Antibiotic prescriptions among dentists across Norway and the impact of COVID-19 pandemic.

BACKGROUND: The prescription of antibiotics in dental practice contributes significantly to the total use of antibiotics in primary healthcare. This study aimed to evaluate antibiotic prescription in dental practice during the years 2016-2021 in Norway and their relative contribution to national outpatient consumption and to investigate the influence of age, gender, geographic region, and COVID-19. A further aim was to review differences in prescribing patterns to verify effect of governmental strategies to reduce over-prescribing of antibiotics. METHODS: This register study investigated the national antibiotic prescription between 2016 and 2021. Data was obtained from the Norwegian prescription register, the Norwegian Institute of Public Health and Statistics Norway. The consumption of 12 common antibiotics was measured using WHO defined daily doses (DDDs), DDD per 1000 inhabitants per day (DIDs 1000). RESULTS: A total of 6,049,445 antibiotic prescriptions of the 12 investigated compounds were issued in primary care during the study period. Dentists accounted for 942,350 prescriptions corresponding to 15.6% of the total. An overall decrease in the number of prescriptions by health professions other than dentists during the 5 years (IRR = 0.92, 95% CI:0.92-0.93, p < 0.001) was observed. For dentists a slight increase in the number of prescriptions (IRR = 1.01, 95% CI: 1.01-1.01, p < 0.001) was seen over the study period. The increase of antibiotic prescriptions in dentistry was more pronounced during the COVID-19 pandemic. The 4 most prescribed type of antibiotics based on average number of DDDs of the total period 2016-2021 were in descending order; phenoxymethylpenicillin (1,109,150) followed by amoxicillin (126,244), clindamycin (72,565), and metronidazole (64,599). An unexpected finding was that the prescription of the combination compound amoxicillin/clavulanic acid had significantly increased in dentistry during the last 5 years. Geographic, gender, and age differences in the rates of prescriptions were also seen. The data revealed that there are seasonal variations in dental prescriptions. CONCLUSIONS: Noticeable differences exist in prescribing patterns of antibiotics in the last 5 years. Restricted access to dental care due to COVID-19 may have resulted in increased antibiotic prescribing in dentistry as opposed to an otherwise downward trend. Despite national guidelines there is still a need for improvement of antibiotic stewardship in dentistry and to define effective methods to disseminate information.

Procedure time and filling quality for bulk-fill base and conventional incremental composite techniques-A randomised controlled in vitro trial.

OBJECTIVES: The aims of this randomised controlled laboratory trial were to determine the procedure time and immediate quality (surface porosity and marginal gaps) of fillings placed using the bulk-fill base technique and the conventional incremental technique in simulated clinical settings. METHODS: Forty-two dentists and dental students were randomly allocated to use either the bulk-fill base technique or the conventional incremental technique to fill an identical class II disto-occlusal cavity in a maxillary left first molar typodont tooth. We recorded the time the participants used to fill the cavity and evaluated the surface porosity and marginal gaps on the approximal surfaces of the fillings using a stereomicroscope and specific probes according to the FDI criteria for restoration evaluation. Data were analysed using the Mann-Whitney U, Kruskal-Wallis, and chi-square tests. RESULTS: The median time ± interquartile range was 186 ± 80 s for the bulk-fill base technique and 463 ± 156 s for the conventional incremental technique (p < 0.001). The quality of the fillings was better for the bulk-fill base technique than for the conventional incremental technique (X2 = 9.5, p = 0.002). Neither operator experience nor the usual technique of choice were associated with the procedure time or the quality of the fillings. CONCLUSIONS: Compared to the conventional incremental technique, the use of the bulk-fill base technique shortened the time to fill a cavity by 59.8 % or 4 min and 36 s, and it improved the immediate surface and marginal quality of the fillings, regardless of the operator's experience or technique preference. CLINICAL SIGNIFICANCE: The use of the bulk-fill base technique instead of the conventional incremental technique leads to significant time-savings when placing large class II composite fillings. Additionally, the use of the bulk-fill base technique instead of the conventional incremental technique improves the immediate quality of large class II composite fillings.

Association between Carbonic Anhydrase VI Gene Copy Number Variations and Dental Caries Experience.

The current study examined the association between the carbonic anhydrase VI (CA VI) copy number variations (CNVs) and dental caries experience in adults. In total, 202 of 35-72 years old subjects participating in the Lithuanian National Oral Health Survey (LNOHS) agreed to provide saliva samples; thus, their data were included in the current study. Information about sociodemographic, environmental, and behavioural determinants was acquired via the self-administered World Health Organization (WHO) questionnaire. Fluoride levels in the drinking water were recorded based on information provided by water suppliers. Dental caries experience was recorded by one calibrated examiner using the WHO criteria for recording caries on smooth (including proximal, buccal, and oral) or occlusal surfaces. Caries experience was measured as the total number of decayed (D3), missing (M), filled (F) surfaces. DNA was extracted from saliva samples to examine CA VI CNVs using the QX200 Droplet Digital PCR system. Negative binomial regression and Poisson regression analyses were employed for data analyses. Based on multivariable regression analyses, higher copy number of CA VI were associated with higher caries experience on smooth surfaces (IRR 1.04, 95% CI: 1.005-1.08) and occlusal surfaces (IRR 1.02, 95% CI: 1.003-1.04). Positive associations between higher copy number of CA VI and higher caries experience on smooth and occlusal surfaces were found, suggesting that the CA VI coding gene may be associated with caries development. Future studies are needed to validate our results and to examine the underlying mechanisms of such associations.

Associations of the activity and concentration of carbonic anhydrase VI with susceptibility to dental caries: A systematic review and meta-analysis.

OBJECTIVES: A number of studies have claimed that carbonic anhydrase VI (CA VI) is associated with dental caries. The aim of this systematic review and meta-analysis was to systematically review and analyze the literature on the association of CA VI (in terms of concentration and activity) with dental caries. MATERIALS AND METHODS: A systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Relevant search terms were employed to search the following databases: PubMed, Web of Science, Scopus, China National Knowledge Infrastructure (CNKI), and Cochrane Library databases. Eligible publications from inception to August 2022 were included. The relevant records were assessed independently by two reviewers, and a meta-analysis was performed using RevMan 5.3. RESULTS: Out of 237 relevant records from the initial search, 9 met the criteria for this review. The 9 papers, including 477 participants, were qualitatively analyzed. Seven studies with 411 participants (203 caries-free) were included in the meta-analysis on CA VI activity, and 2 studies with 141 participants (71 caries-free) were included in the meta-analysis on CA VI concentration. The results showed that CA VI activity was significantly higher among participants with caries than their caries-free counterparts (standardized mean difference (SMD) = 0.894, 95% confidence interval (CI95% ): 0.386 and 1.392; p < 0.001), whereas the CA VI concentration was significantly lower among participants with caries than their caries-free counterparts (SMD = -0.672, CI95% : -1.011 and -0.332; p < 0.001). CONCLUSIONS: This meta-analysis of a relatively small number of studies suggests that the CA VI concentration is lower and CA VI activity is higher in patients with dental caries than in caries-free individuals; however, further studies are needed to determine the exact role of CA VI in dental caries.

Inverse PCR-based detection reveal novel mobile genetic elements and their associated genes in the human oral metagenome.

The human oral cavity is one of the hotspots harboring multiple mobile genetic elements (MGEs), which are segments of DNA that can move either within bacterial genomes or between bacterial cells that can facilitate the spreading of genetic materials, including antimicrobial resistance genes. It is, therefore, important to investigate genes associated with the MGEs as they have a high probability of dissemination within the bacterial population under selective pressure from human activities. As one-third of oral bacteria are not yet culturable in the laboratory condition, therefore, in this work, it is aimed to detect and identify the genetic contexts of MGEs in the oral cavity through an inverse PCR (IPCR)-based approach on the oral metagenomic. The human oral metagenome was extracted from saliva samples collected from healthy individuals in Tromsø, Norway. The extracted DNA was partially digested with the HindIII restriction enzyme and self-circularized by ligation. DNA primers targeting each MGE were designed to amplify outwards from the MGEs and used for the IPCR on the circularized DNA products. The IPCR amplicons were cloned into a pCR-XL-2-TOP vector, screened, and sequenced. Out of 40 IPCR amplicons, we confirmed and verified the genetic contexts of 11 samples amplified with primers targeting integron gene cassettes (GCs), IS431 composite transposons, and Tn916 conjugative transposons (tet(M) and xis-int). Novel integron GCs, MGEs, and variants of Tn916 conjugative transposons were identified, which is the first report using the IPCR technique to detect the genetic contexts of MGEs in the oral metagenomic DNA.

Intracellular Transposition and Capture of Mobile Genetic Elements following Intercellular Conjugation of Multidrug Resistance Conjugative Plasmids from Clinical Enterobacteriaceae Isolates.

Mobile genetic elements (MGEs) are often associated with antimicrobial resistance genes (ARGs). They are responsible for intracellular transposition between different replicons and intercellular conjugation and are therefore important agents of ARG dissemination. Detection and characterization of functional MGEs, especially in clinical isolates, would increase our understanding of the underlying pathways of transposition and recombination and allow us to determine interventional strategies to interrupt this process. Entrapment vectors can be used to capture active MGEs, as they contain a positive selection genetic system conferring a selectable phenotype upon the insertion of an MGE within certain regions of that system. Previously, we developed the pBACpAK entrapment vector that results in a tetracycline-resistant phenotype when MGEs translocate and disrupt the cI repressor gene. We have previously used pBACpAK to capture MGEs in clinical Escherichia coli isolates following transformation with pBACpAK. In this study, we aimed to extend the utilization of pBACpAK to other bacterial taxa. We utilized an MGE-free recipient E. coli strain containing pBACpAK to capture MGEs on conjugative, ARG-containing plasmids following conjugation from clinical Enterobacteriaceae donors. Following the conjugative transfer of multiple conjugative plasmids and screening for tetracycline resistance in these transconjugants, we captured several insertion sequence (IS) elements and novel transposons (Tn7350 and Tn7351) and detected the de novo formation of novel putative composite transposons where the pBACpAK-located tet(A) is flanked by ISKpn25 from the transferred conjugative plasmid, as well as the ISKpn14-mediated integration of an entire 119-kb, blaNDM-1-containing conjugative plasmid from Klebsiella pneumoniae. IMPORTANCE By analyzing transposition activity within our MGE-free recipient, we can gain insights into the interaction and evolution of multidrug resistance-conferring MGEs following conjugation, including the movement of multiple ISs, the formation of composite transposons, and cointegration and/or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow fine-scale experimental studies of factors affecting intracellular transposition and MGE formation in and from ARG-encoding MGEs from multiple species of clinically relevant Enterobacteriaceae.

Functional screening of a human saliva metagenomic DNA reveal novel resistance genes against sodium hypochlorite and chlorhexidine.

OBJECTIVE: Many sections of the health care system are facing a major challenge making infectious disease problematic to treat; antimicrobial resistance (AMR). Identification and surveillance of the resistome have been highlighted as one of the strategies to overcome the problem. This study aimed to screen for AMR genes in an oral microbiota, a complex microbial system continuously exposed to antimicrobial agents commonly used in dental practice. MATERIALS AND METHODS: As a significant part of the oral microbiome cannot be conventionally cultured, a functional metagenomic approach was chosen. The human oral metagenomic DNA was extracted from saliva samples collected from 50 healthy volunteers in Norway. The oral metagenomic library was then constructed by ligating partially digested oral metagenome into pSMART BAC vector and introducing into Escherichia coli. The library was screened against antimicrobials in dental practices. All resistant clones were selected and analyzed. RESULTS: Screening of the oral metagenomic library against different antimicrobials detected multiple clones with resistance against chlorhexidine, triclosan, erythromycin, tetracycline, and sodium hypochlorite. Bioinformatic analysis revealed both already known resistance genes, including msr, mef(A), tetAB(46), and fabK, and genes that were not previously described to confer resistance, including recA and accB conferring resistance to sodium hypochlorite and chlorhexidine, respectively. CONCLUSION: Multiple clones conferring resistance to antimicrobials commonly used in dental practices were detected, containing known and novel resistant genes by functional-based metagenomics. There is a need for more studies to increase our knowledge in the field.

Restorative treatment decisions for carious lesions: Do Russian dentists and dental students apply minimal intervention dentistry?

BACKGROUND: The concept of minimal intervention dentistry (MID) includes both delayed restorative treatment and conservative caries removal, and is now recognised as an evidence-based approach for dental caries management. In order to determine if dental professionals in Russia are incorporating this concept into their clinical practice, we investigated the restorative treatment decisions of Russian dentists and dental students, and the factors associated with these decisions. METHODS: We included 171 general dental practitioners and dental therapists (collectively referred to here as "dentists") from North-West Russia, and 76 dental undergraduate students from the Northern State Medical University in Arkhangelsk (response rate of 11.5% and 67.9%, respectively). Participants completed a questionnaire, which collected background information (sex, region of work, place of dental school graduation, practice type, years of working experience, working in an urban or rural area, and specialisation in restorative dentistry) and information on restorative treatment decisions for proximal and occlusal carious lesions of permanent teeth. Treatment options in accordance with MID were defined as intervention at dentin level and minimally invasive cavity preparation. Multinomial logistic regression was used for statistical analysis. RESULTS: For the proximal carious lesion, 9.4% of participants said they would employ both MID treatment options; 60.7% said they would choose only one; and 29.9% said they would use neither option. For the occlusal carious lesion, the corresponding figures were 37.2%, 52.1%, and 10.7%. No differences in restorative treatment options were observed among general dental practitioners, dental therapists, and dental students. For the proximal carious lesion, dentists from regions outside Arkhangelsk had 4.15 (95% confidence interval [CI] 1.13-15.27) times higher odds of following one versus both MID treatment options. For the occlusal carious lesion, working experience above 15 years was associated with higher odds of using only one versus both MID treatment options (adjusted odds ratio = 3.04, 95% CI 1.33-6.91). Almost all respondents preferred tooth-coloured materials for restorations; more than 75% chose resin-based composite. CONCLUSIONS: The majority of Russian dentists and dental students do not apply the MID concept when treating dental caries in permanent teeth. Clinical protocols on dental caries treatment and dental school curriculums should be updated to place an enhanced focus on evidence-based practice and preventive strategies. Further studies with larger samples of Russian dentists and dental students and alternative methods of recruitment are needed to validate our results.

Prevalence, diversity and transferability of the Tn916-Tn1545 family ICE in oral streptococci.

Background: The Tn916-Tn1545 family of Integrative Conjugative Elements (ICE) are mobile genetic elements (MGEs) that play a role in the spread of antibiotic resistance genes. The Tn916 harbors the tetracycline resistance gene tet(M) and it has been reported in various bacterial species. The increase in the levels of tetracycline resistance among oral streptococci is of great concern primarily due to the abundance of these species in the oral cavity and their ability to act as reservoirs for antibiotic resistance genes.Methods: In the current study, we screened 100 Norwegian clinical oral streptococcal isolates for the presence and diversity of the Tn916-Tn1545 family. In addition, we investigated the transferability the elements, and the associated transfer frequencies.Results: We observed that 21 isolates harboured the Tn916-Tn1545 family and that two of these elements were the novel Tn6815 and Tn6816. The most prevalent member of the Tn916 -Tn1545 family observed in the Norwegian clinical oral streptococcal isolates was the wild type Tn916.Conclusion: The detection of other members of this family of ICE and varying transfer frequencies suggests high versatility of the Tn916 element in oral streptococci in Norway.

Amylase Alpha 1 Gene (AMY1) Copy Number Variation and Dental Caries Experience: A Pilot Study among Adults in Lithuania.

INTRODUCTION: Genetic biomarkers have the potential to be used in personalised dentistry for improved prevention and decision-making in caries management. The amylase alpha 1 gene (AMY1) encodes salivary α-amylase and may be one such biomarker. We examined the association between AMY1 copy number variation (CNV) and dental caries experience in adults. MATERIALS AND METHODS: A stratified random sample of 193 participants from the Lithuanian National Oral Health Survey (LNOHS) agreed to provide saliva samples and were included in this analysis (age 35-44 years; participation rate 43%). Information on socio-demographic and behavioural characteristics was taken from the LNHOS, which used the self-administered World Health Organisation (WHO) questionnaire. Data on fluoride levels in drinking water at the recruitment areas was recorded based on information provided by water suppliers. Dental caries experience was recorded at a surface level (smooth-surface and occlusal-surface decayed, missing, filled surfaces [D3MFS] score) by one trained and calibrated examiner using WHO criteria, and subsequently dichotomised for the statistical analyses. DNA extracted from saliva samples was used to investigate AMY1 CNV using the QX200 droplet digital PCR system. Bivariate and multivariable statistical analyses were employed. RESULTS: When compared to participants with an AMY1 copy number (CN) of 2-3, higher odds of smooth-surface D3MFS >14 was observed for participants with a CN of 4-5 (OR 13.3, 95% CI 2.1-86.3), 6-9 (OR 7.0, 95% CI 1.4-34.1), and 10-16 (OR 5.8, 95% CI 1.2-32.2). Female sex was independently associated with a smooth-surface D3MFS >14 (OR 5.7, 95% CI 1.9-17.2). CONCLUSIONS: Our study demonstrated an association between AMY1 CNV and high smooth-surface caries experience. Studies with larger sample sizes are needed to validate this association.

Salivary Oral Microbiome of Children With Juvenile Idiopathic Arthritis: A Norwegian Cross-Sectional Study.

Background: The oral microbiota has been connected to the pathogenesis of rheumatoid arthritis through activation of mucosal immunity. The objective of this study was to characterize the salivary oral microbiome associated with juvenile idiopathic arthritis (JIA), and correlate it with the disease activity including gingival inflammation. Methods: Fifty-nine patients with JIA (mean age, 12.6 ± 2.7 years) and 34 healthy controls (HC; mean age 12.3 ± 3.0 years) were consecutively recruited in this Norwegian cross-sectional study. Information about demographics, disease activity, medication history, frequency of tooth brushing and a modified version of the gingival bleeding index (GBI) and the simplified oral hygiene index (OHI-S) was obtained. Microbiome profiling of saliva samples was performed by sequencing of the V1-V3 region of the 16S rRNA gene, coupled with a species-level taxonomy assignment algorithm; QIIME, LEfSe and R-package for Spearman correlation matrix were used for downstream analysis. Results: There were no significant differences between JIA and HC in alpha- and beta-diversity. However, differential abundance analysis revealed several taxa to be associated with JIA: TM7-G1, Solobacterium and Mogibacterium at the genus level; and Leptotrichia oral taxon 417, TM7-G1 oral taxon 352 and Capnocytophaga oral taxon 864 among others, at the species level. Haemophilus species, Leptotrichia oral taxon 223, and Bacillus subtilis, were associated with healthy controls. Gemella morbillorum, Leptotrichia sp. oral taxon 498 and Alloprevotella oral taxon 914 correlated positively with the composite juvenile arthritis 10-joint disease activity score (JADAS10), while Campylobacter oral taxon 44 among others, correlated with the number of active joints. Of all microbial markers identified, only Bacillus subtilis and Campylobacter oral taxon 44 maintained false discovery rate (FDR) < 0.1. Conclusions: In this exploratory study of salivary oral microbiome we found similar alpha- and beta-diversity among children with JIA and healthy. Several taxa associated with chronic inflammation were found to be associated with JIA and disease activity, which warrants further investigation.

Comparison of antimicrobial prescribing for dental and oral infections in England and Scotland with Norway and Sweden and their relative contribution to national consumption 2010-2016.

BACKGROUND: Prescribing in dental practice has a relatively small but important contribution to the quantity of antibiotics prescribed in primary care. This study aimed to analyse antibiotic prescribing in dentistry over time (2010-2016) in 4 different Northern European countries and their relative contribution to national outpatients consumption. METHODS: This retrospective study evaluated the frequency and number of national antibiotic prescriptions written by dentists in England, Scotland, Norway and Sweden. The consumption of such antibiotics was measured using WHO defined daily doses (DDDs), DDDs per 100,000 inhabitants per day (DIDs100,000). RESULTS: A total of more than 27 million prescriptions (27,026,599) archived between 2010 and 2016 from the four countries were analysed. The national contribution of Norwegian dentists to the total primary care prescription during this period was 8%. The corresponding figures for Sweden, Scotland and England were 7, 6, and 8%. Dental contribution to National antibiotic use in all four countries has decreased over the study time period for commonly prescribed antibiotics in dentistry, i.e., the beta-lactams (Phenoxymethyl penicillin/Amoxicillin) and metronidazole. There were less numbers of prescriptions by dentists in Norway and Sweden compared to England and Scotland. Marked differences in some classes of antibiotics were noted with Phenoxymethyl penicillin dominating in Sweden/Norway compared to Amoxicillin and Metronidazole in England/Scotland. In England and Scotland, dentists were the largest prescribers of metronidazole in primary care. Clindamycin prescriptions was higher in Norway and Sweden. CONCLUSION: Noticeable differences exist in prescribing patterns for the management of oral infections. High levels of metronidazole use in England and Scotland also require further analysis. All countries over the study period showed a decrease in total numbers of antibiotics prescribed.

Determination of copy number and circularization ratio of Tn916-Tn1545 family of conjugative transposons in oral streptococci by droplet digital PCR.

Background: Tn916 and Tn1545 are paradigms of a large family of related, broad host range, conjugative transposons that are widely distributed in bacteria and contribute to the spread of antibiotic resistance genes (ARGs). Variation in the copy number (CN) of Tn916-Tn1545 elements and the circularization ratio (CR) may play an important role in propagation of ARGs carried by these elements. Objectives and Design: In this study, the CN and CR of Tn916-Tn1545 elements in oral streptococci were determined using droplet digital PCR (ddPCR). In addition, we investigated the influence of tetracycline on the CR of Tn916-Tn1545 elements. Results: The ddPCR assay designed in this study is a reliable way to rapidly determine CN and CR of Tn916-Tn1545 elements. Conclusions: Our data also suggest that Tn916-Tn1545 elements are generally stable without selective pressure in the clinical oral Streptococcus strains investigated in this study.

Co-diversification of Enterococcus faecium Core Genomes and PBP5: Evidences of pbp5 Horizontal Transfer.

Ampicillin resistance has greatly contributed to the recent dramatic increase of a cluster of human adapted Enterococcus faecium lineages (ST17, ST18, and ST78) in hospital-based infections. Changes in the chromosomal pbp5 gene have been associated with different levels of ampicillin susceptibility, leading to protein variants (designated as PBP5 C-types to keep the nomenclature used in previous works) with diverse degrees of reduction in penicillin affinity. Our goal was to use a comparative genomics approach to evaluate the relationship between the diversity of PBP5 among E. faecium isolates of different phylogenomic groups as well as to assess the pbp5 transferability among isolates of disparate clonal lineages. The analyses of 78 selected E. faecium strains as well as published E. faecium genomes, suggested that the diversity of pbp5 mirrors the phylogenomic diversification of E. faecium. The presence of identical PBP5 C-types as well as similar pbp5 genetic environments in different E. faecium lineages and clones from quite different geographical and environmental origin was also documented and would indicate their horizontal gene transfer among E. faecium populations. This was supported by experimental assays showing transfer of large (≈180-280 kb) chromosomal genetic platforms containing pbp5 alleles, ponA (transglycosilase) and other metabolic and adaptive features, from E. faecium donor isolates to suitable E. faecium recipient strains. Mutation profile analysis of PBP5 from available genomes and strains from this study suggests that the spread of PBP5 C-types might have occurred even in the absence of a significant ampicillin resistance phenotype. In summary, genetic platforms containing pbp5 sequences were stably maintained in particular E. faecium lineages, but were also able to be transferred among E. faecium clones of different origins, emphasizing the growing risk of further spread of ampicillin resistance in this nosocomial pathogen.

Antibiotic Prescription Knowledge of Dentists in Kingdom of Saudi Arabia: An Online, Country-wide Survey.

AIM: Dentists are probably contributing to the development of bacterial resistance to certain antibiotics. Campaigns to promote prudent use of antibiotics in dentistry are, thus, needed but require proper identification of dentists' knowledge gaps. The objective here was to comprehensively evaluate antibiotic prescription knowledge of dentists in Saudi Arabia. MATERIAL AND METHODS: A link to an online, previously validated questionnaire was emailed to 5199 dentists registered with the Saudi Dental Society. The questionnaire comprised 42 scorable items measuring antibiotics prescription knowledge in five different domains in addition to nonscorable questions regarding first-choice antibiotics and previous attendance of a course/workshop about antibiotic prescription. Each correct answer was given one mark. Mean scores were calculated as percentages and categorized as good (> 80%), intermediate (60-80%), or poor (< 60%). RESULTS: The response rate was 9.4%; however, only 373 (7.2%) fully completed the questionnaire. Around half of the participants (52%) reported prescribing amoxicillin/clavulanate as the first-choice antibiotic; 62% reported attending a course/workshop in the last 5 years. The average knowledge score was 69%, being highest for nonclinical indications (79%) and lowest for prophylactic use (56%). The worst per-item scores were noted for rheumatic heart disease (19%), trismus (28%), surgical extraction (30%), apicectomy (31%), and periodontal abscess (33%). Female dentists, dentists in governmental sector, and those with higher qualifications had significantly better knowledge. CONCLUSION: The level of knowledge was hardly intermediate and several deficits were identified, indicating an urgent need for educational campaigns and provision of guidelines promoting rational use of antibiotics by dentists. CLINICAL SIGNIFICANCE: Irrational use of antibiotics by dentists can contribute to the problem of antibacterial resistance.

Imipenem Treatment Induces Expression of Important Genes and Phenotypes in a Resistant Acinetobacter baumannii Isolate.

Acinetobacter baumannii has emerged as a notorious multidrug-resistant pathogen, and development of novel control measures is of the utmost importance. Understanding the factors that play a role in drug resistance may contribute to the identification of novel therapeutic targets. Pili are essential for A. baumannii adherence to and biofilm formation on abiotic surfaces as well as virulence. In the present study, we found that biofilm formation was significantly induced in an imipenem-resistant (Imp(r)) strain treated with a subinhibitory concentration of antibiotic compared to that in an untreated control and an imipenem-susceptible (Imp(s)) isolate. Using microarray and quantitative PCR analyses, we observed that several genes responsible for the synthesis of type IV pili were significantly upregulated in the Imp(r) but not in the Imp(s) isolate. Notably, this finding is corroborated by an increase in the motility of the Imp(r) strain. Our results suggest that the ability to overproduce colonization factors in response to imipenem treatment confers biological advantage to A. baumannii and may contribute to clinical success.

Fitness costs of various mobile genetic elements in Enterococcus faecium and Enterococcus faecalis.

OBJECTIVES: To determine the fitness effects of various mobile genetic elements (MGEs) in Enterococcus faecium and Enterococcus faecalis when newly acquired. We also tested the hypothesis that the biological cost of vancomycin resistance plasmids could be mitigated during continuous growth in the laboratory. METHODS: Different MGEs, including two conjugative transposons (CTns) of the Tn916 family (18 and 33 kb), a pathogenicity island (PAI) of 200 kb and vancomycin-resistance (vanA) plasmids (80-200 kb) of various origins and classes, were transferred into common ancestral E. faecium and E. faecalis strains by conjugation assays and experimentally evolved (vanA plasmids only). Transconjugants were characterized by PFGE, S1 nuclease assays and Southern blotting hybridization analyses. Single specific primer PCR was performed to determine the target sites for the insertion of the CTns. The fitness costs of various MGEs in E. faecium and E. faecalis were estimated in head-to-head competition experiments, and evolved populations were generated in serial transfer assays. RESULTS: The biological cost of a newly acquired PAI and two CTns were both host- and insertion-locus-dependent. Newly acquired vanA plasmids may severely reduce host fitness (25%-27%), but these costs were rapidly mitigated after only 400 generations of continuous growth in the absence of antibiotic selection. CONCLUSIONS: Newly acquired MGEs may impose an immediate biological cost in E. faecium. However, as demonstrated for vanA plasmids, the initial costs of MGE carriage may be mitigated during growth and beneficial plasmid-host association can rapidly emerge.

Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K.

BACKGROUND: Biofilms are organized communities of microorganisms embedded in a self-produced extracellular polymeric matrix (EPM), often with great phylogenetic variety. Bacteria in the subgingival biofilm are key factors that cause periodontal diseases; among these are the Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis. The objectives of this study were to characterize the major components of the EPM and to test the effect of deoxyribonuclease I (DNase I) and proteinase K. METHODS: F. nucleatum and P. gingivalis bacterial cells were grown in dynamic and static biofilm models. The effects of DNase I and proteinase K enzymes on the major components of the EPM were tested during biofilm formation and on mature biofilm. Confocal laser scanning microscopy was used in observing biofilm structure. RESULTS: Proteins and carbohydrates were the major components of the biofilm matrix, and extracellular DNA (eDNA) was also present. DNase I and proteinase K enzymes had little effect on biofilms in the conditions used. In the flow cell, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form biofilm alone in similar conditions. F. nucleatum supported the growth of P. gingivalis when they were grown together as dual species biofilm. CONCLUSION: DNase I and proteinase K had little effect on the biofilm matrix in the conditions used. F. nucleatum formed biofilm easily and supported the growth of P. gingivalis, which preferred anaerobic conditions.

Effect of khat chewing on periodontal pathogens in subgingival biofilm from chronic periodontitis patients.

AIMS: Existing in vitro and in vivo data suggest that khat may have a favorable effect on periodontal microbiota. The purpose of this study was to assess the effect of khat chewing on major periodontal pathogens in subgingival plaque samples from subjects with chronic periodontitis. MATERIALS AND METHODS: 40 subgingival plaque samples were obtained from periodontitis and healthy sites of 10 khat chewers (40 y median age) and 10 khat non-chewers (37.5 y median age) with chronic periodontitis. Absolute and relative counts of 6 periodontal pathogens were determined in each sample using highly sensitive and specific Taqman real-time PCR assays. Data were analyzed using an ordinal regression model. RESULTS: Significantly more total bacteria were detected in samples from the periodontitis sites of the khat chewers (OR=20). Treponema denticola was present at significantly higher absolute counts at the healthy as well as periodontitis sites of the khat chewers (OR=3.13 and 13, respectively). However, the khat chewers harbored significantly lower absolute counts of Porphyromonas gingivalis at the healthy sites (OR=0.07). Furthermore, khat chewing was significantly associated with lower relative counts of Porphyromonas gingivalis, fusobacterium ssp., prevotella ssp. and Parvimonas micra-like species in subgingival plaque samples from both healthy and periodontitis sites (OR=0.11-0.33). Only Treponema denticola was found in higher relative counts at the healthy sites of the khat chewers (OR=2.98). CONCLUSIONS: Overall, there was a lower burden of pathogens in the khat chewers. Findings from the current study are suggestive of a potential prebiotic effect for khat on periodontal microbiota.

Fusobacterium nucleatum enters normal human oral fibroblasts in vitro.

BACKGROUND: Fusobacterium nucleatum, a commensal opportunistic oral bacterium, is capable of invading gingival epithelial cells, but the entrance into human primary oral fibroblast cells has not been documented. This study evaluated the ability of three strains of F. nucleatum (F. nucleatum ssp. nucleatum, F. nucleatum ssp. polymorphum, and F. nucleatum ssp. vincentii) to enter gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PLFs). METHODS: GFs and PLFs were cocultured for various periods of time with different strains of F. nucleatum. Scanning and transmission electron microscopy, together with confocal laser scanning microscopy, were used to visualize the entrance and presence of bacteria in host cells. Flow cytometry was performed to compare the load of internalized bacteria in GFs and PLFs exposed for 3 and 5 hours to live F. nucleatum labeled with fluorescein isothiocyanate. RESULTS: All three strains of F. nucleatum were found entering and located in the cytoplasm of GFs and PLFs after 1 hour of exposure. Flow cytometry tests revealed a significant increase in the fluorescent signal, compared to baseline, derived from bacteria internalized in fibroblasts exposed for 3 hours (P <0.001); a further increase was found at 5 hours. The greatest bacterial mass in exposed fibroblasts of both types was of F. nucleatum ssp. polymorphum; the smallest was of F. nucleatum ssp. vincentii. Although not statistically significant, PLFs had a higher bacterial load than corresponding GFs. CONCLUSION: F. nucleatum was capable of entering GFs and PLFs in a manner that is dependent on the cell type and the bacterial strain.