Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression.
BACKGROUND
The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations.


RESULTS
Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI.


CONCLUSIONS
In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene.
Neuroinfection Laboratory, Institute for Infectious Diseases, University of Bern, Bern, Switzerland. denis.grandgirard@ifik.unibe.ch.
Denis
D
Grandgirard
LA.M.M.B., Dip. Biotecnologie Mediche, Università di Siena, Siena, Italy. leofuri@libero.it.
Leonardo
L
Furi
LA.M.M.B., Dip. Biotecnologie Mediche, Università di Siena, Siena, Italy. laciusa@gmail.com.
Maria Laura
ML
Ciusa
Istituto Superiore di Sanità, Roma, Italy. lucilla.baldassarri@iss.it.
Lucilla
L
Baldassarri
Quotient Bioresearch, Fordham, UK. daniel.knight@uwa.edu.au.|Current address: The University of Western Australia, Nedlands, WA, Australia. daniel.knight@uwa.edu.au.
Daniel R
DR
Knight
Quotient Bioresearch, Fordham, UK. i.morrissey@ntlworld.com.|Current address: IHMA Europe Sa'rl, Epalinges, Switzerland. i.morrissey@ntlworld.com.
Ian
I
Morrissey
Institute of Clinical Chemistry, Inselspital, Bern University Hospital, and University of Bern, Bern, Switzerland. carlo.largiader@insel.ch.
Carlo R
CR
Largiadèr
Neuroinfection Laboratory, Institute for Infectious Diseases, University of Bern, Bern, Switzerland. Stephen.leib@ifik.unibe.ch.|Biology Division, Spiez Laboratory, Federal Office for Civil Protection FOCP, Spiez, Switzerland. Stephen.leib@ifik.unibe.ch.
Stephen L
SL
Leib
LA.M.M.B., Dip. Biotecnologie Mediche, Università di Siena, Siena, Italy. mro5@leicester.ac.uk.|Department of Genetics, University of Leicester, Leicester, UK. mro5@leicester.ac.uk.
Marco R
MR
Oggioni
D000891:Anti-Infective Agents, Local / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015536:Down-Regulation / Q000187:drug effects; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D014260:Triclosan / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D000891:Anti-Infective Agents, Local; D001426:Bacterial Proteins; D014260:Triclosan; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)