vicryl Plus (103vs.96). CONCLUSIONS: In terms of the total number of oral bacteria, and especially oral pathogens, that adhered to suture material, no reduction was demonstrated for Vicryl Plus. The use of triclosan-coated suture material offers no advantage in intraoral surgery.

New bacterial composition in primary and persistent/secondary endodontic infections with respect to clinical and radiographic findings.

INTRODUCTION: The aim of the present study was to analyze the microbiota of primary and secondary/persistent endodontic infections of patients undergoing endodontic treatment with respect to clinical and radiographic findings. METHODS: Samples from the root canals of 21 German patients were taken using 3 sequential sterile paper points. In the case of a root canal filling, gutta-percha was removed with sterile files, and samples were taken using sterile paper points. The samples were plated, and microorganisms were then isolated and identified morphologically by biochemical analysis and sequencing the 16S rRNA genes of isolated microorganisms. RESULTS: In 12 of 21 root canals, 33 different species could be isolated. Six (50%) of the cases with isolated microorganisms were primary, and 6 (50%) cases were endodontic infections associated with root-filled teeth. Twelve of the isolated species were facultative anaerobic and 21 obligate anaerobic. Monomicrobial infections were found for Enterococcus faecalis and Actinomyces viscosus. E. faecalis was most frequently isolated in secondary endodontic infections (33%). Moraxella osloensis was isolated from a secondary endodontic infection that had an insufficient root canal filling accompanied by a mild sensation of pain. A new bacterial composition compromising Atopobium rimae, Anaerococcus prevotii, Pseudoramibacter alactolyticus, Dialister invisus, and Fusobacterium nucleatum was recovered from teeth with chronic apical abscesses. CONCLUSIONS: New bacterial combinations were found and correlated to clinical and radiographic findings, particularly to chronic apical abscesses. M. osloensis was detected in root canals for the second time and only in German patients.

Antibiotic resistance and capacity for biofilm formation of different bacteria isolated from endodontic infections associated with root-filled teeth.

INTRODUCTION: To date, a variety of microbial species have been isolated from endodontic infections. However, endodontic clinical bacterial isolates have not been sufficiently characterized with regard to their capacity for antibiotic resistance and biofilm formation. In this study, antibiotic resistance and biofilm formation of 47 different aerobic and anaerobic bacterial isolates, belonging to 32 different species previously isolated from infected filled root canals, were studied. METHODS: Antibiotic sensitivity to 11 antibiotics including penicillin G, amoxicillin, clindamycin, gentamicin, vancomycin, tetracycline, doxycycline, fosfomycin, rifampicin, ciprofloxacin, and moxifloxacin was tested using the standardized Etest method (Bio Merieux, Marcy-1'Etoile, France). The antibiotic sensitivity of 4 control strains was also estimated in parallel. Additionally, the capacity to form biofilms was quantified using the microtiter plate test. RESULTS: Different aerobic and anaerobic bacterial species were either resistant against a number of antibiotics or showed high minimal inhibitory concentrations against clinically relevant antibiotics. Five aerobic and 2 anaerobic isolates, including Enterococcus faecalis, Streptococcus mutans, Lactobacillus fermentum, Actinomyces naeslundii, Actinomyces viscosus, Prevotella buccae, and Propionibacterium acidifaciens, were characterized as being high biofilm producers, whereas 8 aerobic and 3 anaerobic isolates were found to be moderate biofilm producers. Most isolates with resistance or markedly high minimal inhibitory concentration values were also either moderate biofilm producers or high biofilm producers. CONCLUSIONS: These results suggest that the clinical significance of endodontic infections could include that they serve as a reservoir for antibiotic resistance. Furthermore, endodontic treatment should consider the adhesion and biofilm formation by a variety of bacteria.

Concentrations of amoxicillin and clindamycin in teeth following a single dose of oral medication.

OBJECTIVES: The main purpose of this study is the detection of amoxicillin and clindamycin concentrations in teeth. MATERIALS AND METHODS: Eleven patients received 2 g of amoxicillin, and 11 patients received 600 mg of clindamycin in a single dose of oral medication at least 60 min prior to tooth extraction due to systemic diseases. The concentrations were determined in crowns and roots separately using liquid chromatography-tandem mass spectrometry (LC-MS-MS). RESULTS: Amoxicillin (13 samples) and clindamycin (12 samples) were detected in the samples of the root and crown preparations of the extracted teeth. The mean concentration of amoxicillin was 0.502 µg/g in the roots and 0.171 µg/g in the crowns. The mean concentration of clindamycin was 0.270 µg/g in the roots and 0.064 µg/g in the crowns. CONCLUSIONS: A single dose of oral amoxicillin and clindamycin leads to concentrations of both antibiotics in teeth which exceed the minimal inhibition concentration of some oral bacteria. CLINICAL RELEVANCE: The proof of antibacterial activity in dental hard tissue after oral single-dose application is new. The antimicrobial effect of amoxicillin and clindamycin concentrations in roots of teeth may be of clinical relevance to bacterial reinfection from dentinal tubules.

The antibacterial effect of gas ozone after 2 months of in vitro evaluation.

The aim of this study was to evaluate the effect of HealOzone on two microorganisms, 4 and 8 weeks after treatment, using a tooth cavity model. Four groups of caries-free third molars (n = 12) were used (A, B, C and D). Three cavities were prepared into each tooth. After sterilization, groups A and B were inoculated with Streptococcus mutans, and groups C and D, with Lactobacillus casei for 48 h. One cavity of each tooth was used to evaluate the infection. After inoculation, groups B and D were treated with ozone (60 s), and groups A and C were used as controls. Then, the two cavities of each tooth were filled with composite, and the teeth were stored in sucrose medium. The restorations were removed after 4 and 8 weeks, respectively; dentin chips were collected, and the amount of microorganisms was determined. Ozone treatment reduced significantly the amount of S. mutans compared to the control group (p ≤ 0.05). This antibacterial effect was able to be seen after 4 (p = 0.0005) and 8 (p = 0.0002) weeks. No significant difference was found between the control and treated group as far as L. casei is concerned (p > 0.05). HealOzone (60 s) can provide some antibacterial treatment against S. mutans even after 8 weeks. However, an elimination of the microorganisms through HealOzone seems not to be possible. L. casei was more resistant to ozone. Although ozone exerts a significant antibacterial effect against S. mutans, it is probably not enough as the only antibacterial method, during the fillings therapy.

An antimicrobial effect from silver-coated toothbrush heads.

PURPOSE: To examine the antimicrobial effect of silver-coated toothbrush heads in vitro. METHODS: Comparisons were made between 62 silver-coated and 62 non-coated toothbrush heads which were contaminated by different standardized microbial suspensions. The following microorganisms were investigated: Streptococcus oralis, Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus, Lactobacillus casei and Candida albicans. For cultivation of the microorganisms as well as for the subsequent determination of the colony forming units (CFUs), Columbia blood agar plates or Sabouraud agar were used. The cycle of daily toothbrushing was imitated by rinsing the brushes with 200 ml sterile tap water to reduce the number of microorganisms and the brushes were then placed upright to allow drying overnight. Colony counts were done initially (time 0) and again at 20 hours. The rinsing fluid was also examined in order to determine the decrease of microorganisms due to this step. All experiments were done twice and the means were calculated and statistically evaluated. RESULTS: There was no significant reduction in CFUs by silver-coated toothbrushes (P > 0.05) for all of the microorganisms tested. On the contrary, the colony counts for S. sanguis (P = 0.02) and C. albicans (P = 0.01) were significantly higher on silver-coated toothbrushes compared to the controls. Silver-coating in the current form did not improve any antimicrobial effects against residual bacteria present on the toothbrush head.

New bacterial compositions in root-filled teeth with periradicular lesions.

The aim of this study was to isolate and detect microorganisms of root-filled teeth associated with periradicular lesions. Specimens were sampled from patients undergoing root canal retreatment. The bacteria were characterized by morphologic and biochemical analysis and by 16S rRNA gene sequencing. Microorganisms were detected in 10 of 18 teeth. The majority of positive samples revealed a mixed culture of 2-8 species. In 2 teeth Enterococcus faecalis was the only detected species. For the first time Vagococcus fluvialis was detected in root canals. Solobacterium moorei and Fusobacterium nucleatum were the most prevalent species. Presence of F. nucleatum was associated with the presence of S. moorei in 5 of 7 cases. In all teeth with Parvimonas micra and Dialister invisus, F. nucleatum and S. moorei were found. Moreover, members of additional different genera were detected delivering bacterial compositions that have been not described yet.

Characterization of the first oral vagococcus isolate from a root-filled tooth with periradicular lesions.

Strain Endo-EH was isolated from a root-filled tooth associated with periradicular lesions. After subculturing on Columbia blood agar, phenotypic and genomic characterizations using different biochemical test systems, automated ribotyping, MALDI-TOF mass spectronomy, antibiotic susceptibility testing, and 16S rRNA gene sequence analysis were applied for further analysis. Phenotypic characterization identified this strain as Vagococcus fluvialis. Riboprint pattern analysis and 16S rRNA sequencing clearly separated it from relevant genera such as Enterococcus and Tetragenococcus and also from other Vagococcus species. This taxon is a new entry to the list of more than 200 microbial species detected in infected root canal systems.

Analysis of the antimicrobial activity of local anaesthetics used for dental analgesia.

Seven local anaesthetics and their active anaesthetic components [Ultracaine D-S (articaine hydrochloride), Carbostesin (bupivacaine hydrochloride), Scandicaine (mepivacaine hydrochloride), Xylonest (prilocaine hydrochloride), Xylocaine (lidocaine hydrochloride), Hostacaine (butanilicaine phosphate) and Novocaine (procaine hydrochloride)] were tested for their antimicrobial activity against 311 bacterial strains from 52 different species and 14 Candida albicans strains. The tested pathogens were members of the oral flora, and partly members of the skin and intestinal flora. Additionally, the antimicrobial activity of methyl-4-hydroxybenzoate, sodium disulfite, adrenaline hydrogen tartrate and adrenaline (the preservative and vasoconstrictive components of the anaesthetics) was tested. For determination of MIC and minimal bactericidal concentration (MBC), the agar dilution method using Wilkins-Chalgren agar was applied. The trade preparation Ultracaine D-S showed the most prominent antimicrobial activity with regard to both MIC and MBC. Ultracaine D-S and its active substance, articaine hydrochloride, showed similar MIC values, suggesting that the antimicrobial activity is mainly caused by the anaesthetic component. Novocaine showed the lowest antimicrobial activity and did not inhibit 35 of the species tested. The MIC values of all local anaesthetics were between 0.25 and 16 mg ml(-1). The routinely applied concentration of Ultracaine D-S was roughly four times higher, and of Hostacaine was two times higher, than the MBC values for the tested bacteria, whereas for the other anaesthetics, the MBC values were not reached or exceeded with the concentrations used. The MIC range of the preservatives was 0.5-1.0 mg ml(-1) for methyl-4-hydroxybenzoate and 0.2-0.5 mg ml(-1) for sodium disulfite. The articaine MIC values were two to three serial dilution steps lower, and the butanilicaine MIC values one to two serial dilution steps lower, than the MIC of the preservatives. The mepivacaine mean MIC values were slightly lower for Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis and Staphylococcus aureus, but higher for Streptococcus intermedius, compared with the preservative methyl-4-hydroxybenzoate. The same result was found with Streptococcus intermedius and lidocaine. Screening of 20 MIC values of 4 pure anaesthetic substances and the corresponding preservative found 2/20 instances where the MICs of the preservatives against 5 representative species (67 strains) were lower, indicating that the antimicrobial effect was mainly due to the preservative, but 18/20 results where the pure anaesthetic component showed greater antimicrobial effects compared with the preservative. The in vitro results for Carbostesin, Scandicaine and especially for Novocaine indicate that a local disinfection should be done prior to injection of the anaesthetics. Due to the results obtained with nosocomial strains (Escherichia coli, S. aureus and Pseudomonas), disinfection of the mucous membranes should be performed routinely in immunocompromised patients, regardless of the anaesthetic used.

Endogenous endophthalmitis: microorganisms, disposition and prognosis.

PURPOSE: Endogenous endophthalmitis is a severe and potentially blinding complication caused by haematogenous spreading of microorganisms. We evaluated the causative microorganisms, disposition to and prognosis of the disease. METHODS: Thirty-one eyes of 28 patients were treated between 1996 and 2006 as the result of an endogenous endophthalmitis. RESULTS: The microorganisms responsible for infection could be identified in 94% of all eyes investigated. Candida isolates were obtained in 15, gram-positive isolates in 11, gram-negative in one and Aspergillus in two of the 29 eyes studied. The majority of patients suffered from severe general disease (immuno-deficiency, severe surgical procedures, diabetes mellitus) and one third were intravenous drug abusers. Only one patient was otherwise healthy. The prognosis depended on the causative microorganisms. Whereas none of the eyes with Candida infection became blind, all except two of the eyes with gram-positive bacteria, Nocardia or Aspergillus infection lost visual function or had to be enucleated. CONCLUSION: Compared to postoperative endophthalmitis, patients with endogenous endophthalmitis are more likely to have Candida isolates. Visual prognosis depends mainly on the underlying microorganisms, and is particularly poor in the case of infection with gram-positive bacteria or Aspergillus.

Antibacterial effect of an ozone device and its comparison with two dentin-bonding systems.

Microorganisms remaining beneath restorations can cause secondary caries and pulp damage. Because of this, antimicrobial treatment could be useful. The aim of this study was to evaluate the antibacterial effect of the HealOzone device on Streptococcus mutans and to compare it with the already proven activity of two dentin-bonding systems. Thirty-five human molars were divided into 5 groups. Cavities were then cut into the teeth (n = 28 cavities per group). After sterilization, the teeth were left in broth cultures of 10(6) colony-forming units (CFU) ml(-1) of S. mutans at 36 degrees C for 48 h. The appropriate treatment followed (group A, control; group B, Clearfil SE Bond; group C, Clearfil Protect Bond; group D, 40 s of treatment with ozone; and group E, 80 s of treatment with ozone), and the cavities were then filled with composite resin. After 72 h, the restorations were removed, dentin chips were collected with an excavator, and the total number of microorganisms was determined. All treatments significantly reduced the number of S. mutans present compared with the control group. The antimicrobial effect of both bonding systems and treatment with 80 s of ozone was significantly higher than the 40 s ozone treatment. In conclusion, HealOzone and the bonding systems show striking antimicrobial effects against S. mutans.

Cytochemical differences in bacterial glycocalyx.

To examine new cytochemical aspects of the bacterial adhesion, a strain 41452/01 of the oral commensal Streptococcus sanguis and a wild strain of Staphylococcus aureus were grown with and without sucrose supplementation for 6 days. Osmiumtetraoxyde (OsO4), uranyl acetate (UA), ruthenium red (RR), cupromeronic blue (CB) staining with critical electrolytic concentrations (CECs), and the tannic acid-metal salt technique (TAMST) were applied for electron microscopy. Cytochemically, only RR-positive fimbriae in S. sanguis were visualized. By contrast, some types of fimbriae staining were observed in S. aureus glycocalyx: RR-positive, OsO4-positive, tannophilic and CB-positive with ceasing point at 0.3 M MgCl2. The CB staining with CEC, used for the first time for visualization of glycoproteins of bacterial glycocalyx, also reveals intacellular CB-positive substances-probably the monomeric molecules, that is, subunits forming the fimbriae via extracellular assembly. Thus, glycosylated components of the biofilm matrix can be reliably related to single cells. The visualization of intracellular components by CB with CEC enables clear distinction between S. aureus and other bacteria, which do not produce CB-positive substances. The small quantities of tannophilic substances found in S. aureus makes the use of TAMST for the same purpose difficult. The present work protocol enables, for the first time, a partial cytochemical differentiation of the bacterial glycocalyx.

Novel avilamycin derivatives with improved polarity generated by targeted gene disruption.

The oligosaccharide antibiotics avilamycin A and C are produced by Streptomyces viridochromogenes Tu57. Both consist of a heptasaccharide chain, which is attached to a polyketide-derived dichloroisoeverninic acid moiety. They show excellent antibiotic activity against Gram-positive bacteria. Both molecules are modified by O-methylation at different positions, which contributes to poor water solubility and difficulties in galenical drug development. In order to generate novel avilamycin derivatives with improved polarity and improved pharmacokinetic properties, we generated a series of mutants with one, two, or three mutated methyltransferase genes. Based on the structure of the novel avilamycin derivatives, the exact function of three methyltransferases, AviG2, AviG5, and AviG6, involved in avilamycin biosynthesis could be assigned.

Investigations on the influencing of the subgingival microflora in chronic periodontitis. A study in adult patients during fixed appliance therapy.

AIM: The aim of the present study was to investigate changes in the subgingival flora in adults with chronic periodontitis undergoing orthodontic fixed appliance therapy. PATIENTS AND METHODS: In seven adult patients who had undergone nonantibiotic periodontal pretreatment, the subgingival bacteria were subjected to microbiological examination and the number of periodontopathogenic organisms was determined before (T1: prior to treatment being started), during (T2: 6 weeks after orthodontic treatment was started) and after the end of orthodontic treatment (T3: 6 weeks after removal of the fixed appliances). RESULTS AND CONCLUSION: During the fixed appliance therapy (metal brackets, NiTi archwires, stainless steel archwires), a marked reduction was observed in the total bacteria count from the subgingival pocket despite the clinical periodontal parameters remaining almost unchanged. However, the total count of some highly pathogenic bacteria rose again slightly after the end of treatment. We attribute the marked improvement in the periodontopathogenic bacteria spectrum under fixed appliance therapy with metal brackets, NiTi archwires and stainless steel archwires to metal corrosion entailing the release of primarily nickel ions, which have a toxic effect on bacteria and thus enable the regeneration of the physiological bacterial flora. In none of the patients was a deterioration of the periodontal status observed during and after fixed appliance therapy.

Tuboovarian abscess caused by Atopobium vaginae following transvaginal oocyte recovery.

A 39-year-old woman with tubarian sterility fell ill with acute pelvic inflammatory disease 2 months after transvaginal oocyte recovery. Laparotomy revealed a large tuboovarian abscess, from which Atopobium vaginae, an anaerobic gram-positive coccoid bacterium of hitherto unknown clinical significance, was isolated. The microbial etiology and the risk of pelvic infections following transvaginal punctures are discussed.

Small intestinal bacterial overgrowth in human cirrhosis is associated with systemic endotoxemia.

OBJECTIVES: Systemic endotoxemia has been implicated in various pathophysiological sequelae of chronic liver disease. One of its potential causes is increased intestinal absorption of endotoxin. We therefore examined the association of small intestinal bacterial overgrowth with systemic endotoxemia in patients with cirrhosis. METHODS: Fifty-three consecutive patients with cirrhosis (Child-Pugh group A, 23; group B, 18; group C, 12) were included. Jejunal secretions were cultivated quantitatively and systemic endotoxemia determined by the chromogenic Limulus amoebocyte assay. Patients were followed up for 1 yr. RESULTS: Small intestinal bacterial overgrowth, defined as > or = 10(5) total colony forming units per milliliter of jejunal secretions, was present in 59% of patients and strongly associated with acid suppressive therapy. The mean plasma endotoxin level was 0.86 +/- 0.48 endotoxin units/ml (range = 0.03-1.44) and was significantly associated with small intestinal bacterial overgrowth (0.99 vs 0.60 endotoxin units/ml, p = 0.03). During the 1-yr follow-up, seven patients were lost to follow up or underwent liver transplantation and 12 patients died. Multivariate Cox regression showed Child-Pugh group to be the only predictor for survival. CONCLUSIONS: Small intestinal bacterial overgrowth in cirrhotic patients is common and associated with systemic endotoxemia. The clinical relevance of this association remains to be defined.