Biology subjects

Wain, J.

Publications and source records attributed to Wain, J..

10 recordsLinked to original sources

bioRxiv2025

The metallophore staphylopine is essential for survival of Staphylococcus epidermidis in human synovial fluid

Due to extended life expectancies, prosthetic joint infections are an increasing burden on healthcare institutions worldwide. The most commonly isolated causative agents are staphylococci, though the mechanisms underpinning survival and proliferation in synovial fluid are still not fully understood. In this study, we aimed to identify genes important for survival in synovial fluid using a transposon mutant library and RNAseq. We produced a transposon mutant library, containing approximately 57,000 unique insertion mutants, in Staphylococcus epidermidis strain 846. This library was grown in Muller Hinton broth or processed human synovial fluid samples, and Transposon-directed Insertion Sequencing (TraDIS) was used to identify genes involved in survival in synovial fluid. This identified importance of the his, pur and cnt operons. These genes were also upregulated in both Staphylococcus epidermidis 846 and the model Staphylococcus epidermidis strain RP62A when exposed to human synovial fluid. All these key pathways contribute to production of the metallophore staphylopine. To confirm staphyopine production is essential for survival in synovial fluid, a defined transposon insertion in the gene encoding for staphylopine export (cntE) mutant was used. This demonstrated impaired survival in synovial fluid compared to the wild type. RT-qPCR also showed that cntE was more highly expressed after exposure to infected synovial fluid (where metals will be depleted) than non-infected fluid. In conclusion, TraDIS and RNASeq both identified the importance of staphylopine for survival in human synovial fluid. This suggests an opportunity for exploitation for therapeutic or diagnostic use. Author SummaryStaphylococcus epidermidis is a common cause of prosthetic joint infection, however accurate diagnosis remains difficult. In this work we explored the genetic basis of Staphylococcus epidermidis survival in human synovial fluid using a large transposon mutant library, and identified which genes were differentially expressed upon exposure to the fluid. We found crossover between the datasets, which pointed to the importance of the metal acquisition compound staphylopine. The expression levels of the gene required for staphylopine export were shown to be dependent on the infection status of the individual samples were obtained from. We also found the gene cluster to be conserved in a range of staphylococcal species isolated from cases of prosthetic joint infection. Our work provides a valuable resource in the from of a large transposon mutant library, and provides a greater understanding of the requirements for staphylococcal survival in human synovial fluid, providing potential biomarkers for future diagnostic development.

microbiology↗

bioRxiv2025

Genome-scale metabolic modelling of trimethoprim's mode of action reveals free ATP salvaged to restore purine pool

Trimethoprim is a clinically important antibiotic for treating urinary tract infections yet its mechanism of killing remains elusive due to a cascade of effected metabolic reactions. Metabolites available in growth media can selectively counteract trimethoprim which affects the interpretation of results. We sought to understand the full scope of trimethoprims impact on Escherichia coli metabolism and how metabolite availability affects trimethoprim outcomes. We applied flux balance analysis on a genome-scale metabolic model of E. coli to simulate trimethoprim activity under bacteriostatic and bactericidal conditions. Our results suggested that in the absence of environmental purines or nucleosides, trimethoprim induces salvage of ATP to repair DNA. We experimentally validated the result with ATP bioluminescence screening of 96 clinical E. coli isolates. Therefore, the choice of growth media composition significantly changes the outcome of trimethoprim challenge and should be taken into consideration when interpreting trimethoprims activity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/666800v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@cddc46org.highwire.dtl.DTLVardef@138a743org.highwire.dtl.DTLVardef@6d4857org.highwire.dtl.DTLVardef@a8502d_HPS_FORMAT_FIGEXP M_FIG C_FIG Diagrammatic representation of trimethoprim action on susceptible UPEC in bacteriostatic and bactericidal conditions. The diagram flows downwards in a time-irrespective manner with outcomes on cellular functions/properties not in order of events. The half-tone at the end represents slowing down of cellular process, however, the outcome is not clear in bactericidal conditions. Circular, green, glowing element at the end of ATP is a representation of ATP bioluminescence. Further, thinning of the line for viability and ATP denotes a reduction in the respective property. RNA and DNA components are labelled above the trimethoprim line with solid grey-to-yellow lines denoting components unaffected by trimethoprim activity. The components that are affected by trimethoprim are replenished by ATP, adenine, or uracil (U) and are denoted by the flow of lines. Abbreviations: A, adenine; C, cytosine; G, guanine; T, thymidine; U, uracil. Sources: Kwon et al. 1, Amyes and Smith 2, Makino and Munakata 6, this study.

systems biology↗

bioRxiv2025

Plasticity in a bacterial global regulatory switch that drives a shift in antibiotic resistance and virulence

Antibiotic resistance and expression of virulence factors can impact the outcome of infection by Pseudomonas aeruginosa. Pathogenesis is often modelled using the P. aeruginosa PAO1 reference strain but laboratory lineages vary in the sequence and activity of MexT, a global regulator impacting virulence, biofilm formation and ciprofloxacin resistance. We defined the impact of active versus inactive MexT in PAO1 and observed global transcriptomic changes affecting the expression of 900 genes. Phenotyping revealed altered metabolism, antibiotic resistance and virulence - resulting in striking variation across a single model organism. We propose that antibiotic-resistance, introduced during work with the original PAO1 strain, has caused plasticity in mexT that accounts for variation across lineages. To test this, we introduced antibiotic resistance into clinical P. aeruginosa isolates and observed downstream mutations in mexT when selective pressure was removed, supporting the proposed evolutionary pathway. Overall, we have demonstrated the transcriptomic basis of MexT as a phenotypic switch in PAO1 and implicated antibiotic resistance as a cause of downstream changes in mexT in P. aeruginosa. Furthermore, MexS/MexT-regulated efflux is implicated in the antibiotic stress response and virulence, helping identify the mechanisms for rapid phenotypic switching through mexT and confirming that PAO1 is a very different organism compared to most isolates. Improved understanding of the regulatory changes linked to antibiotic resistance is particularly relevant to P. aeruginosa where cycles of antibiotic treatment are common.

microbiology↗

bioRxiv2023

Salmonella Infantis, the emerging human multidrug resistant pathogen - a One Health perspective

Salmonella Infantis presents an ever-increasing threat to public health due to its spread throughout many countries and association with high levels of antimicrobial resistance (AMR). Whole genome sequences of 5,284 S. Infantis strains from 74 countries, isolated between 1989 and 2020 from a wide variety of sources including humans, animals, and food, were analysed to compare genetic phylogeny, AMR determinants and plasmid presence. The global S. Infantis population structure diverged into three clusters: a North American cluster, European cluster and a global cluster. The levels of AMR varied between the S. Infantis clusters and by isolation source; 73% of poultry isolates had multidrug resistance (MDR) compared to 35% of human isolates. This correlated with plasmid of emerging S. Infantis (pESI) presence; 71% of poultry isolates contained pESI versus 32% of human isolates. This provides important information for public health teams engaged in reducing the spread of this pathogen.

microbiology↗

bioRxiv2022

Characterisation of neonatal Staphylococcus capitis NRCS-A isolates compared with non NRCS-A Staphylococcus capitis from neonates and adults.

Staphylococcus capitis is a frequent cause of Late-Onset Sepsis (LOS) in neonates admitted to Neonatal Intensive Care Units (NICU). The NRCS-A clone of S. capitis has been isolated from NICUs globally although the reasons for the global success of this clone are not understood. We analysed a collection of S. capitis colonising babies admitted to two NICUs, one in the UK and one in Germany as well as corresponding pathological clinical isolates. Genome analysis identified 3 groups; non-NRCS-A isolates, NRCS-A isolates, and a group of proto NRCS-A - isolates closely related to NRCS-A but not associated with neonatal infection. All bloodstream isolates belonged to the NRCS-A group and were indistinguishable from strains carried on the skin or in the gut. NRCS-A isolates showed increased tolerance to chlorhexidine and antibiotics relative to the other S. capitis as well as enhanced ability to grow at higher pH values. Analysis of 138 pangenomes of the clades identified characteristic nsr and tarJ genes in the NRCS-A and proto groups with a CRISPR-cas system only seen in NRCS-A isolates which also showed enrichment of genes for metal acquisition and transport. We found evidence for transmission of S. capitis NRCS-A within NICU, with related isolates shared between babies and multiple acquisitions by some babies. Our data show NRCS-A strains commonly colonise uninfected babies in NICU representing a potential reservoir for potential infection. This work provides more evidence that adaptation to survive in the gut and on skin facilitates spread of NRCS-A, and that metal acquisition and tolerance may be important to the biology of NRCS-A. Understanding how NRCS-A survives in NICUs can help develop infection control procedures against this clone.

genomics↗

bioRxiv2022

Impact of Salmonella genome rearrangement on gene expression

In addition to nucleotide variation, many bacteria also undergo changes at a much larger scale via rearrangement of their genome structure around long repeat sequences. These rearrangements result in genome fragments shifting position and/or orientation in the genome without necessarily affecting the underlying nucleotide sequence. To date, scalable techniques have not been applied to genome structure (GS) identification, so it remains unclear how extensive this variation is and the extent of its impact upon gene expression. However, the emergence of multiplexed, long-read sequencing overcomes the scale problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing GS identification. Genome rearrangements were generated in Salmonella enterica serovar Typhi through long-term culture at ambient temperature. Colonies with rearrangements were identified via long-range PCR and subjected to long-read nanopore sequencing to confirm genome variation. Four rearrangements were investigated for differential gene expression using transcriptomics. All isolates with changes in genome arrangement relative to the parent strain were accompanied by changes in gene expression. Rearrangements with similar fragment movements demonstrated similar changes in gene expression. The most extreme rearrangement caused a large imbalance between the origin and terminus of replication and was associated with differential gene expression as a factor of distance moved towards or away from the origin of replication. Genome structure variation may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide level measures of variation.

genomics↗

bioRxiv2021

Known mechanisms account for less than half of antimicrobial resistance in a diverse collection of non-aureus staphylococci.

IntroductionNon-aureus staphylococci (NAS) are implicated in many healthcare-acquired infections and an understanding of the genetics of antimicrobial resistance in NAS is important in relation to both clinical intervention and the role of NAS as a reservoir of resistance genes. Gap statementThe burden of antimicrobial resistance in NAS, particularly to clinically relevant antimicrobials, is under recognised. MethodologyWe sourced 394 NAS isolates from clinical samples, healthy human volunteers, animals and type cultures and subjected them to agar dilution susceptibility testing against eight antimicrobials. Cefoxitin was used to screen for methicillin resistance in S. aureus, as it stimulates expression of mecA. We performed whole genome sequencing on 366 isolates and analysed these genotypically for the presence of genetic mechanisms responsible for the phenotypic levels of reduced antimicrobial susceptibility. ResultsWe observed 175 sequenced isolates with a minimum inhibitory concentration (MIC) of at least 4 g/ml to cefoxitin, of which 50% (87/175) did not harbour a known mec homologue. Eight clinical NAS isolates displayed high daptomycin MICs (>4 g/ml), with no known mechanism identified. Differences in MICs against erythromycin were attributable to the presence of different resistance genes (msrA and ermC). In total, 49% (193 /394) of isolates displayed reduced susceptibility to three or more of the antimicrobials tested. ConclusionsThe widespread presence of reduced antimicrobial susceptibility in NAS is a concern, with an increased likelihood of (1) harder to treat infections caused directly by NAS, and (2) resistance genes being passed on to other bacteria via horizontal gene transfer, both of which have clinical implications for treatment and management of patients.

microbiology↗

bioRxiv2020

Massively parallel transposon mutagenesis identifies temporally essential genes for biofilm formation in Escherichia coli

Biofilms complete a life cycle where cells aggregate, grow and produce a structured community before dispersing to seed biofilms in new environments. Progression through this life cycle requires temporally controlled gene expression to maximise fitness at each stage. Previous studies have largely focused on the essential genome for the formation of a mature biofilm, but here we present an insight into the genes involved at different stages of biofilm formation. We used TraDIS-Xpress; a massively parallel transposon mutagenesis approach using transposon-located promoters to assay the impact of disruption or altered expression of all genes in the genome on biofilm formation. We determined temporal differences in the importance of genes in E. coli growing as a biofilm on glass beads after 12, 24 and 48 hours. A selection of genes identified as important were then validated independently by assaying biofilm biomass, aggregation, curli production and adhesion ability of defined mutants. We identified 48 genes that affected biofilm fitness including genes with known roles and those not previously implicated in biofilm formation. Regulation of type 1 fimbriae and motility were important at all time points. Adhesion and motility were important for the early biofilm, whereas matrix production and purine biosynthesis were only important as the biofilm matured. We found strong temporal contributions to biofilm fitness for some genes including some where expression changed between being beneficial or detrimental depending on the stage at which they are expressed, including dksA and dsbA. Novel genes implicated in biofilm formation included zapE and truA involved in cell division, maoP in DNA housekeeping and yigZ and ykgJ of unknown function. This work provides new insights into the requirements for successful biofilm formation through the biofilm life cycle and demonstrates the importance of understanding expression and fitness through time.

molecular biology↗

bioRxiv2020

CoronaHiT: large scale multiplexing of SARS-CoV-2 genomes using Nanopore sequencing

The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 96 samples at a time. There is a need, however, for a flexible, platform agnostic, method that can provide multiple throughput options depending on changing requirements as the pandemic peaks and troughs. Here we present CoronaHiT, a method capable of multiplexing up to 96 small genomes on a single MinION flowcell or >384 genomes on Illumina NextSeq, using transposase mediated addition of adapters and PCR based addition of barcodes to ARTIC PCR products. We demonstrate the method by sequencing 95 and 59 SARS-CoV-2 genomes for routine and rapid outbreak response runs, respectively, on Nanopore and Illumina platforms and compare to the standard ARTIC LoCost nanopore method. Of the 154 samples sequenced using the three approaches, genomes with [≥] 90% coverage (GISAID criteria) were generated for 64.3% of samples for ARTIC LoCost, 71.4% for CoronaHiT-ONT, and 76.6% for CoronaHiT-Illumina and have almost identical clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 96 SARS-CoV-2 genomes per MinION flowcell and that Illumina sequencing can be performed on the same libraries, which will allow significantly higher throughput. CoronaHiT provides increased coverage for higher Ct samples, thereby increasing the number of high quality genomes that pass the GISAID QC threshold. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic.

genomics↗

bioRxiv2018

Rapid Diagnosis of Lower Respiratory Infection using Nanopore-based Clinical Metagenomics

Lower respiratory infections (LRIs) accounted for three million deaths worldwide in 2016, the leading infectious cause of mortality. The \"gold standard\" for investigation of bacterial LRIs is culture, which has poor sensitivity and is too slow to guide early antibiotic therapy. Metagenomic sequencing potentially could replace culture, providing rapid, sensitive and comprehensive results. We developed a metagenomics pipeline for the investigation of bacterial LRIs using saponin-based host DNA depletion combined with rapid nanopore sequencing. The first iteration of the pipeline was tested on respiratory samples from 40 patients. It was then refined to reduce turnaround and increase sensitivity, before testing a further 41 samples. The refined method was 96.6% concordant with culture for detection of pathogens and could accurately detect resistance genes with a turnaround time of six hours. This study demonstrates that nanopore metagenomics can rapidly and accurately characterise bacterial LRIs when combined with efficient human DNA depletion.

microbiology↗