scholarly journals High-Throughput Screens for Small-Molecule Inhibitors of Pseudomonas aeruginosa Biofilm Development

2007 ◽  
Vol 51 (10) ◽  
pp. 3582-3590 ◽  
Author(s):  
Lauren M. Junker ◽  
Jon Clardy
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Small Molecule ◽  
High Throughput ◽  
Biofilm Formation ◽  
Small Molecule Inhibitors ◽  
Opportunistic Pathogen ◽  
Positive Control ◽  
Strain Pao1 ◽  
Small Molecule Modulators ◽  
Biofilm Development

ABSTRACT Pseudomonas aeruginosa is both a model biofilm-forming organism and an opportunistic pathogen responsible for chronic lung infections in cystic fibrosis (CF) patients and infections in burn patients, among other maladies. Here we describe the development of an efficient high-throughput screen to identify small-molecule modulators of biofilm formation. This screen has been run with 66,095 compounds to identify those that prevent biofilm formation without affecting planktonic bacterial growth. The screen is a luminescence-based attachment assay that has been validated with several strains of P. aeruginosa and compared to a well-established but low-throughput crystal violet staining biofilm assay. P. aeruginosa strain PAO1 was selected for use in the screen both because it forms robust biofilms and because genetic information and tools are available for the organism. The attachment-inhibited mutant, strain PAO1 ΔfliC, was used as a screening-positive control. We have also developed and validated a complementary biofilm detachment assay that can be used as an alternative primary screen or secondary screen for the attachment screening-positive compounds. We have determined the potencies of 61 compounds against biofilm attachment and have identified 30 compounds that fall into different structural classes as biofilm attachment inhibitors with 50% effective concentrations of less than 20 μM. These small-molecule inhibitors could lead to the identification of their relevant biofilm targets or potential therapeutics for P. aeruginosa infections.

scholarly journals A Rapid Phenotypic Whole-Cell Screening Approach for the Identification of Small-Molecule Inhibitors That Counter β-Lactamase Resistance in Pseudomonas aeruginosa

2017 ◽  
Vol 23 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Deanna Collia ◽  
Thomas D. Bannister ◽  
Hao Tan ◽  
Shouguang Jin ◽  
Taimour Langaee ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Laboratory Strain ◽  
Multidrug Resistant ◽  
First Line ◽  
Strain Pao1 ◽  
Whole Cell ◽  
Bacterial Sensitivity ◽  
Opportunistic Human Pathogen

Pseudomonas aeruginosa is an opportunistic human pathogen that is prevalent in hospitals and continues to develop resistance to multiple classes of antibiotics. Historically, β-lactam antibiotics have been the first line of therapeutic defense. However, the emergence of multidrug-resistant (MDR) strains of P. aeruginosa, such as AmpC β-lactamase overproducing mutants, limits the effectiveness of current antibiotics. Among AmpC hyperproducing clinical isolates, inactivation of AmpG, which is essential for the expression of AmpC, increases bacterial sensitivity to β-lactam antibiotics. We hypothesize that inhibition of AmpG activity will enhance the efficacy of β-lactams against P. aeruginosa. Here, using a highly drug-resistant AmpC-inducible laboratory strain PAO1, we describe an ultra-high-throughput whole-cell turbidity assay designed to identify small-molecule inhibitors of the AmpG. We screened 645,000 compounds to identify compounds with the ability to inhibit bacterial growth in the presence of cefoxitin, an AmpC inducer, and identified 2663 inhibitors that were also tested in the absence of cefoxitin to determine AmpG specificity. The Z′ and signal-to-background ratio were robust at 0.87 ± 0.05 and 2.2 ± 0.2, respectively. Through a series of secondary and tertiary studies, including a novel luciferase-based counterscreen, we ultimately identified eight potential AmpG-specific inhibitors.

Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions

2009 ◽  
Vol 58 (6) ◽  
pp. 765-773 ◽  
Author(s):  
Che Y. O'May ◽  
Kevin Sanderson ◽  
Louise F. Roddam ◽  
Sylvia M. Kirov ◽  
David W. Reid
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Low Oxygen ◽  
Anaerobic Conditions ◽  
Chronic Infections ◽  
Strain Pao1 ◽  
Biofilm Inhibition ◽  
Oxygen Conditions ◽  
Biofilm Development ◽  
Chelating Compounds

The success of Pseudomonas aeruginosa in cystic fibrosis (CF) and other chronic infections is largely attributed to its ability to grow in antibiotic-resistant biofilm communities. This study investigated the effects of limiting iron levels as a strategy for preventing/disrupting P. aeruginosa biofilms. A range of synthetic and naturally occurring iron-chelating agents were examined. Biofilm development by P. aeruginosa strain PAO1 and CF sputum isolates from chronically infected individuals was significantly decreased by iron removal under aerobic atmospheres. CF strains formed poor biofilms under anaerobic conditions. Strain PAO1 was also tested under anaerobic conditions. Biofilm formation by this model strain was almost totally prevented by several of the chelators tested. The ability of synthetic chelators to impair biofilm formation could be reversed by iron addition to cultures, providing evidence that these effective chelating compounds functioned by directly reducing availability of iron to P. aeruginosa. In contrast, the biological chelator lactoferrin demonstrated enhanced anti-biofilm effects as iron supplementation increased. Hence biofilm inhibition by lactoferrin appeared to occur through more complex mechanisms to those of the synthetic chelators. Overall, our results demonstrate the importance of iron availability to biofilms and that iron chelators have potential as adjunct therapies for preventing biofilm development, especially under low oxygen conditions such as encountered in the chronically infected CF lung.

scholarly journals Identification of Small Molecule Modulators of Diguanylate Cyclase by FRET-based High-Throughput-Screening

2018 ◽  
Author(s):  
Matthias Christen ◽  
Cassandra Kamischke ◽  
Hemantha D. Kulasekara ◽  
Kathleen C. Olivas ◽  
Bridget R. Kulasekara ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
High Throughput ◽  
Biofilm Formation ◽  
High Throughput Screening ◽  
Diguanylate Cyclase ◽  
Chronic Infections ◽  
Structure Activity ◽  
Diguanylate Cyclases ◽  
Small Molecule Modulators

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which may make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, may be attractive drug targets to control biofilm formation that is part of chronic infections. In this paper, we present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small molecule modulators of the diguanylate cyclase, DgcA, from Caulobacter crescentus. We identified a set of 7 small molecules that in the low µM range regulate DgcA enzymatic activity. Subsequent structure activity studies on selected scaffolds revealed a remarkable diversity of modulatory behaviors, including slight chemical substitutions that revert the effects from allosteric enzyme inhibition to activation. The compounds identified represent novel chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP associated phenotypes. In sum, our studies underline the importance for detailed mechanism of action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

scholarly journals Sodium Salicylate Influences the Pseudomonas aeruginosa Biofilm Structure and Susceptibility Towards Silver

2021 ◽  
Vol 22 (3) ◽  
pp. 1060
Author(s):  
Erik Gerner ◽  
Sofia Almqvist ◽  
Peter Thomsen ◽  
Maria Werthén ◽  
Margarita Trobos
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Sodium Salicylate ◽  
Treatment Strategies ◽  
Cell Aggregation ◽  
Wound Fluid ◽  
Global Challenge ◽  
3D Collagen ◽  
Biofilm Development ◽  
Biofilm Structure

Hard-to-heal wounds are typically infected with biofilm-producing microorganisms, such as Pseudomonas aeruginosa, which strongly contribute to delayed healing. Due to the global challenge of antimicrobial resistance, alternative treatment strategies are needed. Here, we investigated whether inhibition of quorum sensing (QS) by sodium salicylate in different P. aeruginosa strains (QS-competent, QS-mutant, and chronic wound strains) influences biofilm formation and tolerance to silver. Biofilm formation was evaluated in simulated serum-containing wound fluid in the presence or absence of sodium salicylate (NaSa). Biofilms were established using a 3D collagen-based biofilm model, collagen coated glass, and the Calgary biofilm device. Furthermore, the susceptibility of 48-h-old biofilms formed by laboratory and clinical strains in the presence or absence of NaSa towards silver was evaluated by assessing cell viability. Biofilms formed in the presence of NaSa were more susceptible to silver and contained reduced levels of virulence factors associated with biofilm development than those formed in the absence of NaSa. Biofilm aggregates formed by the wild-type but not the QS mutant strain, were smaller and less heterogenous in size when grown in cultures with NaSa compared to control. These data suggest that NaSa, via a reduction of cell aggregation in biofilms, allows the antiseptic to become more readily available to cells.

scholarly journals Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach

2013 ◽  
Vol 170 (1) ◽  
pp. 132-140 ◽  
Author(s):  
Margaret R. Duffy ◽  
Alan L. Parker ◽  
Eric R. Kalkman ◽  
Katie White ◽  
Dmytro Kovalskyy ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Screening Approach
Sign in / Sign up

Export Citation Format

Share Document