Resistencia a daptomicina en el género Staphylococcusanálisis lipídico y genético

Résumé

Daptomycin is a lipopeptide antibiotic with specific activity against Gram-positive organism and used in treatment of complicated infections caused by Gram-positive bacteria, particularly multirresistant Staphylococcus aureus and Enterococcus faecium. Its mechanism of action, depending on calcium ions, is not fully understood, but it is clear its interaction with phosphatidylglycerol, majority lipid in these bacterial membranes. Daptomycin resistance is a rare event, associated with treatments of S. aureus infections. The molecular mechanism of this resistance has been related with genes involved with the synthesis and homeostasis of the cell envelope, such us mprF (synthesis of lisylphosphatidylglycerol), cls2 (synthesis of cardiolipin), yycFG (synthesis of peptidoglycan), vraSR (regulator of the cell wall synthesis), graSR (regulator of the cell envelope charge), etc. In this work we have studied the influence of the lipid composition in the daptomycin resistance and in the molecular mechanisms related with this resistance in serial clinical isolates of S. aureus and S. capitis, got each one of the same patient, that changed their antibiotic susceptibility during the infection. S. capitis, considered as a contaminating microorganism, has emerged in recent years as an important nosocomial pathogen, especially in neonatal ICUs. The lipid composition was studied by mass spectrometry with electrospray ionization and it was analysed by time of fly (ESI-TOF), and we sequenced the chromosome of couples of strains susceptible/resistant of both species to estimate the genetic changes (mutations) between them, checked by PCR and Sanger sequencing. In this couples we studied the lipid composition in presence and absence of daptomycin and we analysed the thickness of the cell wall by transmission electron microscopy. In S. aureus we demonstrated the resistant strain differed from the rest of the isolates in his high content of lisylphosphatidylglycerol (LPG), a cationic phospholipid. This characteristic was accompanied by the P314L mutation in MprF, protein involved in the synthesis of this phospholipid. We also observed mutations in purR, regulator of the purine metabolism, in the Emb adhesin, and a deletion in an hypothetical protein. The susceptible/resistant couple didn´t adapt his lipid composition to the presence of the antibiotic, and we didn´t observe variation into the thickness cell envelope, which is why we considered that the resistance observed in S. aureus could be related con the mutation P314L. The resistant strains of S. capitis showed important lipid variations compared to the susceptible strain, standing out the low content of LPG and an increase of diacylglycerol (DAG) and free fatty acids (AGL). The chromosomic analysis of the susceptible/resistant couples showed eight mutations in the resistant strain, highlighting the mutations observed in rnJ2, ribonuclease of the degradosome complex, and qacB, gen related with the transport of ammonium ions, but neither of this mutations have been related directly with the lipid synthesis or the homeostasis of the cell envelope. We observed as well that the resistant strain showed an increase of the thickness cell envelope. We checked that both susceptible and resistant strains did not adapt his lipid composition to the presence of the antibiotic in the culture, and this strain had two more carbons in his acyl groups than the susceptible strain. In S. capitis, the daptomycin resistance has a great influence by the lipid membrane composition, without any changes in the genes related with his synthesis or homeostasis, something that we could consider unique in the resistance to this antibiotic.