Investigating the compounds to impair initial attachment of bacterial adhesion to biotic/abiotic surfaces could be the foremost effective strategy against the biofilm formation. The current means of controlling such biofilm infections is to physically remove the biofilm or the infected part and using anti-caries compounds mainly fluoride, chlorhexidine, and xylitol (Cui et al., 2019). Therefore, preventing the initial adhesion of bacteria mainly Streptococcus species, instead of attempts to destruct the established biofilms which could be provided by natural anti-adhesive coatings are among the current clinical demand. While only rare studies on dental biofilm inhibition, have focused on marine derived biocompounds (Silva et al., 2020; Stowe et al., 2011), in the present study, marine actinobacterial strains isolated from the Persian Gulf and the Sea of Oman, represented inhibitory metabolites on S.mutans biofilm.
In this research, five potent extracts capable of inhibiting the biofilm formation of S. mutans comparable to diuron were selected and the extract from marine Glycomyces sediminimaris showed the most effective anti-attachment activity with the highest biofilm inhibition value (95.1%) at the lowest concentration (100 𝜇g/mL). The genus Glycomyces is a relatively new genus in the phylum Actinobacteria and so limited reports demonstrating its potent biological activities are present (Bredholdt et al., 2007; Heidarian et al., 2018; W Li et al., 2018; Qian et al., 2020).
The compound walkmycin C from Bacillus subtilis (Eguchi et al., 2011), signermycin B from Streptomyces sp. (Watanabe et al., 2012), carolacton from Sorangium cellulosum (Sudhakar et al., 2014), vizantin obtained from Mycobacterium tuberculosis (Takenaka et al., 2016), outer membrane vesicles (OMV) derived from Burkholderia thailandensis (Y Wang et al., 2021), and the crude extract of marine bacterium Tenacibaculum sp. 20J (Y Wang et al., 2021) are the reported microbial compounds/extracts against biofilm formation of S.mutans. Despite the great potential of microorganisms, especially Actinobacteria in bioactive compounds production, these limited reports indicate the microbial capacity has not been well studied. Besides, despite multiple prevention methods currently being used such as mechanical methods, fluoride treatments, and replacing sugars in the diet, the continuation of dental caries and the side-effects of these treatments prove the demand for small molecules compounds which can selectively inhibit S. mutans biofilms through non-microbicidal (antiadhesion and signal interference) mechanisms (Yang et al., 2021).
Diketopiperazines (DKPs) are the smallest cyclic dipeptides ubiquitously found in nature being produced by non-ribosomal peptide synthetases (NRPS) and cyclodipeptide synthases. DKPs are characterized by a heterocyclic piperazine-2,5-dione synthesized from at least two amino acid residues being biosynthesized by varieties of organisms, including humans, plants, and microorganisms while it is assumed that a higher percentage of Gram-negative bacteria have the potential to produce diketopiperazine structural types compared to Gram-positive bacteria (W Fenical, 1993). Important biological activities contributed to diketopiperazine are not only limited to anti-tumor, anti-viral, anti-fungal, anti-bacterial, anti-prion, anti-hyperglycemic and glycosidase inhibition activities (P de CarvalhoandAbraham, 2012) but also their proposed action in modulating bacterial communication, LuxR based quorum sensing (QS) process and finally in the clinic to control the biofilm infections, indicate the prominent perspective potential of this class of compounds (Sun et al., 2016). The production of diketopiperazine compounds from microorganisms including marine microorganisms has been reported (Song et al., 2021) and its production in the members of Glycomyces spp. other than G. sediminimaris has not been reported previously (Sheida Heidarian, 2018).
Diketopiperazines are reported in studies as potential compounds to control biofilm infections acting as anti-biofilm compounds (P de CarvalhoandAbraham, 2012) which also inhibit biofilm formation in S. mutans. Anti-bacterial and quorum quenching properties of the six out of nine diketopiperazines identified in this extract have already been investigated. These include compounds cyclo-(leucyl-prolyl), cyclo-(phenylalanyl-prolyl), cyclo-(prolyl-valyl), cyclo-(valyl-phenylalanyl), cyclo-(prolyl-tyrosyl) and cyclo-(leucyl-valyl) (Abed et al., 2013; Alshaibani et al., 2017; J Li et al., 2011; P de CarvalhoandAbraham, 2012; RyanandDow, 2008). Among them, the anti-biofilm and quorum-sensing inhibitory activity of the compounds cyclo-(Phe-Pro), cyclo-(Trp-Ser), cyclo (Pro-Leu), cyclo(Pro-Val) and cyclo(Ala-Val) on biofilm forming bacteria (Serratia marcescens, Pseudomonas aeruginosa, Bacillus amyloliquefaciens and Escherichia coli ) and Chromobacterium violaceum (reporter strain for screening QS) has previously reported by (Sulieman et al., 2018), (Sun et al., 2016), (J-H Wang et al., 2016), and (Abed et al., 2013), respectively. Recently, the anti-biofilm activity of 75 synthetic cyclic peptides was assessed against S. mutans (Simon et al., 2019). The obtained results of our study suggest that the inhibition effect of Glycomyces sediminimaris extract probably be attributed to its dominant cocktail of diketopiperazines.
The inhibition potency of the introduced extract in this study implies the lower quantity required of the compound for attaining the same inhibitory effect in comparison with prevalent dentistry fluoride compounds and other potent biocompounds which have been investigated recently.
Furthermore, the impact of G. sediminimaris metabolites on the viability of S. mutans cells in a biofilm state showed 20% reduction in metabolic activity without any anti-bacterial effect on the planktonic state of S. mutans. Considering the anti-adherence activity of a biocompound is independent of its ability to interfere in cell metabolic pathways, this inhibitory performance could be accompanied by metabolic reduction activities and biofilm formation inhibition without affecting the cell viability. In addition to the inhibition of cell attachment, G. sediminimaris extract may be capable of attenuating the integrity of the mature biofilms through diminishing the cellular metabolic activity and even can be considered as an adjuvant compound for the eradication of formed biofilm in infectious oral diseases.
The metabolite extract of G. sediminimaris extract has no anti-bacterial effect on the planktonic state of S. mutans. It seems that the mechanism of action of the extract is predominantly related to adherence and biofilm formation inhibition and it has a negligible negative impact on the proliferation of bacterial cells in the planktonic state while it only diminishes the bacterial cell metabolism up to 20% in the biofilm structure without significant anti-bacterial effect in the biofilm phase. Thus, the type of mediated biofilm inhibition by this extract is not through the inhibition of cell division, which implies its non-toxic effect on oral microflora and the balance of oral ecosystem
The high activity of Glycomyces sediminimaris extract in inhibiting the S. mutans adhesion on polystyrene surfaces by decreasing the surface protein hydrophobicity was comparable with other studies directing to the mechanism underlying the inhibitory effect of G. sediminimaris extract on attachment of S. mutans
It is possible that the Diketopiperazines interconnects in cell surface proteins reducing the net hydrophobicity of the cell surface. Hence, the presence of extract may modify their hydrophobicity as a strategy to defer the initiation of biofilm formation or decrease the affinity of the surface proteins to attach to the surface.
Further, abating exopolymeric substances (EPS) production as a pivotal substance in maintaining the coherency of the biofilms (WatnickandKolter, 1999) and resistance of the cells to a variety of external tension (Wai et al., 1998) not only inhibits the biofilm matrix formation but also will not protect the internal cells of the biofilms from physical or chemical stresses including the antibiotics or biocides. The highly potent anti-adherence extract of G. sediminimaris can also contribute to the inhibition of EPS production by S. mutans and, inhibitors of EPS synthesis hold promise for the development of functional anti-biofilm agents for prophylaxis or therapeutic purposes.
Concludingly, this new attributed biofilm inhibition activity by a new species in the Glycomyces genus demonstrated that G. sediminimaris metabolites can be a competent candidate, not only for biofilm inhibition treatments but also, other unexplored bioactivities that can be discovered from this newly described species in further studies. Furthermore, since the efficacy of diketopiperazine compounds produced by different taxonomic resources is verified in hampering the biofilm formation, the presence of this compound in this non-toxic extract that has not been reported previously, can be considered as a potential candidate as an additive to the anti-septic mouthwash solutions for biofilm suppression, plaque formation and even reduction of other biofilm-forming pathogens on various types of implants.