Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Morales-Soto, Nydia; Anyan, Morgen E.; Mattingly, Anne E.; Madukoma, Chinedu S.; Harvey, Cameron W.; Alber, Mark; Déziel, Éric; Kearns, Daniel B.; Shrout, Joshua D.

doi:10.3791/52338-v

Cited by 5 publications

(3 citation statements)

References 10 publications

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“…Sterile medium (7.5 mL) was gently pipetted into 60 mm polystyrene Petri dishes and allowed to cure for 30 min. After drying, the plates were stab-inoculated with an overnight FAB-glucose broth culture (OD = 1 at 600 nm) and incubated at 30 °C for 48 h. 20…”

Section: Methodsmentioning

confidence: 99%

Multimodal chemical imaging of molecular messengers in emerging Pseudomonas aeruginosa bacterial communities

Baig

Dunham

Morales-Soto

et al. 2015

Analyst

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Two label-free molecular imaging techniques, confocal Raman microscopy (CRM) and secondary ion mass spectrometry (SIMS), are combined for in situ characterization of the spatiotemporal distributions of quinolone metabolites and signaling molecules in communities of the pathogenic bacterium Pseudomonas aeruginosa. Dramatic molecular differences are observed between planktonic and biofilm modes of growth for these bacteria. We observe patterned aggregation and a high abundance of N-oxide quinolines in early biofilms and swarm zones of P. aeruginosa, while the concentrations of these secreted components in planktonic cells and agar plate colonies are below CRM and SIMS detection limits. CRM, in conjunction with principal component analysis (PCA) is used to distinguish between the two co-localized isomeric analyte pairs 4-hydroxy-2-heptylquinoline-N-oxide (HQNO)/2-heptyl-3-hydroxyquinolone (PQS) and 4-hydroxy-2-nonylquinoline-N-oxide (NQNO)/2-nonyl-hydroxyquinolone (C9-PQS) based on differences in their vibrational fingerprints, illustrating how the technique can be used to guide tandem-MS and tandem-MS imaging analysis. Because N-oxide quinolines are ubiquitous and expressed early in biofilms, these analytes may be fundamentally important for early biofilm formation and the growth and organization of P. aeruginosa microbial communities. This study underscores the advantages of using multimodal molecular imaging to study complex biological systems.

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Section: Methodsmentioning

confidence: 99%

Multimodal chemical imaging of molecular messengers in emerging Pseudomonas aeruginosa bacterial communities

Baig

Dunham

Morales-Soto

et al. 2015

Analyst

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show abstract

“…73 There are multiple methods to quantify this phenotype, including thickness and number of tendrils, rhamnolipid quantification, and swarm speeds though these can be tedious to measure and can prove difficult to compare. 13, 61, 63-64, 74 To incorporate these different aspects of motility into a single metric and enable simultaneous evaluation of multiple inhibitors, we quantified the area of overnight growth. 13, 56, 74 Because quantities of inhibitor compounds were limited, each was examined at a single concentration of 200 µM.…”

Section: Resultsmentioning

confidence: 99%

“…13, 61, 63-64, 74 To incorporate these different aspects of motility into a single metric and enable simultaneous evaluation of multiple inhibitors, we quantified the area of overnight growth. 13, 56, 74 Because quantities of inhibitor compounds were limited, each was examined at a single concentration of 200 µM. Given the challenges of effectively penetrating the cell envelope of Gram-negative organisms, such as P. aeruginosa , we hypothesized that even though most members of our compound library were effective protein inhibitors in vitro , there may be large disparities in cell-based assays related to the ability of each molecule to accumulate within the organism.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of Expanded 2-Aminobenzothiazole Library for Inhibition ofPseudomonas aeruginosaVirulence Phenotypes

Fihn

Lembke

Gaulin

et al. 2023

Preprint

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Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

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Evaluation of expanded 2-aminobenzothiazole library as inhibitors of a model histidine kinase and virulence suppressors in Pseudomonas aeruginosa

Fihn,

Lembke,

Gaulin

et al. 2024

Bioorganic Chemistry

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Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Cited by 5 publications

References 10 publications

Multimodal chemical imaging of molecular messengers in emerging Pseudomonas aeruginosa bacterial communities

Multimodal chemical imaging of molecular messengers in emerging Pseudomonas aeruginosa bacterial communities

Evaluation of Expanded 2-Aminobenzothiazole Library for Inhibition ofPseudomonas aeruginosaVirulence Phenotypes

Evaluation of expanded 2-aminobenzothiazole library as inhibitors of a model histidine kinase and virulence suppressors in Pseudomonas aeruginosa

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