Oral Presentation BACPATH 2019

A novel mechanism for translational regulation of linezolid resistance in Staphylococcus aureus (#54)

Riley JT Murphy 1 , Geoffrey W Coombs 2 , Josh P Ramsay 1
  1. Biomedical Sciences, Curtin University, Bentley, WA, Australia
  2. School of Veterinary and Laboratory Sciences, Murdoch University, Perth, Western Australia, Australia

Abstract

Linezolid has become a last defence drug for the treatment of methicillin-resistant Staphylococcus aureus. The cfr gene confers resistance to multiple phenicol antibiotics and linezolid through methylation of the 23s rRNA1,3 and is often carried on multidrug-resistance plasmids. Recently, a new chromosomally-integrated variant of the cfr gene was discovered in pig and human-isolated ST398 S. aureus in Australia2. This allele, dubbed cfrAB, contains a single base-pair deletion resulting in a premature stop codon in the first half of the ORF. Despite this, the cfrAB allele provides low-level linezolid and phenicol resistance, suggesting full-length Cfr methyltransferase is produced. Here we show that the cfrAB allele contains a programmed ribosomal frameshift site capable of inducing translational slippage to the -1 frame, facilitating full translation of Cfr. Fusion of the frameshift site between maltose-binding protein and GFP genes facilitated detection of the frameshifted protein product by SDS-PAGE and mass spectrometry. Fusion of the frameshift site to lacZ allowed quantitative detection of frameshifting through β-galactosidase assays and indicated that in E. coli this site produces around 2% the level of translation compared to the in-frame allele. Preliminary experiments also suggest sub-inhibitory levels of antibiotics such as florfenicol may stimulate frameshifting. Interestingly, the only human-isolated clone of this lineage carries the full-length cfr, suggesting a reversion of cfrAB to cfr may have occurred in situ. Consistent with this, crfAB to cfr mutations were detected at a rate of 1 x 10-8 in stationary-phase cells. We suspect that the crfAB allele may represent a recently evolutionary adaptation to facilitate reduced and potentially regulated resistance, which may confer a competitive fitness benefit. This chromosomally-integrated cfrAB allele may therefore facilitate persistence of this resistance mechanism in ST398 populations – and also may go undetected in routine phenotypic screens for resistance.

  1. Kehrenberg C, Schwarz S, Jacobsen L, Hansen L, Vester B. A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503. Mol Microbiol. 2005;57(4):1064-1073. doi:10.1111/j.1365-2958.2005.04754.x.
  2. Sahibzada S, Abraham S, Coombs G et al. Transmission of highly virulent community-associated MRSA ST93 and livestock-associated MRSA ST398 between humans and pigs in Australia. Sci Rep. 2017;7(1). doi:10.1038/s41598-017-04789-0.
  3. Toh S, Xiong L, Arias C et al. Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid. Mol Microbiol. 2007;64(6):1506-1514. doi:10.1111/j.1365-2958.2007.05744.x.