Biology subjects

Blaskovich, M. A. T.

Publications and source records attributed to Blaskovich, M. A. T..

8 recordsLinked to original sources

bioRxiv2026

Disruption of cAMP Signalling and RNA Regulation Confers Cefiderocol Resistance in Acinetobacter baumannii

Cefiderocol is a siderophore-conjugated cephalosporin used to treat severe multidrug-resistant Gram-negative infections. Its recent prioritisation for US national preparedness underscores its clinical importance, yet resistance can emerge despite apparent in vitro susceptibility. Here, we identify two regulatory routes to cefiderocol resistance in carbapenem-resistant Acinetobacter baumannii (CRAB): mutation of the cAMP-producing adenylate cyclase CavA and truncation of the glycine-rich RNA-binding domain of the post-transcriptional regulator Hfq. These variants occurred in contemporary clinical isolates, conferred resistance in a murine CRAB lung-infection model and imposed minimal fitness costs. In Escherichia coli, deletion of the CavA orthologue cyaA increased competitive fitness during sub-inhibitory cefiderocol exposure. Hfq-CLASH and integrated omics revealed Hfq-dependent regulation of Fur-associated iron homeostasis in A. baumannii. Under iron limitation, truncated-Hfq induced a defence-primed state that attenuated siderophore uptake and reduced cefiderocol activity. These findings identify bacterial regulatory state and iron availability as determinants of cefiderocol susceptibility, with implications for resistance surveillance and susceptibility testing.

microbiology↗

bioRxiv2026

Cellular heterogeneity in the response of Burkholderia thailandensis to antibiotics and phages in synthetic spatial refuges

Understanding the complex interactions between phage, antibiotics and bacteria is vital for the development of rationale phage-antibiotic therapies. These interactions have traditionally been investigated via bulk assays, thus masking eventual cellular heterogeneities and the impact of spatial refuges on phage and antibiotic efficacy. Using microfluidics-based time-lapse microscopy, we reveal large phenotypic heterogeneity in the response of Burkholderia thailandensis to both the bacteriophage {Phi}Bp-AMP1 and sub-inhibitory concentrations of two selected antibiotics ciprofloxacin and trimethoprim. We discover that simultaneous exposure to ciprofloxacin and phage decreases the size of individual B. thailandensis cells, whereas phage and trimethoprim together slow down cell doubling. Exposure to ciprofloxacin also causes extended phage lysis of B. thailandensis which is preceded by a distinguishable pause in cell elongation, whereas exposure to trimethoprim constrains lysis. As a consequence, exposure to ciprofloxacin further hinders the growth of B. thailandensis in the presence of phage, whereas exposure to trimethoprim enhances growth in the presence of phage. These findings help understand the effects of the interactions between antibiotics and phage on bacterial subpopulations in spatial refuges that are notoriously difficult to treat with antibiotics.

microbiology↗

bioRxiv2026

Prophage and metabolic determinants of Staphylococcus aureus survival to vancomycin identified via TraDIS screening

Reduced vancomycin susceptibility phenotypes in Staphylococcus aureus contribute to treatment failure, yet the genetic determinants of survival under inhibitory vancomycin exposure remain incompletely defined. We performed transposon directed insertion-site sequencing (TraDIS) on a methicillin resistant S. aureus (MRSA) ST398 mutant library following exposure to vancomycin at its minimum inhibitory concentration, identifying 52 genes whose disruption was associated with loss of population survival at inhibitory drug concentrations. Prophage associated loci were the largest functional group, spanning predicted structural and regulatory genes as well as multiple conserved hypothetical proteins. Targeted testing of defined transposon mutants in a USA300 background confirmed that disruption of selected loci impaired growth under vancomycin exposure. Our results highlight the contribution of diverse physiological processes, including metabolism, stress responses, and a prominent role for prophage-associated functions, rather than discrete resistance pathways. Together, these findings indicate that vancomycin tolerance is shaped by the general physiological state of the bacterial cell, including metabolic capacity and stress adaptation. ImportanceTreatment failure in Staphylococcus aureus infections often occurs in the absence of known antibiotic resistance determinants, suggesting that additional survival mechanisms influence therapeutic outcomes. In this study, we identify genetic determinants required for survival during inhibitory vancomycin exposure, revealing a broad role for metabolic functions, stress adaptation, and prophage-associated loci. The prominence of these diverse processes highlights that survival reflects global physiological adaptation rather than discrete resistance pathways. This insight underscores the need to consider cellular physiology and stress responses when developing strategies to prevent antibiotic tolerance and improve treatment efficacy.

microbiology↗

bioRxiv2024

Characterisation of in vitro resistance selection against second-/last-line antibiotics in methicillin-resistant Staphylococcus aureus

SYNOPSISO_ST_ABSBackgroundC_ST_ABSThe increasing occurrence of MRSA clinical isolates harbouring reduced susceptibility to mainstay antibiotics has escalated the use of second and last line antibiotics. Hence, it is critical to evaluate the likelihood of MRSA developing clinical resistance to these antibiotics. ObjectivesOur study sought to identify the rate in which MRSA develop resistance to vancomycin, daptomycin and linezolid in vitro and further determine the mechanisms underpinning resistance. MethodsMRSA was exposed to increasing concentrations of vancomycin, daptomycin, and linezolid for 20 days, with eight replicates for each antibiotic conducted in parallel. The resulting day 20 (D20) isolates were subjected to antimicrobial susceptibility testing, whole genome sequencing, autolysis assays, and growth curves to determine bacterial fitness. ResultsExposure to vancomycin or linezolid for 20 days resulted in a subtle two-fold increase in the MIC, whereas daptomycin exposure yielded daptomycin-nonsusceptible isolates with up to 16-fold MIC increase. The MIC increase was accompanied by variable changes in relative fitness and reduced resistance to autolysis in some isolates. D20 isolates harboured mutations in genes commonly associated with resistance to the respective antibiotics (e.g. walK for vancomycin, mprF and rpoB for daptomycin, rplC for linezolid), along with several previously unreported variants. Introduction of key mutations to these identified genes in the parental strain via allelic exchange confirmed their role in the development of resistance. ConclusionsIn vitro selection against vancomycin, daptomycin, or linezolid resulted in the acquisition of mutations similar to those correlated with clinical resistance, including the associated phenotypic alterations.

microbiology↗

bioRxiv2022

A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study mode-of-action and resistance mechanisms in bacteria

Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate mode-of-action studies, we synthesized a fluorescent analogue of the quaternary ammonium compound benzalkonium chloride, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against E. coli, S. aureus and P. aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. Leveraging these findings and NBD-DDAs fluorescent properties, we show that reduced cellular adsorption is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized in the cell envelope of E. coli, which is a primary target of BAC and other QACs. Overall, NBD-DDAs antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study the mode-of-action and the resistance mechanisms of QACs in bacteria.

microbiology↗

bioRxiv2022

Loss of β-ketoacyl acyl carrier protein synthase III activity restores multidrug-resistant Escherichia coli sensitivity to previously ineffective antibiotics

Antibiotic resistance is one of the most prominent threats to modern medicine. In the latest World Health Organization list of bacterial pathogens that urgently require new antibiotics, nine out of 12 are Gram-negative, with four being of Critical Priority. One crucial barrier restricting antibiotic efficacy against Gram-negative bacteria is their unique cell envelope. While fatty acids are a shared constituent of all structural membrane lipids, their biosynthesis pathway in bacteria is distinct from eukaryotes making it an attractive target for new antibiotic development that remains less explored. Here, we interrogated the redundant components of the bacterial Type II Fatty Acid Synthesis (FAS II) pathway, showing that disrupting FAS II homeostasis in Escherichia coli through deletion of the fabH gene damages the cell envelope of antibiotic susceptible and antibiotic resistant clinical isolates. The fabH gene encodes the {beta}-ketoacyl acyl carrier protein synthase III (KAS III), which catalyzes the initial condensation reactions during fatty acid biosynthesis. We show that fabH null mutation potentiated the killing of multi-drug resistant E. coli by a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants, for example, carbapenemase kpc2. Enhanced antibiotic sensitivity was additionally demonstrated in the context of eradicating established biofilms and treating established human cell infection in vitro. Our findings showcase the potential of FabH as a promising target that could be further explored in the development of therapies that may repurpose currently ineffective antibiotics or rescue failing last-resort antibiotics against Gram-negative pathogens. IMPORTANCEGram-negative pathogens are a major concern for global public health due to increasing rates of antibiotic resistance and the lack of new drugs. A major contributing factor towards antibiotic resistance in Gram-negative bacteria is their formidable outer membrane, which acts as a permeability barrier preventing many biologically active antimicrobials from reaching the intracellular targets and thus limiting their efficacy. Fatty acids are the fundamental building blocks of structural membrane lipids and their synthesis constitutes an attractive antimicrobial target as it follows distinct pathways in prokaryotes and eukaryotes. Herein, we identified a component of fatty acid synthesis, FabH, as a gate-keeper of outer membrane barrier function. Without FabH, Gram-negative bacteria become susceptible to otherwise impermeable antibiotics and are re-sensitised to killing by last-resort antibiotics. This study supports FabH as a promising target for inhibition in future antimicrobial therapies.

microbiology↗

bioRxiv2021

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 [A] resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

biophysics↗

bioRxiv2017

Multifactorial Chromosomal Variants Regulate Polymyxin Resistance In Extensively Drug-Resistant Klebsiella pneumoniae

Extensively drug-resistant Klebsiella pneumoniae (XDR-KP) infections cause high mortality and are disseminating globally. Identifying the genetic basis underpinning resistance allows for rapid diagnosis and treatment. XDR isolates sourced from Greece and Brazil, including nineteen polymyxin-resistant and five polymyxin-susceptible strains, underwent whole genome sequencing. Approximately 90% of polymyxin resistance was enabled by alterations upstream or within mgrB. The most common mutation identified was an insertion at nucleotide position 75 in mgrB via an ISKpn26-like element in the ST258 lineage and ISKpn13 in one ST11 isolate. Three strains acquired an IS1 element upstream of mgrB and another strain had an ISKpn25 insertion at 133 bp. Other isolates had truncations (C28STOP, Q30STOP) or a missense mutation (D31E) affecting mgrB. Complementation assays revealed all mgrB perturbations contributed to resistance. Missense mutations in phoQ (T281M, G385C) were also found to facilitate resistance. Several variants in phoPQ co-segregating with the ISKpn26-like insertion were identified as potential partial suppressor mutations. Three ST258 samples were found to contain subpopulations with different resistance conferring mutations, including the ISKpn26-like insertion colonising with a novel mutation in pmrB (P158R), both confirmed via complementation assays. We also characterized a new multi-drug resistant Klebsiella quasipneumoniae strain ST2401 which was susceptible to polymyxins. These findings highlight the broad spectrum of chromosomal modifications which can facilitate and regulate resistance against polymyxins in K. pneumoniae.\n\nDATA SUMMARYO_LIWhole genome sequencing of the 24 clinical isolates has been deposited under BioProject PRJNA307517 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA307517).\nC_LI\n\nIMPACT STATEMENTKlebsiella pneumoniae contributes to a high abundance of nosocomial infections and the rapid emergence of antimicrobial resistance hinders treatment. Polymyxins are predominantly utilized to treat multidrug-resistant infections, however, resistance to the polymyxins is arising. This increasing prevalence in polymyxin resistance is evident especially in Greece and Brazil. Identifying the genomic variations conferring resistance in clinical isolates from these regions assists with potentially detecting novel alterations and tracing the spread of particular strains. This study commonly found mutations in the gene mgrB, the negative regulator of PhoPQ, known to cause resistance in KP. In the remaining isolates, missense mutations in phoQ were accountable for resistance. Multiple novel mutations were detected to be segregating with mgrB perturbations. This was either due to a mixed heterogeneous sample of two polymyxin-resistant strains, or because of multiple mutations within the same strain. Of interest was the validation of novel mutations inphoPQ segregating with a previously known ISKpn26-like element in disrupted mgrB isolates. Complementation of these phoPQ mutations revealed a reduction in minimum inhibitory concentrations and suggests the first evidence of partial suppressor mutations in KP. This research builds upon our current understanding of heteroresistance, lineage specific mutations and regulatory variations relating to polymyxin resistance.

microbiology↗