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1.  In Vitro Efficacy of High-Dose Tobramycin against Burkholderia cepacia Complex and Stenotrophomonas maltophilia Isolates from Cystic Fibrosis Patients 
Burkholderia cepacia complex and Stenotrophomonas maltophilia infections are associated with poor clinical outcomes in persons with cystic fibrosis (CF). The MIC50 based on planktonic growth and the biofilm concentration at which 50% of the isolates tested are inhibited (BIC50) of tobramycin were measured for 180 B. cepacia complex and 101 S. maltophilia CF isolates and were 100 μg/ml for both species. New inhalation devices that deliver high tobramycin levels to the lung may be able to exceed these MICs.
PMCID: PMC4291427  PMID: 25348526
2.  Fosmidomycin Decreases Membrane Hopanoids and Potentiates the Effects of Colistin on Burkholderia multivorans Clinical Isolates 
Burkholderia cepacia complex (Bcc) pulmonary infections in people living with cystic fibrosis (CF) are difficult to treat because of the extreme intrinsic resistance of most isolates to a broad range of antimicrobials. Fosmidomycin is an antibacterial and antiparasitic agent that disrupts the isoprenoid biosynthesis pathway, a precursor to hopanoid biosynthesis. Hopanoids are involved in membrane stability and contribute to polymyxin resistance in Bcc bacteria. Checkerboard MIC assays determined that although isolates of the Bcc species B. multivorans were highly resistant to treatment with fosmidomycin or colistin (polymyxin E), antimicrobial synergy was observed in certain isolates when the antimicrobials were used in combination. Treatment with fosmidomycin decreased the MIC of colistin for isolates as much as 64-fold to as low as 8 μg/ml, a concentration achievable with colistin inhalation therapy. A liquid chromatography-tandem mass spectrometry technique was developed for the accurate quantitative determination of underivatized hopanoids in total lipid extracts, and bacteriohopanetetrol cyclitol ether (BHT-CE) was found to be the dominant hopanoid made by B. multivorans. The amount of BHT-CE made was significantly reduced upon fosmidomycin treatment of the bacteria. Uptake assays with 1-N-phenylnaphthylamine were used to determine that dual treatment with fosmidomycin and colistin increases membrane permeability, while binding assays with boron-dipyrromethene-conjugated polymyxin B illustrated that the addition of fosmidomycin had no impact on polymyxin binding. This work indicates that pharmacological suppression of membrane hopanoids with fosmidomycin treatment can increase the susceptibility of certain clinical B. multivorans isolates to colistin, an agent currently in use to treat pulmonary infections in CF patients.
PMCID: PMC4135860  PMID: 24957830
3.  Swimming Motility in a Longitudinal Collection of Clinical Isolates of Burkholderia cepacia Complex Bacteria from People with Cystic Fibrosis 
PLoS ONE  2014;9(9):e106428.
Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.
PMCID: PMC4159263  PMID: 25203161
4.  Serum Susceptibility in Clinical Isolates of Burkholderia Cepacia Complex Bacteria: Development of a Growth-Based Assay for High Throughput Determination 
Burkholderia cepacia complex (BCC) bacteria can cause devastating chronic infections in people with cystic fibrosis. Of particular concern is “cepacia syndrome,” a rapidly progressive and usually fatal decline in health, characterized by a necrotizing bacteremic pneumonia. An important component of defense against bloodstream infections is the bactericidal action of serum. Traditional methods to determine the capacity of bacterial isolates to resist the bactericidal effects of serum are relatively low-throughput viability assays. In this study, we developed a novel growth-based assay for serum susceptibility, which allows for high throughput analysis. We applied this assay to a range of clinical isolates of BCC as well as isolates comprising the BCC experimental strain panel. Our data demonstrate that isolates from all species of BCC examined can possess serum resistant or serum sensitive/intermediate phenotypes. Of particular clinical significance, we also found no direct link between the last saved pulmonary isolate from patients who subsequently developed “cepacia syndrome” and their capacity to resist the inhibitory effects of human serum, suggesting serum resistance cannot be used as a marker of an isolate’s capacity to escape from the lung and cause bacteremia.
PMCID: PMC3417400  PMID: 22919658
Burkholderia cepacia complex; serum; cystic fibrosis; complement; Bioscreen; cepacia syndrome
5.  The Mucoid Switch in Pseudomonas aeruginosa Represses Quorum Sensing Systems and Leads to Complex Changes to Stationary Phase Virulence Factor Regulation 
PLoS ONE  2014;9(5):e96166.
The opportunistic pathogen Pseudomonas aeruginosa chronically infects the airways of Cystic Fibrosis (CF) patients during which it adapts and undergoes clonal expansion within the lung. It commonly acquires inactivating mutations of the anti-sigma factor MucA leading to a mucoid phenotype, caused by excessive production of the extracellular polysaccharide alginate that is associated with a decline in lung function. Alginate production is believed to be the key benefit of mucA mutations to the bacterium in the CF lung. A phenotypic and gene expression characterisation of the stationary phase physiology of mucA22 mutants demonstrated complex and subtle changes in virulence factor production, including cyanide and pyocyanin, that results in their down-regulation upon entry into stationary phase but, (and in contrast to wildtype strains) continued production in prolonged stationary phase. These findings may have consequences for chronic infection if mucoid P. aeruginosa were to continue to make virulence factors under non-growing conditions during infection. These changes resulted in part from a severe down-regulation of both AHL-and AQ (PQS)-dependent quorum sensing systems. In trans expression of the cAMP-dependent transcription factor Vfr restored both quorum sensing defects and virulence factor production in early stationary phase. Our findings have implications for understanding the evolution of P. aeruginosa during CF lung infection and it demonstrates that mucA22 mutation provides a second mechanism, in addition to the commonly occurring lasR mutations, of down-regulating quorum sensing during chronic infection this may provide a selection pressure for the mucoid switch in the CF lung.
PMCID: PMC4031085  PMID: 24852379
6.  Pseudomonas aeruginosa Enhances Production of a Non-Alginate Exopolysaccharide during Long-Term Colonization of the Cystic Fibrosis Lung 
PLoS ONE  2013;8(12):e82621.
The gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic respiratory infections in individuals with the heritable disease cystic fibrosis (CF). These infections can last for decades, during which time P. aeruginosa has been proposed to acquire beneficial traits via adaptive evolution. Because CF lacks an animal model that can acquire chronic P. aeruginosa infections, identifying genes important for long-term in vivo fitness remains difficult. However, since clonal, chronological samples can be obtained from chronically infected individuals, traits undergoing adaptive evolution can be identified. Recently we identified 24 P. aeruginosa gene expression traits undergoing parallel evolution in vivo in multiple individuals, suggesting they are beneficial to the bacterium. The goal of this study was to determine if these genes impact P. aeruginosa phenotypes important for survival in the CF lung. By using a gain-of-function genetic screen, we found that 4 genes and 2 operons undergoing parallel evolution in vivo promote P. aeruginosa biofilm formation. These genes/operons promote biofilm formation by increasing levels of the non-alginate exopolysaccharide Psl. One of these genes, phaF, enhances Psl production via a post-transcriptional mechanism, while the other 5 genes/operons do not act on either psl transcription or translation. Together, these data demonstrate that P. aeruginosa has evolved at least two pathways to over-produce a non-alginate exopolysaccharide during long-term colonization of the CF lung. More broadly, this approach allowed us to attribute a biological significance to genes with unknown function, demonstrating the power of using evolution as a guide for targeted genetic studies.
PMCID: PMC3855792  PMID: 24324811
7.  Developing an international Pseudomonas aeruginosa reference panel 
MicrobiologyOpen  2013;2(6):1010-1023.
Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents.
PMCID: PMC3892346  PMID: 24214409
Cystic fibrosis; genotype; pathogen; Pseudomonas aeruginosa
8.  Metabolite Profiling to Characterize Disease-related Bacteria 
The Journal of Biological Chemistry  2013;288(21):15098-15109.
Background: Phenotypic profiling of knockout libraries is a possible functional genomics strategy.
Results: Gluconate excretion is a novel phenotype of the Pseudomonas aeruginosa rpoN mutant, which is also weakly associated with antibiotic susceptibility in a clinical strain panel.
Conclusion: The rpoN phenotype results from catabolite repression deregulation of 6-phosphogluconate dehydratase.
Significance: NMR profiling can uncover novel gene functions with potential clinical relevance.
Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles. E.g. for two-component system mutants, the cognate response regulator and sensor kinase genes clustered tightly together. Some strains had metabolic phenotypes (metabotypes) that could be related to the known gene function. E.g. pyruvate dehydrogenase mutants accumulated large amounts of pyruvate in the medium. In other cases, the metabolic phenotypes were not easily interpretable. The rpoN mutant, which lacks the alternative σ factor RpoN (σ54), accumulated high levels of gluconate in the medium. In addition, endometabolome profiling of intracellular metabolites identified a number of systemic metabolic changes. We linked this to indirect regulation of the catabolite repression protein Crc via the non-coding RNA crcZ and found that a crcZ (but not crc) mutant also shared the high-gluconate phenotype. We profiled an additional set of relevant metabolic enzymes and transporters, including Crc targets, and showed that the Crc-regulated edd mutant (gluconate-6-phosphate dehydratase) had similar gluconate levels as the rpoN mutant. Finally, a set of clinical isolates showed patient- and random amplification of polymorphic DNA (RAPD) type-specific differences in gluconate production, which were associated significantly with resistance across four antibiotics (tobramycin, ciprofloxacin, aztreonam, and imipenem), indicating that this has potential clinical relevance.
PMCID: PMC3663530  PMID: 23572517
Functional Genomics; Gene Regulation; Metabolism; Metabolomics; Pseudomonas aeruginosa; Metabolic Footprinting
9.  Functional Quorum Sensing Systems are Maintained during Chronic Burkholderia cepacia Complex Infections in Patients with Cystic Fibrosis 
The Journal of Infectious Diseases  2011;203(3):383-392.
Quorum sensing (QS) contributes to the virulence of Pseudomonas aeruginosa and Burkholderia cepacia complex lung infections. P. aeruginosa QS mutants are frequently isolated from patients with cystic fibrosis. The objective of this study was to determine whether similar adaptations occur over time in B. cepacia complex isolates. Forty-five Burkholderia multivorans and Burkholderia cenocepacia sequential isolates from patients with cystic fibrosis were analyzed for N-acyl-homoserine lactone activity. All but one isolate produced N-acyl-homoserine lactones. The B. cenocepacia N-acyl-homoserine lactone–negative isolate contained mutations in cepR and cciR. Growth competition assays were performed that compared B. cenocepacia clinical and laboratory defined wild-type and QS mutants. Survival of the laboratory wild-type and QS mutants varied, dependent on the mutation. The clinical wild-type isolate demonstrated a growth advantage over its QS mutant. These data suggest that there is a selective advantage for strains with QS systems and that QS mutations do not occur at a high frequency in B. cepacia complex isolates.
PMCID: PMC3071112  PMID: 21208930
10.  In Vitro Susceptibility of Burkholderia vietnamiensis to Aminoglycosides▿ 
Burkholderia cepacia complex (BCC) bacteria are opportunistic pathogens that can cause severe disease in cystic fibrosis (CF) patients and other immunocompromised individuals and are typically multidrug resistant. Here we observed that unlike other BCC species, most environmental and clinical Burkholderia vietnamiensis isolates were intrinsically susceptible to aminoglycosides but not to cationic antimicrobial peptides or polymyxin B. Furthermore, strains acquired aminoglycoside resistance during chronic CF infection, a phenomenon that could be induced under tobramycin or azithromycin pressure in vitro. In comparing susceptible and resistant B. vietnamiensis isolates, no gross differences in lipopolysaccharide structure were observed, all had lipid A-associated 4-amino-4-deoxy-l-arabinose residues, and all were resistant to the permeabilizing effects of aminoglycosides, a measure of drug entry via self-promoted uptake. However, susceptible isolates accumulated 5 to 6 times more gentamicin than a resistant isolate, and aminoglycoside susceptibility increased in the presence of an efflux pump inhibitor. B. vietnamiensis is therefore unusual among BCC bacteria in its susceptibility to aminoglycosides and capacity to acquire resistance. Aminoglycoside resistance appears to be due to decreased cellular accumulation as a result of active efflux.
PMCID: PMC3088185  PMID: 21321142
11.  Hypertonic Saline Therapy in Cystic Fibrosis: Do Population Shifts Caused by the Osmotic Sensitivity of Infecting Bacteria Explain the Effectiveness of this Treatment? 
Cystic fibrosis (CF) is caused by a defect in the CF transmembrane regulator that leads to depletion and dehydration of the airway surface liquid (ASL) of the lung epithelium, providing an environment that can be infected by bacteria leading to increased morbidity and mortality. Pseudomonas aeruginosa chronically infects more than 80% of CF patients and one hallmark of infection is the emergence of a mucoid phenotype associated with a worsening prognosis and more rapid decline in lung function. Hypertonic saline (HS) is a clinically proven treatment that improves mucociliary clearance through partial rehydration of the ASL of the lung. Strikingly, while HS therapy does not alter the prevalence of P. aeruginosa in the CF lung it does decrease the frequency of episodes of acute, severe illness known as infective exacerbations among CF patients. In this article, we propose a hypothesis whereby the positive clinical effects of HS treatment are explained by the osmotic sensitivity of the mucoid sub-population of P. aeruginosa in the CF lung leading to selection against this group in favor of the osmotically resistant non-mucoid variants.
PMCID: PMC3109665  PMID: 21687721
mucoid; mucA; alginate; infective exacerbation; Pseudomonas aeruginosa; metabolomics
12.  Parallel Evolution in Pseudomonas aeruginosa over 39,000 Generations In Vivo 
mBio  2010;1(4):e00199-10.
The Gram-negative bacterium Pseudomonas aeruginosa is a common cause of chronic airway infections in individuals with the heritable disease cystic fibrosis (CF). After prolonged colonization of the CF lung, P. aeruginosa becomes highly resistant to host clearance and antibiotic treatment; therefore, understanding how this bacterium evolves during chronic infection is important for identifying beneficial adaptations that could be targeted therapeutically. To identify potential adaptive traits of P. aeruginosa during chronic infection, we carried out global transcriptomic profiling of chronological clonal isolates obtained from 3 individuals with CF. Isolates were collected sequentially over periods ranging from 3 months to 8 years, representing up to 39,000 in vivo generations. We identified 24 genes that were commonly regulated by all 3 P. aeruginosa lineages, including several genes encoding traits previously shown to be important for in vivo growth. Our results reveal that parallel evolution occurs in the CF lung and that at least a proportion of the traits identified are beneficial for P. aeruginosa chronic colonization of the CF lung.
Deadly diseases like AIDS, malaria, and tuberculosis are the result of long-term chronic infections. Pathogens that cause chronic infections adapt to the host environment, avoiding the immune response and resisting antimicrobial agents. Studies of pathogen adaptation are therefore important for understanding how the efficacy of current therapeutics may change upon prolonged infection. One notorious chronic pathogen is Pseudomonas aeruginosa, a bacterium that causes long-term infections in individuals with the heritable disease cystic fibrosis (CF). We used gene expression profiles to identify 24 genes that commonly changed expression over time in 3 P. aeruginosa lineages, indicating that these changes occur in parallel in the lungs of individuals with CF. Several of these genes have previously been shown to encode traits critical for in vivo-relevant processes, suggesting that they are likely beneficial adaptations important for chronic colonization of the CF lung.
PMCID: PMC2939680  PMID: 20856824
13.  Differential Mucoid Exopolysaccharide Production by Members of the Burkholderia cepacia Complex▿  
Journal of Clinical Microbiology  2008;46(4):1470-1473.
We demonstrate that all nine species of the Burkholderia cepacia complex can express the mucoid phenotype. A survey of clinical isolates showed that strains of B. cenocepacia, the most virulent species of the complex, are most frequently nonmucoid. Additionally, isolates from patients with chronic infections can convert from mucoid to nonmucoid.
PMCID: PMC2292898  PMID: 18256220

Results 1-13 (13)