Restriction-modification systems have shaped the evolution and distribution of plasmids across bacteria.

Many novel traits such as antibiotic resistance are spread by plasmids between species. Yet plasmids have different host ranges. Restriction-modification systems (R-M systems) are by far the most abundant bacterial defense system and therefore represent one of the key barriers to plasmid spread. However, their effect on plasmid evolution and host range has been neglected. Here we analyse the avoidance of targets of the most abundant R-M systems (Type II) for complete genomes and plasmids across bacterial diversity. For the most common target length (6 bp) we show that target avoidance is strongly correlated with the taxonomic distribution of R-M systems and is greater in plasmid genes than core genes. We find stronger avoidance of R-M targets in plasmids which are smaller and have a broader host range. Our results suggest two different evolutionary strategies for plasmids: small plasmids primarily adapt to R-M systems by tuning their sequence composition, and large plasmids primarily adapt through the carriage of additional genes protecting from restriction. Our work provides systematic evidence that R-M systems are important barriers to plasmid transfer and have left their mark on plasmids over long evolutionary time.

Antigen specific T cells in people with obesity at five months following ChAdOx1 COVID-19 vaccination.

BACKGROUND/OBJECTIVES: People with obesity (PWO) face an increased risk of severe outcomes from COVID-19, including hospitalisation, ICU admission and death. Obesity has been seen to impair immune memory following vaccination against influenza, hepatitis B, tetanus, and rabies. Little is known regarding immune memory in PWO following COVID-19 adenovirus vector vaccination. SUBJECTS/METHODS: We investigated SARS-CoV-2 specific T cell responses in 50 subjects, five months following a two-dose primary course of ChAdOx1 nCoV-19 (AZD1222) vaccination. We further divided our cohort into PWO (n = 30) and matched controls (n = 20). T cell (CD4+, CD8+) cytokine responses (IFNγ, TNFα) to SARS-CoV-2 spike peptide pools were determined using multicolour flow cytometry. RESULTS: Circulating T cells specific for SARS-CoV-2 were readily detected across our cohort, with robust responses to spike peptide stimulation across both T cell lines. PWO and controls had comparable levels of both CD4+ and CD8+ SARS-CoV-2 spike specific T cells. Polyfunctional T cells - associated with enhanced protection against viral infection - were detected at similar frequencies in both PWO and controls. CONCLUSIONS: These data indicate that PWO who have completed a primary course of ChAdOx1 COVID-19 vaccination have robust, durable, and functional antigen specific T cell immunity that is comparable to that seen in people without obesity.

Diverse Durham collection phages demonstrate complex BREX defense responses.

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.

The phylogenetic range of bacterial and viral pathogens of vertebrates.

Many major human pathogens are multihost pathogens, able to infect other vertebrate species. Describing the general patterns of host-pathogen associations across pathogen taxa is therefore important to understand risk factors for human disease emergence. However, there is a lack of comprehensive curated databases for this purpose, with most previous efforts focusing on viruses. Here, we report the largest manually compiled host-pathogen association database, covering 2,595 bacteria and viruses infecting 2,656 vertebrate hosts. We also build a tree for host species using nine mitochondrial genes, giving a quantitative measure of the phylogenetic similarity of hosts. We find that the majority of bacteria and viruses are specialists infecting only a single host species, with bacteria having a significantly higher proportion of specialists compared to viruses. Conversely, multihost viruses have a more restricted host range than multihost bacteria. We perform multiple analyses of factors associated with pathogen richness per host species and the pathogen traits associated with greater host range and zoonotic potential. We show that factors previously identified as important for zoonotic potential in viruses-such as phylogenetic range, research effort, and being vector-borne-are also predictive in bacteria. We find that the fraction of pathogens shared between two hosts decreases with the phylogenetic distance between them. Our results suggest that host phylogenetic similarity is the primary factor for host-switching in pathogens.

Microbial Translocation Does Not Drive Immune Activation in Ugandan Children Infected With HIV.

Objective: Immune activation is associated with morbidity and mortality during human immunodeficiency virus (HIV) infection, despite receipt of antiretroviral therapy (ART). We investigated whether microbial translocation drives immune activation in HIV-infected Ugandan children. Methods: Nineteen markers of immune activation and inflammation were measured over 96 weeks in HIV-infected Ugandan children in the CHAPAS-3 Trial and HIV-uninfected age-matched controls. Microbial translocation was assessed using molecular techniques, including next-generation sequencing. Results: Of 249 children included, 142 were infected with HIV; of these, 120 were ART naive, with a median age of 2.8 years (interquartile range [IQR], 1.7-4.0 years) and a median baseline CD4+ T-cell percentage of 20% (IQR, 14%-24%), and 22 were ART experienced, with a median age of 6.5 years (IQR, 5.9-9.2 years) and a median baseline CD4+ T-cell percentage of 35% (IQR, 31%-39%). The control group comprised 107 children without HIV infection. The median increase in the CD4+ T-cell percentage was 17 percentage points (IQR, 12-22 percentage points) at week 96 among ART-naive children, and the viral load was <100 copies/mL in 76% of ART-naive children and 91% of ART-experienced children. Immune activation decreased with ART use. Children could be divided on the basis of immune activation markers into the following 3 clusters: in cluster 1, the majority of children were HIV uninfected; cluster 2 comprised a mix of HIV-uninfected children and HIV-infected ART-naive or ART-experienced children; and in cluster 3, the majority were ART naive. Immune activation was low in cluster 1, decreased in cluster 3, and persisted in cluster 2. Blood microbial DNA levels were negative or very low across groups, with no difference between clusters except for Enterobacteriaceae organisms (the level was higher in cluster 1; P < .0001). Conclusion: Immune activation decreased with ART use, with marker clustering indicating different activation patterns according to HIV and ART status. Levels of bacterial DNA in blood were low regardless of HIV status, ART status, and immune activation status. Microbial translocation did not drive immune activation in this setting. Clinical Trials Registration: ISRCTN69078957.

Children With Cystic Fibrosis Are Infected With Multiple Subpopulations of Mycobacterium abscessus With Different Antimicrobial Resistance Profiles.

BACKGROUND: Children with cystic fibrosis (CF) can develop life-threatening infections of Mycobacterium abscessus. These present a significant clinical challenge, particularly when the strains involved are resistant to antibiotics. Recent evidence of within-patient subclones of M. abscessus in adults with CF suggests the possibility that within-patient diversity may be relevant for the treatment of pediatric CF patients. METHODS: We performed whole-genome sequencing (WGS) on 32 isolates of M. abscessus that were taken from multiple body sites of 2 patients with CF who were undergoing treatment at Great Ormond Street Hospital, United Kingdom, in 2015. RESULTS: We found evidence of extensive diversity within patients over time. A clustering analysis of single nucleotide variants revealed that each patient harbored multiple subpopulations, which were differentially abundant between sputum, lung samples, chest wounds, and pleural fluid. The sputum isolates did not reflect the overall within-patient diversity and did not allow for the detection of subclones with mutations previously associated with macrolide resistance (rrl 2058/2059). Some variants were present at intermediate frequencies before the lung transplants. The time of the transplants coincided with extensive variation, suggesting that this event is particularly disruptive for the microbial community, but the transplants did not clear the M. abscessus infections and both patients died as a result of these infections. CONCLUSIONS: Isolates of M. abscessus from sputum do not always reflect the entire diversity present within the patient, which can include subclones with differing antimicrobial resistance profiles. An awareness of this phenotypic variability, with the sampling of multiple body sites in conjunction with WGS, may be necessary to ensure the best treatment for this vulnerable patient group.

Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

BACKGROUND: Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity. We compared M. tuberculosis whole genome sequences generated from 33 paired clinical samples using two methods. In one method DNA was extracted directly from sputum then enriched with custom-designed SureSelect (Agilent) oligonucleotide baits and in the other it was extracted from mycobacterial growth indicator tube (MGIT) culture. RESULTS: DNA directly sequenced from sputum showed significantly more within-sample diversity than that from MGIT culture (median 5.0 vs 4.5 heterozygous alleles per sample, p = 0.04). Resistance associated variants present as HAs occurred in four patients, and in two cases may provide a genotypic explanation for phenotypic resistance. CONCLUSIONS: Culture-free M. tuberculosis whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating.

Correction to: Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

He authors reported that one of the authors' names was typeset incorrectly in the authorship list.

Oral microbiomes from hunter-gatherers and traditional farmers reveal shifts in commensal balance and pathogen load linked to diet.

Maladaptation to modern diets has been implicated in several chronic disorders. Given the higher prevalence of disease such as dental caries and chronic gum diseases in industrialized societies, we sought to investigate the impact of different subsistence strategies on oral health and physiology, as documented by the oral microbiome. To control for confounding variables such as environment and host genetics, we sampled saliva from three pairs of populations of hunter-gatherers and traditional farmers living in close proximity in the Philippines. Deep shotgun sequencing of salivary DNA generated high-coverage microbiomes along with human genomes. Comparing these microbiomes with publicly available data from individuals living on a Western diet revealed that abundance ratios of core species were significantly correlated with subsistence strategy, with hunter-gatherers and Westerners occupying either end of a gradient of Neisseria against Haemophilus, and traditional farmers falling in between. Species found preferentially in hunter-gatherers included microbes often considered as oral pathogens, despite their hosts' apparent good oral health. Discriminant analysis of gene functions revealed vitamin B5 autotrophy and urease-mediated pH regulation as candidate adaptations of the microbiome to the hunter-gatherer and Western diets, respectively. These results suggest that major transitions in diet selected for different communities of commensals and likely played a role in the emergence of modern oral pathogens.

DNA hairpins destabilize duplexes primarily by promoting melting rather than by inhibiting hybridization.

The effect of secondary structure on DNA duplex formation is poorly understood. Using oxDNA, a nucleotide level coarse-grained model of DNA, we study how hairpins influence the rate and reaction pathways of DNA hybridzation. We compare to experimental systems studied by Gao et al. (1) and find that 3-base pair hairpins reduce the hybridization rate by a factor of 2, and 4-base pair hairpins by a factor of 10, compared to DNA with limited secondary structure, which is in good agreement with experiments. By contrast, melting rates are accelerated by factors of ∼100 and ∼2000. This surprisingly large speed-up occurs because hairpins form during the melting process, and significantly lower the free energy barrier for dissociation. These results should assist experimentalists in designing sequences to be used in DNA nanotechnology, by putting limits on the suppression of hybridization reaction rates through the use of hairpins and offering the possibility of deliberately increasing dissociation rates by incorporating hairpins into single strands.

Distinguishing the Signals of Gingivitis and Periodontitis in Supragingival Plaque: a Cross-Sectional Cohort Study in Malawi.

UNLABELLED: Periodontal disease ranges from gingival inflammation (gingivitis) to the inflammation and loss of tooth-supporting tissues (periodontitis). Previous research has focused mainly on subgingival plaque, but supragingival plaque composition is also known to be associated with disease. Quantitative modeling of bacterial abundances across the natural range of periodontal severities can distinguish which features of disease are associated with particular changes in composition. We assessed a cross-sectional cohort of 962 Malawian women for periodontal disease and used 16S rRNA gene amplicon sequencing (V5 to V7 region) to characterize the bacterial compositions of supragingival plaque samples. Associations between bacterial relative abundances and gingivitis/periodontitis were investigated by using negative binomial models, adjusting for epidemiological factors. We also examined bacterial cooccurrence networks to assess community structure. The main differences in supragingival plaque compositions were associated more with gingivitis than periodontitis, including higher bacterial diversity and a greater abundance of particular species. However, even after controlling for gingivitis, the presence of subgingival periodontitis was associated with an altered supragingival plaque. A small number of species were associated with periodontitis but not gingivitis, including members of Prevotella, Treponema, and Selenomonas, supporting a more complex disease model than a linear progression following gingivitis. Cooccurrence networks of periodontitis-associated taxa clustered according to periodontitis across all gingivitis severities. Species including Filifactor alocis and Fusobacterium nucleatum were central to this network, which supports their role in the coaggregation of periodontal biofilms during disease progression. Our findings confirm that periodontitis cannot be considered simply an advanced stage of gingivitis even when only considering supragingival plaque. IMPORTANCE: Periodontal disease is a major public health problem associated with oral bacteria. While earlier studies focused on a small number of periodontal pathogens, it is now accepted that the whole bacterial community may be important. However, previous high-throughput marker gene sequencing studies of supragingival plaque have largely focused on high-income populations with good oral hygiene without including a range of periodontal disease severities. Our study includes a large number of low-income participants with poor oral hygiene and a wide range of severities, and we were therefore able to quantitatively model bacterial abundances as functions of both gingivitis and periodontitis. A signal associated with periodontitis remains after controlling for gingivitis severity, which supports the concept that, even when only considering supragingival plaque, periodontitis is not simply an advanced stage of gingivitis. This suggests the future possibility of diagnosing periodontitis based on bacterial occurrences in supragingival plaque.

Recent approaches in computational modelling for controlling pathogen threats.

In this review, we assess the status of computational modelling of pathogens. We focus on three disparate but interlinked research areas that produce models with very different spatial and temporal scope. First, we examine antimicrobial resistance (AMR). Many mechanisms of AMR are not well understood. As a result, it is hard to measure the current incidence of AMR, predict the future incidence, and design strategies to preserve existing antibiotic effectiveness. Next, we look at how to choose the finite number of bacterial strains that can be included in a vaccine. To do this, we need to understand what happens to vaccine and non-vaccine strains after vaccination programmes. Finally, we look at within-host modelling of antibody dynamics. The SARS-CoV-2 pandemic produced huge amounts of antibody data, prompting improvements in this area of modelling. We finish by discussing the challenges that persist in understanding these complex biological systems.

The plasmidome associated with Gram-negative bloodstream infections: A large-scale observational study using complete plasmid assemblies.

Plasmids carry genes conferring antimicrobial resistance and other clinically important traits, and contribute to the rapid dissemination of such genes. Previous studies using complete plasmid assemblies, which are essential for reliable inference, have been small and/or limited to plasmids carrying antimicrobial resistance genes (ARGs). In this study, we sequenced 1,880 complete plasmids from 738 isolates from bloodstream infections in Oxfordshire, UK. The bacteria had been originally isolated in 2009 (194 isolates) and 2018 (368 isolates), plus a stratified selection from intervening years (176 isolates). We demonstrate that plasmids are largely, but not entirely, constrained to a single host species, although there is substantial overlap between species of plasmid gene-repertoire. Most ARGs are carried by a relatively small number of plasmid groups with biological features that are predictable. Plasmids carrying ARGs (including those encoding carbapenemases) share a putative 'backbone' of core genes with those carrying no such genes. These findings suggest that future surveillance should, in addition to tracking plasmids currently associated with clinically important genes, focus on identifying and monitoring the dissemination of high-risk plasmid groups with the potential to rapidly acquire and disseminate these genes.

Visualizing and quantifying structural diversity around mobile resistance genes.

Understanding the evolution of mobile genes is important for understanding the spread of antimicrobial resistance (AMR). Many clinically important AMR genes have been mobilized by mobile genetic elements (MGEs) on the kilobase scale, such as integrons and transposons, which can integrate into both chromosomes and plasmids and lead to rapid spread of the gene through bacterial populations. Looking at the flanking regions of these mobile genes in diverse genomes can highlight common structures and reveal patterns of MGE spread. However, historically this has been a largely descriptive process, relying on gene annotation and expert knowledge. Here we describe a general method to visualize and quantify the structural diversity around genes using pangraph to find blocks of homologous sequence. We apply this method to a set of 12 clinically important beta-lactamase genes and provide interactive visualizations of their flanking regions at https://liampshaw.github.io/flanking-regions. We show that nucleotide-level variation in the mobile gene itself generally correlates with increased structural diversity in its flanking regions, demonstrating a relationship between rates of mutational evolution and rates of structural evolution, and find a bias for greater structural diversity upstream. Our framework is a starting point to investigate general rules that apply to the horizontal spread of new genes through bacterial populations.

PanGraph: scalable bacterial pan-genome graph construction.

The genomic diversity of microbes is commonly parameterized as SNPs relative to a reference genome of a well-characterized, but arbitrary, isolate. However, any reference genome contains only a fraction of the microbial pangenome, the total set of genes observed in a given species. Reference-based approaches are thus blind to the dynamics of the accessory genome, as well as variation within gene order and copy number. With the widespread usage of long-read sequencing, the number of high-quality, complete genome assemblies has increased dramatically. In addition to pangenomic approaches that focus on the variation in the sets of genes present in different genomes, complete assemblies allow investigations of the evolution of genome structure and gene order. This latter problem, however, is computationally demanding with few tools available that shed light on these dynamics. Here, we present PanGraph, a Julia-based library and command line interface for aligning whole genomes into a graph. Each genome is represented as a path along vertices, which in turn encapsulate homologous multiple sequence alignments. The resultant data structure succinctly summarizes population-level nucleotide and structural polymorphisms and can be exported into several common formats for either downstream analysis or immediate visualization.

Regulatory fine-tuning of mcr-1 increases bacterial fitness and stabilises antibiotic resistance in agricultural settings.

Antibiotic resistance tends to carry fitness costs, making it difficult to understand how resistance can be maintained in the absence of continual antibiotic exposure. Here we investigate this problem in the context of mcr-1, a globally disseminated gene that confers resistance to colistin, an agricultural antibiotic that is used as a last resort for the treatment of multi-drug resistant infections. Here we show that regulatory evolution has fine-tuned the expression of mcr-1, allowing E. coli to reduce the fitness cost of mcr-1 while simultaneously increasing colistin resistance. Conjugative plasmids have transferred low-cost/high-resistance mcr-1 alleles across an incredible diversity of E. coli strains, further stabilising mcr-1 at the species level. Regulatory mutations were associated with increased mcr-1 stability in pig farms following a ban on the use of colistin as a growth promoter that decreased colistin consumption by 90%. Our study shows how regulatory evolution and plasmid transfer can combine to stabilise resistance and limit the impact of reducing antibiotic consumption.

A longitudinal study reveals persistence of antimicrobial resistance on livestock farms is not due to antimicrobial usage alone.

Introduction: There are concerns that antimicrobial usage (AMU) is driving an increase in multi-drug resistant (MDR) bacteria so treatment of microbial infections is becoming harder in humans and animals. The aim of this study was to evaluate factors, including usage, that affect antimicrobial resistance (AMR) on farm over time. Methods: A population of 14 cattle, sheep and pig farms within a defined area of England were sampled three times over a year to collect data on AMR in faecal Enterobacterales flora; AMU; and husbandry or management practices. Ten pooled samples were collected at each visit, with each comprising of 10 pinches of fresh faeces. Up to 14 isolates per visit were whole genome sequenced to determine presence of AMR genes. Results: Sheep farms had very low AMU in comparison to the other species and very few sheep isolates were genotypically resistant at any time point. AMR genes were detected persistently across pig farms at all visits, even on farms with low AMU, whereas AMR bacteria was consistently lower on cattle farms than pigs, even for those with comparably high AMU. MDR bacteria was also more commonly detected on pig farms than any other livestock species. Discussion: The results may be explained by a complex combination of factors on pig farms including historic AMU; co-selection of AMR bacteria; variation in amounts of antimicrobials used between visits; potential persistence in environmental reservoirs of AMR bacteria; or importation of pigs with AMR microbiota from supplying farms. Pig farms may also be at increased risk of AMR due to the greater use of oral routes of group antimicrobial treatment, which were less targeted than cattle treatments; the latter mostly administered to individual animals. Also, farms which exhibited either increasing or decreasing trends of AMR across the study did not have corresponding trends in their AMU. Therefore, our results suggest that factors other than AMU on individual farms are important for persistence of AMR bacteria on farms, which may be operating at the farm and livestock species level.

Enterobacterales plasmid sharing amongst human bloodstream infections, livestock, wastewater, and waterway niches in Oxfordshire, UK.

Plasmids enable the dissemination of antimicrobial resistance (AMR) in common Enterobacterales pathogens, representing a major public health challenge. However, the extent of plasmid sharing and evolution between Enterobacterales causing human infections and other niches remains unclear, including the emergence of resistance plasmids. Dense, unselected sampling is essential to developing our understanding of plasmid epidemiology and designing appropriate interventions to limit the emergence and dissemination of plasmid-associated AMR. We established a geographically and temporally restricted collection of human bloodstream infection (BSI)-associated, livestock-associated (cattle, pig, poultry, and sheep faeces, farm soils) and wastewater treatment work (WwTW)-associated (influent, effluent, waterways upstream/downstream of effluent outlets) Enterobacterales. Isolates were collected between 2008 and 2020 from sites <60 km apart in Oxfordshire, UK. Pangenome analysis of plasmid clusters revealed shared 'backbones', with phylogenies suggesting an intertwined ecology where well-conserved plasmid backbones carry diverse accessory functions, including AMR genes. Many plasmid 'backbones' were seen across species and niches, raising the possibility that plasmid movement between these followed by rapid accessory gene change could be relatively common. Overall, the signature of identical plasmid sharing is likely to be a highly transient one, implying that plasmid movement might be occurring at greater rates than previously estimated, raising a challenge for future genomic One Health studies.

The evolution of colistin resistance increases bacterial resistance to host antimicrobial peptides and virulence.

Antimicrobial peptides (AMPs) offer a promising solution to the antibiotic resistance crisis. However, an unresolved serious concern is that the evolution of resistance to therapeutic AMPs may generate cross-resistance to host AMPs, compromising a cornerstone of the innate immune response. We systematically tested this hypothesis using globally disseminated mobile colistin resistance (MCR) that has been selected by the use of colistin in agriculture and medicine. Here, we show that MCR provides a selective advantage to Escherichia coli in the presence of key AMPs from humans and agricultural animals by increasing AMP resistance. Moreover, MCR promotes bacterial growth in human serum and increases virulence in a Galleria mellonella infection model. Our study shows how the anthropogenic use of AMPs can drive the accidental evolution of resistance to the innate immune system of humans and animals. These findings have major implications for the design and use of therapeutic AMPs and suggest that MCR may be difficult to eradicate, even if colistin use is withdrawn.