Coupled CFD-DEM modelling to predict how EPS affects bacterial biofilm deformation, recovery and detachment under flow conditions

The deformation and detachment of bacterial biofilm are related to the structural and mechanical properties of the biofilm itself. Extracellular polymeric substances (EPS) play an important role on keeping the mechanical stability of biofilms. The understanding of biofilm mechanics and detachment can help to reveal biofilm survival mechanisms under fluid shear and provide insight about what flows might be needed to remove biofilm in a cleaning cycle or for a ship to remove biofilms. However, how the EPS may affect biofilm mechanics and its deformation in flow conditions remains elusive. To address this, a coupled computational fluid dynamic – discrete element method (CFD-DEM) model was developed. The mechanisms of biofilm detachment, such as erosion and sloughing have been revealed by imposing hydrodynamic fluid flow at different velocities and loading rates. The model, which also allows adjustment of the proportion of different functional group of microorganisms in the biofilm, enables the study of the contribution of EPS towards biofilm resistance to fluid shear stress. Furthermore, the stress-strain curves during biofilm deformation have been captured by loading and unloading fluid shear stress to study the viscoelastic properties of the biofilm.

Optimization of Quantitative Analysis of Biofilm Cell from Pipe Materials

The quantitative analysis of biofilm can be used not only to assess the microbiological stability of tap water but also on its basis can assess: the degree of colonization of materials by bacterial cells, the rate of biofilm formation on the surfaces of pipes and determine their composition and number. The article presents the results of research on the development of an effective method of biofilm detachment from the surface of the galvanized steel. The number of biofilm cells was determined by methods: (1) luminometric ATP determination, (2) flow cytometry and (3) heterotrophic plate counts (HPC). The presence of the biofilm was confirmed by SEM and fractal analysis. The analysis of the obtained results showed that the most effective method of detaching the biofilm cells from the galvanized steel surface was the mechanical separation with a sterile cotton swab. The variant with the use of a sterile swab enables rapid collection of the biofilm from the surface of the ducts forming internal installations or water supply networks. Due to the simplicity and speed of obtaining results, the luminometric ATP measurement has been established as the best method for the quantification of biofilm cells. The results of this study were intended to provide reliable and useful data on the quantification of biofilm cells.

Study of Pseudomonas aeruginosa PPF-1 biofilm formation using microfluidics: effect of magnesium on biofilm detachment in engineered conduits

The bacterium Pseudomonas aeruginosa is an opportunistic pathogen in certain organisms, including humans, but can also survive and proliferate in natural and engineered water systems. Microfluidic technology can address hydrodynamic questions related to bacterial contamination of water flow systems and infrastructure. In this work, a microfluidic approach was devised to study the effect of shear stresses on biofilms from a dental unit waterline (DUWL)-isolated P. aeruginosa strain, PPF-1. During application of relevant shear stress levels to DUWLs, the response of the PPF-1 biofilm was observed and compared to a clinical P. aeruginosa reference strain, PAO1. The response measurements were repeated for biofilms exposed to additional Mg2+ ions. Using a microfluidic approach to transforming optical density maps into three-dimensional images, we applied computational fluid dynamics simulations and determined the critical shear stresses for biofilm sloughing. In the absence of Mg2+, PPF-1 biofilms showed weaker attachment than PAO1 biofilms, resulting in continuous slough/regrowth cycles triggered by applied shear stresses of 1.42 +/- 0.32 Pa. Introducing Mg2+ into the PPF-1 biofilm culture medium seemed to place the biofilm into a viscoplastic mechanical state, thereby increasing mechanical stability, which resulted in elevated tolerances to shear stresses up to a critical value of 5.43 +/- 1.52 Pa. This resulted in a propensity for less frequent but more catastrophic sloughing events like that observed for the PAO1 reference strain. This suggests that in a low ionic environment, biofilms from the PPF-1 strain can result in higher and more continuous ejection of biofilm materials, possibly leading to increased downstream colonization of engineered flow systems.

In vitro Characterization of Biofilm Formation in Prevotella Species

Background: Periodontitis, a chronic inflammatory oral infection is the outcome of disturbances in the homeostasis of the oral biofilm microbiota. A number of studies have found the occurrence of Prevotella species in elevated levels in periodontitis compared to healthy subjects. Even though different aspects of Prevotella as part of oral biofilm have been studied, in vitro biofilms formed by these species have not been characterized systematically. The objective of this study was to characterize biofilms formed by several Prevotella species and further to assess biofilm inhibition and detachment of preformed biofilms.Methods: Biofilms were grown in 24-well plates containing brucella broth in anaerobic conditions for 3 days, and were quantified using crystal violet staining. Images of SYTO 9 Green fluorescent stained biofilms were captured using confocal microscopy. Biofilm inhibition and detachment by proteinase and DNase I was tested. The biochemical characterization included quantification of proteins and DNA in the biofilms and biofilm-supernatants.Results:Prevotella loescheii, Prevotella oralis and Prevotella nigrescens showed highest biofilm formation. P. nigrescens formed significantly higher amounts of biofilms than P. loescheii (P = 0.005) and P. oralis (P = 0.0013). Inhibition of biofilm formation was significant only in the case of P. oralis when treated with proteinase (P = 0.037), whereas with DNase I treatment, the inhibition was not significant (P = 0.531). Overall, proteinase was more effective in biofilm detachment than DNase I. Protein and DNA content were higher in biofilm than the supernatant with the highest amounts found in P. nigrescens biofilm and supernatants. P. oralis biofilms appeared to secrete large amounts of proteins extracellularly into the biofilm-supernatants.Conclusion: Significant differences among Prevotella species to form biofilms may imply their variable abilities to get integrated into oral biofilm communities. Of the species that were able to grow as biofilms, DNase I and proteinase inhibited the biofilm growth or were able to cause biofilm detachment.

Bioguided Isolation of Antibiofilm and Antibacterial Pregnane Glycosides from Caralluma quadrangula: Disarming Multidrug-Resistant Pathogens

Methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRAB) present a serious challenge because of their capability to cause biofilm resistance to commonly used antibiotics producing chronic infections and hindering the process of wound healing. In the current study, we investigated the antibacterial activity of Caralluma quadrangula extracts (MeOH, and its fractions CH2Cl2 and n-butanol) against multidrug-resistant MRSA USA300 and A. baumannii AB5057. In vitro, the MeOH extract and both fractions of C. quadrangula significantly inhibited biofilm formation and disrupted previously established biofilm by MRSA and MDRAB at all the tested concentrations (0.625, 0.313, and 0.156 mg/mL). In vivo, C. quadrangula extracts successfully decreased bacterial loads in MRSA-infected skin lesions in mice. Four pregnane glycosides and one flavone glycoside were isolated from the bioactive n-butanol fraction. The isolated compounds (Rus A–E) were tested for their biofilm inhibition and biofilm detachment activities. The results revealed that Rus C was the most active compound (IC50 = 0.139 mmole), while Rus E was the least active (IC50 = 0.818 mmole). These results support the potential use of C. quadrangula extracts or their isolated compounds for hindering the biofilm attachment and the virulence of MRSA and MDRAB and their application as a topical antimicrobial preparation for MRSA skin infections.

Association of Oral Microbiota Properties on Hyaluronidase and Biofilm Formation

Correlation between hyaluronidase (Hyl) activity and biofilm detachment in a few bacterial species was found. However, it is unclear if this association applies to bacterial species or for more general bacterial characteristics. This study determined the association between biofilm production and Hyl activity among bacterial isolates from the oral cavity of healthy subjects, and its association with Gram staining group, colony surface morphology and bacteria shape. The swab was taken from the tongue, cheek and entire teeth surfaces of 35 subjects, and tested for biofilm through modified microtiter plate assay while Hyl production was screened through HA rapid plate method. Forty-four isolates were found, each 50% are Gram-positive, and Gram-negative bacteria, with the majority are cocci and non-mucoid colony. More than 70% of isolates are moderate and strong; (n= 17, 38.6%) and (n=15, 34.1%) respectively for biofilm production; and 68.2% are Hyl producer. A significant association was found between Hyl and bacterial shape (p=0.018) and colony morphology (p=0.018), while other association is not significantly measured, including between Hyl and biofilm (p=0.659). This study showed that biofilm production is not affected by the characteristics of the bacteria to produce or not produce hyaluronidase. Meanwhile, Hyl production is prone in rod shape and mucoid isolates which need further investigations.

Metabolic Alterations in Shrimp Stomach During Acute Hepatopancreatic Necrosis Disease and Effects of Taurocholate on Vibrio parahaemolyticus

Acute hepatopancreatic necrosis disease (AHPND), a recently emerged bacterial shrimp disease, has increased shrimp mortality and caused huge economic losses in many Asian countries. However, molecular factors underlying pathogenesis of this disease remain largely unknown. Our objective was to characterize metabolic alterations in shrimp stomach during AHPND and determine effects of taurocholate on AHPND-causing Vibrio parahaemolyticus. Based on metabolomics, pathways for lipid metabolism and for primary bile acid (BA) synthesis were majorly affected following AHPND infection. Bile acid metabolites, namely taurocholate, were downregulated in the metabolomics database. This prompted us to study effects of taurocholate on biofilm formation, PirABvp toxin release and biofilm detachment capabilities in AHPND-causing V. parahaemolyticus. Treatment of this bacterium with high concentration of taurocholate, a primary bile acid, induced biofilm formation, PirABvp toxin release and facilitated the dispersion of bacterial cells. Taken together, our findings suggest that AHPND infection can affect the lipid metabolites in shrimp stomach, and further suggest that the primary bile acid taurocholate is important for the virulence of AHPND-causing V. parahaemolyticus.

An evolutionarily diverged mitochondrial protein controls biofilm growth and virulence in Candida albicans

A forward genetic screening approach identified orf19.2500 as a gene controlling Candida albicans biofilm dispersal and biofilm detachment. Three-dimensional (3D) protein modeling and bioinformatics revealed that orf19.2500 is a conserved mitochondrial protein, structurally similar to, but functionally diverged from, the squalene/phytoene synthases family. The C. albicans orf19.2500 is distinguished by 3 evolutionarily acquired stretches of amino acid inserts, absent from all other eukaryotes except a small number of ascomycete fungi. Biochemical assays showed that orf19.2500 is required for the assembly and activity of the NADH ubiquinone oxidoreductase Complex I (CI) of the respiratory electron transport chain (ETC) and was thereby named NDU1. NDU1 is essential for respiration and growth on alternative carbon sources, important for immune evasion, required for virulence in a mouse model of hematogenously disseminated candidiasis, and for potentiating resistance to antifungal drugs. Our study is the first report on a protein that sets the Candida-like fungi phylogenetically apart from all other eukaryotes, based solely on evolutionary “gain” of new amino acid inserts that are also the functional hub of the protein.

Arginine induced Streptococcus gordonii biofilm detachment using a novel rotating-disc rheometry method

Oral diseases are one of the most common pathologies affecting human health. These diseases are typically associated with dental plaque-biofilms, through either build-up of the biofilm or dysbiosis of the microbial community. Arginine can disrupt dental plaque-biofilms, and maintain plaque homeostasis, making it an ideal therapeutic to combat the development of oral disease. Despite our understanding of the actions of arginine towards dental plaque-biofilms, it is still unclear how or if arginine effects the mechanical integrity of the dental plaque-biofilm. Here we adapted a rotating-disc rheometry assay, which is routinely used in marine microbial ecology, to study how arginine treatment of Streptococcus gordonii biofilms influences biofilm detachment from surfaces. We demonstrate that the assay is highly sensitive at quantifying the presence of biofilm and the detachment or rearrangement of the biofilm structure as a function of shear stress. We demonstrate that arginine treatment leads to earlier detachment of the biofilm, indicating that arginine treatment weakens the biofilm, making it more suspectable to removal by shear stresses. Our results add to the understanding that arginine targets biofilms by multifaceted mechanisms, both metabolic and physical, further promoting the potential of arginine as an active compound in dentifrices to maintain oral health.