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Modelling experimentally measured of ciprofloxacin antibiotic diffusion in Pseudomonas aeruginosa biofilm formed in artificial sputum medium.

ABSTRACT: We study the experimentally measured ciprofloxacin antibiotic diffusion through a gel-like artificial sputum medium (ASM) mimicking physiological conditions typical for a cystic fibrosis layer, in which regions occupied by Pseudomonas aeruginosa bacteria are present. To quantify the antibiotic diffusion dynamics we employ a phenomenological model using a subdiffusion-absorption equation with a fractional time derivative. This effective equation describes molecular diffusion in a medium structured akin Thompson's plumpudding model; here the 'pudding' background represents the ASM and the 'plums' represent the bacterial biofilm. The pudding is a subdiffusion barrier for antibiotic molecules that can affect bacteria found in plums. For the experimental study we use an interferometric method to determine the time evolution of the amount of antibiotic that has diffused through the biofilm. The theoretical model shows that this function is qualitatively different depending on whether or not absorption of the antibiotic in the biofilm occurs. We show that the process can be divided into three successive stages: (1) only antibiotic subdiffusion with constant biofilm parameters, (2) subdiffusion and absorption of antibiotic molecules with variable biofilm transport parameters, (3) subdiffusion and absorption in the medium but the biofilm parameters are constant again. Stage 2 is interpreted as the appearance of an intensive defence build-up of bacteria against the action of the antibiotic, and in the stage 3 it is likely that the bacteria have been inactivated. Times at which stages change are determined from the experimentally obtained temporal evolution of the amount of antibiotic that has diffused through the ASM with bacteria. Our analysis shows good agreement between experimental and theoretical results and is consistent with the biologically expected biofilm response. We show that an experimental method to study the temporal evolution of the amount of a substance that has diffused through a biofilm is useful in studying the processes occurring in a biofilm. We also show that the complicated biological process of antibiotic diffusion in a biofilm can be described by a fractional subdiffusion-absorption equation with subdiffusion and absorption parameters that change over time.

SUBMITTER: Kosztolowicz T

PROVIDER: S-EPMC7714214 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Modelling experimentally measured of ciprofloxacin antibiotic diffusion in Pseudomonas aeruginosa biofilm formed in artificial sputum medium.

Kosztołowicz Tadeusz T Metzler Ralf R Wa Sik Sławomir S Arabski Michał M

PloS one 20201203 12

We study the experimentally measured ciprofloxacin antibiotic diffusion through a gel-like artificial sputum medium (ASM) mimicking physiological conditions typical for a cystic fibrosis layer, in which regions occupied by Pseudomonas aeruginosa bacteria are present. To quantify the antibiotic diffusion dynamics we employ a phenomenological model using a subdiffusion-absorption equation with a fractional time derivative. This effective equation describes molecular diffusion in a medium structure ...[more]

PMID: 33270697

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Project description:Pseudomonas aeruginosa airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. In vitro models that closely mimic CF sputum are needed to improve understanding of the pathobiology of P. aeruginosa in the CF airway. We developed an artificial sputum medium (ASMDM) that more closely resembles the composition of CF sputum than current media. In order to validate the utility of ASMDM, we used GeneChip microarrays to compare expression data of P. aeruginosa UCBPP-PA14 (PA14) in ASMDM with published data for this strain grown under the same conditions in an artificial medium containing 10% (v/v) CF sputum. Thirty-seven of 39 nutrition-related genes were differentially expressed in the same manner in both media. However, 24 quorum-sensing (QS) genes, 23 Type III secretion system and several anaerobic respiration genes were more highly expressed in ASMDM than in sputum-containing medium. When grown to stationary phase in ASMDM, PA14 differentially expressed about 50 biologically significant genes compared to stationary phase growth in Luria Broth; genes involved in iron acquisition (pfeA, fepC) and in assimilatory nitrate reduction (nasC, nirD) were upregulated, while 24 QS genes, including the regulator rhlR, lasA, rsaL, aprADEI and phenazine genes phzC2DD2EG2 were downregulated. Downregulation of QS-regulated virulence genes has been noted in chronic P. aeruginosa infection. ASMDM thus appears highly suitable for studies on gene expression of (i) P. aeruginosa strains from acutely and chronically infected CF patients and (ii) established biofilms that are a hallmark of advanced CF lung disease. PA14 was grown in four different ways: 1. Logarithmic growth for early gene expression Cells were grown in MOPS-Glucose and separately in ASMDM. The average of two biological duplicates in each case was compared to the other to determine differential gene expression. 2. Stationary phase growth for gene expression Cells were grown in Luria Broth and separately in ASMDM. The average of two biological duplicates in each case was compared to the other to determine differential gene expression.

Project description:Biofilms are commonly recalcitrant to antibiotics, through incompletely elucidated mechanisms such as tolerance and persistence. We aimed at investigating how a Pseudomonas aeruginosa biofilm escapes ciprofloxacin treatment. P. aeruginosa PA14 in vitro mature biofilms were challenged with supra-MIC ciprofloxacin concentrations. Cell viability was quantified by fluorescein diacetate assay. Population dynamics were determined by counts of surviving culturable cells. Biofilms were analyzed using confocal laser scanning microscopy (CLSM), and the expression of genes involved in stringent response, toxin-antitoxin HigB/HigA, and type 3 secretion system (T3SS) was quantified by RT-qPCR in untreated and treated biofilms. Ciprofloxacin exposure resulted in an initial reduction of bacterial counts following a biphasic time-kill curve. After 24 h of treatment, the overall cell activity and the density of culturable cells significantly decreased as compared to untreated biofilm. No resistant mutant was isolated among the <1% surviving cells. Phenotypic adaptation toward persistence appeared to start after only 1 h of antibiotic exposure, by an overexpression of the genes involved in stringent response and in the toxin-antitoxin system, whereas the expression of genes encoding for the T3SS remained unchanged. After 4 h of ciprofloxacin exposure, stringent response genes returned to their basal level of expression. After a prolonged ciprofloxacin exposure, a deep alteration in the matrix structure that became thinner and lost mushroom-like aggregates was observed, in relation with reduced biovolumes of exopolysaccharides and extracellular DNA. These results support that ciprofloxacin might first induce the bacterial killing of most bacterial cells, but simultaneously activate stringent response mechanisms contributing to the switch of a subpopulation toward a persister phenotype. Once the persister phenotype is expressed, and despite an unexpected alteration of the biofilm matrix, ciprofloxacin fails to eradicate biofilm.

Dataset Information

Modelling experimentally measured of ciprofloxacin antibiotic diffusion in Pseudomonas aeruginosa biofilm formed in artificial sputum medium.

Publications

Modelling experimentally measured of ciprofloxacin antibiotic diffusion in Pseudomonas aeruginosa biofilm formed in artificial sputum medium.

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