Anti-Persisters Activity of Lacticaseibacillus rhamnosus Culture Filtrates against Pseudomonas aeruginosa in Artificial Sputum Medium
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PROVIDER: S-EPMC11241021 | biostudies-literature | 2024 Jun
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC7714214 | biostudies-literature
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.
2010-05-31 | GSE18594 | GEO
Project description:The PANarray design (GPL13324) contains the genes of eight P. aeruginosa genomes in non-redundant format, thus allowing identification of expression of non-PAO1 and other P. aeruginosa genes.
2011-07-16 | GSE28152 | GEO
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.
2010-06-08 | E-GEOD-18594 | biostudies-arrayexpress
Project description:The PANarray design (GPL13324) contains the genes of eight P. aeruginosa genomes in non-redundant format, thus allowing identification of expression of non-PAO1 and other P. aeruginosa genes. For the series GSE28152, isogenic isolates were sequentially collected from two cystic fibrosis (CF) patients several years apart. The isolates had not been eradicated in the meantime and represent persister strains. One was an Australian Epidemic Strain-1 isolate and the other a non-epidemic strain. Strains were cultured in an artificial sputum medium (ASMDM) closely resembling CF sputum.
2011-07-15 | E-GEOD-28152 | biostudies-arrayexpress
Project description:Rapid detection of tuberculosis (TB) infection is paramount to curb further transmission. The gold standard for this remains mycobacterial culture, however emerging evidence confirms the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens. These bacteria do not grow under standard culture conditions and require the presence of culture filtrate (CF), from axenic cultures of Mycobacterium tuberculosis (Mtb), to emerge. It has been hypothesized that molecules such as resuscitation promoting factors (Rpfs), fatty acids and cyclic-AMP (cAMP) present in CF are responsible for the growth stimulatory activity. Herein, we tested the ability of CF from the non-pathogenic bacterium Mycobacterium smegmatis (Msm) to stimulate the growth of DCTB, as this organism provides a more tractable source of CF. We also interrogated the role of Mtb Rpfs in stimulation of DCTB by creating recombinant strains of Msm that express Mtb rpf genes in various combinations. CF derived from this panel of strains was tested on sputum from individuals with drug susceptible TB prior to treatment. CF from wild type Msm did not enable detection of DCTB in a manner akin to Mtb CF preparations and whilst the addition of RpfABMtb and RpfABCDEMtb to an Msm mutant devoid of its native rpfs did improve detection of DCTB compared to the no CF control, it was not statistically different to the empty vector control. To further investigate the role of Rpfs, we compared the growth stimulatory activity of CF from Mtb, with and without Rpfs and found these to be equivalent. Next, we tested chemically diverse fatty acids and cAMP for growth stimulation and whilst some selective stimulatory effect was observed, this was not significantly higher than the media control and not comparable to CF. Together, these data indicate that the growth stimulatory effect observed with Mtb CF is most likely the result of a combination of factors. Future work aimed at identifying the nature of these growth stimulatory molecules may facilitate improvement of culture-based diagnostics for TB.
| S-EPMC7985135 | biostudies-literature
Project description:Research into the cooperative pathogenicity of microbes in cystic fibrosis (CF) lungs is crucial for an understanding of the pathophysiology of infections and the development of novel treatment strategies. This study investigated the impact of the common CF-associated bacterial pathogen Pseudomonas aeruginosa on the black yeast Exophiala dermatitidis. It evaluated the planktonic growth, biofilm formation, morphology, and virulence of the fungus in the presence or absence of P. aeruginosa. It also determined the role of P. aeruginosa quorum-sensing (QS) molecules within these interactions, e.g., by using sterile culture filtrate and QS-deficient mutants. P. aeruginosa is known to inhibit the planktonic growth of E. dermatitidis. We found that fungal biofilm formation increased in the presence of P. aeruginosa after 24 h but is decreased significantly after 48 h. This effect was reversed when, instead of QS wild-type strains, ΔlasR, and ΔrhlR mutants were added to E. dermatitidis biofilm formation. The number and length of hyphae were substantially reduced when E. dermatitidis was co-cultivated with P. aeruginosa, but not when it was co-cultivated with the mutants. Experiments testing the virulence of E. dermatitidis in the greater wax moth Galleria mellonella showed a synergetic effect on larval killing when E. dermatitidis was injected together with P. aeruginosa culture filtrate. Survival rates were decreased when biofilm culture filtrate was added but not when planktonic culture filtrate was added. In summary, P. aeruginosa affects the growth, morphology, biofilm formation, and virulence of E. dermatitidis. N-acyl-L-homoserine lactone (AHL) QS molecules regulated factors that have been shown to contribute to the inhibition of the ability of E. dermatitidis to form filaments and biofilm.
| S-EPMC7100538 | biostudies-literature
Project description:Pseudomonas aeruginosa can cause major infection in immunocompromised patients, and successful antibiotic treatment of the infection relies on accurate and rapid identification of the infectious agents. Here, we reported a culture-free diagnostic method based on the surface-enhanced Raman spectroscopy (SERS) of pyocyanin (PCN), a major biomarker of P. aeruginosa. This platform can detect PCN as low as 5 ppb or 2.38 × 10(-8) mol L(-1) in both aqueous solutions and spiked clinical sputum samples. It has also been used to dynamically monitor the excretion of PCN by P. aeruginosa during its growth. The presence of PCN has been detected by SERS in 15 clinical sputum samples, which indicates P. aeruginosa infection, with 95.6% sensitivity and 93.3% specificity. The system can advantageously process multiple specimens rapidly, overcomes the need for bacterial culture and diagnostic microbiology assays, and have widespread implications in the early detection of P. aeruginosa infection.From the clinical editorA surface enhanced Raman spectroscopy method optimized for the detection of P. aureginosa infections is presented in this paper. The presence of pyocyanin, a marker of this bacterium has been detected in 15 clinical sputum samples utilizing this method. A sensitivity of 95.6% and 93.3% specificity was reported, which suggests that the method may enable culture-free high throughput rapid detection of this infection.
| S-EPMC4232485 | biostudies-literature
Project description:The presence of Pseudomonas aeruginosa biofilms in cystic fibrosis (CF) patients suffering from chronic lung infections contributes to the failure of antimicrobial therapy. Conventionally, the minimal inhibitory concentration (MIC) is determined to assess the antimicrobial susceptibility of a pathogen, however this parameter fails to predict success in treating biofilm-associated infections. In the present study we developed a high throughput method to determine the antimicrobial concentration required to prevent P. aeruginosa biofilm formation, using a synthetic cystic fibrosis sputum medium (SCFM2). Biofilms were grown in SCFM2 for 24 h in the presence of antibiotics (tobramycin, ciprofloxacin or colistin), whereafter biofilms were disrupted and a resazurin staining was used to quantify the number of surviving metabolically active cells. In parallel, the content of all wells was plated to determine the number of colony forming units (CFU). Biofilm preventing concentrations (BPCs) were compared to MICs and minimal bactericidal concentrations (MBCs) determined according to EUCAST guidelines. Correlations between the resazurin-derived fluorescence and CFU counts were assessed with Kendall’s Tau Rank tests. A significant correlation between fluorescence and CFU counts was observed for 9 out of 10 strains investigated, suggesting the fluorometric assay is a reliable alternative to plating for most P. aeruginosa isolates to determine biofilm susceptibility in relevant conditions. For all isolates a clear difference between MICs and BPCs of all three antibiotics was observed, with the BPCs being consistently higher than the MICs. Additionally, the extent of this difference appeared to be antibiotic-dependent. Our findings suggest that this high throughput assay could be a valuable addition to evaluate the antimicrobial susceptibility in P. aeruginosa biofilms in the context of CF.
| S-EPMC9945637 | biostudies-literature
Project description:Defining the essential genome of bacterial pathogens is central to developing an understanding of the biological processes controlling disease. This has proven elusive for Pseudomonas aeruginosa during chronic infection of the cystic fibrosis (CF) lung. In this paper, using a Monte Carlo simulation-based method to analyze high-throughput transposon sequencing data, we establish the P. aeruginosa essential genome with statistical precision in laboratory media and CF sputum. Reconstruction of the global requirements for growth in CF sputum compared with defined growth conditions shows that the latter requires several cofactors including biotin, riboflavin, and pantothenate. Comparison of P. aeruginosa strains PAO1 and PA14 demonstrates that essential genes are primarily restricted to the core genome; however, some orthologous genes in these strains exhibit differential essentiality. These results indicate that genes with similar molecular functions may have distinct genetic roles in different P. aeruginosa strains during growth in CF sputum. We also show that growth in a defined growth medium developed to mimic CF sputum yielded virtually identical fitness requirements to CF sputum, providing support for this medium as a relevant in vitro model for CF microbiology studies.
| S-EPMC4386324 | biostudies-literature