Biology subjects

Turner, P.

Publications and source records attributed to Turner, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Morula complementation restores fetal kidneys in xenocompatible SALL1 null sheep

To meet the global shortage of organs, extensive genome modifications have been performed to "humanize" livestock tissues for xenotransplantation. However, residual immune rejection remains a problem, prompting alternative approaches that explore the use of animals as hosts for growing human organs. This requires genome editing to disable organogenesis in the host and embryo complementation with suitable donor cells to fill the empty organ niche in chimaeric animals. Pigs have been the predominant livestock species investigated for this approach. Here, we used domestic sheep as hosts for donor-derived kidney formation. Spalt-Like Transcription Factor 1 (SALL1) was targeted in male fibroblasts lacking xenoantigens CMAH and GGTA1, using either one gRNA within zinc finger cluster (ZFC) 2 or two gRNAs to remove all ZF domains. Following somatic cell cloning and embryo transfer of triple knockout strains, fetuses were collected on gestational day 48 to analyze the SALL1 KO phenotypes. Single gRNA editing produced a hypomorph with different degrees of metanephric hypoplasia, while the dual-gRNA deletion resulted in a null allele which completely abolished nephrogenesis. Female donor cells carrying high vs low copy numbers of an mCherry transgene, as well as CMAH and GGTA1 edits, were used for morula complementation. Fetal kidney development was anatomically and histologically restored in sex-chimaeric hosts, providing proof-of- concept for using sheep as a new model species for in vivo organ generation.

cell biology↗

bioRxiv2026

Non-canonical histone H3.3 and its chaperones HIRA and DAXX participate in the regulation of KSHV latency

Kaposis sarcoma-associated herpesvirus (KSHV), also named HHV-8, is the etiological agent of Kaposi sarcoma (KS), Primary effusion lymphoma (PEL), and Multicentric Castlemans disease. After de novo infection, KSHV genomes rapidly circularize and acquire a chromatin state that favors latency. During latency, the KSHV episome is decorated with distinct epigenetic marks that segregate the viral genome into transcriptionally active and repressed domains, enabling persistent silencing of lytic genes while retaining the capacity for reactivation. Transcription activity of chromatin is regulated at multiple levels, including the incorporation of histone variants such as H3.3, by a specific set of histone chaperones such as HIRA and DAXX. The interaction between LANA and these interphase active chaperones suggests that H3.3 deposition is a critical driver of early chromatinization and the long-term stability of KSHV latency. We detected rapid H3.3 deposition on KSHV episomes and on episomes within long-term infected cells. Moreover, we demonstrated that genetically disrupting the host H3.3 chaperone HIRA pathway by CRISPR/Cas9-mediated knockout impacted the regulation of LANA and maintenance of viral latency that was not altered in DAXX knockout cells. Collectively, these results support a role for HIRA-mediated H3.3 deposition in the regulation of KSHV latency.

molecular biology↗

bioRxiv2025

Unraveling single-molecule reactions via multiplexed in-situ DNA sequencing

DNA sequence regulates complex reactions and protein-DNA interactions, yet sequence effects remain poorly understood due to the lack of direct, high-throughput approaches to study sequence-dependence at the single-molecule level. Here, we introduce Single-molecule Phenotyping and In-Situ Sequencing (SPIN-Seq), a DNA-based, protein-free method that links functional and structural properties of a single DNA molecule with the sequence of that same molecule. After performing functional assays on immobilized DNA molecules, SPIN-Seq uses sequencing-by-transient-hybridization on the same surface and instrument to read sequences in each immobilized DNA molecule, directly linking phenotype and genotype. Applying SPIN-Seq, we dissected the interaction of a transcription factor with its target sequence, and revealed the sequence-dependence of RNA polymerase pausing and reaction-path branching during initial transcription. Our method provides powerful, systematic ways to understand complex molecular mechanisms and their sequence dependence.

biophysics↗

bioRxiv2025

SMART Sensors for Cell Growth Monitoring in Closed System G-Rex for Scalable, Cost-Conscious Cell and Gene Therapy Manufacturing

Cell and gene-modified cell therapy (CGT), commonly referred to as the fourth pillar of cancer treatment, includes creating advanced cancer killing cells ex vivo and then subsequently putting them into the patients body to selectively attack cancerous cells. Typically, immune cells from a patients body are removed, genetically modified to find and kill cancer, grown to a large army of cancer killing cells, and put back into the patient. This complex process is being simplified through innovation, some of which we describe here. One key innovation is an immune cell manufacturing device configuration called G-Rex (for "gas permeable rapid expansion"), which is now widely used to create armies of cancer killing cells for clinical trials and for commercially approved drug products on a worldwide basis. G-Rex is uniquely simple and popular because it is the only technology that eliminates the need for pumps and flow circuits to deliver oxygen and nutrients. Instead, the material and geometry of G-Rex allow cancer killing cells to get access to oxygen and nutrients on demand. This greatly minimizes complexity and sets the stage for high throughput manufacturing. To further simplify G-Rex relative to all CGT drug product manufacturing alternatives, it would be ideal to eliminate any need of physical intervention to assess cell quantity. With that goal, we conceived a novel approach to determining cell quantity in a non-invasive manner. More specifically, we invented and built a sensor into G-Rex that continuously reports the quantity of cells in G-Rex without any intervention or sampling to an external reader. Herein, we call the sensor a "single-use metabolite absorbing resonant transducer in G-Rex (SMART G-Rex)" and the reader a "SMART Reader." The SMART G-Rex is wirelessly interrogated by the SMART Reader to determine the extent of cell expansion in the G-Rex at any given time. More specifically, as cell quantity increases, the SMART G-Rex absorbs cell secreted metabolites (organic compounds such as eicosanoids) which cause a change in resonant frequency (measured as % of frequency change, or Skroot Growth Index (SGI)). By placing SMART G-Rex in proximity of the SMART Reader, whether in or out of an incubator, changes in resonant frequency are detected and translated into cell quantity with high reliability. For example, using 45 sensors across multiple donor cell samples, we established 99% confidence in SMARTs accuracy at signaling a >60X expansion (3B cells at harvest). The SMART G-Rex provides a non-invasive way of instantly quantifying the number of cells in a CGT drug production process and informing manufacturers when a CGT drug product has reached the desired quantity of cells necessary to proceed with formulation, fill, and finish. By integrating SMART technology, G-Rex is the only technology for CGT manufacturing that can avoid intervention when determining cells quantity or allow real-time knowledge of cell quantity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/676768v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1557118org.highwire.dtl.DTLVardef@740652org.highwire.dtl.DTLVardef@14b0430org.highwire.dtl.DTLVardef@ac0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Quantifying the effects of antibiotic resistance and within-host competition on strain fitness in Streptococcus pneumoniae

Competition plays a key role in shaping the structure and diversity of bacterial populations. In many clinically important bacterial species, strains compete at multiple scales: at the between-host scale for new hosts to colonise, and at the within-host scale during co-colonisation. Characterising these multiple facets of competition plays an important role in understanding bacterial ecology. This is particularly relevant for antibiotic resistance, where competition between antibiotic-susceptible and resistant strains determines resistance dynamics. In this work, we perform survival analyses on a large longitudinal dataset of Streptococcus pneumoniae carriage to quantify how within-host competition affects the rates of clearance and establishment of pneumococcal strains. We find that the presence of a within-host competitor is associated with a 33% increase in clearance and a 54% reduction in establishment. Priority effects and serotype differences partially predict the outcomes of this within-host competition. Further, we quantify the effects of antibiotic resistance on betweenand within-host components of fitness. Antibiotic consumption is associated with increased clearance rate for both susceptible and resistant strains, albeit to a higher extent in susceptible strains. In the absence of antibiotics, we find some evidence that resistance is associated with increased susceptibility to within-host competition, suggesting a fitness cost of resistance. Overall, our work provides quantitative insights into pneumococcal competition across scales and the role of this competition in shaping pneumococcal epidemiology.

ecology↗

bioRxiv2025

Tunable fluorogenic DNA probes drive fast and high-resolution single-molecule fluorescence imaging

A main limitation of single-molecule fluorescence (SMF) measurements is the "high concentration barrier", describing the maximum concentration of fluorescent species tolerable for sufficient signal-to-noise ratio (SNR). To address this barrier in several SMF applications, we design fluorogenic probes based on short ssDNAs, fluorescing only upon hybridising to their complementary target sequence. We engineer the quenching efficiency and fluorescence enhancement upon duplex formation through screening several fluorophore-quencher combinations, label lengths, and sequence motifs, which we utilise as tuning screws to adapt our labels to different experimental designs. Using these fluorogenic probes, we can perform SMF experiments at concentrations of 10 {micro}M fluorescent labels; this concentration is 100-fold higher than the operational limit for standard TIRF experiments. We demonstrate the ease of implementing these probes into existing protocols by performing super-resolution imaging with DNA-PAINT, employing a fluorogenic 6 nt-long imager; through the faster acquisition of binding events, the imaging of viral genome segments could be sped up significantly to achieve extraction of 20-nm structural features with only [~]150 s of imaging. The exceptional tunability of our probe design will overcome concentration barriers in SMF experiments and unlock new possibilities in super-resolution imaging, molecular tracking, and smFRET. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/634148v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d51a20org.highwire.dtl.DTLVardef@1a64fb7org.highwire.dtl.DTLVardef@1bbb4bforg.highwire.dtl.DTLVardef@578c63_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Identification of a large cohort of Enterobacter jumbo phages with broad host ranges across pathogenic Gammaproteobacteria

ESKAPE pathogens cause most hospital-acquired infections globally and often carry antibiotic resistance. Many of them have been the target of bacteriophage therapies. Enterobacter is an ESKAPE pathogen but is less frequently a target for phage therapy due to a relative lack of available phages. We isolated eight jumbo phages with genomes ranging from 223 to 366 kbp targeting Enterobacter spp. and found that they belonged to separate phage clades. Six of them formed nucleus-like structures confirmed by DAPI-staining, and were phylogenetically related to Chimalliviridae. Two jumbo phages did not form nucleus-like structures and did not cluster with Chimalliviridae. Although these jumbo phages were found on Enterobacter, many were closely related to phages with non-Enterobacter hosts. To test whether these phages may have had expanded host ranges, we examined 14 pathogenic Gammaproteobacteria and found that these phages were capable of creating plaques on 8 of them. These species included Escherichia coli, Klebsiella aerogenes, Serratia marcescens, Salmonella spp., Shigella spp., Providencia spp., Citrobacter spp., and Cronobacter sakazakii. We verified that there was phage amplification in these microbes rather than lysis from without by performing qPCR to confirm DNA replication in each species. Phages typically have narrow host ranges, a benefit for microbiome-sparing compared to antibiotics. However, the broad host ranges of these Gammaproteobacteria jumbo phages suggests that not all phages have the same risk/benefit ratios. While this broad range could aid their development as antibiotic alternatives, further study is needed to assess potential microbiome disruption. SignificanceWith the growing threat of antibiotic resistant bacteria, alternative treatments like bacteriophages have emerged. Bacteriophages typically have narrow host ranges, a disadvantage compared to antibiotics. We discovered eight jumbo phages that kill antibiotic resistant Enterobacter, but were diverse phylogenetically. Six of them formed nucleus-like structures and were members of the Chimalliviridae family and the other two did not form nucleus-like structures. We confirmed that each phage had broad host ranges capable of lysing at least eight different human Gammaproteobacteria including pathogens such as E. coli, K. aerogenes, and S. marcescens. By identifying broad host range jumbo phages that attack pathogens, we may have identified phages with spectrums of activity more similar to antibiotics than have been traditionally attributed to phages.

microbiology↗

bioRxiv2024

The major pathogen Haemophilus influenzae experiences pervasive recombination and purifying selection at local and global scales

Haemophilus influenzae is a major opportunistic human pathogen which causes both non-invasive and invasive disease. The H. influenzae type b (Hib) vaccine has led to a significant reduction of invasive Hib disease, but offers no protection against colonisation or disease by unencapsulated non-typeables (NT) or non-b serotypes, and H. influenzae remains a public health burden worldwide, with increasing reports of multi-drug resistance (MDR). Despite this, there is no comprehensive understanding of the species global population structure. To advance understanding about the evolution and epidemiology of the species, we whole-genome sequenced 4,475 isolates of H. influenzae from an unvaccinated paediatric carriage and pneumonia cohort from northwestern Thailand. Despite no Hib immunisation, serotype b was uncommonly found (5.7%), while 91.7% of isolates were NT. We identified a large number of nearly pan-resistant lineages that were mostly NT, and discovered that no lineages were enriched among disease samples, suggesting the ability to cause invasive disease is not restricted to any subpopulation of the species. Extensive population genetic analyses of our data combined with a worldwide collection of 5,976 published genomes revealed a highly admixed population structure, low core genome nucleotide diversity, and evidence of pervasive negative selection. The combined data confirm that MDR lineages are not confined to our cohort, and their establishment globally is an urgent concern.

microbiology↗

bioRxiv2024

Graph-based Nanopore Adaptive Sampling with GNASTy enables sensitive pneumococcal serotyping in complex samples

Serotype surveillance of Streptococcus pneumoniae (the pneumococcus) is critical for understanding the effectiveness of current vaccination strategies. However, existing methods for serotyping are limited in their ability to identify co-carriage of multiple pneumococci and detect novel serotypes. To develop a scalable and portable serotyping method that overcomes these challenges, we employed Nanopore Adaptive Sampling (NAS), an on-sequencer enrichment method which selects for target DNA in real-time, for direct detection of S. pneumoniae in complex samples. Whereas NAS targeting the whole S. pneumoniae genome was ineffective in the presence of non-pathogenic streptococci, the method was both specific and sensitive when targeting the capsular biosynthetic locus (CBL), the operon that determines S. pneumoniae serotype. NAS significantly improved coverage and yield of the CBL relative to sequencing without NAS, and accurately quantified the relative prevalence of serotypes in samples representing co-carriage. To maximise the sensitivity of NAS to detect novel serotypes, we developed and benchmarked a new pangenome-graph algorithm, named GNASTy. We show that GNASTy outperforms the current NAS implementation, which is based on linear genome alignment, when a sample contains a serotype absent from the database of targeted sequences. The methods developed in this work provide an improved approach for novel serotype discovery and routine S. pneumoniae surveillance that is fast, accurate and feasible in low resource settings. GNASTy therefore has the potential to increase the density and coverage of global pneumococcal surveillance. One sentence summaryPangenome graph-based Nanopore Adaptive Sampling, presented in our tool GNASTy, is a sensitive, portable and cost-effective method for Streptococcus pneumoniae surveillance.

genomics↗

bioRxiv2023

Community ecology of phages on a clonal bacterial host

Bacteriophages are the most abundant and diverse biological entities on Earth, yet the ecological mechanisms that sustain this extraordinary diversity remain unclear. Here, we have discovered a general mechanism that allows phage diversity to outstrip the diversity of their bacterial hosts. We assembled and passaged dozens of diverse phage communities on a single, non-coevolving strain of Escherichia coli until the phage communities reached equilibrium. In all cases, we found multiple phage species coexisted stably, despite competition for a single, clonal host population. Coexistence within these communities was supported through host phenotypic heterogeneity, whereby phages specialized on cells adopting different growth phenotypes. Our experiments reveal a rich community ecology of bacteriophages where multiple phage species coexist and interact at the scale of a single bacterial host. One-Sentence SummaryDiverse communities of phages emerge stably and consistently on a clonal bacterial host, enabled by phenotypic heterogeneity.

microbiology↗

bioRxiv2023

Theoretical validation of growth curves for quantifying phage-bacteria interactions

A frequent goal of phage biology is to quantify how well a phage kills a population of host bacteria. Unfortunately, traditional methods to quantify phage success can be time-consuming, limiting the throughput of experiments. Here, we use theory to show how the effects of phages on their hosts can be quantified using bacterial population dynamics measured in a high-throughput microplate reader (automated spectrophotometer). We use mathematical models to simulate bacterial population dynamics where specific phage and bacterial traits are known a priori. We then test common metrics of those dynamics (e.g. growth rate, time and height of peak bacterial density, death rate, extinction time, area under the curve) to determine which best predict: 1) infectivity over the short-term, and 2) phage suppression over the long-term. We find that many metrics predict infectivity and are strongly correlated with one another. We also find that metrics can predict phage growth rate, providing an effective way to quantify the combined effects of multiple phage traits. Finally, we show that peak density, time of peak density, and extinction time are the best metrics when comparing across different bacterial hosts or over longer timescales where plasticity or evolution may play a role. In all, we establish a foundation for using bacterial population dynamics to quantify the effects of phages on their bacterial hosts, supporting the design of in vitro empirical experiments using microplate readers. SignificanceBacteriophages are viruses that infect bacteria, with relevance from basic science to medical application. Frequently we seek to quantify how these viruses negatively impact bacterial growth. Typical methods are labor-intense, limiting the number of experiments that can be done. Here, we show how easily-collectable data (called bacterial population dynamics or growth curves) can be used to quantify virus killing of bacteria across a wide range of conditions. In all, our work suggests that these dynamics provide an effective and high-throughput method to quantify phage effects on their hosts.

microbiology↗

bioRxiv2022

Pneumococcal within-host diversity during colonisation, transmission and treatment

Characterising the genetic diversity of pathogens within the host promises to greatly improve surveillance and reconstruction of transmission chains. For bacteria, it also informs our understanding of inter-strain competition, and how this shapes the distribution of resistant and sensitive bacteria. Here we study the genetic diversity of Streptococcus pneumoniae within individual infants and their mothers by deep sequencing whole pneumococcal populations from longitudinal nasopharyngeal samples. We demonstrate deep sequencing has unsurpassed sensitivity for detecting multiple colonisation, doubling the rate at which highly invasive serotype 1 bacteria were detected in carriage compared to gold-standard methods. The greater resolution identified an elevated rate of transmission from mothers to their children in the first year of the childs life. Comprehensive treatment data demonstrated infants were at an elevated risk of both the acquisition, and persistent colonisation, of a multidrug resistant bacterium following antimicrobial treatment. Some alleles were enriched after antimicrobial treatment, suggesting they aided persistence, but generally purifying selection dominated within-host evolution. Rates of co-colonisation imply that in the absence of treatment, susceptible lineages outcompeted resistant lineages within the host. These results demonstrate the many benefits of deep sequencing for the genomic surveillance of bacterial pathogens.

genomics↗

bioRxiv2021

Genome-wide association, prediction and heritability in bacteria

Advances in whole-genome genotyping and sequencing have allowed genome-wide analyses of association, prediction and heritability in many organisms. However, the application of such analyses to bacteria is still in its infancy, being limited by difficulties including the plasticity of bacterial genomes and their strong population structure. Here we propose, and validate using simulations, a suite of genome-wide analyses for bacteria. We combine methods from human genetics and previous bacterial studies, including linear mixed models, elastic net and LD-score regression, and introduce innovations such as frequency-based allele coding, testing for both insertion/deletion and nucleotide effects and partitioning heritability by genome region. We then analyse three phenotypes of a major human pathogen Streptococcus pneumoniae, including the first analyses of minimum inhibitory concentrations (MIC) for each of two antibiotics, penicillin and ceftriaxone. We show that these are highly heritable leading to high prediction accuracy, which is explained by many genetic associations identified under good control of population structure effects. In the case of ceftriaxone MIC, these results are surprising because none of the isolates was resistant according to the inhibition zone diameter threshold. We estimate that just over half of the heritability of penicillin MIC is explained by a known drug-resistance region, which also contributes around a quarter of the heritability of ceftriaxone MIC. For the within-host survival phenotype carriage duration, no reliable associations were found but we observed moderate heritability and prediction accuracy, indicating a polygenic trait. While generating important new results for S. pneumoniae, we have critically assessed existing methods and introduced innovations that will be useful for future large-scale population genomics studies to help decipher the genetic architecture of bacterial traits. Author summaryGenome-wide association, prediction and heritability analyses in bacteria are beginning to help unravel the genetic underpinnings of traits such as antimicrobial resistance, virulence, within-host survival and transmissibility. Progress to date is limited by challenges including the effects of strong population structure and variable recombination, and the many gaps in sequence alignments including the absence of entire genes in many isolates. More work is required to critically asses and develop methods for bacterial genomics. We address this task here, using a range of existing methods from bacterial and human genetics, such as linear mixed models, elastic net and LD-score regression. Using simulations, we first validate and then adapt these methods to introduce new analyses, including separate assessment of gap and nucleotide effects, a new allele coding for association analyses and a method to partition heritability into genome regions. We analyse within-host survival and two antimicrobial response traits of Streptococcus pneumoniae, identifying many novel associations while demonstrating good control of population structure and accurate prediction. We present both new results for an important pathogen and methodological advances that will be useful in guiding future studies in bacterial population genomics.

genomics↗

bioRxiv2021

Pneumococcal genetic variability influences age-dependent bacterial carriage

The pneumococcal conjugate vaccine (PCV) primarily reduces disease burden in adults through a reduction in carriage prevalence of invasive serotypes in children. Current vaccine formulations are the same for both adults and children, but tailoring these formulations to age category could optimize vaccine efficacy. Identification of specific pneumococcal genetic factors associated with carriage in younger or older age groups may suggest alternative formulations and contribute to a better mechanistic understanding of immunity. Here, we used whole genome sequencing to dissect pneumococcal variation associated with age. We performed genome sequencing in a large carriage cohort, and conducted a meta-analysis with an existing carriage study. We compiled a dictionary of pathogen genetic variation including serotype, sequence cluster, sequence elements, SNPs, burden combined rare variants, and clusters of orthologous genes (COGs) for each cohort - all of which used in a genome-wide association with host age. Age-dependent colonization had some heritability, though this varied between cohorts (h2 = 0.10, 0.00 - 0.69 95% CI in the first; h2 = 0.46, 0.33 - 0.60 95% CI in the second cohort). We found that serotypes and genetic background (strain) explained most of the heritability in each cohort (h2serotype = 0.06 and h2GPSC = 0.04 in the first; h2 serotype = 0.20 and h2 GPSC = 0.23 in the second cohort). We found one candidate association (p = 1.2x10-9) upstream of an accessory Sec-dependent serine-rich glycoprotein adhesin. Overall, association with age was highly cohort and strain dependent, supporting proposals for a future vaccination strategy which is primarily targeted using serotypes rather than proteins, and is tailored towards specific pathogen populations.

microbiology↗

bioRxiv2021

The role of interspecies recombinations in the evolution of antibiotic resistant pneumococci

AbstractThe evolutionary histories of the antibiotic-resistant Streptococcus pneumoniae lineages PMEN3 and PMEN9 were reconstructed using global collections of genomes. In PMEN3, one resistant clade spread worldwide, and underwent 25 serotype switches, enabling evasion of vaccine-induced immunity. In PMEN9, only 9 switches were detected, and multiple resistant lineages emerged independently and circulated locally. In Germany, PMEN9s expansion correlated significantly with the macrolide:penicillin consumption ratio. These isolates were penicillin sensitive but macrolide resistant, through a homologous recombination that integrated Tn1207.1 into a competence gene, preventing further diversification via transformation. Analysis of a species-wide dataset found 183 acquisitions of macrolide resistance, and multiple gains of the tetracycline-resistant transposon Tn916, through homologous recombination, often originating in other streptococcal species. Consequently, antibiotic selection preserves atypical recom- bination events that cause sequence divergence and structural variation throughout the S. pneumoniae chromosome. These events reveal the genetic exchanges between species normally counter-selected until perturbed by clinical interventions.

genomics↗

bioRxiv2020

Metrics for Public Health Perspective Surveillance of Bacterial Antibiotic Resistance in Low- and Middle-Income Countries

Antimicrobial resistance (AMR) is a global health threat, especially in low-/middle-income countries (LMICs), where there is limited surveillance to inform empiric antibiotic treatment guidelines. Enterobacterales are amongst the most important causes of drug-resistant bacterial infections. We developed a novel AMR surveillance approach for Enterobacterales by profiling pooled human faecal metagenomes from three sites (n=563 individuals; Cambodia, Kenya, UK) to derive a taxonomy-adjusted AMR metric ("resistance potential") which could be used to predict the aggregate percentage of resistant invasive Enterobacterales infections within each setting. Samples were sequenced (Illumina); taxonomic and resistance gene profiling was performed using ResPipe. Data on organisms causing bacteraemia and meningitis and antibiotic susceptibility test results from 2010-2017 were collated for each site. Bayesian generalised linear models with a binomial likelihood were fitted to determine the capacity of the resistance potential to predict AMR in Enterobacterales infections in each setting. The most informative model accurately predicted the numbers of resistant infections in the target populations for 14/14 of antibiotics in the UK, 12/12 in Kenya, and 9/12 in Cambodia. Intermittent metagenomics of pooled human samples could represent a powerful pragmatic and economical approach for determining and monitoring AMR in clinical infections, especially in resource-limited settings.

genomics↗

bioRxiv2019

The spread of chloramphenicol-resistant Neisseria meningitidis in Southeast Asia

Invasive disease caused by Neisseria meningitidis is a significant health concern globally, but our knowledge of the prevailing serogroups, antimicrobial susceptibility patterns, and genetics of N. meningitidis in Southeast Asia is limited. Chloramphenicol resistance in N. meningitidis has rarely been reported, but was first described in isolates from Vietnam in 1998. Using whole-genome sequencing of meningococcal isolates from 18 patients collected between 2007 and 2018 from diagnostic microbiology laboratories in Cambodia, Thailand and the Lao Peoples Democratic Republic (Laos), of which eight were non-susceptible to chloramphenicol, we report the spread of this chloramphenicol-resistant lineage of N. meningitidis across Southeast Asia. Strains resistant to penicillin, tetracycline, and ciprofloxacin were also observed, including a chloramphenicol-resistant strain from the previously-described lineage which has acquired penicillin and ciprofloxacin resistance, and most isolates were of serogroup B. This study suggests that chloramphenicol-resistant N. meningitidis is more widespread than previously thought.

microbiology↗

bioRxiv2019

Characterizing the tolerance of phage therapy candidate OMKO1 to environmental stress

Antibiotic resistant bacterial pathogens are increasingly prevalent, driving the need for alternative approaches to chemical antibiotics when treating infections. One such approach is bacteriophage therapy: the use of bacteria-specific viruses that lyse (kill) their host cells. Just as the effect of environmental conditions (e.g. elevated temperature) on antibiotic efficacy is well-studied, the effect of environmental stressors on the potency of phage therapy candidates demands examination. Therapeutic phage OMKO1 infects and kills the opportunistic human pathogen Pseudomonas aeruginosa. Here, we used phage OMKO1 as a model to test how different environments affect the decay rate of a therapeutic virus, and whether exposure to an environmental stressor can damage surviving viral particles. We assessed the effects of elevated temperatures, saline concentrations, and urea concentrations. We observed that OMKO1 particles were highly tolerant to different saline concentrations, but decayed more rapidly at elevated temperatures and under high concentrations of urea. Additionally, we found that exposure to elevated temperature reduced the ability of surviving phage particles to suppress the growth of P. aeruginosa, suggesting a temperature-induced damage. Our findings demonstrate that OMKO1 is highly tolerant to a range of conditions that could be experienced inside and outside the human body, while also showing the need for careful characterization of therapeutic phages to ensure that environmental exposure does not compromise their expected potency, dosing, and pharmacokinetics.

microbiology↗