Biology subjects

Miller, W. R.

Publications and source records attributed to Miller, W. R..

11 recordsLinked to original sources

bioRxiv2025

Loss of the Ferripyochelin Receptor FptA Drives Reduced Cefiderocol Susceptibility and Impairs Fitness in Pseudomonas aeruginosa PA14

Pseudomonas aeruginosa is an opportunistic human pathogen and a frequent cause of multidrug-resistant infections. This organism continues to evade antimicrobial therapy despite the clinical introduction of new anti-pseudomonal antibiotics over the past several years. One of these agents is cefiderocol (FDC), a novel siderophore-cephalosporin conjugate antibiotic that was designed to overcome both intrinsic and acquired {beta}-lactam resistance mechanisms in P. aeruginosa. However, studies have demonstrated that inactivation of TonB-dependent receptors, most notably the catechol siderophore receptor piuA can substantially curtail the drugs ability to permeate the bacterial outer membrane, leading to rapid development of resistance. In this study, we examined the FDC resistance mechanisms of the laboratory strain PA14. We demonstrated that inactivation of the ferripyochelin receptor FptA was a first-step mutation towards FDC resistance. Through transposon mutagenesis, we identified several resistance pathways following fptA inactivation, such as the loss of an additional FDC import porin and overexpression of the MuxABC-OpmB multidrug efflux system. Introduction of clinically-identified mutations analogous to these transposon insertions in the absence of fptA conferred full FDC non-susceptibility while preserving the activity of other antipseudomonal {beta}-lactam antibiotics. We also demonstrated that inactivation of fptA in a pyoverdine biosynthetic mutant disrupted bacterial iron homeostasis and conferred a fitness disadvantage. These FDC resistance mechanisms identified in PA14 highlight the long-term challenges of using FDC treatment for drug-resistant P. aeruginosa infections.

microbiology↗

bioRxiv2025

LiaS-dependent activation of the MadR regulon enables cross-talk between Enterococcus faecalis cell envelope defense systems

Enterococci are gastrointestinal commensals that must defend their cell envelope against antimicrobial peptides derived from the host and other members of the microbiota. The signaling systems LiaFSR and MadRS are pivotal for survival in the presence of antimicrobial peptides and antimicrobial peptide-like antibiotics such as daptomycin. Both systems possess a signaling histidine kinase (LiaS, MadS) and cognate response regulator (LiaR, MadR) that activate transcription of distinct sets of effector genes. Using isogenic deletion strains, we noted differences in daptomycin minimum inhibitory concentration (MIC) between the laboratory strain E. faecalis OG1RF (1.5 {micro}g/mL), OG1RF{Delta}madR (0.38 {micro}g/mL), and OG1RF{Delta}madS (4 {micro}g/mL). Transcriptional analysis of the MadR regulon showed a daptomycin-dependent increase in madG, madL, and dltA gene expression in the OG1RF{Delta}madS background, suggesting activation of the LiaFSR system may provide a cross-regulatory role. Deletion of the liaS gene in OG117{Delta}madS was associated with a significant decrease in daptomycin MIC and loss of madG expression, one of the most differentially expressed genes on activation of the MadR regulon. Using microscale thermophoresis, LiaS showed a similar binding affinity to both LiaR (Kd 2.42 {micro}M) and MadR (Kd 5.02 {micro}M), while MadS showed higher affinity for its cognate regulator MadR (Kd 8.08 {micro}M) than for LiaR (Kd 25.7 {micro}M). Taken together, our findings indicate that the MadR regulon can be expressed independent of MadS-induced signaling, likely through cross-talk between LiaS and MadR. Thus, enterococci have evolved an interconnected network of cell envelope signaling that permits bacterial survival in the presence of antibiotics and antimicrobial peptides.

microbiology↗

bioRxiv2025

Clinical and Genomic Characterization of Recalcitrant Enterococcal Bacteremia: A Multicenter Prospective Cohort Study (VENOUS)

BackgroundPatients with recalcitrant enterococcal bloodstream infections are at greater risk of adverse outcomes. We identified patients in the 2016-2022 Vancomycin-Resistant Enterococcal Bacteremia Outcomes Study (VENOUS) cohort experiencing recalcitrant bloodstream infections for further clinical and genomic characterization. MethodsBacteremia episodes were considered "persistent" if there was a lack of clearance on day four while receiving [≥] 48 hours of active therapy and recurrent if there was clearance during hospitalization with a subsequent positive culture (collectively, "recalcitrant" bacteremia). A matched comparison group of non-recalcitrant bacteremia patients was chosen in a 2:1 control:case ratio. Isolates were subjected to short- and long-read whole-genome sequencing. Hybrid assemblies were created using a custom pipeline. Findings. A total of 46 recalcitrant infections from 41 patients were identified. Patients with persistent bacteremia were more often admitted to the ICU upon admission relative to controls. E. faecalis strains causing persistent infections had a significantly higher proportion of genes associated with carbohydrate utilization relative to controls. Representation of functional groups associated with mutated genes was disparate between E. faecium and E. faecalis index and persistent isolates, suggesting species-specific adaptation. DiscussionEnterococcal isolates causing recalcitrant bacteremia were genomically diverse, indicating that strain-specific signatures are not drivers of persistence. However, comparisons of index vs. persistent isolates revealed that E. faecium may be genetically pre-adapted to cause persistent infection, and site-specific structural variation during infection suggests the role of differential gene expression in adaptation and persistence. This data lays groundwork for future studies to define signatures of enterococcal adaptation during bacteremia.

genomics↗

bioRxiv2024

Cefiderocol heteroresistance associated with mutations in TonB-dependent receptor genes in Pseudomonas aeruginosa of clinical origin

The siderophore-cephalosporin cefiderocol(FDC) presents a promising treatment option for carbapenem-resistant (CR) P. aeruginosa (PA). FDC circumvents traditional porin and efflux mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of P. aeruginosa and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of pirR, pirS, pirA, piuA or piuD from 498 unique isolates collected before the introduction of FDC from 4 clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n=15). Using population analysis profile testing, we found that P. aeruginosa with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.

microbiology↗

bioRxiv2023

Membrane Lipids Augment Cell Envelope Stress Signaling and Resistance to Antibiotics and Antimicrobial Peptides in Enterococcus faecalis

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

microbiology↗

bioRxiv2023

LiaX is a surrogate marker for cell-envelope stress and daptomycin non-susceptibility in Enterococcus faecium

Daptomycin (DAP) is often used as a first line therapy to treat vancomycin-resistant Enterococcus faecium (VREfm) infections but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP MICs have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium (Efm) with an activated LiaFSR system. Unlike E. faecalis, Efm LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX ELISA. We then assessed 86 clinical E. faecium BSI isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-R clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-S isolates by standard MIC determination had elevated LiaX ELISAs above the established cut-off. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many Efm isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

microbiology↗

bioRxiv2023

Molecular Basis of Cell Membrane Adaptation in Daptomycin-Resistant Enterococcus faecalis

Daptomycin is a last-resort lipopeptide antibiotic that disrupts cell membrane (CM) and peptidoglycan homeostasis. Enterococcus faecalis has developed a sophisticated mechanism to avoid daptomycin killing by re-distributing CM anionic phospholipids away from the septum. The CM changes are orchestrated by a three-component regulatory system, designated LiaFSR, with a possible contribution of cardiolipin synthase (Cls). However, the mechanism by which LiaFSR controls the CM response and the role of Cls are unknown. Here, we show that cardiolipin synthase activity is essential for anionic phospholipid redistribution and daptomycin resistance since deletion of the two genes (cls1 and cls2) encoding Cls abolished CM remodeling. We identified LiaY, a transmembrane protein regulated by LiaFSR, as an important mediator of CM remodeling required for re-distribution of anionic phospholipid microdomains via interactions with Cls1. Together, our insights provide a mechanistic framework on the enterococcal response to cell envelope antibiotics that could be exploited therapeutically.

microbiology↗

bioRxiv2021

Evolution of Enterococcus faecium to A Combination of Daptomycin and Fosfomycin Reveals Distinct and Diverse Adaptive Strategies

Infections caused by vancomycin-resistant Enterococcus faecium (VREfm) are an important public health threat. VREfm have become increasingly resistant to the front-line antibiotic, daptomycin (DAP). As such, the use of DAP combination therapies (like fosfomycin [FOS]), has received increased attention. Antibiotic combinations could extend the efficacy of current available antibiotics and potentially delay the onset of further resistance. We investigated the potential for E. faecium HOU503, a clinical VREfm isolate that is DAP and FOS susceptible, to develop resistance to a DAP-FOS combination. Of particular interest was whether the genetic drivers for DAP-FOS resistance might be epistatic and, thus, potentially decrease the efficacy of a combinatorial approach in either inhibiting VREfm or in delaying the onset of resistance. We show that resistance to DAP-FOS could be achieved by independent mutations to proteins responsible for cell wall synthesis for FOS and in altering membrane dynamics for DAP. However, we did not observe genetic drivers that exhibited substantial cross-drug epistasis that could undermine DAP-FOS combination. Of interest was that FOS resistance in HOU503 was largely mediated by changes in phosphoenolpyruvate (PEP) flux as a result of mutations in pyruvate kinase (pyk). Increasing PEP flux could be a readily accessible mechanism for FOS resistance in many pathogens. Importantly, we show that HOU503 were able to develop DAP resistance through a variety of biochemical mechanisms and were able to employ different adaptive strategies. Finally, we showed that the addition of FOS can prolong the efficacy of DAP, significantly extending the timeline to resistance in vitro. ImportanceWhile the discovery of antibiotics was one of the greatest health care advances in history, its success is being challenged by the emergence of multidrug-resistant pathogens, including vancomycin-resistant enterococci (VRE). Daptomycin (DAP), a lipopeptide antibiotic that targets cell membrane, is currently prescribed as a frontline drug to treat VRE infections. However, emergence of daptomycin-resistant VRE is concerning. Consequently, DAP-Fosfomycin (FOS) combination (DF) has been proposed as a potential method to maintain DAP efficacy. Here, we provide evidence that DF successfully delayed the emergence of resistance in vitro. Genetic data indicates that resistance was acquired independently, with little evidence of significant cross-drug epistasis that could undermine a combinatorial approach. We also uncovered a novel FOS resistance mechanism, through changes in phosphoenolpyruvate (PEP) flux, that may potentially be shared with other bacterial species. Additionally, we also have evidence showing that E. faecium was able to employ different resistance mechanisms.

evolutionary biology↗

bioRxiv2020

Daptomycin resistance in Enterococcus faecium can be delayed by disruption of the LiaFSR stress response pathway

The LiaFSR signaling pathway plays a major role in mediating daptomycin (DAP) resistance for both Enterococcus faecalis and Enterococcus faecium. LiaFSR inhibition induces DAP hypersusceptibility but could also potentially delay the acquisition of DAP resistance in a combinatorial therapy of DAP with a LiaFSR inhibitor. To evaluate the potential efficacy of this approach, the adaptation to DAP by both E. faecalis and E. faecium lacking a functional LiaFSR were examined. Here, clinical isolates of E. faecium with liaR deletions were evolved to DAP resistance using in vitro experimental evolution. Genomic analysis of resistant populations was used to identify both the alleles and their relative frequencies in driving DAP resistance. Microscopic and biochemical analyses were then employed to investigate how those adaptive alleles contributed to DAP resistance. We found that deletion of liaR from the E. faecium genome significantly delayed the onset of DAP resistance. Unsurprisingly, resistance strategies emerged eventually. These alternative strategies were influenced by both environment and ancestral genome. The delay in the acquisition of DAP resistance when liaR was deleted supports the concept of developing a LiaFSR pathway inhibitor to prolong DAP efficacy against enterococci. The loss of a functional LiaFSR pathway reset the adaptive landscape and forced adaptation to progress in new ways that were slower in providing DAP tolerance. The observed adaptive trajectories were strongly influenced by both the environment and ancestral genome.

microbiology↗

bioRxiv2019

Environment Shapes the Accessible Daptomycin Resistance Mechanisms in Enterococcus faecium

Daptomycin binds to bacterial cell membranes and disrupts essential cell envelope processes leading to cell death. Bacteria respond to daptomycin by altering their cell envelopes to either decrease antibiotic binding to the membrane or by diverting binding away from vulnerable septal targets to remodeled anionic phospholipid membrane patches. In Enterococcus faecalis, daptomycin resistance is typically coordinated by the three-component cell-envelope-stress-response system, LiaFSR. Here, studying a clinical strain of multidrug-resistant Enterococcus faecium containing alleles associated with activation of the LiaFSR signaling pathway, we found that specific environments selected for different evolutionary trajectories leading to high-level daptomycin resistance. Planktonic environments favored pathways that increased cell surface charge via yvcRS upregulation of dltABCD and mprF, causing a reduction in daptomycin binding. Alternatively, environments favoring complex structured communities, including biofilms, evolved both diversion and repulsion strategies via divIVA and oatA mutations, respectively. Both environments subsequently converged on cardiolipin synthase (cls) mutations, suggesting the importance of membrane modification across strategies. Our findings indicate that E. faecium can evolve diverse evolutionary trajectories to daptomycin resistance that are shaped by the environment to produce a combination of resistance strategies. The accessibility of multiple and different biochemical pathways simultaneously suggests that the outcome of daptomycin exposure results in a polymorphic population of resistant phenotypes making E. faecium a recalcitrant pathogen.

evolutionary biology↗

bioRxiv2019

Extremely Drug-Resistant Pseudomonas aeruginosa ST309 Harboring Tandem GES Enzymes: A Newly Emerging Threat in the United States

Two ST309 Pseudomonas aeruginosa clinical isolates resistant to carbapenems and newer {beta}-lactam/{beta}-lactamase inhibitor combinations were found to harbor the extended spectrum beta-lactamases GES 19 and 26 genetically clustered in tandem. The combination of ceftazidime/avibactam plus aztreonam achieved cure in one patient. Phylogenetic analysis suggests that ST309 P. aeruginosa carrying tandem GES are an emerging lineage in the United States.

microbiology↗