Antibiotics limit adaptation of drug-resistant Staphylococcus aureus to hypoxia

Bacterial pathogens are confronted with a range of challenges at the site of infection, including exposure to antibiotic treatment and harsh physiological conditions, that can alter the fitness benefits and costs of acquiring antibiotic resistance. Here, we develop an experimental system to recapitulate resistance gene acquisition by Staphylococcus aureus and test how the subsequent evolution of the resistant bacterium is modulated by antibiotic treatment and oxygen levels, both of which are known to vary extensively at sites of infection. We show that acquiring tetracycline resistance was costly, reducing competitive growth against the isogenic strain without the resistance gene in the absence of the antibiotic, for S. aureus under hypoxic but not normoxic conditions. Treatment with tetracycline or doxycycline drove the emergence of enhanced resistance through mutations in an RluD-like protein-encoding gene and duplications of tetL, encoding the acquired tetracycline-specific efflux pump. In contrast, evolutionary adaptation by S. aureus to hypoxic conditions, which evolved in the absence of antibiotics through mutations affecting gyrB, was impeded by antibiotic treatment. Together, these data suggest that the horizontal acquisition of a new resistance mechanism is merely a starting point for the emergence of high-level resistance under antibiotic selection but that antibiotic treatment constrains pathogen adaptation to other important environmental selective forces such as hypoxia, which in turn could limit the survival of these highly resistant but poorly adapted genotypes after antibiotic treatment is ended

Xenon ventilation MRI in difficult asthma; initial experience in a clinical setting

Background: Hyperpolarised gas MRI can be used to assess ventilation patterns. Previous studies have shown the image derived metric of ventilation defect percent (VDP) to correlate with FEV1/FVC and FEV1 in asthma. Objectives: To explore the utility of hyperpolarised xenon-129 (129Xe) ventilation MRI in clinical care and examine its relationship with spirometry and other clinical metrics in people seen in a severe asthma service. Methods: 26 people referred from a severe asthma clinic for MRI scanning were assessed by contemporaneous 129Xe MRI and spirometry. A sub-group of 18 patients also underwent reversibility testing with spirometry and MRI. Quantitative MRI measures of ventilation were calculated; VDP and the ventilation heterogeneity index (VHI), and compared to spirometry, ACQ7 and blood eosinophil count. Images were reviewed by a multidisciplinary team. Results: VDP and VHI correlated with FEV1, FEV1/FVC and FEF25–75% but not with ACQ7 or blood eosinophil count. Discordance of MRI imaging and symptoms and/or pulmonary function tests also occurred, prompting diagnostic re-evaluation in some cases. Conclusion Hyperpolarised gas MRI provides a complementary method of assessment in people with difficult to manage asthma in a clinical setting. When used as a tool supporting clinical care in a severe asthma service, occurrences of discordance between symptoms, spirometry and MRI scanning indicate how MRI scanning may add to a management pathway

Enhanced neutrophil extracellular trap formation in COVID-19 is inhibited by the protein kinase C inhibitor ruboxistaurin

Background: Neutrophil extracellular traps (NETs) are web-like DNA and protein lattices which are expelled by neutrophils to trap and kill pathogens, but which cause significant damage to the host tissue. NETs have emerged as critical mediators of lung damage, inflammation and thrombosis in coronavirus disease 2019 (COVID-19) and other diseases, but there are no therapeutics to prevent or reduce NETs that are available to patients. Methods: Neutrophils were isolated from healthy volunteers (n=9) and hospitalised patients with COVID-19 at the acute stage (n=39) and again at 3–4 months post-acute sampling (n=7). NETosis was measured by SYTOX green assays. Results: Here, we show that neutrophils isolated from hospitalised patients with COVID-19 produce significantly more NETs in response to lipopolysaccharide (LPS) compared to cells from healthy control subjects. A subset of patients was captured at follow-up clinics (3–4 months post-acute sampling), and while LPS-induced NET formation is significantly lower at this time point, it remains elevated compared to healthy controls. LPS- and phorbol myristate acetate (PMA)-induced NETs were significantly inhibited by the protein kinase C (PKC) inhibitor ruboxistaurin. Ruboxistaurin-mediated inhibition of NETs in healthy neutrophils reduces NET-induced epithelial cell death. Conclusion: Our findings suggest ruboxistaurin could reduce proinflammatory and tissue-damaging consequences of neutrophils during disease, and since it has completed phase III trials for other indications without safety concerns, it is a promising and novel therapeutic strategy for COVID-19

Incremental Shuttle Walking Test Distance and Autonomic Dysfunction Predict Survival in Pulmonary Arterial Hypertension

Background To ensure effective monitoring of pulmonary arterial hypertension (PAH), a simple, reliable assessment of exercise capacity applicable over a range of disease severity is needed. The aim of this study was to assess the ability of the incremental shuttle walk test (ISWT) to correlate with disease severity, measure sensitivity to change, and predict survival in PAH. Methods We enrolled 418 treatment-naïve patients with PAH with baseline ISWT within 3 months of cardiac catheterization. Clinical validity and prognostic value of ISWT distance were assessed at baseline and 1 year. Results ISWT distance was found to correlate at baseline with World Health Organization functional class, Borg score, and hemodynamics without a ceiling effect (all p 18 beats/min, highest SBP, change in SBP, and 3-minute SBP ratio) were significant predictors of survival (all p < 0.05). Conclusions In patients with PAH, the ISWT is simple to perform, allows assessment of maximal exercise capacity, is sensitive to treatment effect, predicts outcome, and has no ceiling effect. Also, measures of autonomic function made post-exercise predict survival in PAH

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease

The impact of patient choice on survival in chronic thromboembolic pulmonary hypertension

Pulmonary endarterectomy (PEA) is the gold standard treatment for operable chronic thromboembolic pulmonary hypertension (CTEPH). However, a proportion of patients with operable disease decline surgery. There are currently no published data on this patient group. The aim of this study was to identify outcomes and prognostic factors in a large cohort of consecutive patients with CTEPH. Data were collected for consecutive, treatment-naive CTEPH patients at the Pulmonary Vascular Disease Unit of the Royal Hallamshire Hospital (Sheffield, UK) between 2001 and 2014. Of 550 CTEPH patients (mean±sd age 63±15 years, follow-up 4±3 years), 49% underwent surgery, 32% had technically operable disease and did not undergo surgery (including patient choice n=72 and unfit for surgery n=63), and 19% had inoperable disease due to disease distribution. The 5-year survival was superior in patients undergoing PEA (83%) versus technically operable disease who did not undergo surgery (53%) and inoperable due to disease distribution (59%) (p<0.001). Survival was superior in patients following PEA compared with those offered but declining surgery (55%) (p<0.001). In patients offered PEA, independent prognostic factors included mixed venous oxygen saturation, gas transfer and patient decision to proceed to surgery. Outcomes in CTEPH following PEA are excellent and superior to patients declining surgery, and strongly favour consideration of a surgical intervention in eligible patients

Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Background: In this study, we aimed to evaluate the effects of tocilizumab in adult patients admitted to hospital with COVID-19 with both hypoxia and systemic inflammation. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. Those trial participants with hypoxia (oxygen saturation &lt;92% on air or requiring oxygen therapy) and evidence of systemic inflammation (C-reactive protein ≥75 mg/L) were eligible for random assignment in a 1:1 ratio to usual standard of care alone versus usual standard of care plus tocilizumab at a dose of 400 mg–800 mg (depending on weight) given intravenously. A second dose could be given 12–24 h later if the patient's condition had not improved. The primary outcome was 28-day mortality, assessed in the intention-to-treat population. The trial is registered with ISRCTN (50189673) and ClinicalTrials.gov (NCT04381936). Findings: Between April 23, 2020, and Jan 24, 2021, 4116 adults of 21 550 patients enrolled into the RECOVERY trial were included in the assessment of tocilizumab, including 3385 (82%) patients receiving systemic corticosteroids. Overall, 621 (31%) of the 2022 patients allocated tocilizumab and 729 (35%) of the 2094 patients allocated to usual care died within 28 days (rate ratio 0·85; 95% CI 0·76–0·94; p=0·0028). Consistent results were seen in all prespecified subgroups of patients, including those receiving systemic corticosteroids. Patients allocated to tocilizumab were more likely to be discharged from hospital within 28 days (57% vs 50%; rate ratio 1·22; 1·12–1·33; p&lt;0·0001). Among those not receiving invasive mechanical ventilation at baseline, patients allocated tocilizumab were less likely to reach the composite endpoint of invasive mechanical ventilation or death (35% vs 42%; risk ratio 0·84; 95% CI 0·77–0·92; p&lt;0·0001). Interpretation: In hospitalised COVID-19 patients with hypoxia and systemic inflammation, tocilizumab improved survival and other clinical outcomes. These benefits were seen regardless of the amount of respiratory support and were additional to the benefits of systemic corticosteroids. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

Background: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93–1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94–1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93–1·05; p=0·79). Interpretation: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

A fun-guide to innate immune responses to fungal infections

Immunocompromised individuals are at high risk of developing severe fungal infections with high mortality rates, while fungal pathogens pose little risk to most healthy people. Poor therapeutic outcomes and growing antifungal resistance pose further challenges for treatments. Identifying specific immunomodulatory mechanisms exploited by fungal pathogens is critical for our understanding of fungal diseases and development of new therapies. A gap currently exists between the large body of literature concerning the innate immune response to fungal infections and the potential manipulation of host immune responses to aid clearance of infection. This review considers the innate immune mechanisms the host deploys to prevent fungal infection and how these mechanisms fail in immunocompromised hosts. Three clinically relevant fungal pathogens (Candida albicans, Cryptococcus spp. and Aspergillus spp.) will be explored. This review will also examine potential mechanisms of targeting the host therapeutically to improve outcomes of fungal infection