Transposon library screening for identification of genetic loci participating in intrinsic susceptibility and acquired resistance to antistaphylococcal agents

Blake, Katy L.; O’Neill, Alex J.

doi:10.1093/jac/dks373

Cited by 65 publications

(41 citation statements)

References 17 publications

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“…Furthermore, the identification and genomic characterization of a VSSA revertant of BPH0391 confirmed that the IS256 insertion within the walKR 5= UTR was also the cause of the VISA phenotype. A recent report of a S. aureus InsTet Gϩ 2 Cm transposon mutant library also described hVISA/VISA mutants with independent walKR 5=-UTR insertions (37). These findings suggest that altered expression of wild-type WalKR proteins is another relatively common mechanism for VISA formation, as it has previously also been reported in the VISA strain SA137/93A (24).…”

Section: Discussionsupporting

confidence: 69%

Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS 256 Tempering of WalKR Expression

McEvoy

Tsuji

Gao

et al. 2013

Antimicrob Agents Chemother

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f Vancomycin-intermediate Staphylococcus aureus (VISA) strains often arise by mutations in the essential two-component regulator walKR; however their impact on walKR function has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptible S. aureus (VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256 insertions in the walKR 5= untranslated region (5= UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction in walKR gene expression in the IS256 mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256 insertions had replaced two SigA-like walKR promoters with weaker, hybrid promoters. Removal of IS256 reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation of ssaA but no change in expression of sak and sceD in both IS256 mutants. Whole-genome sequencing of the two mutants revealed an additional IS256 insertion within agrC for one mutant, and we confirmed that this mutation abolished agr function. These data provide the first substantial analysis of walKR promoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction in walKR expression; however, the mechanisms by which this occurs remain to be determined.

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Section: Discussionsupporting

confidence: 69%

Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS 256 Tempering of WalKR Expression

McEvoy

Tsuji

Gao

et al. 2013

Antimicrob Agents Chemother

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“…We confirmed the previously established vancomycin intrinsic resistance determinants vraS (Kuroda et al, 2003; Gardete et al, 2012) and vraF of the VraFG ABC transporter system (Meehl et al, 2007). Although our screen identified recognized vancomycin intrinsic resistance determinants, we did not observe mutants of the dlt operon as seen previously in a corresponding vancomycin hypersusceptibility screen (Blake and O’Neill, 2013). No strains in the NTML exist with inactivation of any of the four genes in the dlt operon, which is involved in adding positively charged D -alanine to teichoic acids (Peschel et al, 1999).…”

Section: Resultssupporting

confidence: 52%

“…Equivalent comprehensive genome-wide studies of intrinsic resistance determinants in Gram-positive bacteria have not been performed, except for a single study that determined the intrinsic resistance of S. aureus to vancomycin, nisin and daptomycin (Blake and O’Neill, 2013). Staphylococcus aureus is an opportunistic pathogen with the capability to cause a wide range of diseases, ranging from systemic to skin infections (Lowy, 1998).…”

Section: Introductionmentioning

confidence: 99%

Genome-Wide Identification of Antimicrobial Intrinsic Resistance Determinants in Staphylococcus aureus

et al. 2016

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The emergence of antimicrobial resistance severely threatens our ability to treat bacterial infections. While acquired resistance has received considerable attention, relatively little is known of intrinsic resistance that allows bacteria to naturally withstand antimicrobials. Gene products that confer intrinsic resistance to antimicrobial agents may be explored for alternative antimicrobial therapies, by potentiating the efficacy of existing antimicrobials. In this study, we identified the intrinsic resistome to a broad spectrum of antimicrobials in the human pathogen, Staphylococcus aureus. We screened the Nebraska Transposon Mutant Library of 1920 single-gene inactivations in S. aureus strain JE2, for increased susceptibility to the anti-staphylococcal antimicrobials (ciprofloxacin, oxacillin, linezolid, fosfomycin, daptomycin, mupirocin, vancomycin, and gentamicin). Sixty-eight mutants were confirmed by E-test to display at least twofold increased susceptibility to one or more antimicrobial agents. The majority of the identified genes have not previously been associated with antimicrobial susceptibility in S. aureus. For example, inactivation of genes encoding for subunits of the ATP synthase, atpA, atpB, atpG and atpH, reduced the minimum inhibitory concentration (MIC) of gentamicin 16-fold. To elucidate the potential of the screen, we examined treatment efficacy in the Galleria mellonella infection model. Gentamicin efficacy was significantly improved, when treating larvae infected with the atpA mutant compared to wild type cells with gentamicin at a clinically relevant concentration. Our results demonstrate that many gene products contribute to the intrinsic antimicrobial resistance of S. aureus. Knowledge of these intrinsic resistance determinants provides alternative targets for compounds that may potentiate the efficacy of existing antimicrobial agents against this important pathogen.

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“…In B. subtilis, YtrA binds specifically to an inverted repeat in the ytrA and ywoB promoters, and transcription of the ytr and ywo operons is induced by cell-wall-active antibiotics including the peptide antibiotics bacitracin, vancomycin and ramnoplanin, with ytrA null mutations causing constitutive expression of both operons 27 . Notably, the entire ytrA operon has been shown to be induced by cationic AMPs in S. aureus, where it is under negative regulation by the AMP sensing system aps 28 and has also been implicated in nisin susceptibility in S. aureus SH1000 29 . Although ytrA insertions are not present in the Nebraska Transposon Mutant Library we were able to obtain 2 independent ytr operon transposon mutants with insertions downstream of ytrA which did not show any detectable difference in AMP susceptiblity relative to the wild type (Table S3).…”

Section: Resultsmentioning

confidence: 99%

Genomic signatures of experimental adaptation to antimicrobial peptides in Staphylococcus aureus

Johnston

Dobson

Rolff

2015

Preprint

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ObjectivesThe evolution of resistance against antimicrobial peptides has long been considered unlikely due to their mechanism of action, yet experimental selection with AMPs results in rapid evolution of resistance in several species of bacteria. Although numerous studies have utilized mutant screens to identify loci that determine AMP susceptibility, there is a dearth of data concerning the genomic changes which accompany experimental evolution of AMP resistance.MethodsUsing genome re-sequencing we analysed the mutations which arise during experimental evolution of resistance to the cationic AMPs iseganan, melittin and pexiganan, as well as to a combination of melittin and pexiganan, or to the aminoglycoside antibiotic streptomycin.ResultsAnalysis of 17 independently replicated Staphylococcus aureus selection lines, including unselected controls, showed that each AMP selected for mutations at distinct loci. We identify mutations in genes involved in the synthesis and maintenance of the cell envelope. This includes genes previously identified from mutant screens for AMP resistance, and genes involved in the response to AMPs and cell-wall-active antibiotics. Furthermore, transposon insertion mutants were used to verify that a number of the identified genes are directly involved in determining AMP susceptibility.ConclusionsStrains selected for AMP resistance under controlled experimental evolution displayed consistent AMP-specific mutations in genes which determine AMP susceptibility. This suggests that different routes to evolve resistance are favored within a controlled genetic background.

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Transposon library screening for identification of genetic loci participating in intrinsic susceptibility and acquired resistance to antistaphylococcal agents

Abstract: Tn library screening identified both known and novel modulators of antibacterial susceptibility in S. aureus and therefore represents a useful approach towards delineating the staphylococcal resistome.

Cited by 65 publications

References 17 publications

Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS 256 Tempering of WalKR Expression

Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS 256 Tempering of WalKR Expression

Genome-Wide Identification of Antimicrobial Intrinsic Resistance Determinants in Staphylococcus aureus

Genomic signatures of experimental adaptation to antimicrobial peptides in Staphylococcus aureus

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