Cystic Fibrosis Clinical Isolates Research Articles

EF-hand calcium sensor, EfhP, controls transcriptional regulation of iron uptake by calcium in Pseudomonas aeruginosa.

The human pathogen Pseudomonas aeruginosa (Pa) poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca2+ that are commonly present in CF nasal and lung fluids. In addition, a Ca2+-binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca2+-regulated virulence in P. aeruginosa. Here, we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca2+ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca2+ regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ), and several virulence factors, such as the production of pyocins. The Ca2+ regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the efhP transcription is controlled by Ca2+ and Fe, and this regulation required a Ca2+-dependent two-component regulatory system CarSR. Furthermore, the efhP expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca2+ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca2+ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to the host.IMPORTANCEPseudomonas aeruginosa (Pa) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified Pa rapid and adaptive transcriptional responses to Ca2+ levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca2+ sensor, EfhP, is required for at least 1/3 of the Ca2+ response, including the majority of the iron uptake mechanisms and the production of pyocins. Transcription of efhP itself is regulated by Ca2+ and Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in Pa infections. The findings establish the regulatory interconnectedness between Ca2+ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca2+ and associated regulatory mechanisms will serve in the development of future therapeutics targeting Pa's dangerous infections.

Effect of L-arginine on cystic fibrosis Pseudomonas aeruginosa biofilms.

Cystic fibrosis (CF) airways are L-arginine deficient which may affect susceptibility to infection with certain pathogens. The potential impact of L-arginine supplementation on Pseudomonas aeruginosa, a common CF airway pathogen, is unclear. This study investigated the effects of L-arginine on P. aeruginosa biofilm cultures, using the laboratory strain PAO1 and multi-drug resistant CF clinical isolates. P. aeruginosa biofilms were grown in a chambered cover-glass slide model for 24 h, then exposed to either L-arginine alone or combined with tobramycin for an additional 24 h. Biofilms were visualized using confocal microscopy, and viable cells were measured via plating for colony-forming units. Increasing concentrations of L-arginine in bacterial culture medium reduced the biovolume of P. aeruginosa in a dose-dependent manner. Notably, L-arginine concentrations within the physiological range (50 mM and 100 mM) in combination with tobramycin promoted biofilm growth, while higher concentrations (600 mM and 1200 mM) inhibited growth. These findings demonstrate the potential of L-arginine as an adjuvant therapy to inhaled tobramycin in treating P. aeruginosa infections in people with CF.

Synergistic Antimicrobial Effects of Ibuprofen Combined with Standard-of-Care Antibiotics against Cystic Fibrosis Pathogens.

Cystic fibrosis (CF) is a common life-shortening genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Lungs of CF patients are often colonized or infected with microorganisms requiring frequent courses of antibiotics. Antibiotic-resistant bacterial infections have been a growing concern in CF patients. Chronic bacterial infections and concomitant airway inflammation damage the lungs, ultimately leading to respiratory failure. Several clinical trials have demonstrated that high-dose ibuprofen reduces the rate of pulmonary function decline in CF patients. This beneficial effect has been attributed to the anti-inflammatory properties of ibuprofen. Previously, we have confirmed that high-dose ibuprofen demonstrates antimicrobial activity against P. aeruginosa both in vitro and in vivo. However, no study has examined the antimicrobial effect of combining ibuprofen with standard-of-care antimicrobials. Here, we evaluated the possible synergistic activity of combinations of common nonsteroidal anti-inflammatory drugs (NSAIDs), namely, ibuprofen, naproxen, and aspirin, with commonly used antibiotics for CF patients. The drug combinations were screened against different CF clinical isolates. Antibiotics that demonstrated increased efficacy in the presence of ibuprofen were further tested for potential synergistic effects between these NSAIDS and antimicrobials. Finally, a survival analysis of a P. aeruginosa murine infection model was used to demonstrate the efficacy of the most potent combination identified in in vitro screening. Our results suggest that combinations of ibuprofen with commonly used antibiotics demonstrate synergistic antimicrobial activity against drug-resistant, clinical bacterial strains in vitro. The efficacy of the combination of ceftazidime and ibuprofen against resistant P. aeruginosa was demonstrated in an in vivo pneumonia model.

88 Targeting eukaryotic-like serine/threonine protein kinases in Staphylococcus aureus to combat antimicrobial resistance in cystic fibrosis clinical isolates

88 Targeting eukaryotic-like serine/threonine protein kinases in Staphylococcus aureus to combat antimicrobial resistance in cystic fibrosis clinical isolates

Ceftolozane-Tazobactam against Pseudomonas aeruginosa Cystic Fibrosis Clinical Isolates in the Hollow-Fiber Infection Model: Challenges Imposed by Hypermutability and Heteroresistance.

Pseudomonas aeruginosa remains a challenge in chronic respiratory infections in cystic fibrosis (CF). Ceftolozane-tazobactam has not yet been evaluated against multidrug-resistant hypermutable P. aeruginosa isolates in the hollow-fiber infection model (HFIM). Isolates CW41, CW35, and CW44 (ceftolozane-tazobactam MICs of 4, 4, and 2 mg/L, respectively) from adults with CF were exposed to simulated representative epithelial lining fluid pharmacokinetics of ceftolozane-tazobactam in the HFIM. Regimens were continuous infusion (CI; 4.5 g/day to 9 g/day, all isolates) and 1-h infusions (1.5 g every 8 hours and 3 g every 8 hours, CW41). Whole-genome sequencing and mechanism-based modeling were performed for CW41. CW41 (in four of five biological replicates) and CW44 harbored preexisting resistant subpopulations; CW35 did not. For replicates 1 to 4 of CW41 and CW44, 9 g/day CI decreased bacterial counts to <3 log10 CFU/mL for 24 to 48 h, followed by regrowth and resistance amplification. Replicate 5 of CW41 had no preexisting subpopulations and was suppressed below ~3 log10 CFU/mL for 120 h by 9 g/day CI, followed by resistant regrowth. Both CI regimens reduced CW35 bacterial counts to <1 log10 CFU/mL by 120 h without regrowth. These results corresponded with the presence or absence of preexisting resistant subpopulations and resistance-associated mutations at baseline. Mutations in ampC, algO, and mexY were identified following CW41 exposure to ceftolozane-tazobactam at 167 to 215 h. Mechanism-based modeling well described total and resistant bacterial counts. The findings highlight the impact of heteroresistance and baseline mutations on the effect of ceftolozane-tazobactam and limitations of MIC to predict bacterial outcomes. The resistance amplification in two of three isolates supports current guidelines that ceftolozane-tazobactam should be utilized together with another antibiotic against P. aeruginosa in CF.

WS07.04 Hostile takeover factors of Pseudomonas aeruginosa: contact dependent secretion systems

Objectives: Pseudomonas aeruginosa is the most common cause of chronic pulmonary infections in Cystic Fibrosis (CF) patients, dominating and damaging the lung environment. P. aeruginosa can deliver bacterial-derived toxins into competing neighbours to inhibit growth or kill via the contact-dependent inhibition (CDI) systems. However, the role of CDI systems in Pseudomonas infection in Cystic Fibrosis clinical isolates and dominance during chronic infection remains unclear. Here we bioinformatically and experimentally investigate the CDI systems encoded in P. aeruginosa strain PA14.

Short chain fatty acids reduce the respiratory burst of human neutrophils in response to cystic fibrosis isolates of Staphylococcus aureus

Short chain fatty acids (SCFA) are produced by anaerobic bacteria. The most common SCFAs are acetate, propionate and butyrate. SCFAs have been implicated in several inflammatory diseases including cystic fibrosis (CF) where they are present in the airways at millimolar concentrations. Staphylococcus aureus is one of the main respiratory pathogens in CF. Polymorphonuclear neutrophil granulocytes (PMN) represent the most important immune defense the host uses against S. aureus. However, the reason why PMNs are unable to clear S. aureus in CF remains largely unclear. We hypothesized that SCFAs impair effector functions of PMNs in response to S. aureus. To test this, human PMNs were exposed to CF clinical isolates of S. aureus in vitro in the presence or absence of SCFAs and effector functions of PMNs were assessed. Our data show that SCFAs do not affect the viability of PMNs and do not stimulate the release of neutrophil extracellular traps (NET) from human PMNs. Production of reactive oxygen species (ROS), another important antimicrobial function of PMNs, on the other hand, was significantly inhibited by SCFAs in response to the bacterium. SCFAs did not compromise the ability of PMNs to kill CF isolates of S. aureus in vitro. Overall, our results provide new knowledge into the interactions between SCFAs and the immune system, and indicate that SCFAs produced by anaerobic bacteria in the CF lung could interfere with reactive oxidant production of PMNs in response to S. aureus, one of the prominent respiratory pathogens in this disease.

In vitro Activity of Antivirulence Drugs Targeting the las or pqs Quorum Sensing Against Cystic Fibrosis Pseudomonas aeruginosa Isolates.

The chronic lung infection caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Antivirulence drugs targeting P. aeruginosa quorum sensing (QS) systems are intensively studied as antibiotics substitutes or adjuvants. Previous studies, carried out in non-CF P. aeruginosa reference strains, showed that the old drugs niclosamide and clofoctol could be successfully repurposed as antivirulence drugs targeting the las and pqs QS systems, respectively. However, frequent emergence of QS-defective mutants in the CF lung undermines the use of QS inhibitors in CF therapy. Here, QS signal production and susceptibility to niclosamide and clofoctol have been investigated in 100 P. aeruginosa CF isolates, with the aim of broadening current knowledge on the potential of anti-QS compounds in CF therapy. Results showed that 85, 78, and 69% of the CF isolates from our collection were proficient for the pqs, rhl, and las QS systems, respectively. The ability of both niclosamide and clofoctol to inhibit QS and virulence in vitro was highly variable and strain-dependent. Niclosamide showed an overall low range of activity and its negative effect on las signal production did not correlate with a decreased production of virulence factors. On the other hand, clofoctol displayed a broader QS inhibitory effect in CF isolates, with consequent reduction of the pqs-controlled virulence factor pyocyanin. Overall, this study highlights the importance of testing new antivirulence drugs against large panels of P. aeruginosa CF clinical isolates before proceeding to further pre-clinical studies and corroborates previous evidence that strains naturally resistant to QS inhibitors occur among CF isolates. However, it is also shown that resistance to pqs inhibitors is less frequent than resistance to las inhibitors, thus supporting the development of pqs inhibitors for antivirulence therapy in CF.

Polycationic Glycopolymer Demonstrates Activity Against Persisters and Biofilms of Non-tuberculosis Mycobacteria Cystic Fibrosis Clinical Isolates in vitro.

Non-tuberculosis Mycobacterium (NTM) is a group of opportunistic pathogens associated with pulmonary infections that are difficult to diagnose and treat. Standard treatment typically consists of prolonged combination antibiotic therapy. Antibiotic resistance and the role of biofilms in pathogen communities, such as NTM persister cells, is an important unmet challenge that leads to increased toxicity, frequent relapse, poor clinical management, and an extended treatment period. Infection recurrence and relapse are not uncommon among individuals with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD), where thick mucus supports bacterial biofilm production and impairs mucociliary clearance. The study evaluates a membrane-active cationic glycopolymer [poly (acetyl, arginyl) glucosamine (PAAG)] being developed to support the safe and effective treatment of NTM biofilm infections. PAAG shows antibacterial activity against a wide range of pathogenic bacteria at concentrations non-toxic to human epithelial cells. Time-kill curves demonstrated PAAG’s rapid bactericidal potential at concentrations as low as 1X MIC against all NTM strains tested and compared to the standard of care. PAAG treatment prevents persister formation and eradicates antibiotic-induced persister cells in planktonic NTM cultures below the limit of detection (10 colony-forming unit (CFU)/ml). Further, PAAG showed the ability to penetrate and disperse NTM biofilms formed by both rapidly and slowly growing strains, significantly reducing the biofilm biomass (p < 0.0001) compared to the untreated NTM biofilms. Microscopical examination confirmed PAAG’s ability to disrupt and disperse mycobacterial biofilms. A single PAAG treatment resulted in up to a 25-fold reduction in live-labeled NTM and a 78% reduction in biofilm thickness. Similar to other polycationic molecules, PAAG’s bactericidal and antibiofilm activities employ rapid permeabilization of the outer membrane of the NTM strains, and subsequently, reduce the membrane potential even at concentrations as low as 50 μg/ml (p < 0.001). The outcomes of these in vitro analyses suggest the importance of this polycationic glycopolymer, PAAG, as a potential therapeutic agent for opportunistic NTM infections.

Cystic Fibrosis Clinical Isolates of Aspergillus fumigatus Induce Similar Muco-inflammatory Responses in Primary Airway Epithelial Cells.

Aspergillus is increasingly associated with lung inflammation and mucus plugging in early cystic fibrosis (CF) disease during which conidia burden is low and strains appear to be highly diverse. It is unknown whether clinical Aspergillus strains vary in their capacity to induce epithelial inflammation and mucus production. We tested the hypothesis that individual colonising strains of Aspergillus fumigatus would induce different responses. Ten paediatric CF Aspergillus isolates were compared along with two systemically invasive clinical isolates and an ATCC reference strain. Isolates were first characterised by ITS gene sequencing and screened for antifungal susceptibility. Three clusters (A−C) of Aspergillus isolates were identified by ITS. Antifungal susceptibility was variable, particularly for itraconazole. Submerged CF and non-CF monolayers as well as differentiated primary airway epithelial cell cultures were incubated with conidia for 24 h to allow germination. None of the clinical isolates were found to significantly differ from one another in either IL-6 or IL-8 release or gene expression of secretory mucins. Clinical Aspergillus isolates appear to be largely homogenous in their mucostimulatory and immunostimulatory capacities and, therefore, only the antifungal resistance characteristics are likely to be clinically important.

Murine Model of Sinusitis Infection for Screening Antimicrobial and Immunomodulatory Therapies.

The very common condition of sinusitis is characterized by persistent inflammation of the nasal cavity, which contributes to chronic rhinosinusitis and morbidity of cystic fibrosis patients. Colonization by opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa triggers inflammation that is exacerbated by defects in the innate immune response. Pathophysiological mechanisms underlying initial colonization of the sinuses are not well established. Despite their extensive use, current murine models of acute bacterial rhinosinusitis have not improved the understanding of early disease stages due to analytical limitations. In this study, a model is described that is technically simple, allows non-invasive tracking of bacterial infection, and screening of host-responses to infection and therapies. The model was modified to investigate longer-term infection and disease progression by using a less virulent, epidemic P. aeruginosa cystic fibrosis clinical isolate LESB65. Tracking of luminescent bacteria was possible after intranasal infections, which were sustained for up to 120 h post-infection, without compromising the overall welfare of the host. Production of reactive oxidative species was associated with neutrophil localization to the site of infection in this model. Further, host-defense peptides administered by Respimat® inhaler or intranasal instillation reduced bacterial burden and impacted disease progression as well as cytokine responses associated with rhinosinusitis. Thus, future studies using this model will improve our understanding of rhinosinusitis etiology and early stage pathogenesis, and can be used to screen for the efficacy of emerging therapies pre-clinically.

WS01.2 Impact of estradiol on Pseudomonas aeruginosa biofilms derived from cystic fibrosis clinical isolates

WS01.2 Impact of estradiol on Pseudomonas aeruginosa biofilms derived from cystic fibrosis clinical isolates

(R)-Roscovitine and CFTR modulators enhance killing of multi-drug resistant Burkholderia cenocepacia by cystic fibrosis macrophages

Cystic fibrosis (CF) is characterized by chronic bacterial infections and heightened inflammation. Widespread ineffective antibiotic use has led to increased isolation of drug resistant bacterial strains from respiratory samples. (R)-roscovitine (Seliciclib) is a unique drug that has many benefits in CF studies. We sought to determine roscovitine’s impact on macrophage function and killing of multi-drug resistant bacteria. Human blood monocytes were isolated from CF (F508del/F508del) and non-CF persons and derived into macrophages (MDMs). MDMs were infected with CF clinical isolates of B. cenocepacia and P. aeruginosa. MDMs were treated with (R)-roscovitine or its main hepatic metabolite (M3). Macrophage responses to infection and subsequent treatment were determined. (R)-roscovitine and M3 significantly increased killing of B. cenocepacia and P. aeruginosa in CF MDMs in a dose-dependent manner. (R)-roscovitine-mediated effects were partially dependent on CFTR and the TRPC6 channel. (R)-roscovitine-mediated killing of B. cenocepacia was enhanced by combination with the CFTR modulator tezacaftor/ivacaftor and/or the alternative CFTR modulator cysteamine. (R)-roscovitine also increased MDM CFTR function compared to tezacaftor/ivacaftor treatment alone. (R)-roscovitine increases CF macrophage-mediated killing of antibiotic-resistant bacteria. (R)-roscovitine also enhances other macrophage functions including CFTR-mediated ion efflux. Effects of (R)-roscovitine are greatest when combined with CFTR modulators or cysteamine, justifying further clinical testing of (R)-roscovitine or optimized derivatives.

Proteomic Studies of the Biofilm Matrix including Outer Membrane Vesicles of Burkholderia multivorans C1576, a Strain of Clinical Importance for Cystic Fibrosis.

Biofilms are aggregates of microbial cells encased in a highly hydrated matrix made up of self-produced extracellular polymeric substances (EPS) which consist of polysaccharides, proteins, nucleic acids, and lipids. While biofilm matrix polysaccharides are unraveled, there is still poor knowledge about the identity and function of matrix-associated proteins. With this work, we performed a comprehensive proteomic approach to disclose the identity of proteins associated with the matrix of biofilm-growing Burkholderia multivorans C1576 reference strain, a cystic fibrosis clinical isolate. Transmission electron microscopy showed that B. multivorans C1576 also releases outer membrane vesicles (OMVs) in the biofilm matrix, as already demonstrated for other Gram-negative species. The proteomic analysis revealed that cytoplasmic and membrane-bound proteins are widely represented in the matrix, while OMVs are highly enriched in outer membrane proteins and siderophores. Our data suggest that cell lysis and OMVs production are the most important sources of proteins for the B. multivorans C1576 biofilm matrix. Of note, some of the identified proteins are lytic enzymes, siderophores, and proteins involved in reactive oxygen species (ROS) scavenging. These proteins might help B. multivorans C1576 in host tissue invasion and defense towards immune system assaults.

Optimization of nitric oxide donors for investigating biofilm dispersal response in Pseudomonas aeruginosa clinical isolates

Pseudomonas aeruginosa biofilms contribute heavily to chronic lung infection in cystic fibrosis patients, leading to morbidity and mortality. Nitric oxide (NO) has been shown to disperse P. aeruginosa biofilms in vitro, ex vivo and in clinical trials as a promising anti-biofilm agent. Traditional NO donors such as sodium nitroprusside (SNP) have been extensively employed in different studies. However, the dosage of SNP in different studies was not consistent, ranging from 500 nM to 500 μM. SNP is light sensitive and produces cyanide, which may lead to data misinterpretation and inaccurate predictions of dispersal responses in clinical settings. New NO donors and NO delivery methods have therefore been explored. Here we assessed 7 NO donors using P. aeruginosa PAO1 and determined that SNP and Spermine NONOate (S150) successfully reduced > 60% biomass within 24 and 2 h, respectively. While neither dosage posed toxicity towards bacterial cells, chemiluminescence assays showed that SNP only released NO upon light exposure in M9 media and S150 delivered much higher performance spontaneously. S150 was then tested on 13 different cystic fibrosis P. aeruginosa (CF-PA) isolates; most CF-PA biofilms were significantly dispersed by 250 μM S150. Our work therefore discovered a commercially available NO donor S150, which disperses CF-PA biofilms efficiently within a short period of time and without releasing cyanide, as an alternative of SNP in clinical trials in the future.Key points• S150 performs the best in dispersing P. aeruginosa biofilms among 7 NO donors.• SNP only releases NO in the presence of light, while S150 releases NO spontaneously.• S150 successfully disperses biofilms formed by P. aeruginosa cystic fibrosis clinical isolates.

RhlR-Regulated Acyl-Homoserine Lactone Quorum Sensing in a Cystic Fibrosis Isolate of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhlR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhlR regulon in chronic infection isolates and suggest further investigation of RhlR as a possible target for therapeutic development in chronic infections.IMPORTANCEPseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR in chronic P. aeruginosa infections.

Effects of Signal Disruption Depends on the Substrate Preference of the Lactonase.

Many bacteria produce and use extracellular signaling molecules such as acyl homoserine lactones (AHLs) to communicate and coordinate behavior in a cell-density dependent manner, via a communication system called quorum sensing (QS). This system regulates behaviors including but not limited to virulence and biofilm formation. We focused on Pseudomonas aeruginosa, a human opportunistic pathogen that is involved in acute and chronic lung infections and which disproportionately affects people with cystic fibrosis. P. aeruginosa infections are becoming increasingly challenging to treat with the spread of antibiotic resistance. Therefore, QS disruption approaches, known as quorum quenching, are appealing due to their potential to control the virulence of resistant strains. Interestingly, P. aeruginosa is known to simultaneously utilize two main QS circuits, one based on C4-AHL, the other with 3-oxo-C12-AHL. Here, we evaluated the effects of signal disruption on 39 cystic fibrosis clinical isolates of P. aeruginosa, including drug resistant strains. We used two enzymes capable of degrading AHLs, known as lactonases, with distinct substrate preference: one degrading 3-oxo-C12-AHL, the other degrading both C4-AHL and 3-oxo-C12-AHL. Two lactonases were used to determine the effects of signal disruption on the clinical isolates, and to evaluate the importance of the QS circuits by measuring effects on virulence factors (elastase, protease, and pyocyanin) and biofilm formation. Signal disruption results in at least one of these factors being inhibited for most isolates (92%). Virulence factor activity or production were inhibited by up to 100% and biofilm was inhibited by an average of 2.3 fold. Remarkably, the treatments led to distinct inhibition profiles of the isolates; the treatment with the lactonase degrading both signaling molecules resulted in a higher fraction of inhibited isolates (77% vs. 67%), and the simultaneous inhibition of more virulence factors per strain (2 vs. 1.5). This finding suggests that the lactonase AHL preference is key to its inhibitory spectrum and is an essential parameter to improve quorum quenching strategies.

High Prevalence of Staphylococcus aureus Enterotoxin Gene Cluster Superantigens in Cystic Fibrosis Clinical Isolates.

Background: Staphylococcus aureus is a highly prevalent respiratory pathogen in cystic fibrosis (CF). It is unclear how this organism establishes chronic infections in CF airways. We hypothesized that S. aureus isolates from patients with CF would share common virulence properties that enable chronic infection. Methods: 77 S. aureus isolates were obtained from 45 de-identified patients with CF at the University of Iowa. We assessed isolates phenotypically and used genotyping assays to determine the presence or absence of 18 superantigens (SAgs). Results: We observed phenotypic diversity among S. aureus isolates from patients with CF. Genotypic analysis for SAgs revealed 79.8% of CF clinical isolates carried all six members of the enterotoxin gene cluster (EGC). MRSA and MSSA isolates had similar prevalence of SAgs. We additionally observed that EGC SAgs were prevalent in S. aureus isolated from two geographically distinct CF centers. Conclusions: S. aureus SAgs belonging to the EGC are highly prevalent in CF clinical isolates. The greater prevalence in these SAgs in CF airway specimens compared to skin isolates suggests that these toxins confer selective advantage in the CF airway.

Genomic Analysis Identifies Novel Pseudomonas aeruginosa Resistance Genes under Selection during Inhaled Aztreonam Therapy In Vivo.

Inhaled aztreonam is increasingly used for chronic Pseudomonas aeruginosa suppression in patients with cystic fibrosis (CF), but the potential for that organism to evolve aztreonam resistance remains incompletely explored. Here, we performed genomic analysis of clonally related pre- and posttreatment CF clinical isolate pairs to identify genes that are under positive selection during aztreonam therapy in vivo We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent being ftsI and ampC, and 13 of which increased aztreonam resistance when introduced as single gene transposon mutants. Several previously implicated aztreonam resistance genes were found to be under positive selection in clinical isolates even in the absence of inhaled aztreonam exposure, indicating that other selective pressures in the cystic fibrosis airway can promote aztreonam resistance. Given its potential to confer plasmid-mediated resistance, we further characterized mutant ampC alleles and performed artificial evolution of ampC for maximal activity against aztreonam. We found that naturally occurring ampC mutants conferred variably increased resistance to aztreonam (2- to 64-fold) and other β-lactam agents but that its maximal evolutionary capacity for hydrolyzing aztreonam was considerably higher (512- to 1,024-fold increases) and was achieved while maintaining or increasing resistance to other drugs. These studies implicate novel chromosomal aztreonam resistance determinants while highlighting that different mutations are favored during selection in vivo and in vitro, show that ampC has a high maximal potential to hydrolyze aztreonam, and provide an approach to disambiguate mutations promoting specific resistance phenotypes from those more generally increasing bacterial fitness in vivo.

Hydrogels Embedded With Melittin and Tobramycin Are Effective Against Pseudomonas aeruginosa Biofilms in an Animal Wound Model.

We demonstrate that the antimicrobial peptide, melittin, is effective alone and in combination with the aminoglycosides tobramycin to kill Pseudomonas aeruginosa growing as biofilms both in vitro and in vivo. Melittin and tobramycin show enhanced in vitro activity in combination at micromolar concentrations, resulting in a 2-log10 reduction in the number of cells within mature PAO1 P. aeruginosa biofilms after 6-h of treatment. Alternatively, either agent alone resulted in half-a-log10 reduction. Time-killing assays demonstrated that the combination of melittin and tobramycin was effective at 2-h whereas tobramycin was not effective until after 6-h of treatment. We also found the combination was more effective than tobramycin alone against biofilms of 7 P. aeruginosa cystic fibrosis clinical isolates, resulting in a maximum 1.5-log10 cellular reduction. Additionally, melittin alone was effective at killing biofilms of 4 Staphylococcus aureus isolates, resulting in a maximum 2-log10 cellular reduction. Finally, melittin in combination with tobramycin embedded in an agarose-based hydrogel resulted in a 4-fold reduction in bioluminescent P. aeruginosa colonizing mouse wounds by 4-h. In contrast, tobramycin or melittin treatment alone did not cause a statistically significant reduction in bioluminescence. These data demonstrate that melittin in combination with tobramycin embedded in a hydrogel is a potential treatment for biofilm-associated wound infections.