Mucoidy,Quorum Sensing,Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.     

LasR-deficient Pseudomonas aeruginosa variants increase airway epithelial mICAM-1 expression and enhance neutrophilic lung inflammation

Pseudomonas aeruginosa causes chronic airway infections, a major determinant of lung inflammation and damage in cystic fibrosis (CF). Loss-of-function lasR mutants commonly arise during chronic CF infections, are associated with accelerated lung function decline in CF patients and induce exaggerated neutrophilic inflammation in model systems. In this study, we investigated how lasR mutants modulate airway epithelial membrane bound ICAM-1 (mICAM-1), a surface adhesion molecule, and determined its impact on neutrophilic inflammation in vitro and in vivo. We demonstrated that LasR-deficient strains induce increased mICAM-1 levels in airway epithelial cells compared to wild-type strains, an effect attributable to the loss of mICAM-1 degradation by LasR-regulated proteases and associated with enhanced neutrophil adhesion. In a subacute airway infection model, we also observed that lasR mutant-infected mice displayed greater airway epithelial ICAM-1 expression and increased neutrophilic pulmonary inflammation. Our findings provide new insights into the intricate interplay between lasR mutants, LasR-regulated proteases and airway epithelial ICAM-1 expression, and reveal a new mechanism involved in the exaggerated inflammatory response induced by lasR mutants.     

Study on pyoverdine and biofilm production with detection of LasR gene in MDR Pseudomonas aeruginosa

In cystic fibrosis individuals, chronic lung infections and hospital-acquired pneumonia are caused by Pseudomonas aeruginosa. P. aeruginosa generates siderophores such as pyoverdine (PVD) as iron uptake systems to cover its needs of iron ions for growth and infection. lasR quorum sensing (QS) gene has a crucial function in PVD production and biofilm generation in P. aeruginosa. Fifty isolates of P. aeruginosa were obtained from clinical specimens of sputum (collected from individuals suffering from pulmonary infections). Antibiotic sensitivity test was performed for 50P. aeruginosa isolates by using 10 different types of antibiotics. All isolates of P. aeruginosa showed resistance for all 10 using antibiotics in this study. Ten multidrug resistant isoloates of P. aeruginosa were selected for next tests. Virulence factors of ten multidrug resistant isolates of P. aeruginosa, such as biofilm generation, PVD production, and lasR gene were detected. From results, all 10P. aeruginosa isolates can produce biofilm, PVD, and contain lasR gene. The produced amplicon for the lasR gene was 725 bp. After mice injection by fresh and heated PVD produced by P. aeruginosa PS10 LC619328.2, the fresh PVD caused 100 % mortality within five days using 0.3 ml of its concentration (37.4 µM), while (15.3 µM) of heated PVD (toxoid) caused 50 % mortality.     

Genotypic Diversity within a Single Pseudomonas aeruginosa Strain Commonly Shared by Australian Patients with Cystic Fibrosis

In cystic fibrosis (CF), Pseudomonas aeruginosa undergoes intra-strain genotypic and phenotypic diversification while establishing and maintaining chronic lung infections. As the clinical significance of these changes is uncertain, we investigated intra-strain diversity in commonly shared strains from CF patients to determine if specific gene mutations were associated with increased antibiotic resistance and worse clinical outcomes. Two-hundred-and-one P. aeruginosa isolates (163 represented a dominant Australian shared strain, AUST-02) from two Queensland CF centres over two distinct time-periods (2001–2002 and 2007–2009) underwent mexZ and lasR sequencing. Broth microdilution antibiotic susceptibility testing in a subset of isolates was also performed. We identified a novel AUST-02 subtype (M3L7) in adults attending a single Queensland CF centre. This M3L7 subtype was multi-drug resistant and had significantly higher antibiotic minimum inhibitory concentrations than other AUST-02 subtypes. Prospective molecular surveillance using polymerase chain reaction assays determined the prevalence of the ‘M3L7’ subtype at this centre during 2007–2009 (170 patients) and 2011 (173 patients). Three-year clinical outcomes of patients harbouring different strains and subtypes were compared. MexZ and LasR sequences from AUST-02 isolates were more likely in 2007–2009 than 2001–2002 to exhibit mutations (mexZ: odds ratio (OR) = 3.8; 95% confidence interval (CI): 1.1–13.5 and LasR: OR = 2.5; 95%CI: 1.3–5.0). Surveillance at the adult centre in 2007–2009 identified M3L7 in 28/509 (5.5%) P. aeruginosa isolates from 13/170 (7.6%) patients. A repeat survey in 2011 identified M3L7 in 21/519 (4.0%) P. aeruginosa isolates from 11/173 (6.4%) patients. The M3L7 subtype was associated with greater intravenous antibiotic and hospitalisation requirements, and a higher 3-year risk of death/lung transplantation, than other AUST-02 subtypes (adjusted hazard ratio [HR] = 9.4; 95%CI: 2.2–39.2) and non-AUST-02 strains (adjusted HR = 4.8; 95%CI: 1.4–16.2). This suggests ongoing microevolution of the shared CF strain, AUST-02, was associated with an emerging multi-drug resistant subtype and possibly poorer clinical outcomes.     

Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation

Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting.     

Bacterial cis-2-unsaturated fatty acids found in the cystic fibrosis airway modulate virulence and persistence of Pseudomonas aeruginosa

There is an increasing appreciation of the polymicrobial nature of many bacterial infections such as those associated with cystic fibrosis (CF) and of the potentially important role for interspecies interactions in influencing both bacterial virulence and response to therapy. Patients with CF are often co-infected with Pseudomonas aeruginosa and other pathogens including Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. We have previously shown by in vitro studies that DSF from S. maltophilia leads to altered biofilm formation and increased resistance to antibiotics by P. aeruginosa; these responses of P. aeruginosa require the sensor kinase PA1396. Here we show that DSF signals are present in sputum taken from patients with CF. Presence of these DSF signals was correlated with patient colonization by S. maltophilia and/or B. cenocepacia. Analysis of 50 clinical isolates of P. aeruginosa showed that each responded to the presence of synthetic DSF by increased antibiotic resistance and these strains demonstrated little sequence variation in the PA1396 gene. In animal experiments using CF transmembrane conductance regulator knockout mice, the presence of DSF promoted P. aeruginosa persistence. Furthermore, antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells was enhanced in the presence of DSF. Taken together, these data provide substantial evidence that interspecies DSF-mediated bacterial interactions occur in the CF lung and may influence the efficacy of antibiotic treatment, particularly for chronic infections involving persistence of bacteria.     

Universal soldier: Pseudomonas aeruginosa — an opportunistic generalist

The opportunistic pathogen Pseudomonas aeruginosa commonly causes chronic and ultimately deadly lung infections in individuals with the genetic disease cystic fibrosis (CF). P. aeruginosa is metabolically diverse; it displays a remarkable ability to adapt to and successfully occupy almost any niche, including the ecologically complex CF lung. These P. aeruginosa lung infections are a fascinating example of microbial evolution within a “natural” ecosystem. Initially, P. aeruginosa shares the lung niche with a plethora of other microorganisms and is vulnerable to antibiotic challenges. Over time, adaptive evolution leads to certain commonly-observed phenotypic changes within the P. aeruginosa population, some of which render it resistant to antibiotics and apparently help it to out-compete the other species that co-habit the airways. Improving genomics techniques continue to elucidate the evolutionary mechanisms of P. aeruginosa within the CF lung and will hopefully identify new vulnerabilities in this robust and versatile pathogen.     

Decreased efficacy of antimicrobial agents in a polymicrobial environment

The airways of people with cystic fibrosis (CF) often harbour diverse polymicrobial communities. These airway infections can be impossible to resolve through antibiotic intervention, even though isolates of the individual species present are susceptible to the treatment when tested in vitro. In this work, we investigate how polymicrobial cultures comprised of key CF-associated pathogens respond to challenge with species-specific antimicrobial agents; colistin (targets Pseudomonas aeruginosa), fusidic acid (targets Staphylococcus aureus), and fluconazole (targets Candida albicans). We found that growth in a polymicrobial environment protects the target microorganism (sometimes by several orders of magnitude) from the effect(s) of the antimicrobial agent. This decreased antimicrobial efficacy was found to have both non-heritable (physiological) and heritable (genetic) components. Whole-genome sequencing of the colistin-resistant P. aeruginosa isolates revealed single nucleotide polymorphisms and indels in genes encoding lipopolysaccharide (LPS) biosynthesis and/or pilus biogenesis, indicating that a previously undescribed colistin resistance mechanism was in operation. This was subsequently confirmed through further genetic analyses. Our findings indicate that the polymicrobial nature of the CF airways is likely to have a significant impact on the clinical response to antimicrobial therapy.Subject terms: Clinical microbiology, Antibiotics     

Phenotypic Heterogeneity of Pseudomonas aeruginosa Populations in a Cystic Fibrosis Patient

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lower airways of patients with cystic fibrosis. Throughout the course of infection this organism undergoes adaptations that contribute to its long-term persistence in the airways. While P. aeruginosa diversity has been documented, it is less clear to what extent within-patient diversity contributes to the overall population structure as most studies have been limited to the analysis of only a few isolates per patient per time point. To examine P. aeruginosa population structure in more detail we collected multiple isolates from individual sputum samples of a patient chronically colonized with P. aeruginosa. This strain collection, comprised of 169 clonal isolates and representing three pulmonary exacerbations as well as clinically stable periods, was assayed for a wide selection of phenotypes. These phenotypes included colony morphology, motility, quorum sensing, protease activity, auxotrophy, siderophore levels, antibiotic resistance, and growth profiles. Each phenotype displayed significant variation even within isolates of the same colony morphotype from the same sample. Isolates demonstrated a large degree of individuality across phenotypes, despite being part of a single clonal lineage, suggesting that the P. aeruginosa population in the cystic fibrosis airways is being significantly under-sampled.     

Molecular Epidemiology of Chronic Pseudomonas aeruginosa Airway Infections in Cystic Fibrosis

Background/Methods

The molecular epidemiology of the chronic airway infections with Pseudomonas aeruginosa in individuals with cystic fibrosis (CF) was investigated by cross-sectional analysis of bacterial isolates from 51 CF centers and by longitudinal analysis of serial isolates which had been collected at the CF centers Hanover and Copenhagen since the onset of airway colonization over 30 years.

Results

Genotyping revealed that the P. aeruginosa population in CF is dominated by a few ubiquitous clones. The five most common clones retrieved from the CF host also belonged to the twenty most frequent clones in the environment and in other human disease habitats. Turnover of clones in CF airways was rare. At the Hanover clinic more than half of the patient cohort was still harbouring the initially acquired clone after twenty years of airway colonization. At the Copenhagen clinic, however, two rare clones replaced the initially acquired individual clones in all but one patient.

Conclusion

The divergent epidemiology at the two sites is explained by their differential management of hygiene and antipseudomonal chemotherapy. Hygienic measures to prohibit patient-to-patient transmission and the modalities of antipseudomonal chemotherapy modify the epidemiology of the chronic P. aeruginosa infections in CF.     

Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients

Pseudomonas aeruginosa is the predominant microorganism in chronic lung infection of cystic fibrosis patients. The chronic lung infection is preceded by intermittent colonization. When the chronic infection becomes established, it is well accepted that the isolated strains differ phenotypically from the intermittent strains. Dominating changes are the switch to mucoidity (alginate overproduction) and loss of epigenetic regulation of virulence such as the Quorum Sensing (QS). To elucidate the dynamics of P. aeruginosa QS systems during long term infection of the CF lung, we have investigated 238 isolates obtained from 152 CF patients at different stages of infection ranging from intermittent to late chronic. Isolates were characterized with regard to QS signal molecules, alginate, rhamnolipid and elastase production and mutant frequency. The genetic basis for change in QS regulation were investigated and identified by sequence analysis of lasR, rhlR, lasI and rhlI. The first QS system to be lost was the one encoded by las system 12 years (median value) after the onset of the lung infection with subsequent loss of the rhl encoded system after 17 years (median value) shown as deficiencies in production of the 3-oxo-C12-HSL and C4-HSL QS signal molecules respectively. The concomitant development of QS malfunction significantly correlated with the reduced production of rhamnolipids and elastase and with the occurrence of mutations in the regulatory genes lasR and rhlR. Accumulation of mutations in both lasR and rhlR correlated with development of hypermutability. Interestingly, a higher number of mucoid isolates were found to produce C4-HSL signal molecules and rhamnolipids compared to the non-mucoid isolates. As seen from the present data, we can conclude that P. aeruginosa and particularly the mucoid strains do not lose the QS regulation or the ability to produce rhamnolipids until the late stage of the chronic infection.     

A relationship between Pseudomonal growth behaviour and cystic fibrosis patient lung function identified in a metabolomic investigation

Chronic polymicrobial lung infections in adult cystic fibrosis patients are typically dominated by high levels of Pseudomonas aeruginosa. Determining the impact of P. aeruginosa growth on airway secretion composition is fundamental to understanding both the behaviour of this pathogen in vivo, and its relationship with other potential colonising species. We hypothesised that the marked differences in the phenotypes of clinical isolates would be reflected in the metabolite composition of spent culture media. ¹H NMR spectroscopy was used to characterise the impact of P. aeruginosa growth on a synthetic medium as part of an in vitro CF lower airways model system. Comparisons of 15 CF clinical isolates were made and four distinct metabolomic clusters identified. Highly significant relationships between P. aeruginosa isolate cluster membership and both patient lung function (FEV₁) and spent culture pH were identified. This link between clinical isolate growth behaviour and FEV₁ indicates characterisation of P. aeruginosa growth may find application in predicting patient lung function while the significant divergence in metabolite production and consumption observed between CF clinical isolates suggests dominant isolate characteristics have the potential to play both a selective role in microbiota composition and influence pseudomonal behaviour in vivo.     

Patient-Specific Modeling of Regional Antibiotic Concentration Levels in Airways of Patients with Cystic Fibrosis: Are We Dosing High Enough?

Background

Pseudomonas aeruginosa (Pa) infection is an important contributor to the progression of cystic fibrosis (CF) lung disease. The cornerstone treatment for Pa infection is the use of inhaled antibiotics. However, there is substantial lung disease heterogeneity within and between patients that likely impacts deposition patterns of inhaled antibiotics. Therefore, this may result in airways below the minimal inhibitory concentration of the inhaled agent. Very little is known about antibiotic concentrations in small airways, in particular the effect of structural lung abnormalities. We therefore aimed to develop a patient-specific airway model to predict concentrations of inhaled antibiotics and to study the impact of structural lung changes and breathing profile on local concentrations in airways of patients with CF.

Methods

In- and expiratory CT-scans of children with CF (5–17 years) were scored (CF-CT score), segmented and reconstructed into 3D airway models. Computational fluid dynamic (CFD) simulations were performed on 40 airway models to predict local Aztreonam lysine for inhalation (AZLI) concentrations. Patient-specific lobar flow distribution and nebulization of 75 mg AZLI through a digital Pari eFlow model with mass median aerodynamic diameter range were used at the inlet of the airway model. AZLI concentrations for central and small airways were computed for different breathing patterns and airway surface liquid thicknesses.

Results

In most simulated conditions, concentrations in both central and small airways were well above the minimal inhibitory concentration. However, small airways in more diseased lobes were likely to receive suboptimal AZLI. Structural lung disease and increased tidal volumes, respiratory rates and larger particle sizes greatly reduced small airway concentrations.

Conclusions

CFD modeling showed that concentrations of inhaled antibiotic delivered to the small airways are highly patient specific and vary throughout the bronchial tree. These results suggest that anti-Pa treatment of especially the small airways can be improved.     

Impairment in inflammasome signaling by the chronic Pseudomonas aeruginosa isolates from cystic fibrosis patients results in an increase in inflammatory response

Pseudomonas aeruginosa is a common respiratory pathogen in cystic fibrosis (CF) patients which undergoes adaptations during chronic infection towards reduced virulence, which can facilitate bacterial evasion of killing by host cells. However, inflammatory cytokines are often found to be elevated in CF patients, and it is unknown how chronic P. aeruginosa infection can be paradoxically associated with both diminished virulence in vitro and increased inflammation and disease progression. Thus, we investigated the relationship between the stimulation of inflammatory cell death pathways by CF P. aeruginosa respiratory isolates and the expression of key inflammatory cytokines. We show that early respiratory isolates of P. aeruginosa from CF patients potently induce inflammasome signaling, cell death, and expression of IL-1β by macrophages, yet little expression of other inflammatory cytokines (TNF, IL-6 and IL-8). In contrast, chronic P. aeruginosa isolates induce relatively poor macrophage inflammasome signaling, cell death, and IL-1β expression but paradoxically excessive production of TNF, IL-6 and IL-8 compared to early P. aeruginosa isolates. Using various mutants of P. aeruginosa, we show that the premature cell death of macrophages caused by virulent bacteria compromises their ability to express cytokines. Contrary to the belief that chronic P. aeruginosa isolates are less pathogenic, we reveal that infections with chronic P. aeruginosa isolates result in increased cytokine induction due to their failure to induce immune cell death, which results in a relatively intense inflammation compared with early isolates.Subject terms: Cell death, Immune cell death