Xenia Kostoulias

Conjugative plasmids drive bacterial evolution and niche adaptation, yet how their active acquisition reshapes host transcription remains poorly understood. Most studies focus on stable plasmid carriage, overlooking the dynamic transcriptional changes during conjugation itself. Here, we investigate the active conjugation of RP4, a prototypical broad-host range plasmid, to E. coli , and K. pneumoniae recipients. We observe that an immediate, host- and surface factor-dependent transcriptional response occurs. This includes activation of non-SOS stress pathways, motility, exopolysaccharide production, anaerobic respiration, and metabolic adaptation. These responses do not inhibit conjugation, suggesting they serve to maintain host homeostasis, particularly of envelope encoded functions. Capsule expression prevents these responses and inhibits RP4 activation by physically blocking donor-recipient contact. Unexpectedly, RP4 can bypass the capsular barrier through opportunistic transfer into a sub-population of phenotypically thin-capsulated cells. These findings show how RP4, the hosts species, and surface architecture shape the active conjugation transcriptional landscape, which may have implications for plasmid dissemination and bacterial evolution.

Fast diagnosis of antimicrobial resistance (AMR), especially in high-priority pathogens, is essential to maintain the best-possible patient outcomes. In this communication, we present a new approach for rapid AMR assessment that is able to distinguish between vancomycin-resistant and vancomycin-susceptible enterococci (VRE and VSE, respectively) within 2 hours using ATR-FTIR spectroscopy, chemometrics and chemometrics-based modelling. This study outlined how early spectral markers of effective vancomycin action were used to accurately predict resistance profiles in both known and blinded Enterococcus samples. This paper provides the foundation for a rapid diagnostic tool that enables faster determination of bacterial susceptibility, supporting timely clinical decisions and better patient outcomes.

With a limited number of traditional virulence factors, the success of the nosocomial pathogen Acinetobacter baumannii is largely attributed to its ability to persist and resist. The niches encountered during infection vary significantly from the more commonly studied laboratory setting, and consequently, the genes responsible for in vivo pathogenesis have yet to be fully elucidated. This study utilized the A. baumannii AB5075-UW transposon mutant library with unbiased genome-wide transposon sequencing to identify the genetic basis for survival and fitness during pneumonia and septicaemia infections. We identified 128 genes essential for in-host survival, including 22 required for survival in all tissues. Additionally, 302 genes with significantly altered fitness in vivo were also identified. Tissue specificity was observed, highlighting the importance of genes associated with aa biosynthesis in the lungs, cell shape and structure in the kidneys and metal acquisition during septicaemia. The majority (89%) of the genes with aberrant fitness were constituents of the core A. baumannii genome. The findings were validated using a subset of targeted mutants, including those required for infection ( phoB , cysI and hom ) or specifically septicaemia ( corA , lepA and purN ) or pneumonia ( argC , hisC and leuD ), confirming that these observations were a result of specific in vivo fitness defects rather than aberrant in vitro growth. Taken together, these data provide the first global profile of genes required for in vivo fitness of A. baumannii during different disease states and growth in different tissues.

Preservation and augmentation of existing antimicrobials is crucial in combating antimicrobial resistance. Gram-positive bacteria, exemplified by Staphylococcus aureus, are among the most common human bacterial pathogens, with methicillin-resistant S. aureus (MRSA) now established globally. Daptomycin is a last-line anti-staphylococcal antimicrobial that uniquely targets the bacterial membrane with bactericidal effects. Here, we developed lipid-based nanoparticles, namely cubosomes, to encapsulate daptomycin for targeted delivery via lipid-mediated interactions. Daptomycin-loaded cubosomes synergistically killed 14 clinical MRSA isolates in vitro compared with daptomycin or cubosomes alone. This synergy between daptomycin and cubosome was mediated by cubosomes docking on the S. aureus cell surface, releasing daptomycin for membrane extraction and penetration, followed by lipid cubosome infusion into S. aureus membranes. Using a murine septicemia model, daptomycin-loaded cubosomes significantly reduced the organ bacterial burden of MRSA. Together, these data showed that multifunctional lipid nanocarriers can potentiate the bactericidal activity of daptomycin using a membrane-targeted trojan-horse-like mechanism.

The Alfred Hospital in Melbourne, Australia, has reported an ongoing outbreak of infections caused by multidrug-resistant Enterobacter cloacae complex (ECC). Phage therapy is a promising strategy to treat antimicrobial-resistant infections. Utilizing the hospital’s isolate collection, built over the past decade, we established an initial 3-phage cocktail with 54% ECC coverage. We then iteratively improved this product by enhancing phage killing efficiency using phage adaptation and expanded host range through targeted phage isolation against low-coverage ECC isolates. This optimization yielded Entelli-02, containing five well-characterized virulent phages that target clinical ECC isolates via distinct bacterial cell surface receptors. Entelli-02 exhibits 88% host coverage against The Alfred Hospital’s ECC isolate collection ( n = 206), confirmed by plaque formation and reduced bacterial load in septicaemic mice by >99%. We produced this cocktail as a therapeutic-grade product, ready for clinical use. Entelli-02 represents a hospital-specific phage cocktail with frontline efficacy and on-demand availability.

Objectives: Ventricular assist device driveline infections are difficult-to-treat diseases due to high antimicrobial resistance (AMR) of microbial biofilms. Multiple mechanisms of biofilm AMR have been proposed based on simplified in vitro models and demonstrated limited clinical implications. This study aimed to re-evaluate mechanisms contributing to staphylococcal biofilm AMR encountered in driveline infections and identify translatable therapeutic targets. Methods: A tunnel-based biofilm assay mimicking subcutaneous driveline infections was utilized to grow clinically relevant biofilms. Seven first-line antibiotics were investigated against biofilms formed by two staphylococcal laboratory reference strains. The importance of various biofilm AMR mechanisms was evaluated, including the barrier effect of the biofilm extracellular polymeric substance (EPS) matrix, high-cell-density growth, the presence of persister cells and repressed bacterial metabolism. Results: The presence of the EPS matrix couldn’t decisively explain staphylococcal biofilm AMR in driveline infections. High-density growth and repressed bacterial metabolism appeared to be vital for biofilm AMR. Parallel comparisons of bacterial metabolic states and AMR at different biofilm developmental stages suggested a strong and negative correlation. Population profiling of tunnel-based biofilm cells identified subpopulations with low metabolic activity. Enhancing ATP production of biofilm cells partially restored their susceptibility to antibiotics. Local application of the weakly metabolism-dependent antibiotic gentamicin, but not the strongly metabolism-dependent vancomycin successfully killed staphylococcal biofilms in the simulated driveline tunnel, further underscoring the central role of bacterial metabolic repression in biofilm AMR. Conclusions: This study identified repressed bacterial metabolism as a central mechanism underpinning biofilm AMR and provided an effective therapeutic target for biofilm-associated driveline infections.

Background: Pseudomonas aeruginosa biofilms limit the efficacy of currently available antibacterial therapies and pose significant clinical challenges. Pseudomonal biofilms are complicated further when other markers of persistence such as mucoid and hypermutable phenotypes are present. There is currently a paucity of data regarding the activity of the newer β-lactam/β-lactamase inhibitor combination ceftolozane/tazobactam against P. aeruginosa biofilms. Methods: We evaluated the efficacy of ceftolozane/tazobactam against clinical P. aeruginosa isolates, the laboratory isolate PAO1 and its isogenic mutS-deficient hypermutator derivative (PAOMS) grown under static and dynamic biofilm conditions. The clinical isolate collection included strains with mucoid and hypermutable phenotypes. Results: Ceftolozane/tazobactam exposure led to a bactericidal (≥3 log cfu/cm2) biofilm reduction in 15/18 (83%) clinical isolates grown under static conditions, irrespective of carbapenem susceptibility or mucoid phenotype, with greater activity compared with colistin (P < 0.05). Dynamically grown biofilms were less susceptible to ceftolozane/tazobactam with active biofilm reduction (≥1 log cfu/cm2) observed in 2/3 isolates. Hypermutability did not affect the antibiofilm efficacy of ceftolozane/tazobactam in either static or dynamic conditions when comparing PAO1 and PAOMS. Consistent with the activity of ceftolozane/tazobactam as a potent inhibitor of PBP3, dramatic impacts on P. aeruginosa morphology were observed. Conclusions: Our data demonstrate that ceftolozane/tazobactam has encouraging properties in the treatment of P. aeruginosa biofilm infections, and its activity is not diminished against mucoid or hypermutable variants at the timepoints examined.

Phage therapy is a promising strategy to treat antimicrobial-resistant infections. Currently, phage therapy applications span personalised treatments that are tailored for a given patient’s infection, through to the use of pre-established cocktails of virulent phages against clinically relevant pathogens. However, both approaches face challenges, with personalised phage therapy being time-consuming and requiring a phage match to a patient’s infection, while phage cocktails may not be effective against a patient’s specific strain. The Alfred Hospital in Melbourne, Australia has reported an ongoing outbreak of infections by the Enterobacter cloacae complex (ECC), a group of emerging multidrug-resistant pathogens responsible for considerable morbidity and mortality. Utilising the hospital’s strain collection, built over the last decade, we established an initial three-phage product with 54% ECC coverage that effectively reduced bacterial loads (>99%) in septicaemic mice. We then iteratively improved this product by enhancing phage killing efficiency using phage training and expanded host range through targeted phage isolation against low-coverage ECC strains. This iterative optimisation led to the creation of the product Entelli-02 , containing five well characterised virulent phages that target clinical ECC strains through distinct bacterial cell surface receptors. Importantly, Entelli-02 exhibits broad host coverage (99%) and efficacy (92%) against The Alfred Hospital’s ECC strain collection ( n = 156). We produced this as a therapeutic-grade product, verified and endotoxin unit compliant, ready for use. This approach integrated academic phage research with clinical insights to produce the phage product Entelli-02 as an institution-specific phage cocktail with frontline efficacy and on-demand availability. SUMMARY: In brief: We developed a phage product containing five phages with frontline potential to address infections caused by multidrug-resistant Enterobacter cloacae complex.