Novel agents with inhibitory activity against the Burkholderia cepacia complex

Pulmonary bacterial infections account for 95% of morbidity and mortality in cystic fibrosis (CF) patients, and include a limited spectrum of bacteria; Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, and members of the Burkholderia cepacia complex (Bcc). "B. cepacia" was first recognised in the late 1970's as a cause of life threatening respiratory infections in CF. Initial clinical observations noted that 20% of colonised CF patients developed "cepacia syndrome", a rapid and fatal necrotising pneumonia. In addition, epidemiological evidence highlighted the potential spread of certain "B. cepacia''' strains, that most isolates were highly resistant to conventional antibiotics and consequently, Bcc infections are untreatable. Subsequent taxonomic studies have identified the Bcc that contains ten distinct species ofbacteria previously termed "5. cepacia". Clinical distribution ofBcc species in CF is restricted to mainly B. cenocepacia (50%), B. multivorans (38%) and B. vietnamiensis (7%)The aim of this study was to investigate novel antimicrobial agents against the Bcc and other problematic and emerging CF associated bacterial pathogens including multi-resistant epidemic P. aeruginosa strains, methicillin resistant S. aureus (MRSA), and Stenotrophomonas maltophilia. The novel antimicrobial strategies examined were based on three main themes: First, the use of natural honey, second, the potential use of bacteriophage and their associated lytic enzymes and third, novel mammalian cationic ß-defensins. The project utilised a vast collection of bacterial isolates and included relevant clinical, environmental and epidemic strains. The susceptibility to conventional antibiotics was measured, and resistance was shown to vary across the Bcc. In general, clinical isolates were statistically more resistant to conventional antibiotics than environmental isolates.Members of the Bcc, and an extended panel ofresistant organisms were shown to be sensitive to New Zealand manuka honey (NZMh). The MICs ranged from 9 to 17% (w/v), and the MBCs ranged from 9 to 20% (w/v). The antimicrobial component of NZMh was investigated, and focussed on osmolality, pH and H₂O₂. All were found to contribute to the antibacterial activity, although none were solely responsible for the activity. Killing-curves suggested that NZMh kills within 24 hours. The NZMh preparation was applied to a CF patient infected with B. cenocepacia J2315, and clinical data highlighted possible benefits to the patient.Novel Bcc specific bacteriophages were identified from environmental samples and from lysogeny studies. The spectrum of activity of the novel bacteriophages, and previously reported Bcc bacteriophage (NS1 and NS2), was determined using a panel comprising 66 isolates of the Bcc, 55 isolates representing other pseudomonads, and 40 B. pseudomallei strains. The novel phages were shown to be very promiscuous and had activity across the Bcc, with some active against P. aeruginosa, B. gladioli, and B. pseudomallei. The wide spectrum of activity was detrimental to therapeutic use, therefore, the phage-encoded lytic enzymes were the focus for further study: bacteriophage therapy with a novel twist. Two enzymes were investigated: the B. cepacia bacteriophage Bcep781 endolysin and the P. aeruginosa phage D3 endolysin. The Bcep781 phage and phage DNA was not available, therefore the endolysin gene was synthesized using recursive PCR. Briefly, twenty-two overlapping oligonucleotides encoding the entire gene were synthesized and constructed into the endolysin gene using a single PCR reaction. Both genes were cloned into an expression plasmid and the enzymes were recombinantly expressed as 6-His fusion proteins in BL21 E. coli cells. Bcep781 endolysin was purified using nickel-affinity chromatography, and the D3 lysin was purified using a Resource S® purification protocol. High-resolution mass spectrometry analysis highlighted discrepancies in both lysins, and neither proved to active against relevant bacteria tested.The activity of cationic antimicrobial peptides (CAMPs) including: a synthetic novel murine ß-defensin (Defrl) with 5-cysteine residues, which forms a covalently bound dimer; its 6-cysteine analog (Def-cys); a chemically reduced Defrl; polymyxin B and colistin; were assessed against the Bcc, as well as several multi-resistant bacterial CF pathogens. Two Bcc isolates, B. cepacia type strain ATCC 25416 and B. cenocepacia type strain J2315, were found to be inherently resistant to all CAMPs utilised in this study. Epidemic P. aeruginosa isolates were found to have a MIC of 6 μg/ml for Defrl and a MIC of 50-100 μg/ml for Def-cys, suggesting a possible relationship between defensin structure and function. Similarly, the MIC of 6 μg/ml was also noted for S. maltophilia and Ralstonia sp. that were found to be resistant to polymyxin B and colistin. The recombinant production of Defrl was also attempted in a number of expression systems in E. coli. However, although Defrl was successfully expressed, the recombinant proteins were highly insoluble. This study showed that resistance varies within the Bee. However, the data show that NZMh exerts a bactericidal effect on members of the Bcc, including B. cenocepacia J2315, and that such activity may be utilised clinically. The novel Bcc bacteriophage may prove to be a useful panel for further study, either as vectors for horizontal gene transfer or as therapeutic agents. The data confirm previous observations that the Bcc are inherently resistant to CAMPs, including a novel 5- cysteine defensin. Despite this finding, synthetic Defrl was shown to be active against a panel of multi-resistant pathogens associated with infections in CF. Further research is required to optimise the recombinant expression of Bcep781 endolysin, D3 lysin, and Defrl, to enable their use in the treatment of multiply resistant infections in CF and the wider hospital environment

Pneumococcal and influenza immunization in asplenic persons: a retrospective population-based cohort study 1990-2002

<p>Abstract</p> <p>Background</p> <p>Splenectomy is associated with increased risk for bacteremia, due to impaired clearance of bloodborne agents and to altered phagocytosis and humoral immunity. We conducted a retrospective cohort study of patients at risk for splenectomy for a 13-year period to determine immunization coverage, and outcomes of those with and without splenectomy, and with or without receipt of influenza or pneumococcal vaccine.</p> <p>Methods</p> <p>Data were extracted from the provincial Medical Services Insurance database for insured services rendered by a physician for 1990-2002, and from the Vital Statistics Death database. The eligible cohort was selected based on diagnostic codes for hematologic conditions for which splenectomy might be considered, such as immune thrombocytopenia. Each patient was followed longitudinally from the date of first diagnosis until 31Dec2002, or death, or relocation out-of province. In addition, persons with splenectomy and no hematologic condition were identified and followed for 6 months post-surgery. Infectious illness rates per 100 person-years of observation and death rates were calculated with and without splenectomy. Death rates were determined using splenectomy status as a time-dependent covariate. The relationship between splenectomy and death according to immunization status was examined using Cox proportional hazard ratios.</p> <p>Results</p> <p>Of 38,812 persons in the cohort 427 subjects with a hematologic diagnosis had splenectomy and another 452 subjects without a hematologic diagnosis had this surgery. 72% were > 18 years of age. Pneumococcal immunization was recorded in 16.5% of asplenic patients overall, and was not associated with reduced risk of death in these persons (adjusted Hazard Ratio [HR] = 1.07, 95% CI 0.70 - 1.65). Influenza immunization was recorded in 53.1% of asplenic patients overall, and was associated with reduced risk of death (adjusted HR = 0.46, 0.33-0.62). No pneumococcal or influenza immunization was recorded in patients with a hematologic diagnosis without splenectomy. Infectious illness visits were higher among all patients who had a splenectomy than among those without a splenectomy (151 visits/100 person-years of observation in the post-splenectomy period vs. 120 visits/100 person-years; p < 0.0001).</p> <p>Conclusions</p> <p>In asplenic patients, influenza immunization is associated with a 54% reduced risk of death compared to unimmunized asplenic persons; no reduction in risk was demonstrated with (polysaccharide) pneumococcal vaccine. Vaccine coverage in the entire cohort was less than routinely recommended. Improved delivery of infection prevention programs to this population is warranted. Conjugate pneumococcal vaccines should be urgently studied in this immunocompromised population.</p

Fire, Hurricane and Carbon Dioxide: Effects on Net Primary Production of a Subtropical Woodland

Disturbance affects most terrestrial ecosystems and has the potential to shape their responses to chronic environmental change. Scrub-oak vegetation regenerating from fire disturbance in subtropical Florida was exposed to experimentally elevated carbon dioxide (CO2) concentration (+350ll-1) using open-top chambers for 11yr, punctuated by hurricane disturbance in year 8. Here, we report the effects of elevated CO2 on aboveground and belowground net primary productivity (NPP) and nitrogen (N) cycling during this experiment. The stimulation of NPP and N uptake by elevated CO2 peaked within 2yr after disturbance by fire and hurricane, when soil nutrient availability was high. The stimulation subsequently declined and disappeared, coincident with low soil nutrient availability and with a CO2-induced reduction in the N concentration of oak stems. These findings show that strong growth responses to elevated CO2 can be transient, are consistent with a progressively limited response to elevated CO2 interrupted by disturbance, and illustrate the importance of biogeochemical responses to extreme events in modulating ecosystem responses to global environmental change

Self reporting RNA probes as an alternative to cleavable small molecule mass tags

The large size of biological molecules such as proteins and oligonucleotides makes them inherently problematic to analyse and quantify directly by mass spectrometry. For these molecules, electrospray ionisation produces multiply charged species and associated alkali metal adducts which can reduce sensitivity and complicate quantification. Whereas time-of-flight mass analysers, often coupled to matrix-assisted laser desorption/ionisation, can have insufficient mass resolution to resolve these large molecules in the higher m/z range. This has led to the development of cleavable small molecule mass tag approaches for the indirect analysis of biomolecules such as proteins and oligonucleotides. Existing methodologies require the design and synthesis of a cleavable linker to join the biomolecule and the mass tag. Here, an alternative approach to small molecule mass tags is presented, which exploits the properties of the RNA molecule to afford self-reporting probes which can be easily synthesised using automated phosphoramidite chemistry. The sugar-phosphate backbone of RNA was used as a built-in enzyme cleavable linker and through the use of RNase digestion of bromine labelled oligonucleotides the observation of a range of small molecule mass tags by mass spectrometry is demonstrated. This study provides a proof-of-concept that RNase digestion can be used to produce labelled small molecule mass tags from oligonucleotide probes, thus eliminating the need for custom design and synthesis of a cleavable linker

The Effects of Fixture Congestion on Injury in Professional Male Soccer: A Systematic Review

Methods Following pre-registration on the Open Science Framework (https://osf.io/86m25/) and conforming with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, systematic searches of four electronic databases (PubMed, Scopus, SPORTDiscus, and Web of Science) were conducted by independent researchers from inception until February 2022. Articles were included if they were original articles written in English and contained relevant time-loss injury data (injury that results in unavailability for training and/or match-play) for male professional soccer players regarding periods of fixture congestion (a minimum of two matches with ≤ 4 days recovery). Results A total of eight articles were included in the review. Five studies identified that congested fixture schedules expose players to increased match injury incidence, although layoff duration was typically lower during congested periods. Two studies identified that training and overall injury incidence were higher during congested periods, with another study identifying a lower training injury incidence during congested periods. Conclusion Injury risk is, overall, increased during fixture-congested periods; however, the layoff duration is typically shorter. The current findings have implications for practitioners regarding the management, periodisation, monitoring, and design of training and competition schedules

LIM kinase function and renal growth: potential role for LIM kinases in fetal programming of kidney development

Aims Maternal dietary restriction during pregnancy impairs nephron development and results in offspring with fewer nephrons. Cell turnover in the early developing kidney is altered by exposure to maternal dietary restriction and may be regulated by the LIM-kinase family of enzymes. We set out to establish whether disturbance of LIM-kinase activity might play a role in the impairment of nephron formation. Main methods E12.5 metanephric kidneys and HK2 cells were grown in culture with the pharmacological LIM-kinase inhibitor BMS5. Organs were injected with DiI, imaged and cell numbers measured over 48 h to assess growth. Cells undergoing mitosis were visualised by pH 3 labelling. Key findings Growth of cultured kidneys reduced to 83% of controls after exposure to BMS5 and final cell number to 25% of control levels after 48 h. Whilst control and BMS5 treated organs showed cells undergoing mitosis (100 ± 11 cells/field vs 113 ± 18 cells/field respectively) the proportion in anaphase was considerably diminished with BMS5 treatment (7.8 ± 0.8% vs 0.8 ± 0.6% respectively; P < 0.01). This was consistent with effects on HK2 cells highlighting a severe impact of BMS5 on formation of the mitotic spindle and centriole positioning. DiI labelled cells migrated in 100% of control cultures vs 0% BMS5 treated organs. The number of nephrogenic precursor cells appeared depleted in whole organs and formation of new nephrons was blocked by exposure to BMS5. Significance Pharmacological blockade of LIM-kinase function in the early developing kidney results in failure of renal development. This is likely due to prevention of dividing cells from completion of mitosis with their resultant loss

Treating nontuberculous mycobacteria in children with cystic fibrosis: a multicentre retrospective study

BackgroundRespiratory infection with nontuberculous mycobacteria (NTM) in children with cystic fibrosis (CF) has increased in prevalence. The condition is difficult to diagnose and treatments are complex with limited evidence to guide practice. This study describes the approaches to diagnosis, management and consequences of treatment in a multicentre cohort of children with CF in the UK.MethodsRetrospective data were collected from 11 CF specialist centres from patients less than 17 years old, treated for NTM infection between 2006 and 2017. Descriptive statistics were used to describe the clinical characteristics of children treated. Treatment regimens, adverse events and success of treatment, with respect to lung function and culture conversion, were evaluated.ResultsData from 70 patients treated for NTM pulmonary disease were collated (60 Mycobacterium abscessus complex (MABSC); 10 M. avium complex (MAC)). Older age and previous diagnosis of allergic bronchopulmonary aspergillosis were all significantly associated with NTM. There was a wide variance in drug choice and side effects were reported with all agents. NTM eradication occurred in 80% of patients with MAC and 48% with MABSC, with variable outcomes on lung function.ConclusionsDiagnosis and treatment of NTM infection in children with CF is challenging. Treatment success is not guaranteed, particularly for MABSC. Large clinical trials are urgently required to evaluate treatment regimes and their suitability and efficacy in children.</jats:sec

Indirect effects of the COVID-19 pandemic on paediatric healthcare use and severe disease: a retrospective national cohort study

OBJECTIVES: To determine the indirect consequences of the COVID-19 pandemic on paediatric healthcare utilisation and severe disease at a national level following lockdown on 23 March 2020. DESIGN: National retrospective cohort study. SETTING: Emergency childhood primary and secondary care providers across Scotland; two national paediatric intensive care units (PICUs); statutory death records. PARTICIPANTS: 273 455 unscheduled primary care attendances; 462 437 emergency department attendances; 54 076 emergency hospital admissions; 413 PICU unplanned emergency admissions requiring invasive mechanical ventilation; and 415 deaths during the lockdown study period and equivalent dates in previous years. MAIN OUTCOME MEASURES: Rates of emergency care consultations, attendances and admissions; clinical severity scores on presentation to PICU; rates and causes of childhood death. For all data sets, rates during the lockdown period were compared with mean or aggregated rates for the equivalent dates in 2016–2019. RESULTS: The rates of emergency presentations to primary and secondary care fell during lockdown in comparison to previous years. Emergency PICU admissions for children requiring invasive mechanical ventilation also fell as a proportion of cases for the entire population, with an OR of 0.52 for likelihood of admission during lockdown (95% CI 0.37 to 0.73), compared with the equivalent period in previous years. Clinical severity scores did not suggest children were presenting with more advanced disease. The greatest reduction in PICU admissions was for diseases of the respiratory system; those for injury, poisoning or other external causes were equivalent to previous years. Mortality during lockdown did not change significantly compared with 2016–2019. CONCLUSIONS: National lockdown led to a reduction in paediatric emergency care utilisation, without associated evidence of severe harm