Tobramycin-Treated Pseudomonas aeruginosa PA14 Enhances Streptococcus constellatus 7155 Biofilm Formation in a Cystic Fibrosis Model System

ABSTRACTCystic fibrosis (CF) is a human genetic disorder which results in a lung environment that is highly conducive to chronic microbial infection. Over the past decade, deep-sequencing studies have demonstrated that the CF lung can harbor a highly diverse polymicrobial community. We expanded our existingin vitromodel ofPseudomonas aeruginosabiofilm formation on CF-derived airway cells to include this broader set of CF airway colonizers to investigate their contributions to CF lung disease, particularly as they relate to the antibiotic response of the population. Using this system, we identified an interspecies interaction betweenP. aeruginosa, a bacterium associated with declining lung function and worsening disease, andStreptococcus constellatus, a bacterium correlated with the onset of pulmonary exacerbations in CF patients. The growth rate and cytotoxicity ofS. constellatus7155 andP. aeruginosaPA14 were unchanged when grown together as mixed biofilms in the absence of antibiotics. However, the addition of tobramycin, the frontline maintenance therapy antibiotic for individuals with CF, to a mixed biofilm ofS. constellatus7155 andP. aeruginosaPA14 resulted in enhancedS. constellatusbiofilm formation. Through a candidate genetic approach, we showed thatP. aeruginosarhamnolipids were reduced upon tobramycin exposure, allowing forS. constellatus7155 biofilm enhancement, and monorhamnolipids were sufficient to reduceS. constellatus7155 biofilm viability in the absence of tobramycin. While the findings presented here are specific to a biofilm ofS. constellatus7155 andP. aeruginosaPA14, they highlight the potential of polymicrobial interactions to impact antibiotic tolerance in unanticipated ways.IMPORTANCEDeep-sequencing studies have demonstrated that the CF lung can harbor a diverse polymicrobial community. By recapitulating the polymicrobial communities observed in the CF lung and identifying mechanisms of interspecies interactions, we have the potential to select the best therapy for a given bacterial community and reveal potential opportunities for novel therapeutic interventions. Using anin vitromodel of bacterial infection on CF airway cells, we tested how a particular polymicrobial community grows, damages human cells, and responds to antibiotics in single and mixed infections. We describe here the mechanism of an interspecies interaction between two pathogens in the CF lung,P. aeruginosaandS. constellatus, which is potentiated by a commonly prescribed antibiotic, tobramycin.

Download Full-text

Within-Host Evolution of Pseudomonas aeruginosa Reveals Adaptation toward Iron Acquisition from Hemoglobin

mBio ◽

10.1128/mbio.00966-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 88

Author(s):

Rasmus Lykke Marvig ◽

Søren Damkiær ◽

S. M. Hossein Khademi ◽

Trine M. Markussen ◽

Søren Molin ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Iron Acquisition ◽

Chronic Infections ◽

Content Type ◽

Mortality And Morbidity ◽

Airway Infections ◽

Host Evolution ◽

Promoter Mutations

ABSTRACTPseudomonas aeruginosaairway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist,P. aeruginosadepends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and thePseudomonasheme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation ofP. aeruginosato the host environment. Here we investigated the within-host evolution of the transmissibleP. aeruginosaDK2 lineage. We found positive selection for promoter mutations leading to increased expression of thephusystem. By mimicking conditions of the CF airwaysin vitro, we experimentally demonstrate that increased expression ofphuRconfers a growth advantage in the presence of hemoglobin, thus suggesting thatP. aeruginosaevolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additionalP. aeruginosalineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages,phuRpromoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment ofP. aeruginosainfections in CF patients.IMPORTANCEMost bacterial pathogens depend on scavenging iron within their hosts, which makes the battle for iron between pathogens and hosts a hallmark of infection. Accordingly, the ability of the opportunistic pathogenPseudomonas aeruginosato cause chronic infections in cystic fibrosis (CF) patients also depends on iron-scavenging systems. While the regulation and mechanisms of several such iron-scavenging systems have been well described, not much is known about how the within-host selection pressures act on the pathogens’ ability to acquire iron. Here, we investigated the within-host evolution ofP. aeruginosa, and we found evidence thatP. aeruginosaduring long-term infections evolves toward iron acquisition from hemoglobin. This adaptive strategy might be due to a selective loss of other iron-scavenging mechanisms and/or an increase in the availability of hemoglobin at the site of infection. This information is relevant to the design of novel CF therapeutics and the development of models of chronic CF infections.

Download Full-text

Environment in the lung of cystic fibrosis patients stimulates the expression of biofilm phenotype in Mycobacterium abscessus

Journal of Medical Microbiology ◽

10.1099/jmm.0.001467 ◽

2022 ◽

Vol 71 (1) ◽

Author(s):

Bailey F. Keefe ◽

Luiz E. Bermudez

Keyword(s):

Cystic Fibrosis ◽

Host Cell ◽

Biofilm Formation ◽

Type Species ◽

Divalent Cations ◽

Mycobacterium Abscessus ◽

Content Type ◽

Link Type ◽

Populations At Risk

Introduction. Pulmonary infections caused by organisms of the Mycobacterium abscessus complex are increasingly prevalent in populations at risk, such as patients with cystic fibrosis, bronchiectasis and emphysema. Hypothesis. M. abscessus infection of the lung is not observed in immunocompetent individuals, which raises the possibility that the compromised lung environment is a suitable niche for the pathogen to thrive in due to the overproduction of mucus and high amounts of host cell lysis. Aim. Evaluate the ability of M. abscessus to form biofilm and grow utilizing in vitro conditions as seen in immunocompromised lungs of patients. Methodology. We compared biofilm formation and protein composition in the presence and absence of synthetic cystic fibrosis medium (SCFM) and evaluated the bacterial growth when exposed to human DNA. Results. M. abscessus is capable of forming biofilm in SCFM. By eliminating single components found in the medium, it became clear that magnesium works as a signal for the biofilm formation, and chelation of the divalent cations resulted in the suppression of biofilm formation. Investigation of the specific proteins expressed in the presence of SCFM and in the presence of SCFM lacking magnesium revealed many different proteins between the conditions. M. abscessus also exhibited growth in SCFM and in the presence of host cell DNA, although the mechanism of DNA utilization remains unclear. Conclusions. In vitro conditions mimicking the airways of patients with cystic fibrosis appear to facilitate M. abscessus establishment of infection, and elimination of magnesium from the environment may affect the ability of the pathogen to establish infection.

Download Full-text

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

mSphere ◽

10.1128/msphere.00343-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 5

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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00345-17 ◽

2017 ◽

Vol 200 (1) ◽

Cited By ~ 33

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.

Download Full-text

Polymorphonuclear Leukocytes Restrict Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients

Infection and Immunity ◽

10.1128/iai.01969-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4477-4486 ◽

Cited By ~ 88

Author(s):

Kasper N. Kragh ◽

Morten Alhede ◽

Peter Ø. Jensen ◽

Claus Moser ◽

Thomas Scheike ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Growth Rates ◽

Polymorphonuclear Leukocytes ◽

Growth Patterns ◽

Tissue Samples ◽

Content Type ◽

Growth Physiology

ABSTRACTCystic fibrosis (CF) patients have increased susceptibility to chronic lung infections byPseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate thein vivogrowth physiology ofP. aeruginosawithin lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescencein situhybridization (PNA-FISH)-based method was used to estimate thein vivogrowth rates ofP. aeruginosadirectly in lung tissue samples from CF patients and the growth rates ofP. aeruginosain infected lungs in a mouse model. The growth rate ofP. aeruginosawithin CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect onP. aeruginosaby PMNs was also observedin vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding thatP. aeruginosagrowth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O2consumption, which slows the growth ofP. aeruginosain infected CF lungs. In support of this, the growth ofP. aeruginosawas significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronicP. aeruginosainfection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.

Download Full-text

Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung

Infection and Immunity ◽

10.1128/iai.00437-16 ◽

2016 ◽

Vol 84 (10) ◽

pp. 2995-3006 ◽

Cited By ~ 16

Author(s):

Alex H. Gifford ◽

Sven D. Willger ◽

Emily L. Dolben ◽

Lisa A. Moulton ◽

Dana B. Dorman ◽

...

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Expression Profiles ◽

Clinical Isolates ◽

Probe Design ◽

Content Type ◽

Lung Infections

The discovery of therapies that modulatePseudomonas aeruginosavirulence or that can eradicate chronicP. aeruginosalung infections associated with cystic fibrosis (CF) will be advanced by an improved understanding ofP. aeruginosabehaviorin vivo. We demonstrate the use of multiplexed Nanostring technology to monitor relative abundances ofP. aeruginosatranscripts across clinical isolates, in serial samples, and for the purposes of comparing microbial physiologyin vitroandin vivo. The expression of 75 transcripts encoded by genes implicated in CF lung disease was measured in a variety ofP. aeruginosastrains as well as RNA serial sputum samples from fourP. aeruginosa-colonized subjects with CF collected over 6 months. We present data on reproducibility, the results from different methods of normalization, and demonstrate high concordance between transcript relative abundance data obtained by Nanostring or transcriptome sequencing (RNA-Seq) analysis. Furthermore, we address considerations regarding sequence variation between strains during probe design. Analysis ofP. aeruginosagrownin vitroidentified transcripts that correlated with the different phenotypes commonly observed in CF clinical isolates.P. aeruginosatranscript profiles in RNA from CF sputum indicated alginate productionin vivo, and transcripts involved in quorum-sensing regulation were less abundant in sputum than strains grown in the laboratory.P. aeruginosagene expression patterns from sputum clustered closely together relative to patterns for laboratory-grown cultures; in contrast, laboratory-grownP. aeruginosashowed much greater transcriptional variation with only loose clustering of strains with different phenotypes. The clustering within and between subjects was surprising in light of differences in inhaled antibiotic and respiratory symptoms, suggesting that the pathways represented by these 75 transcripts are stable in chronic CFP. aeruginosalung infections.

Download Full-text

Localization of Burkholderia cepacia Complex Bacteria in Cystic Fibrosis Lungs and Interactions with Pseudomonas aeruginosa in Hypoxic Mucus

Infection and Immunity ◽

10.1128/iai.01876-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4729-4745 ◽

Cited By ~ 57

Author(s):

Ute Schwab ◽

Lubna H. Abdullah ◽

Olivia S. Perlmutt ◽

Daniel Albert ◽

C. William Davis ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Burkholderia Cepacia ◽

Single Cells ◽

Burkholderia Cenocepacia ◽

Burkholderia Cepacia Complex ◽

Test Medium ◽

Fermentation Products ◽

Content Type

ABSTRACTThe localization ofBurkholderia cepaciacomplex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection withPseudomonas aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc andP. aeruginosabacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallelin vitroexperiments examined the growth of two Bcc species,Burkholderia cenocepaciaandBurkholderia multivorans, in environments similar to those occupied byP. aeruginosain the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as “biofilm-like structures.” In contrast,P. aeruginosawas identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions withP. aeruginosain an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions,B. cenocepaciaandB. multivoransused fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence ofP. aeruginosain vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade aP. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibitP. aeruginosabiofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.

Download Full-text

Involvement of Stress-Related GenespolBandPA14_46880in Biofilm Formation of Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.01915-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4746-4757 ◽

Cited By ~ 3

Author(s):

Sahar A. Alshalchi ◽

Gregory G. Anderson

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Acute Infection ◽

Regulatory Gene ◽

Chronic Infections ◽

Content Type ◽

Abiotic Surfaces ◽

Mutant Strains ◽

In The Wild ◽

Airway Cells

ABSTRACTChronic infections ofPseudomonas aeruginosaare generally established through production of biofilm. During biofilm formation, production of an extracellular matrix and establishment of a distinct bacterial phenotype make these infections difficult to eradicate. However, biofilm studies have been hampered by the fact that most assays utilize nonliving surfaces as biofilm attachment substrates. In an attempt to better understand the mechanisms behindP. aeruginosabiofilm formation, we performed a genetic screen to identify novel factors involved in biofilm formation on biotic and abiotic surfaces. We found that deletion of genespolBandPA14_46880reduced biofilm formation significantly compared to that in the wild-type strain PA14 in an abiotic biofilm system. In a biotic biofilm model, wherein biofilms form on cultured airway cells, the ΔpolBand ΔPA14_46880strains showed increased cytotoxic killing of the airway cells independent of the total number of bacteria bound. Notably, deletion mutant strains were more resistant to ciprofloxacin treatment. This phenotype was linked to decreased expression ofalgR, an alginate transcriptional regulatory gene, under ciprofloxacin pressure. Moreover, we found that pyocyanin production was increased in planktonic cells of mutant strains. These results indicate that inactivation ofpolBandPA14_46880may inhibit transition ofP. aeruginosafrom a more acute infection lifestyle to the biofilm phenotype. Future investigation of these genes may lead to a better understanding ofP. aeruginosabiofilm formation and chronic biofilm infections.

Download Full-text

Biological Markers of Pseudomonas aeruginosa Epidemic High-Risk Clones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01481-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5527-5535 ◽

Cited By ~ 65

Author(s):

Xavier Mulet ◽

Gabriel Cabot ◽

Alain A. Ocampo-Sosa ◽

M. Angeles Domínguez ◽

Laura Zamorano ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

High Risk ◽

Multicenter Study ◽

Biological Markers ◽

Biofilm Formation ◽

Specific Treatment ◽

Spontaneous Mutant ◽

Biological Parameters ◽

Content Type

ABSTRACTA limited number ofPseudomonas aeruginosagenotypes (mainly ST-111, ST-175, and ST-235), known as high-risk clones, are responsible for epidemics of nosocomial infections by multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains worldwide. We explored the potential biological parameters that may explain the success of these clones. A total of 20 isolates from each of 4 resistance groups (XDR, MDR, ModR [resistant to 1 or 2 classes], and MultiS [susceptible to all antipseudomonals]), recovered from a multicenter study ofP. aeruginosabloodstream infections performed in 10 Spanish hospitals, were analyzed. A further set of 20 XDR isolates belonging to epidemic high-risk clones (ST-175 [n= 6], ST-111 [n= 7], and ST-235 [n= 7]) recovered from different geographical locations was also studied. When unknown, genotypes were documented through multilocus sequence typing. The biological parameters evaluated included twitching, swimming, and swarming motility, biofilm formation, production of pyoverdine and pyocyanin, spontaneous mutant frequencies, and thein vitrocompetition index (CI) obtained with a flow cytometry assay. All 20 (100%) XDR, 8 (40%) MDR, and 1 (5%) ModR bloodstream isolate from the multicenter study belonged to high-risk clones. No significant differences were observed between clonally diverse ModR and MultiS isolates for any of the parameters. In contrast, MDR/XDR high-risk clones showed significantly increased biofilm formation and mutant frequencies but significantly reduced motility (twitching, swimming, and swarming), production of pyoverdine and pyocyanin, and fitness. The defined biological markers of high-risk clones, which resemble those resulting from adaptation to chronic infections, could be useful for the design of specific treatment and infection control strategies.

Download Full-text

In Vitro Analysis of Tobramycin-Treated Pseudomonas aeruginosa Biofilms on Cystic Fibrosis-Derived Airway Epithelial Cells

Infection and Immunity ◽

10.1128/iai.01373-07 ◽

2008 ◽

Vol 76 (4) ◽

pp. 1423-1433 ◽

Cited By ~ 118

Author(s):

Gregory G. Anderson ◽

Sophie Moreau-Marquis ◽

Bruce A. Stanton ◽

George A. O'Toole

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Epithelial Cells ◽

Biofilm Formation ◽

Airway Epithelial Cells ◽

Bacterial Virulence ◽

Model Systems ◽

Virulence Gene Expression ◽

Airway Cells

ABSTRACT P. aeruginosa forms biofilms in the lungs of individuals with cystic fibrosis (CF); however, there have been no effective model systems for studying biofilm formation in the CF lung. We have developed a tissue culture system for growth of P. aeruginosa biofilms on CF-derived human airway cells that promotes the formation of highly antibiotic-resistant microcolonies, which produce an extracellular polysaccharide matrix and require the known abiotic biofilm formation genes flgK and pilB. Treatment of P. aeruginosa biofilms with tobramycin reduced the virulence of the biofilms both by reducing bacterial numbers and by altering virulence gene expression. We performed microarray analysis of these biofilms on epithelial cells after treatment with tobramycin, and we compared these results with gene expression of (i) tobramycin-treated planktonic P. aeruginosa and (ii) tobramycin-treated P. aeruginosa biofilms on an abiotic surface. Despite the conservation in functions required to form a biofilm, our results show that the responses to tobramycin treatment of biofilms grown on biotic versus abiotic surfaces are different, as exemplified by downregulation of genes involved in Pseudomonas quinolone signal biosynthesis specifically in epithelial cell-grown biofilms versus plastic-grown biofilms. We also identified the gene PA0913, which is upregulated by tobramycin specifically in biofilms grown on CF airway cells and codes for a probable magnesium transporter, MgtE. Mutation of the PA0913 gene increased the bacterial virulence of biofilms on the epithelial cells, consistent with a role for the gene in the suppression of bacterial virulence. Taken together, our data show that analysis of biofilms on airway cells provides new insights into the interaction of these microbial communities with the host.

Download Full-text