Biology subjects

Webber, M. A.

Publications and source records attributed to Webber, M. A..

26 records · Page 2Linked to original sources

bioRxiv2021

Efflux only impacts drug accumulation in actively growing cells

For antibiotics with intracellular targets, effective treatment of bacterial infections requires the drug to accumulate to a high concentration inside cells. Bacteria produce a complex cell envelope and possess drug-export efflux pumps to limit drug accumulation inside cells. Decreasing cell envelope permeability and increasing efflux pump activity can reduce intracellular accumulation of antibiotics, and are commonly seen in antibiotic resistant strains. Here, we show that the balance between influx and efflux differs depending on bacterial growth phase in Gramnegative bacteria. Accumulation of the model fluorescent drug, ethidium bromide (EtBr) was measured in S. Typhimurium SL1344 (wild-type) and efflux deficient ({Delta}acrB) strains during growth. In SL1344, EtBr accumulation remained low, regardless of growth phase and did not correlate with acrAB transcription. EtBr accumulation in {Delta}acrB was high in exponential phase but dropped sharply later in growth, with no significant difference to SL1344 in stationary phase. Low EtBr accumulation in stationary phase was not due to the upregulation of other efflux pumps, but instead, due to decreased permeability of the envelope in stationary phase. RNAseq identified changes in expression of several pathways that remodel the envelope in stationary phase, leading to lower permeability. This study shows that efflux is only important for maintaining low drug accumulation in actively growing cells, and that envelope permeability is the predominant factor dictating the rate of drug entry in stationary phase cells. This conclusion means that (i) antibiotics with intracellular targets may be less effective in complex non-growing or slow-growing bacterial infections where intracellular accumulation may be low, (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing and (iii) the remodelling of the cell envelope prior to stationary phase could provide novel drug targets.

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

Bacterial single-cell genomics enables phylogenetic analysis of in vitro evolutionary studies

Single-cell DNA sequencing has the potential to reveal detailed hierarchical structures in evolving populations of cells. Single cell approaches are increasingly used to study clonal evolution in human ageing and cancer, but have not yet been deployed to study evolving microbial populations. Here, we present an approach for single bacterial genomic analysis using FACS isolation of individual bacteria followed by whole-genome amplification and sequencing. We apply this to in vitro experimental evolution of a hypermutator strain of Salmonella in response to antibiotic stress (ciprofloxacin). By analysing sequence polymorphisms in individual cells from the population we identified the presence and prevalence of sub-populations which have acquired polymorphisms in genes previously demonstrated to be associated with ciprofloxacin susceptibility. We were also able to identify that the population exposed to antibiotic stress was able to both develop resistance whilst maintaining diversity. This population structure could not be resolved from bulk sequence data, and our results show how high-throughput single-cell sequencing can enhance experimental studies of bacterial evolution.

genomics↗

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↗

bioRxiv2019

A genome wide analysis of Escherichia coli responses to fosfomycin using TraDIS-Xpress reveals novel roles for the phosphonate and phosphate transport systems

Fosfomycin is an antibiotic which has seen a revival in use due to its unique mechanism of action and resulting efficacy against isolates resistant to many other antibiotics. Mechanisms of resistance have been elucidated and loss of function mutations within the genes encoding the sugar importers, GlpT and UhpT are commonly selected for by fosfomycin exposure in E. coli. There has however not been a genome wide analysis of the basis for fosfomycin sensitivity reported to date. Here we used TraDIS-Xpress a high-density transposon mutagenesis approach to assay the role of all genes in E. coli in fosfomycin sensitivity. The data confirmed known mechanisms of action and resistance as well as identifying a set of novel loci involved in fosfomycin sensitivity. The assay was able to identify sub domains within genes of importance and also revealed essential genes with roles in fosfomycin sensitivity based on expression changes. Novel genes identified included those involved in glucose metabolism, the phosphonate import and breakdown system, phnC-M and the phosphate importer, pstSACB. The impact of these genes in fosfomycin sensitivity was validated by measuring the susceptibility of defined inactivation mutants. This work reveals a wider set of genes contribute to fosfomycin sensitivity including core sugar metabolism genes and two transport systems previously unrecognised as having a role in fosfomycin sensitivity. The work also suggests new routes by which drugs with a phosphonate moiety may be transported across the inner membrane of Gram-negative bacteria. ImportanceThe emergence and spread of antibiotic resistant bacteria had resulted in increased use of alternative drugs which retain efficacy against isolates resistant to other classes of drugs. One example is fosfomycin; an old drug which has found greatly increased use in recent years. We studied the mechanisms of fosfomycin resistance by applying a genome wide screen based on comparing the fitness of a massive library of transposon mutants in the presence of fosfomycin. This approach identified the previously known mechanisms of resistance but also identified a number of new pathways which contribute to fosfomycin sensitivity including two importer systems. This information advances our knowledge about an increasingly important antibiotic and identifies new potential routes to resistance.

microbiology↗

bioRxiv2018

Opening Pandora box - high level resistance to antibiotics of last resort in Gram negative bacteria from Nigeria

Antimicrobial resistance (AMR) is a global problem but information about the prevalence and mechanisms of resistance in sub-Saharan Africa are lacking. We determined the percentage of drug resistant isolates and resistance mechanisms in 307 Gram negative isolates randomly collected from south western Nigeria. Susceptibility testing revealed 78.1%, 92.2% and 52.6% of all isolates were resistant to fluoroquinolones, third generation cephalosporins and carbapenems respectively. There were more resistant isolates from the stools of uninfected patients than from specimens of patients with symptoms of infections. Only a small proportion of E. coli (10%) and Klebsiella (7%) isolates produced a carbapenemase. Whole genome sequencing of selected isolates identified the presence of globally disseminated clones. This depicts a crisis for the use of first line therapy in Nigerian patients, it is likely that Nigeria is playing a significant role in the spread of AMR due to her high population and mobility across the globe.

microbiology↗

bioRxiv2017

Prevalence, mechanisms and comparison of detection methods of fosfomycin resistance in E. coli from urinary tract infections

As numbers of bacterial isolates resistant to first line antibiotics rise there has been a revival in the use of older drugs such as fosfomycin. Fosfomycin is a cell wall inhibitor with a unique mode of action, increasingly used in the treatment of urinary tract infections. In this study, the prevalence of fosfomycin resistant E. coli in a panel of 1000 urine isolates was investigated. Three different clinically used fosfomycin susceptibility testing methods were assessed and genome sequencing used to characterise resistant isolates.\n\nOf the 1000 isolates, 676 were E. coli of which initial susceptibility testing with the MAST Uri(R)system suggested 81 (12%) were fosfomycin resistant. Of these, 62 were subsequently confirmed as being E. coli. However, using micro-broth dilution, agar dilution and E-test strips, a lower rate of 1.3% (8/62) of E. coli isolates were robustly identified as being truly fosfomycin resistant; a prevalence comparable with other similar studies. The use of E-test and 96-well breakpoint plates gave results that were inconsistent and hard to interpret. Resistant isolates of E. coli belonged to diverse MLST types and each had a unique set of chromosomal alterations in genes associated with fosfomycin resistance. Changes in GlpT and UhpT/UhpA transport systems were commonly identified, with 6/8 of the resistant isolates possessing amino-acid changes or deletions absent in susceptible strains. Fosfomycin resistant isolates were not multiply drug resistance and did not carry plasmidic fosfomycin resistance genes. Therefore, the use of fosfomycin may be unlikely to drive selection of a particular clone or movement of transferrable resistance genes.\n\nFosfomycin remains a viable option for the treatment of E. coli in uncomplicated UTIs, different susceptibility testing platforms can give very different results regarding the prevalence of fosfomycin resistance with false positives a potential problem that may unnecessarily limit use of this agent.

microbiology↗

bioRxiv2017

Increased usage of antiseptics is associated with reduced susceptibility in clinical isolates of S. aureus

Hospital acquired infection is a major cause of morbidity and mortality and regimes to prevent infection are crucial in infection control. These include decolonisation of at-risk patients of carriage of MRSA which is commonly achieved by protocols that include the use of chlorhexidine, or octenidine as biocidal agents. There is however no standardised single decolonisation regime agreed upon in the UK or other countries and protocols include a variety of active agents. Antibiotic resistant bacteria cause major problems in hospital medicine and concern has been raised regarding the development of biocide resistance which would cause decolonisation regimes to become unreliable. In this study, we assembled a panel of isolates of S. aureus including isolates collected before the development of chlorhexidine and octenidine through to a contemporaneous panel of isolates from a major hospital trust in the UK during a period when the decolonisation regime was altered. We observed significant increases in the MIC and MBC of chlorhexidine in isolates collected from periods of high usage of chlorhexidine. No isolates had a significantly altered MIC or MBC of octenidine apart from those collected after octenidine was introduced into the trust where isolates with four-fold decreases in susceptibility emerged. There was no suggestion of cross-resistance between the two biocidal agents. A combination of VNTR, PCR for qac genes and whole genome sequencing was used to type isolates and examine possible mechanisms of resistance. The typing data showed no expansion of a single strain was associated with decreased biocide tolerance and isolates with increased chlorhexidine MIC and MBCs were found from different clonal complexes; CC8, CC22 and CC30. Biocide susceptibility did not correlate with carriage of qac efflux pump genes - carriage of qacA and qacB was detected but, with one exception was restricted to isolates of CC8. Analysis of genome sequence data for closely related pairs of strains with differential biocide susceptibility revealed no common mutations or carriage of accessory elements that correlated with biocide tolerance. Mutations with the NorA or NorB efflux pumps, previously associated with chlorhexidine export were identified suggesting this may be an important mechanism of biocide tolerance. The clinical relevance of decreased biocide tolerance in terms of efficacy of decolonisation therapies remains to be established but we present evidence here that isolates are evolving in the face of biocide challenge in patients and that changes to decolonisation regimes are reflected in changes in susceptibility of isolates. More work is needed to assess the impact of these changes to ensure effective and robust decolonisation protocols remain in place.

microbiology↗