scholarly journals Spatially resolved analysis of Pseudomonas aeruginosa biofilm proteomes measured by laser ablation sample transfer

2021 ◽  
Author(s):  
Aruni Chathurya Pulukkody ◽  
Yeni P. Yung ◽  
Fabrizio Donnarumma ◽  
Kermit K. Murray ◽  
Ross P. Carlson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laser Ablation ◽  
Polyamine Metabolism ◽  
Label Free ◽  
Biofilm Growth ◽  
Chemical Gradients ◽  
Spatially Resolved ◽  
Spatial Differences ◽  
Biofilm Development ◽  
Sample Transfer

AbstractHeterogeneity in the distribution of nutrients and O2 gradients during biofilm growth gives rise to changes in phenotype. There has been long term interest in identifying spatial differences during biofilm development including clues that identify chemical heterogeneity. Laser ablation sample transfer (LAST) allows site-specific sampling combined with label free proteomics to distinguish radially and axially resolved proteomes for Pseudomonas aeruginosa biofilms. Specifically, differential protein abundances on oxic vs. anoxic regions of a biofilm was observed by combining LAST with bottom up proteomics. This study reveals active metabolism in the anoxic region of the biofilm with respect to the oxic region in P. aeruginosa, an aerobe by nature. Protein abundance data related to cellular acclimations to chemical gradients include identification of glucose catabolizing proteins, high abundance of proteins from arginine and polyamine metabolism, and proteins that could also support virulence and environmental stress mediation on the anoxic region. Finally, this methodology requires only a few mm2 of biofilm area to identify hundreds of proteins.

scholarly journals Spatially resolved analysis of Pseudomonas aeruginosa biofilm proteomes measured by laser ablation sample transfer

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0250911
Author(s):  
Aruni Chathurya Pulukkody ◽  
Yeni P. Yung ◽  
Fabrizio Donnarumma ◽  
Kermit K. Murray ◽  
Ross P. Carlson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laser Ablation ◽  
Polyamine Metabolism ◽  
Clinical Strain ◽  
Label Free ◽  
Biofilm Growth ◽  
Chemical Gradients ◽  
Spatially Resolved ◽  
Biofilm Development ◽  
Sample Transfer

Heterogeneity in the distribution of nutrients and oxygen gradients during biofilm growth gives rise to changes in phenotype. There has been long term interest in identifying spatial differences during biofilm development including clues that identify chemical heterogeneity. Laser ablation sample transfer (LAST) allows site-specific sampling combined with label free proteomics to distinguish radially and axially resolved proteomes for Pseudomonas aeruginosa biofilms. Specifically, differential protein abundances on oxic vs. anoxic regions of a biofilm were observed by combining LAST with bottom up proteomics. This study reveals a more active metabolism in the anoxic region of the biofilm with respect to the oxic region for this clinical strain of P. aeruginosa, despite this organism being considered an aerobe by nature. Protein abundance data related to cellular acclimations to chemical gradients include identification of glucose catabolizing proteins, high abundance of proteins from arginine and polyamine metabolism, and proteins that could also support virulence and environmental stress mediation in the anoxic region. Finally, the LAST methodology requires only a few mm2 of biofilm area to identify hundreds of proteins.

scholarly journals Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

2002 ◽  
Vol 184 (4) ◽  
pp. 1140-1154 ◽  
Author(s):  
Karin Sauer ◽  
Anne K. Camper ◽  
Garth D. Ehrlich ◽  
J. William Costerton ◽  
David G. Davies
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Protein Identification ◽  
Electrophoretic Analysis ◽  
Cell Protein ◽  
Biofilm Growth ◽  
Alginate Production ◽  
Multiple Phenotypes ◽  
Reporter Gene Analysis ◽  
Biofilm Development

ABSTRACT Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.

scholarly journals An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jeanyoung Jo ◽  
Krista L Cortez ◽  
William Cole Cornell ◽  
Alexa Price-Whelan ◽  
Lars EP Dietrich
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cytochrome C ◽  
Opportunistic Pathogen ◽  
Bacterial Pathogenesis ◽  
Cytochrome Oxidase Subunit ◽  
Biofilm Growth ◽  
Unique Role ◽  
High Affinity ◽  
Biofilm Model ◽  
Biofilm Development

Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e. ‘orphan’) operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen Pseudomonas aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets.

An FTIR study of Pseudomonas aeruginosa PAO1 biofilm growth and dispersion. An improved ATR method for studying biofilms: the C–H stretch spectral region

Biofilms ◽  
2004 ◽  
Vol 1 (3) ◽  
pp. 157-163 ◽  
Author(s):  
J. Pink ◽  
T. Smith-Palmer ◽  
T. J. Beveridge ◽  
D. A. Pink
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Subsequent Development ◽  
Growth Period ◽  
Flow Cell ◽  
Biofilm Growth ◽  
Chemical Difference ◽  
Qualitative And Quantitative ◽  
Quantitative Changes ◽  
Biofilm Development ◽  
First Time

Pseudomonas aeruginosa PAO1 was grown on 40° ZnSe and 40° AMTIR® (Amorphous Material Transmitting Infrared Radiation, Ge33As12Se55) internal reflection elements in a thermostatted flow cell. Information about the chemistry of the biofilm development was obtained using Fourier transform infrared spectroscopy. The weakly absorbing C–H stretching region of the infrared spectrum was measured for the first time and revealed qualitative and quantitative changes not evident in the amide region studied by previous investigators. There is a chemical difference between the early growth period and subsequent development of the biofilm.

scholarly journals Multidrug Efflux Pumps: Expression Patterns and Contribution to Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

2001 ◽  
Vol 45 (6) ◽  
pp. 1761-1770 ◽  
Author(s):  
Teresa R. De Kievit ◽  
Michael D. Parkins ◽  
Richard J. Gillis ◽  
Ramakrishnan Srikumar ◽  
Howard Ceri ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Agents ◽  
Expression Patterns ◽  
Maximal Activity ◽  
Efflux Pumps ◽  
Active Efflux ◽  
Expression Of Genes ◽  
Multidrug Efflux Pumps ◽  
Spatial Differences ◽  
Biofilm Development

ABSTRACT Pseudomonas aeruginosa biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance ofP. aeruginosa biofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexA andmexC in the developing biofilm. Remarkably, expression ofmexAB-oprM and mexCD-oprJ was not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprM andmexCD-oprJ expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosa biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosa biofilms.

scholarly journals The Pseudomonas aeruginosa CreBC Two-Component System Plays a Major Role in the Response to β-Lactams, Fitness, Biofilm Growth, and Global Regulation

2014 ◽  
Vol 58 (9) ◽  
pp. 5084-5095 ◽  
Author(s):  
Laura Zamorano ◽  
Bartolomé Moyà ◽  
Carlos Juan ◽  
Xavier Mulet ◽  
Jesús Blázquez ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Anaerobic Respiration ◽  
Biofilm Growth ◽  
Content Type ◽  
Comprehensive Collection ◽  
Regulatory Systems ◽  
Two Component ◽  
Life Threatening ◽  
Inner Membrane Protein ◽  
Biofilm Development

ABSTRACTPseudomonas aeruginosais a ubiquitous versatile environmental microorganism with a remarkable ability to grow under diverse environmental conditions. Moreover,P. aeruginosais responsible for life-threatening infections in immunocompromised and cystic fibrosis patients, as the extraordinary capacity of this pathogen to develop antimicrobial resistance dramatically limits our therapeutic arsenal. Its large genome carries an outstanding number of genes belonging to regulatory systems, including multiple two-component sensor-regulator systems that modulate the response to the different environmental stimuli. Here, we show that one of two systems, designated CreBC (carbon source responsive) and BlrAB (β-lactam resistance), might be of particular relevance. We first identified the stimuli triggering the activation of the CreBC system, which specifically responds to penicillin-binding protein 4 (PBP4) inhibition by certain β-lactam antibiotics. Second, through an analysis of a large comprehensive collection of mutants, we demonstrate an intricate interconnection between the CreBC system, the peptidoglycan recycling pathway, and the expression of the concerning chromosomal β-lactamase AmpC. Third, we show that the CreBC system, and particularly its effector inner membrane protein CreD, plays a major role in bacterial fitness and biofilm development, especially in the presence of subinhibitory concentrations of β-lactams. Finally, global transcriptomics reveals broad regulatory functions of CreBC in basic physiological aspects, particularly anaerobic respiration, in both the presence and absence of antibiotics. Therefore, the CreBC system is envisaged as a potentially interesting target for improving the efficacy of β-lactams againstP. aeruginosainfections.

scholarly journals Timing and Localization of Rhamnolipid Synthesis Gene Expression in Pseudomonas aeruginosa Biofilms

2005 ◽  
Vol 187 (1) ◽  
pp. 37-44 ◽  
Author(s):  
Yannick Lequette ◽  
E. P. Greenberg
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Promoter Activity ◽  
Fluorescent Dyes ◽  
Homoserine Lactone ◽  
Biofilm Growth ◽  
Neutral Site ◽  
Biofilm Architecture ◽  
Biofilm Development

ABSTRACT Pseudomonas aeruginosa biofilms can develop mushroom-like structures with stalks and caps consisting of discrete subpopulations of cells. Self-produced rhamnolipid surfactants have been shown to be important in development of the mushroom-like structures. The quorum-sensing-controlled rhlAB operon is required for rhamnolipid synthesis. We have introduced an rhlA-gfp fusion into a neutral site in the P. aeruginosa genome to study rhlAB promoter activity in rhamnolipid-producing biofilms. Expression of the rhlA-gfp fusion in biofilms requires the quorum-sensing signal butanoyl-homoserine lactone, but other factors are also required for expression. Early in biofilm development rhlA-gfp expression is low, even in the presence of added butanoyl-homoserine lactone. Expression of the fusion becomes apparent after microcolonies with a depth of >20 μm have formed and, as shown by differential labeling with rfp or fluorescent dyes, rhlA-gfp is preferentially expressed in the stalks rather than the caps of mature mushrooms. The rhlA-gfp expression pattern is not greatly influenced by addition of butanoyl-homoserine lactone to the biofilm growth medium. We propose that rhamnolipid synthesis occurs in biofilms after stalks have formed but prior to capping in the mushroom-like structures. The differential expression of rhlAB may play a role in the development of normal biofilm architecture.

Bifunctional Composites for Biofilms Modulation on Cervical Restorations

2021 ◽  
pp. 002203452110181
Author(s):  
A.A. Balhaddad ◽  
I.M. Garcia ◽  
L. Mokeem ◽  
M.S. Ibrahim ◽  
F.M. Collares ◽  
...  
Keyword(s):  
Free Energy ◽  
Surface Free Energy ◽  
Contact Angles ◽  
Bacterial Biofilm ◽  
Dental Composite ◽  
Log P ◽  
Rrna Gene ◽  
Cervical Lesions ◽  
Biofilm Growth ◽  
Biofilm Development

Cervical composites treating root carious and noncarious cervical lesions usually extend subgingivally. The subgingival margins of composites present poor plaque control, enhanced biofilm accumulation, and cause gingival irritation. A potential material to restore such lesions should combine agents that interfere with bacterial biofilm development and respond to acidic conditions. Here, we explore the use of new bioresponsive bifunctional dental composites against mature microcosm biofilms derived from subgingival plaque samples. The designed formulations contain 2 bioactive agents: dimethylaminohexadecyl methacrylate (DMAHDM) at 3 to 5 wt.% and 20 wt.% nanosized amorphous calcium phosphate (NACP) in a base resin. Composites with no DMAHDM and NACP were used as controls. The newly formulated 5% DMAHDM–20% NACP composite was analyzed by micro-Raman spectroscopy and transmission electron microscopy. The wettability and surface-free energy were also assessed. The inhibitory effect on the in vitro biofilm growth and the 16S rRNA gene sequencing of survival bacterial colonies derived from the composites were analyzed. Whole-biofilm metabolic activity, polysaccharide production, and live/dead images of the biofilm grown over the composites complement the microbiological assays. Overall, the designed formulations had higher contact angles with water and lower surface-free energy compared to the commercial control. The DMAHDM-NACP composites significantly inhibited the growth of total microorganisms, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum by 3 to 5-log ( P < 0.001). For the colony isolates from control composites, the composition was typically dominated by the genera Veillonella, Fusobacterium, Streptococcus, Eikenella, and Leptotrichia, while Fusobacterium and Veillonella dominated the 5% DMAHDM–20% NACP composites. The DMAHDM-NACP composites contributed to over 80% of reduction in metabolic and polysaccharide activity. The suppression effect on plaque biofilms suggested that DMAHDM-NACP composites might be used as a bioactive material for cervical restorations. These results may propose an exciting path to prevent biofilm growth and improve dental composite restorations’ life span.

scholarly journals An In Vitro Coumarin-Antibiotic Combination Treatment of Pseudomonas aeruginosa Biofilms

2021 ◽  
Vol 16 (1) ◽  
pp. 1934578X2098774
Author(s):  
Jinpeng Zou ◽  
Yang Liu ◽  
Ruiwei Guo ◽  
Yu Tang ◽  
Zhengrong Shi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Resistance ◽  
Combination Treatment ◽  
Crude Extract ◽  
Biofilm Formation ◽  
Bacterial Resistance ◽  
Antibacterial Properties ◽  
Biofilm Growth ◽  
Antibiotic Combination

The drug resistance of Pseudomonas aeruginosa is a worldwide problem due to its great threat to human health. A crude extract of Angelica dahurica has been proved to have antibacterial properties, which suggested that it may be able to inhibit the biofilm formation of P. aeruginosa; initial exploration had shown that the crude extract could inhibit the growth of P. aeruginosa effectively. After the adaptive dose of coumarin was confirmed to be a potential treatment for the bacteria’s drug resistance, “coumarin-antibiotic combination treatments” (3 coumarins—simple coumarin, imperatorin, and isoimperatorin—combined with 2 antibiotics—ampicillin and ceftazidime) were examined to determine their capability to inhibit P. aeruginosa. The final results showed that (1) coumarin with either ampicillin or ceftazidime significantly inhibited the biofilm formation of P. aeruginosa; (2) coumarin could directly destroy mature biofilms; and (3) the combination treatment can synergistically enhance the inhibition of biofilm formation, which could significantly reduce the usage of antibiotics and bacterial resistance. To sum up, a coumarin-antibiotic combination treatment may be a potential way to inhibit the biofilm growth of P. aeruginosa and provides a reference for antibiotic resistance treatment.

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