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.


Introduction
Biofilm consists in a complex microbial community embedded in an exopolymeric matrix based on polysaccharides, glycoproteins, nucleic acids and water 13,26 . Biofilms are found in nature adhered to different surfaces depending on nutritional and environmental conditions, such as oxygen, pH and nutrients. Factors related to the surface itself, like chemical composition, surface energy and roughness, also affect biofilm formation and development 2,13 . In oral environment, biofilm accumulation by pathogenic microorganisms is associated to oral diseases, such as gingivitis, periodontitis, peri-implantitis and caries 13,22 .
Over the last decades, conventional therapy on the use of antimicrobial agents targeting bacterial cell viability has significantly decreased the impact of infectious diseases. Nevertheless, such achievement has stimulated the development of microbial resistance to antibiotics 1,4,5,20 .
The development of new antimicrobial compounds targeting bacterial virulence instead of bacterial viability is a promising strategy for the prevention and treatment of infectious diseases, avoiding the stimulation of microbial resistance. In addition, novel anti-virulence drugs should disturb the adherence of pathogenic species to biotic and abiotic surfaces, eliminating the infection by the host immune system. Furthermore, virulence-targeted drugs can be associated with conventional antibiotics to improve their effectiveness 1,4,5,20 .
Considering oral diseases, antibiofilm compounds can decrease the presence of pathogenic species such Streptococcus mutans on exposed and retentive micro-areas of teeth, dental restorations or implantsupported prostheses 10,21 . For adhesion to oral surfaces, S. mutans produces extracellular polysaccharides that protect the consortia of bacteria against antimicrobial substances (e.g. antibiotics) 3  Previous studies revealed that synthetic lactams and lactones, similar to natural rubrolides, were mainly active against Enterococcus faecalis biofilms 16 .
On another study, a group of 28 lactones and lactams were tested against different biofilms produced by

Statistical analysis
We statistically analyzed the data by one-way ANOVA using the STATISTICA 6.0 ® software (StatSoft, Palo Alto, California, USA). The level of statistical significance was set at 0.05. We performed the T-test for independent samples to discover which lactams significantly affected the inhibition of the S. mutans biofilm.

Results
Results obtained for inhibition of planktonic growth and biofilm formation of S. mutans are shown in Figure   2.   As shown in Figure 2, compounds 1-5, 12, and 13 caused more than 40% biofilm inhibition, at different concentration for each compound. Clearly, the most active compound was the simple lactone 2, with a biofilm inhibition rate of 65% at 0.17 µg/mL -1 . The chlorine analogue 1 was less active, causing 54% biofilm inhibition at 5.44 µg/mL -1 . Statistical analysis confirmed the significance of the results (Figures 3-5).
In Figure 6, scanning electron microscopy images are shown for Streptococcus mutans free of lactam effect, lactam powder particle and S. mutans in the presence of lactam. In our study, the results revealed that twelve of the thirteen compounds tested were able to inhibit from 65% to 5.6% of biofilm formation ( Figure 2). The most active lactam compounds found in the previous study revealed biofilm inhibition percentages at 89, 34, and 31.7%, respectively, while this study revealed biofilm inhibition percentages at 65, 61, and 54.3%.
Previous studies showed reduced CFU counting in the biofilm detached from titanium surfaces exposed to lactam-derived compounds (1.5×10 2 CFU/mL in the presence of lactam and 4×10 2 CFU/ mL in its absence) 26 ( Figure 6). The incorporation of organic antibiofilm compounds based on lactams was successfully carried out in a previous study by functionalizing sPEEK (sulphonated poly-ether-etherketone) 14 . The sulphonation treatment is useful to embed therapeutic compounds such as lactambased antibiofilms. A significant inhibition of biofilm effect was detected on sPEEK surfaces containing lactams and, therefore, no effect was noticed on the physiologic planktonic growth of S. mutans. Such results indicated that the incorporation of lactams into materials used for dental rehabilitations, such as titanium or sPEEK, represents an effective clinical evolution regarding novel biomaterials with specific antibiofilm properties 14 . Additionally, further studies could evaluate the incorporation of antibiofilms in other polymers, such as poly(lactic-co-glycolic) acid, or PLGA, to cover titanium dental abutments or even to produce scaffolds for bone regeneration.
The development of biocompatible lactones and lactam-based compounds is an important strategy to avoid stimulating bacterial resistance 1,2,4,5 . Besides, previous studies had shown that lactams are less cytotoxic than furanones 8,9 and more reactive against biofilm formation than their corresponding lactones 15,16 .
It is important to notice that results presented here do not preclude that other metabolic pathways, indirectly related to biofilm formation, could also be affected at