Professional Documents
Culture Documents
Antibiotic Resistance and Bacterial Biofilm
Antibiotic Resistance and Bacterial Biofilm
To cite this article: Rossella Grande , Valentina Puca & Raffaella Muraro (2020):
Antibiotic resistance and bacterial biofilm, Expert Opinion on Therapeutic Patents, DOI:
10.1080/13543776.2020.1830060
DOI: 10.1080/13543776.2020.1830060
T
IP
a
Department of Pharmacy, University “G. d’Annunzio” of Chieti-Pescara, 66100 Chieti, Italy
R
SC
b
Center for Advanced Studies and Technology (CAST), “G. d’Annunzio” University of Chieti-
Pescara, Chieti, Italy
c
U
Department of Medicine and Aging Science, “G. d’Annunzio” University of Chieti-Pescara, Chieti,
AN
Italy
d
Department of Medical, Oral, and Biotechnological Sciences, “G. d’Annunzio” University of
M
Corresponding author:
TE
Rossella Grande,
Department of Pharmacy, University “G. d’Annunzio” of Chieti-Pescara, Via dei Vestini 31, 66100
EP
Chieti, Italy
C
Email: rossella.grande@unich.it
AC
Extracellular Polymeric Substances (EPS), adhered to a biotic or abiotic surface [1] (Figure 1).
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
T
biofilm is a multistep regulated process in which cell adhesion, EPS production and the detachment
IP
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
R
SC
component, up to 97%, together with polysaccharides, proteins, lipids, nucleic acids, as well as
insoluble components such as amyloids, fimbriae, pili and flagelli [3]. In particular, it has been
U
widely documented that extracellular DNA (eDNA) plays an important role in biofilm
AN
establishment and structural stability [4-8] as well as in the horizontal gene transfer and nutrition as
phosphate source [9]. The origin of eDNA has been hypothesized to result from multiple
M
mechanisms of DNA release, such as the production of extracellular vesicles [10-13], prophage-
D
mediated cell death as in Pseudomonas aeruginosa biofilms [14] and controlled cellular lysis
TE
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
EP
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
C
secretion [3]. The nature and composition of EPS matrix depend on bacterial strains, culture
AC
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
and the secretion of molecules with antimicrobial activity [16-18]. 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
T
IP
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
R
SC
tolerance to antimicrobials trough 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.
U
AN
Tolerance towards antimicrobials, unlike resistance which is genetic-based and can be acquired
through point mutations or horizontal gene transfer mechanisms such as conjugation, transformation
M
or phage transduction, can be defined as that condition in which, in order to kill or inhibit microbial
D
cells sensitive to antimicrobials, but aggregated within a biofilm, concentrations up to 4 times the
TE
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
EP
planktonic phenotype.
The variability in the biofilm composition as well as tolerance versus the antimicrobial drugs
C
commonly used in conventional therapies suggest the need for multi-targeted or combinational
AC
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 surfaces
modification [20].
development and on the EPS degradation in a mature biofilm. The inhibition of extracellular and
intracellular signalling as well as the inhibition of non-signalling 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
T
components like polysaccharides and adhesins. Small molecules as peptides or mannosides have
IP
been proven to be active versus bacterial and fungal biofilm associated with infections [19-22].
R
SC
proven equally effective [23-25]. 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
U
released by neutrophil and microorganisms in the sputum of patients affected by cystic fibrosis. The
AN
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
M
degradation facilitating the diffusion of the antimicrobial drugs into the EPS. The combination of
D
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
EP
contribute to the identification of the best association between degrading enzymes and type/class of
antimicrobials. Microbial cells communicate each other by using small molecules named
C
autoinducers. The Quorum Sensing (QS) system is a mechanism of regulation of cell density and,
AC
molecules while Gram-positive bacteria release small peptides. The use of inhibitors of QS, like
autoinducing peptide I (AIP-I), alone or in combination with antimicrobial drugs, may represent a
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.
T
conditions and increased tolerance to antimicrobial drugs. In H. pylori, the coccoid phenotype
IP
allows to the microorganism to avoid the immune system detection and to promote therapeutic
failures [28]. Antimicrobial peptides (AMP), for example, represent a valid approach in the
R
SC
bacterial and fungal biofilm treatment regardless by cellular metabolic activity, in fact, it has been
demonstrated that the capability of forming pore affects metabolically active, dormant and persister
U
cells [19]. AMPs can increase the effect of conventional antimicrobial therapies, therefore, a
AN
combination with other antibiofilm approaches which target the EPS matrix, as for example the use
Nanoparticles can be considered an innovative and versatile procedure for biofilm eradication.
D
Therefore, the use of them, as a biofilm-targeting strategy is widely studied. Inorganic and organic
TE
nanoparticles can be used alone or in combination with antimicrobial drugs. Silver nanoparticles
demonstrated that Silver Ultra-NanoClusters (SUNCs), showing low toxicity versus human cells,
C
were effective in eradicating H. pylori mature biofilm suggesting that they could represent a novel
AC
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 bacterial outer membrane and directly
release the antibiotic into the cell cytoplasm, increasing therapeutic effects and minimizing
cytotoxicity [34]. In addition, lyposomes can easily penetrate trough the biofilm matrix reaching the
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
coumarins and flavonoids [36], which do not normally cause resistance [37] as well as Essential
T
Oils (EO) from parsley, lovage, basil, thyme and hemp, have been studied for their antimicrobial
IP
and antibiofilm activities [37-40]. 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
R
SC
damage and inhibition of the QS in several bacterial species such as Bacillus cereus, S. aureus, P.
U
AN
M
D
TE
EP
C
Figure 1. A. Illustration of a bacterial biofilm. The microcolonies of bacterial cells are matrix-
AC
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 microbial biofilm is associated with hospital acquire infection, is subtle and often depends by
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
T
components of the biofilm microenvironment, are required [19].
IP
An in-depth study of the content and the amount of the components of EPS matrix developed by the
R
SC
much as possible in vivo conditions such as surface of colonization, pH condition and hypoxia
might help scientists to identify the more appropriate antimicrobial strategies aimed at biofilm
U
eradication. Once confirmed in “in vivo models” this approach would result in time saving, a
AN
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
M
resident microbes and the opportunistic pathogens. The microbiota represents a community of
D
microbes in which mutualistic and commensal microorganisms co-exist with potential pathogens
TE
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
EP
by probiotic strains. In addition, a multi-target approach that include the host immunomodulation
Unfortunately, the effect of new antimicrobial molecules, on the human microbiota is rarely
AC
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
On the basis of these concerns, the collaboration between scientists with different expertise such as
chemists, biologists, clinicians and engineers may contribute to obtain promising results in the
biofilm field.
knowledge obtained in the study of biofilm formed in in vivo-like environments. Such approaches
should comply 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
T
include synthetic and natural quorum sensing inhibitors, antimicrobial peptides, natural compounds,
IP
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
R
SC
health and disease.
Funding
U
AN
This paper was not funded.
M
Declaration of interests
D
The authors have no relevant affiliations or financial involvement with any organization or entity
TE
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
EP
Reviewer disclosures
AC
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.
References
Paper of special note have been highlighted as either of interest (●) or of considerable interest (●●)
to readers
3. Flemming HC, Wingender J, Szewzyk U, et al. Biofilms: an emergent form of bacterial life.
T
Nat Rev Microbiol. 2016;14(9):563-575.
IP
●●
A complete and in-depth review of the biofilm world.
R
SC
choline, extracellular DNA, and capsular polysaccharide in microbial accretion. J Bacteriol.
2006;188(22):7785-7795.
U
5. Izano EA, Amarante MA, Kher WB, et al. Differential roles of poly-N-acetylglucosamine
AN
surface polysaccharide and extracellular DNA in Staphylococcus aureus and Staphylococcus
7. Rice KC, Mann EE, Endres JL, et al. The cidA murein hydrolase regulator contributes to
EP
DNA release and biofilm development in Staphylococcus aureus. Proc Natl Acad Sci U S A.
2007;104(19):8113-8118.
C
alsS in the early development of Staphylococcus aureus UAMS-1 biofilm formation and
2004;150(Pt7):2161-2169.
11. Manning AJ, Kuehn MJ. Functional advantages conferred by extracellular prokaryotic
T
12. Grande R, Di Marcantonio MC, Robuffo I, et al. Helicobacter pylori ATCC 43629/NCTC
IP
11639 Outer Membrane Vesicles (OMVs) from Biofilm and Planktonic Phase Associated
R
SC
13. Puca V, Ercolino E, Celia C, et al. Detection and Quantification of eDNA-Associated
U
14. Webb JS, Lau M, Kjelleberg S. Bacteriophage and phenotypic variation in Pseudomonas
AN
aeruginosa biofilm development. J Bacteriol. 2004;186(23):8066-8073.
15. Yonezawa H, Osaki T, Kurata S, et al. Outer membrane vesicles of Helicobacter pylori
M
16. Rieu A, Aoudia N, Jego G, et al. The biofilm mode of life boosts the anti‐inflammatory
TE
17. Salas-Jara MJ, Ilabaca A, Vega M, et al. Biofilm Forming Lactobacillus: New Challenges
EP
18. Chamignon C, Gueneau V, Medina S, et al. Evaluation of the probiotic properties and the
C
19. Koo H, Allan RN, Howlin RP, et al. Targeting microbial biofilms: current and prospective
20. Parrino B, Schillaci D, Carnevale I, et al. Synthetic small molecules as antibiofilm agents in
22. Nett JE, Cabezas-Olcoz J, Marchillo K, et al. Targeting Fibronectin To Disrupt In Vivo
T
23. Kaplan JB. Biofilm matrix-degrading enzymes. Methods Mol Biol. 2014;1147:203-13.
IP
●
The chapter provides a brief overview of the most commonly used biofilm matrix-
R
degrading enzymes and presents examples of their applications in biofilm research.
SC
24. Fleming D, Chahin L, Rumbaugh K. Glycoside Hydrolases Degrade Polymicrobial Bacterial
U
25. Okshevsky M, Regina VR, Meyer RL. Extracellular DNA as a target for biofilm control.
AN
Curr Opin Biotechnol. 2015;33:73–80.
fibrosis sputum using atomic force and scanning electron microscopy. J Cyst Fibros.
D
2012;11(2): 84-92.
TE
27. Konstan MW, Ratjen F. Effect of dornase alfa on inflammation and lung function: potential
28. Baelo A, Levato R, Julián E, et al. Disassembling bacterial extracellular matrix with DNase-
2015;209:150-158.
AC
2020;9(3):184.
32. Puca V, Traini T, Guarnieri S, et al. The Antibiofilm Effect of a Medical Device Containing
2019;24(12):2280.
T
33. Grande R, Sisto F, Puca V, et al. Antimicrobial and antibiofilm activities of new synthesized
IP
silver ultra-nanoclusters (SUNCs) against Helicobacter pylori. Front Microbiol.
2020;11:1705.
R
SC
34. Forier K, Raemdonck K, De Smedt SC, Demeester J, Coenye T, Braeckmans K. Lipid and
2014;190:607-623.
U
AN
35. Rukavina Z, Vanić Ž. Current Trends in Development of Liposomes for Targeting Bacterial
36. Lewis K, Ausubel FM. Prospects for plant-derived antibacterials. Nat Biotechnol.
D
2006;24(12):1504-1507.
TE
2020;9(8):E480.
Molecules. 2018;23(12):3266.
39. Mocan A, Cairone F, Locatelli M, et al. Polyphenols from Lycium barbarum (Goji) Fruit
oil combinations against Gram-positive and Gram-negative bacteria. J Food Drug Anal.
2017;25(2):403-408.
T
IP
R
SC
U
AN
M
D
TE
EP
C
AC