Biology subjects

Arias, C. A.

Publications and source records attributed to Arias, C. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Multi-lineage emergence of USA300 MRSA in in Chile and a potential role for environmental copper resistance

Methicillin-resistant S. aureus (MRSA) is prevalent worldwide, but individual strains of MRSA tend to spread and expand in relatively confined geographic areas. The USA300 lineage of MRSA is most well-known for its rapid epidemic spread in North America during the first decade of the 2000's leading to an overall increase in serious skin infections and the emergence of methicillin resistance in the majority of community acquired S. aureus infections. A distinct but closely related South American epidemic clade of MRSA USA300 (USA300-SAE), first reported in 2006, proliferated rapidly in Northern South America but was rarely found in other Latin American countries. In a new survey of 274 invasive MRSA isolates from 2022 collected in Chile, we saw a marked increase in USA300-SAE in Southern South America. We found that Chilean USA300-SAE is phylogenetically diverse and was likely introduced multiple times from multiple different South American countries over the course of more than a decade, but only recently became a highly prevalent clone. Using novel molecular diagnostic methods for isolates collected in 2023, we estimate that USA300-SAE made up more than 22% of invasive MRSA strains in Chile in that year and is likely still increasing. We show that copper-associated genes in the signature copper and mercury resistance mobile element (COMER) of USA300-SAE are strongly conserved, and that isogenic mutants of the copper resistance genes in COMER are highly susceptible to copper killing at concentrations seen in tap water. These findings suggest that environmental exposure to copper may provide a fitness advantage that enhances spread of this lineage.

microbiology↗

bioRxiv2026

Functional export of NDM-7 to outer membrane vesicles in Klebsiella pneumoniae compromises imipenem and cefiderocol activity

The carbapenemases KPC and NDM are the most widespread determinants of carbapenem resistance in Klebsiella pneumoniae. Whereas KPC is a soluble periplasmic serine-{beta}-lactamase, NDM is a membrane-anchored metallo-{beta}-lactamase (MBL), a feature that promotes its incorporation into outer membrane vesicles (OMVs). OMVs are naturally released nanoparticles that deliver diverse bioactive cargo, including enzymes, virulence factors, and signaling molecules, and may contribute to antibiotic resistance. Here, we investigated the export and activity of carbapenemases in OMVs produced by carbapenem-resistant Klebsiella pneumoniae clinical isolates expressing NDM-7, an emerging variant, or KPC-2, as well as in isogenic laboratory-derived K. pneumoniae strains producing NDM-1, NDM-7 or KPC-2. NDM enzymes were detected in vesicles released by NDM-producing strains, whereas KPC-2 remained confined to the cellular fraction and was not observed in OMVs. OMVs contained catalytically active NDM enzyme and conferred protection to susceptible K. pneumoniae against imipenem. Importantly, NDM-positive OMVs also partially restored bacterial growth in the presence of cefiderocol, a siderophore cephalosporin used to treat infections caused by MBL producers. This protective effect was more pronounced for NDM-7 than for NDM-1. Together, these findings show that the clinically emerging NDM-7 variant is efficiently packaged into OMVs in K. pneumoniae and remains enzymatically active, allowing extracellular antibiotic degradation and conferring protection to susceptible bacteria exposed to carbapenems and cefiderocol.

microbiology↗

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

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

Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing

Multiple Displacement Amplification (MDA) outperforms conventional PCR in long fragment and whole genome amplification which makes it attractive to couple with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for genome sequence assembly using Oxford Nanopore Technologies (ONT) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Cryptosporidium meleagridis, Staphylococcus aureus, Enterococcus faecium, and Escherichia coli, with the ability to generate high-quality data from samples starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size-increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.

bioinformatics↗

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↗

bioRxiv2023

Heavy Metal Pollution From a Major Earthquake and Tsunami in Chile Is Associated With Geographic Divergence of Clinical Isolates of Methicillin-Resistant Staphylococcus aureus in Latin America

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority pathogen listed by the World Health Organization. The global spread of MRSA is characterized by successive waves of epidemic clones that predominate in specific geographical regions. The acquisition of genes encoding resistance to heavy-metals is thought to be a key feature in the divergence and geographical spread of MRSA. Increasing evidence suggests that extreme natural events, such as earthquakes and tsunamis, could release heavy-metals into the environment. However, the impact of environmental exposition to heavy-metals on the divergence and spread of MRSA clones has been insufficiently explored. We assess the association between a major earthquake and tsunami in an industrialized port in southern Chile and MRSA clone divergence in Latin America. We performed a phylogenomic reconstruction of 113 MRSA clinical isolates from seven Latin American healthcare centers, including 25 isolates collected in a geographic area affected by an earthquake and tsunami that led to high levels of heavy-metal environmental contamination. We found a divergence event strongly associated with the presence of a plasmid harboring heavy-metal resistance genes in the isolates obtained in the area where the earthquake and tsunami occurred. Moreover, clinical isolates carrying this plasmid showed increased tolerance to mercury, arsenic, and cadmium. We also observed a physiological burden in the plasmid-carrying isolates in absence of heavy-metals. Our results are the first evidence that suggests that heavy-metal contamination, in the aftermath of an environmental disaster, appears to be a key evolutionary event for the spread and dissemination of MRSA in Latin America.

microbiology↗

bioRxiv2023

Dynamics of the MRSA Population in A Chilean Hospital: A Phylogenomic Analysis (2000-2016)

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in the last years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary healthcare center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000-2016 in a tertiary healthcare center in Chile. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r=0.8748, p<0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016. The temporal trend analysis revealed that in the period 2000-2003 most of the isolates (94.2%; n=98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013-2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage in Chile. However, this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. ImportanceMethicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or classical typing techniques with several limitations to depict an accurate description of their genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000-2016 in Chile to provide the largest and most detailed study of clonal dynamics of MRSA carried out in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing their frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America.

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↗

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↗

bioRxiv2021

Carbapenem-Resistant Acinetobacter baumannii in US hospitals: diversification of circulating lineages and antimicrobial resistance

Carbapenem-resistant Acinetobacter baumannii (CRAb) are a major cause of healthcare-associated infections. CRAb are typically multidrug-resistant and infection is difficult to treat. Despite the urgent threat that CRAb pose, few systematic studies of CRAb clinical and molecular epidemiology have been conducted. The Study Network of Acinetobacter as a Carbapenem-Resistant Pathogen (SNAP) is designed to investigate the clinical characteristics and contemporary population structure of CRAb circulating in US hospital systems using whole genome sequencing (WGS). Analysis of the initial 120 SNAP patients from four US centers revealed that CRAb remain a significant threat to hospitalized patients, affecting the most vulnerable patients and resulting in 24% all-cause 30-day mortality. The majority of currently circulating isolates belonged to ST2Pas, a part of Clonal Complex 2 (CC2), which is the dominant drug-resistant lineage in the United States and Europe. We identified three distinct sub-lineages within CC2, which differed in their antibiotic resistance phenotypes and geographic distribution. Most concerning, colistin resistance (38%) and cefiderocol (10%) resistance were common within CC2 sub-lineage C (CC2C), where the majority of isolates belonged to ST2Pas/ST281Ox. Additionally, we identified a newly emergent lineage, ST499Pas that was the most common non-CC2 lineage in our study and had a more favorable drug susceptibility profile compared to CC2. Our findings suggest a shift within the CRAb population in the US during the past 10 years, and emphasize the importance of real-time surveillance and molecular epidemiology in studying CRAb dissemination and clinical impact. ImportanceCarbapenem-resistant Acinetobacter baumannii (CRAb) constitute a major threat to public health. To elucidate the molecular and clinical epidemiology of CRAb in the US, clinical CRAb isolates were collected along with data on patient characteristics and outcomes and bacterial isolates underwent whole genome sequencing and antibiotic susceptibility phenotyping. Key findings included emergence of new sub-lineages within the globally predominant clonal complex (CC) 2, increased colistin and cefiderocol resistance within one of the CC2 sub-lineages, and the emergence of ST499Pas as a previously unrecognized CRAb lineage in US hospitals.

microbiology↗

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

Genomic epidemiology of vancomycin 1 resistant Enterococcus faecium (VREfm) in Latin America: Revisiting the global VRE population structure

The prevalence of vancomycin-resistant Enterococcus faecium varies across geographical regions yet little is known about its population structure in Latin America. Here, we provide a complete genomic characterization of 55 representative Latin American VREfm recovered from 1998-2015 in 5 countries. We found that VREfm population in the region is structured into two main clinical clades without geographical clustering. To place our regional findings in context, we reconstructed the global population structure of VREfm by including 285 genomes from 36 countries from 1946-2017. Our results differ from previous studies showing an early branching of animal related isolates and a further split of clinical isolates into two sub-clades, all within clade A. The overall phylogenomic structure was highly dependent on recombination (54% of the genome) and the split between clades A and B is estimated to have occurred more than 3585 years BP. Furthermore, while the branching of animal isolates and clinical clades was predicted to have occur [~]894 years BP, our molecular clock calculations suggest that the split within the clinical clade occurred around [~]371 years BP. By including isolates from Latin America, we present novel insights into the population structure of VREfm and revisit the evolution of this pathogen.

microbiology↗