Individual bacteria in structured environments rely on phenotypic resistance to phage.

Bacteriophages represent an avenue to overcome the current antibiotic resistance crisis, but evolution of genetic resistance to phages remains a concern. In vitro, bacteria evolve genetic resistance, preventing phage adsorption or degrading phage DNA. In natural environments, evolved resistance is lower possibly because the spatial heterogeneity within biofilms, microcolonies, or wall populations favours phenotypic survival to lytic phages. However, it is also possible that the persistence of genetically sensitive bacteria is due to less efficient phage amplification in natural environments, the existence of refuges where bacteria can hide, and a reduced spread of resistant genotypes. Here, we monitor the interactions between individual planktonic bacteria in isolation in ephemeral refuges and bacteriophage by tracking the survival of individual cells. We find that in these transient spatial refuges, phenotypic resistance due to reduced expression of the phage receptor is a key determinant of bacterial survival. This survival strategy is in contrast with the emergence of genetic resistance in the absence of ephemeral refuges in well-mixed environments. Predictions generated via a mathematical modelling framework to track bacterial response to phages reveal that the presence of spatial refuges leads to fundamentally different population dynamics that should be considered in order to predict and manipulate the evolutionary and ecological dynamics of bacteria-phage interactions in naturally structured environments.

Diagnostic MALDI-TOF MS can differentiate between high and low toxic Staphylococcus aureus bacteraemia isolates as a predictor of patient outcome.

Staphylococcus aureus bacteraemia (SAB) is a major cause of blood-stream infection (BSI) in both healthcare and community settings. While the underlying comorbidities of a patient significantly contributes to their susceptibility to and outcome following SAB, recent studies show the importance of the level of cytolytic toxin production by the infecting bacterium. In this study we demonstrate that this cytotoxicity can be determined directly from the diagnostic MALDI-TOF mass spectrum generated in a routine diagnostic laboratory. With further development this information could be used to guide the management and improve the outcomes for SAB patients.

A functional menadione biosynthesis pathway is required for capsule production by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus, we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV, we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione is involved in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

Repeated clinical malaria episodes are associated with modification of the immune system in children.

BACKGROUND: There are over 200 million reported cases of malaria each year, and most children living in endemic areas will experience multiple episodes of clinical disease before puberty. We set out to understand how frequent clinical malaria, which elicits a strong inflammatory response, affects the immune system and whether these modifications are observable in the absence of detectable parasitaemia. METHODS: We used a multi-dimensional approach comprising whole blood transcriptomic, cellular and plasma cytokine analyses on a cohort of children living with endemic malaria, but uninfected at sampling, who had been under active surveillance for malaria for 8 years. Children were categorised into two groups depending on the cumulative number of episodes experienced: high (≥ 8) or low (< 5). RESULTS: We observe that multiple episodes of malaria are associated with modification of the immune system. Children who had experienced a large number of episodes demonstrated upregulation of interferon-inducible genes, a clear increase in circulating levels of the immunoregulatory cytokine IL-10 and enhanced activation of neutrophils, B cells and CD8+ T cells. CONCLUSION: Transcriptomic analysis together with cytokine and immune cell profiling of peripheral blood can robustly detect immune differences between children with different numbers of prior malaria episodes. Multiple episodes of malaria are associated with modification of the immune system in children. Such immune modifications may have implications for the initiation of subsequent immune responses and the induction of vaccine-mediated protection.

Multiscale Immune Selection and the Transmission-Diversity Feedback in Antigenically Diverse Pathogen Systems.

Antigenic diversity is commonly used by pathogens to enhance their transmission success. Within-host clonal antigenic variation helps to maintain long infectious periods, whereas high levels of allelic diversity at the population level significantly expand the pool of susceptible individuals. Diversity, however, is not necessarily a static property of a pathogen population but in many cases is generated by the very act of infection and transmission, and it is therefore expected to respond dynamically to changes in transmission and immune selection. We hypothesized that this coupling creates a positive feedback whereby infection and disease transmission promote the generation of diversity, which itself facilitates immune evasion and further infections. To investigate this link in more detail, we considered the human malaria parasite Plasmodium falciparum, one of the most important antigenically diverse pathogens. We developed an individual-based model in which antigenic diversity emerges as a dynamic property from the underlying transmission processes. Our results show that the balance between stochastic extinction and the generation of new antigenic variants is intrinsically linked to within-host and between-host immune selection. This in turn determines the level of diversity that can be maintained in a given population. Furthermore, the transmission-diversity feedback can lead to temporal lags in the response to natural or intervention-induced perturbations in transmission rates. Our results therefore have important implications for monitoring and assessing the effectiveness of disease control efforts.

Quantitative host resistance drives the evolution of increased virulence in an emerging pathogen.

Emergent infectious diseases can have a devastating impact on host populations. The high selective pressures on both the hosts and the pathogens frequently lead to rapid adaptations not only in pathogen virulence but also host resistance following an initial outbreak. However, it is often unclear whether hosts will evolve to avoid infection-associated fitness costs by preventing the establishment of infection (here referred to as qualitative resistance) or by limiting its deleterious effects through immune functioning (here referred to as quantitative resistance). Equally, the evolutionary repercussions these different resistance mechanisms have for the pathogen are often unknown. Here, we investigate the co-evolutionary dynamics of pathogen virulence and host resistance following the epizootic outbreak of the highly pathogenic bacterium Mycoplasma gallisepticum in North American house finches (Haemorhous mexicanus). Using an evolutionary modelling approach and with a specific emphasis on the evolved resistance trait, we demonstrate that the rapid increase in the frequency of resistant birds following the outbreak is indicative of strong selection pressure to reduce infection-associated mortality. This, in turn, created the ecological conditions that selected for increased bacterial virulence. Our results thus suggest that quantitative host resistance was the key factor underlying the evolutionary interactions in this natural host-pathogen system.

Evolutionary Trade-Offs Underlie the Multi-faceted Virulence of Staphylococcus aureus.

Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.

Identification of immune signatures predictive of clinical protection from malaria.

Antibodies are thought to play an essential role in naturally acquired immunity to malaria. Prospective cohort studies have frequently shown how continuous exposure to the malaria parasite Plasmodium falciparum cause an accumulation of specific responses against various antigens that correlate with a decreased risk of clinical malaria episodes. However, small effect sizes and the often polymorphic nature of immunogenic parasite proteins make the robust identification of the true targets of protective immunity ambiguous. Furthermore, the degree of individual-level protection conferred by elevated responses to these antigens has not yet been explored. Here we applied a machine learning approach to identify immune signatures predictive of individual-level protection against clinical disease. We find that commonly assumed immune correlates are poor predictors of clinical protection in children. On the other hand, antibody profiles predictive of an individual's malaria protective status can be found in data comprising responses to a large set of diverse parasite proteins. We show that this pattern emerges only after years of continuous exposure to the malaria parasite, whereas susceptibility to clinical episodes in young hosts (< 10 years) cannot be ascertained by measured antibody responses alone.

Predicting the virulence of MRSA from its genome sequence.

Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogen's evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolate's toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.

Protein Traffic to the Plasmodium falciparum apicoplast: evidence for a sorting branch point at the Golgi.

Plasmodium falciparum, similar to many other apicomplexan parasites, contains an apicoplast, a plastid organelle of secondary endosymbiotic origin. Nuclear-encoded proteins are targeted to the apicoplast by a bipartite topogenic signal consisting of (i) an endoplasmic reticulum (ER)-type N-terminal secretory signal peptide, followed by (ii) a plant-like transit peptide. Although the signals responsible for transport of most proteins to the apicoplast are well described, the route of trafficking from the ER to the outermost apicoplast membrane is still a matter of debate. Current models of trafficking to the apicoplast suggest that proteins destined for this organelle are, on entry into the lumen of the ER, diverted from the default secretory pathway to a specialized vesicular system which carries proteins directly from the ER to the outer apicoplast membrane. Here, we have re-examined this trafficking pathway. By titrating wild-type and mutant apicoplast transit peptides against different ER retrieval sequences and studying protein transport in a brefeldin A-resistant parasite line, we generated data which suggest a direct involvement of the Golgi in traffic of soluble proteins to the P. falciparum apicoplast.

The Long-Term Safety, Public Health Impact, and Cost-Effectiveness of Routine Vaccination with a Recombinant, Live-Attenuated Dengue Vaccine (Dengvaxia): A Model Comparison Study.

BACKGROUND: Large Phase III trials across Asia and Latin America have recently demonstrated the efficacy of a recombinant, live-attenuated dengue vaccine (Dengvaxia) over the first 25 mo following vaccination. Subsequent data collected in the longer-term follow-up phase, however, have raised concerns about a potential increase in hospitalization risk of subsequent dengue infections, in particular among young, dengue-naïve vaccinees. We here report predictions from eight independent modelling groups on the long-term safety, public health impact, and cost-effectiveness of routine vaccination with Dengvaxia in a range of transmission settings, as characterised by seroprevalence levels among 9-y-olds (SP9). These predictions were conducted for the World Health Organization to inform their recommendations on optimal use of this vaccine. METHODS AND FINDINGS: The models adopted, with small variations, a parsimonious vaccine mode of action that was able to reproduce quantitative features of the observed trial data. The adopted mode of action assumed that vaccination, similarly to natural infection, induces transient, heterologous protection and, further, establishes a long-lasting immunogenic memory, which determines disease severity of subsequent infections. The default vaccination policy considered was routine vaccination of 9-y-old children in a three-dose schedule at 80% coverage. The outcomes examined were the impact of vaccination on infections, symptomatic dengue, hospitalised dengue, deaths, and cost-effectiveness over a 30-y postvaccination period. Case definitions were chosen in accordance with the Phase III trials. All models predicted that in settings with moderate to high dengue endemicity (SP9 ≥ 50%), the default vaccination policy would reduce the burden of dengue disease for the population by 6%-25% (all simulations: -3%-34%) and in high-transmission settings (SP9 ≥ 70%) by 13%-25% (all simulations: 10%- 34%). These endemicity levels are representative of the participating sites in both Phase III trials. In contrast, in settings with low transmission intensity (SP9 ≤ 30%), the models predicted that vaccination could lead to a substantial increase in hospitalisation because of dengue. Modelling reduced vaccine coverage or the addition of catch-up campaigns showed that the impact of vaccination scaled approximately linearly with the number of people vaccinated. In assessing the optimal age of vaccination, we found that targeting older children could increase the net benefit of vaccination in settings with moderate transmission intensity (SP9 = 50%). Overall, vaccination was predicted to be potentially cost-effective in most endemic settings if priced competitively. The results are based on the assumption that the vaccine acts similarly to natural infection. This assumption is consistent with the available trial results but cannot be directly validated in the absence of additional data. Furthermore, uncertainties remain regarding the level of protection provided against disease versus infection and the rate at which vaccine-induced protection declines. CONCLUSIONS: Dengvaxia has the potential to reduce the burden of dengue disease in areas of moderate to high dengue endemicity. However, the potential risks of vaccination in areas with limited exposure to dengue as well as the local costs and benefits of routine vaccination are important considerations for the inclusion of Dengvaxia into existing immunisation programmes. These results were important inputs into WHO global policy for use of this licensed dengue vaccine.

Natural, persistent oscillations in a spatial multi-strain disease system with application to dengue.

Many infectious diseases are not maintained in a state of equilibrium but exhibit significant fluctuations in prevalence over time. For pathogens that consist of multiple antigenic types or strains, such as influenza, malaria or dengue, these fluctuations often take on the form of regular or irregular epidemic outbreaks in addition to oscillatory prevalence levels of the constituent strains. To explain the observed temporal dynamics and structuring in pathogen populations, epidemiological multi-strain models have commonly evoked strong immune interactions between strains as the predominant driver. Here, with specific reference to dengue, we show how spatially explicit, multi-strain systems can exhibit all of the described epidemiological dynamics even in the absence of immune competition. Instead, amplification of natural stochastic differences in disease transmission, can give rise to persistent oscillations comprising semi-regular epidemic outbreaks and sequential dominance of dengue's four serotypes. Not only can this mechanism explain observed differences in serotype and disease distributions between neighbouring geographical areas, it also has important implications for inferring the nature and epidemiological consequences of immune mediated competition in multi-strain pathogen systems.

Role of stochastic processes in maintaining discrete strain structure in antigenically diverse pathogen populations.

Many highly diverse pathogen populations appear to exist stably as discrete antigenic types despite evidence of genetic exchange. It has been shown that this may arise as a consequence of immune selection on pathogen populations, causing them to segregate permanently into discrete nonoverlapping subsets of antigenic variants to minimize competition for available hosts. However, discrete antigenic strain structure tends to break down under conditions where there are unequal numbers of allelic variants at each locus. Here, we show that the inclusion of stochastic processes can lead to the stable recovery of discrete strain structure through loss of certain alleles. This explains how pathogen populations may continue to behave as independently transmitted strains despite inevitable asymmetries in allelic diversity of major antigens. We present evidence for this type of structuring across global meningococcal isolates in three diverse antigens that are currently being developed as vaccine components.

Negative epistasis between the malaria-protective effects of alpha+-thalassemia and the sickle cell trait.

The hemoglobinopathies, disorders of hemoglobin structure and production, protect against death from malaria. In sub-Saharan Africa, two such conditions occur at particularly high frequencies: presence of the structural variant hemoglobin S and alpha(+)-thalassemia, a condition characterized by reduced production of the normal alpha-globin component of hemoglobin. Individually, each is protective against severe Plasmodium falciparum malaria, but little is known about their malaria-protective effects when inherited in combination. We investigated this question by studying a population on the coast of Kenya and found that the protection afforded by each condition inherited alone was lost when the two conditions were inherited together, to such a degree that the incidence of both uncomplicated and severe P. falciparum malaria was close to baseline in children heterozygous with respect to the mutation underlying the hemoglobin S variant and homozygous with respect to the mutation underlying alpha(+)-thalassemia. Negative epistasis could explain the failure of alpha(+)-thalassemia to reach fixation in any population in sub-Saharan Africa.

VespAI: a deep learning-based system for the detection of invasive hornets.

The invasive hornet Vespa velutina nigrithorax is a rapidly proliferating threat to pollinators in Europe and East Asia. To effectively limit its spread, colonies must be detected and destroyed early in the invasion curve, however the current reliance upon visual alerts by the public yields low accuracy. Advances in deep learning offer a potential solution to this, but the application of such technology remains challenging. Here we present VespAI, an automated system for the rapid detection of V. velutina. We leverage a hardware-assisted AI approach, combining a standardised monitoring station with deep YOLOv5s architecture and a ResNet backbone, trained on a bespoke end-to-end pipeline. This enables the system to detect hornets in real-time-achieving a mean precision-recall score of ≥0.99-and send associated image alerts via a compact remote processor. We demonstrate the successful operation of a prototype system in the field, and confirm its suitability for large-scale deployment in future use cases. As such, VespAI has the potential to transform the way that invasive hornets are managed, providing a robust early warning system to prevent ingressions into new regions.

Shifting patterns of dengue three years after Zika virus emergence in Brazil.

In 2015, the Zika virus (ZIKV) emerged in Brazil, leading to widespread outbreaks in Latin America. Following this, many countries in these regions reported a significant drop in the circulation of dengue virus (DENV), which resurged in 2018-2019. We examine age-specific incidence data to investigate changes in DENV epidemiology before and after the emergence of ZIKV. We observe that incidence of DENV was concentrated in younger individuals during resurgence compared to 2013-2015. This trend was more pronounced in Brazilian states that had experienced larger ZIKV outbreaks. Using a mathematical model, we show that ZIKV-induced cross-protection alone, often invoked to explain DENV decline across Latin America, cannot explain the observed age-shift without also assuming some form of disease enhancement. Our results suggest that a sudden accumulation of population-level immunity to ZIKV could suppress DENV and reduce the mean age of DENV incidence via both protective and disease-enhancing interactions.

Markers of prolonged hospitalisation in severe dengue.

BACKGROUND: Dengue is one of the most common diseases in the tropics and subtropics. Whilst mortality is a rare event when adequate supportive care can be provided, a large number of patients get hospitalised with dengue every year that places a heavy burden on local health systems. A better understanding of the support required at the time of hospitalisation is therefore of critical importance for healthcare planning, especially when resources are limited during major outbreaks. METHODS: Here we performed a retrospective analysis of clinical data from over 1500 individuals hospitalised with dengue in Vietnam between 2017 and 2019. Using a broad panel of potential biomarkers, we sought to evaluate robust predictors of prolonged hospitalisation periods. RESULTS: Our analyses revealed a lead-time bias, whereby early admission to hospital correlates with longer hospital stays - irrespective of disease severity. Importantly, taking into account the symptom duration prior to hospitalisation significantly affects observed associations between hospitalisation length and previously reported risk markers of prolonged stays, which themselves showed marked inter-annual variations. Once corrected for symptom duration, age, temperature at admission and elevated neutrophil-to-lymphocyte ratio were found predictive of longer hospitalisation periods. CONCLUSION: This study demonstrates that the time since dengue symptom onset is one of the most significant predictors for the length of hospital stays, independent of the assigned severity score. Pre-hospital symptom durations need to be accounted for to evaluate clinically relevant biomarkers of dengue hospitalisation trajectories.

Multi-layered genome defences in bacteria.

Bacteria have evolved a variety of defence mechanisms to protect against mobile genetic elements, including restriction-modification systems and CRISPR-Cas. In recent years, dozens of previously unknown defence systems (DSs) have been discovered. Notably, diverse DSs often coexist within the same genome, and some co-occur at frequencies significantly higher than would be expected by chance, implying potential synergistic interactions. Recent studies have provided evidence of defence mechanisms that enhance or complement one another. Here, we review the interactions between DSs at the mechanistic, regulatory, ecological and evolutionary levels.

Antigenic variation in Plasmodium falciparum malaria involves a highly structured switching pattern.

Many pathogenic bacteria, fungi, and protozoa achieve chronic infection through an immune evasion strategy known as antigenic variation. In the human malaria parasite Plasmodium falciparum, this involves transcriptional switching among members of the var gene family, causing parasites with different antigenic and phenotypic characteristics to appear at different times within a population. Here we use a genome-wide approach to explore this process in vitro within a set of cloned parasite populations. Our analyses reveal a non-random, highly structured switch pathway where an initially dominant transcript switches via a set of switch-intermediates either to a new dominant transcript, or back to the original. We show that this specific pathway can arise through an evolutionary conflict in which the pathogen has to optimise between safeguarding its limited antigenic repertoire and remaining capable of establishing infections in non-naïve individuals. Our results thus demonstrate a crucial role for structured switching during the early phases of infections and provide a unifying theory of antigenic variation in P. falciparum malaria as a balanced process of parasite-intrinsic switching and immune-mediated selection.

Evolution of the multi-domain structures of virulence genes in the human malaria parasite, Plasmodium falciparum.

The var gene family of Plasmodium falciparum encodes the immunodominant variant surface antigens PfEMP1. These highly polymorphic proteins are important virulence factors that mediate cytoadhesion to a variety of host tissues, causing sequestration of parasitized red blood cells in vital organs, including the brain or placenta. Acquisition of variant-specific antibodies correlates with protection against severe malarial infections; however, understanding the relationship between gene expression and infection outcome is complicated by the modular genetic architectures of var genes that encode varying numbers of antigenic domains with differential binding specificities. By analyzing the domain architectures of fully sequenced var gene repertoires we reveal a significant, non-random association between the number of domains comprising a var gene and their sequence conservation. As such, var genes can be grouped into those that are short and diverse and genes that are long and conserved, suggesting gene length as an important characteristic in the classification of var genes. We then use an evolutionary framework to demonstrate how the same evolutionary forces acting on the level of an individual gene may have also shaped the parasite's gene repertoire. The observed associations between sequence conservation, gene architecture and repertoire structure can thus be explained by a trade-off between optimizing within-host fitness and minimizing between-host immune selection pressure. Our results demonstrate how simple evolutionary mechanisms can explain var gene structuring on multiple levels and have important implications for understanding the multifaceted epidemiology of P. falciparum malaria.