Selective Pressure by Rifampicin Modulates Mutation Rates and Evolutionary Trajectories of Mycobacterial Genomes

Resistance to the frontline antibiotic rifampicin constitutes a challenge to the treatment and control of tuberculosis. Here, we analyzed the mutational landscape of Mycobacterium smegmatis during long-term evolution with increasing concentrations of rifampicin, using a mutation accumulation assay combined with whole-genome sequencing. Antibiotic treatment enhanced the acquisition of mutations, doubling the genome-wide mutation rate of the wild-type cells. While antibiotic exposure led to extinction of almost all wild-type lines, the hypermutable phenotype of the ΔnucS mutant strain (noncanonical mismatch repair deficient) provided an efficient response to the antibiotic, leading to high rates of survival. This adaptative advantage resulted in the emergence of higher levels of rifampicin resistance, an accelerated acquisition of drug resistance mutations in rpoB (β RNA polymerase), and a wider diversity of evolutionary pathways that led to drug resistance. Finally, this approach revealed a subset of adaptive genes under positive selection with rifampicin that could be associated with the development of antibiotic resistance. IMPORTANCE Rifampicin is the most important first-line antibiotic against mycobacterial infections, including tuberculosis, one of the top causes of death worldwide. Acquisition of rifampicin resistance constitutes a major global public health problem that makes the control of the disease challenging. Here, we performed an experimental evolution assay under antibiotic selection to analyze the response and adaptation of mycobacteria, leading to the acquisition of rifampicin resistance. This approach explored the total number of mutations that arose in the mycobacterial genomes under long-term rifampicin exposure, using whole-genome sequencing. Our results revealed the effect of rifampicin at a genomic level, identifying different mechanisms and multiple pathways leading to rifampicin resistance in mycobacteria. Moreover, this study detected that an increase in the rate of mutations led to enhanced levels of drug resistance and survival. In summary, all of these results could be useful to understand and prevent the emergence of drug-resistant isolates in mycobacterial infections.This research was funded by MCIN/AEI/10.13039/501100011033, grant PID2020-112865RB-I00, and Instituto de Salud Carlos III, grant FIS PI17/00159 (ISCIII/FEDER, UE). E.C.-S. is the recipient of a PFIS predoctoral research fellowship (FI18/00036) cofinanced by the Instituto de Salud Carlos III and the European Social Fund. A.C.-G. acknowledges financial support from the Spanish State Research Agency, AEI/10.13039/501100011033, through the “Severo Ochoa” Program for Centers of Excellence in R&D (SEV-2013-0347, SEV-2017-0712). Editorial assistance was provided by Stuart L. Rulten. Statistical consultancy was provided by Applied Statistical Department-SGAI-CSIC.S

SARS-CoV-2 outbreak on a Spanish mink farm: epidemiological, molecular, and pathological studies

Farmed minks have been reported to be highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may represent a risk to humans. In this study, we describe the first outbreak of SARS-CoV-2 occurred on a mink farm in Spain, between June and July 2020, involving 92,700 animals. The outbreak started shortly after some farm workers became seropositive for SARS-CoV-2. Minks showed no clinical signs compatible with SARS-CoV-2 infection throughout the outbreak. Samples from 98 minks were collected for histopathological, serological, and molecular studies. Twenty out of 98 (20.4%) minks were positive by RT-qPCR and 82 out 92 (89%) seroconverted. This finding may reflect a rapid spread of the virus at the farm with most of the animals overcoming the infection. Additionally, SARS-CoV-2 was detected by RT-qPCR in 30% of brain samples from positive minks. Sequencing analysis showed that the mink sequences were not closely related with the other mink SARS-CoV-2 sequences available, and that this mink outbreak has its probable origin in one of the genetic variants that were prevalent in Spain during the first COVID-19 epidemic wave. Histological studies revealed bronchointerstitial pneumonia in some animals. Immunostaining of viral nucleocapsid was also observed in nasal turbinate tissue. Farmed minks could therefore constitute an important SARS-CoV-2 reservoir, contributing to virus spread among minks and humans. Consequently, continuous surveillance of mink farms is needed

Digital inclusion and participation of people with intellectual disabilities during COVID-19: A rapid review and international bricolage

The COVID-19 pandemic has meant a rapid transfer of everyday activities to the online world. Information and communication technologies (ICTs) have become more embedded than ever in people's lives. This investigation addresses how this change has affected the lives of people with intellectual disabilities (ID). A two-step design was used. A rapid review was conducted on empirical studies published between January 2019 and June 2021. Search terms related to ID, ICT use and COVID-19. A qualitative international bricolage was also conducted corresponding to author nationalities. Data gathered from the review and bricolage were analysed separately using thematic analysis and relationally synthesised. Digital solutions to provide access to COVID-19 information and guidance seemed inadequate but were seldom empirically studied. Digital poverty, literacy and exclusion remain significant issues for people with ID internationally. People and their carers experienced reduced and removed service provision, loneliness and impoverished daily lives during the pandemic; amelioration of which was facilitated by digital solutions. One solution often used was videoconferencing. Prior experience of digital participation, adequate finances, connection, support and digital literacy mentoring for both people with ID and those providing services and support facilitated digital inclusion. Digital exclusion during COVID-19 was exacerbated by sociopolitical, structural, individual and support-related barriers. Although awareness of digital exclusion appears to have been raised, the extent to which this has led to action and change remains unclear. Despite digital exclusion and digital participation benefitting continuation of life, social and emotional well-being and autonomy, COVID-19 has not provided the impetus to eradicate digital poverty for people with ID. Governmental support, digital education, creativity and problem solving are required to enable people with ID the human right to be included in the digital world at this essential time and into the future

Digital inclusion and participation of people with intellectual disabilities during COVID‐19: A rapid review and international bricolage

The COVID-19 pandemic has meant a rapid transfer of everyday activities to the online world. Information and communication technologies (ICTs) have become more embedded than ever in people’s lives. This investigation addresses how this change has affected the lives of people with intellectual disabilities (ID). A two-step design was used. A rapid review was conducted on empirical studies published between January 2019 and June 2021. Search terms related to ID, ICT use and COVID-19. A qualitative international bricolage was also conducted corresponding to author nationalities. Data gathered from the review and bricolage were analysed separately using thematic analysis and relationally synthesised. Digital solutions to provide access to COVID-19 information and guidance seemed inadequate but were seldom empirically studied. Digital poverty, literacy and exclusion remain significant issues for people with ID internationally. People and their carers experienced reduced and removed service provision, loneliness and impoverished daily lives during the pandemic; amelioration of which was facilitated by digital solutions. One solution often used was videoconferencing. Prior experience of digital participation, adequate finances, connection, support and digital literacy mentoring for both people with ID and those providing services and support facilitated digital inclusion. Digital exclusion during COVID-19 was exacerbated by sociopolitical, structural, individual and support-related barriers. Although awareness of digital exclusion appears to have been raised, the extent to which this has led to action and change remains unclear. Despite digital exclusion and digital participation benefitting continuation of life, social and emotional well-being and autonomy, COVID-19 has not provided the impetus to eradicate digital poverty for people with ID. Governmental support, digital education, creativity and problem solving are required to enable people with ID the human right to be included in the digital world at this essential time and into the future

Non-invasive ventilation in obesity hypoventilation syndrome without severe obstructive sleep apnoea

Background Non-invasive ventilation (NIV) is an effective form of treatment in patients with obesity hypoventilation syndrome (OHS) who have concomitant severe obstructive sleep apnoea (OSA). However, there is a paucity of evidence on the efficacy of NIV in patients with OHS without severe OSA. We performed a multicentre randomised clinical trial to determine the comparative efficacy of NIV versus lifestyle modification (control group) using daytime arterial carbon dioxide tension (PaCO2) as the main outcome measure. Methods Between May 2009 and December 2014 we sequentially screened patients with OHS without severe OSA. Participants were randomised to NIV versus lifestyle modification and were followed for 2 months. Arterial blood gas parameters, clinical symptoms, health-related quality of life assessments, polysomnography, spirometry, 6-min walk distance test, blood pressure measurements and healthcare resource utilisation were evaluated. Statistical analysis was performed using intention-to-treat analysis. Results A total of 365 patients were screened of whom 58 were excluded. Severe OSA was present in 221 and the remaining 86 patients without severe OSA were randomised. NIV led to a significantly larger improvement in PaCO2 of -6 (95% CI -7.7 to -4.2) mm Hg versus -2.8 (95% CI -4.3 to -1.3) mm Hg, (p<0.001) and serum bicarbonate of -3.4 (95% CI -4.5 to -2.3) versus -1 (95% CI -1.7 to -0.2 95% CI) mmol/L (p<0.001). PaCO2 change adjusted for NIV compliance did not further improve the inter-group statistical significance. Sleepiness, some health-related quality of life assessments and polysomnographic parameters improved significantly more with NIV than with lifestyle modification. Additionally, there was a tendency towards lower healthcare resource utilisation in the NIV group. Conclusions NIV is more effective than lifestyle modification in improving daytime PaCO2, sleepiness and polysomnographic parameters. Long-term prospective studies are necessary to determine whether NIV reduces healthcare resource utilisation, cardiovascular events and mortality

Epidemiological and Genomic Analysis of a Large SARS-CoV-2 Outbreak in a Long-Term Care Facility in Catalonia, Spain

9 páginas, 3 figuras, 1 tabla.Limiting outbreaks in long-term care facilities (LTCFs) is a cornerstone strategy to avoid an excess of COVID-19-related morbidity and mortality and to reduce its burden on the health system. We studied a large outbreak that occurred at an LTCF, combining methods of classical and genomic epidemiology analysis. The outbreak lasted for 31 days among residents, with an attack rate of 98% and 57% among residents and staff, respectively. The case fatality rate among residents was 16% (n = 15). Phylogenetic analysis of 59 SARS-CoV-2 isolates revealed the presence of two closely related viral variants in all cases (B.1.177 lineage), revealing a far more complex outbreak than initially thought and suggesting an initial spread driven by staff members. In turn, our results suggest that resident relocations to mitigate viral spread might have increased the risk of infection for staff members, creating secondary chains of transmission that were responsible for prolonging the outbreak. Our results highlight the importance of considering unnoticed chains of transmission early during an outbreak and making an adequate use and interpretation of diagnostic tests. Outbreak containment measures should be carefully tailored to each LTCF. IMPORTANCE The impact of COVID-19 on long-term care facilities (LTCFs) has been disproportionately large due to the high frailty of the residents. Here, we report epidemiological and genomic findings of a large outbreak that occurred at an LTCF, which ultimately affected almost all residents and nearly half of staff members. We found that the outbreak was initially driven by staff members; however, later resident relocation to limit the outbreak resulted in transmission from residents to staff members, evidencing the complexity and different phases of the outbreak. The phylogenetic analysis of SARS-CoV-2 isolates indicated that two closely related variants were responsible for the large outbreak. Our study highlights the importance of combining methods of classical and genomic epidemiology to take appropriate outbreak containment measures in LTCFsWe thank the CERCA Program/Generalitat de Catalunya for their support of the Germans Trias i Pujol Research Institute (IGTP). We thank the IGTP Translational Genomics Core Facility and staff for their contribution to this publication. Funding from the Instituto de Salud Carlos III project COV20/00140 (SeqCOVID consortium) and the European Commission—Next Generation EU (regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global).Peer reviewe

Roflumilast in moderate-to-severe chronic obstructive pulmonary disease treated with longacting bronchodilators: two randomised clinical trials

Background Patients with chronic obstructive pulmonary disease (COPD) have few options for treatment. The efficacy and safety of the phosphodiesterase-4 inhibitor roflumilast have been investigated in studies of patients with moderate-to-severe COPD, but not in those concomitantly treated with longacting inhaled bronchodilators. The effect of roflumilast on lung function in patients with COPD that is moderate to severe who are already being treated with salmeterol or tiotropium was investigated. Methods In two double-blind, multicentre studies done in an outpatient setting, after a 4-week run-in, patients older than 40 years with moderate-to-severe COPD were randomly assigned to oral roflumilast 500 mu g or placebo once a day for 24 weeks, in addition to salmeterol (M2-127 study) or tiotropium (M2-128 study). The primary endpoint was change in prebronchodilator forced expiratory volume in 1s (FEV(1)). Analysis was by intention to treat. The studies are registered with ClinicalTrials.gov, number NCT00313209 for M2-127, and NCT00424268 for M2-128. Findings In the salmeterol plus roflumilast trial, 466 patients were assigned to and treated with roflumilast and 467 with placebo; in the tiotropium plus roflumilast trial, 371 patients were assigned to and treated with roflumilast and 372 with placebo. Compared with placebo, roflumilast consistently improved mean prebronchodilator FEV(1) by 49 mL (p<0.0001) in patients treated with salmeterol, and 80 mL (p<0.0001) in those treated with tiotropium. Similar improvement in postbronchodilator FEV(1) was noted in both groups. Furthermore, roflumilast had beneficial effects on other lung function measurements and on selected patient-reported outcomes in both groups. Nausea, diarrhoea, weight loss, and, to a lesser extent, headache were more frequent in patients in the roflumilast groups. These adverse events were associated with increased patient withdrawal. Interpretation Roflumilast improves lung function in patients with COPD treated with salmeterol or tiotropium, and could become an important treatment for these patients

Spread of a SARS-CoV-2 variant through Europe in the summer of 2020.

Following its emergence in late 2019, the spread of SARS-CoV-21,2 has been tracked by phylogenetic analysis of viral genome sequences in unprecedented detail3–5. Although the virus spread globally in early 2020 before borders closed, intercontinental travel has since been greatly reduced. However, travel within Europe resumed in the summer of 2020. Here we report on a SARS-CoV-2 variant, 20E (EU1), that was identified in Spain in early summer 2020 and subsequently spread across Europe. We find no evidence that this variant has increased transmissibility, but instead demonstrate how rising incidence in Spain, resumption of travel, and lack of effective screening and containment may explain the variant’s success. Despite travel restrictions, we estimate that 20E (EU1) was introduced hundreds of times to European countries by summertime travellers, which is likely to have undermined local efforts to minimize infection with SARS-CoV-2. Our results illustrate how a variant can rapidly become dominant even in the absence of a substantial transmission advantage in favourable epidemiological settings. Genomic surveillance is critical for understanding how travel can affect transmission of SARS-CoV-2, and thus for informing future containment strategies as travel resumes. © 2021, The Author(s), under exclusive licence to Springer Nature Limited

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of SARS-CoV-2 genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three available genomic nomenclature systems for SARS-CoV-2 to all sequence data from the WHO European Region available during the COVID-19 pandemic until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation. We provide a comparison of the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2.Peer reviewe