Use of ceftazidime/avibactam for the treatment of MDR Pseudomonas aeruginosa and Burkholderia cepacia complex infections in cystic fibrosis: a case series.

BACKGROUND: The efficacy of antibiotic treatment in pulmonary and systemic infections in cystic fibrosis (CF) is limited by the increased prevalence of MDR strains of Pseudomonas aeruginosa and Burkholderia cepacia complex. Ceftazidime/avibactam is a new combination which, in vitro, appears to have good activity against MDR strains of P. aeruginosa and B. cepacia complex. METHODS: A retrospective analysis was performed including adult patients with CF who received at least one course of ceftazidime/avibactam owing to pulmonary exacerbations not responding to conventional antibiotic treatment. RESULTS: Treatment with ceftazidime/avibactam was associated with reduction in inflammatory markers and improvement in lung function. No episodes of acute kidney injury or elevation in transaminase were observed. CONCLUSIONS: Ceftazidime/avibactam appeared to be well tolerated and improved patients' outcomes. Further studies are needed to better assess the role of this new combination in CF.

Dose adjustments of Elexacaftor/Tezacaftor/Ivacaftor in response to mental health side effects in adults with cystic fibrosis.

INTRODUCTION: Deterioration in mental health has been reported in a minority of individuals with cystic fibrosis starting elexacafor/tezacaftor/ivacaftor (ELX/TEZ/IVA). We report our experience of using sweat chloride and markers of clinical stability to titrate dose reduction with the aim of minimising adverse events and maintaining clinical stability. METHOD: Adults (n = 266) prescribed ELX/TEZ/IVA, were included. Adverse events, sweat chloride, lung function and clinical data were collected. RESULTS: Nineteen (7.1%) individuals reported anxiety, low mood, insomnia and "brain fog" with reduced attention and concentration span. Thirteen underwent dose reduction with sweat chloride remained normal (<30 mmol l-1) or borderline (30-60 mmol l-1) in six (46.2%) and seven (53.2%) cases respectively. Improvement or resolution of AEs occurring in 10 of the 13 cases. CONCLUSION: Dose adjustment of ELX/TEZ/IVA was associated with improvement in mental health AEs without significant clinical deterioration. Sweat chloride concentration may prove useful as a surrogate marker of CFTR function.

Why is mock care not a good proxy for predicting hand contamination during patient care?

BACKGROUND: Healthcare worker (HCW) behaviours, such as the sequence of their contacts with surfaces and hand hygiene moments, are important for understanding disease transmission. AIM: To propose a method for recording sequences of HCW behaviours during mock vs actual procedures, and to evaluate differences for use in infection risk modelling and staff training. METHODS: Procedures for three types of care were observed under mock and actual settings: intravenous (IV) drip care, observational care and doctors' rounds on a respiratory ward in a university teaching hospital. Contacts and hand hygiene behaviours were recorded in real-time using either a handheld tablet or video cameras. FINDINGS: Actual patient care demonstrated 70% more surface contacts than mock care. It was also 2.4 min longer than mock care, but equal in terms of patient contacts. On average, doctors' rounds took 7.5 min (2.5 min for mock care), whilst auxiliary nurses took 4.9 min for observational care (2.4 min for mock care). Registered nurses took 3.2 min for mock IV care and 3.8 min for actual IV care; this translated into a 44% increase in contacts. In 51% of actual care episodes and 37% of mock care episodes, hand hygiene was performed before patient contact; in comparison, 15% of staff delivering actual care performed hand hygiene after patient contact on leaving the room vs 22% for mock care. The number of overall touches in the patient room was a modest predictor of hand hygiene. Using a model to predict hand contamination from surface contacts for Staphylococcus aureus, Escherichia coli and norovirus, mock care underestimated micro-organisms on hands by approximately 30%.

Laue diffraction study on the structure of cytochrome c peroxidase compound I.

BACKGROUND: Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group. RESULTS: Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures. CONCLUSIONS: The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.

Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS).

Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C, the committed step in the biosynthesis of cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C terminus of one molecule is inserted into the active site of its neighbor in a cyclical fashion within a trimeric unit. This arrangement has hindered the generation of crystalline enzyme-substrate complexes. Therefore, we constructed a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutarate was significantly uncoupled from oxidation of the penicillin substrate in certain truncated mutants. The extent of uncoupling varied with the number of residues deleted and the penicillin substrate used. Crystal structures were determined for the DeltaR306 mutant complexed with iron(II) and 2-oxoglutarate (to 2.10 A) and the DeltaR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide (to 1.96 A). The latter may mimic a product complex, and supports proposals for a metal-bound CO(2) intermediate during catalysis.

Calcium binding sites in tomato bushy stunt virus visualized by Laue crystallography.

We have collected Laue diffraction data from crystals of tomato bushy stunt virus using the full white X-ray spectrum from the wiggler magnet of the Synchrotron Radiation Source at Daresbury, U.K. A single 24 second exposure of a crystal soaked in EDTA yielded a data set that was 90% complete between 6 and 3.5 A resolution. A large proportion of the data could be measured using an overlap deconvolution routine to separate spatially overlapping reflections in the dense Laue photograph. Reflections with I greater than 2 sigma I (40% of the data set) were subjected to wavelength normalization. A difference Fourier map between these reflections and a monochromatic native set showed, after icosahedral averaging, the three pairs of Ca2+ binding sites related by quasi-symmetry and the movement of a liganding loop in the protein at the A/C subunit interface. The extent and quality of the data obtained from a single Laue photograph of this virus were thus sufficient to detect clearly such small structural alterations. In a second experiment, a Laue photograph was taken from a crystal that was soaked first in EDTA and then in GdCl3. A difference Fourier map between this Laue data set and the Laue data set from the EDTA-soaked crystal showed clearly the Gd3+ sites in the capsid, demonstrating that the Laue technique is a reliable and efficient means for data collection with virus crystals.

Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction.

BACKGROUND: Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the beta-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring. RESULTS: Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme-substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed. CONCLUSIONS: In the absence of its natural reaction partner (the N-H proton of the L-cysteinyl-D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding beta-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.

The treatment of spinal tuberculosis: a retrospective study.

BACKGROUND: There are conflicting guidelines and variations in clinical practice in the management of bone tuberculosis (TB), including spinal TB. A case who received 6 months of treatment in line with current British Thoracic Society (BTS) guidelines, and subsequently relapsed, prompted a survey of treatment and outcomes of spinal and other bone TB. METHODS: A retrospective study examining the clinical features, treatment duration and outcome of patients presenting with spinal and other bone TB to the Leeds Teaching Hospitals National Health Service Trust, between 1998 and 2002. RESULTS: Forty-two patients were identified. Notes from 34 patients with spinal TB and four patients with TB of other bones were reviewed. Of eight patients who received 6 months of therapy, five relapsed. Of 30 patients who received treatment for 9 months or longer, none relapsed (P < 0.05). CONCLUSION: Six months of treatment, as currently recommended by the BTS, may be inadequate for bone TB, including spinal TB.

Crystallization and preliminary X-ray diffraction studies on recombinant isopenicillin N synthase from Aspergillus nidulans.

Recombinant Aspergillus nidulans isopenicillin N synthase was purified from an Escherichia coli expression system. The apoenzyme in the presence of saturating concentrations of MnCl2 could be crystallized by either macro- or microseeding, using the hanging drop vapor diffusion technique with polyethylene glycol 8000 as precipitant. The crystals (0.5-1.0 mm overall dimensions) diffract X-rays to at least 2.0 A resolution at synchrotrons and belong to space group P212121 with unit cell dimensions of a = 59.2 A, b = 127.0 A, and c = 139.6 A. The asymmetric unit contains one dimer, and the solvent content of the crystals is 60%. The crystals are radiation sensitive.

Treatment of resistant distal intestinal obstruction syndrome with a modified antegrade continence enema procedure.

We report a case of a patient with CF who had a long history of recurrent distal intestinal obstruction syndrome. She had been treated with conventional treatment including gastrografin, n-acetyl cysteine, Klean prep and Picolax. She underwent a modified antegrade continence enema procedure. She currently irrigates her conduit every 2-3 days. She has had no further symptoms of distal intestinal obstruction syndrome.

The Effect of Sub-MIC ß-Lactam Antibiotic Exposure of Pseudomonas aeruginosa Strains from People with Cystic Fibrosis in a Desiccation Survival Model.

Prior to modern typing methods, cross-infection of P. aeruginosa between people with cystic fibrosis (CF) was felt to be rare. Recently a number of studies have demonstrated the presence of clonal strains of P. aeruginosa infecting people with CF. The aim of this study was to determine whether strains of P. aeruginosa demonstrated differences in resistance to desiccation and whether preincubation in subminimum inhibitory concentrations (MICs) of ß-lactam affected desiccation resistance. The experimental data were modelled to a first-order decay model and a Weibull decay model using least squares nonlinear regression. The Weibull model was the preferred model for the desiccation survival. The presence of a mucoid phenotype promoted desiccation survival. Preincubation with antibiotics did not have a consistent effect on the strains of P. aeruginosa. Meropenem reduced desiccation resistance, whereas ceftazidime had much less effect on the strains studied.

An aerobiological model of aerosol survival of different strains of Pseudomonas aeruginosa isolated from people with cystic fibrosis.

Pseudomonas aeruginosa is a common and important pathogen in people with cystic fibrosis (CF). Recently epidemic strains of P. aeruginosa associated with increased morbidity, have been identified. The method of transmission is not clear, but there is evidence of a potential airborne route. The aim of this study was to determine whether different strains of P. aeruginosa isolated from people with CF were able to survive within artificially generated aerosols in an aerobiological chamber. Viable P. aeruginosa could still be detected up to 45min after halting generation of the aerosols. All of the strains of P. aeruginosa expressing a non-mucoid phenotype isolated from people with CF had a reduced ability to survive within aerosols compared to an environmental strain. Expression of a mucoid phenotype by the strains of P. aeruginosa isolated from people with CF promoted survival in the aerosol model compared to strains expressing a non-mucoid phenotype.

Acute Burkholderia cenocepacia pyomyositis in a patient with cystic fibrosis.

INTRODUCTION: Extra-pulmonary complications of Burkholderia cepacia complex (Bcc) infection in patients with cystic fibrosis are unusual. To the best of the authors' knowledge no case of pyomyositis secondary to Bcc infection has been reported previously. CASE PRESENTATION: We report a case of pyomyositis of the forearm caused by Bcc infection in a patient with CF. We also briefly discuss the management of pyomyositis. CONCLUSION: Pyomyositis is a potential extra-pulmonary complication of Bcc infection in patients with CF. A high index of clinical suspicion is required to make a prompt diagnosis. Final diagnosis may need MRI. An early diagnosis, aggressive medical therapy, multidisciplinary care and timely surgical intervention are all essential for proper management of this condition.

Mechanisms and structures of crotonase superfamily enzymes--how nature controls enolate and oxyanion reactivity.

Structural and mechanistic studies on the crotonase superfamily (CS) are reviewed with the aim of illustrating how a conserved structural platform can enable catalysis of a very wide range of reactions. Many CS reactions have precedent in the 'carbonyl' chemistry of organic synthesis; they include alkene hydration/isomerization, aryl-halide dehalogenation, (de)carboxylation, CoA ester and peptide hydrolysis, fragmentation of beta-diketones and C-C bond formation, cleavage and oxidation. CS enzymes possess a canonical fold formed from repeated betabetaalpha units that assemble into two approximately perpendicular beta-sheets surrounded by alpha-helices. CS enzymes often, although not exclusively, oligomerize as trimers or dimers of trimers. Two conserved backbone NH groups in CS active sites form an oxyanion 'hole' that can stabilize enolate/oxyanion intermediates. The range and efficiency of known CS-catalyzed reactions coupled to their common structural platforms suggest that CS variants may have widespread utility in biocatalysis.

Ten years of viral and non-bacterial serology in adults with cystic fibrosis.

Viral infections are associated with pulmonary exacerbations in children with cystic fibrosis (CF), but few studies have addressed the frequency in adults. This paper investigates the frequency and impact of viral infections in adults with CF receiving intravenous antibiotics. Pre- and post-treatment spirometry, inflammatory markers and antibody titres against influenza A, influenza B, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, Chlamydia psittaci, and Coxiella burnetti were analysed over a 10-year period. Non-bacterial infections were identified in 5.1% of 3156 courses of treatment. The annual incidence of admissions per patient associated with viral infection was 4.9%. The presence of viral infection in association with a pulmonary exacerbation did not adversely affect lung function or inflammatory markers in the short term. Adults with CF have a lower incidence of respiratory viral infections associated with pulmonary exacerbations requiring intravenous antibiotics compared to children and infants with CF.

Structure of proline 3-hydroxylase. Evolution of the family of 2-oxoglutarate dependent oxygenases.

Iron (II)/2-oxoglutarate (2-OG)-dependent oxygenases catalyse oxidative reactions in a range of metabolic processes including the hydroxylation of proline and lysine residues during the post-translational modification of collagen. 2-OG oxygenases commonly require ascorbate for full activity. In the vitamin C deficient disease, scurvy, reduced activity of 2-OG oxygenases results in impaired formation of collagen. Here we report the crystal structure of bacterial proline 3-hydroxylase from Streptomyces sp., an enzyme which hydroxylates proline at position 3, the first of a 2-OG oxygenase catalysing oxidation of a free alpha-amino acid. Structures were obtained for the enzyme in the absence of iron (to 2.3A resolution, R=20.2%, Rfree=25.3%) and that complexed to iron (II) (to 2.4A resolution, R=19.8%, Rfree=22.6%). The structure contains conserved motifs present in other 2-OG oxygenases including a 'jelly roll' beta strand core and residues binding iron and 2-oxoglutarate, consistent with divergent evolution within the extended family. The structure differs significantly from many other 2-OG oxygenases in possessing a discrete C-terminal helical domain. Analysis of the structure suggests a model for proline binding and a mechanism for uncoupling of proline and 2-OG turnover.

Kinetic and crystallographic analysis of complexes formed between elastase and peptides from beta-casein.

Human beta-casomorphin-7 (NH2-Tyr-Pro-Phe-Val-Glu-Pro-Ile-CO2H) is a naturally occurring peptide inhibitor of elastase that has been shown to form an acyl-enzyme complex stable enough for X-ray crystallographic analysis at pH 5. To investigate the importance of the N-terminal residues of the beta-casomorphin-7 peptide for the inhibition of elastase, kinetic and crystallographic analyses were undertaken to identify the minimum number of residues required for effective formation of a stable complex between truncated beta-casomorphin-7 peptides and porcine pancreatic elastase (PPE). The results clearly demonstrate that significant inhibition of PPE can be effected by simple tri-, tetra-and pentapeptides terminating in a carboxylic acid. These results also suggest that in vivo regulation of protease activity could be mediated via short peptides as well as by proteins. Crystallographic analysis of the complex formed between N-acetyl-Val-Glu-Pro-Ile-CO2H and PPE at pH 5 (to 1.67 A resolution) revealed an active site water molecule in an analogous position to that observed in the PPE/beta-casomorphin-7 structure supportive of its assignment as the 'hydrolytic water' in the deacylation step of serine protease catalysis.