scholarly journals EnhancedIn VitroFormation and Antibiotic Resistance of Nonattached Pseudomonas aeruginosa Aggregates through Incorporation of Neutrophil Products

2014 ◽  
Vol 58 (11) ◽  
pp. 6851-6860 ◽  
Author(s):  
Silvia M. Caceres ◽  
Kenneth C. Malcolm ◽  
Jennifer L. Taylor-Cousar ◽  
David P. Nichols ◽  
Milene T. Saavedra ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Quorum Sensing ◽  
Acquired Resistance ◽  
Biofilm Growth ◽  
Content Type ◽  
Viable Bacteria ◽  
And Function ◽  
Small Clusters

ABSTRACTPseudomonas aeruginosais a major pathogen in cystic fibrosis (CF) lung disease. Children with CF are routinely exposed toP. aeruginosafrom the natural environment, and by adulthood, 80% of patients are chronically infected.P. aeruginosain the CF airway exhibits a unique biofilm-like structure, where it grows in small clusters or aggregates of bacteria in association with abundant polymers of neutrophil-derived components F-actin and DNA, among other components. These aggregates differ substantially in size and appearance compared to surface-attachedin vitrobiofilm models classically utilized for studies but are believed to share properties of surface-attached biofilms, including antibiotic resistance. However, little is known about the formation and function of surface-independent modes of biofilm growth, how they might be eradicated, and quorum sensing communication. To address these issues, we developed a novelin vitromodel ofP. aeruginosaaggregates incorporating human neutrophil-derived products. Aggregates grownin vitroand those found in CF patients' sputum samples were morphologically similar; viable bacteria were distributed in small pockets throughout the aggregate. ThelasAquorum sensing gene was differentially expressed in the presence of neutrophil products. Importantly, aggregates formed in the presence of neutrophils acquired resistance to tobramycin, which was lost when the aggregates were dispersed with DNase, and antagonism of tobramycin and azithromycin was observed. This novel yet simplein vitrosystem advances our ability to model infection of the CF airway and will be an important tool to study virulence and test alternative eradication strategies againstP. aeruginosa.

scholarly journals Antibiotic Resistance Evolution Is Contingent on the Quorum-Sensing Response in Pseudomonas aeruginosa

2019 ◽  
Vol 36 (10) ◽  
pp. 2238-2251 ◽  
Author(s):  
Sara Hernando-Amado ◽  
Fernando Sanz-García ◽  
José Luis Martínez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Quorum Sensing ◽  
Wild Type ◽  
Resistance Mutations ◽  
Epistatic Interactions ◽  
Adaptive Mutations ◽  
Resistance Evolution ◽  
Evolutionary Trajectories

Abstract Different works have explored independently the evolution toward antibiotic resistance and the role of eco-adaptive mutations in the adaptation to a new habitat (as the infected host) of bacterial pathogens. However, knowledge about the connection between both processes is still limited. We address this issue by comparing the evolutionary trajectories toward antibiotic resistance of a Pseudomonas aeruginosa lasR defective mutant and its parental wild-type strain, when growing in presence of two ribosome-targeting antibiotics. Quorum-sensing lasR defective mutants are selected in P. aeruginosa populations causing chronic infections. Further, we observed they are also selected in vitro as a first adaptation for growing in culture medium. By using experimental evolution and whole-genome sequencing, we found that the evolutionary trajectories of P. aeruginosa in presence of these antibiotics are different in lasR defective and in wild-type backgrounds, both at the phenotypic and the genotypic levels. Recreation of a set of mutants in both genomic backgrounds (either wild type or lasR defective) allowed us to determine the existence of negative epistatic interactions between lasR and antibiotic resistance determinants. These epistatic interactions could lead to mutual contingency in the evolution of antibiotic resistance when P. aeruginosa colonizes a new habitat in presence of antibiotics. If lasR mutants are selected first, this would constraint antibiotic resistance evolution. Conversely, when resistance mutations (at least those studied in the present work) are selected, lasR mutants may not be selected in presence of antibiotics. These results underlie the importance of contingency and epistatic interactions in modulating antibiotic resistance evolution.

scholarly journals d-Amino Acids Enhance the Activity of Antimicrobials against Biofilms of Clinical Wound Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

2014 ◽  
Vol 58 (8) ◽  
pp. 4353-4361 ◽  
Author(s):  
Carlos J. Sanchez ◽  
Kevin S. Akers ◽  
Desiree R. Romano ◽  
Ronald L. Woodbury ◽  
Sharanda K. Hardy ◽  
...  
Keyword(s):  
Amino Acids ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Clinical Isolates ◽  
Chronic Infections ◽  
Content Type ◽  
Log Reduction ◽  
Viable Bacteria ◽  
Biofilm Bacteria

ABSTRACTWithin wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluatedin vitrothe dispersive activity ofd-amino acids (d-AAs) on biofilms from clinical wound isolates ofStaphylococcus aureusandPseudomonas aeruginosa; moreover, we determined whether combinations ofd-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin forS. aureusand amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime forP. aeruginosa) enhance activity against biofilms.d-Met,d-Phe, andd-Trp at concentrations of ≥5 mM effectively dispersed preformed biofilms ofS. aureusandP. aeruginosaclinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (d-Met/d-Phe/d-Trp). When combined withd-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates ofS. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 μg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition ofd-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms ofP. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 μg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity ofd-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity.

scholarly journals Studies of Pseudomonas aeruginosa Mutants Indicate Pyoverdine as the Central Factor in Inhibition of Aspergillus fumigatus Biofilm

2017 ◽  
Vol 200 (1) ◽  
Author(s):  
Gabriele Sass ◽  
Hasan Nazik ◽  
John Penner ◽  
Hemi Shah ◽  
Shajia Rahman Ansari ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogens ◽  
Human Pathogens ◽  
Biofilm Growth ◽  
Content Type ◽  
Iron Deprivation

ABSTRACT Pseudomonas aeruginosa and Aspergillus fumigatus are common opportunistic bacterial and fungal pathogens, respectively. They often coexist in airways of immunocompromised patients and individuals with cystic fibrosis, where they form biofilms and cause acute and chronic illnesses. Hence, the interactions between them have long been of interest and it is known that P. aeruginosa can inhibit A. fumigatus in vitro. We have approached the definition of the inhibitory P. aeruginosa molecules by studying 24 P. aeruginosa mutants with various virulence genes deleted for the ability to inhibit A. fumigatus biofilms. The ability of P. aeruginosa cells or their extracellular products produced during planktonic or biofilm growth to affect A. fumigatus biofilm metabolism or planktonic A. fumigatus growth was studied in agar and liquid assays using conidia or hyphae. Four mutants, the pvdD pchE, pvdD, lasR rhlR, and lasR mutants, were shown to be defective in various assays. This suggested the P. aeruginosa siderophore pyoverdine as the key inhibitory molecule, although additional quorum sensing-regulated factors likely contribute to the deficiency of the latter two mutants. Studies of pure pyoverdine substantiated these conclusions and included the restoration of inhibition by the pyoverdine deletion mutants. A correlation between the concentration of pyoverdine produced and antifungal activity was also observed in clinical P. aeruginosa isolates derived from lungs of cystic fibrosis patients. The key inhibitory mechanism of pyoverdine was chelation of iron and denial of iron to A. fumigatus. IMPORTANCE Interactions between human pathogens found in the same body locale are of vast interest. These interactions could result in exacerbation or amelioration of diseases. The bacterium Pseudomonas aeruginosa affects the growth of the fungus Aspergillus fumigatus. Both pathogens form biofilms that are resistant to therapeutic drugs and host immunity. P. aeruginosa and A. fumigatus biofilms are found in vivo, e.g., in the lungs of cystic fibrosis patients. Studying 24 P. aeruginosa mutants, we identified pyoverdine as the major anti-A. fumigatus compound produced by P. aeruginosa. Pyoverdine captures iron from the environment, thus depriving A. fumigatus of a nutrient essential for its growth and metabolism. We show how microbes of different kingdoms compete for essential resources. Iron deprivation could be a therapeutic approach to the control of pathogen growth.

scholarly journals Mep72, a Metzincin Protease That Is Preferentially Secreted by Biofilms of Pseudomonas aeruginosa

2014 ◽  
Vol 197 (4) ◽  
pp. 762-773 ◽  
Author(s):  
Ian J. Passmore ◽  
Kahoko Nishikawa ◽  
Kathryn S. Lilley ◽  
Steven D. Bowden ◽  
Jade C. S. Chung ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Carbohydrate Binding ◽  
Biofilm Growth ◽  
Content Type ◽  
2D Dige ◽  
Binding Domains ◽  
Reverse Transcription Pcr ◽  
Specific Subset

In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures ofPseudomonas aeruginosausing two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) inP. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegradedin vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of manyP. aeruginosabiofilms). The expression of full-lengthmep72inEscherichia coliwas toxic. However, this toxicity could be alleviated by coexpression ofmep72with the adjacent gene,bamI. Mep72 and BamI were found to form a protein-protein complexin vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of anmep72mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors.

scholarly journals Characterization of a Carbapenem-Hydrolyzing Enzyme, PoxB, in Pseudomonas aeruginosa PAO1

2015 ◽  
Vol 60 (2) ◽  
pp. 936-945 ◽  
Author(s):  
Diansy Zincke ◽  
Deepak Balasubramanian ◽  
Lynn L. Silver ◽  
Kalai Mathee
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Inhibitors ◽  
Acquired Resistance ◽  
Opportunistic Pathogen ◽  
Inducible Promoter ◽  
Upstream Open Reading Frame ◽  
Antimicrobial Drugs ◽  
Content Type ◽  
Class D

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen often associated with severe and life-threatening infections that are highly impervious to treatment. This microbe readily exhibits intrinsic and acquired resistance to varied antimicrobial drugs. Resistance to penicillin-like compounds is commonplace and provided by the chromosomal AmpC β-lactamase. A second, chromosomally encoded β-lactamase, PoxB, has previously been reported inP. aeruginosa. In the present work, the contribution of this class D enzyme was investigated using a series of clean in-frameampC,poxB, andoprDdeletions, as well as complementation by expression under the control of an inducible promoter. WhilepoxBdeletions failed to alter β-lactam sensitivities, expression ofpoxBinampC-deficient backgrounds decreased susceptibility to both meropenem and doripenem but had no effect on imipenem, penicillin, and cephalosporin MICs. However, when expressed in anampCpoxB-deficient background, that additionally lacked the outer membrane porin-encoding geneoprD, PoxB significantly increased the imipenem as well as the meropenem and doripenem MICs. Like other class D carbapenem-hydrolyzing β-lactamases, PoxB was only poorly inhibited by class A enzyme inhibitors, but a novel non-β-lactam compound, avibactam, was a slightly better inhibitor of PoxB activity.In vitrosusceptibility testing with a clinical concentration of avibactam, however, failed to reduce PoxB activity against the carbapenems. In addition,poxBwas found to be cotranscribed with an upstream open reading frame,poxA, which itself was shown to encode a 32-kDa protein of yet unknown function.

scholarly journals Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill Pseudomonas aeruginosa

Microbiology ◽  
2020 ◽  
Vol 166 (12) ◽  
pp. 1171-1180 ◽  
Author(s):  
Esther Sweeney ◽  
Akshay Sabnis ◽  
Andrew M. Edwards ◽  
Freya Harrison
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ex Vivo ◽  
Clinical Success ◽  
Biofilm Growth ◽  
Content Type ◽  
The Matrix ◽  
Model Versus ◽  
High Doses

In vivo biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by Pseudomonas aeruginosa when grown in medium that mimics cystic fibrosis (CF) sputum versus standard medium in in vitro biofilm assays, and drastically increased tolerance when grown in an ex vivo CF model versus the in vitro assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into ex vivo biofilms and showed that poor penetration partly explains the high doses of drug necessary to kill bacteria in these biofilms. The ability of antibiotics to penetrate the biofilm matrix is key to their clinical success, but hard to measure. Our results demonstrate both the importance of reduced entry into the matrix in in vivo-like biofilm, and the tractability of using a fluorescent tag and benchtop fluorimeter to assess antibiotic entry into biofilms. This method could be a relatively quick, cheap and useful addition to diagnostic and drug development pipelines, allowing the assessment of drug entry into biofilms, in in vivo-like conditions, prior to more detailed tests of biofilm killing.

scholarly journals Inhibition of Pseudomonas aeruginosa by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
James J. Howard ◽  
Carolyn R. Sturge ◽  
Dina A. Moustafa ◽  
Seth M. Daly ◽  
Kimberly R. Marshall-Batty ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Essential Genes ◽  
Content Type ◽  
Novel Approach ◽  
Phosphorodiamidate Morpholino Oligomers

ABSTRACT Pseudomonas aeruginosa is a highly virulent, multidrug-resistant pathogen that causes significant morbidity and mortality in hospitalized patients and is particularly devastating in patients with cystic fibrosis. Increasing antibiotic resistance coupled with decreasing numbers of antibiotics in the developmental pipeline demands novel antibacterial approaches. Here, we tested peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which inhibit translation of complementary mRNA from specific, essential genes in P. aeruginosa. PPMOs targeted to acpP, lpxC, and rpsJ, inhibited P. aeruginosa growth in many clinical strains and activity of PPMOs could be enhanced 2- to 8-fold by the addition of polymyxin B nonapeptide at subinhibitory concentrations. The PPMO targeting acpP was also effective at preventing P. aeruginosa PAO1 biofilm formation and at reducing existing biofilms. Importantly, treatment with various combinations of a PPMO and a traditional antibiotic demonstrated synergistic growth inhibition, the most effective of which was the PPMO targeting rpsJ with tobramycin. Furthermore, treatment of P. aeruginosa PA103-infected mice with PPMOs targeting acpP, lpxC, or rpsJ significantly reduced the bacterial burden in the lungs at 24 h by almost 3 logs. Altogether, this study demonstrates that PPMOs targeting the essential genes acpP, lpxC, or rpsJ in P. aeruginosa are highly effective at inhibiting growth in vitro and in vivo. These data suggest that PPMOs alone or in combination with antibiotics represent a novel approach to addressing the problems associated with rapidly increasing antibiotic resistance in P. aeruginosa.

scholarly journals Dual bioresponsive antibiotic and quorum sensing inhibitor combination nanoparticles for treatment of Pseudomonas aeruginosa biofilms in vitro and ex vivo

2019 ◽  
Vol 7 (10) ◽  
pp. 4099-4111 ◽  
Author(s):  
Nishant Singh ◽  
Manuel Romero ◽  
Alessandra Travanut ◽  
Patricia F. Monteiro ◽  
Elena Jordana-Lluch ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Ex Vivo ◽  
Biofilm Growth ◽  
Quorum Sensing Inhibitor ◽  
Quorum Sensing Inhibitors ◽  
Inhibitor Combination

Nanoparticles combining Quorum Sensing Inhibitors and anti-bacterials can eradicate biofilm growth in vitro and ex vivo.

scholarly journals A 2.5-Year Within-Patient Evolution of Pseudomonas aeruginosa Isolates with In Vivo Acquisition of Ceftolozane-Tazobactam and Ceftazidime-Avibactam Resistance upon Treatment

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Thibaud Boulant ◽  
Agnès B. Jousset ◽  
Rémy A. Bonnin ◽  
Aurélie Barrail-Tran ◽  
Adrien Borgel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acquired Resistance ◽  
Multidrug Resistant ◽  
Sequencing Analysis ◽  
Nucleotide Polymorphisms ◽  
Related Sequence ◽  
Content Type ◽  
Partial Restoration

ABSTRACT Ceftolozane-tazobactam is considered to be a last-resort treatment for infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. Although resistance to this antimicrobial has been described in vitro, the development of resistance in vivo has rarely been reported. Here, we describe the evolution of resistance to ceftolozane-tazobactam of P. aeruginosa isolates recovered from the same patient during recurrent infections over 2.5 years. Antimicrobial susceptibility testing results showed that 24 of the 27 P. aeruginosa isolates recovered from blood (n = 18), wound (n = 2), pulmonary (n = 1), bile (n = 2), and stool (n = 4) samples from the same patient were susceptible to ceftolozane-tazobactam and ceftazidime-avibactam but resistant to ceftazidime, piperacillin-tazobactam, imipenem, and meropenem. Three clinical isolates acquired resistance to ceftolozane-tazobactam and ceftazidime-avibactam along with a partial restoration of piperacillin-tazobactam and carbapenem susceptibilities. Whole-genome sequencing analysis reveals that all isolates were clonally related (sequence type 111 [ST-111]), with a median of 24.9 single nucleotide polymorphisms (SNPs) (range, 8 to 48 SNPs). The ceftolozane-tazobactam and ceftazidime-avibactam resistance was likely linked to the same G183D substitution in the chromosome-encoded cephalosporinase. Our results suggest that resistance to ceftolozane-tazobactam in P. aeruginosa might occur in vivo upon treatment through an amino acid substitution in the intrinsic AmpC leading to ceftolozane-tazobactam and ceftazidime-avibactam resistance, accompanied by resensitization to piperacillin-tazobactam and carbapenems.

scholarly journals Albumin Inhibits Pseudomonas aeruginosa Quorum Sensing and Alters Polymicrobial Interactions

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Allie Clinton Smith ◽  
Anne Rice ◽  
Bryan Sutton ◽  
Rebecca Gabrilska ◽  
Aimee K. Wessel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Chronic Wounds ◽  
Homoserine Lactone ◽  
Individual Species ◽  
Content Type ◽  
The Individual ◽  
Chronic Wound Infections ◽  
Isolated Species

ABSTRACT Polymicrobial interactions are complex and can influence the course of an infection, as is the case when two or more species exhibit a synergism that produces a disease state not seen with any of the individual species alone. Cell-to-cell signaling is key to many of these interactions, but little is understood about how the host environment influences polymicrobial interactions or signaling between bacteria. Chronic wounds are typically polymicrobial, with Staphylococcus aureus and Pseudomonas aeruginosa being the two most commonly isolated species. While P. aeruginosa readily kills S. aureus in vitro, the two species can coexist for long periods together in chronic wound infections. In this study, we investigated the ability of components of the wound environment to modulate interactions between P. aeruginosa and S. aureus. We demonstrate that P. aeruginosa quorum sensing is inhibited by physiological levels of serum albumin, which appears to bind and sequester some homoserine lactone quorum signals, resulting in the inability of P. aeruginosa to produce virulence factors that kill S. aureus. These data could provide important clues regarding the virulence of P. aeruginosa in albumin-depleted versus albumin-rich infection sites and an understanding of the nature of friendly versus antagonistic interactions between P. aeruginosa and S. aureus.

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