Candida albicans-produced farnesol stimulates Pseudomonas quinolone signal production in LasR-defective Pseudomonas aeruginosa strains

Candida albicans has been previously shown to stimulate the production of Pseudomonas aeruginosa phenazine toxins in dual-species colony biofilms. Here, we report that P. aeruginosa lasR mutants, which lack the master quorum sensing system regulator, regain the ability to produce quorum-sensing-regulated phenazines when cultured with C. albicans. Farnesol, a signalling molecule produced by C. albicans, was sufficient to stimulate phenazine production in LasR− laboratory strains and clinical isolates. P. aeruginosa ΔlasR mutants are defective in production of the Pseudomonas quinolone signal (PQS) due to their inability to properly induce pqsH, which encodes the enzyme necessary for the last step in PQS biosynthesis. We show that expression of pqsH in a ΔlasR strain was sufficient to restore PQS production, and that farnesol restored pqsH expression in ΔlasR mutants. The farnesol-mediated increase in pqsH required RhlR, a transcriptional regulator downstream of LasR, and farnesol led to higher levels of N-butyryl-homoserine lactone, the small molecule activator of RhlR. Farnesol promotes the production of reactive oxygen species (ROS) in a variety of species. Because the antioxidant N-acetylcysteine suppressed farnesol-induced RhlR activity in LasR− strains, and hydrogen peroxide was sufficient to restore PQS production in las mutants, we propose that ROS are responsible for the activation of downstream portions of this quorum sensing pathway. LasR mutants frequently arise in the lungs of patients chronically infected with P. aeruginosa. The finding that C. albicans, farnesol or ROS stimulate virulence factor production in lasR strains provides new insight into the virulence potential of these strains

Nutrient Availability as a Mechanism for Selection of Antibiotic Tolerant Pseudomonas aeruginosa within the CF Airway

Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues

Characterisation of genotypes and phenotypes of Pseudomonas aeruginosa infecting people with cystic fibrosis

Cystic fibrosis (CF) is the most common inherited lethal disorder among Caucasian populations. Chronic pulmonary infections, particularly from Pseudomonas aeruginosa, are the major determinant of the morbidity and mortality of people with CF. It is generally accepted that people with CF acquire this pathogen independently from their surrounding environment, and that individual CF patients carry unique strains different from others. The spread of this pathogen from patient to patient is thought to be rare and occurs particularly among closely contacted cases such as CF siblings. However, over the past decade, there have been several reports of an emergence of clonal P. aeruginosa strains commonly found infecting a number of CF patients. One such report is from the CF paediatric clinic at the Royal Children’s Hospital in Melbourne in which more than half of the patients were infected with a single strain or clone, subsequently called Australian epidemic strain 1 or AES-1. A preliminary survey showed that AES-1 had spread extensively along the Australian eastern seaboard among CF patients attending other CF centres in Melbourne, Sydney and Brisbane, including adult patients at the Royal Prince Alfred Hospital (RPAH), Sydney. Another clonal strain, subsequently called AES-2, was identified in both CF adults and children at the Prince Charles Hospital and the Royal Children’s Hospital, in Brisbane. The total extent of prevalence of the AES-1 and AES-2 strains at the RPAH as well as the clinical status of patients who carried these strains was unknown. Moreover, the pathogenicity of these two clonal strains had not been investigated. The studies presented in this thesis investigated the prevalence of these clonal strains among CF patients attending the adult CF clinic at RPAH, Sydney by using pulsed-field gel electrophoresis. Overall, 50% of 112 patients with P. aeruginosa were found to be infected with clonal strains. The AES-1 and AES-2 strains were identified in 38% and 5% of the patients respectively. Two new clonal strains, called Sydney-1 and Sydney-2, were also identified. Patients with clonal strains had a significant increase in their number of exacerbations and hospitalisation days, and tended to have lower pulmonary functions when compared to patients infected with non-clonal strains. By using a variety of bioassays to examine the pathogenicity of the clonal and non-clonal strains, it was found that both AES-1 and AES-2 produced more virulence factors and were more resistant to antibiotics when compared to the non-clonal strains. AES-1 and AES-2 were associated with increased production of proteases, including elastase, alkaline protease and protease IV. Overall the results presented in this thesis suggest that there may be a link between virulence and transmissibility of this pathogen. The studies presented in this thesis also compared the biofilm forming capacities of the AES-1 and non-clonal isolates. AES-1 was shown to have greater biofilm-forming capacity than the non-clonal strains, when they were grown on a glass surface, suggesting a possible association between clonality and biofilm formation. A model for the study of bacteria grown in conditions similar to CF sputum was also developed. P. aeruginosa grown in this model was found to develop into clumps which may be comparable to the biofilm structure in the CF lung. This model was shown to be beneficial for transcriptomic and proteomic studies which are underway within the research group. AES-1 was also found to have phenotypic variations between isolates. By applying the amplified fragment length polymorphism technique, more subtypes of this clone were revealed. However, these detected subtypes did not correlate with the different phenotypes, suggesting minor mutations such as single point polymorphisms may be responsible for the phenotypic diversity within the clone. The final part of this thesis was devoted to examining the safety of a novel CF treatment: hypertonic saline (HS) inhalation. HS was shown to increase airway mucociliary clearance, while increased osmolarity associated with the use of HS was also shown to have an inhibitory effect on the formation of biofilms. Findings in this study proved that there was no evidence of strain selection in patients who received the long-term treatment with HS. The study also demonstrated that AES-1 was significantly more persistent in the CF lung than the non-clonal strains. The present thesis not only defines the clonal strains of P. aeruginosa and their implications for infected patients, but also provides a general understanding into the pathogenesis of both clonal and non-clonal strains infecting CF lungs

Effect of pre- and post-treatment with Bacopa monnieri (Brahmi) on phencyclidine-induced disruptions in object recognition memory and cerebral calbindin, parvalbumin, and calretinin immunoreactivity in rats

P Piyabhan,1 P Tingpej,2 N Duansak11Division of Physiology, Department of Preclinical Science, Faculty of Medicine, Thammasat University, KlongLuang, Pathumthani, Thailand; 2Division of Microbiology, Department of Preclinical Science, Faculty of Medicine, Thammasat University, KlongLuang, Pathumthani, ThailandBackground: Decreased gamma-aminobutyric acid (GABA)-ergic neurons in the brain of both schizophrenic patients and animal models indicates that impairment of GABAergic function is implicated in pathophysiology of the disorder. Decreased GABAergic neurotransmission might be also involved in cognitive impairment, which is developed in schizophrenia. Brahmi (Bacopa monnieri) could be a new treatment and prevention for this cognitive deficit in schizophrenia by increasing GABAergic neurons to a normal level.Aim: The authors aimed to study cognitive-enhancement- and neuroprotective-effects of Brahmi on novel object recognition memory and GABAergic neuronal density, defined by the presence of calcium binding proteins (CBPs; calbindin (CB), parvalbumin (PV), and calretinin (CR)) in a sub-chronic (2 mg/kg, Bid, ip) phencyclidine (PCP) rat model of schizophrenia.Materials and methods: In the cognitive-enhancement-effect study rats were assigned to three groups; Group-1: Control, Group-2: PCP-administration, and Group-3: PCP+Brahmi. In the neuroprotective-effect study rats were assigned to three groups; Group-1: Control, Group-2: PCP-administration, and Group-3: Brahmi+PCP. A discrimination ratio (DR) representing cognitive ability was obtained from the novel object recognition task. CB, PV, and CR immunodensity were measured in the prefrontal cortex, striatum, and cornuammonis fields 1–3 (CA1–3) using immunohistochemistry.Results: Reduced DR was found in the PCP group, which occurred alongside reduced CB, PV, and CR in all brain regions except for CR in the striatum and CA1–3 in the cognitive-enhancement-effect study. PCP+Brahmi showed a higher DR score with increased CB in the prefrontal cortex and striatum, increased PV in the prefrontal cortex and CA1–3, and increased CR in the prefrontal cortex. The Brahmi+PCP group showed higher DR score with increased CB in all areas, increased PV in the striatum, and increased CR in the prefrontal cortex and striatum.Conclusion: The present study demonstrated the effects, both partial restoration of cognitive deficit and neuroprotection, of Brahmi, and elucidated its underlying mechanism of actions via increasing GABAergic neurons in a PCP-induced schizophrenic-like model.Keywords: schizophrenia, cognitive impairment, Bacopa monnieri, calbindin, parvalbumin, calretini

Effects of Brassica oleracea extract on impaired glucose and lipid homeostasis in high-fat diet-induced obese mice

Objective: To examine the effect of Brassica oleracea extract (BO) on impaired glucose and lipid homeostasis in high-fat diet (HFD)-induced obese mice. Methods: Obesity of ICR mice was induced by feeding a HFD (45 kcal% lard fat) for 16 weeks. During the last 8 weeks of study period, obese mice were additionally administered with BO (100 and 200 mg/kg/day). The metabolic parameters were determined. The gene expressions of hepatic lipogenesis were also studied. Results: After 8 weeks of treatment, BO (100 and 200 mg/kg) significantly reduced hyperglycemia and improved insulin sensitivity (P < 0.05). The serum lipid (total cholesterol, triglyceride, and non-esterified fatty acid) and hepatic triglyceride and non-esterified fatty acid were decreased (P < 0.05). The levels of insulin and leptin in serum were also decreased (P < 0.05). Moreover, the expressions of hepatic lipogenic genes including sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-CoA carboxylase were decreased by BO treatment (P < 0.05). Conclusions: These results suggest that BO is a new therapeutic agent for improving the homeostasis of glucose and lipid in HFD-induced obese mice probably by suppression of lipogenic genes in liver tissue

Anti-insulin resistant effect of ferulic acid on high fat diet-induced obese mice

Objective: To evaluate the insulin sensitivity action of ferulic acid (FA) in skeletal muscle and hypothalamus of high-fat diet (HFD)-induced obese mice. Methods: Obese mouse model was induced by HFD (45 kcal% lard fat) for 16 weeks. After 8 weeks of HFD feeding, these obese mice were orally treated with FA at doses of 25 and 50 mg/kg/day for 8 weeks. At the end of all treatments, the epididymal fat, pancreas, skeletal muscle and hypothalamus were removed for biochemical parameter and protein expression examinations. Results: FA treatment significantly decreased leptin level in fat tissue and insulin level in pancreas (P < 0.05). Interestingly, obese mice treated with FA increased the protein expressions of insulin receptor substrate-1, phosphatidylinositol 3-kinase, and phosphorylated-protein kinase B in both muscle and brain (P < 0.05). The phosphorylations of adenosine monophosphate-activated protein kinase and acetyl-CoA carboxylase in muscle, and leptin receptor protein in hypothalamus were also increased (P < 0.05). The pancreatic islets histology showed smaller size in obese mice treated with FA compared to untreated obese mice. Conclusions: These findings indicate the beneficial effect of FA in improving insulin resistance in HFD-induced obese mice. These effects are probably mediated via modulating the insulin receptor substrate/phosphatidylinositol 3-kinase/protein kinase B or adenosine monophosphate-activated protein kinase pathways

Clinical profile of adult cystic fibrosis patients with frequent epidemic clones of Pseudomonas aeruginosa

Background and objective: Earlier reports suggested that Pseudomonas aeruginosa frequent epidemic clones circulating in cystic fibrosis (CF) centres had increased virulence. However, recent data show no consistent associations with virulence, and suggest attenuation of virulence in chronic infection. Changes to infection control programmes in relation to frequent epidemic clones should be based on their frequency, virulence across all age groups and mode of acquisition. The Australian epidemic strain-1 (AES-1) (or the Melbourne epidemic strain) and AES-2 are common in CF clinics in mainland eastern Australia, but not in the environment. Both have shown increased virulence, but there are no data specifically in adults. This study examines the frequency and virulence of P. aeruginosa frequent epidemic clones in the adult CF clinic at Royal Prince Alfred Hospital, Sydney, Australia. Methods: Two hundred and fifty-eight P. aeruginosa isolates from 112 participants were genotyped by pulsed field gel electrophoresis. Ninety-eight patients were followed up for 1 year and associations sought between infection with a frequent epidemic clone, clinical outcome and antibiotic resistance. Results: Four frequent P. aeruginosa epidemic clones (AES-1, AES-2, S-1, S-2) affected almost 50% of participants. AES-1 predominated (38%). AES-1, AES-2 and S-1 were associated with increased exacerbations and hospital-admission days. AES-1 showed increased resistance to aminoglycosides and ticarcillin-clavulanate. Conclusions: This study supports the potential threat of frequent P. aeruginosa epidemic clones in adult CF populations.7 page(s

Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum

Pseudomonas aeruginosa airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Various in vitro models have been developed to study P. aeruginosa pathobiology in the CF lung. In this study we produced a modified artificial-sputum medium (ASMDM) more closely resembling CF sputum than previous models, and extended previous work by using strain PAO1 arrays to examine the global transcription profiles of P. aeruginosa strain UCBPP-PA14 under early exponential-phase and stationary-phase growth. In early exponential phase, 38/39 nutrition-related genes were upregulated in line with data from previous in vitro models using UCBPP-PA14. Additionally, 23 type III secretion system (T3SS) genes, several anaerobic respiration genes and 24 quorum-sensing (QS)-related genes were upregulated in ASMDM, suggesting enhanced virulence factor expression and priming for anaerobic growth and biofilm formation. Under stationary phase growth in ASMDM, macroscopic clumps resembling microcolonies were evident in UCBPP-PA14 and CF strains, and over 40 potentially important genes were differentially expressed relative to stationary-phase growth in Luria broth. Most notably, QS-related and T3SS genes were downregulated in ASMDM, and iron-acquisition and assimilatory nitrate reductase genes were upregulated, simulating the iron-depleted, microaerophilic/anaerobic environment of CF sputum. ASMDM thus appears to be highly suitable for gene expression studies of P. aeruginosa in CF. © 2010 SGM Printed in Great Britain

Gene expression characteristics of a cystic fibrosis epidemic strain of Pseydomonas aeruginosa during biofilm and planktonic growth

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