In Vivo Efficacy of Antimicrobials against Biofilm-Producing Pseudomonas aeruginosa.
ABSTRACT: Patients suffering from cystic fibrosis (CF) are commonly affected by chronic Pseudomonas aeruginosa biofilm infections. This is the main cause for the high disease severity. In this study, we demonstrate that P. aeruginosa is able to efficiently colonize murine solid tumors after intravenous injection and to form biofilms in this tissue. Biofilm formation was evident by electron microscopy. Such structures could not be observed with transposon mutants, which were defective in biofilm formation. Comparative transcriptional profiling of P. aeruginosa indicated physiological similarity of the bacteria in the murine tumor model and the CF lung. The efficacy of currently available antibiotics for treatment of P. aeruginosa-infected CF lungs, such as ciprofloxacin, colistin, and tobramycin, could be tested in the tumor model. We found that clinically recommended doses of these antibiotics were unable to eliminate wild-type P. aeruginosa PA14 while being effective against biofilm-defective mutants. However, colistin-tobramycin combination therapy significantly reduced the number of P. aeruginosa PA14 cells in tumors at lower concentrations. Hence, we present a versatile experimental system that is providing a platform to test approved and newly developed antibiofilm compounds.
Project description:Cystic 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 existing in vitro model of Pseudomonas aeruginosa biofilm 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 between P. aeruginosa, a bacterium associated with declining lung function and worsening disease, and Streptococcus constellatus, a bacterium correlated with the onset of pulmonary exacerbations in CF patients. The growth rate and cytotoxicity of S. constellatus 7155 and P. aeruginosa PA14 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 of S. constellatus 7155 and P. aeruginosa PA14 resulted in enhanced S. constellatus biofilm formation. Through a candidate genetic approach, we showed that P. aeruginosa rhamnolipids were reduced upon tobramycin exposure, allowing for S. constellatus 7155 biofilm enhancement, and monorhamnolipids were sufficient to reduce S. constellatus 7155 biofilm viability in the absence of tobramycin. While the findings presented here are specific to a biofilm of S. constellatus 7155 and P. aeruginosa PA14, they highlight the potential of polymicrobial interactions to impact antibiotic tolerance in unanticipated ways.Deep-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 an in vitro model 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. aeruginosa and S. constellatus, which is potentiated by a commonly prescribed antibiotic, tobramycin.
Project description:Chronic Pseudomonas biofilm infections are commonly associated in patients afflicted with cystic fibrosis (CF) leading to high degree of morbidity. In a murine tumor model we demonstrated that P. aeruginosa efficiently colonizes and forms biofilms in cancerous tissue post intra-venous injection. Several non-biofilm forming mutants have been identified and incorporated in the present study. Biofilm formation by wild type strains was evident in electron microscopy and immuno-histological studies. Efficacy of currently available CF infection treatment antibiotics such as ciprofloxacin, colistin and tobramycin were tested in this model. We found out that normal doses of these antibiotics were unable to eliminate wild type P. aeruginosa PA14. However, transposon mutants of P. aeruginosa PA14 (pqsA & Pel A) had strong influence on colonization. Subsequently high doses were effective against wild type P. aeruginosa PA14 biofilms. Overall design: The samples of P. aeruginosa PA14 wild type strain and the transposon mutant of pqsA were analyzed by RNA-seq. The control samples were cultivated in the LB medium until the exponential or stationary growth phase. Four biological replicas were pooled in pairs to get two replicas for RNA extraction, library preparation and sequencing. Tumor samples were collected from mice infected with PA14 wild type or PA14 pqsA transposon mutants. There were four mice per group and the material of each group was pooled to assure sufficient bacterial RNA. The tumor samples were split into two technical replicas for sequencing library preparation. Please note that two groups which each processed data file were generated from are indicated in the file names as following; PA14_exponential growth (PA14_e) PA14_stationary growth (PA14_s) WT_murine tumor cells (PA14_t) pqsA_exponential growth (pqsA_e) pqsA_stationary growth (pqsA_s) pqsA_murine tumor cells (pqsA_t)
Project description:MICs and biofilm inhibitory concentrations (BICs) were measured for 68 cystic fibrosis (CF) Achromobacter isolates for amikacin, aztreonam, colistin, levofloxacin, and tobramycin. With the exception of colistin and levofloxacin, the remaining antibiotics had MIC90s, BICs at which 50% of the isolates were susceptible (BIC50s), and BICs at which 90% of the isolates were susceptible (BIC90s) equal to or above the highest concentrations tested. In a biofilm model, tobramycin was able to significantly increase killing of bacterial cells compared to controls, for intermediate-resistant strains only, at concentrations of 1,000 and 2,000 ?g/ml.
Project description:OBJECTIVES:The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D ('DiEthylAmin-Cephalosporin-3'-Diazeniumdiolate') has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. METHODS:?-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. RESULTS:DEA-C3D was confirmed to selectively release NO in response to contact with bacterial ?-lactamase. Despite lacking direct, cephalosporin/?-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. CONCLUSIONS:DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.
Project description:The lungs of cystic fibrosis (CF) patients are often chronically colonized by multiple microbial species that can form biofilms, including the major CF pathogen <i>Pseudomonas aeruginosa</i>. Herewith, lower microbial diversity in CF airways is typically associated with worse health outcomes. In an attempt to treat CF lung infections patients are frequently exposed to antibiotics, which may affect microbial diversity. This study aimed at understanding if common antibiotics that target <i>P. aeruginosa</i> influence microbial diversity. To this end, a microaerophilic multispecies biofilm model of frequently co-isolated members of the CF lung microbiome (<i>Pseudomonas aeruginosa</i>, <i>Staphylococcus aureus</i>, <i>Streptococcus anginosus</i>, <i>Achromobacter xylosoxidans</i>, <i>Rothia mucilaginosa</i>, and <i>Gemella haemolysans</i>) was exposed to antipseudomonal antibiotics. We found that antibiotics that affected several dominant species (i.e. ceftazidime, tobramycin) resulted in higher species evenness compared to colistin, which is only active against <i>P. aeruginosa</i>. Furthermore, susceptibility of individual species in the multispecies biofilm following antibiotic treatment was compared to that of the respective single-species biofilms, showing no differences. Adding three anaerobic species (<i>Prevotella melaninogenica</i>, <i>Veillonella parvula</i>, and <i>Fusobacterium nucleatum</i>) to the multispecies biofilm did not influence antibiotic susceptibility. In conclusion, our study demonstrates antibiotic-dependent effects on microbial community diversity of multispecies biofilms comprised of CF microbiome members.
Project description:While the MerR-like transcriptional regulator BrlR has been demonstrated to contribute to Pseudomonas aeruginosa biofilm tolerance to antimicrobial agents known as multidrug efflux pump substrates, the role of BrlR in resistance to cationic antimicrobial peptides (CAP), which is based on reduced outer membrane susceptibility, is not known. Here, we demonstrate that inactivation of brlR coincided with increased resistance of P. aeruginosa to colistin, while overexpression of brlR resulted in increased susceptibility. brlR expression correlated with reduced transcript abundances of phoP, phoQ, pmrA, pmrB, and arnC. Inactivation of pmrA and pmrB had no effect on the susceptibility of P. aeruginosa biofilms to colistin, while inactivation of phoP and phoQ rendered biofilms more susceptible than the wild type. The susceptibility phenotype of ?phoP biofilms to colistin was comparable to that of P. aeruginosa biofilms overexpressing brlR. BrlR was found to directly bind to oprH promoter DNA of the oprH-phoPQ operon. BrlR reciprocally contributed to colistin and tobramycin resistance in P. aeruginosa PAO1 and CF clinical isolates, with overexpression of brlR resulting in increased tobramycin MICs and increased tobramycin resistance but decreased colistin MICs and increased colistin susceptibility. The opposite trend was observed upon brlR inactivation. The difference in susceptibility to colistin and tobramycin was eliminated by combination treatment of biofilms with both antibiotics. Our findings establish BrlR as an unusual member of the MerR family, as it not only functions as a multidrug transport activator, but also acts as a repressor of phoPQ expression, thus suppressing colistin resistance.
Project description:Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases.
Project description:Aztreonam for inhalation solution (AZLI) was recently approved by the FDA for treating cystic fibrosis (CF) patients infected with Pseudomonas aeruginosa. Here we investigated the effect of aztreonam alone or in combination with tobramycin on P. aeruginosa biofilms grown on CF airway epithelial cells.P. aeruginosa biofilms, produced by laboratory strains or clinical isolates, were formed on confluent CF airway cells before treatment overnight with aztreonam or tobramycin alone or in combination. Alternatively, antibiotics were added 1 h after bacterial inoculation to assess their ability to impair biofilm formation at 5 h. Bacterial cfu remaining after treatment were then determined by plate counting.In the absence of antibiotics, all strains developed biofilms that disrupted CF airway epithelial monolayers overnight. Tobramycin reduced the cfu of all strains grown as biofilms. Aztreonam reduced the cfu of some strains by ?1 log unit without preserving the integrity of cystic fibrosis airway cell monolayers, while decreasing the biofilms of other clinical isolates by ?4 log units and protecting the monolayers from being compromised. The combination of aztreonam and tobramycin reduced the cfu of two strains by an additional 0.5 and 2 log units, respectively. Of all the mechanisms explored, Psl exopolysaccharide production might explain the variations in biofilm tolerance to aztreonam in some of the strains.Effects of aztreonam on P. aeruginosa biofilms in the in vitro co-culture model are strain-dependent. The simultaneous application of aztreonam and tobramycin may be beneficial for a subset of CF patients by eliminating susceptible P. aeruginosa strains.
Project description:Increasing antibiotic resistance and the declining rate at which new antibiotics come into use create a need to increase the efficacy of existing antibiotics. The aminoglycoside tobramycin is standard-of-care for many types of Pseudomonas aeruginosa infections, including those in the lungs of cystic fibrosis (CF) patients. P. aeruginosa is a nosocomial and opportunistic pathogen that, in planktonic form, causes acute infections and, in biofilm form, causes chronic infections. Inhaled bicarbonate has recently been proposed as a therapy to improve antimicrobial properties of the CF airway surface liquid and viscosity of CF mucus. Here we measure the effect of combining tobramycin and bicarbonate against P. aeruginosa, both lab strains and CF clinical isolates. Bicarbonate synergises with tobramycin to enhance killing of planktonic bacteria. In contrast, bicarbonate antagonises with tobramycin to promote better biofilm growth. This suggests caution when evaluating bicarbonate as a therapy for CF lungs infected with P. aeruginosa biofilms. We analyse tobramycin and bicarbonate interactions using an interpolated surface methodology to measure the dose-response function. These surfaces allow more accurate estimation of combinations yielding synergy and antagonism than do standard isobolograms. By incorporating predictions based on Loewe additivity theory, we can consolidate information on a wide range of combinations that produce a complex dose-response surface, into a single number that measures the net effect. This tool thus allows rapid initial estimation of the potential benefit or harm of a therapeutic combination. Software code is freely made available as a resource for the community.
Project description:The lungs of cystic fibrosis (CF) patients are chronically colonized by a polymicrobial biofilm community, leading to difficult-to-treat infections. To combat these infections, CF patients are commonly treated with a variety of antibiotics. Understanding the dynamics of polymicrobial community composition in response to antibiotic therapy is essential in the search for novel therapies. Culture-dependent quantification of individual bacteria from defined multispecies biofilms is frequently carried out by plating on selective media. However, the influence of the selective agents in these media on quantitative recovery before or after antibiotic treatment is often unknown. In the present study we developed selective media for six bacterial species that are frequently co-isolated from the CF lung, i.e. Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans. We show that certain supplementations to selective media strongly influence quantitative recovery of (un)treated biofilms. Hence, the developed media were optimized for selectivity and quantitative recovery before or after treatment with antibiotics of four major classes, i.e. ceftazidime, ciprofloxacin, colistin, or tobramycin. Finally, in a proof of concept experiment the novel selective media were applied to determine the community composition of multispecies biofilms before and after treatment with tobramycin.