Integrated analysis of patient networks and plasmid genomes reveals a regional, multi-species outbreak of carbapenemase-producing Enterobacterales carrying both blaIMP and mcr-9 genes.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of IMP-encoding CPE amongst diverse Enterobacterales species between 2016 and 2019 across a London regional network. METHODS: We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE positive patients. Genomes of IMP-encoding CPE isolates were overlayed with patient contacts to imply potential transmission events. RESULTS: Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, E. coli); 86% (72/84) harboured an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68/72). Phylogenetic analysis of IncHI2 plasmids identified three lineages showing significant association with patient contacts and movements between four hospital sites and across medical specialities, which was missed on initial investigations. CONCLUSIONS: Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multi-modal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks.

Symbiotic Firmicutes establish mutualism with the host via innate tolerance and resistance to control systemic immunity.

The intestinal microbiota regulates immunity across organ systems. Which symbionts control systemic immunity, the mechanisms they use, and how they avoid widespread inflammatory damage are unclear. We uncover host tolerance and resistance mechanisms that allow Firmicutes from the human microbiota to control systemic immunity without inducing immunopathology. Intestinal processing releases Firmicute glycoconjugates that disseminate, resulting in release of cytokine IL-34 that stimulates macrophages and enhances defenses against pneumonia, sepsis, and meningitis. Despite systemic penetration of Firmicutes, immune homeostasis is maintained through feedback control whereby IL-34-mediated mTORC1 activation in macrophages clears polymeric glycoconjugates from peripheral tissues. Smaller glycoconjugates evading this clearance mechanism are tolerated through sequestration by albumin, which acts as an inflammatory buffer constraining their immunological impact. Without these resistance and tolerance mechanisms, Firmicutes drive catastrophic organ damage and cachexia via IL-1ß. This reveals how Firmicutes are safely assimilated into systemic immunity to protect against infection without threatening host viability.

Lipopolysaccharide as an antibiotic target.

Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are amongst the highest priority drug-resistant pathogens, for which new antibiotics are urgently needed. Whilst antibiotic drug development is inherently challenging, this is particularly true for Gram-negative bacteria due to the presence of the outer membrane, a highly selective permeability barrier that prevents the ingress of several classes of antibiotic. This selectivity is largely due to an outer leaflet composed of the glycolipid lipopolysaccharide (LPS), which is essential for the viability of almost all Gram-negative bacteria. This essentiality, coupled with the conservation of the synthetic pathway across species and recent breakthroughs in our understanding of transport and membrane homeostasis has made LPS an attractive target for novel antibiotic drug development. Several different targets have been explored and small molecules developed that show promising activity in vitro. However, these endeavours have met limited success in clinical testing and the polymyxins, discovered more than 70 years ago, remain the only LPS-targeting drugs to enter the clinic thus far. In this review, we will discuss efforts to develop therapeutic inhibitors of LPS synthesis and transport and the reasons for limited success, and explore new developments in understanding polymyxin mode of action and the identification of new analogues with reduced toxicity and enhanced activity.

XerC Is Required for the Repair of Antibiotic- and Immune-Mediated DNA Damage in Staphylococcus aureus.

To survive in the host environment, pathogenic bacteria need to be able to repair DNA damage caused by both antibiotics and the immune system. The SOS response is a key bacterial pathway to repair DNA double-strand breaks and may therefore be a good target for novel therapeutics to sensitize bacteria to antibiotics and the immune response. However, the genes required for the SOS response in Staphylococcus aureus have not been fully established. Therefore, we carried out a screen of mutants involved in various DNA repair pathways to understand which were required for induction of the SOS response. This led to the identification of 16 genes that may play a role in SOS response induction and, of these, 3 that affected the susceptibility of S. aureus to ciprofloxacin. Further characterization revealed that, in addition to ciprofloxacin, loss of the tyrosine recombinase XerC increased the susceptibility of S. aureus to various classes of antibiotics, as well as to host immune defenses. Therefore, the inhibition of XerC may be a viable therapeutic approach to sensitize S. aureus to both antibiotics and the immune response.

Growth Arrest of Staphylococcus aureus Induces Daptomycin Tolerance via Cell Wall Remodelling.

Almost all bactericidal drugs require bacterial replication and/or metabolic activity for their killing activity. When these processes are inhibited by bacteriostatic antibiotics, bacterial killing is significantly reduced. One notable exception is the lipopeptide antibiotic daptomycin, which has been reported to efficiently kill growth-arrested bacteria. However, these studies employed only short periods of growth arrest (<1 h), which may not fully represent the duration of growth arrest that can occur in vivo. We found that a growth inhibitory concentration of the protein synthesis inhibitor tetracycline led to a time-dependent induction of daptomycin tolerance in S. aureus, with an approximately 100,000-fold increase in survival after 16 h of growth arrest, relative to exponential-phase bacteria. Daptomycin tolerance required glucose and was associated with increased production of the cell wall polymers peptidoglycan and wall-teichoic acids. However, while the accumulation of peptidoglycan was required for daptomycin tolerance, only a low abundance of wall teichoic acid was necessary. Therefore, whereas tolerance to most antibiotics occurs passively due to a lack of metabolic activity and/or replication, daptomycin tolerance arises via active cell wall remodelling. IMPORTANCE Understanding why antibiotics sometimes fail to cure infections is fundamental to improving treatment outcomes. This is a major challenge when it comes to Staphylococcus aureus because this pathogen causes several different chronic or recurrent infections. Previous work has shown that a lack of replication, as often occurs during infection, makes bacteria tolerant of most bactericidal antibiotics. However, one antibiotic that has been reported to kill nonreplicating bacteria is daptomycin. In this work, we show that the growth arrest of S. aureus does in fact lead to daptomycin tolerance, but it requires time, nutrients, and biosynthetic pathways, making it distinct from other types of antibiotic tolerance that occur in nonreplicating bacteria.

Synergistic Activity of Repurposed Peptide Drug Glatiramer Acetate with Tobramycin against Cystic Fibrosis Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of people with cystic fibrosis (CF), causing both acute and chronic infections. Intrinsic and acquired antibiotic resistance, coupled with the physical barriers resulting from desiccated CF sputum, allow P. aeruginosa to colonize and persist in spite of antibiotic treatment. As well as the specific difficulties in eradicating P. aeruginosa from CF lungs, P. aeruginosa is also subject to the wider, global issue of antimicrobial resistance. Glatiramer acetate (GA) is a peptide drug, used in the treatment of multiple sclerosis (MS), which has been shown to have moderate antipseudomonal activity. Other antimicrobial peptides (AMPs) have been shown to be antibiotic resistance breakers, potentiating the activities of antibiotics when given in combination, restoring and/or enhancing antibiotic efficacy. Growth, viability, MIC determinations, and synergy analysis showed that GA improved the efficacy of tobramycin (TOB) against reference strains of P. aeruginosa, reducing TOB MICs and synergizing with the aminoglycoside. This was also the case for clinical strains from people with CF. GA significantly reduced the MIC50 of TOB for viable cells from 1.69 mg/L (95% confidence interval [CI], 0.26 to 8.97) to 0.62 mg/L (95% CI, 0.15 to 3.94; P = 0.002) and the MIC90 for viable cells from 7.00 mg/L (95% CI, 1.18 to 26.50) to 2.20 mg/L (95% CI, 0.99 to 15.03; P = 0.001), compared to results with TOB only. Investigation of mechanisms of GA activity showed that GA resulted in significant disruption of outer membranes, depolarization of cytoplasmic membranes, and permeabilization of P. aeruginosa and was the only agent tested (including cationic AMPs) to significantly affect all three mechanisms. IMPORTANCE The antimicrobial resistance crisis urgently requires solutions to the lost efficacy of antibiotics. The repurposing of drugs already in clinical use, with strong safety profiles, as antibiotic adjuvants to restore the efficacy of antibiotics is an important avenue to alleviating the resistance crisis. This research shows that a clinically used drug from outside infection treatment, glatiramer acetate, reduces the concentration of tobramycin required to be effective in treating Pseudomonas aeruginosa, based on analyses of both reference and clinical respiratory isolates from people with cystic fibrosis. The two agents acted synergistically against P. aeruginosa, being more effective combined in vitro than predicted for their combination. As a peptide drug, glatiramer acetate functions similarly to many antimicrobial peptides, interacting with and disrupting the P. aeruginosa cell wall and permeabilizing bacterial cells, thereby allowing tobramycin to work. Our findings demonstrate that glatiramer acetate is a strong candidate for repurposing as an antibiotic resistance breaker of pathogenic P. aeruginosa.

Human serum triggers antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus frequently causes infections that are challenging to treat, leading to high rates of persistent and relapsing infection. Here, to understand how the host environment influences treatment outcomes, we study the impact of human serum on staphylococcal antibiotic susceptibility. We show that serum triggers a high degree of tolerance to the lipopeptide antibiotic daptomycin and several other classes of antibiotic. Serum-induced daptomycin tolerance is due to two independent mechanisms. Firstly, the host defence peptide LL-37 induces tolerance by triggering the staphylococcal GraRS two-component system, leading to increased peptidoglycan accumulation. Secondly, GraRS-independent increases in membrane cardiolipin abundance are required for full tolerance. When both mechanisms are blocked, S. aureus incubated in serum is as susceptible to daptomycin as when grown in laboratory media. Our work demonstrates that host factors can significantly modulate antibiotic susceptibility via diverse mechanisms, and combination therapy may provide a way to mitigate this.

Bacterial Toxin-Triggered Release of Antibiotics from Capsosomes Protects a Fly Model from Lethal Methicillin-Resistant Staphylococcus aureus (MRSA) Infection.

Antibiotic resistance is a severe global health threat and hence demands rapid action to develop novel therapies, including microscale drug delivery systems. Herein, a hierarchical microparticle system is developed to achieve bacteria-activated single- and dual-antibiotic drug delivery for preventing methicillin-resistant Staphylococcus aureus (MRSA) bacterial infections. The designed system is based on a capsosome structure, which consists of a mesoporous silica microparticle coated in alternating layers of oppositely charged polymers and antibiotic-loaded liposomes. The capsosomes are engineered and shown to release their drug payloads in the presence of MRSA toxins controlled by the Agr quorum sensing system. MRSA-activated single drug delivery of vancomycin and synergistic dual delivery of vancomycin together with an antibacterial peptide successfully kills MRSA in vitro. The capability of capsosomes to selectively deliver their cargo in the presence of bacteria, producing a bactericidal effect to protect the host organism, is confirmed in vivo using a Drosophila melanogaster MRSA infection model. Thus, the capsosomes serve as a versatile multidrug, subcompartmentalized microparticle system for preventing antibiotic-resistant bacterial infections, with potential applications to protect wounds or medical device implants from infections.

Quantifying transmissibility of SARS-CoV-2 and impact of intervention within long-term healthcare facilities.

Estimates of the basic reproduction number (R 0) for COVID-19 are particularly variable in the context of transmission within locations such as long-term healthcare (LTHC) facilities. We sought to characterize the heterogeneity of R 0 across known outbreaks within these facilities. We used a unique comprehensive dataset of all outbreaks that occurred within LTHC facilities in British Columbia, Canada as of 21 September 2020. We estimated R 0 in 18 LTHC outbreaks with a novel Bayesian hierarchical dynamic model of susceptible, exposed, infected and recovered individuals, incorporating heterogeneity of R 0 between facilities. We further compared these estimates to those obtained with standard methods that use the exponential growth rate and maximum likelihood. The total size of outbreaks varied dramatically, with range of attack rates 2%-86%. The Bayesian analysis provided an overall estimate of R 0 = 2.51 (90% credible interval 0.47-9.0), with individual facility estimates ranging between 0.56 and 9.17. Uncertainty in these estimates was more constrained than standard methods, particularly for smaller outbreaks informed by the population-level model. We further estimated that intervention led to 61% (52%-69%) of all potential cases being averted within the LTHC facilities, or 75% (68%-79%) when using a model with multi-level intervention effect. Understanding of transmission risks and impact of intervention are essential in planning during the ongoing global pandemic, particularly in high-risk environments such as LTHC facilities.

Polymyxin and lipopeptide antibiotics: membrane-targeting drugs of last resort.

The polymyxin and lipopeptide classes of antibiotics are membrane-targeting drugs of last resort used to treat infections caused by multi-drug-resistant pathogens. Despite similar structures, these two antibiotic classes have distinct modes of action and clinical uses. The polymyxins target lipopolysaccharide in the membranes of most Gram-negative species and are often used to treat infections caused by carbapenem-resistant species such as Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa. By contrast, the lipopeptide daptomycin requires membrane phosphatidylglycerol for activity and is only used to treat infections caused by drug-resistant Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. However, despite having distinct targets, both antibiotic classes cause membrane disruption, are potently bactericidal in vitro and share similarities in resistance mechanisms. Furthermore, there are concerns about the efficacy of these antibiotics, and there is increasing interest in using both polymyxins and daptomycin in combination therapies to improve patient outcomes. In this review article, we will explore what is known about these distinct but structurally similar classes of antibiotics, discuss recent advances in the field and highlight remaining gaps in our knowledge.

Human serum induces daptomycin tolerance in Enterococcus faecalis and viridans group streptococci.

Daptomycin is a membrane-targeting lipopeptide antibiotic used in the treatment of infective endocarditis caused by multidrug-resistant Gram-positive bacteria such as Staphylococcus aureus, enterococci and viridans group streptococci. Despite demonstrating excellent in vitro activity and a low prevalence of resistant isolates, treatment failure is a significant concern, particularly for enterococcal infection. We have shown recently that human serum triggers daptomycin tolerance in S. aureus, but it was not clear if a similar phenotype occurred in other major infective endocarditis pathogens. We found that Enterococcus faecalis, Streptococcus gordonii or Streptococcus mutans grown under standard laboratory conditions were efficiently killed by daptomycin, whereas bacteria pre-incubated in human serum survived exposure to the antibiotic, with >99 % cells remaining viable. Incubation of enterococci or streptococci in serum led to peptidoglycan accumulation, as shown by increased incorporation of the fluorescent d-amino acid analogue HADA. Inhibition of peptidoglycan accumulation using the antibiotic fosfomycin resulted in a >tenfold reduction in serum-induced daptomycin tolerance, demonstrating the important contribution of the cell wall to the phenotype. We also identified a small contribution to daptomycin tolerance in E. faecalis from cardiolipin synthases, although this may reflect the inherent increased susceptibility of cardiolipin-deficient mutants. In summary, serum-induced daptomycin tolerance is a consistent phenomenon between Gram-positive infective endocarditis pathogens, but it may be mitigated using currently available antibiotic combination therapy.

Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic.

Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug-resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to bacterial death and lysis. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilized colistin-resistance (mcr) genes. Both these colistin-resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other mcr genes, or resistance due to chromosomal mutations. Using a panel of clinical E. coli that had acquired mcr -1, -1.5, -2, -3, -3.2 or -5, or had acquired polymyxin resistance independently of mcr genes, we found that almost all isolates were susceptible to colistin-mediated permeabilization of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilization of the outer membrane of colistin-resistant isolates by the polymyxin is in turn sufficient to sensitize bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in these E. coli isolates is due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.

DNA Repair in Staphylococcus aureus.

Staphylococcus aureus is a common cause of both superficial and invasive infections of humans and animals. Despite a potent host response and apparently appropriate antibiotic therapy, staphylococcal infections frequently become chronic or recurrent, demonstrating a remarkable ability of S. aureus to withstand the hostile host environment. There is growing evidence that staphylococcal DNA repair makes important contributions to the survival of the pathogen in host tissues, as well as promoting the emergence of mutants that resist host defenses and antibiotics. While much of what we know about DNA repair in S. aureus is inferred from studies with model organisms, the roles of specific repair mechanisms in infection are becoming clear and differences with Bacillus subtilis and Escherichia coli have been identified. Furthermore, there is growing interest in staphylococcal DNA repair as a target for novel therapeutics that sensitize the pathogen to host defenses and antibiotics. In this review, we discuss what is known about staphylococcal DNA repair and its role in infection, examine how repair in S. aureus is similar to, or differs from, repair in well-characterized model organisms, and assess the potential of staphylococcal DNA repair as a novel therapeutic target.

RexAB Promotes the Survival of Staphylococcus aureus Exposed to Multiple Classes of Antibiotics.

Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and, in some cases, cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA, leading to induction of the mutagenic SOS response associated with the emergence of drug resistance. However, the type of DNA damage that arises and how this triggers the SOS response are largely unclear. We found that several different classes of antibiotic triggered dose-dependent induction of the SOS response in Staphylococcus aureus, indicative of DNA damage, including some bacteriostatic drugs. The SOS response was heterogenous and varied in magnitude between strains and antibiotics. However, in many cases, full induction of the SOS response was dependent upon the RexAB helicase/nuclease complex, which processes DNA double-strand breaks to produce single-stranded DNA and facilitate RecA nucleoprotein filament formation. The importance of RexAB in repair of DNA was confirmed by measuring bacterial survival during antibiotic exposure, with most drugs having significantly greater bactericidal activity against rexB mutants than against wild-type strains. For some, but not all, antibiotics there was no difference in bactericidal activity between wild type and rexB mutant under anaerobic conditions, indicative of a role for reactive oxygen species in mediating DNA damage. Taken together, this work confirms previous observations that several classes of antibiotics cause DNA damage in S. aureus and extends them by showing that processing of DNA double-strand breaks by RexAB is a major trigger of the mutagenic SOS response and promotes bacterial survival.

Assessing the impact of silicon nanowires on bacterial transformation and viability of Escherichia coli.

We investigated the biomaterial interface between the bacteria Escherichia coli DH5α and silicon nanowire patterned surfaces. We optimised the engineering of silicon nanowire coated surfaces using metal-assisted chemical etching. Using a combination of focussed ion beam scanning electron microscopy, and cell viability and transformation assays, we found that with increasing interfacing force, cell viability decreases, as a result of increasing cell rupture. However, despite this aggressive interfacing regime, a proportion of the bacterial cell population remains viable. We found that the silicon nanowires neither resulted in complete loss of cell viability nor partial membrane disruption and corresponding DNA plasmid transformation. Critically, assay choice was observed to be important, as a reduction-based metabolic reagent was found to yield false-positive results on the silicon nanowire substrate. We discuss the implications of these results for the future design and assessment of bacteria-nanostructure interfacing experiments.

Lattice Boltzmann Simulations of Multiphase Dielectric Fluids.

The dynamic effect of an electric field on dielectric liquids is called liquid dielectrophoresis. It is widely used in several industrial and scientific applications, including inkjet printing, microfabrication, and optical devices. Numerical simulations of liquid-dielectrophoresis are necessary to understand the fundamental physics of the phenomenon, but also to explore situations that might be difficult or expensive to implement experimentally. However, such modeling is challenging, as one needs to solve the electrostatic and fluid dynamics equations simultaneously. Here, we formulate a new lattice-Boltzmann method capable of modeling the dynamics of immiscible dielectric fluids coupled with electric fields within a single framework, thus eliminating the need of using separate algorithms to solve the electrostatic and fluid dynamics equations. We validate the numerical method by comparing it with analytical solutions and previously reported experimental results. Beyond the benchmarking of the method, we study the spreading of a droplet using a dielectrowetting setup and quantify the mechanism driving the variation of the apparent contact angle of the droplet with the applied voltage. Our method provides a useful tool to study liquid-dielectrophoresis and can be used to model dielectric fluids in general, such as liquid-liquid and liquid-gas systems.

Practical and Clinical Approaches Using Pacing to Improve Selfregulation in Special Populations such as Children and People with Mental Health or Learning Disabilities.

For special populations such as people with a mental health issue or learning disability, a disconnect between the ability to accurately monitor and regulate exercise behaviour can lead to reduced levels of physical activity, which, in turn, is associated with additional physical or mental health problems. Activity pacing is a strategy used in clinical settings to address issues of pain amelioration, while self-pacing research is now well addressed in sport and exercise science literature. It has been proposed recently that these overlapping areas of investigation collectively support the development of self-regulatory, lifestyle exercise skills across broad population groups. Activity pacing appears to have substantial application in numerous development and rehabilitation settings and, therefore, the purpose of this short communication is to articulate how an activity pacing approach could be utilized among population groups in whom self-regulatory skills may require development. This paper provides specific examples of exercise practice across 2 discrete populations: children, and people with mental health and learning difficulties. In these cases, homeostatic regulatory processes may either be altered, or the individual may require extrinsic support to appropriately self-regulate exercise performance. A support-based exercise environment or approach such as programmatic activity (lifestyle) pacing would be beneficial to facilitate supervised and education-based self-regulation until such time as fully self-regulated exercise is feasible.

How much leeway is there to relax COVID-19 control measures?

Following successful non-pharmaceutical interventions (NPI) aiming to control COVID-19, many jurisdictions reopened their economies and borders. As little immunity had developed in most populations, re-establishing higher contact carried substantial risks, and therefore many locations began to see resurgence in COVID-19 cases. We present a Bayesian method to estimate the leeway to reopen, or alternatively the strength of change required to re-establish COVID-19 control, in a range of jurisdictions experiencing different COVID-19 epidemics. We estimated the timing and strength of initial control measures such as widespread distancing and compared the leeway jurisdictions had to reopen immediately after NPI measures to later estimates of leeway. Finally, we quantified risks associated with reopening and the likely burden of new cases due to introductions from other jurisdictions. We found widely varying leeway to reopen. After initial NPI measures took effect, some jurisdictions had substantial leeway (e.g., Japan, New Zealand, Germany) with > 0.99 probability that contact rates were below 80% of the threshold for epidemic growth. Others had little leeway (e.g., the United Kingdom, Washington State) and some had none (e.g., Sweden, California). For most such regions, increases in contact rate of 1.5-2 fold would have had high (> 0.7) probability of exceeding past peak sizes. Most jurisdictions experienced June-August trajectories consistent with our projections of contact rate increases of 1-2-fold. Under such relaxation scenarios for some regions, we projected up to ∼100 additional cases if just one case were imported per week over six weeks, even between jurisdictions with comparable COVID-19 risk. We provide an R package covidseir to enable jurisdictions to estimate leeway and forecast cases under different future contact patterns. Estimates of leeway can establish a quantitative basis for decisions about reopening. We recommend a cautious approach to reopening economies and borders, coupled with strong monitoring for changes in transmission.