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

2024 , Stephanie L. Egge , Samie A. Rizvi , Shelby R. Simar , ALCALDE RICO, MANUEL , JOSE RODRIGO WALDEMAR MARTINEZ SOLIS , Blake M. Hanson , An Q. Dinh , Rodrigo P. Baptista , Truc T. Tran , Samuel A. Shelburne , MUNITA SEPULVEDA, JOSE MANUEL , Cesar A. Arias , Morgan Hakki , William R. Miller , Ryan K. Shields

ABSTRACT 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 four 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.

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Multisite Detection of Tn 1549 -Mediated vanB Vancomycin Resistance in Multidrug-Resistant Enterococcus faecalis ST6 in Texas and Florida

2023 , Shelby R. Simar , Truc T. Tran , Kirsten B. Rydell , Diana Panesso , German A. Contreras , MUNITA SEPULVEDA, JOSE MANUEL , Renzo O. Cifuentes , Lilian M. Abbo , Pranoti Sahasrabhojane , An Q. Dinh , Dierdre B. Axell-House , Tor Savidge , Samuel A. Shelburne , Blake M. Hanson , Cesar A. Arias

In the United States, vanB -mediated resistance in enterococci is rare. We characterized three sequence type (ST) 6, vancomycin-resistant Enterococcus faecalis isolates causing bacteremia in unique patients in spatiotemporally distinct settings.

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Mechanisms of drug resistance: daptomycin resistance

2015 , Truc T. Tran , MUNITA SEPULVEDA, JOSE MANUEL , Cesar A. Arias

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A liaR Deletion Restores Susceptibility to Daptomycin and Antimicrobial Peptides in Multidrug-Resistant Enterococcus faecalis

2014 , Jinnethe Reyes , Diana Panesso , Truc T. Tran , Nagendra N. Mishra , Melissa R. Cruz , MUNITA SEPULVEDA, JOSE MANUEL , Kavindra V. Singh , Michael R. Yeaman , Barbara E. Murray , Yousif Shamoo , Danielle Garsin , Arnold S. Bayer , Cesar A. Arias

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In Vivo Resistance to Ceftolozane/Tazobactam in Pseudomonas aeruginosa Arising by AmpC- and Non-AmpC-Mediated Pathways

2018 , Erik Skoglund , Henrietta Abodakpi , Rafael Rios , Lorena Diaz , Elsa De La Cadena , An Q. Dinh , Javier Ardila , William R. Miller , MUNITA SEPULVEDA, JOSE MANUEL , Cesar A. Arias , Vincent H. Tam , Truc T. Tran

Two pairs of ceftolozane/tazobactam susceptible/resistant P. aeruginosa were isolated from 2 patients after exposure to β-lactams. The genetic basis of ceftolozane/tazobactam resistance was evaluated, and β-lactam-resistant mechanisms were assessed by phenotypic assays. Whole genome sequencing identified mutations in AmpC including the mutation (V213A) and a deletion of 7 amino acids (P210–G216) in the Ω-loop. Phenotypic assays showed that ceftolozane/tazobactam resistance in the strain with AmpCV213A variant was associated with increased β-lactamase hydrolysis activity. On the other hand, the deletion of 7 amino acids in the Ω-loop of AmpC did not display enhanced β-lactamase activity. Resistance to ceftolozane/tazobactam in P. aeruginosa is associated with changes in AmpC; however, the apparent loss of β-lactamase activity in AmpC∆7 suggests that non-AmpC mechanisms could play an important role in resistance to β-lactam/β-lactamase inhibitor combinations.

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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 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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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 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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Ceftaroline-Resistant, DaptomycinTolerant, and Heterogeneous Vancomycin-Intermediate MethicillinResistant Staphylococcus aureus Causing Infective Endocarditis

2017 , Masayuki Nigo , Lorena Diaz , Lina P. Carvajal , Truc T. Tran , Rafael Rios , Diana Panesso , Juan D. Garavito , William R. Miller , Audrey Wanger , George Weinstock , MUNITA SEPULVEDA, JOSE MANUEL , Cesar A. Arias , Henry F. Chambers

ABSTRACT We report a case of infective endocarditis (IE) caused by ceftaroline-resistant, daptomycin-tolerant, and heterogeneous vancomycin-intermediate methicillin-resistant S. aureus (MRSA). Resistance to ceftaroline emerged in the absence of drug exposure, and the E447K substitution in the active site of PBP2a previously associated with ceftaroline resistance was identified. Additionally, we present evidence of patient-to-patient transmission of the strain within the same unit. This case illustrates the difficulties in treating MRSA IE in the setting of a multidrug-resistant phenotype.

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