Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid

Abstract

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Figure 1

An unusual posttranscriptional modification of A2503 in 23S rRNA of the clinical MRSA isolate. Primer extension analysis of 23S rRNA isolated from a control wild type strain RN6390B (wt) and clinical linezolid-resistant S. aureus strain CM05 (CM05) or a laboratory strain RN4220 transformed with an empty vector pLI50 (vector) or cfr-expressing plasmid pLXM1 (pLXM1). Sequencing lanes are marked (G, A). The position of the reverse transcriptase stop corresponding to characteristic posttranscriptional modifications at A2058 and A2503 are indicated by arrows. A, ErmB-specific dimethylation of A2058. B and C, Cfr-specific modification of A2503. D. PCR amplification of a 330 bp portion of the cfr gene from the genome of CM05 cells. Similar amount of template DNA prepared from wild type S. aureus cells was used in the control (wt).

Figure 2

Localization of the cfr gene on the chromosome of the S. aureus CM05 strain. A), Restriction analysis of the 32-kb plasmid isolated from the CM05 strain. B), C), Southern blotting analysis of the total genomic (G) or plasmid (P) DNA prepared from the CM05 cells. The full-length cfr probe was used for hybridization. The sizes of DNA fragments in the DNA marker are indicated.

Figure 3

The gene cfr and its environment on the chromosome of the S. aureus strain CM05. The complete genes present within the sequenced regions are shown as solid arrows and incomplete genes are shown as solid rectangles. The unsequenced parts of the identified genes are shown as contoured arrows. The presence of the presumably complete istAS and istBS genes constituting the IS21–558 insertion sequence was verified by PCR (Smith and Mankin, unpublished). The location of the ermB and cfr leader ORFs (ermBL and cfrL, respectively) which might be involved in translation attenuation is shown. The ermB promoter (P_erm) upstream of the ermB gene and a putative terminator (term) downstream from the cfr gene which might control transcription of the mlr operon are shown. The 290 bp-long inverted repeats flanking the ermB gene, which include the 28 bp-long transposon tn917 repeats, are shown by arrows. The scale is in bp.

Figure 4

The proximity of A2503, which is the target of Cfr-modification, to linezolid bound in the A-site of the bacterial ribosome. The orientation and placement of linezolid within its binding site in the translating bacterial ribosome was taken from the reference (Leach et al., 2007). RNA nucleotides constituting the linezolid binding site are shown as lines (blue) and are also rendered as a surface representation. Linezolid and A2503 are shown as sticks. A 2-methyl group has been computationally added to A2503. In linezolid, carbon atoms are colored orange, nitrogens - blue, oxygens - red and fluorine is light-blue. The proximity of 2’O and C8 of A2503 with the linezolid C5 side chain is indicated by dashed lines.