Background Older age is associated with increased severity and death from respiratory infections, including coronavirus disease 2019 (Covid-19). The tuberculosis vaccine Bacille Calmette-Guérin (BCG) may provide heterologous protection against non-tuberculous infections, and has been proposed as a potential preventive strategy against Covid-19. Methods In this multicenter, placebo-controlled trial, we randomly assigned elderly individuals (60 years or older, n=2014) to intracutaneous vaccination with BCG (n=1008) or placebo (n=1006). The primary endpoint was the cumulative incidence of respiratory tract infections that required medical intervention, during 12 months of follow-up. Secondary endpoints included the incidence of Covid-19, and the effect of BCG vaccination on the cellular and humoral immune responses. Results The cumulative incidence of respiratory tract infection requiring medical intervention was 0.029 in the BCG-vaccinated group and 0.024 in the control group (subdistribution hazard ratio [SHR], 1.26; 98.2% confidence interval [CI], 0.65 to 2.44). 51 and 48 individuals tested positive for SARS-CoV-2 by PCR in the BCG and placebo group, respectively (SHR, 1.053; 95% CI, 0.71 to 1.56). No difference was observed in the frequency of adverse events. BCG vaccination was associated with enhanced cytokines responses after influenza, and partially also after SARS-CoV-2 stimulation. In patients diagnosed with Covid-19, antibody responses after infection were significantly stronger if the volunteers had previously received BCG. Conclusions BCG-vaccination had no effect on the incidence of respiratory tract infections, including SARS-CoV-2 infection, in elderly volunteers. However, BCG vaccination improved cytokine responses stimulated by influenza and SARS-CoV-2, and induced stronger antibody titers after Covid-19 infection.

dKlebsiella pneumoniae is emerging as an important nosocomial pathogen due to its rapidly increasing multidrug resistance, which has led to a renewed interest in polymyxin antibiotics, such as colistin, as antibiotics of last resort. However, heteroresistance (i.e., the presence of a subpopulation of resistant bacteria in an otherwise susceptible culture) may hamper the effectiveness of colistin treatment in patients. In a previous study, we showed that colistin resistance among extendedspectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates emerged after the introduction of selective digestive tract decontamination (SDD) in an intensive care unit (ICU). In this study, we investigated heteroresistance to colistin among ESBL-producing K. pneumoniae isolates by using population analysis profiles (PAPs). We used whole-genome sequencing (WGS) to identify the mutations that were associated with the emergence of colistin resistance in these K. pneumoniae isolates. We found five heteroresistant subpopulations, with colistin MICs ranging from 8 to 64 mg/liter, which were derived from five clonally related, colistin-susceptible clinical isolates. WGS revealed the presence of mutations in the lpxM, mgrB, phoQ, and yciM genes in colistin-resistant K. pneumoniae isolates. In two strains, mgrB was inactivated by an IS3-like or ISKpn14 insertion sequence element. Complementation in trans with the wild-type mgrB gene resulted in these strains reverting to colistin susceptibility. The MICs for colistin-susceptible strains increased 2-to 4-fold in the presence of the mutated phoQ, lpxM, and yciM alleles. In conclusion, the present study indicates that heteroresistant K. pneumoniae subpopulations may be selected for upon exposure to colistin. Mutations in mgrB and phoQ have previously been associated with colistin resistance, but we provide experimental evidence for roles of mutations in the yciM and lpxM genes in the emergence of colistin resistance in K. pneumoniae.

Their induction of immunogenic tumor cell death and association with pro-inflammatory signals make OVs attractive immunotherapeutic modalities. The first promising clinical results with immunologically armed OVs warrant their further optimization and development. OVs should be modified to avoid detrimental effects of pre-existent anti-OV immunity as well as for increased tumor targeting and selectivity, so as to ultimately allow for systemic administration while achieving local immune potentiation and tumor elimination in the TME. In particular, a combination of trans-genes encoding bispecific T-cell engagers, immune checkpoint blockers and antigen-presenting cell enhancers will remove suppressive hurdles in the TME and allow for optimal antitumor efficacy of armed OVs.

Objectives The objectives of these two separate trials are: (1) to reduce health care workers (HCWs) absenteeism; and (2) to reduce hospital admission among the elderly during the COVID-19 pandemic through BCG vaccination. Trial design Two separate multi-centre placebo-controlled parallel group randomized trials Participants (1) Health care personnel working in the hospital or ambulance service where they will take care of patients with the COVID-19 infection and (2) elderly ≥60 years. The HCW trial is being undertaken in 9 hospitals. The elderly trial is being undertaken in locations in the community in Nijmegen, Utrecht, and Veghel, in the Netherlands, using senior citizen organisations to facilitate recruitment. Intervention and comparator For both trials the intervention group will be randomized to vaccination with 0.1 ml of the licensed BCG vaccine (Danish strain 1331, SSI, Denmark, equivalent to 0.075 mg attenuated M. bovis). The placebo group consists of 0.1 ml 0.9% NaCl, which is the same amount, and has the same colour and appearance as the suspended BCG vaccine. Main outcomes (1) Number of days of unplanned work absenteeism in HCWs for any reason which can be continuously measured on a bi-weekly basis, and (2) the cumulative incidence of hospital admission due to documented COVID-19. Randomisation Participants will be randomized to BCG vaccine or placebo (1;1) centrally using a computer- based system, stratified by study centre. Blinding (masking) Subjects, investigators, physicians and outcome assessors are blinded for the intervention. Only the pharmacist assistant that prepares- and research personnel that administers- study medicines are unblinded. Numbers to be randomised (sample size) (1) The sample size for the first trial is N=1500 HCWs randomised 1:1 to either BCG vaccine (n=750) and placebo (n=750) and (2) The sample size for the second trial is N=1600 elderly persons randomised to BCG vaccine (n=800) and the placebo group (n=800). Trial Status HCW: version 4.0, 24-04-2020. Recruitment began 25-03-2020 and was completed on the 23-04-2020. Elderly: version 3.0, 04-04-2020. Recruitment began 16-04- 2020 and is ongoing. Trial registration The HCWs trial was registered 31-03-2020 at clinicaltrials.gov (identifier: NCT04328441) and registered 20-03-2020 at the Dutch Trial Registry (trialregister.nl, identifier Trial NL8477). The elderly trial was registered 22-04-2020 at the Dutch trial registry with number NL8547. Full protocol The full protocols will be attached as additional files, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.