Efficacy of Natural and Synthetic Biofilm Inhibitors Associated with Antibiotics in Eradicating Biofilms Formed by Multidrug-Resistant Bacteria

Abstract

Biofilms formed by multidrug resistant (MDR) bacteria like methicillin-resistant Staphylococcus aureus (MRSA) and others are the main causes of infections that represent a serious public health issue. Persistent MDR infections are mostly derived from biofilm formation which in turn leads to resistance to conventional antimicrobial therapy. Inhibition of bacterial surface attachment is the new alternative strategy without affecting the bacterial growth. Thus, the discovery of compounds that interfere with biofilm production, virulence factors release and quorum sensing (QS) detection in pathogens is a promising processus. Among these compounds, natural and synthetic molecules are a compelling alternative to attenuate pathogenicity. The combination of these compounds with antibiotics makes the bacteria more vulnerable to the later, once used alone. This combination can restore antibiotic effectiveness against MDR bacteria. Among these molecules, 3-phenylpropan-1-amine (3-PPA) has been found to inhibit Serratia marcescens biofilm formation, PAβN has been proven to inhibit biofilm prodcution in A. baumannii, while brominated Furanone C-30 has been reported to be a potent inhibitor of the QS system and P. aeruginosa biofilm. Therefore, the combination between biofilm-inhibitors and antibiotics represents a promising strategy to mitigate antibiotic resistance in MDR pathogens, which has become a major threat to public healthcare around the globe.

1. Introduction

Diseases that are caused by pathogens producing bacterial biofilms are increasingly spread, which represent a real threat to human health. Therefore, floating or swimming bacteria are more vulnerable to antibiotics. However, they can be reorganized into clusters of very complex structure, composed of a matrix of self-synthesized exopolymers, which forms the notorious biofilm that is hard to eradicate because bacteria embedded in this structure become highly resistant to many antimicrobial agents. In fact, when trapped in biofilms, biofilm-producing bacteria can be over 1000-fold more resistant to antimicrobials than their planktonic equivalents [1]. In addition, the massive use of antimicrobials has led to an increment in multi-drug resistance (MDR) of pathogenic bacteria, rendering the failure of antibiotic treatment. The six main multidrug-resistant and fatal pathogens are known as “ESKAPE” pathogens: Escherichia coli, S. aureus, Klebsiella pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp. These bacterial agents are responsible for polymicrobial infections that cause diseases such as cystic fibrosis, ear and urinary tract infections, respiratory tract infections, diabetic ulcers, wounds, in addition to the contamination of certain medical devices [2]. Furthermore, majority of chronic and nosocomial infections are associated with mono- or polymicrobial biofilms, having a significant impact on the survival rates of patients. Although the use of medical devices revolutionized health care services and significantly improved patient outcomes, it also led to complications due to the associations with biofilms and the emergence of multidrug resistant bacteria.

In particular, MDR bacteria poses a major challenge as current antimicrobial therapies are often associated with poor outcomes [3]. Based on the progress of the mechanism of biofilm development in MDR bacteria, many anti-biofilm molecules are being discovered with diverse modes of action such as quorum quenching (QQ) and cell adhesion inhibition, dispersion of extracellular polymeric substance, and interference with c-di-GMP signaling pathways, etc. [4]. Taking these factors into account, it is clear that new strategies are required to weaken the biofilm, inhibiting its proliferation and making it less resistant to antibiotics. These strategies involve targetting the resistance mechanisms of pathogenic bacteria like the production of biofilms by controlling quorum sensing (QS) since it is an intercellular communication system, which influences microbial virulence [5]. Therefore, interference in QS system of bacterial pathogens can reduce drug resistance which is considered as a suitable alternative that attenuates pathogenicity and protects the host from infection due to biofilm formation [1].

This issue has prompted researchers to find new microbial biofilm inhibitors that could be combined with existing antibiotics to improve their efficacy in bacterial eradication. In recent years, researchers have increasingly sought alternative therapeutic strategies for effective treatment of biofilm-producing pathogens. The target is to overcome the drawbacks of conventional antimicrobial therapies as microbial infections involving biofilms become quite challenging because of their high antibiotic resistance capacities. Within this framework, the present study has evaluated the anti-biofilm characteristics of natural and synthetic molecules against MDR bacteria.

2. Anti-biofilm activity of peptides and organic compounds

2.1 Anti-biofilm activity of 3-Phenylpropan-1-amine (3-PPA)

Phenylpropane-1-amine (3-PPA) is known to be an antibiotic adjuvant that interferes with QS and disrupts signaling between bacteria without posing a threat to the bacteria themselves, potentially resolving resistance in pathogenic bacteria [6]. In this recent and unique study, 3-phenylpropan-1-amine (3-PPA) was determined to inhibit biofilm formation. Furthermore, the inhibitory effect rises along with high drug concentrations. Notably, at 50 μg/mL, 3-PPA treatment reduces biofilm formation by 48%. Moreover, 3-PPA probably acts on virulence factors. They also studied the expression of genes related to detoxification enzymes and found a 37% inhibition in the expression of sodB gene, which encodes superoxide dismutase (SOD). Given the inhibitory effects of 3-PPA on biofilm formation, they also explored whether 3-PPA can increase the vulnerability of biofilms to traditional antibiotics. Thus, biofilms were exposed to 3-PPA and antibiotics in combination. In fact, 3-PPA (50 μg/mL) or ofloxacin (0.2 μg/mL) alone had weak effects on biofilm eradication, but relatively strong effects when used in combination, with a biofilm erasure rate of 44%. They also confirmed that by scanning electron microscopy (SEM), treatment with the combination of 3-PPA and ofloxacin resulted in the significant dispersal, destruction, and reduction of the preformed biofilm. Therefore, 3-PPA was used as an antibiotic adjuvant to interfere with the QS and interrupt the signaling between bacteria while not being a threat to the microorganism, which could help solve the problem of resistance in disease-causing bacteria. This is the only work to develop a strategy to by-pass multidrug-resistant S. marcescens and improve treatment outcomes for recalcitrant infections (Table 1).

Biofilm Inhibitors	Composition or structure	Dose	MDR	Target	Associated antibiotic	References
3-phenylpropan-1-amine (3-PPA)	Peptide	50 mg/mL	Serratia marescens; NJ01	Biofilm inhibition (48%) Virulence factors: Prodigiosin Protease Lipase Superoxide dismutase Hemolysine Swimming	PPA (50 μ g/ml) + Ofloxacine; (0.2 μ g/ml)	[6]
AS10	Peptide	0.22 M	Candida albicans; and; various Gram-positive and Gram-negative bacteria; Mixed biofilm	Biofilm inhibition	AS10 (0.22 M) +0 Caspofungin or Amphotericin B	[7]
CRAMP	peptide	NM	P. aeruginosa	91.05% of biofilm inhibition QS regulated genes: Pyocyanin Rhamnolipid production	CRAMP + Colistin (1/4 MIC)	[8, 10]
NP16	Peptide	NM	Methicillin resistant S. aureus (MRSA)	Antistaphyloxantin production Dehydrosqualene desaturase (CrtN) Anti-biofilm Expolysacharides reduction Membrane integrity	NA	[11]
Celastrol	Organic compound: Pentacyclic triterpenoid	1 μg/mL			Membrane targeting antibiotics: Polymixin B	[12]
Meta-bromo-thiolactone (mBTL)	Synthetic molecule	NM	P. aeruginosa	1.Quorum-sensing receptors, LasR and RhlR RhiR is the most relevant target genes encoding pyocyanin	NA	[13]
Malondialdehyde (MDA)	Natural/Synthetic molecule	90 μg/ml; 180 μg/ml	Staphylococcus xylosus; Lactiplantibacillus. plantarum	LDH release Ca^2+ and Mg^2+ leakage ATP reduction	NA	[14]
Psammaplin A	Marine-derived bromotyrosine compounds	NM	P. aeruginosa PAO1 lasB-gfp; P. aeruginosa PAO1 rhlA-gfp	Quorum-sensing inhibitor	IC50 value at 30.69 for PAO1 lasB-gfp and; IC50 value at 2.64 μM for PAO1 rhlA-gfp	[15]
Bisaprasin					IC50 value at 8.70 μM for PAO1 lasB-gfp and; IC50 value at 8.53 μM for PAO1 rhlA-gfp
Maipomycin A (MaiA)	Marine and bacterial-derived products	NM	Gram negative bacteria Actinomycete strain Kibdelosporangium phytohabitans XY-R10	Undefined pathway	Colistin	[8]
Piperine	Naturally occurring alkaloid	NM	K. pneumoniae	Preformed biofilm (MBEC)	Kanamycine + piperine (MBEC reduced by 8- to 16-fold)	[16]
Thymol (2-Isopropyl-5-methylphenol)	Essential oil: natural volatile monoterpenoid phenol	NM	Methicillin resistant S. aureus (MRSA)	Staphyloxanthin biosynthesis (CrtM)	90% of staphyloxanthin inhibition at 100 μg/mL + Polymixin B	[17]
			K. pneumoniae	Biofilm formation (MBIC)	Streptomycin + thymol (MBIC reduced by 16- to 64-fold)
					Kanamycin+ piperine (MBIC reduced by 4-fold)
			K. pneumoniae	Preformed biofilm (MBEC)	Streptomycin + thymol (MBEC reduced by 16- to 128-fold)	[16]
					Amikacine+ thymol (MBEC reduced by 4- to 128-fold)
					Kanamycine + thymol (MBEC reduced by 8- to 256-fold)
			Methicillin resistant S. aureus (MRSA)	Biofilm formation	Rifampicine +thymol (88% MRSA biofilm reduction)	[18]

Table 1.

List of biofilm inhibitors.

Abbrevations: NM: Not mentioned; NA: Not associated; QS: Quorum sensing; MBIC: Minimal biofilm inhibition concentration; MBEC: Minimal biofilm eradication concentration.

2.2 Anti-biofilm activity of cathelicidin-related antimicrobial peptide (CRAMP)

De Brucker et al. [7] identified AS10 (Peptide Sequene: KLKKIAQKIKNFFQKLVP) as the most potent anti-biofilm peptide at 0.22 M. This peptide inhibits biofilm formation of the fungus C. albicans and also various Gram-positive and Gram-negative bacteria in a mixed biofilm and acts synergistically with caspofungin or amphotericin B against mature C. albicans biofilm. Recently, in the study by Zhang et al. [8], the best synergistic activity of CRAMP combined with colistin at 62.5 μg/ml was confirmed for P. aeruginosa, with a significant inhibition of the biomass of preformed biofilms reaching 91.05%, confirmed by confocal laser scanning microscopy (CLSM) images. It was also confirmed that the combination (CRAMP-1/4 MIC colistin) also down-regulated the expression of QS regulated genes, including pyocyanin and rhamnolipid production [9]. In 2022, the same research team also elucidated the specific mechanism by which CRAMP was able to eradicate P. aeruginosa biofilms using an integrative analysis of transcriptomic, proteomic and metabolomic data [10]. Somal data showed that CRAMP acts on P. aeruginosa biofilms through a range of pathways, which include the Pseudomonas quinolone signaling system (PQS), the cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway, and the exopolysaccharide and rhamnolipid synthesizing pathways [10]. These studies provide new possibilities for the development of CRAMP as a potentially effective anti-biofilm dispersant or even a biofilm-preventive coating for implants (Table 1).

2.3 Anti-staphyloxantin activity of NP16 and Celastrol in S. aureus biofilm

In the recent study by Gao et al. [11], it was demonstrated a novel inhibitor (NP16) of S. aureus staphyloxantin (STX) production. This inhibitor targets the dehydrosqualene desaturase (CrtN) which catalyzes the first step of the staphyloxantin biosynthetic pathway. Staphyloxantin inhibition can reduce the survival of S. aureus under oxidative stress conditions and limits biofilm formation. This newly discovered CrtN inhibitor NP16 may represent an effective strategy for combating S. aureus biofilms. This molecule is not the only one to have an anti-staphyloxantin activity, as the study of Yehia et al. [12] demonstrated that celastrol efficiently STX biosynthesis in S. aureus through its effect on CrtM efficiently, confirmed by liquid chromatography-mass spectrometry (LC-MS) and molecular docking. In addition to its anti-pigment capability, celastrol exhibits significant anti-biofilm activity with its inhibitory effect on bacterial cell exopolysaccharides. Similarly, inhibition of STX upon celastrol treatment rendered S. aureus more susceptible to membrane targeting antibiotics. As a novel anti-virulent agent against S. aureus, Celastrol provides a prospective therapeutic role as a anti-pathogenic agent with multi-targets (Table 1).

3. Anti-biofilm activity of synthetic molecules

3.1 Anti-biofilm activity of meta-bromo-thiolactone (mBTL) via QS inhibition

In the study of O’Loughlin et al. [13], synthetic molecules were analyzed to prove their inhibitor effects on the two P. aeruginosa quorum-sensing receptors, LasR and RhlR. It was found that the most effective compound, is the meta-bromo-thiolactone (mBTL). It was also confirmed that both LasR and RhlR are partially inhibited by mBTL in vivo and in vitro; however, RhlR, not LasR, is the relevant in vivo target. Therefore, this work, that explores interference with QS, demonstrates that mBTL, an analogue of the native self-inducers of P. aeruginosa, suppresses the expression of genes encoding the virulence factor pyocyanin, on the one hand, and prevents biofilm formation on the other hand, which protects C. elegans and human lung epithelial cells from attack by P. aeruginosa. Taken together, these data about mBTL provide a strong argument for the efficacy of QS modulators for attenuation of QS-controlled phenotypes in pathogenic bacteria, such as biofilm formation (Table 1).

3.2 Anti-biofilm effect of malondialdehyde (MDA) via cell membrane injury

Malondialdehyde (MDA), one of the most representative reactive carbonyl species (RCSs) produced by lipid oxidation in bacteria [19] and in food [14], has received extensive attention recently. However, the inhibitory effect of MDA on microorganisms has received little attention. The study of Zhang et al. [10] proved the antibacterial effects of MDA on S. xylosus and Lactiplantibacillus plantarum with the MICs of 90 and 180 μg/ml, respectively. In addition, the antibacterial mechanisms of MDA on these two bacteria were associated with LDH activity changes as the LDH release is indicator of cell wall injury, accompanied with Ca²⁺ and Mg²⁺ leakage. Overall, the emission of Ca²⁺ and Mg²⁺ demonstrated that MDA enhanced the permeability of S. xylosus and L. plantarum cell membrane and further affected bacterial metabolism. In addition, MDA treatment induces cell membrane depolarization, indicating severe membrane damage with important impact on cell development and differentiation. This result has been confirmed by combination of CLSM and FEGSEM observations which have affirmed that MDA disrupts the cell membrane of S. xylosus and L. plantarum. It was also shown that MDA treatment significantly reduced the ATP concentration in S. xylosus and L. plantarum, suggesting that MDA may inhibit their growth by affecting the metabolic functions or cell membrane permeability of bacteria. Moreover, FT-IR studies showed that MDA might affect the molecular composition of S. xylosus and L. plantarum cells. These changes indicated the negative influence of MDA on cell membrane and cellular homeostasis [14].

4. Anti-biofilm Furanone activity through anti-QS activity

4.1 Furanones in general

Furanones are a family of structurally related molecules characterized by the presence of a five-membered heterocyclic furan ring. Furanones are available in a number of natural sources like marine and terrestrial plants, strawberries, coffee and fungi, and can also be chemically synthesized. Both natural and synthetic furanones have been shown to effectively inhibit QS, but synthetic furanones offer the possibility of precise control over compound structure and, therefore, control of any potential off-target effects [20, 21].

4.2 Anti-QS activity of natural furanones

Among the widely studied natural furanones, (5Z)-4-bromo-5- (bromomethylene)-3-butyl-2(5H)-furanone and Furanone 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), Furanone F202 show strong anti-biofilm activity by up to 55%. Based on the study of Ren et al. [22], a natural furanone, known as (5Z)-4-bromo-5- (bromomethylene)-3-butyl-2(5H)-furanone was demonstrated to attenuate biofilm formation in E. coli, reducing average biofilm thickness by 55%. Moreover, lower furanone concentrations (64.5 μM) significantly decreased E. coli swarming motility. On the other hand, investigations by Witsø et al. [23] into the impact of synthetic brominated furanones demonstrated that these compounds could also decrease E. coli biofilm wall thickness and surface area coverage by up to 50%. Brominated furanones, added at 50 μM, could suppress swarming motility and lower biofilm production by up to 40% in the foodborne pathogen E. coli 0103:H2. These important works [23] clearly demonstrated that natural furanones can interfere with QS processes and that the phenomenon could be used to combat virulence in human pathogens. Moreover, the study of Choi et al. [24] proved that natural furanones greatly reduce the production of P. aeruginosa virulence factors, including protease (up to 43%), chitinase and pyoverdine by almost 100% (Table 2) [24].

Furanones	Origin	Dose	MDR	Biofilm inhibition	Target	References
(5Z)-4-bromo-5- (bromomethylene)-3-butyl-2(5H)-furanone	Algae Delisea pulchra	164 μM; 64.5 μM	E. coli	55% biofilm thickness reduction; Swarming inhibition	QS process	[22, 25]
		[16.13–32.26 μM]	Vibrio harveyi	NM
		64.5–322.5 μM	P. aeruginosa PAO1 and JB2	NM	increase in siderophore production Protease reduction (43%) Chitinase, pyoverdine reduction (100%)
Furanone 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF)	Variety of fruits	0.1 or 1 μM	P. aeruginosa; PAO1	27.8% (0.1 μM) and 42.6% (1 μM) of biofilm inhibition	Reduced rhamnolipid (40.9%), pyocyanin (51.4%), LasA protease (53.8%) production	[24]
Furanone F202	Algae Delisea pulchra	50 μM	E. coli; 0103:H2	50% biofilm inhibition	QS process	[23]

Table 2.

List of natural furanone inhibitors.

4.3 Anti-QS activity of synthetic furanones

The process of developing synthetic furanones began in the 1980s and it usually starts with relative simple compounds, such as dimethyl ketones and acetals or other straight forward organic precursors. Then, it is also possible to modify existing furanone compounds and add existing furanone structures, as it is highlighted in Table 3. Recently, the majority of research on furanones-mediated QS inhibitors has been conducted on the effects of these compounds on human pathogens, especially on the model organisms E. coli and P. aeruginosa [20]. It was the synthesis of a range of structurally diverse bromine-, chlorine and iodine-containing furanones using a variety of palladium-catalysed coupling reactions was recently described [1]. The finding of this study [1] is interesting, as furanone is an ideal QS disruptor. Various compounds from the furanone library were screened for their inhibitory effects on biofilm production in opportunistic human pathogens and were found to potently suppress bacterial biofilm formation in S. enterica, S. aureus, P. aeruginosa, and, to a lesser extent, E. coli. Compounds which inhibited biofilm formation do not generally impact bacterial growth, highlighting their potential as QS inhibitors. According to the Furanone Library [1], tribromofuranone was found to be the most active compound decreasing biofilm formation in S. enterica by 72% and S. aureus by 71% at a concentration of 50 μM, whereas methyl-substituted dibromofuranone was the most potent inhibitor, which reduces biofilm growth P. aeruginosa PAR7244 by 44% compared to a 70% reduction in PAO1. For E. coli biofilm, bis-4-methoxyphenylacetylene was the most active compound, which inhibits E. coli ATCC9637 biofilm growth by 31%. Moreover, it was tested whether synthesized furanones, with relevant anti-biofilm activity, were able to disturb mixed fungal-bacterial biofilms. It was confirmed that the chosen bromofuranones and chloroiodofuranones were initially subjected to testing for their effect on monospecific biofilms of P. aeruginosa and C. albicans with confocal laser scanning microscopy (CLSM). Thus, it was found that all of them decrease the biomass of both microorganisms within the mixed biofilms [1].

Synthetic Furanones	Dose	MDR	Biofilm Inhibition	References
Tribromofuranone	50 μM	S. enterica	72% biofilm reduction; Via Quorum sensing	[1]
		S. aureus	71% biofilm inhibition
		C. albicans	51% biofilm inhibition
Bis-4-methoxyphenylacetylene-substituted	NM	E.coli ATCC9637	31% biofilm inhibition
Control monobromofuranone	NM	P. aeruginosa PAO1	75% biofilm inhibition
Methyl-containing dibromofuranones	NM	P. aeruginosa; PAO1; PAR7244	70%; 44% biofilm inhibition
Dibromofuranone; Chloroiodofuranone	50 μM	C. albicans M2396 Mixed biofilm: C. albicans M2396 with P. aeruginosa PAO1	92% biofilm inhibition
Brominated Furanone C-30	3.125–50 μM.	P. aeruginosa	Near total prevention of pyoverdine production Significant inhibition of LasR RhIR	[26]; [21]
Furanone C-56	5 μg ml − 1 (28.5 μM)	P. aeruginosa	37% reduction in biofilm thickness	[26]

Table 3.

List of synthetic furanone inhibitors.

5. Anti-biofilm activity of natural compounds

Natural products exhibit higher structural and biochemical variety than synthetic compounds, making them useful for the advancement of anti-biofilm agents [27]. More recently, there have been reviews of bacterial products that include small molecules, enzymes, exopolysaccharides and isolated peptides displaying anti-biofilm activities toward different pathogens [28]. Moreover, several studies have demonstrated solid evidences that plants [29] and marine-derived products [15] are an excellent source to provide abundant natural compounds for the development of preventative and therapeutic agents against biofilm-based infections (Table 1).

5.1 Anti-biofilm activity of marine and bacterial-derived products

Concerning antibiofilm activity of marine-derived products, the study by Oluwabusola et al. [15] proved that psammaplin A and bisaprasin, isolated from marine sponges, could be a potent QS inhibitory agents by preventing P. aeruginosa PAO1 biofilm formation. The present results indicated that psammaplin a showed moderate to significant inhibition against QS gene promoters, with IC50 values ranging from 30.69 to 2.64 μM. In contrast, bisaprasin showed significant inhibition for both biosensor strains, with equal IC50 values. Hence, using marine sources to find novel QS inhibitors as antipathogenic drugs to combat antimicrobial resistance has high potentials. Concerning antibiofilm activity of bacterial-derived products, the study of Zhang et al. (2021) described a novel and effective anti-biofilm compound named maipomycin A (MaiA), which was isolated from the metabolites of a rare actinomycete strain Kibdelosporangium phytohabitans XY-R10. This compound demonstrated a broad spectrum of anti-biofilm activities against Gram-negative bacteria [8].

5.2 Anti-biofilm activity of thymol

The study of Valliammai et al. [17] demonstrated the anti-biofilm potential of thymol against methicillin resistant S. aureus (MRSA) by inhibition of staphyloxanthin biosynthesis. The staphyloxanthin inhibitory potential of thymol was assessed against MRSA in terms of quality and quantity. It was demonstrated that 100 μg/mL concentration of thymol brings about 90% of staphyloxanthin inhibition. In addition, it was confirmed that thymol treatment makes MRSA more susceptible to reactive oxygen species. Experimental analyses were also confirmed by molecular docking analysis and in vitro measurement of metabolic intermediates of staphyloxanthin. It was also revealed that thymol could possibly interact with CrtM, which is involved in staphyloxanthin biosynthesis to inhibit production. In addition, reduction in staphyloxanthin by thymol treatment increases the membrane fluidity and makes MRSA cells more susceptible to Polymyxin B, an antibiotic targeting membrane. Thus, the present study suggests thymol as a potential alternative to antibiotics to combat MRSA infections. It can also be used as adjuvant in antimicrobial treatments [17]. Likewise, Ndezo et al. [16] showed the synergistic effect of the anti-biofilm potential of thymol and piperine either alone or combined with three aminoglycoside antibiotics were evaluated against the biofilm of K. pneumoniae. Their effect were also tested on either formed or pre-formed biofilms. It was found that the minimal biofilm inhibition concentration (MBIC) of streptomycin was reduced 16- to 64-fold when associated with thymol, whereas the MBIC of kanamycin was decreased 4-fold when associated with piperine. In addition, the minimal biofilm eradication concentration (MBEC) values of streptomycin, amikacin, and kanamycin were 16- to 128-fold, 4- to 128-fold, and 8- to 256-fold higher than the planktonic minimum inhibitory concentration (MIC), respectively. Therefore, thymol, in combination with antibiotics, has shown a broad synergistic activity in both inhibiting biofilm formation and destroying pre-formed biofilm of K. pneumoniae [16].

The synergistic effects associated with the combination of thymol or piperine along with the three considered aminoglycoside antibiotics indicate that thymol and piperine are very promising agents for the development of new antibacterial combination therapies to combat biofilm-associated infections. The study by Valliammai et al. (2020) aimed to decrypt the molecular mechanism for the anti-biofilm activity of thymol toward MRSA and to evaluate the ability of thymol to enhance the antibacterial activity of rifampicin. Thymol markedly inhibited 88% of MRSA biofilm formation at 100 μg/mL and decreased MRSA adhesion to human plasma-coated glass, stainless steel, and titanium surfaces, as demonstrated by microscopic analysis. In fact, thymol reinforced the antibacterial efficacy and biofilm eradication of rifampicin against MRSA and also minimized the formation of persisters. Thus, the present study suggests that thymol is a very promising combinatory agent candidate to enhance the antibacterial activity of rifampicin for persistent MRSA infections [18].

6. Conclusion

Bacterial biofilms appear in many infections that are related to diverse medical implants and well defined body sites such as the urinary tract, lungs, wounds and their resistance to antimicrobial treatments is a serious problem in clinical settings. It is, therefore, imperative to study efficient solutions to this problem and to find an alternative to our current armory of antibiotics. The challenges related to biofilm infections have prompted researchers to seek a better understanding of the molecular mechanisms involved in biofilm formation, which has led to the identification of several steps in biofilm formation that could be targeted to eradicate these serious infections. Within this context, the combination of current antibiotics with potential anti-biofilm and anti-toxic agents that interfere with the QS without stimulating the incidence of resistance is a new therapeutic strategy aiming to reduce the antibiotic dosages. In this study, a screening of the most studied molecules with anti-biofilm activity, associated with or not with antibiotics, is performed. The different anti-biofilm molecules investigated here have various modes of action including (i) inhibition via interference in QS pathways by 3-PPA, AS10, mBTL, natural and synthetic furanones and natural compounds, (ii) adhesion mechanism, (iii) disruption of extracellular DNA, proteins, lipopolysaccharides, exopolysaccharides and secondary messengers involved in various signaling pathways like small molecule DGC-inhibitors of c-di-GMP signaling. As QS and c-di-GMP signaling govern the production of virulence factors and some of the protective mechanisms operating in the biofilm mode, development of chemical compounds capable of preventing formation of biofilms by targeting these two major systems could be used to treat biofilm-associated infections. However, studies on the structural modifications on these molecules and their minimal effective concentration without posing harmful side effects should be made in future studies in order to improve their efficacy (Figure 1).

Figure 1.

Various modes of action of the studied anti-biofilm molecules in ESKAPE pathogenes.

Abbreviations

STX	staphyloxantin
QS	quorum sensing
EPS	exopolysaccharides
MDA	malondialdehyde
ATB	antibiotics
3-PPA	3-phenylpropan-1-amine
c-di-GMP	cyclic dimeric guanosine monophosphate
mBTL	meta-bromo-thiolactone