Antibiotic-Induced Autoactivation of IS 256 in Staphylococcus aureus

Abstract

The 3= end of rsbU, encoding the positive regulator of the stress factor sigma B, was identified as a hot spot for spontaneous IS256 insertion in Staphylococcus aureus SA137/93G. Interestingly, subinhibitory concentrations of chloramphenicol in combination with heat stress, as well as linezolid and spectinomycin at physiological temperatures, selected for such rsbU:: IS256 insertion mutants. In consequence of the inactivation of rsbU, the IS256 transposition frequency was increased 4-fold in S. aureus HG001. Staphylococcus aureus is a major human pathogen and has been able to acquire resistance to nearly all clinically used antibiotics. Insertion sequences (IS elements) have been instrumental in the development of resistance, since they represent the active component of transposons and, thus, mobilize resistance genes for horizontal gene transfer (1) or modulate the expression of resistance-conferring genes (2). Therefore, investigation of the transposition process of IS elements, especially in the presence of subinhibitory concentrations of antibiotics, will help us to understand and ultimately, perhaps, interfere with resistance development.IS256 is a highly active insertion sequence that, as a component of the composite transposon Tn4001, has played a pivotal role in the development of aminoglycoside resistance in S. aureus (3, 4). IS256 has been detected in the genome of several clinical isolates of enterococci and staphylococci (5, 6), e.g., in the clinical methicillin-resistant S. aureus (MRSA) isolates of sequence type 228 (ST228) (1 copy), ST239 (8 to 21 copies), and ST247 (at least 14 copies) (7-11). The accumulation of multiple IS256 copies in the genomes is due to its transposition process, which is mediated by a copy-and-paste mechanism (12). The effect of multiple IS256 elements on genomic flexibility and resistance development is exemplified by S. aureus SA137/93G (more than 14 copies), an ST247 strain that displays homogeneous vancomycin-intermediate resistance (vancomycin-intermediate S. aureus [VISA] phenotype) (13), which is mainly caused by the insertion of IS256 into the tcaA gene (11,14). A variety of intrinsic and extrinsic mechanisms regulate the transposition activity of mobile elements in bacteria (15). Whereas subinhibitory concentrations of ciprofloxacin and vancomycin lead to the activation of IS256 transposition (16), the alternative sigma factor B, which controls the expression of genes involved in stress response (17), is a negative regulator of IS256 transposition in S. aureus (18). In the present study, we demonstrate that frequent insertion of IS256 into the rsbU gene, which encodes a positive regulator of SigB (19), resulted in the autoactivation of IS256 transposition in S. aureus. rsbU is a hot spot for spontaneous IS256 insertions. All strains, plasmids, and oligonucleotides are shown in Tables 1 and 2. Spontaneous insertion of IS256 into rsbU was first detected during attempts to integrate temperature-sensitive plasmids harboring a chloramphenicol resistance gene into the...

Impact of efflux in the development of multidrug resistance phenotypes in Staphylococcus aureus

“…Multiple IS256 transpositions have been observed in the genome of S. aureus and correlated with reduced susceptibility to vancomycin, as a copy of IS256 was found in the promoter region of genes associated with glycopeptide resistance originating hybrid promoters, probably responsible for an increased gene expression [ 34 ]. Other studies have also shown that exposure to antibiotics such as CIP, vancomycin, chloramphenicol, linezolid and spectinomycin activates spontaneous transposition of IS256 which could be associated with a decrease of antibiotic susceptibility [ 35 – 37 ]. Moreover, it has been shown that IS256 insertion in the promoter regions of the genes mecA in Staphylococcus sciuri [ 38 ] and llm in S. aureus [ 39 ] is involved in higher transcription rates of both genes with a concomitant increased resistance to methicillin.…”

Molecular Characterization of a Prevalent Ribocluster of Methicillin-Sensitive Staphylococcus aureus from Orthopedic Implant Infections. Correspondence with MLST CC30

“…Among these, bone sialoprotein-binding protein (Bbp) and collagen adhesin (Cna) play a crucial role in the onset of device-related infections (Arciola et al, 2005b ; Xu et al, 2005 ; Campoccia et al, 2009 ; Vazquez et al, 2011 ; Post et al, 2014 ). S. aureus can also yield a variety of other virulence factors, such as Panton-Valentine leukocidin (PVL), often associated with necrotizing pneumonia and skin infections (Rasigade et al, 2011 ), toxic shock syndrome toxin (TSST), a super-antigenic toxin more common in MSSA lineages (He et al, 2013 ), and the insertion sequence IS 256 , widespread in genomes of multi-resistant staphylococci (Byrne et al, 1989 ; Depardieu et al, 2007 ; Schreiber et al, 2013 ).…”

Dynamics of bacterial insertion sequences: can transposition bursts help the elements persist?

“…Bursts in IS transposition have been reported to be associated with harsh environmental conditions [ 45 , 48 , 50 ]. As an example, antibiotic treatment results in an increased frequency of IS transposition which may accelerate the development of resistance [ 33 , 52 ]. Under our model, strong transposition bursts increase the mean fitness of organisms through selection of beneficial mutations.…”

“…The rate of IS transposition is often suppressed through such negative regulation [ 3 ]. The release from regulation of IS transpositions in response to environmental challenges has been observed [ 33 , 45 , 47 – 52 ]. We call this elevation of the transposition rate a transposition burst .…”