standardized-vitro-assays-to-visualize-quantify-interactions-between

Standardized <em>In vitro</em> Assays to Visualize and Quantify Interactions between Human Neutrophils and <em>Staphylococcus aureus</em> Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

The Ohio State University

Pseudomonas aeruginosa evolves during chronic pulmonary infections of cystic fibrosis (CF) patients, forming pathoadapted variants that are persistent. Mucoid and rugose small-colony variants (RSCVs) are typically isolated from sputum of CF patients. These variants overproduce exopolysaccharides in the biofilm extracellular polymeric substance (EPS). Currently, changes to the biophysical properties of RSCV and mucoid biofilms due to variations in EPS are not well understood. This knowledge may reveal how lung infections resist host clearance mechanisms. Here, we used mechanical indentation and shear rheometry to analyse the viscoelasticity of RSCV and mucoid colony-biofilms compared to their isogenic parent at 2-, 4-, and 6-d. While the viscoelasticity of parental colony-biofilms underwent fluctuating temporal changes, in contrast, RSCV and mucoid colony-biofilms showed a gradual progression to more elastic-solid behaviour. Theoretical indices of mucociliary and cough clearance predict that mature 6-d parental and RSCV biofilms may show reduced cough clearance from the lung, while early mucoid biofilms may show reduced clearance by both mechanisms. We propose that viscoelasticity be considered a virulence property of biofilms.

Viscoelastic properties of Pseudomonas aeruginosa variant biofilms.

Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse  infections. Wounds were infected with a mixed community of six  strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the  pathway, conferring hyperbiofilm phenotypes. Several of the  mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the  wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes. Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of  in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection,  experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.

Pseudomonas aeruginosa Interstrain Dynamics and Selection of Hyperbiofilm Mutants during a Chronic Infection.

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic  HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic , nontypeable  influenzae, and  Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Dental plaque biofilms are the causative agents of caries, gingivitis and periodontitis. Both mechanical and chemical strategies are used in routine oral hygiene strategies to reduce plaque build-up. If allowed to mature biofilms can create anoxic microenvironments leading to communities which harbor pathogenic Gram-negative anaerobes. When subjected to high velocity fluid jets and sprays biofilms can be fluidized which disrupts the biofilm structure and allows the more efficient delivery of antimicrobial agents.

Use of an oxygen planar optode to assess the effect of high velocity microsprays on oxygen penetration in a human dental biofilms in-vitro.

An amendment to this paper has been published and can be accessed via the original article.

Correction to: Use of an oxygen planar optode to assess the effect of high velocity microsprays on oxygen penetration in a human dental biofilms in-vitro.

It has long been recognized that biofilms are viscoelastic materials, however the importance of this attribute to the survival and persistence of these microbial communities is yet to be fully realized. Here we review work, which focuses on understanding biofilm mechanics and put this knowledge in the context of biofilm survival, particularly for biofilm-associated infections. We note that biofilm viscoelasticity may be an evolved property of these communities, and that the production of multiple extracellular polymeric slime components may be a way to ensure the development of biofilms with complex viscoelastic properties. We discuss viscoelasticity facilitating biofilm survival in the context of promoting the formation of larger and stronger biofilms when exposed to shear forces, promoting fluid-like behavior of the biofilm and subsequent biofilm expansion by viscous flow, and enabling resistance to both mechanical and chemical methods of clearance. We conclude that biofilm viscoelasticity contributes to the virulence of chronic biofilm infections.

Biofilm mechanics: Implications in infection and survival.

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 (Ma) and rough (Ma). These two variants display distinct colony morphologies due to the presence (Ma) or absence (Ma) of cell wall glycopeptidolipids (GPLs). We hypothesized that Ma and Ma variant biofilms might have different mechanical properties. To test this hypothesis, we performed uniaxial mechanical indentation, and shear rheometry on Ma and Ma colony-biofilms. We identified that Ma biofilms were significantly stiffer than Ma under a normal force, while Ma biofilms were more pliant compared to Ma, under both normal and shear forces. Furthermore, using theoretical indices of mucociliary and cough clearance, we identified that M. abscessus biofilms may be more resistant to mechanical forms of clearance from the lung, compared to another common pulmonary pathogen, Pseudomonas aeruginosa. Thus, the mechanical properties of M. abscessus biofilms may contribute to the persistent nature of pulmonary infections caused by this organism.

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

 (Mtb) and SARS-CoV-2 (CoV2) are the leading causes of death due to infectious disease. Although Mtb and CoV2 both cause serious and sometimes fatal respiratory infections, the effect of Mtb infection and its associated immune response on secondary infection with CoV2 is unknown. To address this question we applied two mouse models of COVID19, using mice which were chronically infected with Mtb. In both model systems, Mtb-infected mice were resistant to secondary CoV2 infection and its pathological consequences, and CoV2 infection did not affect Mtb burdens. Single cell RNA sequencing of coinfected and monoinfected lungs demonstrated the resistance of Mtb-infected mice is associated with expansion of T and B cell subsets upon viral challenge. Collectively, these data demonstrate that Mtb infection conditions the lung environment in a manner that is not conducive to CoV2 survival.

Mice infected with  are resistant to secondary infection with SARS-CoV-2.

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, a method used to quantify marine biofilm fouling, to study how arginine treatment of  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 susceptible to removal by shear stresses. Finally, we demonstrate that the biofilm disrupting affect is specific to arginine, and not a general property of amino acids, as  biofilms treated with either glycine or lysine had mechanical properties similar to untreated biofilms. 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.

Arginine Induced  Biofilm Detachment Using a Novel Rotating-Disc Rheometry Method.

Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 (CoV2) are the leading causes of death due to infectious disease. Although Mtb and CoV2 both cause serious and sometimes fatal respiratory infections, the effect of Mtb infection and its associated immune response on secondary infection with CoV2 is unknown. To address this question we applied two mouse models of COVID19, using mice which were chronically infected with Mtb. In both model systems, Mtb-infected mice were resistant to the pathological consequences of secondary CoV2 infection, and CoV2 infection did not affect Mtb burdens. Single cell RNA sequencing of coinfected and monoinfected lungs demonstrated the resistance of Mtb-infected mice is associated with expansion of T and B cell subsets upon viral challenge. Collectively, these data demonstrate that Mtb infection conditions the lung environment in a manner that is not conducive to CoV2 survival.

Article	Year
Viscoelastic properties of Pseudomonas aeruginosa variant biofilms. Scientific reports\| PubMed ID: 29946126
Pseudomonas aeruginosa Interstrain Dynamics and Selection of Hyperbiofilm Mutants during a Chronic Infection. mBio\| PubMed ID: 31409682
The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31767757
Use of an oxygen planar optode to assess the effect of high velocity microsprays on oxygen penetration in a human dental biofilms in-vitro. BMC oral health\| PubMed ID: 32825831	2020
Correction to: Use of an oxygen planar optode to assess the effect of high velocity microsprays on oxygen penetration in a human dental biofilms in-vitro. BMC oral health\| PubMed ID: 32887584	2020
Biofilm mechanics: Implications in infection and survival. Biofilm\| PubMed ID: 33447803	2020
Mycobacterium abscessus biofilms have viscoelastic properties which may contribute to their recalcitrance in chronic pulmonary infections. Scientific reports\| PubMed ID: 33658597	2021
Mice infected with are resistant to secondary infection with SARS-CoV-2. bioRxiv : the preprint server for biology\| PubMed ID: 34790981	2021
Arginine Induced Biofilm Detachment Using a Novel Rotating-Disc Rheometry Method. Frontiers in cellular and infection microbiology\| PubMed ID: 34805002	2021
Mice infected with Mycobacterium tuberculosis are resistant to acute disease caused by secondary infection with SARS-CoV-2. PLoS pathogens\| PubMed ID: 35325013	2022
Author Correction: Viscoelastic properties of Pseudomonas aeruginosa variant biofilms. Scientific reports\| PubMed ID: 35585192	2022

Erin S. Gloag

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