Development of Antibiofilm Substances by Endophytic Microorganisms with an Emphasis on Medicine

Abstract

The growing antimicrobial resistance and persistence of pathogenic microorganisms in infections–particularly in nosocomial infections–have become a major problem for public health worldwide. One of the main causes of these issues is the formation of biofilms, which are microbial communities associated with extracellular polymeric substances (EPS) that form a slimy extracellular matrix, causing the bacteria to become more tolerant to usual drugs in these structures. Thus, the search for new antibiofilm compounds is part of a strategy to deal with this problem. Endophytic microorganisms such as bacteria and fungi, mutualistically associated with plants, are sources of compounds with biological properties, including antimicrobials, and can be important allies in the synthesis of antibiofilm. These secondary metabolites can interfere with cell-to-cell communication and cell adhesion ability, promoting the dispersal of bacterial colonies and affecting biofilm. Since endophytes are cultivable in laboratory conditions, these microorganisms are environmentally friendly, as they do not contribute to pollution, are easy to handle and are produced on a large scale. Furthermore, metabolites from endophytes are of natural origin and may contribute to the reduced use of synthetic drugs. Considering these aspects, this chapter will focus on the characterization of endophytic microorganisms as potential active sources of antibiofilm and antimicrobial compounds with applications in medicine.

1. Introduction

One of the most worrisome problems in public health nowadays is antimicrobial resistance and multi-resistance (AMR and MDR). This natural process has been accelerated by the unrestrained and irrational use of antimicrobials, such as antibiotics and antifungals [1]. One of the biggest challenges to overcome this problem is to equate the speed of development of new drugs with the adaptation of pathogens to current drugs, since the development of new compounds does not follow the growing resistance of microorganisms [2]. In addition, there is a large number of resistant pathogens involved in healthcare-related infections (HAI), making the treatment of diseases more difficult and expensive as well as increasing mortality and morbidity rates [3, 4]. Among the most common pathogens in nosocomial infections, bacteria from the ESKAPE group–an acronym used to refer to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter are the most problematic, as they have mechanisms potentially involved in antimicrobial resistance [5]. Nevertheless, it is argued that the main cause of resistance may not be related to the classic mechanisms of microbial adaptation, but to the formation of a structure called biofilm [6].

Biofilms are organized, complex and dynamic communities of microorganisms adhered to a biotic or abiotic surface and protected by a polymeric extracellular matrix, which is composed of nucleic acids, polysaccharides, lipids, and proteins, generally called polymeric extracellular substances (EPS) [7]. This characteristic of adhering to different surfaces makes biofilms well disseminated in nature and easily found in different environments, including hospitals [8]. What makes biofilms so problematic for health is the fact that they allow the microorganisms inside them to thrive and persist in their environment. When related to infections, these structures tend to increase the tolerance of pathogens to treatments with conventional antimicrobial drugs, as they often prevent these compounds from reaching target cells [9]. Additionally, biofilms harbor different species of microorganisms that when acting together can lead to the development of chronic diseases [10] as well as to antimicrobial resistance due to horizontal gene transfer [11]. Another important point is the form of communication within biofilms. Through the so-called quorum sensing (QS), an intra and extracellular communication channel of microorganisms, they are able to coordinately regulate their activities in biofilms [12, 13]. Based on these considerations, the search for new compounds with antibiofilm activity becomes essential for combating resistant microorganisms.

A niche that has been gaining space because of its diversified production of biomolecules is endophytic microorganisms. By definition, endophytic microorganisms are bacteria and fungi that live symbiotically associated with healthy plant tissues without causing any apparent damage to their host [14]. Endophytes are a source of several secondary metabolites with, for example, antimicrobial [15], antitumor [16], enzymatic [17], anti-COVID [18], and antibiofilm activities. The main antibiofilm compounds currently sought are those capable of i) preventing or inhibiting microbial adhesion to avoid biofilm formation, ii) dispersing the already formed biofilm, and iii) interfering with intra/extracellular communication for biofilm formation (anti-QS) [19]. It is already known that natural products, such as those produced by endophytes, have advantages over synthetic compounds [20], for instance, rigidity, which provides better protein-protein interactions [21], and the possibility of being structurally shaped by evolution to be used by/in living beings [22]. Endophytic microorganisms can also be used in the synthesis of nanoparticles with antibiofilm activity. Nanoparticles can be defined as particles ranging from 1 to 100 nm and with size-related properties [23], being important allies in public health, as they can be applied in medicine [24]. Thus, the eco-friendliest method for the production of nanoparticles is precisely through the so-called green synthesis, which uses products from biological sources for the biosynthesis of nanoparticles [25].

This book chapter discusses the use of endophytic microorganisms and their compounds as potential tools for controlling and combating pathogenic biofilms, which are closely linked to antimicrobial resistance.

2. Natural antibiofilm and anti-quorum sensing products synthesized by endophytic microorganisms

Several studies have reported antibiofilm and anti-QS compounds produced by endophytes, reinforcing and highlighting the potential application of these microorganisms in various areas of health. Some of these studies are presented in Table 1 and will be fully discussed throughout this chapter.

Host Plant	Endophyte	Target	Compound	Reference
Datura metel	Streptomyces californicus ADR1	S. aureus ATCC 25923, S. aureus ATCC 29213, MR S. aureus ATCC 43300, MR S. aureus 562	Metabolite extract	[26]
Casuarina spp.	Frankia casuarinae DDNSF-02	Candida sp., Pseudomonas sp.	Ethyl Acetate Extract	[27]
Ventilago madraspatana	Enterobacter aerogenes VT66	P. aeruginosa PAO1	N-acyl homoserine lactone-lactonase	[28]
Coscinium fenestratum	Enterobacter sp.	Aeromonas hydrophila	Metallo-protein AHL-lactonase	[29]
Caulerpa racemosa	Nocardiopsis sp. DMS 2	Klebsiella pneumoniae	Crude Extract; 1, 4-diaza-2, 5-dioxo-3-isobutyl bicyclo [4.3.0] nonane	[30]
Ocimum Sanctum	Lasiodiplodiapseudotheobromae IBRL OS-64	MR S. aureus ATCC 33591	Ethyl Acetate Extract	[31]
Acanthus ilicifolius	Aspergillus flavipes AIL8	S. aureus	Flavipesin A	[32]
Ammi majus L	Aspergillus amstelodami (MK215708)	S. aureus, E. coli	Dihydroauroglaucin	[33]
Ocimum Sanctum	L. pseudotheobromae IBRL OS-64	Streptococcus mutans	Ethyl Acetate Extract	[34]
Carica papaya	Phomopsis tersa	P. aeruginosa PAO1	Crude Extract	[35]
Delonix regia	Talaromyces sp.	P. aeruginosa PAO1	Dextranase	[36]
Ocimum Sanctum	L. pseudotheobromae IBRL OS-64	P. aeruginosa PAO1	Ethyl Acetate Extract	[37]
Anredera cordifolia	P. aeruginosa CP043328.1	C. violacuem ATCC 12472	Diisooctyl phthalate and [1, 2, 4] oxadiazole, 5-benzyl-3	[38]
Ventilago madraspatana	Fusarium graminearum; Lasidiplodia sp	C. violaceum; CV026	Crude Extract	[39]
Silybum marianum	Penicillium restrictum	MR S. aureus	Polyhydroxyanthraquinones	[40]
Diploria strigosa	Fusarium sp.	Chromobacterium violaceum CVO26	Crude Extract	[41]
Solanum nigrum	Setosphaeria rostrata	P. aeruginosa	Mycosilver nanoparticles	[42]
C. papaya	Diaporthe phaseolorum SSP12	P. aeruginosa PAO1	Crude Extract	[43]
Ipomoea carnea	Aspergillus terreus AH1	P. aeruginosa,; S. aureus,; E. coli,; B. subtilis	Asterrelenin, Periplanamide, Butyrolacton, Pyranterron, Arenarin A	[44]

Table 1.

Antibiofilm and anti-QS activities of natural compounds produced by endophytic fungi and bacteria isolated from different host plants.

2.1 Natural antibiofilm agents from endophytic bacteria

Endophytic bacteria play a significant role in the production of a variety of secondary metabolites with potential applications in medicine [45], opening up new perspectives for the prospection of different bacterial species towards the discovery of novel antibiofilm agents against pathogenic microorganisms.

El-Gendy et al. [46] isolated 51 Streptomyces strains from the inner healthy tissue of Sarcophyton convolutum and determined the antibiofilm activity of ethyl acetate extracts of these endophytes onto 96-well polystyrene plates against seven methicillin-resistant S. aureus (MRSA) strains and nine multidrug-resistant Pseudomonas species (MRD). The Streptomyces strain MORSY 22 showed destructive activity of the biofilm produced by all S. aureus strains (MRSA1 to MRSA7), with values ranging from 87.46 to 95.75%, and all Pseudomonas species (MRD 1 to MRD9), with values ranging from 96.58 to 70.38%. These results revealed the potential of the strain MORSY 22 to prevent biofilm formation by bacterial pathogens and to develop antibiotic resistance.

Theodora et al. [47] screened the antibiofilm activity of endophytic bacteria against the pathogenic bacteria Bacillus cereus ATCC 14579, S. aureus ATCC 29213, Enterococcus faecalis ATCC 33186, P. aeruginosa ATCC 27853, Salmonella typhimurium and Vibrio cholerae. Crude extracts of isolates JB 19B and JB 18B showed the highest biofilm inhibition activity (90%) and biofilm destruction (76%), respectively, against S. aureus. Through scanning electron microscopy (SEM) analysis it was possible to verify a reduction in the extracellular matrix of the biofilms of B. cereus and S. typhimurium after treatment with extracts of isolates JB 18B and JB 19 B. The isolate JB 3B also showed inhibition activity against biofilm formation by all pathogenic bacteria. These findings confirmed the potential use of antibiofilm inhibitors from endophytic bacteria as a strategy for the control of bacterial infections.

Sabu et al. [48] isolated 14 endophytic actinomycetes from the rhizomes of Zingiber officinale. The crude extract of Nocardiopsis sp. ZoA1 at 200 μg/mL caused a reduction of more than 90% biofilm formation by multidrug-resistant coagulase-negative Staphylococcus capitis 267 and Staphylococcus haemolyticus 41 strains. GC-MS/MS analysis of Nocardiopsis sp. also revealed the presence of various compounds with antimicrobial activity, such as phenol, 2,4-bis (1,1-dimethylethyl), and trans-cinnamic acid. These results pointed to the inhibition of the synthesis of exopolysaccharide and proteinaceous factors by tested crude extracts and their potential to prevent biofilm formation by multidrug-resistant biofilm-forming strains.

Biosurfactants are an important class of natural antibiofilm agents produced by microorganisms. They comprise a structural and heterogeneous group of amphipathic molecules, which include glycolipids, lipopeptides, phospholipids, fatty acids and neutral lipids, polymeric and particulate biosurfactants [49, 50]. These microbial molecules can interfere with cell-to-cell communication mediated by QS and cell adhesion ability, promoting the dispersal of bacterial colonies and affecting biofilm formation through distinct mechanisms, such as cell membrane damage, inhibition of electron transport chain and energy restriction [51, 52]. Additionally, microbial surfactants have been considered an eco-friendly alternative with low toxicity and high biodegradability, selectivity and compatibility when compared to chemically synthesized surfactants [53].

Recently, Ashitha et al. [54] studied the endophyte Burkholderia sp. WYAT7 was isolated from the medicinal plant Artemisia nilagirica (Clarke) Pamp. in order to evaluate its antibiofilm activity. The biosurfactant present in the culture supernatant was identified and characterized as a glycolipid, and an inhibitory effect on the S. aureus (MTCC 1430) biofilm formation was observed. The percentage of biofilm formation suppression by MTCC 1430 was 41.79% and 79.22% when treated with 1 mg/ml and 2 mg/ml, respectively. These results suggested that the surfactant produced by Burkholderia sp. WYAT7 could be explored as a therapeutic agent for the control of pathogenic bacteria.

Ceresa et al. [55] reported that lipopeptide biosurfactants produced by the endophytic B. subtilis AC7 (AC7BS) isolated from Robinia pseudoacacia efficiently reduced Candida albicans adhesion to and biofilm formation on medical-grade silicone elastomeric disks (SEDs) by 57–62% and 46–47%, respectively. Chemical analysis of the crude extract revealed the presence of surfactin and fengycin. Since the fungus C. albicans is considered responsible for colonizing medical implants and causing a high mortality rate, the authors suggested the potential use of these biosurfactants to coat silicone medical devices in order to limit colonization of the pathogen and prevent infections. Later, Ceresa et al. [56] studied the synergistic effect of lipopeptides of B. subtilis AC7 (AC7BS) combined with the QS molecule farnesol to counteract C. albicans biofilms on silicone elastomer in simulated physiological conditions. There was a significant reduction of up to 74% in the pathogen adhesion within 1.5 hours and up to 93% and 60% in the biofilm formation within 24 and 48 hours, respectively. These effects were confirmed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). According to the authors, these findings opened up new perspectives for the combination of biosurfactants and farnesol to counteract C. albicans adhesion to and biofilm formation on materials for medical use.

Cochis et al. [57] evaluated the preventive anti-adhesion activity of biosurfactants extracted from endophytes from R. pseudoacacia (AC5 and AC7) and Nerium oleander (OC5) against C. albicans biofilm on acrylic resin and disks of silicon. The effective concentrations for C. candida biofilm inhibition without cytotoxic effects on mouse fibroblasts (ATCC L929) and human keratinocytes (ATCC HeLa S3) were 156.3 g/ml and 78.1 g/ml, respectively. These results demonstrated the potential use of these biosurfactants for the prevention of C. albicans biofilm adhesion to catheter and prosthesis materials.

2.2 Natural antibiofilm agents from endophytic fungi

Several recent studies have shown the potential of endophytic fungi as producers of biomolecules with antimicrobial activity [58]. Historically, fungi are known for their diverse production, including penicillin–the first antibiotic discovered [59]. For such reason, over the years researchers have focused on the discovery of new fungal antimicrobials, such as clavatol, sordaricin, jesterone, and javanicin [60]. Based on this, it is evident how interesting endophytic fungi can be in terms of the production of antimicrobial compounds.

May Zin et al. [61] obtained several bioactive metabolites from the endophytic fungus Eurotium chevalieri KUFA 0006 isolated from Rhizophora mucronata. The new compounds were tested to verify their antibiofilm activity against E. coli ATCC 25922, E. faecalis ATCC 29212, and S. aureus ATCC 25923. Thirteen metabolites effectively inhibited the growth of biofilms, whereas eight inhibited the biofilm formation by E. coli ATCC 25922, six by S. aureus ATCC 25923 and only one by E. faecalis ATCC 29212. This work also highlighted compound 3, which showed antibiofilm activity against E. coli ATCC 25922 and S. aureus ATCC 25923, causing a reduction of about 80% in the staphylococcal biofilm. The authors also performed tests to evaluate the antibiotic activity of these metabolites against the same pathogenic strains and found a positive result in only one compound. This is a very interesting finding, because even though certain compounds did not present an inhibitory effect against the pathogen alone, they had an inhibitory activity against the biofilm.

Narmani et al. [62] isolated the fungus Chaetosphaeronema achilleae from Taxus baccata and reported the production of seven compounds from the endophyte. In general, the metabolites were tested at different concentrations against S. aureus DSM 1104 biofilms and all of them presented some inhibitory activity even at lower concentrations. Among them, compound 4 stood out, showing strong biofilm inhibitory activity of about 96.82% at a concentration of 256 μg/mL and approximately 91.95% at 128 μg/mL. In addition, compound 7 was able to inhibit about 96.18% at 256 μg/mL of the biofilm, which represents a quite positive result. In the same work, it was observed that not all compounds exhibited antimicrobial activity against S. aureus DSM 1104 alone, as only metabolites 2 and 7 were positive.

Kaur et al. [63] isolated the fungus Alternaria destruens (AKL-3) from Calotropis gigantea and observed antibiofilm activity of the active fractions AF1 and AF2 during biofilm formation and in the preformed biofilm. The test microorganisms were P. aeruginosa, C. albicans, E. coli and Salmonella enterica, and two different concentrations of each active fraction were tested. In the case of AF1, all biofilms had their formation relatively inhibited, in addition to having been moderately reduced in the preformed biofilm. With regard to AF2, the same could be observed, that is, all biofilms were inhibited in the initial phase and in the preformed biofilm. Nonetheless, according to the authors AF1 was more promising and showed significantly greater activity than AF2 in all tests with the pathogenic strains.

Kaur et al. [64] evaluated the antibiofilm activity of the chloroform extract of the endophytic Aspergillus fumigatus isolated from Moringa oleifera against S. aureus MTCC 740, K. pneumoniae MTCC 109, and C. albicans MTCC 227. In this study, the authors performed tests at different stages of the biofilm, namely, the initial cell fixation phase and the preformed biofilm. In the initial fixation tests, the fungal extract was able to inhibit the formation of S. aureus, K. pneumoniae, and C. albicans biofilms by 69.2%, 57.66%, and 55%, respectively, with the standard antimicrobials showing similar results. The authors also argued that the inhibition of the initial fixation of the C. albicans biofilm by the fungal extract was better than that of the standard antifungal (amphotericin B) since the value obtained was approximately 53.3%. Regarding the tests against preformed biofilms, the extract reduced by about 51%, 53.4% and 47.6% of the S. aureus, K. pneumoniae and C. albicans biofilms.

Elkhouly et al. [65] studied the metabolism of the endophytic fungus Aspergillus Tubenginses ASH4 isolated from Hyoscyamus muticus in order to understand the production of antibiofilm compounds. During the study, pathogenic biofilms of S. aureus ATCC6538-P, Bacillus subtilis, P. aeruginosa ATCC27853 and E. coli were bioindicators of the extract as well as of the pure compound. The endophytic extract was able to suppress the formation of the S. aureus, B. subtilis, P. aeruginosa, and E. coli biofilms by 60.8%, 50.06%, 28.44%, and 37.68%, respectively. Subsequently, the pure compound identified as anophinic acid was tested against the same strains, reaching an inhibition of 61.39%, 54.93%, 69.51%, and 34.45%, respectively. Based on these results, it is possible to observe that the values are similar between them, except in the case of P. aeruginosa.

Qader et al. [66] isolated the marine endophytic fungi Epicoccum nigrum M13 and Alternaria alternata 13A from Thalassia hemprichii and tested 16 pure compounds obtained from them. The bioindicators for the antibiofilm activity test were E. coli, S. aureus, B. subtilis, and P. aeruginosa, all clinically isolated from hospitals in Egypt. Among the tested compounds of E. nigrum M13, three showed antibiofilm activity against pathogenic strains ranging from moderate to weak. The authors pointed out that some compounds such as 1 exhibited moderate activity against S. aureus and B. subtilis, but weak activity against E. coli and P. aeruginosa. In addition, compounds 3 and 5 showed moderate activity against Gram-positive bacteria, but weak activity against Gram-negative ones. As for the compounds isolated from A. alternata 13A, five of them presented activity against the biofilms of the indicator strains. Unlike what was seen in E. nigrum M13, compounds 7, 8, 9 and 10 from A. alternata 13A inhibited by 70–80% the S. aureus and B. subtilis biofilms, indicating an excellent activity. The same compounds also showed moderate activity against E. coli and P. aeruginosa biofilms. On the other hand, compound 11 exhibited weak activity against S. aureus, E. coli and P. aeruginosa but moderate activity against B. subtilis.

2.3 Anti-quorum sensing activity of natural agents from endophytic microorganisms

Quorum sensing (QS) is a complex density-dependent microbial cell communication system that occurs in single or mixed populations through autoinducers (AIs) or QS molecules. It is a population-dependent signaling mechanism in which microorganisms activate some signaling molecules according to the cell density. This behavior can be observed in several species of fungi and bacteria [67, 68, 69], being considered an inter- and intraspecies communication behavior that leads to genetic responses to autoinducers. This allows the microbial community to perceive and respond to various factors, including the presence of threats. The QS activity is responsible for the regulation of several bacterial physiological activities, such as pathogenesis, biofilm formation, swarming motility, bioluminescence, pigment disposal, polysaccharide production, and virulence, transforming the QS molecules into an important target for alternative antimicrobial therapy and antibiofilm activity [70].

After their production, when AIs reach an optimal concentration they bind to receptors on microbial cells, causing an alteration in gene expression. This ability gives biofilms adaptability to the environment as well as greater resistance to elimination, which in turn increases their virulence [71, 72]. In addition, QS molecules are also considered responsible for inhibiting or delaying the growth of other bacteria or fungi that are not part of their biofilm.

It is known that QS molecules are different for each microbial species. Furthermore, the type of communication in mixed biofilms also differs, that is, it can be either inter or intraspecies. There are four main categories of AIs: AI-1, AI-2, AI-3, and AIP. According to Schauder et al. [73], the molecules AI-2 are responsible for interspecies communication, while Smith et al. [74] argue that the molecules AI-1, AI-3, and AIP are in charge of the intraspecies communication.

Figure 1 shows the QS mechanism in a fungal cell in a simplified way. AIs (named signal molecules) are synthesized by fungal cells and released to the outside of the cell. Signal receptor proteins detect AIs and stimulate the expression of various genes, such as virulence, growth, and morphogenesis regulators.

Figure 1.

QS mechanism scheme adapted from Sharma et al. [75].

Since the QS mechanism is responsible for the survival and increased virulence of biofilms, the development of QS inhibition strategies has been of great importance. Most QS inhibition mechanisms use one of the following strategies: i) degradation and/or inactivation of AIs; ii) inhibition of AI synthesis; iii) inhibition of AI detector; and iv) antibiotics as QS inhibitors [76]. In the context of QS mechanisms of biofilms, endophytic microorganisms–considered to be synthesizers of QS inhibitors–have gained increasing attention. According to Mookherjee et al. [76], as endophytic microorganisms need to constantly produce defenses against competing microbial populations, they become an interesting source of QS inhibitors. QS inhibitor molecules can be produced by either endophytic fungi or bacteria [40, 77, 78].

Since QS can regulate the expression of virulence factors, QS inhibitors (QSIs) appear to be a promising antimicrobial strategy. As they act by imitating the QS autoinducers, they can be used to attenuate bacterial virulence, thus requiring lower doses, being more susceptible to the host immune system and reducing the use of antibiotics [39]. There are several studies reporting the QSI activity of biofilms.

2.3.1 QSIs produced by endophytes

It is known that endophytic fungi are responsible for the control and regulation of physiological activities of pathogens in animals and plants. Several studies have identified the production of QS inhibitors by endophytic fungi. Rajesh and Rai [39] isolated the endophytic fungus Fusarium graminearum from Ventilago madraspatana and measured the enzyme production using spectrophotometric and plate assay methods. Its anti-QS activity was analyzed against Chromobacterium violaceum CVO26, yielding strong positive results. Additionally, the extract of the endophytic fungus was able to inhibit the production of violacein pigment in the bacterium tested without any changes in bacterial growth. The authors then concluded that there was production of QS inhibitors by the endophytic fungus from Ventilago madraspatana, which in turn can be used for the development of anti-QS drugs–mainly against drug-resistant microorganisms.

Anti-QS molecules of Lasiodiplodia sp. from marine plants were also tested against C. violaceum CVO26 by Martín-Rodríguez et al. [41]. Four strains of the endophytic fungus stood out for their strong anti-QS activity. These strains were identified as belonging to four genera: Sarocladium (LAEE06), Fusarium (LAEE13), Epicoccum (LAEE14), and Khuskia (LAEE21). The authors reported that this was the first time that QS inhibitors were found in endophytic fungi extracted from marine plants.

Mishra et al. [70] showed that 2,4-di-tert-butylphenol (2,4-DBP), a component isolated from the endophytic fungus Daldinia eschscholtzii, is capable of inhibiting the QS activity of P. aeruginosa–one of the top three gram-negative bacteria considered a global threat due to its multiple drug resistance. They noticed that when exposed to 2,4-DBP, P. aeruginosa reduced the biofilm production and its virulence factors, as well as the expression of QS-related genes, confirming that 2,4-DBP can be used in combination with antibiotics to combat P. aeruginosa.

Zhou et al. [79] conducted a study that identified the QSI activity of 1-(4-amino-2-hydroxyphenyl) ethanone (AHE) isolated from the endophytic fungus Phomopsis liquidambari S47 from the leaves of Punica granatum against P. aeruginosa PAO1. The compound acted by suppressing the expression of genes related to QS, inhibiting the activity of antioxidant enzymes and enhancing oxidative stress. Pellissier et al. [80] explored the QSI activity of endophytic fungi extracted from the tropical palm Astrocaryum sciophilum against P. aeruginosa. Two pyran derivatives extracted from the endophytic strain Laccophilus venezuelensis showed activity affecting QS-regulated virulence factors.

Like endophytic fungi, bacteria are able to interact with each other (intra- and interspecies communication) through AIs. Kusari et al. [77] studied how endophytic bacteria from Cannabis sativa plants use QS inhibition as an antivirulence strategy in C. violaceum. A total of 13 endophytic bacteria were isolated from C. sativa, and their extracts were prepared and tested against C. violaceum. Four of them (Bacillus sp. strain B3, Bacillus megaterium strain B4, Brevibacillus borstelensis strain B8, and Bacillus sp. strain B11) exhibited the significant potential to weaken C. violaceum cell QS signals in a concentration-dependent manner.

Endophytic isolates of the phylum Actinobacteria previously isolated from common bean (Phaseolus vulgaris) were tested against pathogenic microorganisms by Lopes et al. [81]. Among them, Microbacterium testaceum BAC1065 and BAC1093 were found to inhibit QS of C. violaceum and E. coli. Kiarood et al. [82] found two strains (Bacillus cereus Si-Ps1 and Pseudomonas nitrogenformans La-Pot3–3) among 64 endophytic bacteria isolated from Citrus sinensis able to reduce the detection of QS molecules in Pseudomonas syringae. The B. cereus extract strongly inhibited P. syringae biofilm formation. An interesting fact reported by the authors was the increased number of cells in planktonic cultures treated with anti-QS molecules compared to control groups. This demonstrates that the molecules directly affect biofilm formation, but do not interfere with population growth.

3. Metal-based nanoparticles (NPs) synthesized from endophytic microorganisms as antibiofilm agents

The biosynthesis of metal-based NPs using endophytic microorganisms is a promising green synthetic route, considering the way to obtain these NPs and their final environmental impact [83]. These NPs can be used in many different technology sectors with emphasis on health [84, 85]. The biosynthesis of these NPs can occur intra- and/or extracellularly. The intracellular biosynthesis occurs through electrostatic interaction between positive charges from metal ions in a solution and negative charges from the bacterial/fungal cell wall [86]. In this process, microbial reductases dependent on NADH and NADPH are responsible for the transport of electrons, working as biocatalysts for redox reactions [87, 88]. In contrast, in extracellular synthesis, the culture supernatant, biomass, or cell-free extract is mixed with the metal ion solution, and the NPs are produced outside the microbial cell [89]. This process is performed by reductases produced and secreted into the culture medium by microbial cells and other cofactors [89, 90]. Therefore, biosynthesis through endophytic microorganisms can be used to obtain a series of different NPs, being the most common metallic/metallic oxides.

Noble metal NPs such as Ag has been widely used since ancient times for medicinal purposes due to their antimicrobial action [91]. Thus, it is natural that most of the works in the literature on the production of nanoparticles from endophytic microorganisms for microbial elimination are focused on Ag NPs. When these NPs are used for the inhibition of biofilms, the interaction between the NPs and the biofilm occurs in a succession of steps: first, the NPs are transferred to the biofilm surroundings; then, their superficial fixation occurs, followed by their migration to the biofilm interior [92]. Metal NPs can generate high local oxidative stress as a result of the production of reactive oxygen species (ROS), in addition to releasing M⁺ ions, which can interact with various functional groups of microorganisms, such as proteins, lipids, and DNA [93]. Furthermore, they can bind to the cell membrane surface by electrostatic interactions and penetrate by endocytosis and direct diffusion [94]. Metal oxide NPs can generate a high concentration of ROS even in the dark, interacting similarly with metal NPs, and cause secondary effects due to both local contact of metal oxide NPs with microorganisms and ionic release (depending on the stability of the oxide in the reaction medium used) [95, 96]. Figure 2 illustrates the mechanism of action of the nanoparticles on the biofilm.

Figure 2.

Schematic illustration of antibiofilm effects of metal and metal oxide NPs.

Bakhtiari-Sardari et al. [97] biosynthesized Ag NPs from the inoculum of two strains of Streptomyces sp. (OSIP1 and OSNP14) using the cell-free supernatant from these cultures to inhibit P. aeruginosa ATCC 27853 biofilms, resulting in Ag NPs with a spherical shape and an average size of 8 and 15 nm, respectively. The growth of P. aeruginosa biofilms was inhibited by up to 85% at a minimum concentration of 125 μg/mL of Ag NPs. The highest activity of the Ag NPs synthesized by the strain of Streptomyces sp. OSIP1 was attributed to the smaller size of Ag NPs obtained. Ranjani et al. [98] used the same Ag NP biosynthesis strategy to inhibit the growth of P. aeruginosa ATCC 27853. Using the cell extract of the fungus L. theobromae (MK942601), it was possible to obtain agglomerated Ag particles with an average size of 163.3 nm. The result of biofilm growth inhibition was 70% at a concentration of 50 μg/mL of Ag NPs. Bagur et al. [99] biosynthesized Ag NPs with an average size of 16.1 nm through a cell extract of the fungus E. rostrata due to its crucial role in the growth inhibition of P. aeruginosa and S. aureus. It was observed that there was a significant decrease in the growth of both pathogens at a concentration of 5 μg/mL of Ag NPs.

Neethu et al. showed in two different works the effectiveness of Ag NPs against the biofilm growth of the multidrug-resistant bacterium A. baumanii [100, 101]. In their first work, the biomass of the fungus Peridinium polonicum was used to synthesize spherical Ag NPs with sizes between 10 and 15 nm. It was observed that after 5 hours of exposure to the Ag NPs there was a reduction of more than 99.9% (3 log reduction) in the number of viable bacteria at a concentration of 15.6 μg/mL [100]. In their other work, the authors [101] produced a bionanocomposite coating with biosynthesized Ag NPs for a central venous catheter (CVC) using polydopamine as an adherent film of Ag NPs. Like in their previous work, it was observed that the CVC functionalized with Ag NPs eradicated the A. baumanii biofilm.

Ranjani et al. [102] synthesized Ag NPs nanocolloids and used them for the elimination of E. coli ATCC 25922 biofilms, commonly present in intensive care units (ICUs). The cell extract of the fungus L. theobromae (LtNc’s) was able to produce Ag particles with an average size of 436.5 nm. At a concentration of 12.5 μg/mL of these Ag NPs, there was a 50% reduction in E. coli biofilm formation. In another work, Chandankere et al. [103] synthesized Ag NPs with sizes between 4 and 26 nm using the fungus Colletotrichum sp. DM16.3 to inhibit the growth of biofilms of bacteria B. cereus (Gram-positive) and Vibrio cholerae (gram-negative). At a concentration of 10 μg/mL of these Ag NPs, it was possible to observe an inhibition of biofilm growth of 45.6% for B. cereus and 85.1% for V. cholerae. Ibrahim et al. [104] used the cell extract of the bacterium B. siamensis to synthesize Ag NPs with sizes between 25 and 50 nm. It was observed that at a concentration of 20 μg/mL these Ag NPs were able to inhibit the growth of biofilms of Xanthomonas oryzae pv. oryzae LND0005 and Acidovorax oryzae RS-1 by 86.31 and 80.59%, respectively.

Metal oxide NPs can also be synthesized by endophytic microorganisms and used to inhibit biofilm growth. Dhandapani et al. [105] synthesized TiO₂ NPs (10–30 nm) from the biomass of the bacterium B. subtilis (FJ460362). Tests were performed using microorganisms present in local aquatic sources and in the presence of light so that TiO₂ produced more ROS, causing high oxidative stress to microorganisms. The Se and SeO₂ particles (75–225 nm) were synthesized from the extract of the bacterium Bacillus sp. MSh-1 and tested against the biofilms of P. mirabilis, S. aureus, and P. aeruginosa, resulting in inhibitions of 53.4, 48.1, and 55.1%, respectively [106]. Balaji et al. synthesized ZrO₂ particles using the bacterium B. niancini and used them to remove the biofilms of E. coli (91.5%), Klebsiella aerogenes (71%), P. vulgaris (83.25%), S. aureus (92.5%) and S. mutant (90.5%) at a concentration of 40 μg/ml [107].

4. Conclusions

Biofilms are known to be closely linked to the growing resistance of pathogens, posing a threat to public health. Based on this fact, endophytic microorganisms considered as potential and eco-friendly producers of compounds with antibiofilm activity may be a source for the discovery of new biomolecules to combat these pathogens since they can synthesize compounds with anti-adherent properties, being capable of dispersing pre-synthesized biofilms.

These microorganisms also produce QS inhibitors that can harm the communication between pathogens in biofilm and, consequently, interrupt its formation. There are several researches showing the capacity of endophytic production in the prevention and dispersion of biofilms of, for example, ESKAPE pathogens, and this is really relevant because these microorganisms had been causing such a considerable problem to public health.

In addition, the microbial products of endophytes can also be used in the biosynthesis of metal-based nanoparticles, which have been demonstrating an interesting activity against biofilms. Some studies showed that metal-based nanoparticles can allocate on the surface of biofilm and migration to its interior, interacting directly with the pathogens inside, causing their death in different ways.

Thus, endophytic microorganisms deserve a position in the discussion about the development of new antimicrobial and antibiofilm medicines, mainly because several researches described in this review showed the potential of endophytes against harmful pathogens and their biofilms.

Acknowledgments

This work was supported by grants from the São Paulo Research Foundation (FAPESP) No. 2020/16299-9 to SHR, Nos. 2016/13423-5 and 2017/12905-9 to CPS and No. 2013/07296-2 to EL, and from the Coordination for the Improvement of Higher Education Personnel (CAPES) under finance code (001) to MA.