Previous studies of fusidic acid-resistance in clinical isolates have mostly focused on methicillin-susceptible S. aureus (MSSA) and other staphylococci [17, 20, 26]. Chen et al. recently reported that the prevalence of fusidic acid-resistance determinants was quite different between MRSA and MSSA groups . In northern Taiwan collections, the fusA mutations were the major determinant (84%) followed by fusC with 16% fusidic acid-resistance in MRSA isolates . In the present study based in central Taiwan, we found that the fusidic acid-resistant predominant determinant in MRSA was a high prevalence of fusC with 74% in clinical isolates. Furthermore, one isolate carried the fusB determinant on the plasmid and fusC determinant on the chromosome in a clinical fusidic acid-resistant S. aureus isolate. The FusC protein has a 45% amino acid similarity to FusB. The fusC gene was originally identified in the genome sequence of S. aureus MSSA476, and has been reported in fusidic acid-resistant S. intermedius and S. epidermidis [18, 20]. In most European collections, fusC has been shown to be responsible for resistance to fusidic acid in all S. aureus strains examined that do not carry fusB or resistance mutations in fusA [17, 18]. Moreover, the fusB gene has only been detected in MSSA, not in MRSA in most clinical collections in Taiwan . Therefore, the present study shows the spread of fusC in Taiwan and for the first time demonstrates the presence of both fusB and fusC in a MRSA clinical isolate.
The most common mutation in fusA that conferred resistance to fusidic acid was the substitution H457Y in our study (Table 1). We reviewed the English literature and did not find any reports of two amino acid substitutions in EF-G of G556S and R659L relative to the resistance of fusidic acid. Mutations in EF-G are associated with fitness cost in the fusidic acid-resistance of S. aureus in vitro and in vivo [12, 14]. The resistance mutations with amino acid substitutions occur mostly in structural domain III of EF-G, but some occur in domains I and V [28, 29]. We identified a novel substitution present in fusidic acid-resistant S. aureus (isolates 9 and 33), which conferred an identical resistance mutation in fusA (G556S). The two isolates exhibited resistance to fusidic acid with MIC = 16 μg/ml and carried neither fusB nor fusC. In addition, substitution G556S was found in isolates 10 and 21 and was accompanied by mutations in fusA (H457Y). Another novel substitution amino acid substitution R659L located in domain V of EF-G was found to be accompanied with fusC mutations in our study. The role of this newly found amino acid substitution in fusA on the level of resistance is unknown and needs further investigation. Of the 34 isolates that were studied completely, isolate 4 harboured fusC and a resistance mutation in fusA (H457Y). This indicates that the fusidic acid-resistance in these MRSA clinical isolates had multiple genetic lineages.
The isolates with fusB and fusC determinants usually displayed higher level resistance to fusidic acid (> 16 μg/ml) [8, 17]. The MICs of fusidic acid in our collections carrying fusC ranged from 2-64 μg/ml. It is not clear the reason why in non-selective subcultures, isolate 29 with one mutation site of the fusC gene lost the resistance to fusidic acid. We hypothesized that the mutation may result in FusC truncated after amino acid 174, and thus isolate 29 became susceptible. In this study, the single-amino-acid substitutions in EF-G substitution did not result in a high level fusidic acid resistance which is similar to previous report in MRSA strains belonging to CC8, H457Y mutation was associated with MIC of 64 μg/L and H457Q was associated with MIC of 4 μg/L . The level of fusidic acid resistance in the isolate 4 with two fusidic acid resistance determinants couldn't be accounted for by their genotypes when compared with other clinical isolates with one of the determinants. A previous study showed a similar result that a laboratory strain containing both fusA resistance mutation and fusB failed to increase the level of fusidic acid resistance . The chromosomal gene fusC confer resistance to fusidic acid on S. aureus or S. intermedius is identified with 45% amino acid similarity to FusB, protect EF-G from the antibiotic . Genes for FusB-type resistance (fusB and fusC) are thought to act by the same mechanism of protection the drug target . It remains unclear whether these resistance mechanisms of a strain do act in combination or not. The precise action mode of FusB-type resistance awaits further investigation. The level of fusidic acid resistance in isolate 32 did not decrease after curing the pUB101 plasmid. The result may indicate that the resistance mechanisms do not act synergistically or additively.
In this study, all MRSA isolates met the criteria of being health-care associated. PFGE patterns revealed that there was greater than 80% similarity among the isolates. MLST and SCCmec typing showed that all isolates belonged to ST239 and carried SCCmec III elements, which is the most prevalent health care-associated strain of MRSA in Taiwan . A previous study conducted in 2002-2007 in northern Taiwan also revealed that most of fusidic acid-resistant MRSA isolates carried SCCmec type III . The two studies results suggest that a clonal strain had disseminated in Taiwan during the period of the study. In contrast to our findings, a previous European study finding indicated that the majority of fusidic acid-resistant MRSA isolates belonged to CC80-MRSA-IV clone carrying fusB and CC5 clone harbouring fusC .