Biology subjects

Shelburne, S. A.

Publications and source records attributed to Shelburne, S. A..

11 recordsLinked to original sources

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

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure

BackgroundIt remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to becoming carbapenem resistant. MethodsCarbapenem mutation frequency was measured in multiple subclades of extended-spectrum {beta}-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/H30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation. ResultsAll thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P-value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) encoding genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing blaCTX-M-15 copy numbers via modular, insertion sequence 26 (IS26) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. ConclusionsESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/H30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

microbiology↗

bioRxiv2024

Emergent emm4 group A Streptococcus evidences a survival strategy during interaction with immune effector cells

The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence amongst emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. Through the creation and analysis of isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates relative to the historic strains. Via creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found high ratio of mucosal (i.e., pharyngeal) relative to invasive infections amongst emm4 GAS. Inasmuch as ever-increasing virulence is unlikely to be evolutionary advantageous for a microbial pathogen, our data furthers understanding of the well described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.

microbiology↗

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

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

Genetic Determinants Underlying the Progressive Phenotype of Beta-lactam/Beta-lactamase Inhibitor Resistance in Escherichia coli

Currently, whole genome sequencing (WGS) data has not shown strong concordance with E. coli susceptibility profiles to the commonly used {beta}-lactam/{beta}-lactamase inhibitor (BL/BLI) combinations: ampicillin-sulbactam (SAM), amoxicillin-clavulanate (AMC), and piperacillin-tazobactam (TZP). Progressive resistance to these BL/BLIs in absence of cephalosporin resistance, also known as extended-spectrum resistance to BL/BLI (ESRI), has been suggested to primarily result from increased copy numbers of blaTEM variants, which is not routinely assessed in WGS data. We sought to determine whether addition of gene amplification could improve genotype-phenotype associations through WGS analysis of 147 E. coli bacteremia isolates with increasing categories of BL/BLI non-susceptibility ranging from ampicillin-susceptible to fully resistant to all three BL/BLIs. Consistent with a key role of blaTEM in ESRI, 112/134 strains (84%) with at least ampicillin non-susceptibility encoded blaTEM. Evidence of blaTEM amplification (i.e., blaTEM gene copy number estimates > 2x) was present in 40/112 (36%) strains. There were positive correlations between blaTEM copy numbers with minimum inhibitory concentrations (MICs) of AMC and TZP (P-value < 0.05), but not for SAM (P-value = 0.09). The diversity of {beta}-lactam resistance mechanisms, including non-ceftriaxone hydrolyzing blaCTX-M variants, blaOXA-1, as well as ampC and blaTEM strong promoter mutations, were greater in AMC and TZP non-susceptible strains but rarely observed within SAM and AMP non-susceptible isolates. Our study indicates a comprehensive analysis of WGS data, including {beta}-lactamase encoding gene amplification, can help categorize E. coli with AMC or TZP non-susceptibility but that discerning the transition from SAM susceptible to non-susceptible using genetic data requires further refinement. ImportanceThe increased feasibility of whole genome sequencing has generated significant interest in using such molecular diagnostic approaches to characterize difficult-to-treat, antimicrobial resistant (AMR) infections. Nevertheless, there are current limitations in the accurate prediction of AMR phenotypes based on existing AMR gene database approaches, which primarily correlate a phenotype with the presence/absence of a single AMR gene. Our study utilized a large cohort of cephalosporin-susceptible E. coli bacteremia samples to determine how increasing dosage of narrow-spectrum {beta}-lactamase encoding genes in conjunction with other diverse BL/BLI genetic determinants contribute to progressively more severe BL/BLI phenotypes. We were able to characterize the complexity of the genetic mechanisms underlying progressive BL/BLI resistance including the critical role of {beta}-lactamase encoding gene amplification. For the diverse array of AMR phenotypes with complex mechanisms involving multiple genomic factors, our study provides an example of how composite risk scores may improve understanding of AMR genotype/phenotype correlations.

genomics↗

bioRxiv2023

Dynamic Periodicity of Extended-Spectrum Cephalosporin Resistant Bloodstream Escherichia coli Infections Driven by Non-CC131 Strains

Extended-spectrum cephalosporin resistant Escherichia coli (ESC-R-Ec) is an urgent public health threat with sequence type clonal complex 131 (STc131), phylogroup B2 strains being particularly concerning as the dominant cause of ESC-R-Ec infections. To address the paucity of recent ESC-R-Ec molecular epidemiology data in the United States, we used whole genome sequencing (WGS) to fully characterize a large cohort of invasive ESC-R-Ec at a tertiary care cancer center in Houston, Texas collected from 2016-2020. During the study timeframe, there were 1154 index E. coli bloodstream infections (BSIs) of which 389 (33.7%) were ESC-R-Ec. Using time series analyses, we identified a temporal dynamic of ESC-R-Ec distinct from ESC-susceptible E. coli (ESC-S-Ec), with cases peaking in the last six months of the calendar year. WGS of 297 ESC-R-Ec strains revealed that while STc131 strains accounted for [~]45% of total BSIs, the proportion of STc131 strains remained stable across the study time frame with infection peaks driven by genetically heterogeneous ESC-R-Ec clonal complexes. BlaCTX-M variants accounted for most {beta}-lactamases conferring the ESC-R phenotype (89%; 220/248 index ESC-R-Ec), and amplification of blaCTX-M genes was widely detected in ESC-R-Ec strains, particularly in carbapenem non-susceptible, recurrent BSI strains. BlaCTX-M-55 was significantly enriched within phylogroup A strains, and we identified blaCTX-M-55 plasmid-to-chromosome transmission occurring across non-B2 strains. Our data provide important information regarding the current molecular epidemiology of invasive ESC-R-Ec infections at a large tertiary care cancer center and provide novel insights into the genetic basis of observed temporal variability for these clinically important pathogens. IMPORTANCEGiven that E. coli is the leading cause of worldwide ESC-R Enterobacterales infections, we sought to assess the current molecular epidemiology of ESC-R-Ec using a WGS analysis of many BSIs over a five-year period. We identified fluctuating temporal dynamics of ESC-R-Ec infections, which has also recently been identified in other geographical regions such as Israel. Our WGS data allowed us to visualize the stable nature of STc131 over the study period and demonstrate a limited, but genetically diverse group of ESC-R-Ec clonal complexes are detected during infection peaks. Additionally, we provide a widespread assessment of {beta}-lactamase gene copy number in ESC-R-Ec infections and delineate mechanisms by which such amplifications are achieved in a diverse array of ESC-R-Ec strains. These data suggest that serious ESC-R-Ec infections are driven by a diverse array of strains in our cohort and impacted by environmental factors suggesting that community-based monitoring could inform novel preventative measures.

microbiology↗

bioRxiv2023

High-level ceftazidime-avibactam resistance in Escherichia coli conferred by the novel plasmid-mediated beta-lactamase CMY-185 variant

ObjectivesTo characterize a blaCMY variant associated with ceftazidime-avibactam (CZA) resistance from a serially collected Escherichia coli isolate. MethodsA patient with an intra-abdominal infection due to recurrent E. coli was treated with CZA. On day 48 of CZA therapy, E. coli with a CZA MIC of >256 mg/L was identified from abdominal drainage. Illumina WGS was performed on all isolates to identify potential resistance mechanisms. Site-directed mutants of CMY {beta}-lactamase were constructed to identify amino acid residues responsible for CZA resistance. ResultsWGS revealed that all three isolates were E. coli ST410. The CZA-resistant strain uniquely acquired a novel CMY {beta}-lactamase gene, herein called blaCMY-185, harbored on an IncI{gamma}-type conjugative plasmid. The CMY-185 enzyme possessed four amino acid substitutions relative to CMY-2 including A114E, Q120K, V211S, and N346Y and conferred high-level CZA resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced CZA susceptibility. However, double and triple mutants containing N346Y previously associated with CZA resistance in other AmpC enzymes, conferred CZA MICs ranging between 4 and 32 mg/L as well as reduced susceptibility to the newly developed cephalosporin, cefiderocol. Molecular modelling suggested that the N346Y substitution confers the reduction of avibactam inhibition due to the steric hindrance between the side chain of Y346 and the sulfate group of avibactam. ConclusionWe identified CZA resistance in E. coli associated with a novel CMY variant. Unlike other AmpC enzymes, CMY-185 appears to require an additional substitution on top of N346Y to confer CZA resistance.

microbiology↗

bioRxiv2022

Systematic Analysis of Mobile Genetic Elements Mediating β-lactamase Gene Amplification in Non-Carbapenemase-Producing Carbapenem Resistant Enterobacterales Bloodstream Infections

Non-carbapenemase-producing carbapenem resistant Enterobacterales (non-CP-CRE) are increasingly recognized as important contributors to prevalent carbapenem resistant Enterobacterales (CRE) infections. However, there is limited understanding of mechanisms underlying non-CP-CRE causing invasive disease. Long- and short-read whole genome sequencing (WGS) was used to elucidate carbapenem non-susceptibility determinants in Enterobacterales bloodstream isolates at MD Anderson Cancer Center in Houston, Texas. We investigated carbapenem non-susceptible Enterobacterales (CNSE) mechanisms through a combination of phylogenetic analysis, antimicrobial resistant (AMR) gene detection/copy number quantification, porin assessment, and mobile genetic element (MGE) characterization. Most CNSE isolates sequenced were non-CP-CRE (41/79; 51.9%) whereas 25.3% (20/79) were carbapenem intermediate Enterobacterales (CIE) and 22.8% (18/79) were carbapenemase producing Enterobacterales (CPE). Statistically significant copy number variants (CNVs) of extended-spectrum {beta}-lactamase (ESBL) genes (Wilcoxon Test; p-value < 0.001) were present in both non-CP-CR E. coli (median CNV = 2.6X; n= 17) and K. pneumoniae (median CNV = 3.2X, n = 17). All non-CP-CR E. coli and K. pneumoniae had predicted reduced expression of at least one outer membrane porin gene (i.e., ompC/ompF or ompK36/ompK35). Completely resolved CNSE genomes revealed that IS26 and ISEcp1 structures harboring blaCTX-M variants along with other AMR elements were the primary drivers of gene amplification, occurring in mostly IncFIB/IncFII plasmid contexts. MGE mediated {beta}-lactamase gene amplifications resulted in either tandem arrays, primarily mediated by IS26 translocatable units, or segmental duplication, typically due to ISEcp1 transposition units. Non-CP-CRE strains were the most prevalent cause of CRE bacteremia with carbapenem non-susceptibility driven by concurrent porin loss and MGE-mediated amplification of blaCTX-M genes. IMPORTANCECarbapenem resistant Enterobacterales (CRE) are considered urgent antimicrobial resistance (AMR) threats. The vast majority of CRE research has focused on carbapenemase producing Enterobacterales (CPE) even though non-carbapenemase-producing CRE (non-CP-CRE) comprise 50% or more of isolates in some surveillance studies. Thus, carbapenem resistance mechanisms in non-CP-CRE remain poorly characterized. To address this problem, we applied a combination of short- and long-read sequencing technologies to a cohort of CRE bacteremia isolates and used these data to unravel complex mobile genetic element structures mediating {beta}- lactamase gene amplification. By generating complete genomes of 65 carbapenem non-susceptible Enterobacterales (CNSE) covering a genetically diverse array of isolates, our findings both generate novel insights into how non-CP-CRE overcome carbapenem treatments and provide researchers scaffolds for characterization of their own non-CP-CRE isolates. Improved recognition of mechanisms driving development of non-CP-CRE could assist with design and implementation of future strategies to mitigate the impact of these increasingly recognized AMR pathogens.

genomics↗

bioRxiv2019

VAMPr: VAriant Mapping and Prediction of antibiotic resistance via explainable features and machine learning

Antimicrobial resistance (AMR) is an increasing threat to public health. Current methods of determining AMR rely on inefficient phenotypic approaches, and there remains incomplete understanding of AMR mechanisms for many pathogen-antimicrobial combinations. Given the rapid, ongoing increase in availability of high density genomic data for a diverse array of bacteria, development of algorithms that could utilize genomic information to predict phenotype could both be useful clinically and assist with discovery of heretofore unrecognized AMR pathways. To facilitate understanding of the connections between DNA variation and phenotypic AMR, we developed a new bioinformatics tool, variant mapping and prediction of antibiotic resistance (VAMPr), to (1) derive gene ortholog-based sequence features for variants; (2) interrogate these explainable gene-level variants for their known or novel associations with AMR; and (3) build accurate models to predict AMR based on whole genome sequencing data. Following the Clinical & Laboratory Standards Institute (CLSI) guidelines, we curated the publicly available sequencing data for 3,393 bacterial isolates from 9 species along with AMR phenotypes for 29 antibiotics. We detected 14,615 variant genotypes and built 93 association and prediction models. The association models confirmed known genetic antibiotic resistance mechanisms, such as blaKPC and carbapenem resistance consistent with the accurate nature of our approach. The prediction models achieved high accuracies (mean accuracy of 91.1% for all antibiotic-pathogen combinations) internally through nested cross validation and were also validated using external clinical datasets. The VAMPr variant detection method, association and prediction models will be valuable tools for AMR research for basic scientists with potential for clinical applicability.

bioinformatics↗

bioRxiv2018

Identification of a Chimeric M4 Protein and Novel Emm Pattern in Currently Circulating Strains of Emm4 Group A Streptococcus

Group A Streptococcus (GAS) is classified by sequence of the gene encoding the M protein (emm) and the patterns into which emm types are grouped. We discovered a novel emm pattern in emm4 GAS, historically considered pattern E, arising from a fusion event between emm and the adjacent enn gene. We identified the emm/enn fusion event in 51/52 emm4 GAS strains isolated by national surveillance in 2015. GAS isolates with an emm/enn fusion event completely replaced pattern E emm4 strains over a 4-year span in Houston (2013-2017). The novel emm/enn gene fusion and new emm pattern has potential vaccine implications.

genomics↗