The concept for the antivirulence therapeutics approach as alternative to antibiotics: hope or still a fiction?

Abstract While the development of antibiotics since their first discovery brought about a revolutionary step forward in the fight against infectious microorganisms, unfortunately its side effect was the highly increasing risks of antibiotic resistance. Resistance development poses the urgent task for discovery of novel prospective approaches in the fight against multidrug resistant bacteria. Together with the search for new antibacterials, there is a growing interest in novel non-traditional approaches. Such non-traditional approaches are the attempts to suppress bacterial virulence and the development of virulence-related phenotypes, instead of killing the bacteria. The focus of this review falls on the bacterial regulatory mechanisms of virulence expression via quorum sensing (QS), and the formation of multicellular communities—biofilms, that protect bacteria from the host defenses and the antibacterial substances. The review gives a general outline of two types of approaches for control of bacterial virulence-related phenotypes. One is the search for reagents with expected antivirulence efficacy via the inhibition of QS, for example among low molecular weight metabolites of different medicinal plants. The other is directed to the possible prevention and/or destruction of bacterial biofilms, which are a well-recognized source of chronic, persistent and recurrent infections. The concerns regarding possible practical applications are considered as well.


Introduction
Infectious diseases are among the top 10 causes of death worldwide and the leading cause of disability-accompanied life for years. Among these, acute lower respiratory tract infections, diarrhoeal diseases and tuberculosis are responsible for significant global morbidity and mortality, while the development of antibiotics since their first discovery brought about a revolutionary step forward in the fight against infectious microorganisms, unfortunately its side effect was the highly increasing risks of antibiotic resistance among gram-positive and gram-negative pathogenic bacteria. In response to this and in line with the Global Action Plan on Antimicrobial Resistance to support the identification of pathogens of greatest concern, the World Health Organization (WHO) formulated the development of novel anti-bacterial drugs and strategies as a high health priority worldwide, and updates a priority list of antibiotic resistant bacteria. The list of bacterial species that are recently in focus, according to their priority risk level, include as Priority 1. Critical: Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacteriaceae (all -carbapenem resistant); as Priority 2. High: Enterococcus faecium (vancomycin-resistant), Staphylococcus aureus (methicillin-resistant), Helicobacter pylory (clarythromycin-resistant), Campylobacter spp. ( f l u o r o q u i n o l o n e -r e s i s t a n t ) , S a l m o n e l l a e (fluoroquinolone-resistant) and Neisseria gonorhoeae (caphalosporin-resistant, fluoroquinolone-resistant); and as Priority 3. Medium: Streptococcus pneumoniae (penicillin-non-susceptible), Haemophilus influenzae ( a m p i c i l l i n -re s i s t a n t ) a n d Shigella s p p. (fluoroquinolone-resistant) [1].
The increasing antimicrobial resistance, facilitated by overprescribing by medical doctors, use of antibiotics as growth factors in livestock breeding, discharge in wastewaters thus contaminating various environmental sites [2], etc., seems to have become out of control. This necessitated the formulation of novel anti-infectious strategies. While the occurrence of new effective antibiotics has been quite limited lately, the re-purposed administration of approved compounds is only one and more or less routine aspect of the fight. Of concern, the use of such substances might be just a temporary solution and could, again, create resistance, by killing sensitive bacteria and opening niches for the spread of the evolved resistant strains. Together with this, novel approaches occur, among which the application of antimicrobial peptides [3], phage therapy [4], low frequency ultrasound, photodynamic inactivation [5], toxin neutralization [6] and improvement of immunotherapy approaches with the development of monoclonal antibodies against bacterial toxins and the protein machinery responsible for toxin release [7].
An emerging but rapidly expanding area of research is the search for ways and compounds capable of suppressing bacterial virulence [8]. This strategy known as 'anti-virulence therapy' is a novel approach in the fight against the bacterial pathogens [6,8]. It aims at suppressing the expression of bacterial virulence-related phenotypes rather than killing the bacteria. Virulence factors are microbial components (biomolecules and structures) or more complicated behavioural phenotypes (like biofilm formation) used by pathogens to colonize, invade and persist in a susceptible host [7].
How the anti-virulence approach should work: When you apply a substance with a strong antibacterial action to a genetically variable bacterial population, it exerts a selective pressure, i.e. kills the sensitive bacteria leaving more sources for the reproduction and spread of the resistant bacteria [2]. This can be achieved by both occupancy of the liberated living niches and the transfer of resistance genes from the resistant bacteria to some of the drug-sensitive ones. When, on the other hand, we suppress the bacterial virulence mechanisms, without killing the microorganisms, we could protect the host from the harmful actions without promoting the emergence of resistance. It remains to be seen whether such an 'anti-virulence therapy' is a good prospect or fiction presenting other hazards.
The aim of this mini-review is to provide a general outline of some not widely familiar areas related with antibacterial drug tolerance and the novel approaches for overcoming it. The paper presents the emerging trend of the anti-virulence approaches against bacterial infections to a wider audience of researchers in fields other than medical microbiology. The interdisciplinary joint efforts of microbiologists with chemists, physicists, phytochemists, biotechnologists, specialists in bioinformatics, etc., will be decisive in the struggle against the spread of antibiotic resistance and in the efforts to suppress bacterial virulence.

What are virulence and virulence factors?
Virulence is the ability of an organism to infect the host and cause disease. Bacterial characteristics that contribute to disease are called 'virulence factors' . These can be secretory, membrane-bound or cytosolic. Cytosolic factors facilitate the bacterium to start adaptive struc tural and physiological shif ts. Membrane-associated ones, like flagella and pili, are responsible for motility and promote adhesion and host colonization. Secretory products like toxins and enzymes cause harm to host cells and tissues and are important components of the armamentarium used by bacteria in order to evade the host's innate and adaptive immune response [7,9,10]. Together with these, an important multifactorial virulence factor is the ability of pathogenic bacteria to form biofilms [11].
Virulence factors are specific for each bacterial species. They have evolved in relation with the characteristics of the occupied host niches and determine the relationship with the host. Thus, toxins released from different pathogens are among the most harmful to the host organism. For example, the α-hemolysin, the enterotoxin and the toxic shock syndrome toxin of S. aureus, the Shiga toxin of Shigella and enterohemorrhagic E. coli, streptolysin O and pneumolysin of Streptococcus pneumoniae are known to cause serious exacerbations during infection [12]. Together with this, some toxins causing high lethality have urged the development of non-antibiotic, immunological approaches to therapy. Examples: the antisera against the toxins from Clostridium botulinum, C. difficile and Bacillus anthracis [7]. The membrane-associated factors that determine swimming, swarming and twitching motility of bacteria are associated with enhanced virulence as well [13]. They are responsible for the capacity of the pathogens to reach, occupy and invade different host niches -cellular, extracellular as well as tissue-related ones, and also to migrate through the endothelium and via the blood to finally disseminate to host organs far from the initial site of the bacterial penetration.
Together with the vast variety of virulence factors, there is one thing in common: the expression of these factors is regulated by a cell-density dependent mechanism named quorum sensing (QS) [14].

Quorum sensing: a target for interference with bacterial virulence
The quorum sensing process is defined as cell-to-cell communication that allows bacteria to obtain information about microbial cell density. Shortly, through their lifespan, bacterial cells release various signal molecules. Once the quantity of these molecules in the environment reaches a threshold, this is a signal to bacterial cells that the living resources have changed, so it is necessary to respond and adapt to the changes by adjusting gene expression. Among the processes controlled by quorum sensing, which are of special importance for the interaction between the host and the pathogenic bacteria, is the expression of virulence factors [15][16][17]. For this reason, the QS system has lately been recognized as one of the important targets for antivirulence therapeutics [18]. A recent focus is the search for methods to interfere with QS. It can be directed to each of the steps of the QS cascade ( Figure 1): synthesis of the QS signals, their release in the environment, their stability in the environment, their recognition by the cognate receptors and the generation of the response -the expression of the virulence-related phenotypes [19][20][21]. At present, it is considered that blocking the QS signalling cascades is less likely to develop resistance [22].
The approaches to QS inhibition are various. One direction is re-purposing of old drugs for new therapeutic use. Example: the FDA-approved drugs niclosamide (anti-helminthic) and the synthetic antibacterial drug clofoctol were found to inhibit QS responses [23]. Other strategies include synthesis of structural analogues of QS signal molecules, or isolation of natural ones that will competitively bind to the receptor; application or modulation of enzymes capable of degrading the signals, known as quorum quenching (QQ) enzymes (lactonases, acylases, reductases and oxidases), blocking the signal transduction cascades, etc. [23,24].
Together with these, a promising scientific area is the search for QS inhibiting agents from natural sources, e.g. honey, medicinal plants, other microorganisms, etc. It unites the efforts of botanists, phytochemists and microbiologists. Thus, medicinal herbal products are believed to be a prospective source of pharmaceuticals or phytochemicals for the treatment of QS-mediated bacterial virulence [25,26]. The screening among a vast variety of samples of plant origin has revealed the QS interference potential, resulting in suppression of virulence factor expression by flavonoids, tannins, phenolics and polyphenolics, peptides, sesquiterpene lactones [27][28][29][30][31][32]. There are also some promising results on QS suppression obtained with enriched samples.
Anti-virulence substances, including QS inhibitors, can be applied either alone, or in combination with traditional antimicrobials to promote their effect [19].
For example, studies have demonstrated synergistic effects of QS inhibitors in combination with traditional antibiotics [33,34]. These approaches are expected to diminish the use of antibiotics. They are thus planned to reduce the selective pressure exerted by killing bacteria with antibiotics which leads to resistance [19, 35,].

Biofilms as a drug-tolerant virulence-related phenotype
A quorum sensing-related phenotype [15] intrinsically characterized by drug resistance and/or tolerance is the formation of sessile bacterial communities-biofilms. They are attached to surfaces, and the bacterial contamination of some surfaces may be associated with serious health hazards. The surfaces may be natural (including various external and internal surfaces in plants and animals) or artificial (e.g. surfaces in food processing or hospital environments, as well as of indwelling medical devices).
Biofilm formation is a multifactorial process. Its detailed mechanisms, as well as the structures (molecules, organelles, etc.) involved in it may largely vary between microbial species and strains. However, there are several common steps in the biofilm-formation (Figure 2). Free-floating (planktonic) bacteria may use their surface appendages (pilli or flagella) or other adhesins, to approach a surface and attach to it. Single bacterial cells then start reproducing and forming microcolonies. Together with increasing in number, the bacteria lose their motility and release 'glue' , i.e. species-specific combinations of extracellular polymeric substances (EPS) like polysaccharides, proteins, extracellular DnA and a variety of low-molecular weight substances.
Once the number of biofilm cells in the microcolonies increases to a threshold, QS regulation mechanisms start to operate in the control of the biofilm-formation cycle: increase in the size of the microbial colonies, formation of biofilm architecture, which is species-and even strain-specific, and, finally, the stage when some of the sessile bacteria re-gain their motility and detach from the biofilm to disseminate to new niches. To the present moment, some mechanisms of the regulation of biofilm formation have become elucidated. Thus, studies on gram-negative bacteria have underlined the role of two-component systems of signal transduction and of the diguanylate cyclase in biofilm control [11,36]. The third group of regulatory mechanisms comprises the QS cascades. The role of different QS signals in biofilm development differs. Thus, in P. aeruginosa and Aeromonas hydrophyla the AI-1 signals, acyl homoserine lactones, promote biofilm growth, while the activation of the interspecies and inter-kingdom signalling systems mediated by autoinducers AI-2 and AI-3 suppress it [23,36].
After attachment to surfaces, bacteria acquire properties different from the characteristics of the same strain grown in liquid media [37]. They lose motility and secrete EPS materials. The EPS forms a heterogeneous extracellular surrounding that, in combination with the genetic characteristics of the bacteria, determines the peculiarities of the biofilm [38]. Various gradients may arise within a biofilm, e.g. pH, oxygen, water and nutrient supply, and all these may represent physical or chemical signals for the bacterial cells [39,40]. The emerging heterogeneous microenvironments are related with the emergence of different bacterial sub-populations, like persistent, non-conformist (not responsive to quorum sensing), etc. [39]. The presence of different microorganism phenotypes together with Figure 2. antibiofilm approaches. Suppression of biofilm growth may influence the whole biofilm development cycle, and can facilitate the natural processes of biofilm dispersion. the detachment of mature biofilms requires agents that loosen/decompose extracellular polymeric substances (epS) or agents that can penetrate through the epS to reach and attack the bacterial cells.
the ECM characterized with low permeability makes the biofilm a very efficient escape mechanism from antibiotics [41][42][43]. Different references provide evidence for 10 to 1000 times higher antibiotic amounts needed for the successful treatment of biofilm bacteria if compared with the necessary amounts for killing liquid-cultivated bacteria from the same species [44]. Together with the high antibiotic tolerance, biofilm structure creates a barrier which prevents the access of the host antibodies and immune cells which determines a tolerance to the immune system [45].
The biofilm phenotype of pathogenic bacteria is recently gaining more concern. Biofilms are considered to contribute up to 80% of chronic and healthcare-associated infections [43,46,47]. Among these: atopic dermatitis, chronic wounds, burn infections, osteomyelitis, endocarditis, catheter-associated bloodstream and urinary tract infections, sinusitis, chronic lung infections, etc. [45, 48,]. Biofilms are considered one of the leading causes for a shift from acute-phase to chronic diseases [49]. There is growing evidence that the predominant amount of chronic infections is related with biofilms [50,51]. In biofilms, the inhibitory doses of antibiotics are much higher than these determined in liquid media, and exceed significantly these in planktonic forms [52]. Together with this, the effects of antibiotics on biofilm growth may be difficult to predict. Thus, sub-inhibitory amounts of antibiotics may have various effects, from suppression to stimulation [53]. The effects depend on the bacterial species and strains. This draws the attention to the risk of biofilm occurrence during antibiotic application especially in chronic infections.

Inhibition of biofilm development and destruction of mature biofilms
Both the biofilm-related infections of humans and the hazards caused by biofilm contamination of medical, food-processing and in-house surfaces necessitate the development of novel effective anti-biofilm strategies. These have been in the focus of several reviews (e.g. [42, 46,]). Two general antibiofilm approaches can be outlined: (i) Prevention of biofilm formation by identifying of inhibitors of biofilm growth and/or developing antiadhesive surfaces, especially such that can applied as coatings on indwelling medical devices; (ii) Destruction of pre-formed biofilms. Both these strategies can be applied either alone, or in combination with traditional antibiotics [54].
The prevention and/or inhibition of biofilm growth may be directed to practically all steps of the biofilm formation cycle (Figure 2): initial adherence of bacteria to a substratum, formation of microcolonies, growth and development of a 3 D structure, and biofilm dispersal as a final step. Bacterial adhesion was one of the earliest identified targets, and it was accompanied by the search for anti-adhesive agents and the development of various antimicrobial coatings for medical application [7, 24, 47, 55,]. Among possible biofilm suppression approaches, QS inhibition is also considered an important target [54].
The extensive screening of biofilm suppression substances has lately included trials using synthetic and natural substances. natural sources of putative effective biofilm-modulation activities have been sought among antibacterial peptides, essential oils, plant extracts and purified molecules, probiotic bacteria and the products they release, etc. [12,54]. Many of the tested preparations have been chosen because of their antibacterial action. However, the presence of antibacterial activity of a substance does not necessarily determine antibiofilm effects; sometimes it can be the opposite. Thus, released products from some strains of lactic acid bacteria with established antibacterial activity against E. coli stimulated biofilm growth [56]. On the contrary, sesquiterpene lactone-rich fractions from the plant Arnica montana suppressed biofilm development while having no antibacterial activity [27]. Also, strain-and species-related effects have been reported in these studies as well. Since these effects are sometimes diverse, it is important to supplement the initial screening for anti-biofilm activities by more detailed insights and the combination of various experimental approaches.

Destruction of mature biofilms
The natural trend in biofilm development includes a final step at which bacteria become motile and escape from the biofilm matrix ( Figure 2). This is an active mechanism, named dispersion, aiming the dissemination and the occupation of new niches by biofilm bacteria. On the other hand, certain external factors and treatments can cause a passive escape -detachment, of the bacteria from the biofilm [36]. Pre-formed mature biofilms, including medically important ones, are difficult to eradicate [24]; hence, the elaboration of effective detachment strategies is lately the focus of many studies. Efforts are made in the development of various approaches in biofilm destruction. These approaches include enzymatic treatments directed to EPS components, antimicrobial photodynamic therapy, antimicrobial peptides, electrical methods, etc. [47, 57,]. The destruction of mature biofilms depends on the possibility to overcome the permeability barrier created by the EPS [58]. Once it is destroyed, this would enhance the access of antibiotics to the bacterial cells in the biofilm and its detachment [59]. Various approaches to destroy or at least loosen the EPS have been examined. The predominantly anionic character of the EPS motivated the attempts for its permeabilization with cationic substances [60,61] like these included in some anti-biofilm hydrogels [62]. Amphiphilic molecules like surfactants [62,63] and biosurfactants [42, 64,] have also been tested, with variable success. The major EPS components have been in the focus of some strategies, with attempts for enzymatic dissolution of eDnA, proteins and polysaccharides [65].
A promising novel approach is the facilitated drug penetration inside the biofilm by specifically designed drug-delivery systems represents. Promising candidates are the various types of nanoparticles that are applicable as biofilm-dispersal agents (for review, see [41,66]). Some of these, like metal nanoparticles, may possess intrinsic antimicrobial activity [37,58]. Thus silver nanoparticles have been extensively studied with the aim to combine antibacterial and antibiofilm activities [67][68][69][70]. Other nanostructured materials may have the capacity to enhance the penetration of antibacterials via the host mucus and the biofilm EPS. A point of concern regarding nanoparticle applications are the possible toxic effects to the cells of the host [60,71], and this, for instance, is one of the leading limitations for the application of metal nanoparticles. A more recent focus is the applicability of biodegradable high-performance polymeric materials. Many of them have initially been developed for other medical purposes, e.g. as anticancer-drug carriers. Their possible drug-delivery targets have lately been expanded to include also anti-biofilm applications. A type of nanocomposites is polymeric cationic micelles, which are expected to interact with the anionic moieties in the biofilm extracellular matrix. Cationic micelles have been shown to disintegrate biofilms without having an additional antibacterial action [37,49,72,73], others have been successful in biofilm-loosening activity combined with drug delivery [40,60], or delivery of other antimicrobial materials like silver ions [74].

Critical points
Together with the already significant amount of reports on laboratory experiments with promising results, there remain still more open questions. Many of the studies in search for natural QS inhibitors include screening of extracts, with only rarely providing identification of the active substance and even more rarely -the mode of action. Also, while it is generally considered that the antivirulence approach is less likely to develop resistance than the methods that kill bacteria, resistance cannot be completely excluded. There are some already known possible mechanisms for pathogens to avoid antivirulence treatments, as evidenced by the registration of QS-disfunctional mutants [18], and strains able to exclude QS inhibitors by overproducing efflux pumps [75]. Presently, the existence of bacterial strains capable of avoiding an antivirulence drug does not seem very likely to exert enough selection pressure for the establishment and vast spread of resistance mutants. nevertheless, there are concerns that side effects might occur on the useful microbiome, similarly to the side effects of traditional antibiotics [18]. When the biofilm virulence-related phenotype is considered, strain-specific effects in biofilm modulation should be taken into account [53]. Another question that is not traditionally addressed in the lab is what happens to the biofilm when antivirulence therapy stops, will the biofilm increase again or will it not [75]? Importantly, the biocompatibility of the potential antivirulence preparations should be tested in cytotoxicity trials. As a step further, in vivo experiments are needed prior to clinical trials. At present, only a few in vivo experiments have been performed, and even fewer pre-clinical experiments. So far, there is not a clinically approved anti-virulence drug [26,76,77]. This indicates the need to speed up and expand the research on the possibilities to suppress bacterial virulence and thus protect the host. This could be achieved via a complex standardized multidisciplinary methodological approach for selecting, characterizing and determining the range of applications of antivirulence drugs.

Conclusions
The global trend for rapid increase in antibiotic resistance and the occurrence and spread of numerous resistant strains of pathogenic bacteria necessitates that scientists from the fields of biomedicine, chemistry, pharmaceutics, phytochemistry, and other areas of research join their efforts in search for novel ways of overcoming the problem. One promising approach, defined as 'antivirulence therapeutics' , is based on the possibility for suppressing the virulence of bacteria, rather than killing them. While the expression of virulence phenotypes like toxins, adhesins, biofilms, etc., may vary between bacterial species, they share principally similar regulatory pathways. An important focus in the antivirulence therapeutics is to interfere with the regulatory mechanisms of virulence expression. We hope that the development of successful antivirulence preparations, applicable either alone or in combination with traditional antibiotics, would help in overcoming, or, at least, in reducing, the speed of spread of resistant pathogens.

Data availability statement
Data sharing is not applicable to this article, as no new data were created or analyzed in this study.