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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Clinical characteristics, microbiology and outcomes of a cohort of patients treated with ceftolozane/tazobactam in acute care inpatient facilities, Houston, Texas, USA

2022 , Truc T Tran , Nicolo L Cabrera , Anne J Gonzales-Luna , Travis J Carlson , Faris Alnezary , William R Miller , Aki Sakurai , An Q Dinh , Kirsten Rydell , Rafael Rios , Lorena Diaz , Blake M Hanson , MUNITA SEPULVEDA, JOSE MANUEL , Claudia Pedroza , Samuel A Shelburne , Samuel L Aitken , Kevin W Garey , Ryan Dillon , Laura Puzniak , Cesar A Arias

Abstract Background Ceftolozane/tazobactam is a β-lactam/β-lactamase inhibitor combination with activity against a variety of Gram-negative bacteria, including MDR Pseudomonas aeruginosa. This agent is approved for hospital-acquired and ventilator-associated bacterial pneumonia. However, most real-world outcome data come from small observational cohorts. Thus, we sought to evaluate the utilization of ceftolozane/tazobactam at multiple tertiary hospitals in Houston, TX, USA. Methods We conducted a multicentre retrospective study of patients receiving at least 48 h of ceftolozane/tazobactam therapy from January 2016 through to September 2019 at two hospital systems in Houston. Demographic, clinical and microbiological data were collected, including the infecting bacterial isolate, when available. The primary outcome was composite clinical success at hospital discharge. Secondary outcomes included in-hospital mortality and clinical disposition at 14 and 30 days post ceftolozane/tazobactam initiation. Multivariable logistic regression analysis was used to identify predictors of the primary outcome and mortality. Recovered isolates were tested for susceptibility to ceftolozane/tazobactam and underwent WGS. Results A total of 263 patients were enrolled, and composite clinical success was achieved in 185 patients (70.3%). Severity of illness was the most consistent predictor of clinical success. Combination therapy with ceftolozane/tazobactam and another Gram-negative-active agent was associated with reduced odds of clinical success (OR 0.32, 95% CI 0.16–0.63). Resistance to ceftolozane/tazobactam was noted in 15.4% of isolates available for WGS; mutations in ampC and ftsI were common but did not cluster with a particular ST. Conclusions Clinical success rate among this patient cohort treated with ceftolozane/tazobactam was similar compared with previous experiences. Ceftolozane/tazobactam remains an alternative agent for treatment of susceptible isolates of P. aeruginosa.

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The Cefazolin Inoculum Effect Is Associated With Increased Mortality in Methicillin-Susceptible Staphylococcus aureus Bacteremia

2018 , William R Miller , Carlos Seas , Lina P Carvajal , Lorena Diaz , Aura M Echeverri , Carolina Ferro , Rafael Rios , Paola Porras , Carlos Luna , Eduardo Gotuzzo , MUNITA SEPULVEDA, JOSE MANUEL , Esteban Nannini , Cesar Carcamo , Jinnethe Reyes , Cesar A Arias

Abstract Background Recent studies have favored the use of cefazolin over nafcillin for the treatment of methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. The clinical influence of the cefazolin inoculum effect (CzIE) in the effectiveness of cephalosporins for severe MSSA infections has not been evaluated. Methods We prospectively included patients from 3 Argentinian hospitals with S. aureus bacteremia. Cefazolin minimum inhibitory concentrations (MICs) were determined at standard (105 colony-forming units [CFU]/mL) and high (107 CFU/mL) inoculum. The CzIE was defined as an increase of MIC to ≥16 µg/mL when tested at high inoculum. Whole-genome sequencing was performed in all isolates. Results A total of 77 patients, contributing 89 MSSA isolates, were included in the study; 42 patients (54.5%) had isolates with the CzIE. In univariate analysis, patients with MSSA exhibiting the CzIE had increased 30-day mortality (P = .034) and were more likely to have catheter-associated or unknown source of bacteremia (P = .033) compared with patients infected with MSSA isolates without the CzIE. No statistically significant difference between the groups was observed in age, clinical illness severity, place of acquisition (community vs hospital), or presence of endocarditis. The CzIE remained associated with increased 30-day mortality in multivariate analysis (risk ratio, 2.65; 95% confidence interval, 1.10–6.42; P = .03). MSSA genomes displayed a high degree of heterogeneity, and the CzIE was not associated with a specific lineage. Conclusions In patients with MSSA bacteremia where cephalosporins are used as firstline therapy, the CzIE was associated with increased 30-day mortality. Clinicians should be cautious when using cefazolin as firstline therapy for these infections.

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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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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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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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Clinical outcomes and bacterial characteristics of carbapenem-resistant Klebsiella pneumoniae complex among patients from different global regions (CRACKLE-2): a prospective, multicentre, cohort study

2022 , Minggui Wang , Michelle Earley , Liang Chen , Blake M Hanson , Yunsong Yu , Zhengyin Liu , Soraya Salcedo , Eric Cober , Lanjuan Li , Souha S Kanj , Hainv Gao , MUNITA SEPULVEDA, JOSE MANUEL , Karen Ordoñez , Greg Weston , Michael J Satlin , Sandra L Valderrama-Beltrán , Kalisvar Marimuthu , Martin E Stryjewski , Lauren Komarow , Courtney Luterbach , Steve H Marshall , Susan D Rudin , Claudia Manca , David L Paterson , Jinnethe Reyes , Maria V Villegas , Scott Evans , Carol Hill , Rebekka Arias , Keri Baum , Bettina C Fries , Yohei Doi , Robin Patel , Barry N Kreiswirth , Robert A Bonomo , Henry F Chambers , Vance G Fowler , Cesar A Arias , David van Duin , Lilian M Abbo , Deverick J Anderson , Rebekka Arias , Cesar A Arias , Keri Baum , Robert A Bonomo , Henry F Chambers , Liang Chen , Kean Lee Chew , Eric Cober , Heather R Cross , Partha Pratim De , Samit Desai , Sorabh Dhar , Valentina Di Castelnuovo , Lorena Diaz , AN Q Dinh , Yohei Doi , Michelle Earley , Brandon Eilertson , Beth Evans , Scott Evans , Vance G Fowler Jr , Bettina C Fries , Hainv Gao , Julia Garcia-Diaz , Omai B Garner , Kerryl Greenwood-Quaintance , Blake Hanson , Erica Herc , Carol Hill , Jesse T Jacob , Jianping Jiang , Robert C Kalayjian , Souha S Kanj , Keith S Kaye , Angela Kim , Lauren Komarow , Barry N Kreiswirth , Courtney Lauterbach , Lanjuan Li , Zhengyin Liu , Claudia Manca , Kalisvar Marimuthu , Steven H Marshall , Todd McCarty , Oon Tek Ng , Jose Millan Oñate Gutierrez , Karen Ordoñez , Robin Patel , David L Paterson , Anton Peleg , Jinnethe Reyes , Susan D Rudin , Robert A Salata , Soraya Salcedo , Michael J Satlin , Suzannah Schmidt-Malan , Nares Smitasin , Maria Spencer , Martin Stryjewski , Jiachun Su , Paul Ananth Tambyah , Sandra Valderrama , David van Duin , Maria Virginia Villegas Botero , Minggui Wang , Mary Waters , Greg Weston , Darren Wong , Glenn Wortmann , Yang Yang , Yunsong Yu , Fujie Zhang

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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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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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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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