title: Two novel point mutations in clinical Staphylococcus aureus reduce linezolid susceptibility and switch on the stringent response to promote persistent infection.
abstract: Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3', 5'-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens.
authors.affiliation: Department of Microbiology, Monash University, Clayton, Victoria, Australia.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.forename: Kyra
authors.initials: K
authors.lastname: Chua
authors.forename: John K
authors.initials: JK
authors.lastname: Davies
authors.forename: Hayley J
authors.initials: HJ
authors.lastname: Newton
authors.forename: Torsten
authors.initials: T
authors.lastname: Seemann
authors.forename: Paul F
authors.initials: PF
authors.lastname: Harrison
authors.forename: Natasha E
authors.initials: NE
authors.lastname: Holmes
authors.forename: Hyun-Woo
authors.initials: HW
authors.lastname: Rhee
authors.forename: Jong-In
authors.initials: JI
authors.lastname: Hong
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Hartland
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Stinear
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Howden
mesh_terms: D000081:Acetamides / Q000494:pharmacology*; D000368:Aged; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D020869:Gene Expression Profiling; D006801:Humans; D008025:Ligases / Q000235:genetics*; D000069349:Linezolid; D008297:Male; D008780:Methyltransferases / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D023303:Oxazolidinones / Q000494:pharmacology*; D017354:Point Mutation / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013203:Staphylococcal Infections / Q000188:drug therapy / Q000235:genetics* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D014774:Virulence / Q000235:genetics
chemical_list: D000081:Acetamides; D000900:Anti-Bacterial Agents; D015415:Biomarkers; D023303:Oxazolidinones; D012333:RNA, Messenger; D012338:RNA, Ribosomal, 23S; D008780:Methyltransferases; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases; D000069349:Linezolid