Mutations affecting the Rossman fold of isoleucyl-tRNA synthetase are correlated with low-level mupirocin resistance in Staphylococcus aureus.
ABSTRACT: The isoleucyl-tRNA synthetase (ileS) gene was sequenced in toto from 9 and in part from 31 Staphylococcus aureus strains with various degrees of susceptibility to mupirocin. All strains for which the mupirocin MIC was greater than 8 microg/ml contained point mutations affecting the Rossman fold via Val-to-Phe changes at either residue 588 (V588F) or residue 631 (V631F). The importance of the V588F mutation was confirmed by an allele-specific PCR survey of 32 additional strains. Additional mutations of uncertain significance were found in residues clustered on the surface of the IleS protein.
Project description:Emergence and spread of low-level mupirocin resistance in staphylococci have been increasingly reported in recent years. The aim of this study was to characterize missense mutations within the chromosomal isoleucyl-tRNA synthetase gene (ileS) among clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) with low-level mupirocin resistance. A total of 20 isolates of MRSA with low-level mupirocin resistance (minimal inhibitory concentration, 16-64 microg/mL) were collected from 79 patients in intensive care units for six months. The isolates were analyzed for isoleucyl-tRNA synthetase (IleS) mutations that might affect the binding of mupirocin to the three-dimensional structure of the S. aureus IleS enzyme. All isolates with low-level mupirocin resistance contained the known V588F mutation affecting the Rossman fold, and some of them additionally had previously unidentified mutations such as P187F, K226T, F227L, Q612H, or V767D. Interestingly, Q612H was a novel mutation that was involved in stabilizing the conformation of the catalytic loop containing the KMSKS motif. In conclusion, this study confirms that molecular heterogeneity in ileS gene is common among clinical MRSA isolates with low-level mupirocin resistance, and further study on clinical mutants is needed to understand the structural basis of low-level mupirocin resistance.
Project description:Mupirocin is an antibiotic commonly used in selective media for the isolation of bifidobacteria. However, little is known about the genetic traits responsible for bifidobacterial resistance to mupirocin. Our investigation demonstrates that all of the bifidobacteria tested exhibit a phenotype of generally high resistance to this antibiotic. The genotypic reason for bifidobacterial mupirocin resistance was further characterized by sequencing of the isoleucyl-tRNA synthetase gene (ileS) coupled with three-dimensional modeling of the encoded protein and cloning of the ileS gene of Bifidobacterium bifidum PRL2010 in a mupirocin-sensitive Escherichia coli strain. These analyses revealed key amino acid residues of the IleS protein that apparently are crucial for conferring a mupirocin resistance phenotype to bifidobacteria.
Project description:Mupirocin is a topical antibiotic used for the treatment of skin infections and the eradication of methicillin-resistant Staphylococcus aureus carriage. It inhibits bacterial protein synthesis by interfering with isoleucyl-tRNA synthetase activity. High-level mupirocin resistance (MIC of ≥ 512 μg/ml) is mediated by the expression of mupA (ileS2), which encodes an alternate isoleucyl-tRNA synthetase. In this study, we describe high-level mupirocin resistance mediated by a novel locus, mupB. The mupB gene (3,102 bp) shares 65.5% sequence identity with mupA but only 45.5% identity with ileS. The deduced MupB protein shares 58.1% identity (72.3% similarity) and 25.4% identity (41.8% similarity) with MupA and IleS, respectively. Despite this limited homology, MupB contains conserved motifs found in class I tRNA synthetases. Attempts to transfer high-level mupirocin resistance via conjugation or transformation (using plasmid extracts from an mupB-containing strain) were unsuccessful. However, by cloning the mupB gene into a shuttle vector, it was possible to transfer the resistance phenotype to susceptible S. aureus by electroporation, proving that mupB was responsible for the high-level mupirocin resistance. Further studies need to be done to determine the prevalence of mupB and to understand risk factors and outcomes associated with resistance mediated by this gene.
Project description:BACKGROUND:The data on the prevalence of resistance to mupirocin (MUP), fusidic acid (FA) and retapamulin (RET) in methicillin-resistant Staphylococcus aureus (MRSA) from China are still limited. This study aimed to examine these three antibiotics resistance in 1206 MRSA clinical isolates from Eastern China. Phenotypic MUP, FA and RET resistance was determined by minimum inhibitory concentrations (MICs), and genotypic by PCR and DNA sequencing of the mupA/B, fusB-D, cfr, vgaA/Av/ALC/B/C/E, lsaA-C/E and salA and mutations in ileS, fusA/E, rplC, and 23S RNA V domain. The genetic characteristics of resistance isolates were conducted by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS:Overall MRSA MUP, FA and RET resistance was low (5.1, 1.0 and 0.3%, respectively). MupA was the mechanism of high-level MUP resistance. All low-level MUP resistance isolates possessed an equivocal mutation N213D in IleS; of these, 2 reported an additional V588F mutation with an impact on the Rossman fold. FusA mutations, such as L461K, H457Q, H457Y and V90I were the primary FA mechanisms among high-level resistance isolates, most of which also contained fusC; however, all low-level resistance strains carried fusB. Except lsaE gene detected in one isolate, no other resistance mechanisms tested were found among RET-resistant isolates. Additionally, sixteen PFGE types (A-P) were observed, among which type B was the most common (49/76, 64.5%), followed by types E and G (4/76, 5.3% each) and types C and M (3/76, 3.9% each). All resistant strains were divided into 15 ST types by MLST. ST764 (24/76, 31.6%), ST630 (11/76, 14.5%), ST239 (9/76, 11.8%) and ST5 (7/76, 9.2%) were the major types. PFGE type B isolates with the aforementioned STs were mainly found in mupirocin resistant isolates. CONCLUSIONS:MUP, FA and RET exhibited highly activity against the MRSA isolates. Acquired genes and chromosome-borne genes mutations were responsible for MUP and FA resistance; however, the mechanism for some RET-resistant isolates remains to be further elucidated. Also, the surveillance to MUP in MRSA should be strengthened to prevent elevated resistance due to the expansion of clones.
Project description:Chromosomal resistance to mupirocin in clinical isolates of Staphylococcus aureus arises from V(588)F or V(631)F mutations in isoleucyl-tRNA synthetase (IRS). Whether these are the only IRS mutations that confer mupirocin resistance or simply those that survive in the clinic is unknown. Mupirocin-resistant mutants of S. aureus 8325-4 were therefore generated to examine their ileS genotypes and the in vitro and in vivo fitness costs associated with them before and after compensatory evolution. Most spontaneous first-step mupirocin-resistant mutants carried V(588)F or V(631)F mutations in IRS, but a new mutation (G(593)V) was also identified. Second-step mutants carried combinations of previously identified IRS mutations (e.g., V(588)F/V(631)F and G(593)V/V(631)F), but additional combinations also occurred involving novel mutations (R(816)C, H(67)Q, and F(563)L). First-step mupirocin-resistant mutants were not associated with substantial fitness costs, a finding that is consistent with the occurrence of V(588)F or V(631)F mutations in the IRS of clinical strains. Second-step mutants were unfit, but fitness could be restored by subculture in the absence of mupirocin. In most cases, this was the result of compensatory mutations that also suppressed mupirocin resistance (e.g., A(196)V, E(190)K, and E(195)K), despite retention of the original mutations conferring resistance. Structural explanations for mupirocin resistance and loss of fitness were obtained by molecular modeling of mutated IRS enzymes, which provided data on mupirocin binding and interaction with the isoleucyl-AMP reactive intermediate.
Project description:The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase (IleRS), contains a long mRNA leader region. This region exhibits many of the features of the gram-positive synthetase gene family, including the T box and leader region terminator and antiterminator. The terminator was shown to be functional in vivo, and readthrough increased during growth in the presence of mupirocin, an inhibitor of IleRS activity. The S. aureus ileS leader structure includes several critical differences from the other members of the T-box family, suggesting that regulation of this gene in S. aureus may exhibit unique features.
Project description:BACKGROUND:Antimicrobial resistance is an increasingly serious problem in public health globally. Monitoring resistance levels within healthcare and community settings is critical to combat its ongoing increase. This study aimed to describe the rates and molecular mechanisms of mupirocin resistance in clinical Staphylococcus aureus isolates from Tygerberg Hospital, and to describe its association with strain types. METHODS:We retrospectively selected 212?S. aureus isolates which were identified from blood samples and pus swabs during the years 2009-2011 and 2015-2017. The isolates were identified using conventional microbiological methods and genotyping was done using spa typing. Cefoxitin (30??g) disc diffusion and the two disc strategy (5??g and 200??g) were used to determine susceptibility to methicillin and mupirocin, respectively. Isolates with high-level resistance were screened for the plasmid mediated genes mupA and mupB by PCR, and sequencing of the ileS gene was done for all isolates exhibiting low-level resistance to describe the mutations associated with this phenotype. Chi-square test was used to assess the associations between mupirocin resistance and S. aureus genotypes. RESULTS:Of 212?S. aureus isolates, 12% (n?=?25) were resistant to mupirocin, and 44% (n?=?93) were methicillin resistant. Strain typing identified 73 spa types with spa t045 being the most predominant constituting 11% of the isolates. High-level mupirocin resistance was observed in 2% (n?=?5), and low-level resistance in 9% (n?=?20) of the isolates. The prevalence of high-level mupirocin resistance amongst MRSA and MSSA was 4 and 1% respectively, while the prevalence of low-level mupirocin resistance was significantly higher in MRSA (18%) compared to MSSA (3%), (p?=?0.032). mupA was the only resistance determinant for high-level resistance, and the IleS mutation V588F was identified in 95% of the isolates which showed low-level resistance. A significant association was observed between spa type t032 and high-level mupirocin resistance, and types t037 and t012 and low-level resistance (p?<? 0.0001). CONCLUSION:The study reported higher rates of low-level mupirocin resistance compared to high-level resistance, and in our setting, mupirocin resistance was driven by certain genotypes. Our study advocates for the continuous screening for mupirocin resistance in S. aureus in clinical settings to better guide treatment and prescribing practices.
Project description:The nucleotide sequence of the ileS gene conferring high-level resistance to mupirocin in Staphylococcus aureus J2870 has been determined. The gene sequence is substantially different from that of the native ileS gene of S. aureus, indicating that high-level resistance to mupirocin results from the acquisition of a novel ileS gene.
Project description:Of 137 Staphylococcus lugdunensis isolates collected from two nephrology centers in Hong Kong, 10 (7.3%) and 3 (2.2%) isolates had high-level and low-level mupirocin resistance, respectively. Isolates with high-level resistance contained the plasmid-mediated ileS2 gene, while isolates with low-level resistance contained the mutation V588F within the chromosomal ileS gene. All but one of the ileS2-positive isolates belong to the predominating clone HKU1. Plasmids carrying the ileS2 gene were mosaic and also cocarry multiple other resistance determinants.
Project description:In the present study, we analyzed the response of S. aureus to mupirocin, the drug of choice for nasal decolonization. Mupirocin selectively inhibits the bacterial isoleucyl-tRNA synthetase (IleRS), leading to the accumulation of uncharged isoleucyl-tRNA and eventually the synthesis of (p)ppGpp. The alarmone (p)ppGpp induces the stringent response, an important global transcriptional and translational control mechanism that allows bacteria to adapt to nutritional deprivation. To identify proteins with an altered synthesis pattern in response to mupirocin treatment, we used the highly sensitive 2-dimensional gel electrophoresis technique in combination with mass spectrometry. The results were complemented by DNA microarray, Northern blot, and metabolome analyses. Whereas expression of genes involved in nucleotide biosynthesis, DNA metabolism, energy metabolism, and translation was significantly downregulated, expression of isoleucyl-tRNA synthetase, the branched-chain amino acid pathway, and genes with functions in oxidative-stress resistance (ahpC and katA) and putative roles in stress protection (the yvyD homologue SACOL0815 and SACOL1759 and SACOL2131) and transport processes was increased. A comparison of the regulated genes to known regulons suggests the involvement of the global regulators CodY and SigB in shaping the response of S. aureus to mupirocin. Of particular interest was the induced transcription of genes encoding virulence-associated regulators (i.e., arlRS, saeRS, sarA, sarR, sarS, and sigB), as well as genes directly involved in the virulence of S. aureus (i.e., fnbA, epiE, epiG, and seb).