Pseudomonas Aeruginosa Biofilm Research Articles

Exploring roles of the chitinase ChiC in modulating Pseudomonas aeruginosa virulence phenotypes.

Chitinases are ubiquitous enzymes involved in biomass degradation and chitin turnover in nature. Pseudomonas aeruginosa (PA), an opportunistic human pathogen, expresses ChiC, a secreted glycoside hydrolase 18 family chitinase. Despite speculation about ChiC's role in PA disease pathogenesis, there is scant evidence supporting this hypothesis. Since PA cannot catabolize chitin, we investigated the potential function(s) of ChiC in PA pathophysiology. Our findings show that ChiC exhibits activity against both insoluble (α- and β-chitin) and soluble chitooligosaccharides. Enzyme kinetics toward (GlcNAc)4 revealed a kcat of 6.50 s-1 and a KM of 1.38 mM, the latter remarkably high for a canonical chitinase. In our label-free proteomics investigation, ChiC was among the most abundant proteins in the Pel biofilm, suggesting a potential contribution to PA biofilm formation. Using an intratracheal challenge model of PA pneumonia, the chiC::ISphoA/hah transposon insertion mutant paradoxically showed slightly increased virulence compared to the wild-type parent strain. Our results indicate that ChiC is a genuine chitinase that contributes to a PA pathoadaptive pathway.IMPORTANCEIn addition to performing chitin degradation, chitinases from the glycoside hydrolase 18 family have been found to play important roles during pathogenic bacterial infection. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing pneumonia in immunocompromised individuals. Despite not being able to grow on chitin, the bacterium produces a chitinase (ChiC) with hitherto unknown function. This study describes an in-depth characterization of ChiC, focusing on its potential contribution to the bacterium's disease-causing ability. We demonstrate that ChiC can degrade both polymeric chitin and chitooligosaccharides, and proteomic analysis of Pseudomonas aeruginosa biofilm revealed an abundance of ChiC, hinting at a potential role in biofilm formation. Surprisingly, a mutant strain incapable of ChiC production showed higher virulence than the wild-type strain. While ChiC appears to be a genuine chitinase, further investigation is required to fully elucidate its contribution to Pseudomonas aeruginosa virulence, an important task given the evident health risk posed by this bacterium.

A New Class of Polyion Complex Vesicles (PIC-somes) to Improve Antimicrobial Activity of Tobramycin in Pseudomonas Aeruginosa Biofilms.

Pseudomonas aeruginosa (PA) is a major healthcare concern due to its tolerance to antibiotics when enclosed in biofilms. Tobramycin (Tob), an effective cationic aminoglycoside antibiotic against planktonic PA, loses potency within PA biofilms due to hindered diffusion caused by interactions with anionic biofilm components. Loading Tob into nano-carriers can enhance its biofilm efficacy by shielding its charge. Polyion complex vesicles (PIC-somes) are promising nano-carriers for charged drugs, allowing higher drug loadings than liposomes and polymersomes. In this study, a new class of nano-sized PIC-somes, formed by Tob-diblock copolymer complexation is presented. This approach replaces conventional linear PEG with brush-like poly[ethylene glycol (methyl ether methacrylate)] (PEGMA) in the shell-forming block, distinguishing it from past methods. Tob paired with a block copolymer containing hydrophilic PEGMA induces micelle formation (PIC-micelles), while incorporating hydrophobic pyridyldisulfide ethyl methacrylate (PDSMA) monomer into PEGMA chains reduces shell hydrophilicity, leads to the formation of vesicles (PIC-somes). PDSMA unit incorporation enables unprecedented dynamic disulfide bond-based shell cross-linking, significantly enhancing stability under saline conditions. Neither PIC-somes nor PIC-micelles show any relevant cytotoxicity on A549, Calu-3, and dTHP-1 cells. Tob's antimicrobial efficacy against planktonic PA remains unaffected after encapsulation into PIC-somes and PIC-micelles, but its potency within PA biofilms significantly increases.

Tobramycin-mediated self-assembly of DNA nanostructures for targeted treatment of Pseudomonas aeruginosa-infected lung inflammation.

Pseudomonas aeruginosa (PA) is one of the most common multidrug-resistant pathogens found in clinics, often manifesting as biofilms. However, due to the emergence of superbugs in hospitals and the overuse of antibiotics, the prevention and treatment of PA infections have become increasingly challenging. Utilizing DNA nanostructures for packaging and delivering antibiotics presents an intervention strategy with significant potential. Nevertheless, construction of functional DNA nanostructures with multiple functionalities and enhanced stability in physiological settings remains challenging. In this study, the authors propose a magnesium-free assembly method that utilizes tobramycin (Tob) as a mediator to assemble DNA nanostructures, allowing for the functionalization of DNA nanostructures by combining DNA and antibiotics. Additionally, our study incorporates maleimide-modified DNA into the nanostructures to act as a targeting moiety specifically directed towards the pili of PA. The targeting ability of the constructed functional DNA nanostructure significantly improves the local concentration of Tob, thereby reducing the side effects of antibiotics. Our results demonstrate the successful construction of a maleimide-decorated Tob/DNA nanotube (NTTob-Mal) for the treatment of PA-infected lung inflammation. The stability and biocompatibility of NTTob-Mal are confirmed, highlighting its potential for clinical applications. Furthermore, its specificity in recognizing and adhering to PA has been validated. In vitro experiments have shown its efficacy in inhibiting PA biofilm formation, and in a murine model, NTTob-Mal has exhibited significant therapeutic effectiveness against PA-induced pneumonia. In summary, the proposed antibiotic drug-mediated DNA nanostructure assembly approach holds promise as a novel strategy for targeted treatment of PA infections.

Study on the inhibitory effect of quercetin combined with gentamicin on the formation of Pseudomonas aeruginosa and its bioenvelope

ObjectiveThe potential effects of quercetin and gentamicin combination on the bacteriostatic activity and biofilm formation of Pseudomonas aeruginosa (PA) were examined, and the findings provided a theoretical basis for the development of quercetin as a new biofilm inhibitor. MethodsThe minimum inhibitory concentration (MIC) of eight PAs was determined by microdilution method and the partial inhibitory concentration index (FICI) of the combined drug was analyzed by micro-dilution method. Thereafter, the lowest film inhibitory concentration (MBIC) of quercetin and gentamicin alone and in combination was evaluated by crystal violet staining. Finally, scanning electron microscopy (SEM) and laser confocal microscopy (CLSM) were used to decipher the inhibitory effect of the combination on biofilm formation. OutcomeThe antibacterial activity of quercetin alone was relatively weak, but after combination with gentamicin, the antibacterial activity was significantly enhanced, as evident by FICI of 0.28 and 0.53 and manifested as synergistic or additive effect, which indicated that quercetin can enhance gentamicin antibacterial activity. The results of crystal violet staining revealed that quercetin and gentamicin alone exhibited a similar biofilm formation inhibitory effect, but the inhibitory effect was substantially weaker, and the antibiofilm activity was stronger and exhibited a dose-dependent response after the combination of the two with 1/2FICI. The results of scanning electron microscopy and laser confocal microscopy also showed that the treatment of PA biofilm after combining quercetin and gentamicin with 1/2FICI could completely destroy the spatial structure of the complete biofilm, significantly reduce the thickness of bacteria, and markedly reduce the proportion of viable bacteria in the membrane. ConclusionThe combination of quercetin and gentamicin can effectively inhibit the formation of PA as well as its biofilm, and exhibit synergistic and additive effects.

In-silico and in-vitro assessment of the antibiofilm potential of azo dye, carmoisine against Pseudomonas aeruginosa

Biofilm is a community of microorganisms attached to the substrate and plays a significant role in microbial pathogenesis and medical device-related infection. Pseudomonas aeruginosa (PA) is a highly infectious gram-negative opportunistic biofilm-forming bacterium with high antibiotic resistance. Several reports underscore the antimicrobial activity of natural and synthetic food coloring agents, including carmoisine, turmeric dye, red amaranth dye, and phloxine B. However, their ability to suppress the PA biofilm is not clearly understood. Carmoisine is a red-colored synthetic azo dye containing naphthalene subunits and sulfonic groups and is widely used as a food coloring agent. This study investigated the antibiofilm potential and possible mechanism of biofilm inhibition by carmoisine against PA. Computational studies through molecular docking revealed that carmoisine strongly binds to QS regulator LasR (-12.7) and relatively less strongly but significantly with WspR (-6.9). Further analysis of the docked LasR-carmoisine complex using 100 ns MD simulation (Desmond, Schrödinger) validated the bonding strength and stability. Crystal violet assay, triphenyl tetrazolium chloride salt assay, and confocal microscopic studies were adopted for biofilm quantification, and the results indicated the dose-dependent antibiofilm activity of carmoisine against PA. We hypothesise that the carmoisine-mediated reduction of biofilm in PA is due to its interaction with LasR and interference with the QS system. The computational and biochemical analysis of another compound, 1,2-naphthoquinone-4-sulphonic acid, reiterated the role of the naphthalene ring in biofilm inhibition. Hence, this work will pave the way for the future discovery of antibiofilm drugs based on naphthalene ring-based lead compounds. Communicated by Ramaswamy H. Sarma

Degree of Gelatination on Ag-Nanoparticles to Inactivate Multi-drug Resistant Bacterial Biofilm Isolated from Sewage Treatment Plant.

Overuse and improper dosage of antibiotics have generated antimicrobial resistance (AMR) worldwide. Pseudomonas aeruginosa (PA), a well-known bacterial strain can establish MDR leading to a variety of infections in humans. Furthermore, these PA strains hold the ability to form biofilms by generating extracellular polymeric substances on the surface of medical tools and critical care units. To supersede the infectious effect of MDR organisms, silver nanoparticles have been known to be the choice. Hence, the present study concentrates on the engineering of varying concentrations of gelatin-based polymeric hydrogel embedded with silver nanoparticles (G-AgNPs) for controlled bactericidal activity against MDR PA biofilms. Biofilms formation by MDR PA was confirmed microscopically and spectroscopy was taken as a tool to characterize and analyze the efficacy at every stage of experiments. When MDR PA biofilms were treated with G-AgNPs prepared with 5 % gelatin concentration (AgNP3), they exhibited superior bactericidal activity. Furthermore, a dose-dependent study showed that 800 nM of AgNP3 could inhibit the growth of MDR PA. Hence it can be concluded that silver nanoparticles synthesized in the presence of 5% gelatin can act as a bactericidal agent in the inactivation of MDR PA biofilms, thereby controlling the infections caused by these biofilms.

Material Substrate Physical Properties Control Pseudomonas aeruginosa Biofilm Architecture.

In the wild, bacteria are most frequently found in the form of multicellular structures called biofilms. Biofilms grow at the surface of abiotic and living materials with wide-ranging mechanical properties. The opportunistic pathogen Pseudomonas aeruginosa forms biofilms on indwelling medical devices and on soft tissues, including burn wounds and the airway mucosa. Despite the critical role of substrates in the foundation of biofilms, we still lack a clear understanding of how material mechanics regulate their architecture and the physiology of resident bacteria. Here, we demonstrate that physical properties of hydrogel material substrates define P. aeruginosa biofilm architecture. We show that hydrogel mesh size regulates twitching motility, a surface exploration mechanism priming biofilms, ultimately controlling the organization of single cells in the multicellular community. The resulting architectural transitions increase P. aeruginosa's tolerance to colistin, a last-resort antibiotic. In addition, mechanical regulation of twitching motility affects P. aeruginosa clonal lineages, so that biofilms are more mixed on relatively denser materials. Our results thereby establish material properties as a factor that dramatically affects biofilm architecture, antibiotic efficacy, and evolution of the resident population. IMPORTANCE The biofilm lifestyle is the most widespread survival strategy in the bacterial world. Pseudomonas aeruginosa biofilms cause chronic infections and are highly recalcitrant to antimicrobials. The genetic requirements allowing P. aeruginosa to grow into biofilms are known, but not the physical stimuli that regulate their formation. Despite colonizing biological tissues, investigations of biofilms on soft materials are limited. In this work, we show that biofilms take unexpected forms when growing on soft substrates. The physical properties of the material shape P. aeruginosa biofilms by regulating surface-specific twitching motility. Physical control of biofilm morphogenesis ultimately influences the resilience of biofilms to antimicrobials, linking physical environment with tolerance to treatment. Altogether, our work established that the physical properties of a surface are a critical environmental regulator of biofilm biogenesis and evolution.

Effects of biofilms on the retention and transport of PFOA in saturated porous media.

Understanding the fate and transport of perfluorooctanoic acid (PFOA) in soil and groundwater is essential to reliable assessments of its risks. This study investigated the impacts of Gram-positive Bacillus subtilis (BS), Gram-negative Pseudomonas aeruginosa (PA) and wild microbiota (WM) biofilm on the transport of PFOA in saturated sand columns at two ionic strengths (i.e., 1.0 and 20.0mM NaCl). The retention of PFOA in biofilm-coated sand columns was higher than that in uncoated sand columns, due to biofilm-induced reinforced hydrophobic interactions and surface roughness, and decreased zeta potential. However, the retention effects varied among biofilm bacterial species with PFOA retardation factors in PA, WM and BS columns of 1.29-1.38, 1.21-1.29 and 1.11-1.15, respectively. Notably, PA biofilm had the most pronounced effect on PFOA retention. While increasing ionic strength promoted the retention of PFOA in BS biofilm-coated sand, it had no significant impact on PFOA transport in PA and WM biofilm-coated sand. This could be attributed to the differences in biofilm composition, deviating the ionic strengths effects on electrostatic double layer compression. The advection dispersion equation coupled with two-site kinetic retention model well described the transport of PFOA in all saturated columns. Our findings reveal that biofilm plays important roles in PFOA transport in porous media, instructive for risk assessment and remediation of PFOA contamination.

Towards Translation of PqsR Inverse Agonists: From In Vitro Efficacy Optimization to In Vivo Proof-of-Principle.

Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, which is involved in a wide range of dangerous infections. It develops alarming resistances toward antibiotic treatment. Therefore, alternative strategies, which suppress pathogenicity or synergize with antibiotic treatments are in great need to combat these infections more effectively. One promising approach is to disarm the bacteria by interfering with their quorum sensing (QS) system, which regulates the release of various virulence factors as well as biofilm formation. Herein, this work reports the rational design, optimization, and in-depth profiling of a new class of Pseudomonas quinolone signaling receptor (PqsR) inverse agonists. The resulting frontrunner compound features a pyrimidine-based scaffold, high in vitro and in vivo efficacy, favorable pharmacokinetics as well as clean safety pharmacology characteristics, which provide the basis for potential pulmonary as well as systemic routes of administration. An X-ray crystal structure in complex with PqsR facilitated further structure-guided lead optimization. The compound demonstrates potent pyocyanin suppression, synergizes with aminoglycoside antibiotic tobramycin against PA biofilms, and is active against a panel of clinical isolates from bronchiectasis patients. Importantly, this in vitro effect translated into in vivo efficacy in a neutropenic thigh infection model in mice providing a proof-of-principle for adjunctive treatment scenarios.

603. Comparing assays for Clinical Phage Microbiology in biofilm

Abstract Background Non-resolving infections are commonly associated with bacterial biofilm formation. One promising solution is the use of lytic bacterial bacteriophages, as an adjunctive approach with antibiotics to penetrate biofilms and cure infections. A major challenge in the use of phages for therapy is the need for accurate personal matching of the most effective phage-antibiotic combination against the target bactera. This matching, termed “Clinical Phage Microbiology” (CPM) is even more problematic regarding biofilm and there are no standard methods for that. Here we compared several approaches for CPM in biofilms. Methods The efficacy of phages on the biofilm was tested in 8 methods using 5 phages at two concentrations. To this end five Pseudomonas aeruginosa (PA) specific phages were grown for biofilm establishment. Next, we followed the metabolic activity of the cultures using: (1)Agilent Seahorse XF Analyzers., and (2) Symcel’s calScreener calorimeter. In addition, we measured the levels of extracellular DNA in the biofilm supernatant using (3) rtPCR and (4) online by SYBR Green without amplification. The 5th method used GFP PA14 with an allure-red dye that allows the assessment of the adherent bacteria only. Last, we used the commonly used (6) Colony Forming Units (CFU) counts, (7) Crystal Violet (CV), and (8) Live/dead stain as our gold standard controls. Results Out of these methods, the calScreener (Figure 1A-F), and Seahorse were able to differentiate between phages real-time. The calSreener also showed a clear difference between biofilm and planktonic growth curves. The Real-Time PCR and the Live dead stain methods have shown a significant differentiation at the treatment endpoint. The other methods, CFU, CV, Syber Green, and Allura red did not show a significant difference between the phages. It should be noted that phages biofilm ranking did not correlate with the planktonic one. Figure 1 heat flow was measured using calScreener over 21 hours, comparing 5 different phages at a PFU of 108 and 106 on Pseudomonas aeruginosa planktonic (A) or biofilm (B-F). (A) planktonic bacteria and PASA16 phage treated, (B) KLEIN 3 phage, (C) PASA16 phage, (D) PB2F phage, (E) D9 phage, (F) PB2C phage. Figure 2 Comparison, using rtPCR, of 5 different phages at a PFU of 10^8 and 10^6 treated for 24 hours, and then their supernatant was filtered. Primers for housekeeping genes (A) ldhD, (B) rpoD. Conclusion We have compared 8 different methods as assays for phage screening. In this model of PA biofilm and phages, we found that calScreener and Seahorse are superior while CFU, CV, Syber Green, Allura red are not as useful. Disclosures Ran Nir-Paz, MD, BiomX: Advisor/Consultant|Technophage: Advisor/Consultant.

Characterization of Distinct Biofilm Cell Subpopulations and Implications in Quorum Sensing and Antibiotic Resistance.

ABSTRACTBacteria change phenotypically in response to their environment. Free swimming cells transition to biofilm communities that promote cellular cooperativity and resistance to stressors and antibiotics. We uncovered three subpopulations of cells with diverse phenotypes from a single-species Pseudomonas aeruginosa PA14 biofilm, and used a series of steps to isolate, characterize, and map these cell subpopulations in a biofilm. The subpopulations were distinguishable by size and morphology using dynamic light scattering (DLS) and scanning electron microscopy (SEM). Additionally, growth and dispersal of biofilms originating from each cell subpopulation exhibited contrasting responses to antibiotic challenge. Cell subpopulation surface charges were distinctly different, which led us to examine the ionizable surface molecules associated with each subpopulation using mass spectrometry. Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of cell subpopulations revealed ions unique to each subpopulation of cells that significantly co-localized with ions associated with quorum sensing. Transcript levels of algR, lasR, and rhlI in subpopulations isolated from biofilms differed from levels in planktonic stationary and mid-log cell subpopulations. These studies provide insight into diverse phenotypes, morphologies, and biochemistries of PA14 cell subpopulations for potential applications in combating bacterial pathogenesis, with medical, industrial, and environmental complications.

Antimicrobial Resistance in Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa (PA) is part of a group of common nosocomial pathogens that exhibit multidrug resistance, thus proving to be a significant threat to healthcare. This study analyzes the ability of four commonly used antibiotics to observe eradication of the PA biofilm growth. Ceftazidime (CAZ), Tobramycin (TOB), Ofloxacin (OFLX), Meropenem (MEM), were tested against overnight cultures of PA strain PA01. The minimal inhibitory concentrations (MIC) of planktonic cells for all the four antibiotics were determined using broth microdilution while the minimal bactericidal concentrations (MBCs) were determined by colony count after antibiotic treatment and regrowth. Biofilm growth inhibition was performed by treating cells with antibiotic at the time of inoculation while eradication was determined by adding antibiotics 24 hours after inoculation, allowing mature biofilm formation, followed by the measurement of absorbance. PA planktonic cells exhibited highest susceptibility to MEM compared to overnight grown PA biofilm which demonstrated resistance to CAZ, complete sensitivity to ofloxacin, and minimal sensitivity to TOB and MEM. PA biofilm displayed dose-dependent sensitivity to TOB, MEM and OFLX, and a significant level of resistance to CAZ during inhibition phase. However, in the eradication phase, PA showed significant resistance to TOB followed by CAZ while PA biofilm showed sensitivity at higher concentrations of MEM. Our study exhibits that PA strain PA01 is resistant to ceftazidime in both planktonic and biofilm phases. While ofloxacin proved to be the most effective even at lower concentrations when compared with other antibiotics, tobramycin was most effective at higher concentrations for eradicating and inhibiting PA biofilms.

Nano- and Macroscale Imaging of Cholesterol Linoleate and Human Beta Defensin 2-Induced Changes in Pseudomonas aeruginosa Biofilms.

The biofilm production of Pseudomonas aeruginosa (PA) is central to establishing chronic infection in the airways in cystic fibrosis. Epithelial cells secrete an array of innate immune factors, including antimicrobial proteins and lipids, such as human beta defensin 2 (HBD2) and cholesteryl lineolate (CL), respectively, to combat colonization by pathogens. We have recently shown that HBD2 inhibits biofilm production by PA, possibly linked to interference with the transport of biofilm precursors. Considering that both HBD2 and CL are increased in airway fluids during infection, we hypothesized that CL synergizes with HBD2 in biofilm inhibition. CL was formulated in phospholipid-based liposomes (CL-PL). As measured by atomic force microscopy of single bacteria, CL-PL alone and in combination with HBD2 significantly increased bacterial surface roughness. Additionally, extracellular structures emanated from untreated bacterial cells, but not from cells treated with CL-PL and HBD2 alone and in combination. Crystal violet staining of the biofilm revealed that CL-PL combined with HBD2 effected a significant decrease of biofilm mass and increased the number of larger biofilm particles consistent with altered cohesion of formed biofilms. These data suggest that CL and HBD2 affect PA biofilm formation at the single cell and community-wide level and that the community-wide effects of CL are enhanced by HBD2. This research may inform future novel treatments for recalcitrant infections in the airways of CF patients.

A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry‐driven hit‐to‐lead optimization and in‐depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter‐gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI‐tobramycin (Tob) combination against PA biofilms using a tailor‐made squalene‐derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32‐fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker‐mediated therapy against PA infections opening up avenues for preclinical development.

Lactobacillus cell-free supernatant as a novel bioagent and biosurfactant against Pseudomonas aeruginosa in the prevention and treatment of orthopedic implant infection.

The hypothesis was that probiotic Lactobacillus species (spp.) or their cell-free supernatant (CFS) are effective in inhibiting (a) planktonic growth of Pseudomonas aeruginosa (PA), (b) its adhesion to a Ti6Al4V-alloy surface, and (c) in dispersing biofilm once formed. (a) A planktonic co-culture containing PA(104 colony-forming unit [CFU]/ml) was combined with either Lactobacillus acidophilus, Lactobacillus plantarum (LP), or Lactobacillus fermentum (LF) at a suspension of 104 (1:1) or 108 CFU/ml (1:2). Lactobacillus and PA CFUs were then quantified. (b) Ti-6Al-4V discs were inoculated with PA followed by supplementation with CFS and adherent PA quantified. (c) Biofilm covered discs were supplemented with Lactobacillus CFS and remaining PA activity quantified. Results showed that whole-cell cultures were ineffective in preventing PA growth; however, the addition of CFS resulted in a 99.99 ± 0.003% reduction in adherent PA in all Lactobacillus groups (p < .05 in all groups) with no viable PA growth measured in the LF and LP groups. Following PA biofilm formation, CFS resulted in a significant reduction in PA activity in all Lactobacillus groups (p ≤ .05 in all groups) with a 29.75 ± 15.98% increase measured in control samples. Supplementation with CFS demonstrated antiadhesive, antibiofilm, and toxic properties to PA.

In Vitro Newly Isolated Environmental Phage Activity against Biofilms Preformed by Pseudomonas aeruginosa from Patients with Cystic Fibrosis.

As disease worsens in patients with cystic fibrosis (CF), Pseudomonas aeruginosa (PA) colonizes the lungs, causing pulmonary failure and mortality. Progressively, PA forms typical biofilms, and antibiotic treatments determine multidrug-resistant (MDR) PA strains. To advance new therapies against MDR PA, research has reappraised bacteriophages (phages), viruses naturally infecting bacteria. Because few in vitro studies have tested phages on CF PA biofilms, general reliability remains unclear. This study aimed to test in vitro newly isolated environmental phage activity against PA isolates from patients with CF at Bambino Gesù Children’s Hospital (OBG), Rome, Italy. After testing in vitro phage activities, we combined phages with amikacin, meropenem, and tobramycin against CF PA pre-formed biofilms. We also investigated new emerging morphotypes and bacterial regrowth. We obtained 22 newly isolated phages from various environments, including OBG. In about 94% of 32 CF PA isolates tested, these phages showed in vitro PA lysis. Despite poor efficacy against chronic CF PA, five selected-lytic-phages (Φ4_ZP1, Φ9_ZP2, Φ14_OBG, Φ17_OBG, and Φ19_OBG) showed wide host activity. The Φ4_ZP1-meropenem and Φ14_OBG-tobramycin combinations significantly reduced CF PA biofilms (p < 0.001). To advance potential combined phage-antibiotic therapy, we envisage further in vitro test combinations with newly isolated phages, including those from hospital environments, against CF PA biofilms from early and chronic infections.

Aerosolized drug-loaded nanoparticles targeting migration inhibitory factors inhibit Pseudomonas aeruginosa-induced inflammation and biofilm formation.

Aim: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, which has been shown to promote disease severity in cystic fibrosis. Methods: In this study, aerosolized drug-loaded nanoparticlescontaining SCD-19, an inhibitor of MIF's tautomerase enzymatic activity, were developed and characterized. Results: The aerosolized nanoparticles had an optimal droplet size distribution for deep lung deposition, with a high degree of biocompatibility and significant cellular uptake. Conclusion: For the first time, we have developed an aerosolized nano-formulation against MIF's enzymatic activity that achieved a significant reduction in the inflammatory response of macrophages, and inhibited Pseudomonas aeruginosabiofilm formation on airway epithelial cells. This represents a potential novel adjunctive therapy for the treatment of P. aeruginosa infection in cystic fibrosis.

Role of Tobramycin in the Induction and Maintenance of Viable but Non-Culturable Pseudomonas aeruginosa in an In Vitro Biofilm Model

The recurrence of Pseudomonas aeruginosa (PA) biofilm infections is a major issue in cystic fibrosis (CF) patients. A pivotal role is played by the presence of antibiotic-unresponsive persisters and/or viable but non-culturable (VBNC) forms, whose development might be favored by subinhibitory antibiotic concentrations. The involvement of tobramycin and ciprofloxacin, widely used to treat CF PA lung infections, in the abundance of VBNC cells was investigated in PA biofilms models. In vitro biofilms of the laboratory strain PAO1-N and the clinical strain C24 were developed and starved by subculture for 170 days in a non-nutrient (NN) broth, unsupplemented or supplemented with one-quarter minimal inhibitory concentration (MIC) of tobramycin or ciprofloxacin. VBNC cells abundance, estimated as the difference between total live (detected by qPCR and flow cytometry) and colony forming unit (CFU) counts, showed a strain- and drug-specific pattern. A greater and earlier abundance of VBNC PAO1-N cells was detected in all conditions. Exposure of the C24 strain to NN and NN + ciprofloxacin induced only a transient VBNC subpopulation, which was more abundant and stable until the end of the experiment in tobramycin-exposed biofilms. The same response to tobramycin was observed in the PAO1-N strain. These findings suggest that low tobramycin concentrations might contribute to PA infection recurrence by favoring the development of VBNC forms.

657 Preliminary Study on the Effect of Various Antimicrobial Formulations Containing Silver Oxynitrate on Reducing Pseudomonas Aeruginosa Using an in-vivo Porcine Burn Wound Model

Abstract Introduction Silver has long been known for its antimicrobial effects and has been commonly applied topically to burn wounds for years. More recently, wound dressings compounded with silver ions, have been developed to prevent and treat wound infection in both burn and chronic wounds. Methods This preliminary study evaluates the effect of a proprietary silver oxynitrate creams on Pseudomonas aeruginosa (PA) biofilms using a well-established burn wound porcine model model. Swine were used due to their skins similarities to humans and response to wound treatments. Briefly, second degree burn wounds were created and inoculated with PA. Wounds were then covered for 24 hours with a polyurethane dressing to allow for biofilm formation. The polyurethane dressing was removed and wounds were randomly assigned to one of the following treatment groups: 1) silver oxynitrate cream 0%, 2) silver oxynitrate cream 4%, 3) silver oxynitrate cream 7%, 4) silver oxynitrate cream * 10%, 5) silver oxynitrate powder, 6) silver sulfadiazine cream (SSD)~, or 7) untreated control. All treatments groups were covered with a polyurethane dressing to prevent any cross contamination. On days 3 and 7 after wounding wounds were cultured using an established scrub technique. Results Silver oxynitrate powder was the most effective treatment group at reducing PA counts. Silver oxynitrate 10% formulation had a high percentage of bacterial reduction. On day 7, compared to untreated control the silver oxynitrate 4, 7 and 10% treatments showed a 3.45, 4.05, and 4.30 log CFU/ml reduction, respectively. Conclusions These studies suggest that the silver oxynitrate formulations can reduce the bacterial bioburden in vivo against wounds that have PA biofilms. Additional animals are needed to substantiate these findings. Applicability of Research to Practice Gram-negative bacteria such as PA pose a challenge for wound care practitioners and new effective therapies are needed.

Pseudomonas aeruginosa antibody response in cystic fibrosis decreases rapidly following lung transplantation

BackgroundSpecific Pseudomonas aeruginosa (PA) precipitating immunoglobulin G antibodies in serum are correlated with PA biofilm infection and are used as diagnostic and prognostic markers in cystic fibrosis (CF). The aim of this study was to examine the change of PA antibody response in CF patients after bilateral sequential lung transplantation (LTx). MethodsPA antibodies and airway bacteriology were retrospectively evaluated in 20 chronically infected CF patients, who underwent LTx between 2001 and 2016 at Rigshospitalet, Copenhagen. Yearly precipitin counts from one year before LTx and up to five years after LTx were compared. Monthly airway cultures were examined in the five-year period after LTx. In addition, crossed immunoelectrophoresis (CIE) were analysed for each patient for antigenic similarities from time of infection, pre-LTx and post-LTx. ResultsAll patients experienced a significant drop in PA antibodies from one year pre-LTx to one year post-LTx (p < 0.0001). The PA antibody level did not differ between those, who became reinfected immediately after LTx, and those, who did not. No patients regained the high pre-LTx precipitin levels in the following five years. The antigenic specificities of the sera post-LTx were in each patient similar to the antigenic specificities at the beginning of infection indicating a decades long memory of their immune response like an “immunological fingerprint”. ConclusionsAfter LTx a significant and continuous reduction in PA antibodies was observed. The reduction was independent of immediate reinfection after LTx. A novel three-factor explanatory model is presented.