Browsing by Author "Murray, Barbara"
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Item A liaF codon deletion abolishes daptomycin bactericidal activity against vancomycin-resistant enterococcus faecali(American Society for Microbiology, 2013) Munita, José; Tran, Truc; Diaz, Lorena; Panesso, Diana; Reyes, Jinnethe; Murray, Barbara; Arias, CesarThe genetic bases for antibiotic tolerance are obscure. Daptomycin (DAP) is a lipopeptide antibiotic with bactericidal activity against enterococci. Using time-kill assays, we provide evidence for the first time that a deletion of isoleucine in position 177 of LiaF, a member of the three-component regulatory system LiaFSR involved in the cell envelope response to antimicrobials, is directly responsible for a DAP-tolerant phenotype and is likely to negatively affect response to DAP therapy.Item A liaR deletion restores susceptibility to daptomycin and antimicrobial peptides in multidrug-resistant Enterococcus faecalis(The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America, 2015) Reyes, Jinnethe; Panesso, Diana; Mishra, Nagendra; Cruz, Melissa; Munita, José; Singh, Kavindra; Yeaman, Michael; Murray, Barbara; Shamoo, Yousif; Garsin, Danielle; Bayer, Arnold; Arias, CesarDaptomycin is a lipopeptide antibiotic that is used clinically against many gram-positive bacterial pathogens and is considered a key frontline bactericidal antibiotic to treat multidrug-resistant enterococci. Emergence of daptomycin resistance during therapy of serious enterococcal infections is a major clinical issue. In this work, we show that deletion of the gene encoding the response regulator, LiaR (a member of the LiaFSR system that controls cell envelope homeostasis), from daptomycin-resistant Enterococcus faecalis not only reversed resistance to 2 clinically available cell membrane-targeting antimicrobials (daptomycin and telavancin), but also resulted in hypersusceptibility to these antibiotics and to a variety of antimicrobial peptides of diverse origin and with different mechanisms of action. The changes in susceptibility to these antibiotics and antimicrobial peptides correlated with in vivo attenuation in a Caenorhabditis elegans model. Mechanistically, deletion of liaR altered the localization of cardiolipin microdomains in the cell membrane. Our findings suggest that LiaR is a master regulator of the enterococcal cell membrane response to diverse antimicrobial agents and peptides; as such, LiaR represents a novel target to restore the activity of clinically useful antimicrobials against these organisms and, potentially, increase susceptibility to endogenous antimicrobial peptides.Item Correlation between mutations in liaFSR of enterococcus faecium and MIC of daptomycin: revisiting daptomycin breakpoints(American Society for Microbiology, 2012) Munita, José; Panesso, Diana; Diaz, Lorena; Tran, Truc; Reyes, Jinnethe; Wanger, Audrey; Murray, Barbara; Arias, CesarMutations 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.Item Daptomycin for the treatment of bacteraemia due to vancomycin-resistant enterococci(Elsevier, 2014) Munita, José; Murray, Barbara; Arias, CesarTreatment of severe infections caused by vancomycin-resistant enterococci (VRE) is challenging due to the scarcity of reliable therapeutic alternatives. In this context, daptomycin (DAP), a lipopeptide antibiotic, has emerged as an interesting alternative as it is one of the few compounds that retain in vitro bactericidal activity against VRE isolates, although it has not been approved for this purpose by regulatory agencies. In this review, we will summarise the clinical, animal and in vitro evidence evaluating the efficacy of DAP for the management of deep-seated VRE infections. In addition, we will address important clinical concerns such as the emergence of DAP resistance during therapy and reports of therapeutic failure with DAP monotherapy. Finally, we will discuss possible future strategies (such as the use of higher doses and/or combination therapies) to optimise the use of this antibiotic against VRE.Item Daptomycin-resistant enterococcus faecalis diverts the antibiotic molecule from the division septum and remodels cell membrane phospholipids(American Society for Microbiology, 2013) Tran, Truc; Panesso, Diana; Mishra, Nagendra; Mileykovskaya, Eugenia; Guan, Ziqianq; Munita, José; Reyes, Jinnethe; Diaz, Lorena; Weinstock, George; Murray, Barbara; Shamoo, Yousif; Dowhan, William; Bayer, Arnold; Arias, CesarTreatment 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.Item Dissecting the mechanisms of linezolid resistance in a drosophila melanogaster infection model of staphylococcus aureus(Oxford University Press, 2013) Diaz, Lorena; Kontoyiannis, Dimitrios; Panesso, Diana; Albert, Nathaniel; Singh, Kavindra; Tran, Truc; Munita, José; Murray, Barbara; Arias, CesarBACKGROUND: Mini-host models are simple experimental systems to study host-pathogen interactions. We adapted a Drosophila melanogaster infection model to evaluate the in vivo effect of different mechanisms of linezolid (LNZ) resistance in Staphylococcus aureus. METHODS: Fly survival was evaluated after infection with LNZ-resistant S. aureus strains NRS119 (which has mutations in 23S ribosomal RNA [rRNA]), CM-05 and 004-737X (which carry cfr), LNZ-susceptible derivatives of CM-05 and 004-737X (which lack cfr), and ATCC 29213 (an LNZ-susceptible control). Flies were then fed food mixed with LNZ (concentration, 15-500 µg/mL). Results were compared to those in mouse peritonitis, using LNZ via oral gavage at 80 and 120 mg/kg every 12 hours. RESULTS: LNZ at 500 µg/mL in fly food protected against all strains, while concentrations of 15-250 µg/mL failed to protect against NRS119 (survival, 1.6%-20%). An in vivo effect of cfr was only detected at concentrations of 30 and 15 µg/mL. In the mouse peritonitis model, LNZ (at doses that mimic human pharmacokinetics) protected mice from challenge with the cfr+ 004-737X strain but was ineffective against the NRS119 strain, which carried 23S rRNA mutations. CONCLUSIONS: The fly model offers promising advantages to dissect the in vivo effect of LNZ resistance in S. aureus, and findings from this model appear to be concordant with those from the mouse peritonitis model.Item Enterococcal endocarditis: can we win the war?(Springer, 2012) Munita, José; Arias, Cesar; Murray, BarbaraTreatment of enterococcal infections has long been recognized as an important clinical challenge, particularly in the setting of infective endocarditis (IE). Furthermore, the increase prevalence of isolates exhibiting multidrug resistance (MDR) to traditional anti-enterococcal antibiotics such as ampicillin, vancomycin and aminoglycosides (high-level resistance) poses immense therapeutic dilemmas in hospitals around the world. Unlike IE caused by most isolates of Enterococcus faecalis, which still retain susceptibility to ampicillin and vancomycin, the emergence and dissemination of a hospital-associated genetic clade of multidrug resistant Enterococcus faecium, markedly limits the therapeutic options. The best treatment of IE MDR enterococcal endocarditis is unknown and the paucity of antibiotics with bactericidal activity against these organisms is a cause of serious concern. Although it appears that we are winning the war against E. faecalis, the battle rages on against isolates of multidrug-resistant E. faecium.Item Gram-positive bacterial infections: research priorities, accomplishments, and future directions of the antibacterial resistance leadership group(Oxford University Press, 2017) Doernberg, Sarah; Lodise, Thomas; Thaden, Joshua; Munita, José; Cosgrove, Sara; Arias, Cesar; Boucher, Helen; Corey, G. Ralph; Lowy, Franklin; Murray, Barbara; Miller, Loren; Holland, ThomasAntimicrobial resistance in gram-positive bacteria remains a challenge in infectious diseases. The mission of the Gram-Positive Committee of the Antibacterial Resistance Leadership Group (ARLG) is to advance knowledge in the prevention, management, and treatment of these challenging infections to improve patient outcomes. Our committee has prioritized projects involving methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) due to the scope of the medical threat posed by these pathogens. Approved ARLG projects involving gram-positive pathogens include (1) a pharmacokinetics/pharmacodynamics study to evaluate the impact of vancomycin dosing on patient outcome in MRSA bloodstream infection (BSI); (2) defining, testing, and validating innovative assessments of patient outcomes for clinical trials of MRSA-BSI; (3) testing new strategies for "step-down" antibiotic therapy for MRSA-BSI; (4) management of staphylococcal BSIs in neonatal intensive care units; and (5) defining the impact of VRE bacteremia and daptomycin susceptibility on patient outcomes. This article outlines accomplishments, priorities, and challenges for research of infections caused by gram-positive organisms.Item Methicillin-Susceptible, Vancomycin-Resistant Staphylococcus aureus, Brazil(Centers for Disease Control and Prevention, 2015) Panesso, Diana; Planet, Paul; Diaz, Lorena; Hugonnet, Jean-Emmanuel; Tran, Truc; Narechania, Apurva; Munita, José; Rincon, Sandra; Carvajal, Lina; Reyes, Jinnethe; Londoño, Alejandra; Smith, Hannah; Sebra, Robert; Deikus, Gintaras; Weinstock, George; Murray, Barbara; Rossi, Flavia; Arthur, Michel; Arias, CesarWe report characterization of a methicillin-susceptible, vancomycin-resistant bloodstream isolate of Staphylococcus aureus recovered from a patient in Brazil. Emergence of vancomycin resistance in methicillin-susceptible S. aureus would indicate that this resistance trait might be poised to disseminate more rapidly among S. aureus and represents a major public health threat.Publication Priorities and Progress in Gram-positive Bacterial Infection Research by the Antibacterial Resistance Leadership Group: A Narrative Review(2023) Doernberg, Sarah; Arias, Cesar; Altman, Deena; Babiker, Ahmed; Boucher, Helen; Creech, C Buddy; Cosgrove, Sara; Evans, Scott; Fowler, Vance; Fritz, Stephanie; Hamasaki, Toshimitsu; Kelly, Brendan; Leal, Sixto; Liu, Catherine; Lodise, Thomas; Miller, Loren; Munita, Jose M.; Murray, Barbara; Pettigrew, Melinda; Ruffin, Felicia; Scheetz, Marc; Shopsin, Bo; Tran, Truc; Turne, Nicholas; Williams, Derek; Zaharoff, Smitha; Holland, Thomas; Antibacterial Resistance Leadership GroupThe Antibacterial Resistance Leadership Group (ARLG) has prioritized infections caused by gram-positive bacteria as one of its core areas of emphasis. The ARLG Gram-positive Committee has focused on studies responding to 3 main identified research priorities: (1) investigation of strategies or therapies for infections predominantly caused by gram-positive bacteria, (2) evaluation of the efficacy of novel agents for infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci, and (3) optimization of dosing and duration of antimicrobial agents for gram-positive infections. Herein, we summarize ARLG accomplishments in gram-positive bacterial infection research, including studies aiming to (1) inform optimal vancomycin dosing, (2) determine the role of dalbavancin in MRSA bloodstream infection, (3) characterize enterococcal bloodstream infections, (4) demonstrate the benefits of short-course therapy for pediatric community-acquired pneumonia, (5) develop quality of life measures for use in clinical trials, and (6) advance understanding of the microbiome. Future studies will incorporate innovative methodologies with a focus on interventional clinical trials that have the potential to change clinical practice for difficult-to-treat infections, such as MRSA bloodstream infections.Item Transferable vancomycin resistance in a community-associated MRSA lineage(Massachusetts Medical Society, 2014) Rossi, Flávia; Diaz, Lorena; Wollam, Aye; Panesso, Diana; Zhou, Yanjiao; Rincon, Sandra; Narechania, Apurva; Xing, Galen; Di Gioia, Thais; Doi, André; Tran, Truc; Reyes, Jinnethe; Munita, José; Carvajal, Lina; Hernandez-Roldan, Alejandra; Brandão, Denise; van der Heijden, Inneke Marie; Murray, Barbara; Planet, Paul; Weinstock, George; Arias, CesarWe report the case of a patient from Brazil with a bloodstream infection caused by a strain of methicillin-resistant Staphylococcus aureus (MRSA) that was susceptible to vancomycin (designated BR-VSSA) but that acquired the vanA gene cluster during antibiotic therapy and became resistant to vancomycin (designated BR-VRSA). Both strains belong to the sequence type (ST) 8 community-associated genetic lineage that carries the staphylococcal chromosomal cassette mec (SCCmec) type IVa and the S. aureus protein A gene (spa) type t292 and are phylogenetically related to MRSA lineage USA300. A conjugative plasmid of 55,706 bp (pBRZ01) carrying the vanA cluster was identified and readily transferred to other staphylococci. The pBRZ01 plasmid harbors DNA sequences that are typical of the plasmid-associated replication genes rep24 or rep21 described in community-associated MRSA strains from Australia (pWBG745). The presence and dissemination of community-associated MRSA containing vanA could become a serious public health concern.Item What's New in the Treatment of Enterococcal Endocarditis?(Springer, 2014) Nigo, Masayuki; Munita, José; Arias, Cesar; Murray, BarbaraEnterococcus spp. are among the common pathogens causing infective endocarditis (IE). Despite major medical advances and new potent antimicrobial agents, the mortality has not significantly improved for several decades. The usual lack of bactericidal activity of penicillin or ampicillin, the toxicity from the combination of penicillin plus aminoglycosides, and the increased reports of high-level resistance to aminoglycosides have led to the exploration of other regimens for treatment of Enterococcus faecalis IE. As an example, ampicillin plus ceftriaxone is now a well-recognized regimen for this organism. However, the emerging of new drug resistances in Enterococcus faecium dramatically reduces the therapeutic alternatives for this organism in IE which continues to be an immense challenge for clinicians even with the availability of newer antimicrobial agents. This article summarizes the current treatment options for enterococcal endocarditis and reviews of recent publications on the topic.Item Whole-Genome Analyses of Enterococcus faecium Isolates with Diverse Daptomycin MICs(American Society for Microbiology, 2014) Diaz, Lorena; Tran, Truc; Munita, José; Miller, William; Rincon, Sandra; Carvajal, Lina; Wollam, Aye; Reyes, Jinnethe; Panesso, Diana; Rojas, Natalia; Shamoo, Yousif; Murray, Barbara; Weinstock, George; Arias, CesarDaptomycin (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.Item Whole-genome analysis of a daptomycin-susceptible enterococcus faecium strain and its daptomycin-resistant variant arising during therapy(American Society for Microbiology, 2013) Tran, Truc; Panesso, Diana; Gao, Hongyu; Roh, Jung; Munita, José; Reyes, Jinnethe; Diaz, Lorena; Lobos, Elizabeth; Shamoo, Yousif; Mishra, Nagendra; Bayer, Arnold; Murray, Barbara; Weinstock, George; Arias, CesarDevelopment 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.