Whole Genome Characterization of the Mechanisms of Daptomycin Resistance in Clinical and Laboratory Derived Isolates of Staphylococcus aureus

Abstract

BackgroundDaptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus.MethodsWhole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates.ResultsOn average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol.ConclusionPoint mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus.

The action mechanism of daptomycin

“…Mutations that reduce its activity reduce membrane PG levels and daptomycin susceptibility in Bacillus subtilis and Staphylococcus aureus. 64,90,91 …”

“…This suggests that increased synthesis of CL could mediate bacterial resistance to daptomycin. Indeed, cardiolipin synthase mutants have been isolated from resistant mutants of Staphylococcus aureus 91 as well Enterococcus species. 99 Some of these mutants have been shown to have increased activity, 100 but their substitution into the genome of a daptomycin-susceptible strain did not detectably increase resistance.…”

Resistencia a antibióticos de última línea en cocos Gram positivos: la era posterior a la vancomicina

“…El sistema regulador de dos componentes YycFG se ha relacionado con el desarrollo de la resistencia a la daptomicina en cepas clínicas y de laboratorio (92,100). Otro sistema regulador de dos componentes relacionado con el desarrollo de la resistencia a la daptomicina es el vraSR (también relacionado con el desarrollo de S. aureus de sensibilidad intermedia a la vancomicina), el cual se ha encontrado sobreexpresado en cepas resistentes a la daptomicina.…”

“…De hecho, S. aureus posee dos genes que codifican para la sintetasa de cardiolipina (cls) y es importante anotar que el cls2 es el que se ha relacionado con la resistencia a la daptomicina en S. aureus. La enzima Cls contiene dos dominios transmembrana y dos dominios putativos de la sintetasa de cardiolipina (104); los cambios aminoacídicos en estos dominios se han asociado con el desarrollo de la resistencia a la daptomicina; más aún, la presencia simultánea de sustituciones en Cls y MprF parece ser importante en el mecanismo de 'repulsión' de la superficie celular exhibido por la daptomicina, aunque también se ha postulado que puede contribuir a la alteración de la interacción del compuesto con los fosfolípidos de la membrana celular (105). Otro de los genes implicados en la resistencia a la daptomicina es el pgsA, el cual codifica para una CDP-diacilglicerolglicerol-3-fosfato-3-fosfatidiltransferasa, enzima involucrada en la síntesis de FG en la que Peleg, et al, identificaron una mutación puntual en pgsA que incrementó la CIM frente a la daptomicina en S. aureus (de 0,5 µg/ml a 2 µg/ml) (105).…”

Antibacterial photosensitization through activation of coproporphyrinogen oxidase

“…Phenotypic high-throughput screens using small-molecule libraries often result in the identification of numerous molecules with unknown targets. Several approaches have been successful in identifying intracellular targets of small molecules (26)(27)(28)(29)(30)(31)(32). Here, we report a genetic selection strategy based on the creation of a suicide strain that enables the identification of spontaneous resistant mutants to an activating compound that is typically nontoxic.…”