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.

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.

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.

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.

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.

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.

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.