Metabolic adaptations of Pseudomonas aeruginosa during cystic fibrosis chronic lung infections.

Pseudomonas aeruginosa forms chronic infections in the lungs of cystic fibrosis (CF) patients, and is the leading cause of morbidity and mortality in patients with CF. Understanding how this opportunistic pathogen adapts to the CF lung during chronic infections is important to increase the efficacy of treatment and is likely to increase insight into other long-term infections. Previous studies of P. aeruginosa adaptation and divergence in CF infections have focused on the genetic level, both identifying characteristic mutations and patterns of gene expression. However, these approaches are not sufficient to fully understand the metabolic changes that occur during long-term infection, as metabolic regulation is complex and takes place on different biological levels. We used untargeted metabolic profiling (metabolomics) of cell supernatants (exometabolome analysis, or metabolic footprinting) to compare 179 strains, collected over time periods ranging from 4 to 24 years for the individual patients, representing a series of mostly clonal lineages from 18 individual patients. There was clear evidence of metabolic adaptation to the CF lung environment: acetate production was highly significantly negatively associated with length of infection. For amino acids, which are available to the bacterium in the lung environment, the tendency of isolates to evolve more efficient uptake was related to the biosynthetic cost of producing each metabolite; conversely, for the non-mammalian metabolite trehalose, isolates had significantly reduced tendency to utilize this compound with length of infection. However, as well as adaptation across patients, there was also a striking degree of metabolic variation between the different clonal lineages: in fact, the patient the strains were isolated from was a greater source of variance than length of infection for all metabolites observed. Our data highlight the potential for metabolomic investigation of complex phenotypic adaptations during infection.

Sub-lethal concentrations of antibiotics increase mutation frequency in the cystic fibrosis pathogen Pseudomonas aeruginosa.

Approximately 80% of adult patients with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa and consequently require antibiotic therapy at intervals throughout their lives. Achieving lethal concentrations of antibiotics in the lung remains a challenge. Recent evidence from Escherichia coli and Staphylococcus aureus suggests that the generation of hydroxyl radicals by sublethal concentrations of antibiotics may induce mutagenesis and confer bacteria with resistance to a wide range of antimicrobials. As Ps. aeruginosa can persist for many years following colonization of the airways and during this time it is repeatedly exposed to bactericidal antibiotics, we tested whether its exposure to sublethal levels increases mutation frequency. We demonstrate that sublethal levels of three classes of bactericidal antibiotics commonly used against Ps. aeruginosa infections, ß-lactams, aminoglycosides and quinolones lead to an increase in mutation frequency, varying between c. threefold increase with aminoglycosides and a c. 14-fold increase in mutation frequency with ß-lactam antibiotics. These findings could be clinically significant because exposure to sublethal concentrations of antibiotics during chronic infection leading to increased mutation frequency may facilitate adaptive radiation of pathogenic bacteria in the heterogeneous environment of the CF lung.

Differences in strategies to combat osmotic stress in Burkholderia cenocepacia elucidated by NMR-based metabolic profiling.

AIMS: To investigate mechanisms of osmotic tolerance in Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc) of closely related strains, which is of clinical as well as environmental importance. METHODS AND RESULTS: We employed NMR-based metabolic profiling (metabolomics) to elucidate the metabolic consequences of high osmotic stress for five isolates of B. cenocepacia. The strains differed significantly in their levels of osmotic stress tolerance, and we identified three different sets of metabolic responses with the strains least impacted by osmotic stress exhibiting higher levels of the osmo-protective metabolites glycine-betaine and/or trehalose. Strains either increased concentrations or had constitutively high levels of these metabolites. CONCLUSIONS: Even within the small set of B. cenocepacia isolates, there was a surprising degree of variability in the metabolic responses to osmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic responses, and hence osmotic stress tolerance, vary between different B. cenocepacia isolates. This study provides a first look into the potentially highly diverse physiology of closely related strains of one species of the Bcc and illustrates that physiological or clinically relevant phenotypes are unlikely to be inferable from genetic relatedness within this species group.

Cyanogenesis by the entomopathogenic bacterium Pseudomonas entomophila.

AIMS: To investigate whether the entomopathogenic bacterium Pseudomonas entomophila can synthesize hydrogen cyanide (HCN). METHODS AND RESULTS: Cyanide production was assayed for during the growth of P. entomophila in liquid culture and during colonial growth. Pseudomonas entomophila produced HCN at a concentration of up to 40 micromol l(-1) during growth in liquid cultures and its production was found to be affected by oxygen availability, with levels increasing as the oxygen-transfer coefficient decreased. Pseudomonas entomophila made HCN during colonial growth at levels greater (approximately threefold) than those made by the well studied cyanogenic bacterium Pseudomonas aeruginosa. CONCLUSIONS: This study demonstrated unequivocally that P. entomophila can synthesize HCN, placing it among the small number of cyanogenic bacteria. Our data indicate that HCN production in P. entomophila is regulated by oxygen availability. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas entomophila was recently identified to be the only pseudomonad that naturally infects and induces lethality of Drosophila melanogaster. The virulence factors which contribute to entomopathogenicity exerted by this species are largely unknown. In this study, we demonstrate that P. entomophila produces HCN, a secondary metabolite implicated in biocontrol properties and pathogenicity exerted by other bacteria.

In vitro and in vivo properties of the bovine antimicrobial peptide, Bactenecin 5.

Antimicrobial peptides (AMP), part of the innate immune system, are well studied for their ability to kill pathogenic microorganisms. However, many also possess important immunomodulatory effects, and this area has potential for the development of novel therapies to supplement traditional methods such as the use of antibiotics. Here, we characterise the microbicidal and immunomodulatory potential of the proline-rich bovine AMP, Bactenecin 5 (Bac5). We demonstrate broad antimicrobial activity, including against some mycobacterial species, which are important pathogens of fish, cattle and humans. Bac5 is able to activate macrophage-like THP-1 cells and can synergistically trigger the upregulation of tnf-α when co-stimulated with M. marinum. Furthermore, Bac5 sensitises A549 epithelial cells to stimulation with TNF-α. For the first time, we characterise the activity of Bac5 in vivo, and show it to be a potent chemokine for macrophages in the zebrafish (Danio rerio) embryo model of infection. Bac5 also supports the early recruitment of neutrophils in the presence of M. marinum. In the absence of host adaptive immunity, exogenous injected Bac5 is able to slow, although not prevent, infection of zebrafish with M. marinum.

Pseudomonas aeruginosa, cyanide accumulation and lung function in CF and non-CF bronchiectasis patients.

In patients with cystic fibrosis (CF) and non-CF bronchiectasis, Pseudomonas aeruginosa is the most important respiratory pathogen. It is able to synthesise hydrogen cyanide, a potent inhibitor of cellular respiration. The present study investigated whether cyanide is present in the sputum of CF and non-CF bronchiectasis patients infected with P. aeruginosa, and whether the detection of cyanide affected lung function. Cyanide was measured in sputum using a cyanide ion selective electrode. Cyanide was detected in sputum from 15 out of 25 CF and non-CF bronchiectasis patients with current P. aeruginosa infection; however, it was not detected in any of the 10 patients without this organism. Maximum levels were 130 microM (mean+/-SE 72+/-6.6 microM). Concurrent lung function data were available on all 21 P. aeruginosa-infected CF patients; the group with measurable sputum cyanide (n = 11) was not different from those without (n = 10) on the basis of age or sex. However, those with detectable cyanide had significantly poorer lung function than those without (forced expiratory volume in one second (% predicted) 26.8+/-3.8 versus 46.0+/-6.7%; forced vital capacity (% pred) 44.4+/-4.9 versus 60.1+/-7.7%). Cyanide is detectable in sputum from cystic fibrosis and non-cystic fibrosis bronchiectasis patients infected with Pseudomonas aeruginosa, and is also associated with impaired lung function.

Development of strategies for the use of anti-growth factor treatments.

Aberrant signalling through the epidermal growth factor receptor (EGFR) is associated with increased cancer cell proliferation, reduced apoptosis, invasion and angiogenesis. Over-expression of the EGFR is seen in a variety of tumours and is a rational target for antitumour strategies. Among the classes of agent targeting the EGFR are small-molecule inhibitors, which include gefitinib (IRESSA), which acts by preventing EGFR phosphorylation and downstream signal transduction. De novo and acquired resistance, however, have been reported to gefitinib and here we describe evidence which indicates that the type II receptor tyrosine kinases (RTKs) insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (InsR) play important roles in the mediation of responses to gefitinib in the de novo- and acquired-resistance phenotypes in several cancer types. Moreover, combination strategies that additionally target the IGF-IR/InsR can enhance the antitumour effects of gefitinib.

Formation of the 680 nm-absorbing form of the cytochrome bd oxidase complex of Escherichia coli by reaction of hydrogen peroxide with the ferric form.

Reduced minus aerated difference spectra of membranes from Escherichia coli (grown under oxygen-limited conditions) show, in addition to the 650 nm trough attributed to the oxygenated form of cytochrome d, a smaller trough centred at about 680 nm of unknown origin. When the reference spectrum is that of a sample oxidized with ferricyanide and to which hydrogen peroxide was added, the trough proportions changed, the 680 nm species being more dominant. Similarly, when 8.8 mM hydrogen peroxide is added to a persulphate-oxidized sample, a peak at 680 nm is immediately formed. No such compound is observed when peroxide is added to persulphate-oxidized membranes from a cytochrome d-deficient mutant. It is concluded that the 680 nm species represents a peroxy form of haem d, which is stable at room temperature and is probably an intermediate in the reaction mechanism of this oxidase.

Proposal that the function of the membrane-bound cytochrome a1-like haemoprotein (cytochrome b-595) in Escherichia coli is a direct electron donation to cytochrome d.

The cytochrome d-containing oxidase of oxygen-limited Escherichia coli comprises cytochromes d, cytochrome b-558 and cytochrome b-595, previously called cytochrome a1. The reaction of the fully reduced complex with oxygen involves ligand binding to the ferrous haem d to form an oxygenated species, followed by oxidation of two b-type cytochromes, whose identity is unclear. Here we report kinetic studies on cytochrome b-595 oxidation and suggest that these results, together with optical and EPR data on the oxidase complex and its reaction with oxygen, are consistent with the hypothesis that the role of cytochrome b-595 is further reduction of the oxygen bound to cytochrome d.

The effects of mutation of the anr gene on the aerobic respiratory chain of Pseudomonas aeruginosa.

The anr gene of Pseudomonas aeruginosa encodes a transcriptional regulator of anaerobic gene expression, homologous to the Fnr protein of Escherichia coli. We report here that Anr has a role in regulating the activity of the aerobic respiratory chain of P. aeruginosa. Strains with internal deletions in their anr gene had lowered levels of membrane bound cytochromes whilst the activity of the cytochrome c oxidase, cytochrome co (likely to be a cytochrome cbb3-type oxidase), and the cyanide-insensitive respiratory pathway was markedly higher than in the wild-type strains. These data, and the finding that provision of multiple copies of the anr gene led to severe repression of these respiratory activities, suggest that Anr is a repressor of aerobic respiratory pathways and possibly the terminal oxidases themselves. In contrast, Anr activated cytochrome c peroxidase, a respiratory chain linked enzyme induced under low oxygen conditions.

Investigation of the role of the cydD gene product in production of a functional cytochrome d oxidase in Escherichia coli.

The cydD gene is needed for the formation of a functional cytochrome d oxidase in the aerobic respiratory chain of Escherichia coli. In this paper we demonstrate that transcription from a cydA-lacZ gene fusion is not significantly affected in a cydD mutant. This, together with the finding that subunit I of cytochrome d is present in cytoplasmic membranes of a cydD mutant, rules out a role for CydD in the regulation of cytochrome d expression or the assembly of its polypeptides into the membrane. The activity of the haem d-containing catalase HP II was found to be similar in a cydD mutant and the wild-type. Therefore, if CydD has a role in haem d biosynthesis it must be in a unique step only associated with the synthesis of the haem d prosthetic group of cytochrome d.

A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase.

Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c-type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway's terminal oxidase is a quinol oxidase.

Catecholamine alterations attending spinal cord injury: a reanalysis.

The catecholamine response of injured tissue after severe spinal cord injury (SPI) remains a puzzling controversy. This study was undertaken in an attempt to resolve that controversy. The influence of the biochemical assay method, the magnitude of injury, and the spinal cord region injured on catecholamine levels was determined in the cat spinal cord. It was found that the concentration of norepinephrine (NE) in the traumatized spinal cord is dependent on both the magnitude and the region of injury. The relatively large tissue samples necessitated by the older, less sensitive assay methodology show little or no change in NE levels after a 500-g/cm injury in the cat. When regional samples are analyzed with more sensitive methods, a net depression in the NE level of local tissue is observed. The results of earlier studies from this laboratory indicating an increase in tissue NE after trauma were apparently artifactual, presumably due to the nonselective nature of the biochemical assay used at that time. Dopamine levels were not elevated after SCI, and previous reports from other laboratories indicating an increase in dopamine levels were probably also errant due to methodology-related problems.

Oxygenated fluorocarbon nutrient solution in the treatment of experimental spinal cord injury.

We employed an extravascular perfusion system through the subarachnoid space of the traumatized spinal cord of the cat for the delivery of oxygen utilizing a fluorocarbon emulsion containing essential nutrients, termed the oxygenated fluorocarbon nutrient solution (OFNS). Animals perfused for 2 hours with saline after impact injury of the spinal cord had significantly less edema at 1 cm below this site of injury than injured, untreated animals. However, in injured animals perfused with OFNS there was significant protection from spinal cord edema at both 1 and 2 cm below the site of injury. OFNS perfusion reduced the magnitude of hemorrhagic necrosis in both the gray and the white matter and protected the anterior horn cells against lysis at the site of injury. Adenosine triphosphate (ATP) is decreased within 1 minute and remains suppressed for 1 hour in gray and white matter of unperfused, injured animals. The level of ATP in both gray and white matter was significantly higher in injured OFNS-perfused animals than in saline-treated animals at the site below the spinal cord injury. Our data show that OFNS perfusion of the injured spinal cord reduced necrosis and edema and tended to normalize the levels of high energy ATP and intact anterior horn cells. These results demonstrate the feasibility of treating ischemic hypoxia of the spinal cord after trauma through an extravascular perfusion route that utilizes a fluorocarbon emulsion as a vehicle for the delivery of oxygen and other cellular nutrients.

Severe cerebral ischemia treatment by ventriculosubarachnoid perfusion with an oxygenated fluorocarbon emulsion.

Global hemispheric ischemia was produced in cats by bilateral carotid ligation and bleeding to a mean arterial pressure of 30 +/- 2 (SE) mm Hg. Total electrocerebral silence, as determined by computer-based power analysis, was obtained and maintained for 15 minutes. After this severe cerebral ischemic episode, the heparinized blood was reinfused and the carotid clamps were removed. After the cerebral ischemia, the ventriculosubarachnoid space was perfused with an oxygenated fluorocarbon nutrient solution (OFNS) or modified Elliott's B solution (ES) (control perfusion). The OFNS perfusate contained 400 to 640 mm Hg pO2 (produced by means of a bubble oxygenator pump system) as well as electrolytes, glucose, and amino acids, all of which are known to be important in cerebral metabolism. Flow rates of the perfusion were maintained at either 3 or 6 ml/minute and intracranial pressures were never permitted to exceed 10 mm Hg. During passage through the ventriculosubarachnoid space, oxygen, carbon dioxide, and electrolytes were exchanged between the brain and the OFNS perfusate. In addition, the OFNS perfusate was capable of picking up pCO2, lactate, and pyruvate. This produced a significant return of electrocerebral activity (P less than 0.01) and oxidative metabolism (P less than 0.01), as evidenced by a decline in the lactate/pyruvate ratio in the OFNS-treated cats, but not in nonperfused animals or those perfused with ES. In this study the ventriculosubarachnoid system served as an alternate vascular tree and enabled the perfusate to accomplish many of the functions of blood. Substantial penetration of the perfusate products into the brain occurred, enabling oxidative metabolism, removal of waste products, and electrocerebral activity to be reestablished.

The Exeter universal cemented femoral component at 8 to 12 years. A study of the first 325 hips.

We describe our experience with the implantation of 325 Exeter Universal hips. The fate of every implant was known. The procedures were undertaken by surgeons of widely differing experience. At follow-up at 12 years, survivorship with revision of the femoral component for aseptic loosening as the endpoint was 100% (95% CI 98 to 100). Survivorship with revision of the acetabular component for aseptic loosening as the endpoint was 96.86% (95% CI 93.1 to 98.9) and that with any reoperation as the endpoint 91.74% (95% CI 87.7 to 95.8). No adverse features have emerged as a consequence of the modular connection between the head and neck of the implant.

Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro.

Mycobacterium tuberculosis has 10 universal stress proteins, whose function is unknown. However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines. Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial species, suggests a potential role in the persistence and/or intracellular survival of Mtb. Knock-out mutants of individual usp genes encoding the USPs Rv1996, Rv2005c, Rv2026c and Rv2028c were generated and their growth and survival under hypoxic and other stress conditions examined. Although the majority of usp genes are highly induced in hypoxic conditions, mutation did not affect the long term survival of Mtb under these conditions, or in response to a range of stress conditions chosen to represent the environmental onslaughts experienced by the bacillus during an infection, nor during infection of mouse and human - derived macrophage cell lines. The possibility remains that these USPs are functionally redundant in Mtb.

Adaptation to nutrient starvation in Rhizobium leguminosarum bv. phaseoli: analysis of survival, stress resistance, and changes in macromolecular synthesis during entry to and exit from stationary phase.

The nitrogen-fixing bacterium Rhizobium leguminosarum bv. phaseoli often has to survive long periods of starvation in the soil, when not in a useful symbiotic relationship with leguminous plants. We report that it can survive carbon, nitrogen, and phosphorus starvation for at least 2 months with little loss of viability. Upon carbon starvation, R. leguminosarum cells were found to undergo reductive cell division. During this period, they acquired the potential for long-term starvation-survival, levels of protein, DNA, and RNA synthesis were decreased to base levels, and pool mRNA was stabilized. The starved cells are ready to rapidly restart growth when nutrients become available. Upon addition of fresh nutrients, there is an immediate increase in the levels of macromolecular synthesis, pool mRNA destabilizes, and the cultures enter exponential growth within 5 to 8 h. The starved cells were cross-protected against pH, heat, osmotic, and oxidative shock. These results provide evidence for a general starvation response in R. leguminosarum similar to that previously found in other bacteria such as Escherichia coli and Vibrio sp.