Mohammed Al-Haroni

Knowledge of prescribing antimicrobials among Yemeni general dentists

Objective. Overuse of antimicrobial agents is closely related to an increase in bacterial resistance. A sound knowledge of appropriate prescribing of antimicrobials among health professionals is thus critical in combating the resistance. The objectives of this study were to assess the rationale for and patterns of antimicrobial prescriptions by general dental practitioners in Yemen. Material andMethods. A questionnaire containing 65 closed questions was used for this cross-sectional study and distributed to 280 dentists in the three major governorates in Yemen. The anonymously completed questionnaires sought answers to demographic questions and to questions on the therapeutic and prophylactic use of antimicrobial agents in dentistry. Correct and incorrect answers were defined according to information available in the current authoritative literature. Each correct answer was given a score of 1 while an incorrect answer scored 0. Thus, the total score had an attainable range from 0 to 65. Frequencies, means, and associations were assessed statistically. Results. Out of 181 collected forms (response rate 64.6%), 150 were appropriately completed and used for data analyses. Penicillins were the most frequently prescribed drugs (72%), followed by spiramycin (10%). It was found that up to 84% of practitioners were likely to prescribe an antimicrobial agent when there was no clinical indication for such a medication. Many respondents (70%) would consider antibiotics for at least one of the given non-clinical factors. Conclusions. The results suggest that dental practitioners in Yemen lack uniformity in the rationale for appropriate prescribing of antimicrobials to their patients. Consequently, to reduce overuse, there is an urgent need for the dental community in the country to be informed about evidence-based guidelines and the appropriate use of antimicrobial agents in clinical dental practice.

Caries experience and quantification of Streptococcus mutans and Streptococcus sobrinus in saliva of Sudanese schoolchildren.

Streptococcus mutans and Streptococcus sobrinus are among the most commonly isolated bacterial species implicated as etiological agents of dental caries. Details of the composition of the oral microflora related to dental caries should aid in assessing the prevalence and risk of disease at an individual level. The aim of the present study was to determine the presence and relative amounts of S. mutans and S. sobrinus in the saliva samples obtained from schoolchildren in Khartoum State, the Sudan, and to study the association of the amounts of S. mutans and S. sobrinus with caries experience, socioeconomic status and sugar-sweetened snacks in this population. 140 samples, 30 of which were from individuals with caries experience, were examined using quantitative real-time polymerase chain reaction (qRT-PCR) with specific oligonucleotide primers. The mean ratio of fold differences of S. mutans to S. sobrinus was 0.77 (SD 5.4) and 2.29 (SD 6.0) for samples obtained from caries-free and caries-active individuals, respectively. This suggested that the proportion of S. sobrinus was higher than S.mutans in the caries-active group when compared to the caries-free group. An association was found between children with caries-active lesions and the frequent consumption of sticky desserts and higher socioeconomic status. S. sobrinus seems to be associated with caries experience in the studied population. A proposal of caries screening programs designed to test for S. sobrinus in this population may be developed.

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.

Proteomic analysis of ampicillin-resistant oral Fusobacterium nucleatum.

INTRODUCTION Fusobacterium nucleatum represents one of the predominant anaerobic species in the oral microbiota. Penicillin-resistant F. nucleatum have been isolated from intra- and extraoral infections. This study aimed to assess ampicillin resistance in F. nucleatum by investigating the synthesis of resistance-associated proteins. METHODS Ampicillin-resistant and ampicillin-susceptible F. nucleatum isolates were obtained from 22 dental plaque samples. Two-dimensional gel electrophoresis and mass spectrometry were used to investigate bacterial protein synthesis. Proteins exhibiting statistically significant quantitative changes between sensitive and resistant isolates were identified using peptide mass mapping and matrix-assisted laser desorption/ionization - time of flight/time of flight (MALDI-TOF/TOF) mass spectrometry. RESULTS Twenty-three F. nucleatum isolates were recovered from plaque samples and their ampicillin minimum inhibitory concentrations ranged between 0.125 microg/ml and 256 microg/ml. Analysis of the bacterial cellular proteins by two-dimensional gel electrophoresis resolved 154-246 distinct protein spots (mean 212, n = 9). Between 32% and 83% of the protein spots were common for the F. nucleatum isolates. Comparisons of the protein profiles of sensitive and resistant isolates revealed the presence of a 29 kDa protein and significant increases in the synthesis of two proteins at 37 and 46 kDa in the ampicillin-resistant F. nucleatum isolates. These proteins were identified as a class D beta-lactamase, ATP-binding cassette (ABC) transporter ATP-binding protein and enolase, respectively. CONCLUSION Synthesis of a class D beta-lactamase by ampicillin-resistant F. nucleatum isolates could complicate antimicrobial treatment because these enzymes might confer resistance to many classes of beta-lactam antibiotics. The differences observed in protein synthesis between ampicillin-resistant and ampicillin-susceptible F. nucleatum may contribute to the antibiotic resistance and virulence of these bacteria.

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.

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

ABSTRACT 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.