Biology subjects

Holden, E. R.

Publications and source records attributed to Holden, E. R..

8 recordsLinked to original sources

bioRxiv2026

Unsupervised decomposition of massively parallel TraDIS-Xpress fitness data from Escherichia coli identifies distinct functional and expression modules of antimicrobial resistance determinants

Linking genotype to phenotype is a central goal in biology, one way to do this is using transposon insertion sequencing (TIS) where the fitness impacts of mutations across a genome are assessed in different conditions. Traditional analyses use comparison of each gene response independently in control versus test conditions, so a compendium of hundreds of test conditions returns hundreds of separate ranked gene lists. To unlock the potential information hidden within large data sets, we applied independent component (ICA) analysis to 754 Escherichia coli BW25113 TraDIS-Xpress samples. These samples included a series of antibiotic and other stress condition exposures. ICA is designed to identify hidden structure within complex data and to extract meaningfully linked components. In this analysis each annotated gene was resolved into a gene body and 198 bp 5' and 3' features so that expression effects driven by the transposon's outwards facing promoter enter the matrix separately from gene disruption effects. The decomposition returned 99 components, which we term fxModules, which were defined as containing elements with similar responses across the whole compendium rather than per any individual experiment. Of the 754 stress samples, the antibiotic arm included 110 samples derived from exposure to 14 separate agents at multiple concentrations. Many of these have been highly studied allowing us to explore the accuracy of predicted responsive fxModules. This analysis showed excellent predictive power of the approach and the fxModules responsive to individual drugs typically reproduced known mechanisms at a resolution superior to analysis of single gene sets. For example, exposure to fosfomycin identified responsive fxModules containing genes which are known to be involved in uptake of the drug, cAMP-CRP control and exposure to gentamicin identified oxidative phosphorylation split across three fxModules responding with different magnitudes and directions at different concentrations. This recapitulates recent work studying the landscape of E. coli genes sensitive to this drug with high precision without any prior knowledge of functional annotation. Similarly precise predictions were made for other drugs. Responsive modules also identified novel groups of genes and linkages beyond the boundaries of defined pathways as being responsive to various stresses demonstrating the ability to predict new genotype-phenotype links. Inclusion of the regulatory regions also resolved fxModules which were entirely contributed by promoter regions, for example for chloramphenicol genes responsible for efflux were identified by signals in intergenic control regions, and unidirectional insertions in gene bodies, a signal that a gene level matrix would not be able to predict with accuracy. These data show that ICA decomposition provides an efficient and accurate annotation independent approach to identifying sets of genes with related functional impacts in any given condition in the compendium. The functional modular association of genes also help in predicting gene functions of unknown genes based on the group function. This approach allows rapid identification of novel genotype-phenotype links and unlocks the power of multiple comparisons of transposon mutagenesis data.

microbiology↗

bioRxiv2026

Identification of genes important for response of Pseudomonas aeruginosa biofilms to ciprofloxacin exposure

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections in immunocompromised individuals, such as patients with cystic fibrosis where it commonly forms biofilms. Ciprofloxacin is used extensively to treat P. aeruginosa infections, but its effectiveness can be significantly reduced due to biofilm formation. Although many individual genes associated with biofilm formation or ciprofloxacin resistance have been characterised, the genetic basis of P. aeruginosa biofilm fitness related to antibiotic challenge remains incompletely understood. In this study we employed a whole genome screen to assay the impact of gene disruptions or altered gene expression on survival of P. aeruginosa biofilms exposed to different concentrations of ciprofloxacin. Genes impacting fitness in the biofilm context were identified by comparing the biofilm samples to planktonic samples harvested at 12h, 24h and 48h with and without ciprofloxacin. Genes associated with c-di-GMP regulation and Gac/Rsm signalling were identified as primary regulators for biofilm formation in the presence and absence of ciprofloxacin. In addition, a group of genes involved in respiration, metabolism (especially polyamine metabolism), and various transporter and efflux systems were identified as important for biofilm fitness. Ciprofloxacin specifically imposed a selective pressure on flagellar function and Psl production which were essential for survival in early biofilms. Moreover, transposon insertions within the CPA gene clusters (PA5448-PA5451 and PA5455-PA5456) and the salvage peptidoglycan recycling pathway showed reduced fitness in late biofilms at high concentration of ciprofloxacin, indicating that cell envelope integrity is beneficial for mature biofilms. This study identifies important determinants of survival for biofilms at different stages of maturity in the presence and absence of ciprofloxacin and implicates potential therapeutic targets for antibiofilm drug development.

genomics↗

bioRxiv2023

Investigating how Salmonella colonise alfalfa using a whole genome screen.

Enteropathogenic bacteria including Salmonella regularly cause outbreaks of infection from fresh produce posing a significant public health threat. Salmonellas ability to persist on fresh produce for extended periods is partly attributed to its capacity to form biofilms, which poses a challenge to food decontamination and facilitates persistence in the food chain. Preventing biofilm formation on food products and in food processing environments is crucial for reducing the incidence of foodborne diseases. Understanding the mechanisms of colonisation and establishment on fresh produce will inform the development of decontamination approaches. We used Transposon-directed Insertion site sequencing (TraDIS-Xpress) to investigate the mechanisms employed by Salmonella enterica serovar Typhimurium to colonise and establish itself on fresh produce at critical timepoints following infection. We established an alfalfa infection model and compared the findings to those obtained from glass surfaces. Our research revealed dynamic changes in the pathways associated with biofilm formation over time, with distinct plant-specific and glass-specific mechanisms for biofilm formation, alongside the identification of shared genes playing pivotal roles in both contexts. Notably, we observed variations in the significance of factors such as flagella biosynthesis, lipopolysaccharide (LPS) production, and stringent response regulation in biofilm development on plant versus glass surfaces. Understanding the genetic underpinnings of biofilm formation on both biotic and abiotic surfaces offers valuable insights that can inform the development of targeted antibacterial therapeutics, ultimately enhancing food safety throughout the food processing chain. FundingThe authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC); ERH, JAA, HAK, MAW and ET were supported by the BBSRC Institute Strategic Programme Microbes and Food Safety BB/X011011/1 and its constituent project BBS/E/F/000PR13635. NV was supported by the Food Safety Research Network grant BB/X002985/1 awarded to ET. Data availabilityNucleotide sequence data supporting the analysis in this study has been deposited in ArrayExpress under the accession number E-MTAB-13495. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2023

Common food preservatives impose distinct selective pressures on Salmonella Typhimurium planktonic and biofilm populations

Food preservatives are crucial in controlling microbial growth in processed foods to maintain food safety. Bacterial biofilms pose a threat in the food chain by facilitating persistence on a range of surfaces and food products. Cells in a biofilm are often highly tolerant of antimicrobials and can evolve in response to antimicrobial exposure. Little is known about the efficacy of preservatives against biofilms and their potential impact on the evolution of antimicrobial resistance. In this study we investigated how the common food pathogen Salmonella enterica serovar Typhimurium responded to subinhibitory concentrations of four common food preservatives (sodium chloride, potassium chloride, sodium nitrite or sodium lactate) when grown planktonically and in biofilms. We found that each preservative exerted a unique selective pressure on S. Typhimurium populations grown planktonically and in a biofilm. Biofilm formation itself seemed to confer protection when exposed to each of the four preservatives, more so than previous exposure to sub-inhibitory concentrations of preservatives. There was a trade-off between biofilm formation and growth in the presence of three of the four preservatives, where prolonged preservative exposure resulted in reduced biofilm biomass and matrix production over time. Despite the differences in biofilm formation and preservative tolerance seen following three preservative stresses, they selected for mutations in global stress response regulators rpoS and crp. There was no evidence for any selection of cross-resistance to antibiotics after preservative exposure, and some evidence that antagonism between preservatives can be exploited in compound cocktails to reduce contamination in the food chain. HighlightsO_LIPreservative-specific evolutionary adaptation of Salmonella was shown over time. C_LIO_LIA trade-off between adaptation and biofilm formation was observed. C_LIO_LINo cross-resistance to antibiotics was seen after preservative exposure. C_LIO_LIMutations were found to be preservative-specific, with some common ones like rpoS and crp. C_LI

microbiology↗

bioRxiv2023

Mechanisms of action and synergies of a novel lipid IVA biosynthesis inhibitor

The development of novel antimicrobials provides additional treatment options for infectious diseases, including antimicrobial resistant infections. There are many hurdles to antimicrobial development and identifying an antimicrobials mechanism of action is a crucial step in progressing candidate molecules through the drug discovery pipeline. We used the genome wide screening method TraDIS-Xpress to identify genes in two model Gram-negative bacteria that affected sensitivity to three analogues of a novel antimicrobial compound (OPT-2U1). TraDIS-Xpress identified that all three analogues targeted the lipid IVA biosynthetic pathway in E. coli and Salmonella Typhimurium. Specifically, we determined that the antimicrobial target was likely to be LpxD, and validated this by finding a 5 log2-fold increase in the MIC of the OPT-2U1 analogues in E. coli when lpxD was overexpressed. Synergies were identified between OPT-2U1 analogues combined with rifampicin or colistin, to varying strengths, in both E. coli and S. Typhimurium. LPS composition was a likely reason for differences between E. coli and S.Typhimurium, as perturbation of LPS synthesis affected synergy between antibiotics and OPT-2U1 analogues. Finally, genes involved in ATP synthesis and membrane signalling functions were also found to affect the synergy between colistin and OPT-2U1 analogues. TraDIS-Xpress has proven a powerful tool to rapidly assay all genes (and notably, essential genes) within a bacterium for roles in dictating antimicrobial sensitivity. This study has confirmed the predicted target pathway of OPT-2U1 and identified synergies which could be investigated for development of novel antimicrobial formulations. Data SummaryNucleotide sequence data supporting the analysis in this study has been deposited in ArrayExpress under the accession number E-MTAB-13250. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

molecular biology↗

bioRxiv2023

Tazobactam selects for multidrug resistance

Piperacillin-Tazobactam is a {beta}-lactam/{beta}-lactamase inhibitor combination which is amongst the most prescribed antimicrobials in hospital medicine. Piperacillin is inactivated by commonly carried resistance enzymes, but tazobactam inhibits these allowing successful treatment. The effect of piperacillin on Gram-negative bacteria has been widely studied, but less attention has been paid to the effects of tazobactam. We used a massive transposon mutagenesis approach (TraDIS-Xpress) to determine the genes in Escherichia coli that affect survival when exposed to piperacillin and tazobactam, separately and together. We found significant differences in the selective pressure of the two drugs: a striking finding was that multiple efflux pump families and regulators were essential for survival in the presence of tazobactam, but only one efflux system was beneficial for piperacillin. Additionally, we identified the shikimate kinase AroK as a potential target for tazobactam. This method also found that genes involved in DNA replication and repair reduced E. coli susceptibility to a combination of piperacillin and tazobactam, not seen from either drug treatment alone. Treatment of E. coli and Klebsiella pneumoniae with piperacillin and/or tazobactam selected for mutants with reduced susceptibility, and SNP analyses supported the TraDIS-Xpress findings that tazobactam selects for changes in membrane permeability and maintenance associated with multidrug-resistance. Increased efflux activity is an important foundation of multidrug resistance in human pathogens, therefore the finding that tazobactam can select for this is concerning. These findings could have consequences for antibiotic prescription and should inform the development of future {beta}-lactamase inhibitors to reduce the global increase in multidrug-resistant infections.

molecular biology↗

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↗

bioRxiv2019

Antibiotics select for novel pathways of resistance in biofilms

Most bacteria in nature exist in aggregated communities known as biofilms. Bacteria within biofilms are inherently highly resistant to antibiotics. Current understanding of the evolution and mechanisms of antibiotic resistance is largely derived from work from cells in liquid culture and it is unclear whether biofilms adapt and evolve in response to sub-inhibitory concentrations of drugs. Here we used a biofilm evolution model to show that biofilms of a model food borne pathogen, Salmonella Typhimurium rapidly evolve in response to exposure to three clinically important antibiotics. Whilst the model strongly selected for improved biofilm formation in the absence of any drug, once antibiotics were introduced the need to adapt to the drug was more important than the selection for improved biofilm formation. Adaptation to antibiotic stress imposed a marked cost in biofilm formation, particularly evident for populations exposed to cefotaxime and azithromycin. We identified distinct resistance phenotypes in biofilms compared to corresponding planktonic control cultures and characterised new mechanisms of resistance to cefotaxime and azithromycin. Novel substitutions within the multidrug efflux transporter, AcrB were identified and validated as impacting drug export as well as changes in regulators of this efflux system. There were clear fitness costs identified and associated with different evolutionary trajectories. Our results demonstrate that biofilms adapt rapidly to low concentrations of antibiotics and the mechanisms of adaptation are novel. This work will be a starting point for studies to further examine biofilm specific pathways of adaptation which inform future antibiotic use.

microbiology↗