DSpace Repository :: Browsing by Author "Panesso, Diana"

Browsing by Author "Panesso, Diana"

Now showing 1 - 17 of 17

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, Cesar
The 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.
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, Cesar
Daptomycin 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.
A prospective cohort multicenter study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine Latin American countries
(2017) Arias, Cesar A; Reyes, Jinnethe; Carvajal, Lina; Rincon, Sandra; Diaz, Lorena; Panesso, Diana; Ibarra, Gabriel; Rios, Rafael; Munita, José; Salles, Mauro J; Alvarez-Moreno, Carlos; Labarca, Jaime; Garcia, Coralith; Luna, Carlos M; Mejia-Villatoro, Carlos; Zurita, Jeanette; Guzman-Blanco, Manuel; Rodriguez-Noriega, Eduardo; Narechania, Apurva; Rojas, Laura J; Planet, Paul J; Weinstock, George M; Gotuzzo, Eduardo; Seasg, Carlos
Staphylococcus aureus is an important pathogen causing a spectrum of diseases ranging from mild skin and soft tissue infections to life-threatening conditions. Bloodstream infections are particularly important, and the treatment approach is complicated by the presence of methicillin-resistant S. aureus (MRSA) isolates. The emergence of new genetic lineages of MRSA has occurred in Latin America (LA) with the rise and dissemination of the community-associated USA300 Latin American variant (USA300-LV). Here, we prospectively characterized bloodstream MRSA recovered from selected hospitals in 9 Latin American countries. All isolates were typed by pulsed-field gel electrophoresis (PFGE) and subjected to antibiotic susceptibility testing. Whole-genome sequencing was performed on 96 MRSA representatives. MRSA represented 45% of all (1,185 S. aureus) isolates. The majority of MRSA isolates belonged to clonal cluster (CC) 5. In Colombia and Ecuador, most isolates (72%) belonged to the USA300-LV lineage (CC8). Phylogenetic reconstructions indicated that MRSA isolates from participating hospitals belonged to three major clades. Clade A grouped isolates with sequence type 5 (ST5), ST105, and ST1011 (mostly staphylococcal chromosomal cassette mec [SCCmec] I and II). Clade B included ST8, ST88, ST97, and ST72 strains (SCCmec IV, subtypes a, b, and c/E), and clade C grouped mostly Argentinian MRSA belonging to ST30. In summary, CC5 MRSA was prevalent in bloodstream infections in LA with the exception of Colombia and Ecuador, where USA300-LV is now the dominant lineage. Clonal replacement appears to be a common phenomenon, and continuous surveillance is crucial to identify changes in the molecular epidemiology of MRSA.
Ceftaroline-resistant, daptomycin-tolerant, and heterogeneous vancomycin-intermediate methicillin-resistant staphylococcus aureus causing infective endocarditis
(American Society for Microbiology, 2017) Nigo, Masayuki; Diaz, Lorena; Carvajal, Lina P.; Tran, Truc T.; Rios, Rafael; Panesso, Diana; Garavito, Juan D; Miller, William; Wagner, Audrey
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.
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, Cesar
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.
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, Cesar
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.
Deletion of liaR reverses daptomycin resistance in enterococcus faecium independent of the genetic background
(American Society for Microbiology, 2015) Panesso, Diana; Reyes, Jinnethe; Gaston, Elizabeth; Deal, Morgan; Londoño, Alejandra; Nigo, Masayuki; Munita, José; Miller, William; Shamoo, Yousif; Tran, Truc; Arias, Cesar
We have shown previously that changes in LiaFSR, a three-component regulatory system predicted to orchestrate the cell membrane stress response, are important mediators of daptomycin (DAP) resistance in enterococci. Indeed, deletion of the gene encoding the response regulator LiaR in a clinical strain of Enterococcus faecalis reversed DAP resistance (DAP-R) and produced a strain hypersusceptible to antimicrobial peptides. Since LiaFSR is conserved in Enterococcus faecium, we investigated the role of LiaR in a variety of clinical E. faecium strains representing the most common DAP-R genetic backgrounds. Deletion of liaR in DAP-R E. faecium R446F (DAP MIC of 16 μg/ml) and R497F (MIC of 24 μg/ml; harboring changes in LiaRS) strains fully reversed resistance (DAP MICs decreasing to 0.25 and 0.094 μg/ml, respectively). Moreover, DAP at concentrations of 13 μg/ml (achieved with human doses of 12 mg/kg body weight) retained bactericidal activity against the mutants. Furthermore, the liaR deletion derivatives of these two DAP-R strains exhibited increased binding of boron-dipyrromethene difluoride (BODIPY)-daptomycin, suggesting that high-level DAP-R mediated by LiaR in E. faecium involves repulsion of the calcium-DAP complex from the cell surface. In DAP-tolerant strains HOU503F and HOU515F (DAP MICs within the susceptible range but bacteria not killed by DAP concentrations of 5× the MIC), deletion of liaR not only markedly decreased the DAP MICs (0.064 and 0.047 μg/ml, respectively) but also restored the bactericidal activity of DAP at concentrations as low as 4 μg/ml (achieved with human doses of 4 mg/kg). Our results suggest that LiaR plays a relevant role in the enterococcal cell membrane adaptive response to antimicrobial peptides independent of the genetic background and emerges as an attractive target to restore the activity of DAP against multidrug-resistant strains.
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, Cesar
BACKGROUND: 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.
Failure of High-Dose Daptomycin for Bacteremia Caused by Daptomycin-Susceptible Enterococcus faecium Harboring LiaSR Substitutions
(2014) Munita, José; Mishra, Nagendra N.; Alvarez, Danya; Tran, Truc T; Diaz, Lorena; Panesso, Diana; Reyes, Jinnethe; Murray, Barbara E; Adachi, Javier A; Bayer, Arnold S; Arias, Cesar A
High-dose daptomycin (DAP) therapy failed in a neutropenic patient with bloodstream infection caused by a DAP-susceptible Enterococcus faecium (minimum inhibitory concentration, 3 µg/mL) harboring genetic changes associated with DAP resistance, with persistent bacteremia and selection of additional resistances. Daptomycin monotherapy should be used cautiously against DAP-susceptible E. faecium strains with minimum inhibitory concentrations >2 µg/mL.
Genomic Epidemiology of Vancomycin-Resistant Enterococcus faecium (VREfm) in Latin America: Revisiting The Global VRE Population Structure
(2020) Rios, Rafael; Reyes, Jinnethe; Carvajal, Lina P.; Rincon, Sandra; Panesso, Diana; Echeverri, Aura M.; Dinh, An; Kolokotronis, Sergios-Orestis; Narechania, Apurva; Tran, Truc T.; Munita, José; Murray, Bárbara E.; Planet, Paul J.; Arias, Cesar A.; Díaz, Lorena
Little 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.
Influence of minimum inhibitory concentration in clinical outcomes of enterococcus faecium bacteremia treated with daptomycin: is it time to change the breakpoint?
(Oxford University Press, 2016) Shukla, Bhavarth; Shelburne, Samuel; Reyes, Katherine; Kamboj, Mini; Lewis, Jessica; Rincon, Sandra; Reyes, Jinnethe; Carvajal, Lina; Panesso, Diana; Sifri, Costi; Zervos, Marcus; Pamer, Eric; Tran, Truc; Adachi, Javier; Munita, José; Hasbun, Rodrigo; Arias, Cesar
BACKGROUND: Daptomycin has become a front-line antibiotic for multidrug-resistant Enterococcus faecium bloodstream infections (BSIs). We previously showed that E. faecium strains with daptomycin minimum inhibitory concentrations (MICs) in the higher end of susceptibility frequently harbor mutations associated with daptomycin resistance. We postulate that patients with E. faecium BSIs exhibiting daptomycin MICs of 3-4 µg/mL treated with daptomycin are more likely to have worse clinical outcomes than those exhibiting daptomycin MICs ≤2 µg/mL. METHODS: We conducted a multicenter retrospective cohort study that included adult patients with E. faecium BSI for whom initial isolates, follow-up blood culture data, and daptomycin administration data were available. A central laboratory performed standardized daptomycin MIC testing for all isolates. The primary outcome was microbiologic failure, defined as clearance of bacteremia ≥4 days after the index blood culture. The secondary outcome was all-cause in-hospital mortality. RESULTS: A total of 62 patients were included. Thirty-one patients were infected with isolates that exhibited daptomycin MICs of 3-4 µg/mL. Overall, 34 patients had microbiologic failure and 25 died during hospitalization. In a multivariate logistic regression model, daptomycin MICs of 3-4 µg/mL (odds ratio [OR], 4.7 [1.37-16.12]; P = .014) and immunosuppression (OR, 5.32 [1.20-23.54]; P = .028) were significantly associated with microbiologic failure. Initial daptomycin dose of ≥8 mg/kg was not significantly associated with evaluated outcomes. CONCLUSIONS: Daptomycin MICs of 3-4 µg/mL in the initial E. faecium blood isolate predicted microbiological failure of daptomycin therapy, suggesting that modification in the daptomycin breakpoint for enterococci should be considered.
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, Cesar
We 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.
Multisite Detection of Tn 1549-Mediated vanB Vancomycin Resistance in Multidrug-Resistant Enterococcus faecalis ST6 in Texas and Florida
(2023) Simar, Shelby; Tran, Truc; Rydell, Kirsten; Panesso, Diana; Contreras, German; Munita, Jose M.; Cifuentes. Renzo; Abbo, Lilian; Sahasrabhojane, Pranoti; Dinh, An; Axell-House, Dierdre; Savidge, Tor; Shelburne, Samuel; Hanson, Blake; Arias, Cesar
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. Isolates were recovered between 2018 and 2020 in two cities in the United States (Houston, TX; Miami, FL). The isolates harbored the vanB operon on a chromosomally located Tn1549 transposon, and epidemiological data suggested multiple introductions of the vanB gene cluster into ST6 E. faecalis.
Resistance to last resort antibiotics in gram-positive cocci: the post-vancomycin era
(Instituto Nacional de Salud de Colombia, 2014) Rincon, Sandra; Panesso, Diana; Diaz, Lorena; Carvajal, Lina; Reyes, Jinnethe; Munita, José; Arias, Cesar
New therapeutic alternatives have been developed in the last years for the treatment of multidrug-resistant Gram-positive infections. Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are considered a therapeutic challenge due to failures and lack of reliable antimicrobial options. Despite concerns related to the use of vancomycin in the treatment of severe MRSA infections in specific clinical scenarios, there is a paucity of solid clinical evidence that support the use of alternative agents (when compared to vancomycin). Linezolid, daptomycin and tigecycline are antibiotics approved in the last decade and newer cephalosporins (such as ceftaroline and ceftobiprole) and novel glycopeptides (dalvavancin, telavancin and oritavancin) have reached clinical approval or are in the late stages of clinical development. This review focuses on discussing these newer antibiotics used in the "post-vancomycin" era with emphasis on relevant chemical characteristics, spectrum of antimicrobial activity, mechanisms of action and resistance, as well as their clinical utility.
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, Cesar
We 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.
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, Cesar
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.
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, Cesar
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.

Browsing by Author "Panesso, Diana"

Results Per Page

Sort Options