Detection of a New cfr-Like Gene, cfr(B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program

Abstract

Two linezolid-resistant Enterococcus faecium isolates (MICs, 8 μg/ml) from unique patients of a medical center in New Orleans were included in this study. Isolates were initially investigated for the presence of mutations in the V domain of 23S rRNA genes and L3, L4, and L22 ribosomal proteins, as well as cfr. Isolates were subjected to pulsed-field gel electrophoresis (just one band difference), and one representative strain was submitted to whole-genome sequencing. Gene location was also determined by hybridization, and cfr genes were cloned and expressed in a Staphylococcus aureus background. The two isolates had one out of six 23S rRNA alleles mutated (G2576T), had wild-type L3, L4, and L22 sequences, and were positive for a cfr-like gene. The sequence of the protein encoded by the cfr-like gene was most similar (99.7%) to that found in Peptoclostridium difficile, which shared only 74.9% amino acid identity with the proteins encoded by genes previously identified in staphylococci and non-faecium enterococci and was, therefore, denominated Cfr(B). When expressed in S. aureus, the protein conferred a resistance profile similar to that of Cfr. Two copies of cfr(B) were chromosomally located and embedded in a Tn6218 similar to the cfr-carrying transposon described in P. difficile. This study reports the first detection of cfr genes in E. faecium clinical isolates in the United States and characterization of a new cfr variant, cfr(B). cfr(B) has been observed in mobile genetic elements in E. faecium and P. difficile, suggesting potential for dissemination. However, further analysis is necessary to access the resistance levels conferred by cfr(B) when expressed in enterococci.

FIG 1

PFGE of E. faecium genomic DNA digests and Southern blot hybridization with a cfr-specific digoxigenin-labeled probe. (A) SmaI digests; (B) EcoRI digests; (C) ICeuI digests. Lanes λ, 1, and 2 represent lambda DNA molecular marker, E. faecium 18203R, and E. faecium 18961R, respectively.

FIG 2

Evolutionary relationships predicted from the Cfr, Cfr(B), and RlmN sequence alignment. The length of each branch represents the distance between sequences, while the units at the bottom of the tree indicate the number of substitution events. Reference information and accession numbers are given in parentheses here for the following proteins: Cfr(B) (KR610408) from E. faecium strains 18203R and 18961R (this study; USA), Cfr(B) (GenBank accession number EFQ16578) from E. faecalis TX0635/WH245 (USA) (37), Cfr(B) (GenBank accession number EOI25076) from E. faecalis WH571 (USA) (37), Cfr(B) (GenBank accession number EOI92877) from E. faecalis TX0635/WH245 (USA) (37), Cfr(B) (GenBank accession number EJX57584) from E. faecium R497 (USA) (38), Cfr(B) (GenBank accession number AIX48091) from P. difficile 11107643 (Spain) (23), Cfr(B) (GenBank accession number CDF47262) from P. difficile Ox2167 (England) (32), Cfr(B) (GenBank accession number EQF87020) from P. difficile 824 (USA; not available), Cfr (GenBank accession number NP_899167) from S. sciuri (Germany) (39), Cfr (GenBank accession number ACC77590) from S. aureus 737X (USA) (13), RlmN (GenBank accession number AIL04523) from E. faecalis ATCC 29212 (USA; not available), RlmN (GenBank accession number EFS10488) from E. faecium TX0082 (USA) (40), and RlmN (GenBank accession number ZP_06924569) from S. aureus ATCC 51811 (USA) (41).

FIG 3

Schematic representation of DNA sequences (8.4 kb) surrounding cfr(B).