With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), bacteria can be identified quickly and reliably. This accounts especially for anaerobic bacteria. ...Because growth rate and oxygen sensitivity differ among anaerobic bacteria, we aimed to study the influence of incubation time, exposure to oxygen and sample preparation on the quality of the spectrum using the Bruker system. Also, reproducibility and inter-examiner variability were determined. Twenty-six anaerobic species, representing 17 genera, were selected based on gram-stain characteristics, growth rate and colony morphology. Inter-examiner variation showed that experience in the preparation of the targets can be a significant variable. The influence of incubation time was determined between 24 and 96 h of incubation. Reliable species identification was obtained after 48 h of incubation for gram-negative anaerobes and after 72 h for gram-positive anaerobes. Exposure of the cultures to oxygen did not influence the results of the MALDI-TOF MS identifications of all tested gram-positive species. Fusobacterium necrophorum and Prevotella intermedia could not be identified after >24 h and 48 h of exposure to oxygen, respectively. Other tested gram-negative bacteria could be identified after 48 h of exposure to oxygen. Most of the tested species could be identified using the direct spotting method. Bifidobacterium longum and Finegoldia magna needed on-target extraction with 70% formic acid in order to obtain reliable species identification and Peptoniphilus ivorii a full extraction. Spectrum quality was influenced by the amount of bacteria spotted on the target, the homogeneity of the smear and the experience of the examiner.
Abstract Periodontitis is a bacterial disease that can be treated with systemic antibiotics. The aim of this study was to establish the antibiotic susceptibility profiles of five periodontal ...pathogens to six commonly used antibiotics in periodontics. A total of 247 periodontal bacterial isolates were tested for susceptibility to the six antibiotics using the Etest method. MIC50 and MIC90 values (minimum inhibitory concentrations for 50% and 90% of the organisms, respectively) were calculated. Both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints were used in the study to interpret results. β-Lactamase production was tested when amoxicillin resistance was found. MIC90 values of the anaerobic bacteria were all well below breakpoint values, except for three isolates of Prevotella intermedia and one isolate of Fusobacterium nucleatum that were resistant to amoxicillin (CLSI breakpoints); these isolates were β-lactamase-positive. Two isolates of the capnophilic Aggregatibacter actinomycetemcomitans appeared to be amoxicillin-resistant but failed to show β-lactamase activity. Comparison with a previous study from The Netherlands showed minor differences in susceptibility profiles, but the MIC90 values of A. actinomycetemcomitans for amoxicillin, clindamycin, azithromycin and tetracycline were higher. Geographical differences in the susceptibility profiles of Porphyromonas gingivalis and A. actinomycetemcomitans between European countries were noted. Comparison of European susceptibility profiles with that of a South American country (Colombia) revealed a much higher resistance in the latter. Owing to these differences in susceptibility profiles, it is of concern to regularly perform surveillance studies on antibiotic resistance.
Porphyromonas gingivalis is one of the major oral pathogens implicated in the widespread inflammatory disorder periodontitis. Moreover, in recent years, P. gingivalis has been associated with the ...autoimmune disease rheumatoid arthritis. The peptidylarginine deiminase enzyme of P. gingivalis (PPAD) is a major virulence factor that catalyzes the citrullination of both bacterial and host proteins, potentially contributing to production of anticitrullinated protein antibodies. Considering that these antibodies are very specific for rheumatoid arthritis, PPAD appears to be a link between P. gingivalis, periodontitis, and the autoimmune disorder rheumatoid arthritis. PPAD was thus far considered unique among prokaryotes, with P. gingivalis being the only bacterium known to produce and secrete it. To challenge this hypothesis, we investigated the possible secretion of PPAD by 11 previously collected Porphyromonas isolates from a dog, 2 sheep, 3 cats, 4 monkeys, and a jaguar with periodontitis. Our analyses uncovered the presence of secreted PPAD homologues in 8 isolates that were identified as Porphyromonas gulae (from a dog, monkeys, and cats) and Porphyromonas loveana (from sheep). In all 3 PPAD-producing Porphyromonas species, the dominant form of the secreted PPAD was associated with outer membrane vesicles, while a minor fraction was soluble. Our results prove for the first time that the citrullinating PPAD exoenzyme is not unique to only 1 prokaryotic species. Instead, we show that PPAD is produced by at least 2 other oral pathogens.
Background: The aim of this case‐control study is to compare oral microbiologic characteristics of patients with healthy peri‐implant conditions and patients with peri‐implantitis and to explore the ...influence of various patient‐ and implant‐related factors on microbiologic characteristics.
Methods: Peri‐implant submucosal microbial samples were collected from 85 patients with peri‐implantitis (cases) and from 69 patients with only implants with healthy peri‐implant conditions (controls). Samples were analyzed using culturing techniques. Multivariable logistic regression was used to explore the association of disease status and various patient‐ and implant‐related factors (sex, patient age, smoking, number of remaining teeth, percentage of teeth with bone loss, implant function time, implant surface, and presence of plaque) with microbiologic characteristics.
Results: Peri‐implant disease status was significantly associated with the submucosal presence of Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythia (Tf), and Fusobacterium nucleatum (Fn). The association with disease status was most obvious for Pi (odds ratio OR: 15.1; 95% confidence interval CI: 5.1 to 45.3) and Tf (OR: 13.3; 95% CI: 5.4 to 32.5). The prevalence of Aggregatibacter actinomycetemcomitans and Staphylococcus species was very low.
Conclusions: The periodontal pathogens Pg, Pi, Tf, and Fn are associated with peri‐implantitis. A. actinomycetemcomitans and Staphylococcus species do not seem to play an important role in peri‐implantitis.
Aim
The objective of this randomized, double‐blind, placebo‐controlled trial was to study the effect of implant surface decontamination with chlorhexidine (CHX)/cetylpyridinium chloride (CPC) on ...microbiological and clinical parameters.
Material & Methods
Thirty patients (79 implants) with peri‐implantitis were treated with resective surgical treatment consisting of apically re‐positioned flap, bone re‐contouring and surface debridement and decontamination. Patients were randomly allocated to decontamination with 0.12% CHX + 0.05% CPC (test‐group) or a placebo‐solution (without CHX/CPC, placebo‐group). Microbiological parameters were recorded during surgery; clinical and radiographical parameters were recorded before (pre‐) treatment (baseline), and at 3, 6 and 12 months after treatment.
Results
Nine implants in two patients in the placebo‐group were lost due to severe persisting peri‐implantitis. Both decontamination procedures resulted in significant reductions of bacterial load on the implant surface, but the test‐group showed a significantly greater reduction than the placebo‐group (log 4.21 ± 1.89 versus log 2.77 ± 2.12, p = 0.006). Multilevel analysis showed no differences between both groups in the effect of the intervention on bleeding, suppuration, probing pocket depth and radiographical bone loss over time.
Conclusion
Implant surface decontamination with 0.12% CHX + 0.05% CPC in resective surgical treatment of peri‐implantitis leads to a greater immediate suppression of anaerobic bacteria on the implant surface than a placebo‐solution, but does not lead to superior clinical results. The long‐term microbiological effect remains unknown.
Objective
The objective of this randomized, double‐blind, controlled trial was to evaluate the clinical, radiographic, and microbiological effects of implant surface decontamination with a 2% ...chlorhexidine (CHX) solution in comparison with a 0.12% chlorhexidine + 0.05% cetylpyridinium chloride (CPC) solution during resective surgical peri‐implantitis treatment.
Material and methods
Forty‐four patients (108 implants) with peri‐implantitis were treated with resective surgical treatment consisting of bone re‐contouring, surface debridement and chemical decontamination, and apically repositioned flap. Patients were randomly allocated to decontamination with a 2% CHX solution (test group) or 0.12% CHX + 0.05% CPC (control group). Clinical and radiographic parameters were recorded before treatment (baseline), and at 3, 6, and 12 months after treatment. Microbiological parameters were recorded during surgery.
Results
Multilevel analysis showed no significant differences in bleeding, suppuration, probing pocket depth, and radiographic bone loss between control and test group over three follow‐up measurements (3, 6, and 12 months) from baseline. Both decontamination procedures resulted in significant reductions in anaerobic bacterial counts on the implant surface, but no significant difference was noted between control and test group (mean log 3.37 ± 2.34 vs. 3.65 ± 2.87, P = 0.99).
Conclusions
The use of a 2% CHX solution for implant surface decontamination during resective peri‐implantitis therapy does not lead to improved clinical, radiographic, or microbiological results compared with a 0.12% CHX + 0.05% CPC solution. Overall, the additional use of CHX reduces anaerobic bacterial load on the implant surface better than mechanical debridement alone, but does not seem to enhance clinical treatment outcomes (ClinicalTrials.gov number NCT01852253).
Background and Objective
Beta‐lactam antibiotics prescribed in periodontal therapy are vulnerable to degradation by bacterial β‐lactamases. This study evaluated the occurrence of β‐lactamase‐positive ...subgingival bacteria in chronic periodontitis subjects of USA origin, and assessed their in vitro resistance to metronidazole at a breakpoint concentration of 4 μg/mL.
Material and Methods
Subgingival plaque specimens from deep periodontal pockets with bleeding on probing were removed from 564 adults with severe chronic periodontitis before treatment. The samples were transported in VMGA III and then plated onto: (i) nonselective enriched Brucella blood agar (EBBA) and incubated anaerobically for 7 d; and (ii) selective trypticase soy‐bacitracin‐vancomycin (TSBV) and incubated for 3 d in air + 5% CO2. At the end of the incubation periods, the bacterial test species were identified and quantified. Specimen dilutions were also plated onto EBBA plates supplemented with 2 μg/mL of amoxicillin, a combination of 2 μg/mL of amoxicillin plus 2 μg/mL of the β‐lactamase inhibitor clavulanic acid, or 4 μg/mL of metronidazole, followed by anaerobic incubation for 7 d. Bacterial test species presumptively positive for β‐lactamase production were identified by growth on EBBA primary isolation plates supplemented with amoxicillin alone and no growth on EBBA primary isolation plates containing both amoxicillin plus clavulanic acid. A subset of such isolates was subjected to nitrocefin‐based chromogenic disk testing to confirm the presence of β‐lactamase activity. In vitro resistance to 4 μg/mL of metronidazole was noted when growth of test species occurred on metronidazole‐supplemented EBBA culture plates.
Results
Two‐hundred and ninety‐four (52.1%) of the study subjects yielded β‐lactamase‐producing subgingival bacterial test species, with Prevotella intermedia/nigrescens, Fusobacterium nucleatum and other Prevotella species most frequently identified as β‐lactamase‐producing organisms. Of the β‐lactamase‐producing bacterial test species strains recovered, 98.9% were susceptible in vitro to metronidazole at 4 μg/mL.
Conclusion
The occurrence of β‐lactamase‐positive subgingival bacterial species in more than half of the subjects with severe chronic periodontitis raises questions about the therapeutic potential of single‐drug regimens with β‐lactam antibiotics in periodontal therapy. The in vitro effectiveness of metronidazole against nearly all recovered β‐lactamase‐producing subgingival bacterial species further supports clinical periodontitis treatment strategies involving the combination of systemic amoxicillin plus metronidazole.
Objectives
Objective of this study was to identify prognostic indicators for the outcome of resective peri‐implantitis treatment, by an analysis of the pooled data of two previously conducted ...randomized controlled trials.
Material and methods
Data of 74 patients with peri‐implantitis (187 implants) who had received resective surgical treatment were available. Primary outcome variable was failure of peri‐implantitis treatment after 12 months. Multilevel univariable and multiple logistic regression analyses were performed to evaluate the effect of various potentially prognostic indicators on the primary outcome.
Results
Peri‐implantitis treatment was unsuccessful in 106 implants (57%) and 48 patients (67%) after 12 months. In the multiple regression analysis, the variables “order of inclusion” (P = 0.016) and mean bone loss at baseline (P = 0.030) were significant prognostic indicators for treatment failure. To eliminate the effect of “order of inclusion,” post hoc analyses were carried out in a subgroup of patients. The univariable post hoc analysis showed a significant association for smoking (P = 0.015), maximum pocket depth at baseline (P = 0.073), mean bone loss at baseline (P = 0.003), and presence of plaque (P = 0.100). In the multiple regression post hoc analysis, only the variables smoking (P = 0.044) and mean bone loss (P = 0.043) remained statistically significant.
Conclusions
The outcome of surgical peri‐implantitis treatment is influenced by the experience of the surgical team with the surgical procedure. The observed learning effect has consequences for clinical practice and for conducting and interpreting clinical trials on peri‐implantitis treatment. Other prognostic indicators are amount of peri‐implant bone loss at baseline and smoking, and to a lesser extent, probing pocket depth at baseline and presence of plaque during follow‐up. Early diagnosis of peri‐implantitis and control of behavioral factors are crucial in achieving peri‐implantitis treatment success.
Objectives
The aim of the present study was to compare the composition of the periodontal microflora at baseline (T0) with the submucosal microflora at least 1 year after implant placement (T1) in ...periodontally healthy patients.
Material and methods
For all 169 consecutive patients that visited our clinic during 1 year, we determined their periodontal parameters, implant mucosal index, and presence of implant calculus. At T0, self-reported smoking status was recorded and subgingival and submucosal biofilm samples were obtained and analyzed for the presence and numbers of selected periodontal pathogens. All measurements were repeated at T1.
Results
One hundred twenty patients completed the study. Periodontal parameters were stable or had improved at T1. The total bacterial load was lower at implant sites (
P
< 0.05). The prevalence of
Porphyromonas gingivalis
was low at baseline, but at T1, detection rate and numbers were higher at implant sites compared to dentate sites. At T1, the frequency of detection of
P. gingivalis
(
P
= 0.01),
Parvimonas micra
(
P
= 0.018), and
Fusobacterium nucleatum
(
P
= 0.035) was higher in smoking patients (
n
= 23) than in non-smokers (
n
= 97).
Conclusions
Colonization of the submucosal peri-implant area is similar to the composition of subgingival microbiota. Smoking has a measurable effect on the colonization of implant-associated biofilms and may select for
P. gingivalis
,
P. micra
, and
F. nucleatum
.
Clinical relevance
The colonization of implants by well-known periodontal pathogens is very similar to that in normal dentition, also in a healthy cohort. Smoking status was related with the prevalence of periodontal pathogens where smokers harbored more often periodontal pathogens such as
P. gingivalis
,
P. micra
, and
F. nucleatum
.
Gram-positive anaerobic cocci (GPAC) account for 24%–31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS ...database. Profiles of new species have yet to be added. We present the optimization of the matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) database for the identification of GPAC. Main spectral profiles (MSPs) were created for 108 clinical GPAC isolates. Identity was confirmed using 16S rRNA gene sequencing. Species identification was considered to be reliable if the sequence similarity with its closest relative was ≥98.7%. The optimized database was validated using 140 clinical isolates. The 16S rRNA sequencing identity was compared with the MALDI-TOF MS result. MSPs were added from 17 species that were not yet represented in the MALDI-TOF MS database or were under-represented (fewer than five MSPs). This resulted in an increase from 53.6% (75/140) to 82.1% (115/140) of GPAC isolates that could be identified at the species level using MALDI-TOF MS. An improved log score was obtained for 51.4% (72/140) of the strains. For strains with a sequence similarity <98.7% with their closest relative (n = 5) or with an inconclusive sequence identity (n = 4), no identification was obtained by MALDI-TOF MS or in the latter case an identity with one of its relatives. For some species the MSP of the type strain was not part of the confined cluster of the corresponding clinical isolates. Also, not all species formed a homogeneous cluster. It emphasizes the necessity of adding sufficient MSPs of human clinical isolates.
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