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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
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.
2Scopus© Citations 108
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Correlation between Mutations in
<i>liaFSR</i>
of Enterococcus faecium and MIC of Daptomycin: Revisiting Daptomycin Breakpoints
(2012)
MUNITA SEPULVEDA, JOSE MANUEL
;
Diana Panesso
;
Lorena Diaz
;
Truc T. Tran
;
Jinnethe Reyes
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
14Scopus© Citations 104
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Evolving Resistance Among Gram-positive Pathogens
(2015)
MUNITA SEPULVEDA, JOSE MANUEL
;
Arnold S. Bayer
;
Cesar A. Arias
8Scopus© Citations 98
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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
<jats:title>ABSTRACT</jats:title> <jats:p> Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a “last-resort” antibiotic against vancomycin-resistant <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Enterococcus faecium</jats:named-content> (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 <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">E. faecium</jats:named-content> . Thus, in order to gain insights into the genetic bases of DAP resistance in <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">E. faecium</jats:named-content> , we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">E. faecium</jats:named-content> 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 <jats:italic>liaFSR</jats:italic> or <jats:italic>yycFGHIJ</jats:italic> 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. </jats:p>
4 1Scopus© Citations 123

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