Mechanisms Of Biofilm Formation Research Articles

High Adhesion and Increased Cell Death Contribute to Strong Biofilm Formation in Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp), is a frequent cause of hospital and community-acquired infections and WHO had declared it as a “priority pathogen”. Biofilm is a major virulence factor of Kp and yet the mechanism of strong biofilm formation in Kp is unclear. A key objective of the present study is to investigate the differences between strong and weak biofilms formed by clinical isolates of Kp on various catheters and in different media conditions and to identify constituents contributing to strong biofilm formation. Quantification of matrix components (extracellular DNA (eDNA), protein, exopolysaccharides (EPS), and bacterial cells), confocal laser scanning microscopy (CLSM), field emission gun scanning electron microscopy (FEG-SEM) and flow-cytometry analysis were performed to compare strong and weak biofilm matrix. Our results suggest increased biofilm formation on latex catheters compared to silicone and silicone-coated latex catheters. Higher amounts of eDNA, protein, EPS, and dead cells were observed in the strong biofilm of Kp. High adhesion capacity and cell death seem to play a major role in formation of strong Kp biofilms. The enhanced eDNA, EPS, and protein in the biofilm matrix appear as a consequence of increased cell death.

Mechanisms of Immune Evasion and Bone Tissue Colonization That Make Staphylococcus aureus the Primary Pathogen in Osteomyelitis.

Staphylococcus aureus is the primary pathogen responsible for osteomyelitis, which remains a major healthcare burden. To understand its dominance, here we review the unique pathogenic mechanisms utilized by S. aureus that enable it to cause incurable osteomyelitis. Using an arsenal of toxins and virulence proteins, S. aureus kills and usurps immune cells during infection, to produce non-neutralizing pathogenic antibodies that thwart adaptive immunity. S. aureus also has specific mechanisms for distinct biofilm formation on implants, necrotic bone tissue, bone marrow, and within the osteocyte lacuno-canicular networks (OLCN) of live bone. In vitro studies have also demonstrated potential for intracellular colonization of osteocytes, osteoblasts, and osteoclasts. S. aureus has evolved a multitude of virulence mechanisms to achieve life-long infection of the bone, most notably colonization of OLCN. Targeting S. aureus proteins involved in these pathways could provide new targets for antibiotics and immunotherapies.

Carbon Starvation Induces the Expression of PprB-Regulated Genes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa can cause severe infections in humans. This bacterium often adopts a biofilm lifestyle that is hard to treat. In several previous studies, the PprA-PprB two-component system (TCS), which controls the expression of type IVb pili, BapA adhesin, and CupE fimbriae, was shown to be involved in biofilm formation (M. Romero, H. Silistre, L. Lovelock, V. J. Wright, K.-G. Chan, et al., Nucleic Acids Res 46:6823-6840, 2018, https://doi.org/10.1093/nar/gky324; S. de Bentzmann, C. Giraud, C. S. Bernard, V. Calderon, F. Ewald F, et al., PLoS Pathog 8:e1003052, 2012, https://doi.org/10.1371/journal.ppat.1003052). However, signals or environmental conditions that can trigger the PprA-PprB TCS are still unknown, and the molecular mechanisms of PprB-mediated biofilm formation are poorly characterized. Here, we report that carbon starvation stress (CSS) can induce the expression of pprB and genes in the PprB regulon. CSS-induced pprB transcription is mediated by the stress response sigma factor RpoS rather than the two-component sensor PprA. We also observed a strong negative regulation of PprB on the transcription of itself. Further experiments showed that PprB overexpression greatly enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Specifically, under the background of PprB overexpression, both the BapA adhesin and CupE fimbriae displayed positive effects on CCA and CSA, while the type IVb pili showed an unexpected negative effect on CCA and no effect on CSA. In addition, expression of the PprB regulon genes were significantly increased in 3-day colony biofilms, indicating a possible carbon limitation state. The CSS-RpoS-PprB-Bap/Flp/CupE pathway identified in this study provides a new perspective on the process of biofilm formation in carbon-limited environments.IMPORTANCE Typically, the determination of the external signals that can trigger a regulatory system is crucial to understand the regulatory logic and inward function of that system. The PprA-PprB two-component system was reported to be involved in biofilm formation in Pseudomonas aeruginosa, but the signals triggering this system are unknown. In this study, we found that carbon starvation stress (CSS) induces transcription of pprB and genes in the PprB regulon through an RpoS-dependent pathway. Increased PprB expression leads to enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Both CCA and CSA are largely dependent on the Bap secretion system and are moderately dependent on the CupE fimbriae. Our findings suggest that PprB reinforces the structure of biofilms under carbon-limited conditions, and the Bap secretion system and CupE fimbriae are two potential targets for biofilm treatment.

Vibrio cholerae Biofilm: Mechanisms, Regulating Formation and Signals of External Environment, Factoring Its Production

Currently it is a common knowledge that the ability of cholera agent to form biofilm increases the survival rate and persistence both in external environment and macroorganism. V. cholerae cells in associated state are better protected from the effect of a range of factors, more effectively consume nutrient substances and exchange genetic information. The process of biofilm formation by cholera vibrio is investigated in sufficient detail. However, taking into consideration the significant role of this structure in the life cycle of V. cholerae, researchers obtain new data and clarify earlier gathered information on the molecular mechanisms that lie at the bottom of this process, using advanced analytical methods. Herewith, close attention is paid to studies of regulatory mechanisms of biofilm formation, as well as external environment signals that trigger it. This review presents previously obtained data and new information on V cholerae regulatory network, controlling the process of biofilm formation, including transcriptional activators, repressors, alternative sigma-factors, regulatory RNA, and a range of signal molecules. The role of regulatory mechanisms in biofilm formation inside a macroorganism is also considered in the paper. Given are the data on external environment signals (availability of nutrient substances (carbohydrates), bile, non-organic substances; change in osmolarity of the media), stimulating/suppressing its formation. Taking into account the critical role of exopolysaccharide in mature biofilm formation, as well as significant role of signal molecules of Quorum Sensing system and 3'-5'-cyclic diguanylate monophosphate in the process, a particular attention is drawn to mechanisms of exopolysaccharide biosynthesis and effect of the mentioned molecules.

Genotypic and phenotypic characterization of biofilm production by Staphylococcus aureus strains isolated from bovine intramammary infections in Colombian dairy farms

The ability of Staphylococcus aureus to form biofilms is an important virulence factor because this has been associated with persistent bovine intramammary infections. Different mechanisms of biofilm formation have been described in S. aureus; however, the process has been found to be mainly driven by the ica and bap genes. The presence of the ica and bap genes, as well as the biofilm formation in vitro were evaluated in 229 S. aureus strains isolated from bovine milk collected from different regions of Department of Antioquia, Colombia. Three different genotypes grouped into three separate clusters were identified from in vitro assays. Genotype 1 (ica positive and bap negative) was the most prevalent (78.17%), followed by genotype 2 (ica and bap positive) (12.66%) and genotype 0 (ica and bap negative) (9.17%). Biofilm formation was observed in 81.26% of the strains from which 100% of genotype 2 isolates showed biofilm formation. The biofilms formed by genotype 2 isolates were also found to have the highest optical density (>2.4). These results showed that most of the S. aureus strains were capable of biofilm formation, suggesting the virulence potential particularly in bap-positive strains.

Streptococcus pyogenes Capsule Promotes Microcolony-Independent Biofilm Formation.

Biofilms play an important role in the pathogenesis of group A streptococcus (GAS), a Gram-positive pathogen responsible for a wide range of infections and with a significant public health impact. Although most GAS serotypes are able to form biofilms, there is a large amount of heterogeneity between individual strains in biofilm formation, as measured by standard crystal violet assays. It is generally accepted that biofilm formation includes the initial adhesion of bacterial cells to a surface followed by microcolony formation, biofilm maturation, and extensive production of extracellular matrix that links together proliferating cells and provides a scaffold for the three-dimensional (3D) biofilm structure. However, our studies show that for GAS strain JS95, microcolony formation is not an essential step in static biofilm formation, and instead, biofilm can be effectively formed from slow-growing or nonreplicating late-exponential- or early-stationary-phase planktonic cells via sedimentation and fixation of GAS chains. In addition, we show that the GAS capsule specifically contributes to the alternative sedimentation-initiated biofilms. Microcolony-independent sedimentation biofilms are similar in morphology and 3D structure to biofilms initiated by actively dividing planktonic bacteria. We conclude that GAS can form biofilms by an alternate noncanonical mechanism that does not require transition from microcolony formation to biofilm maturation and which may be obscured by biofilm phenotypes that arise via the classical biofilm maturation processes.IMPORTANCE The static biofilm assay is a common tool for easy biomass quantification of biofilm-forming bacteria. However, Streptococcus pyogenes biofilm formation as measured by the static assay is strain dependent and yields heterogeneous results for different strains of the same serotype. In this study, we show that two independent mechanisms, for which the protective capsule contributes opposing functions, may contribute to static biofilm formation. We propose that separation of these mechanisms for biofilm formation might uncover previously unappreciated biofilm phenotypes that may otherwise be masked in the classic static assay.

Biofilms: The Microbial "Protective Clothing" in Extreme Environments.

Microbial biofilms are communities of aggregated microbial cells embedded in a self-produced matrix of extracellular polymeric substances (EPS). Biofilms are recalcitrant to extreme environments, and can protect microorganisms from ultraviolet (UV) radiation, extreme temperature, extreme pH, high salinity, high pressure, poor nutrients, antibiotics, etc., by acting as “protective clothing”. In recent years, research works on biofilms have been mainly focused on biofilm-associated infections and strategies for combating microbial biofilms. In this review, we focus instead on the contemporary perspectives of biofilm formation in extreme environments, and describe the fundamental roles of biofilm in protecting microbial exposure to extreme environmental stresses and the regulatory factors involved in biofilm formation. Understanding the mechanisms of biofilm formation in extreme environments is essential for the employment of beneficial microorganisms and prevention of harmful microorganisms.

Nanoparticles for Oral Biofilm Treatments.

Pathogenic oral biofilms are universal, chronic, and costly. Despite advances in understanding the mechanisms of biofilm formation and persistence, novel and effective treatment options remain scarce. Nanoparticle-mediated eradication of the biofilm matrix and resident bacteria holds great potential. In particular, nanoparticles that target specific microbial and biofilm features utilizing nontoxic materials are well-suited for clinical translation. However, much work remains to characterize the local and systemic effects of therapeutic agents that are topically applied to chronic biofilms, such as those that cause dental caries. In this Perspective, we summarize the pathogenesis of oral biofilms, describe current and future nanoparticle-mediated treatment approaches, and highlight outstanding questions that are paramount to answer for effectively targeting and treating oral biofilms.

Membrane Vesicles Are the Dominant Structural Components of Ceftazidime-Induced Biofilm Formation in an Oxacillin-Sensitive MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) has received increasing attention in recent years. However, the characteristics and relevant mechanisms of biofilm formation in oxacillin-sensitive MRSA (OS-MRSA) are poorly understood. This study was designed to characterize biofilm formation in OS-MRSA BWSA15 in response to ceftazidime (TZ) by comparing the methicillin-sensitive S. aureus (MSSA) strain BWSA23 and the oxacillin-resistant MRSA (OR-MRSA) strain BWSA11. The biofilms and biofilm-forming cells were observed by electron microscopy. Biofilms grown on microtiter plates were chemically decomposed and analyzed by Fourier transform infrared spectroscopy. The transcriptional regulation of genes associated with methicillin resistance, surface adhesion, fatty acid biosynthesis, and global regulation (sigma B) was investigated. A significant increase in biofilm formation ability (10.21-fold) and aggregation ability (2.56-fold) was observed in BWSA15 upon the treatment with TZ (16 μg/ml). The TZ-induced biofilm formation in BWSA15 was characterized by a disappearance of polysaccharide-like extracellular substances and an appearance of a large number of intercellular MVs from extracellular matrix. Few MVs were identified in the biofilms formed by BWSA11 and BWSA23. There was a significant upregulation of mecA, sigB, and fatty acid biosynthesis-associated genes and downregulation of icaA, icaD, clfA, clfB, and fnaA in BWSA15 upon the treatment with TZ. The formation of intracellular junctions of MVs in the biofilms of BWSA15 was mediated by a significant increase in the proportion of proteins as well as by an increase in the proportion of non-ionized carboxyl groups in fatty acids. This study demonstrated that beta-lactam antibiotics can induce biofilm formation in OS-MRSA, and the biofilm induction in OS-MRSA can mainly be attributed to exposed MVs with increased hydrophobicity rather than polysaccharide intercellular adhesins, cell wall-anchored surface proteins, and extracellular DNA.

Adaptive antibacterial biomaterial surfaces and their applications.

Bacterial infections on the implant surface may eventually lead to biofilm formation ​and thus threaten the use of implants in body. Despite efficient host immune system, the implant surface can be rapidly occupied by bacteria, resulting in infection persistence, implant failure, and even death of the patients. It is difficult to cope with these problems because bacteria exhibit complex adhesion mechanisms to the implants that vary according to bacterial strains. Different biomaterial coatings have been produced to release antibiotics to kill bacteria. However, antibiotic resistance occurs very frequently. Stimuli-responsive biomaterials have gained much attention in recent years ​but are not effective enough in killing the pathogens because of the complex mechanisms in bacteria. This review is focused on the development of highly efficient and specifically targeted biomaterials that release the antimicrobial agents or respond to bacteria on demands in body. The mechanisms of bacterial adhesion, biofilm formation, and antibiotic resistance are discussed, and the released substances accounting for implant infection are described. Strategies that have been used in past for the eradication of bacterial infections are also discussed. Different types of stimuli can be triggered only upon the existence of bacteria, leading to the release of antibacterial molecules that in turn kill the bacteria. In particular, the toxin-triggered, pH-responsive, and dual stimulus-responsive adaptive antibacterial biomaterials are introduced. Finally, the state of the art in fabrication of dual responsive antibacterial biomaterials and tissue integration in medical implants is discussed.

Influence of some parameters on the ability of Listeria monocytogenes, Listeria innocua, and Escherichia coli to form biofilms.

Aim:The present study was conducted to evaluate the capacity of Listeria monocytogenes (L.m), Listeria innocua (L.i), and Escherichia coli to form biofilms on polystyrene support under different parameters by performing crystal violet (CV) staining technique.Materials and Methods:Different suspensions were prepared with single strains and with multiple combinations of strains including two serogroups of L.m (IIa and IIb), L.i, and E. coli strains at different microbial load. Selected strains and combinations were grown in biofilms for 6 days attached to polystyrene microplates under aerobic and microaerophilic conditions. The evaluation of the power of adhesion and biofilm formation was determined by CV staining followed by the measurement of optical density at 24 h, 72 h, and 6 days incubation time with and without renewal of the culture medium.Results:All the strains tested, presented more or less adhesion power depending on the variation of the studied parameters as well as the ability to form multispecies biofilms. Their development is more important by renewing the culture medium and increasing the initial load of bacteria. The ability to adhere and form biofilms differs from one serogroup to another within the same species. In bacterial combination, strains and species of bacteria adopt different behaviors.Conclusion:The ability to form biofilms is a key factor in the persistence of tested strains in the environment. Our study showed that L.m, L.i, and E. coli could adhere to polystyrene and form biofilms under different conditions. More researches are necessary to understand the mechanisms of biofilm formation and the influence of different parameters in their development.

Scalable fabrication of anti-biofouling membranes through 2-aminoimidazole incorporation during polyamide casting

A proof-of-concept for the fabrication of novel anti-biofouling water purification membranes through the incorporation of a 2-aminoimidazole (2-AI) during membrane casting is presented. 2-AI molecules are known to inhibit biofouling through the disruption of biofilm formation mechanisms, not through the inactivation of bacteria. Three approaches to incorporation were evaluated, adding the 2-AI to either one of the two monomer solutions, (a) m-phenylene diamine or (b) trimesoyl chloride, that polymerize to make a polyamide active layer, or by (c) reacting the active layer with a post-polymerization-2-AI-soak solution. These methods of incorporation are directly translatable to current membrane fabrication practices without the addition of other chemicals aside from the 2-AIs themselves. Results showed that the 2-AI was incorporated into the active layer of the membranes at concentrations (0.16–0.93 M) orders of magnitude higher than what is required for biofilm inhibition (IC50 = 162–420 μM). The 2-AI membranes significantly (p = 0.002–0.04) inhibited Pseudomonas aeruginosa biofilms (49–90% on average) due to the presence and action of 2-AI, not physico-chemical changes. The 2-AI-soak approach produced membranes that had the most stable incorporation, with no loss of compound during use and cleaning, and the highest biofilm inhibition, at 90% inhibition on average. The incorporation of 2-AI into the membranes decreased water permeability by 26–44% and salt rejection by 1.2–4.3% points, as compared to control membranes. No attempt was made to optimize 2-AI membrane preparation toward minimization of changes in water permeability and salt rejection. Given the substantial biofilm formation inhibition exhibited by the 2-AI membranes, and the limited decrease observed in water permeability and salt rejection, the 2-AI membranes presented in this study support 2-AI incorporation into polyamide active layers as a promising avenue to enhance current water purification membranes.

Biofilm-producing ability of Staphylococcus spp isolated from different foodstuff products.

In recent times, microbial-biofilm contamination has attracted considerable attention to the food industry. Pathogenic microorganisms can attach to food surfaces, grow on them, and form biofilm that cause an increase in the food safety risk. The mechanisms of biofilm formation have become an important issue in the food-processing industry, therefore, the aim of this study is to determine the biofilm formation and profiles of genes involved in biofilm production of staphylococci isolated from various foodstuff products. This cross-sectional study was conducted at some grocery stores and confectionaries from September 2015 to October 2016 in different areas of Isfahan, Iran. Staphylococcus spp were isolated from different foodstuff samples including sweet pastries, cakes and similar baked goods, dairy products such as cheese and yogurt, meat products such as sausages, and hamburgers. Standard microbiological methods were used for identification of Staphylococcus spp isolates. Antibiotic susceptibility pattern was determined by the disc diffusion method and icaA/icaD genes have been investigated as PCR target because of their role in the expression of intercellular adhesions involved in biofilm formation by S. aureus. From a total of 194 different foodstuffs samples, 84 Staphylococcus spp were isolated. Out of the 84 Staphylococcus isolates, 95.2% (80/84) were positive to the ability of biofilm formation. Overall, 35.7% (30/84) and 26.2% (22/84) of Staphylococcus spp isolates were positive for icaA and icaD genes, respectively. The results of the present study indicate that the remarkable rate of biofilm formation with the emergence of antibiotic resistance still remains a significant risk for the food safety, especially in foodstuff samples.

Silencing of cyt-c4 led to decrease of biofilm formation in Aeromonas hydrophila.

Aquaculture suffers from a number of diseases caused by Aeromonas hydrophila. Biofilm can protect bacteria from antibiotic therapy. To identify the genes those play crucial roles in A. hydrophila biofilm formation, a library of mini-Tn10 transposon insertion mutants of A. hydrophila B11has been constructed, and 10 mutants were subjected to biofilm formation assay. The biofilm formation ability of mutant (B188) was significantly decreased compared with B11. The DNA sequence flanking the mini-Tn10 transposon inserted showed that an ORF of approximately 576 bp of the mutant strain B188 was inserted. This ORF putatively displays the highest identity (92%) with the cytochrome c4gene (cyt-c4) of A. hydrophila subsp. hydrophila ATCC 7966. Silencing cyt-c4 led to deficiencies in biofilm formation, adhesion, drug resistance and pathogenicity of A. hydrophila, which suggests that cyt-c4 plays crucial role in the biofilm formation and virulence mechanisms of A. hydrophila. ABBREVIATIONS: GEN: gentamycin; SDZ: sulfadiazine; AK: amikacin; P: penicillin; CFP: cefoperazone; LEV: levofloxacin; MH: minocycline; FFC: florfenicol; TE: tetracycline; AMP: ampicillin; KAN: kanamycin; STR: streptomycin; SXT: sulfamethoxazole/trimethoprim; DO: doxycycline; OT: Oxytetracycline.

Candida glabrata: A Lot More Than Meets the Eye

Candida glabrata is an opportunistic human fungal pathogen that causes superficial mucosal and life-threatening bloodstream infections in individuals with a compromised immune system. Evolutionarily, it is closer to the non-pathogenic yeast Saccharomyces cerevisiae than to the most prevalent Candida bloodstream pathogen, C. albicans. C. glabrata is a haploid budding yeast that predominantly reproduces clonally. In this review, we summarize interactions of C. glabrata with the host immune, epithelial and endothelial cells, and the ingenious strategies it deploys to acquire iron and phosphate from the external environment. We outline various attributes including cell surface-associated adhesins and aspartyl proteases, biofilm formation and stress response mechanisms, that contribute to the virulence of C. glabrata. We further discuss how, C. glabrata, despite lacking morphological switching and secreted proteolytic activity, is able to disarm macrophage, dampen the host inflammatory immune response and replicate intracellularly.

Two-Component Signal Transduction Systems: A Major Strategy for Connecting Input Stimuli to Biofilm Formation.

Biofilms are multicellular communities of microbes that are encased within an extracellular matrix. Environmental factors induce bacteria to form biofilm. Bacteria have several regulatory mechanisms in response to environmental changes, and the two-component signal transduction system (TCS) is a major strategy in connecting changes in input signals to changes in cellular physiological output. The TCS employs multiple mechanisms such as cross-regulation, to integrate and coordinate various input stimuli to control biofilm formation. In this mini-review, we demonstrate the roles of TCS on biofilm formation, illustrating these input signals and modulation modes, which may be utilized by future investigations in elucidating the regulatory signals and underlying the mechanisms of biofilm formation.

Complete Genome Sequence of Staphylococcus epidermidis CSF41498.

Staphylococcus epidermidis CSF41498 is amenable to genetic manipulation and has been used to study mechanisms of biofilm formation. We report here the whole-genome sequence of this strain, which contains 2,427 protein-coding genes and 82 RNAs within its 2,481,008-bp-long genome, as well as three plasmids.

Biofilm and Quorum Sensing in Archaea

In the article, we presented a brief description of the Archaea, considering their structure, physiology and systematics. Based on the analysis of the literature, we bring closer the mechanism of biofilm formation, including extracellular polymeric substances as well as cellular organelles, such as archaella, pili and ‘hami’. The method of forming a biofilm depends on the type of Archaea and the environment in which it naturally lives. We are also introducing the phenomenon of quorum-sensing, as a mechanism of communication of Archaea in the environment. This phenomenon corresponds to similar molecules as bacteria, namely acylated homoserines lactones, QS peptides, autoinducer-2 and -3 and others. In the case of biofilms and the occurrence of the phenomenon of quorum sensing, it can be concluded that these phenomena are very important for the life of Archaea. The phenomena described seem to be conservative, because both in Archaea and Bacteria are regulated by the same mechanisms.

The ArlR-MgrA regulatory cascade regulates PIA-dependent and protein-mediated biofilm formation in Rbf-dependent and Rbf-independent pathways

The two-component system response regulator ArlR and the global regulator MgrA in Staphylococcus aureus participated in numerous biological processes including biofilm formation inhibition. Previous studies have shown that these two regulators could function as a regulatory cascade. Rbf is a positive regulator of biofilm formation enhancing the production of PIA (polysaccharide intercellular adhesin). Here we have demonstrated that both ArlR and MgrA can directly bind to the promoter of rbf and repress its expression. ArlR and MgrA can also directly bind to the promoter of ica operon and enhance the expression of icaA and PIA production, revealing that the ArlR-MgrA regulatory cascade controls PIA-dependent biofilm formation. In addition, we have found that Rbf can directly bind to the aur promoter and repress the expression of aur, which encodes a protease initiating a protease cascade to inhibit protein-mediated biofilm formation. Moreover, our data indicate that the ArlR-MgrA regulatory cascade can promote the expression of aur by directly binding to its promoter and inhibit protein-mediated biofilm formation. These findings shed light on the molecular mechanisms of both PIA-dependent and protein-mediated biofilm formation modulated by the ArlR-MgrA regulatory cascade and the new role of Rbf in protein-mediated biofilm formation, and broaden our understanding of the biofilm formation regulation in S. aureus.

Microbial biofilm: a “sticky” problem

Bacteria can form, on virtually any surface, single- and multispecies biofilms intrinsically resistant/tolerant to antibiotics and elusive of the host immune response. The study of bacterial biofilm development has, therefore, received great interest over the past 20 years and is motivated by the well-recognized role of these multicellular communities in infectious diseases. In this review article, we provide a synopsis of (i) biofilm formation mechanisms; (ii) biofilm clinical significance and underlying mechanisms; (iii) the current methodologies for microbiological diagnosis of biofilm-related infections; and (iv) current and future therapeutic strategies to combat biofilm-associated infections.