Establishment of Diagnostic Reference Levels in Portuguese Interventional Radiology departments.

PURPOSE: To establish Portuguese Diagnostic Reference Levels (DRLs), for six body fluoroscopy guided interventional procedures (FGIP). METHOD: A retrospective study was conducted in five interventional departments most representative of Interventional Radiology (IR) practice. Dose values, in terms of air kerma area product (PKA in Gy.cm2), air kerma at the patient entrance reference point (Ka,r in mGy), and exposure parameters (fluoroscopy time (FT) and number of cine runs) were collected. Examinations were selected per procedure (at least 20), according to the antero-posterior and lateral diameter mean value (±5 cm), measured on previous Computed Tomography (CT) examinations. RESULTS: Data of 489 body FGIP show a large variation on dose values per procedure and per department. National DRLs in terms of PKA were 20.2 Gy.cm2 for Percutaneous transhepatic biliary drainage (PTBD), 98.2 Gy.cm2 for Bronchial artery embolisation (BAE), 247.7 Gy.cm2 for Transarterial chemoembolisation (TACE), 331.6 Gy.cm2 for Inferior epigastric arteries embolisation (IEAE), 312.0 Gy.cm2 for Transjugular intrahepatic portosystemic shunt (TIPS) and 19.3 Gy.cm2 for Endovascular treatment of femoral popliteal arteries (ETFPA). CONCLUSIONS: This is the first study reporting Interventional Radiology DRLs in Portugal and we propose preliminary national estimates for the six more common body FGIP. The results of this study will be presented and discussed with all Portuguese IR departments, to promote procedures optimisation.

Recent Updates on Microbial Biofilms in Periodontitis: An Analysis of In Vitro Biofilm Models.

The development of oral biofilm models has been extremely important to study the specific role of most microbial species at the early stages of periodontitis. The current knowledge on monospecies or multispecies biofilms originates mainly from the observation of in vitro dynamic or static biofilm model systems, which were engineered to mimic clinical oral conditions. In the last few decades, mounting evidence has confirmed that biofilms are the major form of bacterial lifestyle, and more importantly, that microorganisms dwelling in sessile mixed-species aggregates display completely different phenotypes and physiological characteristics than when living in planktonic pure cultures. Interspecies interactions within these communities, mediated by chemical communication systems, have been shown to affect biofilm physiology and increase antimicrobial resistance by up to 1000 fold. These aspects reinforce the importance of developing multispecies biofilm models to better understand and control biofilms. Literature reports demonstrate that while monospecies models are still most commonly used in caries research, authors have used different multispecies models to study periodontal diseases. Periodontitis is a polymicrobial biofilm-dependent disease mainly associated with Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Interestingly, these species hardly adhere to substrates commonly used for biofilm formation, which makes multispecies models essential for an accurate analysis of periodontitis-related biofilms. The multispecies models currently available are generally composed of 6-10 species, but a more recent 34-species model was developed to better examine the dynamics within oral biofilms. The complexity of such polymicrobial biofilm models mimics more consistently the oral microbiome and different aspects of the oral environment. Collectively, the evidence on multispecies biofilm models described herein may support future studies on the use of antimicrobials for biofilm control as well as provide research opportunities to expand the current knowledge on interspecies interactions. The present manuscript reviews the most recent updates on in vitro biofilm model systems for periodontitis.

Comparison of results from two different leakage models to evaluate the apical sealing of endodontic materials

Introduction: The methods used to evaluate the apical sealing of root canal filling materials have some limitations and great variability of results can be observed. Aim: The aim of this study was to compare, in an ex vivo apexification model, the results from bacterial and glucose leakage tests, which were applied in mineral trioxide aggregate (MTA) apical plugs. Materials and Methods: Sixty root segments (12mm) were randomly divided into 2 experimental groups (n=30): G1) MTA; G2) MTA + phosphate-buffered saline intracanal. Half of the specimens in each group were submitted to bacterial leakage test with E. faecalis for 70 days. The other half was submitted to the glucose leakage test under pressure (103KPa) for 60 min. The results from the two tests were compared based on the number of specimens presenting leakage. Data were analyzed by Fisher's test (p < 0.05). Results: There was no significant difference between tests for both groups analyzed (p > 0.05).Conclusion: The results of the present ex vivo study demonstrated that there was no difference between glucose and bacteria leakage evaluation methods, within the parameters of the present study and regardless

Early Biofilm Formation on Rough and Smooth Titanium Specimens: a Systematic Review of Clinical Studies.

OBJECTIVES: There is a concern whether the enhancement on implant surface roughness is responsible for higher biofilm formation, which acts as an aetiological factor for peri-implant diseases. The aim of the present systematic review was to answer the following question: "Are rough surfaces more susceptible to early biofilm formation when compared to smoother surfaces on titanium specimens?". MATERIAL AND METHODS: The research was performed on PubMed, Web of Science and Scopus, up to August 2021. Eligibility criteria included studies that analysed human biofilm formation on titanium specimens with distinct surface roughness (smooth vs minimally, moderate, or rough) over the experimental times of 1 or 3 days. Roughness average (Ra) and biofilm analysis parameters were extracted from selected articles. Risk of bias was evaluated using the Checklist for Quasi-Experimental Studies. RESULTS: Of 5286 papers, 5 were included and analysed. Smooth titanium surfaces included machined and anodized titanium/Ti-6Al-4V; machined and acid etched TiZr. Minimally, moderately, or rough surfaces comprised titanium and titanium alloys (TiZr, Ti-6Al-4V), that received surface treatments (anodization, acid-etching, blasting, hydroxyapatite-coating). No statistically significant difference on biofilm formation on rough and smooth titanium surfaces was reported by 3 studies, while more contamination on rough titanium surfaces was stated by 2 investigations. An isolated smooth surface has also been associated to higher contamination. Moderate to high quality methodological assessment of studies were identified. CONCLUSIONS: It is not possible to assume that rough surfaces are more susceptible to early biofilm formation than smooth titanium surfaces. Additional studies are required to study this multifarious interaction.

Strategies to Reduce Biofilm Formation in PEEK Materials Applied to Implant Dentistry-A Comprehensive Review.

Polyether-ether-ketone (PEEK) has emerged in Implant Dentistry with a series of short-time applications and as a promising material to substitute definitive dental implants. Several strategies have been investigated to diminish biofilm formation on the PEEK surface aiming to decrease the possibility of related infections. Therefore, a comprehensive review was carried out in order to compare PEEK with materials widely used nowadays in Implant Dentistry, such as titanium and zirconia, placing emphasis on studies investigating its ability to grant or prevent biofilm formation. Most studies failed to reveal significant antimicrobial activity in pure PEEK, while several studies described new strategies to reduce biofilm formation and bacterial colonization on this material. Those include the PEEK sulfonation process, incorporation of therapeutic and bioactive agents in PEEK matrix or on PEEK surface, PEEK coatings and incorporation of reinforcement agents, in order to produce nanocomposites or blends. The two most analyzed surface properties were contact angle and roughness, while the most studied bacteria were Escherichia coli and Staphylococcus aureus. Despite PEEK's susceptibility to biofilm formation, a great number of strategies discussed in this study were able to improve its antibiofilm and antimicrobial properties.

Effectiveness of nanoparticles solutions and conventional endodontic irrigants against Enterococcus faecalis biofilm.

CONTEXT: To overcome the challenge imposed by the presence of biofilm and reach significant bacterial reduction of the root canals, many irrigants have been indicated during endodontic treatment, among them nanoparticles solutions. AIMS: This study aims to evaluate the effectiveness of experimental solutions containing silver and zinc oxide nanoparticles (ZnO Np) and conventional endodontic irrigants against Enterococcus faecalis biofilm, in root canals. METHODS: Seventy-six extracted human teeth were biomechanically prepared and sterilized. The root canal surface was exposed to E. faecalis suspension to form a 7-day-old biofilm. Four teeth were analyzed by scanning electron microscopy (SEM) to confirm the presence of biofilm. The remaining teeth were randomly divided into 6 groups (n = 12) and treated with passive ultrasonic irrigation and different solutions: G1 - 0.85% saline (control); G2 - 2% chlorhexidine gluconate (CHX); G3 - 5% sodium hypochlorite (NaOCl); G4 - 1% NaOCl; G5 - 1% silver nanoparticles (Ag Np) solution; and G6 - 26% ZnO Np solution. The susceptibility of E. faecalis biofilms to disinfecting solutions (n = 10) was determined by quantification of colony-forming units. SEM analysis was also carried out to examine the biofilm structure after treatments (n = 2). Data were analyzed by Kruskal-Wallis and Dunn post hoc tests (P < 0.05). RESULTS: All tested solutions showed superior effectiveness compared to 0.85% saline (P < 0.05). Overall, 2% CHX presented the most effective action against E. faecalis biofilm, followed by 5% NaOCl, 1% Ag Np, 26% ZnO Np, and 1% NaOCl. CONCLUSIONS:: 1% Ag Np and 26% ZnO Np were effective against E. faecalis biofilm similarly to conventional endodontic irrigants.

Effect of γ-lactones and γ-lactams compounds on Streptococcus mutans biofilms.

Considering oral diseases, antibiofilm compounds can decrease the accumulation of pathogenic species such as Streptococcus mutans at micro-areas of teeth, dental restorations or implant-supported prostheses. OBJECTIVE: To assess the effect of thirteen different novel lactam-based compounds on the inhibition of S. mutans biofilm formation. MATERIAL AND METHODS: We synthesized compounds based on γ-lactones analogues from rubrolides by a mucochloric acid process and converted them into their corresponding γ-hydroxy-γ-lactams by a reaction with isobutylamine and propylamine. Compounds concentrations ranging from 0.17 up to 87.5 µg mL-1 were tested against S. mutans. We diluted the exponential cultures in TSB and incubated them (37°C) in the presence of different γ-lactones or γ-lactams dilutions. Afterwards, we measured the planktonic growth by optical density at 630 nm and therefore assessed the biofilm density by the crystal violet staining method. RESULTS: Twelve compounds were active against biofilm formation, showing no effect on bacterial viability. Only one compound was inactive against both planktonic and biofilm growth. The highest biofilm inhibition (inhibition rate above 60%) was obtained for two compounds while three other compounds revealed an inhibition rate above 40%. CONCLUSIONS: Twelve of the thirteen compounds revealed effective inhibition of S. mutans biofilm formation, with eight of them showing a specific antibiofilm effect.

Effects of three γ-alkylidene-γ-lactams on the formation of multispecies biofilms.

This study evaluated the inhibitory effects of lactams on Streptococcus mutans, Enterococcus faecalis, and Candida glabrata multispecies biofilm formation. γ-Alkylidene-γ-lactams 1, 2, and 3 [solubilized in 3.5% dimethyl sulfoxide (DMSO)] were tested. Glass coverslips were conditioned with either the lactams or 3.5% DMSO (control) for 1 h, inoculated with microbial cultures, and incubated for 48 h. To assess the effect of the lactams on biofilm formation, the following parameters were determined: the biofilm biomass (by both crystal violet staining and protein determination); the amount of insoluble polysaccharides of the extracellular matrix; and the number of viable and total cells [by both colony-forming unit counting and quantitative real-time PCR (qPCR)]. Data were analysed using one-way anova and post-hoc Tukey tests. Lactams 1, 2, and 3 promoted a statistically significant reduction in the amount of biofilm biomass, but only lactam 3 resulted in a statistically significant reduction in the number of attached viable E. faecalis. Both total protein content and the amount of extracellular polysaccharides decreased significantly. The effects of γ-alkylidene-γ-lactams 1, 2, and 3 on the inhibition of multispecies biofilm formation were evident by their ability to reduce the amount of protein and extracellular polysaccharides.

Effect of γ-lactones and γ-lactams compounds on Streptococcus mutans biofilms

Abstract Considering oral diseases, antibiofilm compounds can decrease the accumulation of pathogenic species such as Streptococcus mutans at micro-areas of teeth, dental restorations or implant-supported prostheses. Objective To assess the effect of thirteen different novel lactam-based compounds on the inhibition of S. mutans biofilm formation. Material and methods We synthesized compounds based on γ-lactones analogues from rubrolides by a mucochloric acid process and converted them into their corresponding γ-hydroxy-γ-lactams by a reaction with isobutylamine and propylamine. Compounds concentrations ranging from 0.17 up to 87.5 μg mL-1 were tested against S. mutans. We diluted the exponential cultures in TSB and incubated them (37°C) in the presence of different γ-lactones or γ-lactams dilutions. Afterwards, we measured the planktonic growth by optical density at 630 nm and therefore assessed the biofilm density by the crystal violet staining method. Results Twelve compounds were active against biofilm formation, showing no effect on bacterial viability. Only one compound was inactive against both planktonic and biofilm growth. The highest biofilm inhibition (inhibition rate above 60%) was obtained for two compounds while three other compounds revealed an inhibition rate above 40%. Conclusions Twelve of the thirteen compounds revealed effective inhibition of S. mutans biofilm formation, with eight of them showing a specific antibiofilm effect.

Biofilm behavior on sulfonated poly(ether-ether-ketone) (sPEEK).

Poly(ether-ether-ketone) (PEEK) has also shown to be very attractive for incorporating therapeutic compounds thanks to a sulfonation process which modifies the material structure resulting in a sulfonated-PEEK (sPEEK). Concerning biomedical applications, the objective of this work was to evaluate the influence of different sulfonation degree of sPEEK on the biofilm growth. PEEK samples were functionalized by using sulphuric acid (98%) and then dissolved into dimethyl-sulfoxide. A dip coating technique was used to synthesize sPEEK thin films. The sulfonation degree of the materials was analyzed by FT-IR, H NMR, TG and IEC. The surfaces were characterized by scanning electron microscopy, profilometry and contact angle analyses. Subsequently, the biofilm formation on sulfonated-PEEK based on Streptococcus mutans and Enterococcus faecalis was measured by spectrophotometry, colony forming units (CFUmL-1) and SEM. Results obtained from thermal and chemical analyses showed an intensification in sulfonation degree for sPEEK at 2 and 2.5h. The E. faecalis or S. mutans biofilm growth revealed statistically significant differences (p<0.05) between 2 and 3h sulfonation groups. A significant decrease (p<0.05) in CFUmL-1 was recorded for S. mutans or E. faecalis biofilm grown on 2.5 or 3h sPEEK. Regarding the thermal-chemical and microbiologic analyses, the sulfonation degree of sPEEK ranging from 2 up to 3h was successful capable to decrease the biofilm growth. That revealed an alternative strategy to embed anti-biofilm and therapeutic compounds into PEEK avoiding infections in biomedical applications.

Chemical, microscopic, and microbiological analysis of a functionalized poly-ether-ether-ketone-embedding antibiofilm compounds.

Poly-ether-ether-ketone (PEEK) is currently introduced as an alternative material for orthopedic implants due to its biocompatibility and low elastic modulus compared to titanium. Also, a sulphonation treatment can functionalize PEEK to embed therapeutical substances. The objective of this work was to functionalize a PEEK film to incorporate novel lactam-based antibiofilms compounds. PEEK samples were functionalized by sulphuric acid treatment and then dissolved in dimethylsulfoxide, where lactams were added to be incorporated into the polymer. A dip-coating technique was used to synthesize a thin film on a glass-based substrate. The degree of sulfonation (DS) and the incorporation of lactams into sulphonated PEEK (sPEEK) were analyzed by Fourier transform infrared spectroscopy, nuclear magnetic resonance, thermogravimetric analysis (TGA), and scanning electron microscopy. A DS of 65% was obtained and TGA curves confirmed the presence of SO3 H and lactams in the sPEEK structure. The growth of Streptococcus mutans biofilm decreased on sPEEK surface containing lactams when compared to sPEEK free of lactams. That indicated the antibiofilm activity of those compounds was maintained after incorporation into sPEEK. Planktonic growth analysis showed no long distant effects of sPEEK containing lactams, indicating that no systemic effects should be expected upon clinical uses of medical devices produced with lactam-treated sPEEK. Results revealed that inclusion of lactams into sPEEK represents a good alternative for the production of biomaterials resistant to bacterial accumulation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3015-3020, 2016.

A laboratory assessment of bacterial leakage in MTA apical plugs exposed to phosphate-buffered saline.

This study evaluated the influence of the exposure of mineral trioxide aggregate (MTA) - with and without calcium chloride (CaCl2) -to phosphate-buffered saline (PBS) on apical microleakage. Sixty root segments were divided into 4 experimental groups (n=15). Apical cavities were filled with MTA with or without CaCl2, and the root canals dressed with a moistened cotton pellet or PBS: 1) MTA/cotton pellet; 2) MTA/PBS; 3) MTA+ 10%CaCl2/cotton pellet; 4) MTA+10%CaCl2/PBS. After 2 months, E. faecalis penetration was analyzed along the apical plugs. Samples were observed weekly for 70 days, and leakage was detected by turbidity of the medium in contact with the root segment. Teeth in the control groups (n=2) were either made completely impermeable or kept without an apical plug. The Kaplan-Meier method was used to analyze survival and the Logrank test was used to compare the survival curves (p<0.05). All specimens in the positive control group showed evidence of leakage within 24h, while none in the negative control group showed leakage up to 70 days. There was no statistically significant difference among the experimental groups (p=0.102). The use of PBS as intracanal dressing may improve MTA sealing ability, but cannot prevent bacterial leakage. The addition of CaCl2 to the MTA did not improve MTA sealing ability.

Pesquisa a serviço da sociedade: área de concentração de Implantodontia do PPGO/UFSC / Scientific research for social benefits: oral Implantology at the PPGO/UFSC

Este manuscrito descreve as atividades desenvolvidas na PPGO/UFSC, concentradas em três linhas de pesquisa: 1) neoformação óssea peri-implantar; 2) engenharia tecidual; e 3) microbiologia aplicada à Implantodontia. Na linha um, diversos métodos analíticos quantitativos e qualitativos (SEM, AFM, FTIR, OCP, EIS, FEG-SEM) têm sido usados para investigação de diferentes intensidades de corrente elétrica na superfície do Ti-cp e Ti-6Al-4V, quando imersos em meios fisiológicos simulados, na adsorção de proteínas, proliferação celular com fibroblastos, queratinócitos e osteoblastos. Na linha dois, o desenvolvimento in vitro de um arcabouço poroso de PLGA e cerâmica bifásica, incorporado com sinvastatina, sera testado in vivo na resposta de fibroblastos gengivais provenientes de cultura primaria, em meio de cultura com plasma rico em plaquetas (PRP), depositado sobre matriz dérmica acelular e matriz de celulose bacteriana (BC). A linha três concentra a atuação com dois centros internacionais (Belgica e EUA) para a incorporação de compostos antibiofilme em polímeros biocompatíveis, e verificação da eficiência de um método de liberação prolongada de antibióticos incorporados em superfícies nanoestruturadas. Estes projetos devem resultar em melhoria de saúde aos pacientes e no desenvolvimento econômico e social da comunidade.

This paper describes the activities developed at PPGO/UFSC divided into three research lines: 1) peri-implant bone neoformation; 2) tissue engineering, and 3) microbiology applied to implant dentistry. On the first line, several quantitative and qualitative (SEM, AFM, FTIR, OCP, EIS, FEG-SEM) analytical methods have been used to investigate the different electric current intensities on Ti c.p. and Ti6Al4V implant surfaces when immersed into simulated physiological media, protein adsorption, cell proliferation with fibroblasts, keratinocytes, and osteoblasts. On the second line, the in vitro development of a 3D PLGA and biphasic ceramic, simvastatin-incorporated scaffold will be tested in vivo for gingival fibroblast response from primary culture in platelet-rich plasma (PRP) enriched vehicle over an acellular dermal (Sure- Derm) and bacterial cellulose matrix (BC). For line three, two international research centers (Belgium and USA) joined common efforts to incorporate polymer anti-biofi lm compounds and to verify the efficiency of a prolonged release method for antibiotics attached to nanostructured surfaces. All those projects must result in better health conditions and social and economic development of our community.

A laboratory assessment of bacterial leakage in MTA apical plugs exposed to phosphate-buffered saline / Avaliação laboratorial da infiltração bacteriana em plugs apicais de mta expostos ao tampão fosfato-salino

O presente estudo avaliou a influência da exposição do agregado de trióxido mineral (MTA) – com e sem cloreto decálcio (CaCl2) – ao tampão fosfato-salino (PBS) sobre a microinfiltração apical. Sessenta segmentos radiculares foram divididos em 4 grupos experimentais (n=15). As cavidades apicais foram preenchidas com MTA, com ou sem CaCl2, e os canais radiculares receberam uma bolinha de algodão umedecida ou PBS, como medicação intracanal: 1) MTA/bolinha de algodão umedecida; 2) MTA/PBS; 3) MTA+10%CaCl2/ bolinha de algodão umedecida; 4) MTA+10% CaCl2/PBS. Após 2 meses, a penetração de E. faecalis ao longo dos plugs apicais foi avaliada. As amostras foram observadas semanal -mente durante 70 dias e a infiltração detectada através da turbidez do meio em contato com os segmentos radiculares. Dentes pertencentes aos grupos controle (n=2) foram mantidos completamente impermeáveis ou sem plug apical. A análise de sobrevivência e a comparação das curvas foram realizadas por meio dos testes Kaplan-Meier e Log-rank (p<0.05), respectiva -mente. Todas as amostras do grupo controle positivo apresentaram evidência de infiltração dentro de 24h, enquanto nenhuma amostra do grupo controle negativo apresentou infiltração aolongo dos 70 dias. Não houve diferença significativa entre os grupos experimentais (p=0.102). O uso do PBS como medicação intracanal pode melhorar a capacidade de selamento do MTA,mas não é capaz de impedir a infiltração bacteriana. A adição de CaCl2 ao MTA não melhora sua capacidade de selamento.

This study evaluated the influence of the exposure of mineral trioxide aggregate (MTA) - with and without calcium chloride(CaCl2) -to phosphate-buffered saline (PBS) on apical microleakage. Sixty root segments were divided into 4 experimental groups (n=15). Apical cavities were filled with MTA with or without CaCl2, and the root canals dressed with a moistened cotton pellet or PBS: 1) MTA/cotton pellet; 2) MTA/PBS; 3) MTA+10%CaCl2/cotton pellet; 4) MTA+10%CaCl2/PBS. After 2months, E. faecalis penetration was analyzed a long the apical plugs. Samples were observed weekly for 70 days, and leakage was detected by turbidity of the medium in contact with the root segment. Teeth in the control groups (n=2) were either made completely impermeable or kept without an apical plug. The Kaplan–Meier method was used to analyze survival and the Log-rank test was used to compare the survival curves (p<0.05). All specimens in the positive control group showed evidence of leakage within 24h, while none in the negative control group showed leakage up to 70 days. There was no statisticall y significant difference among the experimental groups (p=0.102).The use of PBS as intracanal dressing may improve MTA sealing ability, but cannot prevent bacterial leakage. The addition of CaCl2 to the MTA did not improve MTA sealing ability.

Inhibition of Enterococcus faecalis biofilm formation by highly active lactones and lactams analogues of rubrolides.

Seven ß-aryl substituted γ-alkylidene-γ-lactones analogues of rubrolides were synthesized from mucobromic acid and converted through a lactamization with isobutylamine into their corresponding γ-hydroxy-γ-lactams (76-85%). These lactams were converted into (Z)- and (E)-γ-alkylidene-γ-lactams (23-45%). All compounds were fully characterized by IR, NMR ((1)H and (13)C), COSY and HETCOR bidimensional experiments, and NOE difference spectroscopy experiments when necessary. Evaluation of these three different classes of compounds against Enterococcus faecalis biofilm formation showed that all classes are active and the highest biofilm inhibition activity was caused by lactam 13f (IC50 = 0.76 µg/mL). Moreover, in almost all cases at least one of the lactams is more active than its correspondent γ-alkylidene-γ-lactone. The use of rubrolides as a lead structure has proven successful for the identification of new compounds displaying novel antibacterial activities, namely biofilm inhibition, which have the potential for the development of antimicrobial drugs targeted to inhibition of the initial stages of bacterial infections, rather than bacterial viability. Such drugs are less prompt to induce bacterial resistance, being therefore a more cost-effective investment for pharmaceutical research.

γ-Alkylidene-γ-lactones and isobutylpyrrol-2(5H)-ones analogues to rubrolides as inhibitors of biofilm formation by gram-positive and gram-negative bacteria.

Several molecules have been discovered that interfere with formation of bacterial biofilms, opening a new strategy for the development of more efficient treatments in case of antibiotic resistant bacteria. Amongst the most active compounds are some natural brominated furanones from marine algae Delisea pulchra that have proven to be able to control pathogenic biofilms. We have recently reported that some rubrolide analogues are able to inhibit biofilm formation of Enterococcus faecalis. In the present Letter we describe results of the biological evaluation of a small library of 28 compounds including brominated furanones and the corresponding lactams against biofilm formation of Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis and Streptococcus mutans. Our results showed that in general these compounds were more active against biofilms of S. epidermidis and P. aeruginosa, with little or no inhibition of planktonic bacterial growth. In some cases they were able to prevent biofilm formation of P. aeruginosa at concentrations as low as 0.6 µg/mL (1.3 µM, compound 3d) and 0.7 µg/mL (1.3 µM, 3f). Results also indicate that, in general, lactams are more active against biofilms than their precursors, thus designating this class of molecules as good candidates for the development of a new generation of antimicrobial drugs targeted to biofilm inhibition.

Synthetic organic compounds with potential for bacterial biofilm inhibition, a path for the identification of compounds interfering with quorum sensing.

New unconventional approaches to the development of antimicrobial drugs must target inhibition of infection stages leading to host colonisation or virulence itself, rather than bacterial viability. Amongst the most promising unconventional targets for the development of new antimicrobial drugs is bacterial adherence and biofilm formation as well as their control system, the quorum-sensing (QS) system, a mechanism of communication used to co-ordinate bacterial activities. Here we describe the evaluation of synthetic organic compounds as bacterial biofilm inhibitors against a panel of clinically relevant Gram-positive and Gram-negative bacterial strains. This approach has successfully allowed the identification of five compounds (GEt, GHex, GOctad, G19 and C33) active not only against bacterial biofilms but also displaying potential to be used as antagonists and/or inhibitors of bacterial QS.

From insects to human hosts: Identification of major genomic differences between entomopathogenic strains of Photorhabdus and the emerging human pathogen Photorhabdus asymbiotica.

Pathogenic bacteria of the genus Photorhabdus are naturally found in symbiotic association with soil entomopathogenic nematodes, and are of increasing economic interest in view of their potential for the development of novel biopesticides. This bipartite natural system is currently used for the biological control of crop pests in several countries. However, an increasing number of Photorhabdus strains have recently been isolated from human clinical specimens in both the United States and Australia, associated with locally invasive soft tissue infections and disseminated bacteraemia. In view of their growing use in biological control, which increases the potential rate of exposure of humans to these pathogens, we decided to undertake a comparative study of the genomic differences between insect and human pathogenic strains of Photorhabdus, in an attempt to understand the genetic mechanisms involved in the apparent change of host specificity, presumably responsible for their recently acquired capacity to infect humans. The data presented here demonstrates that major genomic differences exist between strains of Photorhabdus exhibiting virulence against insects or humans. Several individual genes, coding for virulence factors, were isolated and shown to be specific to the Photorhabdus asymbiotica human pathogens. One of these genes, sopB, encoding a host cell invasion factor translocated via the type III secretion system, has been cloned and the comparison of its genomic context in different pathogens strongly indicates that horizontal gene transfer is implicated in the acquisition of these virulence factors specific to the human pathogens. The precise role of this and other virulence factors identified here in the pathogenicity of P. asymbiotica towards humans is currently under investigation.

Positive correlation between in vivo and in vitro assays for the evaluation of Pseudomonas virulence.

Some bacterial phenotypes measured in vitro can be used to access bacterial virulence, on the premise that they are positively correlated with data from in vivo experiments. We show here that in vitro assessment of bacterial phenotypes, such as adherence and cytotoxicity, are positively correlated with data from in vivo experiments in Drosophila and can be used to assess bacterial virulence in vivo. Manipulation of environmental parameters, such as iron availability, induced changes in the phenotypes measured in vitro that correlated with changes in vivo virulence of all strains tested. Applying these assays, we demonstrate the pathogenic potential of a Pseudomonas fluorescens strain, initially isolated as a non-pathogenic milk contaminant. This strain displayed adherence and cytotoxicity comparable to those of the Pseudomonas aeruginosa pathogenic strain PAK, and colonized the infected flies as rapidly as the PAK strain. These results indicate that this "a priori" non-pathogenic bacterium is capable of escaping the host immune response, supporting the use of in vitro tests for screening of potential pathogens.

In vitro identification of two adherence factors required for in vivo virulence of Pseudomonas fluorescens.

By enriching a random transposon insertion bank of Pseudomonas fluorescens for mutants affected in their adherence to the human extracellular matrix protein fibronectin, we isolated 23 adherence minus mutants. Mutants showed a defect in their ability to develop a biofilm on an abiotic surface and were impaired for virulence when tested in an in vivo virulence model in the fruit fly, Drosophila melanogaster. Molecular characterisation of these mutants showed that the transposon insertions localised to two distinct chromosomal locations, which were subsequently cloned and characterised from two mutants. A search in the databanks identified two loci in the Pseudomonas aeruginosa PAO1 genome with significant homology to the genes interrupted by the transposon insertions. Mutant IVC6 shows homology to gmd, coding for the enzyme GDP-mannose dehydratase, involved in the synthesis of A-band- O-antigen-containing lipopolysaccharide (LPS). Mutant IVG7 is significantly similar to a probable outer membrane protein of strain PAO1, with no specific function attributed thus far, yet with significant homology to Escherichia coli FadL, involved in long-chain fatty acid transport. We propose that this protein, together with LPS, is involved in the first steps of P. fluorescens adherence leading to host colonisation. Results presented here also demonstrate the pathogenic potential of P. fluorescens, assessed in an in vivo Drosophila model system, correlated with its ability to adhere to the human extracellular matrix protein, fibronectin. Correlation between the mutant phenotypes with identified virulence factors and their actual role in the virulence of P. fluorescens is discussed.