Genetic basis for in vitro and in vivo resistance to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) in Enterococcus faecium

Abstract

As opposed to Enterococcus faecalis, which is intrinsically resistant to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) by production of the ABC protein Lsa(A), Enterococcus faecium is naturally susceptible. Since this phenotype may be selected for in vivo by quinupristin-dalfopristin (Q-D), the aim of this study was to investigate the molecular mechanism of acquired LSAP resistance in E. faecium. Six LSAP-resistant in vitro mutants of E. faecium HM1070 as well as three different pairs of clinical isolates (pre- and postexposure to Q-D) were studied. The full genome sequence of an in vitro mutant (E. faecium UCN90B) was determined by using 454 sequencing technology and was compared with that of the parental strain. Single-nucleotide replacement was carried out to confirm the role of this mutation. By comparative genomic analysis, a point mutation was found within a 1,503-bp gene coding for an ABC homologue showing 66% amino acid identity with Lsa(A). This mutation (C1349T) led to an amino acid substitution (Thr450Ile). An identical mutation was identified in all in vitro and in vivo resistant strains but was not present in susceptible strains. The wild-type allele was named eat(A) (for Enterococcus ABC transporter), and its mutated allelic variant was named eat(A)v. The introduction of eat(A)v from UCN90B into HM1070 conferred the LSAP phenotype, whereas that of eat(A) from HM1070 into UCN90B restored susceptibility entirely. This is the first description of the molecular mechanism of acquired LSAP resistance in E. faecium. Characterization of the biochemical mechanism of resistance and the physiological role of this ABC protein need further investigations.

Fig 1

Amino acid sequence comparison of the Eat(A) and Eat(A)_v ABC proteins with Lsa-like proteins involved in MLS resistance, Lsa(A), Lsa(B), Lsa(C), and Lsa(E). The two copies of Walker A and B motifs and ABC signatures are boxed. Similarities in amino acid sequences are marked by asterisks (same amino acid), colons (strong similarity), and dots (family similarity). Multiple-sequence alignment was done with ClustalX 1.83 software. The location of the Thr450Ile substitution is indicated by an arrow.

Fig 2

Schematic map of genetic environment of eat(A) (i.e., EFAU004_00630) in the genome of E. faecium Aus0004 (GenBank accession no. NC_017022). Open reading frames are shown as arrows indicating the orientation of their coding sequence. The genes EFAU004_00628, EFAU004_00629, and EFAU004_00631 code for a 2-dehydropentoate 2-reductase, a glycerate kinase, and a phosphoglycerate mutase, respectively. The nucleotide sequence corresponding to the upstream region of the eat(A) gene is represented in detail. The −35 and −10 promoter boxes are underlined, and the transcription start site is represented by an arrow. The start codon of eat(A) and its putative ribosome-binding site (RBS) as well as the stop codon of EFAU004_00629 are also indicated.