Antibiotic resistance and bacterial biofilm

KEYWORDS:

• Antibiofilm strategies
• AMPs
• biofilm
• drug resistance
• EPS-degrading enzymes
• nanosystems
• persisters
• quorum-sensing
• tolerance

1. Introduction

A biofilm can be defined as a microbial community embedded in a self-produced matrix of extracellular polymeric substances (EPS), adhered to a biotic or abiotic surface [1] (Figure 1).

Figure 1. (a) Illustration of a bacterial biofilm. The microcolonies of bacterial cells are matrix-enclosed communities that may be composed of 10–25% cells and 75–90% EPS matrix. Bacterial cells within the matrix do not have Brownian movement, and show a tower-like shape. Most of the cells are localized in the top of the tower and very few, among them, the persisters are localized in the bottom. (b) Representative image of H. pylori biofilm stained with Live/Dead BacLight kit and analyzed using fluorescence microscopy. The green fluorescence indicates the live cells. Scale bar: 5 µm

The biofilm phenotype represents a microbial survival strategy that arises as a result of stressful conditions that induce cells to organize and cooperate with each other [2]. The development of a biofilm is a multistep regulated process in which cell adhesion, EPS production, and the detachment of microorganisms from the mature biofilm involve the expression of specific genes. Biofilm is a complex, highly hydrated three-dimensional structure, in which water represents the main component, up to 97%, together with polysaccharides, proteins, lipids, nucleic acids, as well as insoluble components such as amyloids, fimbriae, pili, and flagella [3]. In particular, it has been widely documented that extracellular DNA (eDNA) plays an important role in biofilm establishment and structural stability [4,5,6,8] as well as in the horizontal gene transfer and nutrition as a phosphate source [9]. The origin of eDNA has been hypothesized to result from multiple mechanisms of DNA release, such as the production of extracellular vesicles [10,11,13], prophage-mediated cell death as in Pseudomonas aeruginosa biofilms [14], and controlled cellular lysis occurring during biofilm formation, as in the case of Staphylococcus aureus [7]. In Helicobacter pylori, the eDNA associated with extracellular vesicles might also has a role in cell aggregation as well as in biofilm formation [12,15]. The production of a matrix is a dynamic process and is influenced by the availability of nutrients, the microbial competition, and the mechanism of secretion [3]. The nature and composition of the EPS matrix depend on bacterial strains, culture conditions, and biofilm age [2].

Microbial biofilm has been described as an ‘arcane behavior of bacterial populations,’ therefore, cannot only be considered as an enemy to fight against. Many studies, in fact, demonstrated the key important role of biofilms developed by the microorganisms of the human microbiota. It has been demonstrated that the development of Lactobacillus spp. biofilm is associated with beneficial properties such as a stable, long-term colonization of the microorganisms which protects the host by pathogenic bacteria colonization through different mechanisms, including the immunomodulation and the secretion of molecules with antimicrobial activity [16,17,18]. At the same time, biofilm has been recognized also as ‘a principle virulence factor in many localized chronic infections’ that are recalcitrant and generally recur after long periods of clinical quiescence [19]. The biofilm persistence in the environment and in the host is due to the high microbial cell density which also includes ‘persister cells,’ characterized by a dormancy state. The EPS matrix preserves microbial cells from external stressful stimuli and promotes the horizontal genetic exchange. Therefore, the microorganisms residing in a biofilm develop protection from the host immune system and tolerance to antimicrobials through different mechanisms such as slow penetration of drugs through the biofilm matrix, the presence of cells in a dormancy state, and the presence of altered microenvironments.

Tolerance toward antimicrobials, unlike resistance which is genetic-based and can be acquired through point mutations or horizontal gene transfer mechanisms such as conjugation, transformation, or phage transduction, can be defined as that condition in which, in order to kill or inhibit microbial cells sensitive to antimicrobials, but aggregated within a biofilm, concentrations up to 4 times the Minimal Inhibitory Concentration (MIC) are required. Tolerance, which we can also define as ‘phenotypic resistance’ to drugs can be lost when biofilm disperse and microbial cells reacquire the planktonic phenotype.

The variability in the biofilm composition as well as tolerance versus the antimicrobial drugs commonly used in conventional therapies suggest the need for multi-targeted or combinational therapies aimed at the eradication of biofilms; furthermore, polymicrobial biofilms represent a further concern that needs to be addressed. Among the different approaches aimed at biofilm inhibition and/or eradication, we can distinguish different strategies like the ones that target the EPS or dormant cells or the quorum-sensing mechanism as well as the use of nanoparticles or surface modification [20,21].

EPS-targeting strategies are based on both the inhibition of the cellular adhesion affecting biofilm development and on the EPS degradation in a mature biofilm. The inhibition of extracellular and intracellular signaling as well as the inhibition of non-signaling mechanisms, which are involved in the secretion of the EPS, could surely represent an effective strategy. For example, cyclic-di-GMP and cyclic-di-AMP are regulated enzymes involved in the production of biofilm matrix components like polysaccharides and adhesins. Small molecules as peptides or mannosides have been proven to be active versus bacterial and fungal biofilm associated with infections [19–22]. Furthermore, the use of EPS-degrading enzymes as glucanohydrolases, dispersin B, or DNase I have proven equally effective [23,24,25]. In particular, the recombinant human DNase I (dornase alfa) is used in clinical therapy because it is capable of reducing the viscosity associated with the DNA released by neutrophils and microorganisms in the sputum of patients affected by cystic fibrosis. The degradation of the DNA promotes a significant improvement of lung function in the abovementioned patients [26,27]. The degrading enzymes can be used for the biofilm matrix degradation facilitating the diffusion of the antimicrobial drugs into the EPS. The combination of antimicrobials and Exopolysaccharides (EP)-degrading enzymes has been demonstrated a successful strategy in the eradication of a mature biofilm [28]. Therefore, the knowledge of the composition of the EPS matrix developed by a single or a mixture of microbial species/genera may contribute to the identification of the best association between degrading enzymes and type/class of antimicrobials. Microbial cells communicate with each other by using small molecules named autoinducers. The Quorum Sensing (QS) system is a mechanism of regulation of cell density and, thus, biofilm formation. Gram-negative bacteria produce Acyl-Homoserine Lactone (AHL) molecules while Gram-positive bacteria release small peptides. The use of inhibitors of QS, like RNA III inhibiting peptide (RIP) or benzamidine-benzimidazole derivative (M64) or the autoinducing peptide I (AIP-I), alone or in combination with antimicrobial drugs, may represent a valid and promising therapeutic treatment.

Persister cells within a biofilm showed tolerance to antimicrobials due to their dormancy status, therefore, targeting dormant cells trough the disruption or inhibition of key molecules, may represent an alternative approach to overcome one of the antibiotic tolerance mechanisms exhibited by the biofilms.

Morphological variability is related to the adaptation of microorganisms to stressful environmental conditions and increased tolerance to antimicrobial drugs. In H. pylori, the coccoid phenotype allows the microorganism to avoid the immune system detection and to promote therapeutic failures [28,29]. Antimicrobial peptides (AMP), for example, represent a valid approach in the bacterial and fungal biofilm treatment regardless of cellular metabolic activity, in fact, it has been demonstrated that the capability of forming pore affects metabolically active, dormant, and persister cells [19]. AMPs can increase the effect of conventional antimicrobial therapies; therefore, a combination with other antibiofilm approaches that target the EPS matrix, as, for example, the use of digestive enzymes, might increase the diffusion of AMPs inside a biofilm.

Nanoparticles can be considered an innovative and versatile procedure for biofilm eradication. Therefore, the use of them, as a biofilm-targeting strategy is widely studied. Inorganic and organic nanoparticles can be used alone or in combination with antimicrobial drugs. Silver nanoparticles displayed a strong antimicrobial activity against several Gram-positive and Gram-negative microorganisms as well as an antibiofilm effect [30,31,32]. In particular, it has been recently demonstrated that Silver Ultra-NanoClusters (SUNCs), showing low toxicity versus human cells, were effective in eradicating H. pylori mature biofilm suggesting that they could represent a novel strategy for the treatment of H. pylori infections both alone and in combination with metronidazole [33]. Among organic nanoparticles, liposomes, made up of phospholipid bilayers, are biocompatible and are widely used for drug delivery since they fuse with the bacterial outer membrane and directly release the antibiotic into the cell cytoplasm, increasing therapeutic effects and minimizing cytotoxicity [34]. In addition, liposomes can easily penetrate through the biofilm matrix reaching the target cells and protecting the antimicrobial drug from degradation or enzymatic inactivation. It has been shown that they are effective against biofilms developed by several bacterial species [34,35].

Finally, to eradicate biofilms and limit the spread of antibiotic resistance, the study of compounds or molecules of natural origin is increasingly widespread. Alkaloids, terpenoids, tannins, steroids, coumarins, and flavonoids [36], which do not normally cause resistance [37] as well as Essential Oils (EO) from parsley, lovage, basil, thyme, and hemp, have been studied for their antimicrobial and antibiofilm activities [37,38,39,40]. The latter cause an increase in cell permeability, alterations in the bacterial cell wall and membrane, ATP loss, inhibition of protein synthesis, pH alterations, DNA damage, and inhibition of the QS in several bacterial species such as Bacillus cereus, S. aureus, P. aeruginosa, Escherichia coli, and Salmonella enterica serovar typhimurium [40].

2. Expert opinion

The microbial biofilm associated with hospital acquired infection, is subtle and often depends on an interaction between the opportunistic pathogens, the host immune system, and microbiota.

The high variability and multi-factoriality of the microbial biofilms represent a limit and make biofilms clinically difficult to treat; therefore, combination therapies that target different components of the biofilm microenvironment are required [19].

An in-depth study of the content and the amount of the components of EPS matrix developed by the single microorganism or a mix of microorganisms under experimental conditions that reproduce as much as possible in vivo conditions such as the surface of colonization, pH condition, and hypoxia might help scientists to identify the more appropriate antimicrobial strategies aimed at biofilm eradication. Once confirmed in ‘in vivo models’ this approach would result in time saving, a rational use of drugs, the outline of treatment guidelines, and a reduction of healthcare costs.

Not less important is the role of the human microbiota and the possible interplay between the resident microbes and the opportunistic pathogens. The microbiota represents a community of microbes in which mutualistic and commensal microorganisms co-exist with potential pathogens and when this balance fails infection might develop. Therefore, new results could be obtained by the study of the human microbiome as well as by the use of new molecules or compounds produced by probiotic strains. In addition, a multi-target approach that includes the host immunomodulation therapies might be significantly effective.

Unfortunately, the effect of new antimicrobial molecules, on the human microbiota is rarely evaluated; thus, the identification of molecules that possess a selective toxicity between pathogens and some components of the human microbiota might represent an important turning point in the field of microbiological research.

On the basis of these concerns, the collaboration between scientists with different expertises such as chemists, biologists, clinicians, and engineers may contribute to obtain promising results in the biofilm field.

Future directions should focus on identifying of multi-target approaches, developed on the basis of knowledge obtained in the study of biofilm formed in in vivo-like environments. Such approaches should comply with requirements such as stability, selectivity, minimal toxicity, and low-cost formulations.

The present special issue is publishing a series of research articles on anti-biofilm strategies which include synthetic and natural quorum-sensing inhibitors, antimicrobial peptides, natural compounds, carbonic anhydrase inhibitors, nanosystems, and biomaterials. The large numbers of papers published in the biofilm research field confirm the key role of the microbial biofilms in human health and disease.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.