Biology subjects

Stoodley, P.

Publications and source records attributed to Stoodley, P..

7 recordsLinked to original sources

bioRxiv2022

Differential metabolism between biofilm and suspended Pseudomonas aeruginosa cultures in bovine synovial fluid by 2D NMR-based metabolomics

Total joint arthroplasty is a common surgical procedure resulting in improved quality of life; however, a leading cause of surgery failure is periprosthetic joint infection. Periprosthetic infection often involves biofilms, making treatment challenging. Periprosthetic joint infections are difficult to diagnose by traditional culturing methods and there are no clinical biomarkers for the presence of biofilms. Further, the metabolic state of pathogens in the joint space is difficult to diagnose, the mechanism of their tolerance to antibiotics and host defenses is not well understood, and their culturing in the laboratory is challenging. Thus, there is a critical need for improved pathogen- and phenotype-specific diagnosis as well as improved treatment strategies toward better patient outcomes. Here, we present a quantitative, untargeted NMR-based metabolomics strategy for Pseudomonas aeruginosa suspended culture and biofilm phenotypes grown in bovine synovial fluid. We identified 21 unique metabolites as potential markers of P. aeruginosa and one unique marker of the biofilm phenotype in synovial fluid. Significant differences in metabolic pathways were found between the suspended culture and biofilm phenotypes including creatine, glutathione, alanine, and choline metabolism and the tricarboxylic acid cycle. These unique metabolite and pathway differences have the potential to serve as targets for P. aeruginosa and specifically biofilm diagnosis and biofilm control in synovial fluid. Author SummaryJoint replacement surgery is a common procedure frequently required in later stages of life due to damage in the joint. Over one million joint replacement surgeries are performed annually with rates increasing every year. A devastating complication associated with joint replacement is the development of infection around the implant device in the joint space, known as a periprosthetic joint infection. Bacteria in the joint space can form a biofilm, which is a gel-like matrix encasing the cells that increases resistance to treatment and exacerbates chronic infections. A particular challenge for the diagnosis of biofilm-mediated periprosthetic joint infections is the slowly growing nature of biofilm-mediated phenotypes, resulting in frequent failure to detect these bacteria by clinical microbiological culturing methods. Small molecule metabolites are uniquely produced by strains of bacteria in the biofilm versus planktonic or suspended culture phenotype. Identification of metabolites as specific markers of infection and biofilm could allow a new culture-free diagnostic approach to diagnose infection by biofilm. Furthermore, knowledge of metabolic pathway populations in biofilm in joint fluid could point to specific targets to prevent biofilm formation in the joint space.

microbiology↗

bioRxiv2022

Genomic and Transcriptomic Profiling of Phoenix Colonies

Pseudomonas aeruginosa is a Gram-negative bacterium responsible for numerous human infections. Previously, novel antibiotic tolerant variants known as phoenix colonies as well as variants similar to viable but non-culturable (VBNC) colonies were identified in response to high concentrations of aminoglycosides. In this study, the mechanisms behind phoenix colony and VBNC-like colony emergence were further explored using both whole genome sequencing and RNA sequencing. Phoenix colonies were found to have a single nucleotide polymorphism (SNP) in the PA4673 gene, which is predicted to encode a GTP-binding protein. No SNPs were identified within VBNC-like colonies compared to the founder population. RNA sequencing did not detect change in expression of PA4673 but revealed multiple differentially expressed genes that may play a role in phoenix colony emergence. One of these differentially expressed genes, PA3626, encodes for a tRNA pseudouridine synthase which when knocked out led to a complete lack of phoenix colonies. Although not immediately clear whether the identified genes in this study may have interactions which have not yet been recognized, they may contribute to the understanding of how phoenix colonies are able to emerge and survive in the presence of antibiotic exposure.

microbiology↗

bioRxiv2022

Rapid aggregation of Staphylococcus aureus in synovial fluid is influenced by synovial fluid concentration, viscosity, and fluid dynamics-with evidence of polymer bridging

Early bacterial survival in the post-surgical joint is still a mystery. Recently, synovial fluid-induced aggregation was proposed as a potential mechanism of bacterial protection upon entry into the joint. As synovial fluid is secreted back into the joint cavity following surgery, rapid fluctuations in synovial fluid concentration, composition, and viscosity occur. These changes, along with fluid movement from post-operative joint motion, will modify the environment and potentially affect the kinetics of aggregate formation. Through this work, we sought to evaluate the influence of exposure time, synovial fluid concentration, viscosity, and fluid dynamics on aggregation. Furthermore, we aimed to elucidate the primary mechanism of aggregate formation by assessing the interaction of bacterial adhesins with synovial fluid polymer, fibrinogen. Following incubation in each simulated post-operative joint condition, the aggregates were imaged using confocal microscopy. Our analysis revealed the formation of two distinct aggregate phenotypes dependent on whether the incubation was conducted under static or dynamic conditions. Using a surface adhesin mutant, we have narrowed down the genetic determinants for synovial fluid aggregate formation and identified essential host polymers required. We report here that synovial fluid-induced aggregation is influenced by various changes specific to the post-surgical joint environment. While we now have evidence that select synovial fluid polymers facilitate bridging aggregation through essential bacterial adhesins, we suspect that their utility is limited by the increasing viscosity under static conditions. Furthermore, dynamic fluid movement recovers the ability of the bacteria with present surface proteins to aggregate under high viscosity conditions, yielding large, globular aggregates. ImportanceInfection is a major complication of knee and hip joint replacement surgery which is used to treat arthritis or joint damage. We have shown that Staphylococcus aureus, a common bacterial pathogen, aggregates upon contact with synovial fluid. Within seconds, the bacterial cells will interact with synovial fluid polymers in the joint fluid through their cell wall adhesins. The rapid formation of these aggregates likely aids in early bacterial survival in the joint-potentially contributing to the likelihood of developing an infection. By strengthening our basic understanding of the mechanics of synovial fluid aggregate formation under clinically relevant conditions, we hope to expand the knowledge of how to prevent or disrupt aggregation and reduce and more successfully treat these joint infections.

microbiology↗

bioRxiv2021

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.

microbiology↗

bioRxiv2020

Mycobacterium abscessus biofilms have viscoelastic properties which may contribute to their recalcitrance in chronic pulmonary infections.

Mycobacterium abscessus is emerging as a cause of recalcitrant chronic pulmonary infections, particularly in people with cystic fibrosis (CF). Biofilm formation has been implicated in the pathology of this organism, however the role of biofilm formation in infection is unclear. Two colony-variants of M. abscessus are routinely isolated from CF samples, smooth (MaSm) and rough (MaRg). These two variants display distinct colony morphologies due to the presence (MaSm) or absence (MaRg) of cell wall glycopeptidolipids (GPLs). We hypothesized that MaSm and MaRg variant biofilms might have different biophysical and mechanical properties, including stiffness, viscosity and elasticity. To test this hypothesis, we performed uniaxial mechanical indentation, and shear rheometry on MaSm and MaRg colony-biofilms. We identified that MaRg biofilms were significantly stiffer than MaSm under a normal force, while MaSm biofilms were more pliant compared to MaRg, under both normal and shear forces. Furthermore, using theoretical indices of mucociliary and cough clearence, we identified that M. abscessus biofilms may be more resistant to mechanical forms of clearance from the lung, compared to other common pulmonary pathogens, such as P. aeruginosa. Thus, the mechanical properties of M. abscessus biofilms may contribute to the persistent nature of pulmonary infections caused by this organism.

microbiology↗

bioRxiv2019

Computational and experimental investigation of biofilm disruption dynamics induced by high velocity gas jet impingement

Experimental data showed that high-speed micro-sprays can effectively disrupt biofilms on their support substratum, producing a variety of dynamic reactions such as elongation, displacement, ripples formation and fluidization. However, the mechanics underlying the impact of high-speed turbulent flows on biofilm structure is complex in such extreme conditions, since direct measurements of viscosity at these high shear rates are not possible using dynamic testing instruments. Here we used computational fluid dynamics simulations to assess the complex fluid interactions of ripple patterning produced by high-speed turbulent air jets impacting perpendicular to the surface of Streptococcus mutans biofilms, a dental pathogen causing caries, captured by high speed imaging. The numerical model involved a two-phase flow of air over a non-Newtonian biofilm, whose viscosity as a function of shear rate was estimated using the Herschel-Bulkley model. The simulation suggested that inertial, shear and interfacial tension forces governed biofilm disruption by the air jet. Additionally, the high shear rates generated by the jet impacts coupled with shear-thinning biofilm property resulted in rapid liquefaction (within milliseconds) of the biofilm, followed by surface instability and travelling waves from the impact site. Our findings suggest that rapid shear-thinning in the biofilm reproduces dynamics under very high shear flows that elasticity can be neglected under these conditions, behaving the biofilm as a Newtonian fluid. A parametric sensitivity study confirmed that both applied force intensity (i.e. high jet-nozzle air velocity) and biofilm properties (i.e. low viscosity, low air-biofilm surface tension and thickness) intensify biofilm disruption, by generating large interfacial instabilities.\n\nIMPORTANCEKnowledge of mechanisms promoting disruption though mechanical forces is essential in optimizing biofilm control strategies which rely on fluid shear. Our results provide insight into how biofilm disruption dynamics is governed by applied forces and fluid properties, revealing a mechanism for ripples formation and fluid-biofilm mixing. These findings have important implications for the rational design of new biofilms cleaning strategies with fluid jets, such as determining optimal parameters (e.g. jet velocity and position) to remove the biofilm from a certain zone (e.g. in dental hygiene or debridement of surgical site infections), or using antimicrobial agents which could increase the interfacial area available for exchange, as well as causing internal mixing within the biofilm matrix, thus disrupting the localized microenvironment which is associated with antimicrobial tolerance. The developed model also has potential application in predicting drag and pressure drop caused by biofilms on bioreactor, pipeline and ship hull surfaces.

microbiology↗

bioRxiv2018

Increased rates of genomic mutation in a biofilm co-culture model of P. aeruginosa and S. aureus

Biofilms are major contributors to disease chronicity and are typically multi-species in nature. Pseudomonas aeruginosa and Staphylococcus aureus are leading causes of morbidity and mortality in a variety of chronic diseases but current in vitro dual-species biofilms models involving these pathogens are limited by short co-culture times (24 to 48 hours). Here, we describe the establishment of a stable (240 hour) co-culture biofilm model of P. aeruginosa and S. aureus that is reproducible and more representative of chronic disease.\n\nThe ability of two P. aeruginosa strains, (PAO1 and a cystic fibrosis isolate, PA21), to form co-culture biofilms with S. aureus was investigated. Co-culture was stable for longer periods using P. aeruginosa PA21 and S. aureus viability within the model improved in the presence of exogenous hemin. Biofilm co-culture was associated with increased tolerance of P. aeruginosa to tobramycin and increased susceptibility of S. aureus to tobramycin and a novel antimicrobial, HT61, previously shown to be more effective against non-dividing cultures of Staphylococcal spp. Biofilm growth was also associated with increased short-term mutation rates; 10-fold for P. aeruginosa and 500-fold for S. aureus.\n\nBy describing a reproducible 240 hour co-culture biofilm model of P. aeruginosa and S. aureus, we have shown that interspecies interactions between these organisms may influence short-term mutation rates and evolution, which could be of importance in understanding the adaptive processes that lead to the development of antimicrobial resistance.

microbiology↗