Craig Winstanley

Graphical Pseudomonas aeruginosa survival in a commercially available contact lens disinfection solution is increased by exposure to sub-inhibitory concentrations of the solution via induction of lipid A modification pathways. Created in BioRender. Hilliam, Y. (2025) https://BioRender.com/w7jkvrj.

Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the 2-component regulatory system sensor kinase gene pmrB in P. aeruginosa from CF lung infections and from experimental infection of mice. Here, we demonstrate that, while such mutations lowered in vitro minimum inhibitory concentrations for multiple antimicrobial classes, this was not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impaired aminoarabinose modification of LPS, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this could be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coated the surface of PmrB - deficient P. aeruginosa , reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine was elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilized.

The Liverpool epidemic strain (LES) is an important transmissible clonal lineage of Pseudomonas aeruginosa that chronically infects the lungs of people with cystic fibrosis (CF). Previous studies have focused on the genomics of the LES in a limited number of isolates, mostly from one CF centre in the UK, and from studies highlighting identification of the LES in Canada. Here we significantly extend the current LES genome database by genome sequencing 91 isolates from multiple CF centres across the UK, and we describe the comparative genomics of this large collection of LES isolates from the UK and Canada. Phylogenetic analysis revealed that the 145 LES genomes analysed formed a distinct clonal lineage when compared with the wider P. aeruginosa population. Notably, the isolates formed two clades: one associated with isolates from Canada, and the other associated with UK isolates. Further analysis of the UK LES isolates revealed clustering by clinic geography. Where isolates clustered closely together, the association was often supported by clinical data linking isolates or patients. When compared with the earliest known isolate, LESB58 (from 1988), many UK LES isolates shared common loss-of-function mutations, such as in genes gltR and fleR. Other loss-of-function mutations identified in previous studies as common adaptations during CF chronic lung infections were also identified in multiple LES isolates. Analysis of the LES accessory genome (including genomic islands and prophages) revealed variations in the carriage of large genomic regions, with some evidence for shared genomic island/prophage complement according to clinic location. Our study reveals divergence and adaptation during the spread of the LES, within the UK and between continents.

This study investigated the genetic basis of multidrug resistance in two strains of Achromobacter xylosoxidans isolated from patients attending a hospital in Thailand in 2012. These isolates were highly resistant to cephalosporins, aminoglycosides, fluoroquinolones, co-trimoxazole and carbapenems. Whole genome sequencing revealed that the two isolates were not clonally related and identified a carbapenem resistance gene-habouring integron (In687), residing in a novel genomic island, AcGI1. This In687 shares 100% identical nucleotide sequence with ones found in Acinetobacter baumannii Aci 16, isolated from the same hospital in 2007. We report the first analysis of multidrug-resistant A. xylosoxidans isolated in Thailand, and the first example of this island in A. xylosoxidans. Our data support the idea that resistance has spread in Thailand via horizontal gene transfer between species and suggest the possibility of A. xylosoxidans may serve as a reservoir of antibiotic resistance, especially in hospital setting.

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. The type III secretion system of P. aeruginosa delivers the toxin ExoU into the cytosol of target host cells, where its plasma membrane directed phospholipase activity induces rapid cell lysis. Therefore, inhibition of the phospholipase activity of ExoU would be an important treatment strategy in P. aeruginosa infections. We evaluated a panel of ExoU small molecule inhibitors, previously identified from high throughput cellular based assays, and analysed their inhibition of ExoU phospholipase activity in vitro . A corneal epithelial (HCE-T) scratch and infection model using florescence microscopy, and cell viability assays, were used to test the efficacy of compounds to inhibit ExoU from P. aeruginosa . Compounds Pseudolipasin A, compound A and compound B were effective at mitigating ExoU mediated cytotoxicity after infection at concentrations as low as 0.5 μM. Importantly, by using the antimicrobials moxifloxacin and tobramycin to control bacterial load, these assays were extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration (MIC) for 24 h, but, when used in combination with either PSA, compound A or compound B, partial scratch healing was observed. These results provide evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat clinical infections of ExoU producing P. aeruginosa .

Multidrug resistance (MDR) represents a global threat to health. Here, we used whole genome sequencing to characterise Pseudomonas aeruginosa MDR clinical isolates from a hospital in Thailand. Using long-read sequence data we obtained complete sequences of two closely related megaplasmids (>420 kb) carrying large arrays of antibiotic resistance genes located in discrete, complex and dynamic resistance regions, and revealing evidence of extensive duplication and recombination events. A comprehensive pangenomic and phylogenomic analysis indicates that: 1) these large plasmids comprise an emerging family present in different members of the Pseudomonas genus, and associated with multiple sources (geographical, clinical or environmental); 2) the megaplasmids encode diverse niche-adaptive accessory traits, including multidrug resistance; 3) the accessory genome of the megaplasmid family is highly flexible and diverse. The history of the megaplasmid family, inferred from our analysis of the available database, suggests that members carrying multiple resistance genes date back to at least the 1970s.

Campylobacter jejuni is the most common bacterial cause of human infectious intestinal disease. We genome sequenced 601 human C. jejuni isolates, obtained from two large prospective studies of infectious intestinal disease (IID1 [isolates from 1993–1996; n = 293] and IID2 [isolates from 2008–2009; n = 93]), the INTEGRATE project [isolates from 2016–2017; n = 52] and the ENIGMA project [isolates from 2017; n = 163]. There was a significant increase in the prevalence of the T86I mutation conferring resistance to fluoroquinolone between each of the three later studies (IID2, INTEGRATE and ENIGMA) and IID1. Although the distribution of major multilocus sequence types (STs) was similar between the studies, there were changes in both the abundance of minority STs associated with the T86I mutation, and the abundance of clones within single STs associated with the T86I mutation. Four population-based studies of community diarrhoea over a 25 year period revealed an increase over time in the prevalence of the T86I amongst isolates of C. jejuni associated with human gastrointestinal disease in the UK. Although associated with many STs, much of the increase is due to the expansion of clones associated with the resistance mutation.

Pseudomonas aeruginosa , a versatile Gram-negative pathogen that can cause a wide range of infections, is the most common causative agent in cases of bacterial keratitis associated with contact-lens use. Corneal infections with P. aeruginosa often have poor clinical outcomes and can result in long and costly treatments. During the infection process, the pathogen exploits its large genome, encoding complex regulatory networks and a wide range of virulence factors, including motility and the secretion of various proteases and toxins. Although antibiotic resistance levels in the UK are low, higher levels have been seen in some other countries. In the face of increasing antibiotic resistance, alternative therapeutic approaches such as antivirulence strategies and phage therapy are being developed. There is increasing evidence to suggest that keratitis infections are associated with a phylogenetic subgroup of P. aeruginosa isolates carrying the gene encoding the potent cytotoxin exotoxin U, one of two mutually exclusive exotoxins secreted via the type III secretion system. The mechanisms behind this association are unclear, but understanding the genetic differences that predispose P. aeruginosa to cause corneal infections may allow for the development of targeted and more effective future treatments to reduce the morbidity of P. aeruginosa keratitis. In order to minimize the risk of severe P. aeruginosa eye infections, a wide range of contact-lens disinfection solutions are available. Constant exposure to biocides at a range of concentrations, from sub-inhibitory to inhibitory, could contribute to the development of resistance to both antibiotics and disinfectants.

The opportunistic pathogen Pseudomonas aeruginosa employs the type III secretion system (T3SS) and four effector proteins, ExoS, ExoT, ExoU, and ExoY, to disrupt cellular physiology and subvert the host’s innate immune response. Of the effector proteins delivered by the T3SS, ExoU is the most toxic. In P. aeruginosa infections, where the ExoU gene is expressed, disease severity is increased with poorer prognoses. This is considered to be due to the rapid and irreversible damage exerted by the phospholipase activity of ExoU, which cannot be halted before conventional antibiotics can successfully eliminate the pathogen. This review will discuss what is currently known about ExoU and explore its potential as a therapeutic target, highlighting some of the small molecule ExoU inhibitors that have been discovered from screening approaches.