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Antimicrobial Agents and Chemotherapy, May 2007, p. 1840-1843, Vol. 51, No. 5
0066-4804/07/$08.00+0     doi:10.1128/AAC.01283-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mutations in fusA Associated with Posttherapy Fusidic Acid Resistance in Clostridium difficile{triangledown}

T. Norén,1* T. Åkerlund,4 M. Wullt,3 L. G. Burman,4 and M. Unemo2

Department of Infectious Diseases,1 Department of Clinical Microbiology,2 Örebro University Hospital, Örebro, Department of Clinical Sciences, Malmö, Lund University, Lund,3 Swedish Institute for Infectious Disease Control, Solna, Sweden4

Received 13 October 2006/ Returned for modification 10 November 2006/ Accepted 2 February 2007


   ABSTRACT
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 ABSTRACT
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In silico, we identified fusA (2,067 bp) in Clostridium difficile 630. Sequencing of fusA in posttherapy fusidic acid-resistant C. difficile isolates from 12 patients with C. difficile-associated diarrhea (CDAD) identified fusA mutations, one or two nonsynonymous substitutions, or in one case a deletion of one codon associated with resistance. Five of these mutations have previously been described in fusA of fusidic acid-resistant Staphylococcus aureus, but seven were novel fusA mutations. Fusidic acid monotherapy for CDAD seemed to rapidly select conserved resistant mutants.


   TEXT
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Fusidic acid, derived from Fusidium coccineum (7), is used for the treatment of staphylococcal bone and soft tissue infections; and resistance in Staphylococcus aureus is mainly due to mutations in fusA (2, 14). This gene encodes a ribosomal translocation enzyme, elongation factor G (EF-G), which is crucial to bacterial protein synthesis and which represents the target of fusidic acid (10). fusA has also been described in, e.g., Bacillus subtilis (9), Bacillus cereus (23), Clostridium acetobutylicum (15), Clostridium perfringens (25), Clostridium tetani (3), and Salmonella enterica serovar Typhimurium (8, 12).

A decline in the rate of cure of C. difficile-associated diarrhea (CDAD) with metronidazole (6, 13) opened the way for fusidic acid as an alternative treatment, found to have a clinical efficacy similar to that of metronidazole (5, 27, 29). The in vitro susceptibility of C. difficile to fusidic acid is generally excellent (4, 11), and like for metronidazole, the high fusidic acid absorption (98%) (24) presumably results in sufficient therapeutic concentrations in feces from an inflammatory mucosa. However, we recently found that 55% of the fusidic acid-treated CDAD patients with posttherapy persistent C. difficile carried fusidic acid-resistant isolates of identical PCR ribotypes at follow-up (16). Our aims were to identify fusA of C. difficile, to explore the heterogeneity of the gene, and to find mutations associated with the emergence of fusidic acid resistance.

Origin, culture, MIC determination, and PCR ribotyping of C. difficile isolates. Of 44 patients treated with fusidic acid in our previous study (16), 20 (45%) had C. difficile persisting at follow-up, and 11 (55%) of these patients carried fusidic acid-resistant C. difficile isolates (n = 12) (Table 1). These isolates were selected for study, together with the resistant isolates recovered both pre- and posttherapy from one patient randomized to metronidazole (Table 1, patient 12) but who had received fusidic acid 2 weeks before inclusion in the study. Thus, the putative fusA genes of 27 paired C. difficile isolates from 12 patients were sequenced. Culture, MIC determination, and PCR ribotyping of the isolates were performed as described previously (16, 17).


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  		TABLE 1. Mutations in fusA associated with posttherapy fusidic acid-resistant Clostridium difficilea

 
DNA isolation and PCR of putative fusA. C. difficile isolates cultured anaerobically in peptone yeast medium for 24 h were pelleted, dissolved in 5% Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA), and boiled for 10 min. After centrifugation, the supernatant was used for PCR. The first segment (approximately 1.2 kbp) of fusA and the remaining segment of the gene (approximately 0.9 kbp), identified as described below, were amplified separately by using a LightCycler real-time PCR system (Roche Molecular Biochemicals, Mannheim, Germany) and the primers described in Table 2.


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  		TABLE 2. Primers used for PCR amplification and sequencing of fusA alleles

 
Sequencing, alignments, and phylogenetic analysis. The amplicons of fusA were purified by using a High Pure PCR product purification kit (Roche) and were sequenced with the primers described in Table 2 by using the BigDye Terminator (version 3.1) cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI Prism 3100 genetic analyzer (Applied Biosystems). Alignments of entire fusA sequences, encoding mature EF-G, and the deduced amino acid sequences were performed with BioEdit (version 5.0.9) software. Phylogenetic analysis was performed with TREECON (version 1.3b) software, as described previously (26).

Identification of fusA in C. difficile. A BLAST search of the genome of C. difficile strain 630 (http://www.sanger.ac.uk/Projects/C_difficile/) by using fusA of C. perfringens (GenBank accession no. NC_003366) identified a putative fusA (2,067 bp) in the C. difficile genome (nucleotide positions 98757 to 100823) comprising an open reading frame encoding 688 amino acids. This gene displayed 72%, 70%, and 67% identities to fusA of C. perfringens, C. acetobutylicum, and B. subtilis, respectively. By using the putative fusA of C. difficile strain 630, primers for PCR and sequencing of the gene were constructed.

Comparison of fusA sequences. Among the 27 C. difficile isolates studied, 18 unique fusA alleles and a total of 44 polymorphic nucleotide sites (2.1 per 100 sites) were identified. These comprised synonymous substitutions (n = 26), nonsynonymous substitutions (n = 15), and one 3-bp deletion (i.e., a codon). Alignment of the deduced EF-G amino acid sequences showed 13 polymorphic sites (1.9 per 100 sites).

The wild-type fusA sequences, i.e., those present in the fusidic acid-susceptible C. difficile isolates obtained on day 1, comprised 26 synonymous substitutions and 4 nonsynonymous substitutions.

However, each fusidic acid-resistant isolate had a fusA sequence identical to that of its corresponding susceptible day 1 isolate, except for one or two nonsynonymous substitutions or in one case a deletion of one codon (Table 1). A total of 10 distinct EF-G sequence variants were identified among all the 14 resistant isolates. Whereas the deletion of one amino acid (A375) resulted in a moderately increased MIC (4 mg/liter), the amino acid alterations in EF-G yielded MICs of 64 mg/liter (1 isolate) and >256 mg/liter (12 isolates).

Growth characteristics, morphology, and stability of resistant mutants. Neither colony numbers nor colony morphologies differed between the fusidic acid-susceptible isolate and the corresponding resistant mutant after subcultivation of the 27 isolates 10-fold over 22 days (data not shown). The fusidic acid MICs measured initially and after the last subcultivation were identical for 20 isolates and differed by a maximum of ±1 log2 (mg/liter) in 7 isolates.

We identified fusA in C. difficile and found that one or two nonsynonymous mutations in fusA represented the genetic key to fusidic acid resistance in C. difficile.

Five of the resistance-associated mutations identified, i.e., A374->V, D432->N, T434->I, H455->N, and H455->Q, have been described at corresponding positions of the S. aureus fusA (1, 14, 18, 19). The remaining ones (n = 7) were novel mutations associated with fusidic acid resistance and comprised either a variant substitution at a classical position (1, 18, 19), i.e., the S449->P, H455->R, and I459->R substitutions, or mutations not previously identified in any species, i.e., the M16->I, S116->L, and V119->A mutations and the deletion of A375 (Table 1). The fusidic acid resistance-associated mutations in C. difficile were mainly located in clusters, and as described for S. aureus (18), distinct locations in FusA appeared to be especially important for resistance, such as amino acid residues 432 to 459 (Table 1). This region also comprises the amino acids, centered on H455, that presumably interact directly with the fusidic acid molecule (10). In contrast to the variable levels of fusidic acid resistance associated with fusA mutations in S. aureus, all but one (deletion of A375) conferred high-level resistance. Nevertheless, additional mechanisms of fusidic acid resistance may exist in C. difficile, as reported for S. aureus (14, 18, 22). Interestingly, the in vitro growth characteristics did not differ between the resistant and the susceptible isolates, and no reversion of resistance was seen in subcultured mutant isolates. This finding, together with the late isolation of resistant C. difficile on days 35 to 40 in the absence of antimicrobial therapy, supported the notion of the frequent selection of stable fusidic acid-resistant mutants, as previously found for, e.g., S. aureus (20, 21, 28). Primary fusidic acid resistance in C. difficile is seldom seen (4, 11). However, this is probably due to the limited use of this antimicrobial in clinical practice.


   ACKNOWLEDGMENTS
 
We thank Ingegerd Alriksson and Ingela Persson for technical assistance and Inga Odenholt and Erik Bäck for continuous support.

The present study was supported by grants from the Research Committee of Örebro County.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Diseases, Örebro University Hospital, SE-701 85 Örebro, Sweden. Phone: 46-19-602 10 42. Fax: 46-19-18 48 55. E-mail: torbjorn.noren{at}orebroll.se Back

{triangledown} Published ahead of print on 16 February 2007. Back


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Antimicrobial Agents and Chemotherapy, May 2007, p. 1840-1843, Vol. 51, No. 5
0066-4804/07/$08.00+0     doi:10.1128/AAC.01283-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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