scholarly journals Building a better biofilm - formation of in vivo-like biofilm structures by Pseudomonas aeruginosa in a porcine model of cystic fibrosis lung infection

2019 ◽  
Author(s):  
Niamh E. Harrington ◽  
Esther Sweeney ◽  
Freya Harrison
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Ex Vivo ◽  
Lung Infection ◽  
Transposon Insertion ◽  
Prevention Methods ◽  
Biofilm Infection

AbstractPseudomonas aeruginosa biofilm infections in the cystic fibrosis (CF) lung are highly resistant to current antimicrobial treatments and are associated with increased mortality rates. The existing models for such infections are not able to reliably mimic the clinical biofilms observed. We aimed to further optimise an ex vivo pig lung (EVPL) model for P. aeruginosa CF lung infection that can be used to increase understanding of chronic CF biofilm infection. The EVPL model will facilitate discovery of novel infection prevention methods and treatments, and enhanced exploration of biofilm architecture. We investigated purine metabolism and biofilm formation in the model using transposon insertion mutants in P. aeruginosa PA14 for key genes: purD, gacA and pelA. Our results demonstrate that EVPL recapitulates a key aspect of in vivo P. aeruginosa infection metabolism, and that the pathogen forms a biofilm with a clinically realistic structure not seen in other in vitro studies. Two pathways known to be required for in vivo biofilm infection - the Gac regulatory pathway and production of the Pel exopolysaccharide - are essential to the formation of this mature, structured biofilm on EVPL tissue. We propose the high-throughput EVPL model as a validated biofilm platform to bridge the gap between in vitro and CF lung infection.

scholarly journals Synergistic Activity of Berberine with Azithromycin against Pseudomonas Aeruginosa Isolated from Patients with Cystic Fibrosis of Lung In Vitro and In Vivo

2017 ◽  
Vol 42 (4) ◽  
pp. 1657-1669 ◽  
Author(s):  
YongTao Li ◽  
JianRong Huang ◽  
LanJuan Li ◽  
LinSheng Liu
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lung Infection ◽  
Extracellular Dna ◽  
Pcr Analysis ◽  
Infection Model ◽  
Synergistic Activity ◽  
Time Kill

Background/Aims: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. Methods: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. Results: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. Conclusion: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.

scholarly journals Hyperencapsulated mucoid pneumococcal isolates from patients with cystic fibrosis have increased biofilm density and persistence in vivo

2018 ◽  
Vol 76 (7) ◽  
Author(s):  
Evida A Dennis ◽  
Mamie T Coats ◽  
Sarah Griffin ◽  
Bing Pang ◽  
David E Briles ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Continuous Flow ◽  
Biofilm Formation ◽  
Capsular Polysaccharides ◽  
Wild Type ◽  
Pneumococcal Colonization ◽  
Phase Variants

AbstractMucoid bacteria, predominately Pseudomonas aeruginosa, are commonly associated with decline in pulmonary function in children with cystic fibrosis (CF), and are thought to persist at least in part due to a greater propensity toward forming biofilms. We isolated a higher frequency of mucoid Streptococcus pneumoniae (Sp) expressing high levels of capsular polysaccharides from sputa from children with CF, compared to those without CF. We compared biofilm formation and maturation by mucoid and non-mucoid isolates of Sp collected from children with and without CF. Non-mucoid Sp serotype 19A and 19F isolates had significantly higher levels of biofilm initiation and adherence to CF epithelial cells than did serotype 3 isolates. However, strains expressing high levels of capsule had significantly greater biofilm maturation, as evidenced by increased density and thickness in static and continuous flow assays via confocal microscopy. Finally, using a serotype 3 Sp strain, we showed that highly encapsulated mucoid phase variants predominate during late adherence and better colonize CFTR–/– as compared to wild-type mice in respiratory infection studies. These findings indicate that overexpression of capsule can enhance the development of mature pneumococcal biofilms in vitro, and may contribute to pneumococcal colonization in CF lung disease.

scholarly journals Transcriptome analysis of Pseudomonas aeruginosa biofilm infection in an ex vivo pig model of the cystic fibrosis lung.

2021 ◽  
Author(s):  
Niamh E. Harrington ◽  
Jenny L. Littler ◽  
Freya Harrison
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Resistance ◽  
Transcriptome Analysis ◽  
Ex Vivo ◽  
Clinical Diagnostics ◽  
Growth Environment ◽  
Key Aspects ◽  
Biofilm Infection

Pseudomonas aeruginosa is the predominant cause of chronic biofilm infections that form in the lungs of people with cystic fibrosis (CF). These infections are highly resistant to antibiotics and persist for years in the respiratory tract. One of the main research challenges is that current laboratory models do not accurately replicate key aspects of a P. aeruginosa biofilm infection, highlighted by previous RNA-sequencing studies. We compared the P. aeruginosa PA14 transcriptome in an ex vivo pig lung (EVPL) model of CF and a well-studied synthetic cystic fibrosis sputum medium (SCFM). P. aeruginosa was grown in the EVPL model for 1, 2 and 7 days, and in vitro in SCFM for 1 and 2 days. The RNA was extracted and sequenced at each time point. Our findings demonstrate that expression of antimicrobial resistance genes was cued by growth in the EVPL model, highlighting the importance of growth environment in determining accurate resistance profiles. The EVPL model created two distinct growth environments: tissue-associated biofilm and the SCFM surrounding tissue, each cued a transcriptome distinct from that seen in SCFM in vitro . The expression of quorum sensing associated genes in the EVPL tissue-associated biofilm at 48 h relative to in vitro SCFM was similar to CF sputum versus in vitro conditions. Hence, the EVPL model can replicate key aspects of in vivo biofilm infection that are missing from other current models. It provides a more accurate P. aeruginosa growth environment for determining antimicrobial resistance that quickly drives P. aeruginosa into a chronic-like infection phenotype. Importance Pseudomonas aeruginosa lung infections that affect people with cystic fibrosis are resistant to most available antimicrobial treatments. The lack of a laboratory model that captures all key aspects of these infections hinders not only research progression but also clinical diagnostics. We used transcriptome analysis to demonstrate how a model using pig lungs can more accurately replicate key characteristics of P. aeruginosa lung infection, including mechanisms of antibiotic resistance and infection establishment. Therefore, this model may be used in the future to further understand infection dynamics to develop novel treatments and more accurate treatment plans. This could improve clinical outcomes as well as quality of life for individuals affected by these infections.

scholarly journals Transcriptome analysis of Pseudomonas aeruginosa biofilm infection in an ex vivo pig model of the cystic fibrosis lung

2021 ◽  
Author(s):  
Niamh E Harrington ◽  
Jenny L Littler ◽  
Freya Harrison
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Resistance ◽  
Ex Vivo ◽  
Surrounding Tissue ◽  
Growth Environment ◽  
Antimicrobial Resistance Genes ◽  
Key Aspects ◽  
Biofilm Infection

Pseudomonas aeruginosa is the predominant cause of chronic biofilm infections that form in the lungs of people with cystic fibrosis (CF). These infections are highly resistant to antibiotics and persist for years in the respiratory tract. One of the main research challenges is that current laboratory models do not accurately replicate key aspects of a chronic P. aeruginosa biofilm infection, highlighted by previous RNA-sequencing studies. We compared the P. aeruginosa PA14 transcriptome in an ex vivo pig lung (EVPL) model of CF and a well-studied synthetic cystic fibrosis sputum medium (SCFM). P. aeruginosa was grown in the EVPL model for 1, 2 and 7 days, and in vitro in SCFM for 1 and 2 days. The RNA was extracted and sequenced at each time point. Our findings demonstrate that expression of antimicrobial resistance genes was cued by growth in the EVPL model, highlighting the importance of growth environment in determining accurate resistance profiles. The EVPL model created two distinct growth environments: tissue-associated biofilm and the SCFM surrounding tissue, each of which cued a transcriptome distinct from that seen in SCFM in vitro. The expression of quorum sensing associated genes in the EVPL tissue-associated biofilm at 48 h relative to in vitro SCFM was found to be similar to CF sputum versus in vitro conditions. Hence, the EVPL model can replicate key aspects of in vivo biofilm infection that are missing from other current models and provides a more accurate P. aeruginosa growth environment for determining antimicrobial resistance.

scholarly journals Type-4 Phosphodiesterase (PDE4) Blockade Reduces NETosis in Cystic Fibrosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Licia Totani ◽  
Concetta Amore ◽  
Antonio Piccoli ◽  
Giuseppe Dell’Elba ◽  
Angelo Di Santo ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lung Disease ◽  
Pde4 Inhibitor ◽  
Free Dna ◽  
Impaired Lung Function ◽  
Cellular Integrity ◽  
Net Release

Neutrophilic inflammation is a key determinant of cystic fibrosis (CF) lung disease. Neutrophil-derived free DNA, released in the form of extracellular traps (NETs), significantly correlates with impaired lung function in patients with CF, underlying their pathogenetic role in CF lung disease. Thus, specific approaches to control NETosis of neutrophils migrated into the lungs may be clinically relevant in CF. We investigated the efficacy of phosphodiesterase (PDE) type-4 inhibitors, in vitro, on NET release by neutrophils from healthy volunteers and individuals with CF, and in vivo, on NET accumulation and lung inflammation in mice infected with Pseudomonas aeruginosa. PDE4 blockade curbed endotoxin-induced NET production and preserved cellular integrity and apoptosis in neutrophils, from healthy subjects and patients with CF, challenged with endotoxin, in vitro. The pharmacological effects of PDE4 inhibitors were significantly more evident on CF neutrophils. In a mouse model of Pseudomonas aeruginosa chronic infection, aerosol treatment with roflumilast, a selective PDE4 inhibitor, gave a significant reduction in free DNA in the BALF. This was accompanied by reduced citrullination of histone H3 in neutrophils migrated into the airways. Roflumilast-treated mice showed a significant improvement in weight recovery. Our study provides the first evidence that PDE4 blockade controls NETosis in vitro and in vivo, in CF-relevant models. Since selective PDE4 inhibitors have been recently approved for the treatment of COPD and psoriasis, our present results encourage clinical trials to test the efficacy of this class of drugs in CF.

scholarly journals Identification of gmhA, a Yersinia pestis Gene Required for Flea Blockage, by Using a Caenorhabditis elegans Biofilm System

2005 ◽  
Vol 73 (11) ◽  
pp. 7236-7242 ◽  
Author(s):  
Creg Darby ◽  
Sandya L. Ananth ◽  
Li Tan ◽  
B. Joseph Hinnebusch
Keyword(s):  
Caenorhabditis Elegans ◽  
Yersinia Pestis ◽  
Biofilm Formation ◽  
Yersinia Pseudotuberculosis ◽  
C Elegans ◽  
Transposon Insertion ◽  
Insertion Mutants ◽  
Random Screening

ABSTRACT Yersinia pestis, the cause of bubonic plague, blocks feeding by its vector, the flea. Recent evidence indicates that blockage is mediated by an in vivo biofilm. Y. pestis and the closely related Yersinia pseudotuberculosis also make biofilms on the cuticle of the nematode Caenorhabditis elegans, which block this laboratory animal's feeding. Random screening of Y. pseudotuberculosis transposon insertion mutants with a C. elegans biofilm assay identified gmhA as a gene required for normal biofilms. gmhA encodes phosphoheptose isomerase, an enzyme required for synthesis of heptose, a conserved component of lipopolysaccharide and lipooligosaccharide. A Y. pestis gmhA mutant was constructed and was severely defective for C. elegans biofilm formation and for flea blockage but only moderately defective in an in vitro biofilm assay. These results validate use of the C. elegans biofilm system to identify genes and pathways involved in Y. pestis flea blockage.

scholarly journals Adaptive resistance of Pseudomonas aeruginosa induced by aminoglycosides and killing kinetics in a rabbit endocarditis model.

1997 ◽  
Vol 41 (4) ◽  
pp. 823-826 ◽  
Author(s):  
Y Q Xiong ◽  
J Caillon ◽  
M F Kergueris ◽  
H Drugeon ◽  
D Baron ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Single Dose ◽  
Ex Vivo ◽  
Day Treatment ◽  
Total Daily Dose ◽  
Once Daily ◽  
Adaptive Resistance ◽  
Dosing Regimens

Adaptive resistance following the first exposure to aminoglycosides is a recently described in vitro phenomenon in Pseudomonas aeruginosa and other aerobic gram-negative bacilli. We investigated the in vivo relevance of adaptive resistance in P. aeruginosa following a single dose of amikacin in the experimental rabbit endocarditis model. Rabbits with P. aeruginosa endocarditis received either no therapy (control) or a single intravenous (i.v.) dose of amikacin (80 mg/kg of body weight) at 24 h postinfection, after which they were sacrificed at 5, 8, 12, 16, or 24 h postdose. Excised aortic vegetations were subsequently exposed ex vivo to amikacin at 2.5, 5, 10 or 20 times the MIC for 90 min. In vivo adaptive resistance was identified when amikacin-induced pseudomonal killing within excised aortic vegetations was less in animals receiving single-dose amikacin in vivo than in vegetations from control animals not receiving amikacin in vivo. Maximal adaptive resistance occurred between 8 and 16 h after the in vivo amikacin dose, with complete refractoriness to ex vivo killing by amikacin seen at 12 h postdose. By 24 h postdose, bacteria within excised vegetations had partially recovered their initial amikacin susceptibility. In a parallel treatment study, we demonstrated that amikacin given once daily (but not twice daily) at a total dose of 80 mg/kg i.v. for 1-day treatment significantly reduced pseudomonal densities within aortic vegetations versus those in untreated controls. When therapy was continued for 3 days with the same total daily dose (80 mg/kg/day), amikacin given once or twice daily significantly reduced intravegetation pseudomonal densities versus those in controls. However, amikacin given once daily was still more effective than the twice-daily regimen. These data confirm the induction of aminoglycoside adaptive resistance in vivo and further support the advantages of once-daily aminoglycoside dosing regimens in the treatment of serious pseudomonal infections.

scholarly journals Disruption of Cross-Feeding Inhibits Pathogen Growth in the Sputa of Patients with Cystic Fibrosis

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Jeffrey M. Flynn ◽  
Lydia C. Cameron ◽  
Talia D. Wiggen ◽  
Jordan M. Dunitz ◽  
William R. Harcombe ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Susceptibility ◽  
Susceptibility Testing ◽  
Anaerobic Bacteria ◽  
Content Type ◽  
Growth Environment ◽  
Polymicrobial Infections

ABSTRACT A critical limitation in the management of chronic polymicrobial infections is the lack of correlation between antibiotic susceptibility testing (AST) and patient responses to therapy. Underlying this disconnect is our inability to accurately recapitulate the in vivo environment and complex polymicrobial communities in vitro. However, emerging evidence suggests that, if modeled and tested accurately, interspecies relationships can be exploited by conventional antibiotics predicted to be ineffective by standard AST. As an example, under conditions where Pseudomonas aeruginosa relies on cocolonizing organisms for nutrients (i.e., cross-feeding), multidrug-resistant P. aeruginosa may be indirectly targeted by inhibiting the growth of its metabolic partners. While this has been shown in vitro using synthetic bacterial communities, the efficacy of a “weakest-link” approach to controlling host-associated polymicrobial infections has not yet been demonstrated. To test whether cross-feeding inhibition can be leveraged in clinically relevant contexts, we collected sputa from cystic fibrosis (CF) subjects and used enrichment culturing to isolate both P. aeruginosa and anaerobic bacteria from each sample. Predictably, both subpopulations showed various antibiotic susceptibilities when grown independently. However, when P. aeruginosa was cultured and treated under cooperative conditions in which it was dependent on anaerobic bacteria for nutrients, the growth of both the pathogen and the anaerobe was constrained despite their intrinsic antibiotic resistance profiles. These data demonstrate that the control of complex polymicrobial infections may be achieved by exploiting obligate or facultative interspecies relationships. Toward this end, in vitro susceptibility testing should evolve to more accurately reflect in vivo growth environments and microbial interactions found within them. IMPORTANCE Antibiotic efficacy achieved in vitro correlates poorly with clinical outcomes after treatment of chronic polymicrobial diseases; if a pathogen demonstrates susceptibility to a given antibiotic in the lab, that compound is often ineffective when administered clinically. Conversely, if a pathogen is resistant in vitro, patient treatment with that same compound can elicit a positive response. This discordance suggests that the in vivo growth environment impacts pathogen antibiotic susceptibility. Indeed, here we demonstrate that interspecies relationships among microbiotas in the sputa of cystic fibrosis patients can be targeted to indirectly inhibit the growth of Pseudomonas aeruginosa. The therapeutic implication is that control of chronic lung infections may be achieved by exploiting obligate or facultative relationships among airway bacterial community members. This strategy is particularly relevant for pathogens harboring intrinsic multidrug resistance and is broadly applicable to chronic polymicrobial airway, wound, and intra-abdominal infections.

scholarly journals Microbial Growth Inhibition by Alternating Electric Fields in Mice with Pseudomonas aeruginosa Lung Infection

2010 ◽  
Vol 54 (8) ◽  
pp. 3212-3218 ◽  
Author(s):  
Moshe Giladi ◽  
Yaara Porat ◽  
Alexandra Blatt ◽  
Esther Shmueli ◽  
Yoram Wasserman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Growth Inhibition ◽  
Electric Fields ◽  
Bacterial Growth ◽  
Lung Infection ◽  
Alternating Electric Fields ◽  
Microbial Growth Inhibition ◽  
Bacterial Growth Inhibition

ABSTRACT High-frequency, low-intensity electric fields generated by insulated electrodes have previously been shown to inhibit bacterial growth in vitro. In the present study, we tested the effect of these antimicrobial fields (AMFields) on the development of lung infection caused by Pseudomonas aeruginosa in mice. We demonstrate that AMFields (10 MHz) significantly inhibit bacterial growth in vivo, both as a stand-alone treatment and in combination with ceftazidime. In addition, we show that peripheral (skin) heating of about 2°C can contribute to bacterial growth inhibition in the lungs of mice. We suggest that the combination of alternating electric fields, together with the heat produced during their application, may serve as a novel antibacterial treatment modality.

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