Ali Al-Ahmad

Biodegradability of some antibiotics, elimination of the genotoxicity and affection of wastewater bacteria in a simple test

Most antibiotics and their metabolites are excreted by humans after administration and therefore reach the municipal sewage with the excretions. Only little is known about their biodegradability in aquatic environments. It was recognised that genotoxic substances may represent a health hazard to humans but also may affect organisms in the environment. Therefore, the biodegradability of some clinically important antibiotic drugs (ciprofloxacin, ofloxacin, metronidazole) and hereby the elimination of their genotoxicity was investigated as the first step of an environmental risk assessment using the Closed Bottle test (CBT) (OECD 301 D) and the SOS chromotest. Additionally, to assess toxicity of the antibiotics tested against aquatic bacteria (i) a growth inhibition test (GIT) with Pseudomonas putida was conducted, (ii) a toxicity control was used in the CBT and (iii) the colony forming units (CFUs) were monitored in the test vessels. Worst case concentrations of the antibiotics in hospital effluents were estimated and compared with minimum inhibitory concentrations for susceptible pathogenic bacteria and with the genotoxic potency in the SOS chromotest. Both the concentrations calculated for hospital effluents and the adverse effects in bacteria were in the same order of magnitude. None of the test compounds were biodegraded. The genotoxicity was not eliminated.

Visualization of adherent micro-organisms using different techniques

The visualization and quantification of adherent bacteria is still one of the most relevant topics in microbiology. Besides electron microscopic techniques such as transmission electron microscopy, scanning electron microscopy and environmental scanning electron microscopy, modern fluorescence microscopic approaches based on fluorogenic dyes offer detailed insight into bacterial biofilms. The aim of the present review was to provide an overview of the advantages and disadvantages of different methods for visualization of adherent bacteria with a special focus on the experiences gained in dental research.

Biodegradability of antineoplastic compounds in screening tests: influence of glucosidation and of stereochemistry.

Some pharmaceuticals such as antineoplastics are carcinogenic, mutagenic, teratogenic and fetotoxic. Antineoplastics and their metabolites are excreted by patients into waste water. In laboratory testing the frequently used isomeric anti-tumour agents cyclophosphamide (CP) and ifosfamide (IF) were shown to be not biodegradable. They are not eliminated in municipal sewage treatment plants and therefore detected in their effluents. Structural related compounds are beta-D-glucosylisophosphoramidmustard (beta-D-Glc-IPM; INN = glufosfamide) and beta-L-glucosylisophosphoramidmustard (beta-L-Glc-IPM). beta-L-Glc-IPM has no antineoplastic effects whereas beta-D-Glc-IPM is active against tumours. In contrast to IF and CP and almost all other investigated antineoplastics beta-D-Glc-IPM is inherently biodegradable. Improved biodegradability of beta-D-Glc-IPM compared to IF shows that reducing the impact of pharmaceuticals on the aquatic environment is feasible by changing the chemical structure of a given compound exerting a similar mode of action and therapeutic activity. Stereochemistry may be crucial for pharmaceutical activity of the compounds as well as for its biodegradability in the environment.

New Bacterial Compositions in Root-filled Teeth with Periradicular Lesions

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

Biofilm formation and composition on different implant materials in vivo

Biofilm formation was evaluated on the following titanium and zirconia implants in vivo: machined titanium (Ti-m), modified titanium (TiUnite), modified zirconia (ZiUnite), machined alumina-toughened zirconia (ATZ-m), sandblasted alumina-toughened zirconia (ATZ-s), and machined zirconia (TZP-A-m). Bovine enamel slabs were used as controls. Surface morphologies were examined by atomic force (AFM) and scanning electron microscopy (SEM). The surface wettability was also determined. Twelve healthy volunteers wore a splint system with the tested materials. After 3 and 5 days the materials were examined by fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). The levels of Streptococcus spp., Veillonella spp., Fusobacteriaum nucleatum, and Actinomyces naeslundii were quantitatively determined. The biofilm thickness was found to be between 19.78 and 36.73 μm after 3 days and between 26.11 and 32.43 μm after 5 days. With the exception of Ti-m the biofilm thickness after 3 days was correlated with surface roughness. In addition to Streptococcus spp. as the main component of the biofilm (11.23-25.30%), F. nucleatum, A. naeslundii, and Veillonella spp. were also detected. No significant differences in biofilm composition on the implant surfaces could be observed. In total, the influence of roughness and material on biofilm formation was compensated by biofilm maturation.

Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization.

Oral biofilms are one of the greatest challenges in dental research. The present study aimed to investigate initial bacterial colonization of enamel surfaces in situ using fluorescence in situ hybridization (FISH) over a 12 h period. For this purpose, bovine enamel slabs were fixed on buccal sites of individual splints worn by six subjects for 2, 6 and 12 h to allow biofilm formation. Specimens were processed for FISH and evaluated with confocal laser-scanning microscopy, using probes for eubacteria, Streptococcus species, Veillonella species, Fusobacterium nucleatum and Actinomyces naeslundii. The number of adherent bacteria increased with time and all tested bacterial species were detected in the biofilm formed in situ. The general percentage composition of the eubacteria did not change over the investigated period, but the number of streptococci, the most frequently detected species, increased significantly with time (2 h: 17.7+/-13.8 %; 6 h: 20.0+/-16.6 %; 12 h: 24.7+/-16.1 %). However, < or =1 % of the surface was covered with bacteria after 12 h of biofilm formation in situ. In conclusion, FISH is an appropriate method for quantifying initial biofilm formation in situ, and the proportion of streptococci increases during the first 12 h of bacterial adherence.

New Bacterial Composition in Primary and Persistent/Secondary Endodontic Infections with Respect to Clinical and Radiographic Findings

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

In vivo study of the initial bacterial adhesion on different implant materials.

OBJECTIVE Biofilm formation on implant materials plays a major role in the aetiology of periimplantitis. The aim of this study was to examine in vivo the initial bacterial adhesion on six different implant materials. METHODS The implant materials Ti-m, TiUnite®, ZiUnite®, ATZ-m, ATZ-s, TZP-A-m were tested using bovine enamel slabs as controls. All materials, fixed on splint systems, were examined after 30 min and 120 min of oral exposure. DAPI staining was used for quantitative analysis of the initially adherent microorganisms. Initial adherent microorganisms were visualised by fluorescence In situ-hybridisation (FISH) and quantified by confocal laser scanning microscopy (CLSM). The targets of the oligonucleotide probes were Eubacteria, Veillonella spp., Fusobacterium nucleatum, Actinomyces naeslundii and Streptococcus spp. RESULTS DAPI analysis showed that increasing the time of oral exposure resulted in an increasing amount of initial adherent bacteria. The highest level of colonisation was on ZiUnite®, with the lowest occurring on the bovine enamel, followed by Ti-m. This early colonisation correlated significantly with the surface roughnesses of the materials. FISH and CLSM showed no significant differences relating to total bacterial composition. However, Streptococcus spp. was shown to be the main colonisers on each of the investigated materials. CONCLUSION it could be shown that within an oral exposure time of 30 min and 120 min, despite the salivary acquired pellicle initial biofilm formation is mainly influenced directly or indirect by the material surface topography. Highly polished surfaces should minimise the risk of biofilm formation, plaque accumulation and possibly periimplantitis.

Comprehensive Analysis of Secondary Dental Root Canal Infections: A Combination of Culture and Culture-Independent Approaches Reveals New Insights

Persistence of microorganisms or reinfections are the main reasons for failure of root canal therapy. Very few studies to date have included culture-independent methods to assess the microbiota, including non-cultivable microorganisms. The aim of this study was to combine culture methods with culture-independent cloning methods to analyze the microbial flora of root-filled teeth with periradicular lesions. Twenty-one samples from previously root-filled teeth were collected from patients with periradicular lesions. Microorganisms were cultivated, isolated and biochemically identified. In addition, ribosomal DNA of bacteria, fungi and archaea derived from the same samples was amplified and the PCR products were used to construct clone libraries. DNA of selected clones was sequenced and microbial species were identified, comparing the sequences with public databases. Microorganisms were found in 12 samples with culture-dependent and -independent methods combined. The number of bacterial species ranged from 1 to 12 in one sample. The majority of the 26 taxa belonged to the phylum Firmicutes (14 taxa), followed by Actinobacteria, Proteobacteria and Bacteroidetes. One sample was positive for fungi, and archaea could not be detected. The results obtained with both methods differed. The cloning technique detected several as-yet-uncultivated taxa. Using a combination of both methods 13 taxa were detected that had not been found in root-filled teeth so far. Enterococcus faecalis was only detected in two samples using culture methods. Combining the culture-dependent and –independent approaches revealed new candidate endodontic pathogens and a high diversity of the microbial flora in root-filled teeth with periradicular lesions. Both methods yielded differing results, emphasizing the benefit of combined methods for the detection of the actual microbial diversity in apical periodontitis.

Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro

OBJECTIVE Sodium benzoate (SB), potassium sorbate (PS) and sodium nitrite (SN) are commonly used food preservatives. In this in vitro study, the effects of these substances on biofilm formation of Streptococcus mutans were analysed. METHODS In addition to the microtiter plate test (MPT), a biofilm reactor containing bovine enamel slabs (BES) was used to study the influence of food preservatives on biofilm formation in 5 independent periods of 4 days each. These included one period with chlorhexidine digluconate (CHX) as a positive control as well as a period with growth medium alone as a negative control. The vitality of the biofilm on BES was detected using live/dead staining and confocal laser scanning microscopy. Additionally, the number of colony forming units (CFU) was determined. RESULTS In MPT 0.12% SN significantly reduced the biofilm formation. PS at a concentration of 0.4% tended to inhibit biofilm formation, whereas the inhibition for 0.8% PS was significant. Less inhibition was caused by 0.8% SB. In the biofilm reactor 0.06% of SN, 0.1% of SB and 0.1% PS significantly reduced the covering grade as well as the CFU of the biofilm. Biofilm vitality was reduced significantly by CHX to a level of 32.5% compared to the control. Only SB reduced the vitality to a level of 19.1%. SN and PS showed no influence on biofilm vitality. CONCLUSION This study indicates the potential of food preservatives as inhibitory agents in S. mutans biofilm formation, which should be kept in mind when studying the effects of conserved food on dental plaque biofilm in situ.