Dissemination of fusidic acid resistance among Staphylococcus aureus clinical isolates

A significant trend towards increased fusidic acid (FA) resistance among Staphylococcus aureus with increased duration of use is of concern. The aim of the present study is to investigate the dissemination of fusidic acid resistance among Staphylococcus aureus clinical isolates. The susceptibility of S. aureus isolates to antimicrobial agents was determined by disc-diffusion method. The minimal inhibitory concertrations(MICs) of fusidic acid and vacomycin for fusidic acid-resisitant isolates were determined by ager dillution method. FA resistance determinants were determined by PCR and DNA sequencing. SCCmec typing, spa typing and multi-locus sequence typing were used for the determination of molecular characteristics for S. aureus isolates. A total of 392 non-duplicate S. aureus isolates including 181 methicillin-resistant S. aureus (MRSA) isolates, which were isolated from the clinical specimens of patients at a Chinese tertiary hospital from January, 2012 to September, 2013, were collected for investigating FA resistance. Among 392 S. aureus clinical isolates tested, 56 (14.3 %) with FA MIC values ranging from 2 μg/ml to ≥128 μg/ml were resistant to FA. The proportions of FA resistance among MRSA and MSSA isolates were 27.1 % (49/181) and 3.3 % (7/211). There was a trend of rapidly increased FA resistance among S. aureus and MRSA isolates from 5.2 % and 8.9 % in 2012 to 24.9 % and 45.1 % in 2013. Acquired FA resistance gene, fusB, was present in 73.2 % (41/56) of FA-resistant S. aureus isolates. fusC and fusA mutation were not found in any of tested isolates. A total of 9 sequence types (STs) and 12 spa types were identified among the 56 FA-resistant S. aureus isolates. ST5 accounting for 66.1 % (37/56) was the most prevalent ST. The majority (92.9 %, 52/56) of the isolates tested belonged to clonal complex 5(CC5). t2460 was the most prevalent spa type, accounting for 67.9 % (38/56) . ST5-MRSA- II-t2460 was predominant clone, accounting for 75.5 % (37/49) of FA-resistant MRSA isolates and 66.1 % (37/56) of FA-resistant S. aureus isolates. Five of 7 FA-resistant MSSA isolates belonged to ST630-MSSA. Increased FA resistance among S. aureus isolates was found in China. fusB was predominant FA resistance determinant. The spread of CC5 clone, especially novel ST5-MRSA- II-t2460 clone with high-level resistance to FA, was responsible for the increase of FA resistance.


Background
Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important and often encountered human pathogen responsible for many infectious diseases from mild superficial skin infection to life-threatening diseases, including skin and soft tissue infections (SSTIs), foreign-body infections, pneumonia, septic arthritis, endovascular infections, osteomyelitis, sepsis, and bloodstream infections (BSI) in both hospitals and community settings [1]. Since the first European isolate of MRSA was detected in 1960s, MRSA with resistance to all available penicillins and other β-lactam antimicrobial agents has become a leading cause of nosocomial infections in hospitals and other healthcare facilities. In addition to β-lactam, MRSA isolates are often resistant to erythromycin, clindamycin, aminoglycosides and quinolones. Since the mid-1990s, however, MRSA isolates are found to be responsible for severe infections in individuals lacking risk factors for exposure to the health care system [1,2]. MRSA infections has become a global concern [1,3]. As vancomycin has relatively clean safety profile and durability against the development of resistance, it has been considered to be the optimal option for the treatment of invasive MRSA infections [4]. However, the emergence of vancomycin-heteroresistant S. aureus (hVRSA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) limits the use of vancomycin for the treatment of serious MRSA infections [5][6][7]. In addition, the drawbacks of vancomycin, including poor tissue penetration, serious nephrotoxicity and neutropenia and unpredictable synergy with other antimicrobial agents, also limit the selection of the alternatives for the treatment of MRSA infections. Fusidic acid (FA), is an effective agent for the treatment of SSTIs, acute osteomyelitis, chronic osteomyelitis, vertebral infection, septic arthritis, and prosthetic and other device related infections caused by MRSA and methicillinsusceptible S. aureus (MSSA) [8][9][10]. FA dose of 250-500 mg every 8-12 h was recommended for the treatment of MRSA infections [8]. However, recent study reported that front-loaded dosing of ≥1200 mg every 12 h (q12h) has better activity against MRSA infections than nonfront-loaded dosing (600 mg q12 h) [11]. Although FA has not been approved for use in the USA by the US FDA, this drug has been widely used in Europe, Canada, Australia and some Asian countries for decades [8]. FA is a valuable alternative to vancomycin for the treatment of MRSA infections. In the 1990s, the prevalence of resistance to FA in S. aureus remained low. But there is a concern that there is a significant trend towards increased FA resistance among S. aureus with increased duration of use. In a surveillance report on FA resistance in 13 European countries, the prevalence of resistance to FA among S. aureus isolates was 10.7 %, with the highest resistance rates of 62.4 % among the isolates from Greece [12]. Although many Asian countries have reported FA resistance among S. aureus isolates, these data in China are limited. The aim of the present study is to investigate the dissemination of fusidic acid resistance among S. aureus clinical isolates from a tertiary hospital between 2012 and 2013 in Wenzhou, east China.

Collection of S. aureus clinical isolates
From Jan. 2012 to Sep. 2013, a total of 392 non-duplicate S. aureus isolates (single isolate per patient) from the various specimens of patients at the first Affiliated Hospital of Wenzhou Medical University locating in Wenzhou, east China, were selected for investigating the prevalence of FA resistance. The numbers of S. aureus isolates included in 2012 and 2013 were 211 and 181. Isolates were identified as S. aureus by using Gram's stain, positive catalase and coagulase test results, and Vitek microbiology analysis (BioMérieux, Marcy l'Etoile, France). S. aureus ATCC25923 was used as a control strain for the identification of bacteria. Because the present study focused on bacteria, the Ethics Committee of the first Affiliated Hospital of Wenzhou Medical University exempted this investigation from review. Informed consents were required from patients for sample collection.

DNA extraction
S. aureus isolates with FA resistance were cultured on blood agar overnight at 35°C. Then, three to four bacterial colonies were suspended in 150 μl sterile distilled water with lysostaphin (1 mg/mL) (Sangon, Shanghai, China) and incubated at 37°C for an hour. Finally, DNA was extracted following the instructions of the Genomic DNA Extraction kit (Sangon, Shanghai, China), stored at −20°C and prepared for PCR assays.

MRSA identification
A simplex PCR was used for the detection of mecA using MRSA N315 as positive control strain. The isolates positive for mecA were identified as MRSA. And cefoxitin disk diffusion test was also used for the detection of MRSA in accordance with the guideline provided by the Clinical and Laboratory Standards Institute (CLSI) [13].

Detection of FA resistance determinants by PCR
Detection of fusA mutations and acquired FA resistance determinants including fusB and fusC was detected by PCR assays with primers and reaction conditions described previously [15]. DNA sequencing was used for the identification of genotypes of acquired FA resistance determinants. The fusA mutations were determined by sequencing entire fusA gene.

spa typing
The spa variable repeat region from each S. aureus isolate was amplified using simplex PCR oligonucleotide primers as previously described [17,18]. Following their purification and sequencing, spa types were assigned using the spa database website (http://spaserver.ridom.de).

Multilocus sequence typing (MLST)
MLST typing of S. aureus isolates was performed using amplification of internal fragments of the seven housekeeping genes(arcc, aroe, glpf, gmk, pta, tpi and yqil) of S. aureus as described previously [19]. Following purification and sequencing of these genes, the sequences were compared with the existing sequences available on the MLST website for S. aureus (http://saureus.mlst.net), and STs were assigned according to the allelic profiles.  [20,21], the prevalence of FA resistance among S. aureus clinical isolates in the present study was very high. The resistance rates to FA among S. aureus isolates are different from country to country. The resistance rates of S. aureus isolates to FA were very low in the USA (0.3 %) and relatively high in Canada (7.0 %) and Australia (7.0 %) [22]. In an updated surveillance report on FA resistance in 13 European countries, Israel, Italy, Poland, Spain and Sweden had low rates (1.4-3.1 %), while Greece (62.4 %) and Ireland (19.9 %) had the highest resistance rates [12]. In most Asian countries, FA resistance rates are relatively low (<10 %) [8]. FA resistance was observed more frequently among MSSA isolates (0.6 %) than among MRSA isolates from the United States [22]. On the contrary, the prevalence of FA resistance among MRSA isolates was significantly higher than that among MSSA isolates in the present study. There is a significant trend towards increased FA resistance among S. aureus since FA has been used widely. In Kuwait, the rate of FA resistance increased rapidly from 22 % in 1994 to 92 % in 2004 [23]. There was a trend of rapidly increased FA resistance among S. aureus and MRSA isolates from 5.2 % and 8.9 % in 2012 to 24 nitrofurantoin. The antimicrobials with the resistance rates of more than 50 % among FA-resistant S. aureus isolates were penicillin (100 %), erythromycin (92.9 %), clindamycin (89.3 %), ciprofloxacin (82.1 %), and tetracycline (75.0 %), respectively. The resistance rates of the 56 isolates to gentamycin, rifampin, trimethoprim/sulfamethoxazole and chloramphenicol were relatively low. In contrast to the report from Taiwan among which the resistance rate to rimethoprim/sulfamethoxazole among FA-resistant MRSA isolates was high to 97 % [24], only 4.9 % (4/81) of FA-resistant MRSA isolates were resistant to this antimicrobial.

Prevalence of FA resistance determinants
Two major FA resistance mechanisms have been reported in S. aureus, including the alteration of the drug target site, which is due to mutations in fusA encoding elongation factor G (EF-G) or rplF encoding ribosome protein L6), and the protection of the drug target site by FusB family proteins including FusB, FusC, and FusD [8]. In staphylococci, high-level FA resistance is usually associated with mutations in fusA encoding EF-G, while lowlevel resistance is generally caused by the horizontally transferable genes including fusB, fusC and fusD [25]. The spontaneous mutations in fusA lead to modification of the drug target and reduced susceptibility [26]. The fusB-type resistance involves the recruitment of an exogenous resistance determinant whose product binds EF-G and protects it from FA [27]. In this study, the results from PCR and DNA sequencing demonstrated that acquired resistance gene, fusB was present in 73.2 % (41/56) of FA-resistant isolates tested. However, fusC and fusA mutations were not found in any of tested isolates. The previous investigation from China found that all 4 clinical isolates with FA resistance were positive for fusB and negative for fusC and fusA mutations [20]. These data indicated that fusB is the predominant determinant responsible for FA resistance among S. aureus in China. In Netherlands, fusB was also found to be predominant FA resistance determinant which was detected in about 90 % of FA-resistant S. aureus isolates [28]. On the contrary, a report from northern Taiwan found that 84 % of FA-resistant MRSA isolates had fusA mutations and another report from a Taiwanese hospital found that fusC was the most common FA resistance determinant [15,24]. S. aureus isolates from Australia with FA MIC values ranging from 2 to 32 μg/ml were predominantly fusC positive [22]. In U.S. and European collections, fusC was also more prevalent than fusB in FA-resistant S. aureus isolates [12,22]. In contrast to previous report from Taiwan among which the distribution of FA resistance determinants including fusA mutations, fusB and fusC was quite different between MRSA and MSSA groups and no MRSA isolates carried fusB [15].

Molecular characteristics of FA-resistant S. aureus isolates
The molecular characteristics of FA-resistant S. aureus isolates were listed in Table 2. A total of 9 STs were identified among the 56 FA-resistant S. aureus isolates, among which ST5 accounting for 66.1 % (37/56) was the most common ST, followed by ST630 (12.5 %, 7/56). Only 3 MRSA isolates belonged to ST239 which is the predominant ST in China. The STs for two isolates were not identified. The antimicrobial resistance profiles of ST5 and ST239 FA-resistant MRSA isolates were showed in Table 3. The clonal complex 5 (CC5) was the  [30]. ST5-MRSA-II-t002 was one of two major clones in China [30]. However, ST5-MRSA-II was linked to t2460 in this study. This fairly unusual clone was only found among 6 MRSA isolates from Shanghai in China [30]. FA MIC values for 36 of 37 ST5-MRSA-II-t2460 isolates were ≥128 μg/ml. ST5-MRSA-II-t2460 clone first emerged in 2012. However, this newly emerging clone with FA resistance increased dramatically in 2013, accounting for 77.8 % (35/45) of FA-resistant isolates. The origin of 35 isolates belonged to ST5-MRSA-II-t2460 in 2013 was confined to only 4 wards including neurosurgery ward (28 isolates), orthopaedic ward (three isolates), intensive care unit (three isolates) and hematology ward (one isolate), indicating that outbreak of infections caused by this newly clone has occurred in our hospital, mainly in neurosurgery ward. Clonal spread is responsible for the increase in FA resistance in UK, France and other European countries [31,32]. CC80-MRSA-IV clone carrying fusB has made a major contribution to the prevalence of FA resistance among MRSA isolates in Europe [25]. The highest prevalence of FA resistance in 2013 in this investigation was associated with the spread of ST5-MRSA-II-t2460 clone with high-level FA resistance. Among 7 FAresistant MSSA isolates, 5 belonged to ST630-MSSA.

Conclusions
In conclusion, increased FA resistance among S. aureus isolates was found in China. fusB was predominant FA resistance determinant. The spread of CC5 clone, especially novel ST5-MRSA-II-t2460 clone with high-level resistance to FA, was responsible for the increase of FA resistance.