Fast and simple tool for the quantification of biofilm-embedded cells sub-populations from fluorescent microscopic images

Bogachev, Mikhail I.; Volkov, V.; Markelov, Oleg A.; Trizna, Elena Y.; Baydamshina, D.R.; Melnikov, Vladislav; Murtazina, Regina; Zelenikhin, P. V.; Sharafutdinov, Irshad; Kayumov, Airat R.

doi:10.1371/journal.pone.0193267

Cited by 80 publications

(33 citation statements)

References 25 publications

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“…The biofilms were imaged with confocal laser scanning microscopy (CLSM; A1R, Nikon, Tokyo, Japan) and biofilm thickness quantified using ImageJ (NIH, Bethesda, MD) by referencing at least 4 different images per condition. The proportions of live and dead bacteria were also quantified using BioFilmAnalyzer version 1.0 (https://bitbucket.org/rogex/biofilmanalyzer/downloads/) by counting fluorescence‐specific pixels in digital fluorescent images. Five different images were selected for analysis per condition.…”

Section: Methodsmentioning

confidence: 99%

In‐vitro evaluation of a ciprofloxacin‐ and ivacaftor‐coated sinus stent against Pseudomonas aeruginosa biofilms

Cho

Lim

Mackey

et al. 2019

Int Forum Allergy Rhinol

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Background:We recently developed a novel ciprofloxacincoated sinus stent capable of releasing antibiotics over a sustained period of time. Ivaca or is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that has synergistic bactericidal activity with ciprofloxacin and also enhances sinus mucociliary clearance. The objective of this study was to optimize and evaluate the efficacy of a ciprofloxacin-and ivaca or-releasing biodegradable sinus stent (CISS) in vitro. Methods: A CISS was created by coating ciprofloxacin/ivacaor-embedded nanoparticles with an acrylate and ammonium methacrylate copolymer onto a biodegradable poly-L-lactic acid stent. In-vitro evaluation of the CISS included:(1) assessment of drug stability in nanoparticles by zeta potential, and drug-coating stability within the CISS using scanning electron microscopy (SEM); (2) determination of ciprofloxacin-and ivaca or-release kinetics; and (3) assessment of anti-Pseudomonas aeruginosa biofilm formation by calculating relative optical density units (RODUs) compared with control stents at 590-nm optical density. Results:The presence of drugs and a uniform coating on the stent were confirmed by zeta potential and SEM.Sustained drug release was observed through 21 days without an initial burst release. Anti-biofilm formation was observed a er placing the CISS for 3 days onto a preformed 1-day P aeruginosa biofilm. The CISS significantly reduced biofilm mass compared with bare stents and controls (RO-DUs at 590-nm optical density; CISS, 0.31 ± 0.01; bare stent, 0.78 ± 0.12; control, 1.0 ± 0.00; p = 0.001; n = 3). Conclusion:The CISS maintains a uniform coating and sustained delivery of drugs providing a marked reduction in P aeruginosa biofilm formation. Further studies evaluating the efficacy of CISS in a preclinical model are planned. C 2019 ARS-AAOA, LLC.

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Section: Methodsmentioning

confidence: 99%

In‐vitro evaluation of a ciprofloxacin‐ and ivacaftor‐coated sinus stent against Pseudomonas aeruginosa biofilms

Cho

Lim

Mackey

et al. 2019

Int Forum Allergy Rhinol

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“…The analysis of TEM images was based on the shape-based algorithm of globular vs. brillar structures selection 55 with subsequent sub-populations quantication using an in-house developed algorithm and soware. 56…”

Section: Discussionmentioning

confidence: 99%

N- and C-terminal regions of the small heat shock protein IbpA from Acholeplasma laidlawii competitively govern its oligomerization pattern and chaperone-like activity

et al. 2020

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“…Experiments were carried out in six biological repeats with newly prepated cultures and medium in each of them. The fraction of non-viable cells in microscopic images was estimated as the relative fraction of the red cells among all cells in the combined images obtained by overlaying of the green and the red fluorescence microphotographs (10 images per each sample) by using BioFilmAnalyzer software 92 .The statistical significance of the discrepancy between monoculture and mixed biofilms treatment efficacy was determined using the Kruskal-Wallis statistical test with significance threshold at p < 0.05.…”

Section: Methodsmentioning

confidence: 99%

Bidirectional alterations in antibiotics susceptibility in Staphylococcus aureus - Pseudomonas aeruginosa dual-species biofilm

Kayumov

Trizna

Yarullina

et al. 2018

Preprint

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16While in biofilms bacteria are embedded into an extracellular matrix which forms 17 inaccessible barrier for antimicrobials thereby drastically increasing the concentrations 18 of antibiotics required for treatment. Here we show that the susceptibility of S. aureus 19 and P. aeruginosa to antibiotics in mixed biofilms significantly differs from monoculture 20 biofilms depending on both conditions and chosen antimicrobial agents. While S. aureus 21 could completely avoid vancomycin, ampicillin and ceftriaxone by embedding into the 22 biofilm of P. aeruginosa, the very same consortium was characterized by 10-fold 23 increase in susceptibility to broad-spectrum antimicrobials like ciprofloxacin and 24 aminoglycosides compared to monocultures. These data clearly indicate that efficient 25 treatment of biofilm-associated mixed infections requires antimicrobials active against 26 both pathogens, since the interbacterial antagonism would enhance the efficacy of 27 treatment. Moreover, similar increase in antibiotics efficacy was observed when 28 P. aeruginosa suspension was added to the mature S. aureus biofilm, compared to 29 S. aureus monoculture, and vice versa. These findings open promising perspectives to 30 increase the antimicrobial treatment efficacy of the wounds infected with nosocomial 31 pathogens by the transplantation of the skin residential microflora.32 42 polymicrobial biofilms 11,12 which drastically reduce their susceptibility to both antimicrobials 43 and the immune system of the host 13,14 . Current data suggests that bacterial pathogenicity is 44 promoted during polymicrobial infections and recovery is delayed in comparison with 45 monoculture infections 15-17 . Accordingly, interspecies interactions between S. aureus and 46 P. aeruginosa within mixed biofilms attracted major attention in recent years including both in 47 vitro 15 and in vivo studies 16 . 48 P. aeruginosa is known as a common dominator in polymicrobial biofilm-associated 49 infections due to multiple mechanisms allowing its rapid adaptation to the specific conditions of 50 the host. In particular, P. aeruginosa produces multiple molecules to compete with other 51 microorganisms for space and nutrients. The main anti-staphylococcal tools of P. aeruginosa are 52 siderophores and 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), the inhibitor of the electron 53 transport chain of S. aureus. Their presence shifts S. aureus to a fermentative mode of growth, 54 eventually leading to reduced S. aureus viability 18-22 , forces increased S. aureus biofilm 55 formation 23,24 and transition of S. aureus into small-colony variants (SCVs) 25 . SCV is a well-56 characterized phenotype detected in various diseases, including cystic fibrosis and device-related 57 infections 23-26 . SCVs appear as small, smooth colonies on a culture plate and grow significantly 58 slower compared to wild type colonies. Remarkably, switch to the SCV phenotype improves the 59 survival of S. aureus under unfavorable conditions, as it exhibits increased vancomycin and...

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Fast and simple tool for the quantification of biofilm-embedded cells sub-populations from fluorescent microscopic images

Cited by 80 publications

References 25 publications

In‐vitro evaluation of a ciprofloxacin‐ and ivacaftor‐coated sinus stent against Pseudomonas aeruginosa biofilms

In‐vitro evaluation of a ciprofloxacin‐ and ivacaftor‐coated sinus stent against Pseudomonas aeruginosa biofilms

N- and C-terminal regions of the small heat shock protein IbpA from Acholeplasma laidlawii competitively govern its oligomerization pattern and chaperone-like activity

Bidirectional alterations in antibiotics susceptibility in Staphylococcus aureus - Pseudomonas aeruginosa dual-species biofilm

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