MUNITA SEPULVEDA, JOSE MANUEL
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MUNITA SEPULVEDA, JOSE MANUEL
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josemunita@udd.cl
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24825037700

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2020 2020 2019 2019 2018 2018 2017 2017 2016 2016 2015 2015 2014 2014 2013 2013 2012 2012 0.0 0.0 1.0 1.0 2.0 2.0 3.0 3.0 4.0 4.0 5.0 5.0 6.0 6.0

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Dissecting the Mechanisms of Linezolid Resistance in a Drosophila melanogaster Infection Model of Staphylococcus aureus

2013 , Lorena Diaz , Dimitrios P. Kontoyiannis , Diana Panesso , Nathaniel D. Albert , Kavindra V. Singh , Truc T. Tran , MUNITA SEPULVEDA, JOSE MANUEL , Barbara E. Murray , Cesar A. Arias

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Whole-Genome Analyses of Enterococcus faecium Isolates with Diverse Daptomycin MICs

2014 , Lorena Diaz , Truc T. Tran , MUNITA SEPULVEDA, JOSE MANUEL , William R. Miller , Sandra Rincon , Lina P. Carvajal , Aye Wollam , Jinnethe Reyes , Diana Panesso , Natalia L. Rojas , Yousif Shamoo , Barbara E. Murray , George M. Weinstock , Cesar A. Arias

ABSTRACT Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a “last-resort” antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium . Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium , we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 μg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120→Ala and Trp73→Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.

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What's New in the Treatment of Enterococcal Endocarditis?

2014 , Masayuki Nigo , MUNITA SEPULVEDA, JOSE MANUEL , Cesar A. Arias , Barbara E. Murray

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Transferable Vancomycin Resistance in a Community-Associated MRSA Lineage

2014 , Flávia Rossi , Lorena Diaz , Aye Wollam , Diana Panesso , Yanjiao Zhou , Sandra Rincon , Apurva Narechania , Galen Xing , Thais S.R. Di Gioia , André Doi , Truc T. Tran , Jinnethe Reyes , MUNITA SEPULVEDA, JOSE MANUEL , Lina P. Carvajal , Alejandra Hernandez-Roldan , Denise Brandão , Inneke Marie van der Heijden , Barbara E. Murray , Paul J. Planet , George M. Weinstock , Cesar A. Arias

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Daptomycin-Resistant Enterococcus faecalis Diverts the Antibiotic Molecule from the Division Septum and Remodels Cell Membrane Phospholipids

2013 , Truc T. Tran , Diana Panesso , Nagendra N. Mishra , Eugenia Mileykovskaya , Ziqianq Guan , MUNITA SEPULVEDA, JOSE MANUEL , Jinnethe Reyes , Lorena Diaz , George M. Weinstock , Barbara E. Murray , Yousif Shamoo , William Dowhan , Arnold S. Bayer , Cesar A. Arias , Steven J. Projan

ABSTRACT Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides. IMPORTANCE The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.

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Whole-Genome Analysis of a Daptomycin-Susceptible Enterococcus faecium Strain and Its Daptomycin-Resistant Variant Arising during Therapy

2013 , Truc T. Tran , Diana Panesso , Hongyu Gao , Jung H. Roh , MUNITA SEPULVEDA, JOSE MANUEL , Jinnethe Reyes , Lorena Diaz , Elizabeth A. Lobos , Yousif Shamoo , Nagendra N. Mishra , Arnold S. Bayer , Barbara E. Murray , George M. Weinstock , Cesar A. Arias

Development of daptomycin (DAP) resistance in Enterococcus faecalis has recently been associated with mutations in genes encoding proteins with two main functions: (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase [cls]). However, the genetic bases for DAP resistance in Enterococcus faecium are unclear. We performed whole-genome comparative analysis of a clinical strain pair, DAP-susceptible E. faecium S447 and its DAP-resistant derivative R446, which was recovered from a single patient during DAP therapy. By comparative whole-genome sequencing, DAP resistance in R446 was associated with changes in 8 genes. Two of these genes encoded proteins involved in phospholipid metabolism: (i) an R218Q substitution in Cls and (ii) an A292G reversion in a putative cyclopropane fatty acid synthase enzyme. The DAP-resistant derivative R446 also exhibited an S333L substitution in the putative histidine kinase YycG, a member of the YycFG system, which, similar to LiaFSR, has been involved in cell envelope homeostasis and DAP resistance in other Gram-positive cocci. Additional changes identified in E. faecium R446 (DAP resistant) included two putative proteins involved in transport (one for carbohydrate and one for sulfate) and three enzymes predicted to play a role in general metabolism. Exchange of the "susceptible" cls allele from S447 for the "resistant" one belonging to R446 did not affect DAP susceptibility. Our results suggest that, apart from the LiaFSR system, the essential YycFG system is likely to be an important mediator of DAP resistance in some E. faecium strains.

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Correlation between Mutations in liaFSR of Enterococcus faecium and MIC of Daptomycin: Revisiting Daptomycin Breakpoints

2012 , MUNITA SEPULVEDA, JOSE MANUEL , Diana Panesso , Lorena Diaz , Truc T. Tran , Jinnethe Reyes , Audrey Wanger , Barbara E. Murray , Cesar A. Arias

Mutations in liaFSR, a three-component regulatory system controlling cell-envelope stress response, were recently linked with the emergence of daptomycin (DAP) resistance in enterococci. Our previous work showed that a liaF mutation increased the DAP MIC of a vancomycin-resistant Enterococcus faecalis strain from 1 to 3 μg/ml (the DAP breakpoint is 4 μg/ml), suggesting that mutations in the liaFSR system could be a pivotal initial event in the development of DAP resistance. With the hypothesis that clinical enterococcal isolates with DAP MICs between 3 and 4 μg/ml might harbor mutations in liaFSR, we studied 38 Enterococcus faecium bloodstream isolates, of which 8 had DAP MICs between 3 and 4 μg/ml by Etest in Mueller-Hinton agar. Interestingly, 6 of these 8 isolates had predicted amino acid changes in the LiaFSR system. Moreover, we previously showed that among 6 DAP-resistant E. faecium isolates (MICs of >4 μg/ml), 5 had mutations in liaFSR. In contrast, none of 16 E. faecium isolates with a DAP MIC of ≤2 μg/ml harbored mutations in this system (P<0.0001). All but one isolate with liaFSR changes exhibited DAP MICs of ≥16 μg/ml by Etest using brain heart infusion agar (BHIA), a medium that better supports enterococcal growth. Our findings provide a strong association between DAP MICs within the upper susceptibility range and mutations in the liaFSR system. Concomitant susceptibility testing on BHIA may be useful for identifying these E. faecium first-step mutants. Our results also suggest that the current DAP breakpoint for E. faecium may need to be reevaluated

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Enterococcal Endocarditis: Can We Win the War?

2012 , MUNITA SEPULVEDA, JOSE MANUEL , Cesar A. Arias , Barbara E. Murray

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Influence of Inoculum Effect on the Efficacy of Daptomycin Monotherapy and in Combination with beta-Lactams against Daptomycin-Susceptible Enterococcus faecium Harboring LiaSR Substitutions

2018 , Razieh Kebriaei , Seth A. Rice , Kavindra V. Singh , Kyle C. Stamper , An Q. Dinh , Rafael Rios , Lorena Diaz , Barbara E. Murray , MUNITA SEPULVEDA, JOSE MANUEL , Truc T. Tran , Cesar A. Arias , Michael J. Rybak

Enterococcus faecium isolates that harbor LiaFSR substitutions but are phenotypically susceptible to daptomycin (DAP) by current breakpoints are problematic, since predisposition to resistance may lead to therapeutic failure. Using a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model, we investigated DAP regimens (6, 8, and 10 mg/kg of body weight/day) as monotherapy and in combination with ampicillin (AMP), ceftaroline (CPT), or ertapenem (ERT) against E. faecium HOU503, a DAP-susceptible strain that harbors common LiaS and LiaR substitutions found in clinical isolates (T120S and W73C, respectively).

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Genomic Epidemiology of Vancomycin-Resistant Enterococcus faecium (VREfm) in Latin America: Revisiting The Global VRE Population Structure

2020 , Rafael Rios , Jinnethe Reyes , Lina P. Carvajal , Sandra Rincon , Diana Panesso , Aura M. Echeverri , An Dinh , Sergios-Orestis Kolokotronis , Apurva Narechania , Truc T. Tran , MUNITA SEPULVEDA, JOSE MANUEL , Barbara E. Murray , Paul J. Planet , Cesar A. Arias , Lorena Diaz

AbstractLittle is known about the population structure of vancomycin-resistant Enterococcus faecium (VREfm) in Latin America (LATAM). Here, we provide a complete genomic characterization of 55 representative Latin American VREfm recovered from 1998–2015 in 5 countries. The LATAM VREfm population is structured into two main clinical clades without geographical clustering. Using the LATAM genomes, we reconstructed the global population of VREfm by including 285 genomes from 36 countries spanning from 1946 to 2017. In contrast to previous studies, our results show an early branching of animal related isolates and a further split of clinical isolates into two sub-clades within clade A. The overall phylogenomic structure of clade A was highly dependent on recombination (54% of the genome) and the split between clades A and B was estimated to have occurred more than 2,765 years ago. Furthermore, our molecular clock calculations suggest the branching of animal isolates and clinical clades occurred ~502 years ago whereas the split within the clinical clade occurred ~302 years ago (previous studies showed a more recent split between clinical an animal branches around ~74 years ago). By including isolates from Latin America, we present novel insights into the population structure of VREfm and revisit the evolution of these pathogens.

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