Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening

Harrison, Joe J.; Stremick, Carol A.; Turner, Raymond J.; Allan, Nick; Olson, Merle E.; Ceri, Howard

doi:10.1038/nprot.2010.71

Cited by 264 publications

(240 citation statements)

References 51 publications

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“…Since PS80 is well tolerated on human tissues at concentrations of 1% or more, this is a clinically relevant finding (17). While the mechanism of action by which PS80 inhibits biofilm formation has not yet been elucidated, PS80's ability to inhibit bacterial biofilm formation has been noted in other independent publications (1,13,26).Some clinical isolates of P. aeruginosa are resistant to PS80 biofilm inhibition, prompting us to investigate the basis of this resistance in order to determine if PS80 formulations can be enhanced or modified to increase their spectrum of activity. As we have reported, one mechanism of PS80 resistance by some strains of P. aeruginosa is overexpression of a secreted lipase, LipA, resulting in cleavage of PS80 at its ester bond.…”

mentioning

confidence: 99%

Pseudomonas aeruginosa Exopolysaccharide Psl Promotes Resistance to the Biofilm Inhibitor Polysorbate 80

Zegans

Wozniak

Griffin

et al. 2012

Antimicrob Agents Chemother

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Polysorbate 80 (PS80) is a nonionic surfactant and detergent that inhibits biofilm formation by Pseudomonas aeruginosa at concentrations as low as 0.001% and is well tolerated in human tissues. However, certain clinical and laboratory strains (PAO1) of P. aeruginosa are able to form biofilms in the presence of PS80. To better understand this resistance, we performed transposon mutagenesis with a PS80-resistant clinical isolate, PA738. This revealed that mutation of algC rendered PA738 sensitive to PS80 biofilm inhibition. AlgC contributes to the biosynthesis of the exopolysaccharides Psl and alginate, as well as lipopolysaccharide and rhamnolipid. Analysis of mutations downstream of AlgC in these biosynthetic pathways established that disruption of the psl operon was sufficient to render the PA738 and PAO1 strains sensitive to PS80-mediated biofilm inhibition. Increased levels of Psl production in the presence of arabinose in a strain with an arabinose-inducible psl promoter were correlated with increased biofilm formation in PS80. In P. aeruginosa strains MJK8 and ZK2870, known to produce both Pel and Psl, disruption of genes in the psl but not the pel operon conferred susceptibility to PS80-mediated biofilm inhibition. The laboratory strain PA14 does not produce Psl and does not form biofilms in PS80. However, when PA14 was transformed with a cosmid containing the psl operon, it formed biofilms in the presence of PS80. Taken together, these data suggest that production of the exopolysaccharide Psl by P. aeruginosa promotes resistance to the biofilm inhibitor PS80. Bacterial biofilm formation on prosthetic medical devices, such as contact lenses, indwelling catheters, and artificial joints, is a common cause of serious infections (8,9,12,22,32). The formation of biofilms in these clinical settings has prompted interest in developing inhibitors that are compatible with use on human tissues. We have previously reported that polysorbate 80 (PS80), a nonionic detergent and surfactant, inhibits biofilm formation of the pathogen Pseudomonas aeruginosa and many other bacteria at concentrations as low as 0.001% (39). Since PS80 is well tolerated on human tissues at concentrations of 1% or more, this is a clinically relevant finding (17). While the mechanism of action by which PS80 inhibits biofilm formation has not yet been elucidated, PS80's ability to inhibit bacterial biofilm formation has been noted in other independent publications (1,13,26).Some clinical isolates of P. aeruginosa are resistant to PS80 biofilm inhibition, prompting us to investigate the basis of this resistance in order to determine if PS80 formulations can be enhanced or modified to increase their spectrum of activity. As we have reported, one mechanism of PS80 resistance by some strains of P. aeruginosa is overexpression of a secreted lipase, LipA, resulting in cleavage of PS80 at its ester bond. Based on these findings, we identified a compound related to PS80, polyethoxylated(20) oleyl alcohol, containing a lipase-resistant ether bond rather...

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mentioning

confidence: 99%

Pseudomonas aeruginosa Exopolysaccharide Psl Promotes Resistance to the Biofilm Inhibitor Polysorbate 80

Zegans

Wozniak

Griffin

et al. 2012

Antimicrob Agents Chemother

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“…Innovotech has successfully cultured over 65 different bacterial species in this manner in the past (21). The S. aureus (ATCC 29213) and P. aeruginosa (ATCC 27853) strains for the present study had been optimized previously by Innovotech for this assay.…”

Section: Not Done 39mentioning

confidence: 99%

“…The MBEC assay is a highthroughput screening assay used to determine the efficacy of therapeutic agents against biofilms (4,21). Conical plastic pegs are arrayed in a 96-well format, allowing serial dilutions and replicates to be processed in parallel for several experimental conditions.…”

Section: Gonorrhoeae)mentioning

confidence: 99%

A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model

Estellés

Woischnig

Liu

et al. 2016

Antimicrob Agents Chemother

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Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observed in vitro at an antibody concentration of 1.2 g/ml over 12 h. The effect of TRL1068 in vivo was evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections.T he understanding of bacterial physiology has fundamentally changed since the discovery of biofilms in the bacterial life cycle (1-3). Biofilms provide an anchor and physical protection for bacterial cells and the physiology and genetic programming of the bacteria shift from the planktonic (free-floating) to a sessile (adherent) state. This shift can result in a substantial reduction of antibiotic sensitivity in the biofilm (4). As much as 65 to 80% of clinically significant bacterial infections resistant to antibiotics are associated with biofilm (5, 6), including those of implants and catheters, infective endocarditis, lung infections associated with cystic fibrosis and chronic obstructive pulmonary disease (COPD), persistent infections of the ears and urinary tract, osteomyelitis, and surgery-associated nosocomial infections. Accordingly, a promising approach to treatment is to disrupt biofilms so that the freed bacteria become sensitive to available antibiotics as well as more fully subject to immune control (7).Biofilms are not simply random assemblies of bacterial and host components. Rather, the polymers in a biofilm form a multinode scaffolding with a semirigid, three-dimensional web-like architecture (8) which serves to exclude host immune cells while allowing the diffusion of nutrients and waste. Comparative genomic studies have identified tens of proteins a...

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“…Sonication was used to quantitate the number of cells in a biofilm using a procedure adapted from Harrison, et al [115]. After the 96-well plate had been incubated for the desired amount of time, the foil and sealing film were removed and the liquid culture dumped into a 10% (v/v) bleach solution.…”

Section: Sonication and Drip Dilutionsmentioning

confidence: 99%

“…The plate was subjected to sonication at 60 Hz for 10 min to disrupt biofilms, as suggested in the protocol by Harrison, et al [115].…”

Section: Sonication and Drip Dilutionsmentioning

confidence: 99%

Burkholderia cenocepacia J2315-mediated destruction of Staphylococcus aureus NRS77 biofilms.

Thompson¹

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Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening

Cited by 264 publications

References 51 publications

Pseudomonas aeruginosa Exopolysaccharide Psl Promotes Resistance to the Biofilm Inhibitor Polysorbate 80

Pseudomonas aeruginosa Exopolysaccharide Psl Promotes Resistance to the Biofilm Inhibitor Polysorbate 80

A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model

Burkholderia cenocepacia J2315-mediated destruction of Staphylococcus aureus NRS77 biofilms.

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