Journal of Antimicrobial Chemotherapy Advance Access published October 13, 2014
J Antimicrob Chemother doi:10.1093/jac/dku408
A novel fusidic acid resistance determinant, fusF, in Staphylococcus cohnii Hsiao-Jan Chen1, Wei-Chun Hung1, Yu-Tzu Lin1, Jui-Chang Tsai2,3, Hao-Chieh Chiu1,4, Po-Ren Hsueh4,5 and Lee-Jene Teng1,4* 1
Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan; Center for Optoelectronic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan; 3Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan; 4Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan; 5Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan 2
Received 29 May 2014; returned 1 July 2014; revised 22 September 2014; accepted 22 September 2014 Objectives: To determine MICs of fusidic acid for and identify genetic determinants of resistance in Staphylococcus cohnii isolates. Methods: Susceptibility to fusidic acid was determined by the standard agar dilution method in 24 S. cohnii subsp. urealyticus clinical isolates, 7 S. cohnii subsp. cohnii clinical isolates and 2 reference strains. Sequencing of a novel resistance determinant, fusF, and its flanking regions was performed by long and accurate PCR and inverse PCR. To evaluate the function of fusF, the MIC of fusidic acid was determined for recombinant Staphylococcus aureus carrying a plasmid expressing fusF. Results: A total of 25 S. cohnii subsp. urealyticus (24 clinical isolates and 1 reference strain) and 2 S. cohnii subsp. cohnii displayed low-level resistance to fusidic acid (MICs 2– 16 mg/L). Sequencing of a 4259 bp fragment from S. cohnii subsp. urealyticus ATCC 49330 revealed a novel resistance gene, designated fusF, which displayed 70.5% nucleotide and 67.3% amino acid identity to fusD. Expression of fusF in S. aureus confers resistance to fusidic acid. Conclusions: A novel FusB-family gene, fusF, was identified as a major resistance determinant in S. cohnii clinical isolates resistant to fusidic acid. Keywords: fusD, FusB family, staphylococci
Introduction
Materials and methods
Staphylococcus cohnii subsp. urealyticus and S. cohnii subsp. cohnii, members of the CoNS, have been described as pathogens in human diseases.1,2 Antibiotic-resistant S. cohnii collected from ward environments has been reported.3 However, susceptibility testing with fusidic acid has not yet been reported in clinical isolates and any resistance determinants remain to be identified. Fusidic acid acts by binding elongation factor G (EF-G) and blocks bacterial protein synthesis.4,5 Resistance to fusidic acid may result from alteration of the drug target site6 – 8 or may be due to the protection of the drug target site by genes encoding FusB-family proteins.9,10 Binding of FusB-family proteins to EF-G promotes the release of the EF-G/guanosine 5′ -diphosphate complex in the presence of fusidic acid and thus rescues translation.11,12 The aim of this study was to describe susceptibility to fusidic acid in S. cohnii and identify any resistance determinants. A novel fusB-type gene was identified in the fusidic acid-resistant S. cohnii.
Bacterial strains A total of 24 S. cohnii subsp. urealyticus and 7 S. cohnii subsp. cohnii collected between 2000 and 2011 in the Bacteriology Laboratory of the National Taiwan University Hospital, a 2500 bed teaching hospital in northern Taiwan, as well as S. cohnii subsp. urealyticus ATCC 49330 and S. cohnii subsp. cohnii ATCC 29974, were used. The clinical isolates were initially identified by the Vitek 2 automated system (bioMe´rieux SA, Marcy-l’E´toile, France) and were then further divided into two subspecies based on the PCR– RFLP analysis of dnaJ 13 and biochemical assays including lactose fermentation and the urease test.
Antimicrobial susceptibility testing Antimicrobial susceptibility testing was performed by the standard agar dilution method according to the guidelines of the CLSI.14 The breakpoint used to indicate fusidic acid resistance was 2 mg/L.15
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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*Corresponding author. Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan. Tel: +886-2-23123456, ext. 66918; Fax: +886-2-23711574; E-mail: [email protected]
Chen et al.
Detection of fusidic acid resistance determinants
Results
The DNA of the isolates was amplified with primers specific for fusB, fusC or fusD 16 and a pair of degenerate primers for genes encoding FusB-family proteins (uni-fusF and uni-fusR; Table S1, available as Supplementary data at JAC Online). After DNA sequencing of fusF, a pair of specific primers (fusF 119-141F and fusF 546-527R; Table S1) was designed. To detect fusA mutations, the DNA was amplified with primers fusA -68_-4916 and fusA-CR and then sequenced (Table S1) in four S. cohnii subsp. urealyticus (all resistant) and four S. cohnii subsp. cohnii (two resistant and two susceptible).
Susceptibility and fusidic acid resistance determinants
Sequencing of fusF and flanking regions
Sequence analysis of the fusF element Construction and expression of plasmids with fusF, fusB or fusD To construct plasmids with fusF, fusB or fusD, specific PCR products were amplified from S. cohnii subsp. urealyticus ATCC 49330, Staphylococcus epidermidis NTUH-7778 (JF808726) and Staphylococcus saprophyticus ATCC 15305 (AP008934), respectively (Table S1). The constructions were generated by using pRMC2 under the control of the xyl/tetO promoter,17 then transformed into Escherichia coli K12 ER2925 and confirmed by sequencing. The recombinant pRMC2 derivatives were then transformed into Staphylococcus aureus RN4220 and S. aureus ATCC 29213 with a Gene pulser Xcell (Bio-Rad Laboratories, Hercules, CA, USA).18 Ampicillin (100 mg/L) and chloramphenicol (10 mg/L) were used for the selection of recombinant E. coli and S. aureus, respectively. Anhydrotetracycline hydrochloride (0.16 mg/L) was used to induce gene expression from the xyl/tetO promoter.17
Nucleotide sequence accession numbers The sequences of fusF elements from S. cohnii subsp. urealyticus ATCC 49330 and S. cohnii subsp. cohnii ATCC 29974 were deposited in GenBank under accession numbers AB934903 and AB934908, respectively.
Sequencing of a 4259 bp fragment from S. cohnii subsp. urealyticus ATCC 49330 revealed a novel resistance gene, designated fusF, which displayed highest similarity to fusD in S. saprophyticus ATCC 15305, with 70.5% nucleotide and 67.3% amino acid identity (Figure 1 and Table S2). The sequences of the flanking genes were also most similar to those in S. saprophyticus ATCC 15305 (AP008934) (Figure 1). Sequence similarities between fusF and other fusB-type genes ranged from 50.4% to 70.5% in nucleotide sequence and 39.5% to 67.3% in amino acid sequence (Table S2). Previous studies indicated that four conserved amino acids in the C-terminal domain of FusB-family proteins (Phe-156, Lys-184, Tyr-187 and Phe-208) are key regions for the interaction with EF-G.19 In FusF, three of the above residues were found; the missing residue was Phe-156, which was replaced by tyrosine (Tyr) (Figure S1).
Truncated fusF element in S. cohnii subsp. cohnii Sequencing of a 4886 bp fragment revealed a truncated fusF in S. cohnii subsp. cohnii ATCC 29974. The truncated fusF (ORF3) was only 396 bp, showing 86.4% nucleotide identity to the 5′ end of
Figure 1. Comparison of the genetic organization of the fusF gene and surrounding regions in S. saprophyticus ATCC 15305 (AP008934), S. cohnii subsp. urealyticus ATCC 49330 (AB934903) and S. cohnii subsp. cohnii ATCC 29974 (AB934908). Arrows represent putative ORFs. Homologous regions (.70% nucleotide identity) are shaded grey and the numbers in the shaded sections represent the percentage homologies of nucleotide sequences between the corresponding ORFs. aSusceptibility to fusidic acid: R, resistant; and S, susceptible.
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To determine the sequences of the entire fusF gene and its flanking regions, a long and accurate PCR in vitro cloning kit (Takara Shuzo Co. Ltd, Japan) and inverse PCR were used (Table S1) with subsequent sequencing.
All isolates of 25 S. cohnii subsp. urealyticus (MICs ranging from 4 to 16 mg/L) and 2 of 8 of S. cohnii subsp. cohnii (MICs ranging from 0.125 to 4 mg/L) were resistant to fusidic acid. None of the fusidic acid-resistant S. cohnii possessed fusB, fusC or fusD. However, a 228 bp amplicon was generated by a pair of degenerate primers designed to amplify fusB-type genes. Each of the two subspecies of S. cohnii had the same fusA sequence. However, a total of seven amino acid differences between S. cohnii subsp. urealyticus and S. cohnii subsp. cohnii at positions 127, 213, 246, 288, 597, 691 and 695 in EF-G were found.
JAC
Novel fusidic acid resistance determinant
Table 1. MICs of fusidic acid for recombinant S. aureus strains Strain S. aureus RN4220 ATCC 29213
MIC (mg/L)
0.125 0.25
Recombinant S. aureus RN4220 expressing FusB-family protein RN4220/pRMC2 0.0625 RN4220/pRMC2:fusB 16 RN4220/pRMC2:fusD 1 RN4220/pRMC2:fusF 16 Recombinant S. aureus ATCC 29213 expressing FusB-family protein ATCC 29213/pRMC2 0.25 ATCC 29213/pRMC2:fusB 16 ATCC 29213/pRMC2:fusD 1 ATCC 29213/pRMC2:fusF 16
Funding
fusF (645 bp) in S. cohnii subsp. urealyticus (Figure 1). Downstream sequences of ORF3 also differed from those in the fusF element.
Transparency declarations
fusF mediates resistance to fusidic acid To determine whether FusF is functionally similar to other FusB-family proteins, fusF, fusB and fusD were cloned into the plasmid pRMC2 and introduced into S. aureus RN4220 (MIC¼0.125 mg/L) and ATCC 29213 (MIC¼0.25 mg/L). The MICs of fusidic acid for the recombinant S. aureus containing fusF (MIC ¼ 16 mg/L), fusB (MIC ¼ 16 mg/L) and fusD (MIC ¼ 1 mg/L) were increased (Table 1), indicating that the function of FusF was similar to that of other FusB proteins that are associated with fusidic acid resistance.
Discussion In the present study, a novel fusidic acid resistance determinant, fusF, was identified in fusidic acid-resistant S. cohnii. The fusD-like gene was designated fusF based on the cut-off value of ≤80% amino acid identity and represents a novel fusidic acidresistance determinant that belongs to the FusB family of proteins. Flanking sequences of fusF suggested that the location of fusF may not be related to mobile genetic elements such as plasmids or pathogenicity islands, although more studies are required to confirm this. The fusF gene was not detected in other staphylococcal species, such as S. epidermidis, S. saprophyticus, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus caprae, Staphylococcus warneri and Staphylococcus capitis. Moreover, our results suggested that fusF was an intrinsic factor in S. cohnii subsp. urealyticus and may not be conserved in another subspecies, S. cohnii subsp. cohnii. A truncated fusF was found in susceptible strains of S. cohnii subsp. cohnii, lacking the C-terminal regions that were considered important for resistance to fusidic acid (Figure S1). The correlation of fusF structure with subspecies is of unknown significance, but may be due to genetic evolution. The function of fusF was confirmed by the increase in MICs of fusidic acid from 0.125 or 0.25 to 16 mg/L for the recombinant S. aureus. Different from other FusB-family proteins, the amino
This work was supported by a grant from the National Science Council of Taiwan (NSC 100-2320-B-002-014-MY3).
Conflicts of interest: none to declare. The manuscript has been edited by the professional language editors from American Journal Experts (http://www.aje.com/).
Supplementary data Table S1, Table S2 and Figure S1 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 Yamashita S, Yonemura K, Sugimoto R et al. Staphylococcus cohnii as a cause of multiple brain abscesses in Weber-Christian disease. J Neurol Sci 2005; 238: 97– 100. 2 Soldera J, Nedel WL, Cardoso PR et al. Bacteremia due to Staphylococcus cohnii ssp. urealyticus caused by infected pressure ulcer: case report and review of the literature. Sao Paulo Med J 2013; 131: 59– 61. 3 Szewczyk EM, Piotrowski A, Rozalska M. Predominant staphylococci in the intensive care unit of a paediatric hospital. J Hosp Infect 2000; 45: 145–54. 4 Gao YG, Selmer M, Dunham CM et al. The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 2009; 326: 694–9. 5 Chen Y, Koripella RK, Sanyal S et al. Staphylococcus aureus elongation factor G-structure and analysis of a target for fusidic acid. FEBS J 2010; 277: 3789– 803. 6 Norstrom T, Lannergard J, Hughes D. Genetic and phenotypic identification of fusidic acid-resistant mutants with the small-colony-variant phenotype in Staphylococcus aureus. Antimicrob Agents Chemother 2007; 51: 4438– 46. 7 O’Neill AJ, Larsen AR, Henriksen AS et al. A fusidic acid-resistant epidemic strain of Staphylococcus aureus carries the fusB determinant, whereas fusA mutations are prevalent in other resistant isolates. Antimicrob Agents Chemother 2004; 48: 3594– 7. 8 Lannergard J, Norstrom T, Hughes D. Genetic determinants of resistance to fusidic acid among clinical bacteremia isolates of Staphylococcus aureus. Antimicrob Agents Chemother 2009; 53: 2059 –65. 9 O’Neill AJ, Chopra I. Molecular basis of fusB-mediated resistance to fusidic acid in Staphylococcus aureus. Mol Microbiol 2006; 59: 664– 76.
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acid at position 156 (Phe), the key residue related to interaction of FusB-family proteins and EF-G,19 was replaced by Tyr, a hydroxyl derivative of Phe, in FusF. Although fusA mutations in S. aureus can confer high-level resistance to fusidic acid,16 it is unknown whether fusA in S. cohnii subsp. urealyticus behaves in the same way. Since all of the S. cohnii subsp. urealyticus isolates tested in this study were resistant to fusidic acid, there was no susceptible fusA-carrying S. cohnii subsp. urealyticus that could be used for comparison. Thus, it is unclear whether fusA is responsible for fusidic acid resistance in S. cohnii subsp. urealyticus. In conclusion, a novel fusidic acid resistance determinant, fusF, was found in fusidic acid-resistant S. cohnii.
Chen et al.
10 O’Neill AJ, McLaws F, Kahlmeter G et al. Genetic basis of resistance to fusidic acid in staphylococci. Antimicrob Agents Chemother 2007; 51: 1737–40. 11 Cox G, Thompson GS, Jenkins HT et al. Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid. Proc Natl Acad Sci USA 2012; 109: 2102–7. 12 Guo X, Peisker K, Backbro K et al. Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling. Open Biol 2012; 2: 120016–28. 13 Hauschild T, Stepanovic S. Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism analysis of dnaJ gene. J Clin Microbiol 2008; 46: 3875– 9.
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16 Chen HJ, Hung WC, Tseng SP et al. Fusidic acid resistance determinants in Staphylococcus aureus clinical isolates. Antimicrob Agents Chemother 2010; 54: 4985– 91. 17 Corrigan RM, Foster TJ. An improved tetracycline-inducible expression vector for Staphylococcus aureus. Plasmid 2009; 61: 126– 9. 18 Schenk S, Laddaga RA. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett 1992; 73: 133– 8. 19 Cox G, Edwards TA, O’Neill AJ. Mutagenesis mapping of the proteinprotein interaction underlying FusB-type fusidic acid resistance. Antimicrob Agents Chemother 2013; 57: 4640– 4.
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14 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplement M100-S23. CLSI, Wayne, PA, USA, 2013.
15 Coutant C, Olden D, Bell J et al. Disk diffusion interpretive criteria for fusidic acid susceptibility testing of staphylococci by the National Committee for Clinical Laboratory Standards method. Diagn Microbiol Infect Dis 1996; 25: 9 –13.
J Antimicrob Chemother doi:10.1093/jac/dku408
A novel fusidic acid resistance determinant, fusF, in Staphylococcus cohnii Hsiao-Jan Chen1, Wei-Chun Hung1, Yu-Tzu Lin1, Jui-Chang Tsai2,3, Hao-Chieh Chiu1,4, Po-Ren Hsueh4,5 and Lee-Jene Teng1,4* 1
Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan; Center for Optoelectronic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan; 3Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan; 4Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan; 5Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan 2
Received 29 May 2014; returned 1 July 2014; revised 22 September 2014; accepted 22 September 2014 Objectives: To determine MICs of fusidic acid for and identify genetic determinants of resistance in Staphylococcus cohnii isolates. Methods: Susceptibility to fusidic acid was determined by the standard agar dilution method in 24 S. cohnii subsp. urealyticus clinical isolates, 7 S. cohnii subsp. cohnii clinical isolates and 2 reference strains. Sequencing of a novel resistance determinant, fusF, and its flanking regions was performed by long and accurate PCR and inverse PCR. To evaluate the function of fusF, the MIC of fusidic acid was determined for recombinant Staphylococcus aureus carrying a plasmid expressing fusF. Results: A total of 25 S. cohnii subsp. urealyticus (24 clinical isolates and 1 reference strain) and 2 S. cohnii subsp. cohnii displayed low-level resistance to fusidic acid (MICs 2– 16 mg/L). Sequencing of a 4259 bp fragment from S. cohnii subsp. urealyticus ATCC 49330 revealed a novel resistance gene, designated fusF, which displayed 70.5% nucleotide and 67.3% amino acid identity to fusD. Expression of fusF in S. aureus confers resistance to fusidic acid. Conclusions: A novel FusB-family gene, fusF, was identified as a major resistance determinant in S. cohnii clinical isolates resistant to fusidic acid. Keywords: fusD, FusB family, staphylococci
Introduction
Materials and methods
Staphylococcus cohnii subsp. urealyticus and S. cohnii subsp. cohnii, members of the CoNS, have been described as pathogens in human diseases.1,2 Antibiotic-resistant S. cohnii collected from ward environments has been reported.3 However, susceptibility testing with fusidic acid has not yet been reported in clinical isolates and any resistance determinants remain to be identified. Fusidic acid acts by binding elongation factor G (EF-G) and blocks bacterial protein synthesis.4,5 Resistance to fusidic acid may result from alteration of the drug target site6 – 8 or may be due to the protection of the drug target site by genes encoding FusB-family proteins.9,10 Binding of FusB-family proteins to EF-G promotes the release of the EF-G/guanosine 5′ -diphosphate complex in the presence of fusidic acid and thus rescues translation.11,12 The aim of this study was to describe susceptibility to fusidic acid in S. cohnii and identify any resistance determinants. A novel fusB-type gene was identified in the fusidic acid-resistant S. cohnii.
Bacterial strains A total of 24 S. cohnii subsp. urealyticus and 7 S. cohnii subsp. cohnii collected between 2000 and 2011 in the Bacteriology Laboratory of the National Taiwan University Hospital, a 2500 bed teaching hospital in northern Taiwan, as well as S. cohnii subsp. urealyticus ATCC 49330 and S. cohnii subsp. cohnii ATCC 29974, were used. The clinical isolates were initially identified by the Vitek 2 automated system (bioMe´rieux SA, Marcy-l’E´toile, France) and were then further divided into two subspecies based on the PCR– RFLP analysis of dnaJ 13 and biochemical assays including lactose fermentation and the urease test.
Antimicrobial susceptibility testing Antimicrobial susceptibility testing was performed by the standard agar dilution method according to the guidelines of the CLSI.14 The breakpoint used to indicate fusidic acid resistance was 2 mg/L.15
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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*Corresponding author. Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan. Tel: +886-2-23123456, ext. 66918; Fax: +886-2-23711574; E-mail: [email protected]
Chen et al.
Detection of fusidic acid resistance determinants
Results
The DNA of the isolates was amplified with primers specific for fusB, fusC or fusD 16 and a pair of degenerate primers for genes encoding FusB-family proteins (uni-fusF and uni-fusR; Table S1, available as Supplementary data at JAC Online). After DNA sequencing of fusF, a pair of specific primers (fusF 119-141F and fusF 546-527R; Table S1) was designed. To detect fusA mutations, the DNA was amplified with primers fusA -68_-4916 and fusA-CR and then sequenced (Table S1) in four S. cohnii subsp. urealyticus (all resistant) and four S. cohnii subsp. cohnii (two resistant and two susceptible).
Susceptibility and fusidic acid resistance determinants
Sequencing of fusF and flanking regions
Sequence analysis of the fusF element Construction and expression of plasmids with fusF, fusB or fusD To construct plasmids with fusF, fusB or fusD, specific PCR products were amplified from S. cohnii subsp. urealyticus ATCC 49330, Staphylococcus epidermidis NTUH-7778 (JF808726) and Staphylococcus saprophyticus ATCC 15305 (AP008934), respectively (Table S1). The constructions were generated by using pRMC2 under the control of the xyl/tetO promoter,17 then transformed into Escherichia coli K12 ER2925 and confirmed by sequencing. The recombinant pRMC2 derivatives were then transformed into Staphylococcus aureus RN4220 and S. aureus ATCC 29213 with a Gene pulser Xcell (Bio-Rad Laboratories, Hercules, CA, USA).18 Ampicillin (100 mg/L) and chloramphenicol (10 mg/L) were used for the selection of recombinant E. coli and S. aureus, respectively. Anhydrotetracycline hydrochloride (0.16 mg/L) was used to induce gene expression from the xyl/tetO promoter.17
Nucleotide sequence accession numbers The sequences of fusF elements from S. cohnii subsp. urealyticus ATCC 49330 and S. cohnii subsp. cohnii ATCC 29974 were deposited in GenBank under accession numbers AB934903 and AB934908, respectively.
Sequencing of a 4259 bp fragment from S. cohnii subsp. urealyticus ATCC 49330 revealed a novel resistance gene, designated fusF, which displayed highest similarity to fusD in S. saprophyticus ATCC 15305, with 70.5% nucleotide and 67.3% amino acid identity (Figure 1 and Table S2). The sequences of the flanking genes were also most similar to those in S. saprophyticus ATCC 15305 (AP008934) (Figure 1). Sequence similarities between fusF and other fusB-type genes ranged from 50.4% to 70.5% in nucleotide sequence and 39.5% to 67.3% in amino acid sequence (Table S2). Previous studies indicated that four conserved amino acids in the C-terminal domain of FusB-family proteins (Phe-156, Lys-184, Tyr-187 and Phe-208) are key regions for the interaction with EF-G.19 In FusF, three of the above residues were found; the missing residue was Phe-156, which was replaced by tyrosine (Tyr) (Figure S1).
Truncated fusF element in S. cohnii subsp. cohnii Sequencing of a 4886 bp fragment revealed a truncated fusF in S. cohnii subsp. cohnii ATCC 29974. The truncated fusF (ORF3) was only 396 bp, showing 86.4% nucleotide identity to the 5′ end of
Figure 1. Comparison of the genetic organization of the fusF gene and surrounding regions in S. saprophyticus ATCC 15305 (AP008934), S. cohnii subsp. urealyticus ATCC 49330 (AB934903) and S. cohnii subsp. cohnii ATCC 29974 (AB934908). Arrows represent putative ORFs. Homologous regions (.70% nucleotide identity) are shaded grey and the numbers in the shaded sections represent the percentage homologies of nucleotide sequences between the corresponding ORFs. aSusceptibility to fusidic acid: R, resistant; and S, susceptible.
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To determine the sequences of the entire fusF gene and its flanking regions, a long and accurate PCR in vitro cloning kit (Takara Shuzo Co. Ltd, Japan) and inverse PCR were used (Table S1) with subsequent sequencing.
All isolates of 25 S. cohnii subsp. urealyticus (MICs ranging from 4 to 16 mg/L) and 2 of 8 of S. cohnii subsp. cohnii (MICs ranging from 0.125 to 4 mg/L) were resistant to fusidic acid. None of the fusidic acid-resistant S. cohnii possessed fusB, fusC or fusD. However, a 228 bp amplicon was generated by a pair of degenerate primers designed to amplify fusB-type genes. Each of the two subspecies of S. cohnii had the same fusA sequence. However, a total of seven amino acid differences between S. cohnii subsp. urealyticus and S. cohnii subsp. cohnii at positions 127, 213, 246, 288, 597, 691 and 695 in EF-G were found.
JAC
Novel fusidic acid resistance determinant
Table 1. MICs of fusidic acid for recombinant S. aureus strains Strain S. aureus RN4220 ATCC 29213
MIC (mg/L)
0.125 0.25
Recombinant S. aureus RN4220 expressing FusB-family protein RN4220/pRMC2 0.0625 RN4220/pRMC2:fusB 16 RN4220/pRMC2:fusD 1 RN4220/pRMC2:fusF 16 Recombinant S. aureus ATCC 29213 expressing FusB-family protein ATCC 29213/pRMC2 0.25 ATCC 29213/pRMC2:fusB 16 ATCC 29213/pRMC2:fusD 1 ATCC 29213/pRMC2:fusF 16
Funding
fusF (645 bp) in S. cohnii subsp. urealyticus (Figure 1). Downstream sequences of ORF3 also differed from those in the fusF element.
Transparency declarations
fusF mediates resistance to fusidic acid To determine whether FusF is functionally similar to other FusB-family proteins, fusF, fusB and fusD were cloned into the plasmid pRMC2 and introduced into S. aureus RN4220 (MIC¼0.125 mg/L) and ATCC 29213 (MIC¼0.25 mg/L). The MICs of fusidic acid for the recombinant S. aureus containing fusF (MIC ¼ 16 mg/L), fusB (MIC ¼ 16 mg/L) and fusD (MIC ¼ 1 mg/L) were increased (Table 1), indicating that the function of FusF was similar to that of other FusB proteins that are associated with fusidic acid resistance.
Discussion In the present study, a novel fusidic acid resistance determinant, fusF, was identified in fusidic acid-resistant S. cohnii. The fusD-like gene was designated fusF based on the cut-off value of ≤80% amino acid identity and represents a novel fusidic acidresistance determinant that belongs to the FusB family of proteins. Flanking sequences of fusF suggested that the location of fusF may not be related to mobile genetic elements such as plasmids or pathogenicity islands, although more studies are required to confirm this. The fusF gene was not detected in other staphylococcal species, such as S. epidermidis, S. saprophyticus, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus caprae, Staphylococcus warneri and Staphylococcus capitis. Moreover, our results suggested that fusF was an intrinsic factor in S. cohnii subsp. urealyticus and may not be conserved in another subspecies, S. cohnii subsp. cohnii. A truncated fusF was found in susceptible strains of S. cohnii subsp. cohnii, lacking the C-terminal regions that were considered important for resistance to fusidic acid (Figure S1). The correlation of fusF structure with subspecies is of unknown significance, but may be due to genetic evolution. The function of fusF was confirmed by the increase in MICs of fusidic acid from 0.125 or 0.25 to 16 mg/L for the recombinant S. aureus. Different from other FusB-family proteins, the amino
This work was supported by a grant from the National Science Council of Taiwan (NSC 100-2320-B-002-014-MY3).
Conflicts of interest: none to declare. The manuscript has been edited by the professional language editors from American Journal Experts (http://www.aje.com/).
Supplementary data Table S1, Table S2 and Figure S1 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 Yamashita S, Yonemura K, Sugimoto R et al. Staphylococcus cohnii as a cause of multiple brain abscesses in Weber-Christian disease. J Neurol Sci 2005; 238: 97– 100. 2 Soldera J, Nedel WL, Cardoso PR et al. Bacteremia due to Staphylococcus cohnii ssp. urealyticus caused by infected pressure ulcer: case report and review of the literature. Sao Paulo Med J 2013; 131: 59– 61. 3 Szewczyk EM, Piotrowski A, Rozalska M. Predominant staphylococci in the intensive care unit of a paediatric hospital. J Hosp Infect 2000; 45: 145–54. 4 Gao YG, Selmer M, Dunham CM et al. The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 2009; 326: 694–9. 5 Chen Y, Koripella RK, Sanyal S et al. Staphylococcus aureus elongation factor G-structure and analysis of a target for fusidic acid. FEBS J 2010; 277: 3789– 803. 6 Norstrom T, Lannergard J, Hughes D. Genetic and phenotypic identification of fusidic acid-resistant mutants with the small-colony-variant phenotype in Staphylococcus aureus. Antimicrob Agents Chemother 2007; 51: 4438– 46. 7 O’Neill AJ, Larsen AR, Henriksen AS et al. A fusidic acid-resistant epidemic strain of Staphylococcus aureus carries the fusB determinant, whereas fusA mutations are prevalent in other resistant isolates. Antimicrob Agents Chemother 2004; 48: 3594– 7. 8 Lannergard J, Norstrom T, Hughes D. Genetic determinants of resistance to fusidic acid among clinical bacteremia isolates of Staphylococcus aureus. Antimicrob Agents Chemother 2009; 53: 2059 –65. 9 O’Neill AJ, Chopra I. Molecular basis of fusB-mediated resistance to fusidic acid in Staphylococcus aureus. Mol Microbiol 2006; 59: 664– 76.
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acid at position 156 (Phe), the key residue related to interaction of FusB-family proteins and EF-G,19 was replaced by Tyr, a hydroxyl derivative of Phe, in FusF. Although fusA mutations in S. aureus can confer high-level resistance to fusidic acid,16 it is unknown whether fusA in S. cohnii subsp. urealyticus behaves in the same way. Since all of the S. cohnii subsp. urealyticus isolates tested in this study were resistant to fusidic acid, there was no susceptible fusA-carrying S. cohnii subsp. urealyticus that could be used for comparison. Thus, it is unclear whether fusA is responsible for fusidic acid resistance in S. cohnii subsp. urealyticus. In conclusion, a novel fusidic acid resistance determinant, fusF, was found in fusidic acid-resistant S. cohnii.
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16 Chen HJ, Hung WC, Tseng SP et al. Fusidic acid resistance determinants in Staphylococcus aureus clinical isolates. Antimicrob Agents Chemother 2010; 54: 4985– 91. 17 Corrigan RM, Foster TJ. An improved tetracycline-inducible expression vector for Staphylococcus aureus. Plasmid 2009; 61: 126– 9. 18 Schenk S, Laddaga RA. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett 1992; 73: 133– 8. 19 Cox G, Edwards TA, O’Neill AJ. Mutagenesis mapping of the proteinprotein interaction underlying FusB-type fusidic acid resistance. Antimicrob Agents Chemother 2013; 57: 4640– 4.
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14 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplement M100-S23. CLSI, Wayne, PA, USA, 2013.
15 Coutant C, Olden D, Bell J et al. Disk diffusion interpretive criteria for fusidic acid susceptibility testing of staphylococci by the National Committee for Clinical Laboratory Standards method. Diagn Microbiol Infect Dis 1996; 25: 9 –13.
