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

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

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

Association between Carbonic Anhydrase VI (CA VI) gene copy number 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 Organisation (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 (D3MFS). 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

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 bulkfill base technique instead of the conventional incremental technique improves the immediate quality of large class II composite fillings

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 characterisation 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 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 utilisation of pBACpAK to other bacterial species. We utilised 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 IS elements and novel transposons (Tn7350 and Tn7351), we 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 and we also detected the ISKpn14 mediated integration of an entire 119kb, blaNDM-1 containing conjugative plasmid from Klebsiella pneumoniae. Importance By analysing 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 the cointegration and, or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow for fine-scale experimental studies into factors affecting intracellular transposition and MGE formation in, and from, ARG encoding MGEs from multiple species of clinically relevant Enterobacteriaceae

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

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) 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

Antibiotic resistance in oral microbiota : a study on prevalence, molecular analysis, and possible contributing factors in Yemen and Norway

Worldwide antibiotic resistance among oral microbiota is an increasing problem and information regarding such resistance is completely lacking for Yemen and very limited data is available for Norway. The aims of the current thesis were to (1) disclose the prevalence of ampicillin and metronidazole resistance among selected subgingival microbial species obtained from individuals in Yemen and Norway (paper 1), (2) determine the susceptibility pattern of Fusobacterium nucleatum isolated from Yemen and characterize the aminopenicillins-resistance determinant of F. nucleatum (paper 2), and (3) assess if antimicrobial prescription practices by dentists in Yemen and Norway could possibly contribute to the current prevalence and the emergence of bacterial resistance in these geographically separate locations (papers 3 and 4). Materials: Thirty-four and 21 subgingival plaque samples from Yemen and Norway, respectively, were cultivated on fastidious anaerobic blood agar containing 2 μg/mL of either ampicillin or metronidazole. The bacterial growth from each plate was then screened using DNA-DNA checkerboard hybridization technique for the presence of ampicillin and metronidazole resistance among 18 selected subgingival species (paper 1). Ampicillin-resistant F. nucleatum strains were isolated from Yemen by cultivating subgingival plaque samples on crystal violet erythromycin (CVE) plates supplemented with or without 2 μg/mL ampicillin. The molecular basis of ampicillin resistance among F. nucleatum strains was studied using two-dimensional gel electrophoresis and mass spectrometry (paper 2).The antimicrobials prescription knowledge of Yemeni dentists was investigated by distributing a structured questionnaire to all working dentists in the three major governorates in Yemen. The questionnaire aimed at investigating the therapeutic and prophylactic use of antimicrobials with relevant clinical and non-clinical parameters (paper 3). The Norwegian dentists’ antimicrobial prescription practices were revealed by analyzing aggregated data obtained from the Norwegian Prescription Database (NorPD) on the basis of their total prescriptions of 11 antibiotics issued in 2004 and 2005. Consumptions of these antibiotics in dental practice were measured using the WHO measurement unit, the Defined Daily Dose (DDD) (paper 4). Results: A statistically significant higher resistance to metronidazole and ampicillin among nine and seven species (P <0.05), respectively, was found in Yemeni isolates compared with Norwegian ones. The molecular characterization of ampicillin-resistant F. nucleatum isolates showed that ampicillin-resistant F. nucleatum isolates harboured a class D β-lactamase enzyme. In addition, increased synthesis of two proteins, enolase and ABC transporter ATP-binding proteins, was associated with β-lactamase enzyme production. A sound knowledge of antimicrobials prescription among Yemeni dentists was lacking with a trend of overuse. NorPD data showed antimicrobial prescribing trend in favour of narrow-spectrum penicillins among Norwegian dentists. Conclusions: The findings indicate that high prevalence of resistance among oral bacteria in Yemen maybe a potential threat in the management of dental infections in the region. Antimicrobial overuse by dentists in Yemen could exacerbate the existing dilemma of antimicrobial resistance of oral bacteria. In contrast, the low prevalence of resistance among subgingival species in Norway is most probably a result of the judicious use of antimicrobials, that is, more restrictive prescribing practices in the country. The presence of class D β-lactamase among ampicillin- resistant F. nucleatum strains increases their virulence and cost of treatments as these enzymes might present resistance to several classes of β-lactam antibiotics. Recommendations: Halting resistance development and saving effectiveness of antimicrobials need strict, practical, and feasible approaches. There is an urgent need to formulate an action plan to counter the revealed situation of antimicrobial resistance in Yemen. A proposed strategy to be adopted in the country for the control of antimicrobial resistance should be based on the prevention of communicable diseases and infection control to reduce the need for antimicrobial agents. An antimicrobial resistance surveillance system and a multidisciplinary committee that monitors antimicrobial use in the country should form integral parts of a structured approach to reduce antimicrobial resistance by improving antimicrobial prescribing. Finally, such an effective strategy requires close cooperation and consultation between Yemen and other involved parties, both at national and international levels

Investigation of fitness cost, copy number variation and excision rate of Tn916/Tn916-like elements in oral streptococci and enterococci

The Tn916 and other related elements are broad host range conjugative transposons that are widely spread in bacteria. These elements contribute to the spread of antibiotic resistance genes (ARG) between bacterial species. Variation in the Tn916/Tn916-like elements copy number (Tn916CN) and their excision frequency (Tn916ExFr) from their host genome could play a role in propagation of ARG. In this study, the Tn916CN and the Tn916ExFr in oral streptococci and enterococci were determined using droplet digital PCR (ddPCR). In addition, we investigated the fitness cost associated with new acquisition of Tn916 in a selected oral streptococci and enterococci strains. Amelioration of fitness cost and mechanism(s) to mitigate fitness loss were evaluated by ddPCR and next generation sequencing (NGS). We found the ddPCR is superior to southern blot to determine Tn916CN. It was also found that both the excision rate of Tn916 and its autonomous replication increase because of antimicrobial challenge. Loss of bacterial fitness was usually associated with multiple acquisitions of Tn916. It is concluded that amelioration of fitness cost is achieved after a few hundred generations and by maintaining one copy of Tn916 in bacterial genome under no selection pressure