The Dissemination of Fusidic Acid Resistance Among Staphylococcus epidermidis Clinical Isolates in Wenzhou, China

Introduction

Staphylococcus epidermidis is a member of the coagulase-negative staphylococci (CoNS), considered a component of the normal commensal microflora in human.¹ Colonization by S. epidermidis contributes to the maintenance of a healthy skin flora, especially by competing with potentially harmful microorganisms such as Staphylococcus aureus.² However, in recent decades, with the increased use of indwelling or implanted medical devices and the increase of sickly, elderly and immunocompromised patients, S. epidermidis has emerged as an important opportunistic pathogen responsible for hospital-acquired infections, particularly biofilm-associated infections.³

Fusidic acid, employed for the treatment of superficial and systemic disease caused by staphylococci, is a valuable alternative to vancomycin for the infections caused by multi-drug resistant Staphylococci, especially the infection of methicillin-resistant Staphylococcus aureus.^4–6 But there is a concern that there is a significant trend towards increased FA resistance among Staphylococci with increased duration of use, which is an even greater problem for limiting our therapeutic options.^1,7 Fusidic acid inhibits elongation factor G (EF-G) release from ribosomes and eventually blocks the synthesis of bacterial proteins.⁸ As in S. aureus, resistance to FA in S. epidermidis results either from the mutation of fusA, or acquiring fusB or fusC resistance genes.^9,10

Strikingly, studies have shown the proportion of fusidic acid-resistant S. epidermidis higher than that in S. aureus.^11,12 Molecular analysis for S. epidermidis isolates associated with nosocomial infections has shown considerable clonal diversity and is much less studied than that for S. aureus.¹³ Multi-locus sequence typing (MLST) based on nucleotide sequencing of seven housekeeping genes revealed distinct related clones of MRSE from clinically significant isolates and showed a worldwide predominance of only a few hospital-associated, epidemic clonal lineages.¹³ Clonal complex 2 (CC2) was major genetic lineage among the population structure of hospital-acquired S. epidermidis worldwide.^14,15 Limited information of the resistance of S. epidermidis clinical isolates to FA is available. The purpose of our study is to investigate the dissemination of FA resistance and molecular characteristics of S. epidermidis isolates from hospitalized patients in a tertiary hospital in Wenzhou, east China.

Materials and Methods

Collection of S. Epidermidis Clinical Isolates

Seven hundred and seventy-one non-duplicate S. epidermidis isolates were collected consecutively from various specimens of inpatients at the first Affiliated Hospital of Wenzhou Medical University. The isolates were identified as S. epidermidis by using Gram’s stain, catalase test and VITEK automatic microbiology analyzer (bioMe´rieux, Marcy l’Etoile, France).

Screening for Fusidic Acid-Resistant S. Epidermidis

The minimum inhibitory concentration (MIC) of FA for S. epidermidis isolates was determined by the agar dilution method. The interpretive criterion of FA susceptibility for Staphylococci was in accordance with the European Committee for Antimicrobial Susceptibility Testing (EUCAST)/British Society of Antimicrobial Chemotherapy (BSAC) criteria (susceptible, MIC < 2 μg/mL; resistant, MIC ≥ 2 μg/mL).

Antimicrobial Susceptibility Testing

In vitro antimicrobial susceptibility testing was determined by the disk diffusion method with antimicrobials in accordance with the standards recommended by the Clinical and Laboratory Standards Institute (CLSI).¹⁶ Antibiotic discs were tested as follows: penicillin (10U), vacomycin (30 μg), erythromycin (15 μg), clindamycin (2 μg), linezolid (30 μg), tetracycline (30 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), gentamicin (10 μg), ciprofloxacin (5 μg), and levofloxacin (5 μg). All disks were obtained from Oxoid Ltd. S. aureus ATCC25923 was used as quality control strain for antimicrobial susceptibility testing. The minimum inhibitory concentration of vancomycin was determined using the agar dilution method according to the guidelines of the CLSI.^16,17

Detection of Fusidic Acid Resistance Determinants by PCR

Isolates were tested for the presence of fusB and fusC by PCR, as previously described.¹⁸ Then, the amplified DNA fragments were sequenced for further confirmation.

Multi-Locus Sequence Typing (MLST)

Multi-locus sequence typing for S. epidermidis isolates was performed by amplification of internal fragments of the seven housekeeping genes including arcC, aroE, gtr, mutS, pyrR, tpiA, and yqiL of S. epidermidis as described previously.¹⁹ The PCR products of seven housekeeping genes tested for MLST typing were purified and sequenced. The numbers of alleles and sequence types were assigned using online database (http://sepidermidis.mlst.net/). The eBURST algorithm was used to infer the evolutionary relatedness of the STs.

Staphylococcal Cassette Chromosome (SCCmec) Typing

Primers for SCCmec typing were designed according to literature.²⁰ The 8 pairs of primers of CCr gene were CIF, KDP, MECI, DCS, RIF4, RIF5 and PUBl10R1, and the 8 pairs of primers (Type I ~ Type V) sequence were detected for mec complex. The strains harmany21 (SCCmec I), harmany24 (SCCmec II), harmany20 (SCCmec III), harmany22 (SCCmec IV) and HS662 (SCCmec V) of SCCmec were used as positive control for SCCmec typing.

Pulsed-Fifield Gel Electrophoresis (PFGE)

Our test was slightly modified from the previously described method.²¹ The restriction enzyme SmaI was used and the following electrophoresis conditions: switch time, 4 s to 40s; running time, 19 h in a 1% agarose gel.

Results

A total of 711 strains of Staphylococcus epidermidis isolated from patients with clinical and infectious symptoms were considered for invasive isolates and included in the investigation. Among 711 S. epidermidis isolates, 55 (7.7%) FA-resistant S. epidermidis were isolated from blood (n = 18), catheter (n = 14), sputum (n = 5), ascites (n = 3), tissue (n = 3), drainage (n = 2), wound exudate (n = 2), urine (n = 2), dialysate (n = 2), marrow (n = 1), and other sources (n = 2).

Antimicrobial Susceptibility of FA-Resistant S. epidermidis Clinical Isolates

The distribution of FA MIC levels among FA-resistant S. epidermidis is shown in Table 1. The MIC values ranging from 4 to 32 μg/mL were resistant to FA. Among 55 isolates, 50 (90.9%, 50/55) and 5 (9.1%, 5/55) FA-resistant isolates were MRSE and MSSE, respectively. Besides FA resistance, the 55. S. epidermidis were also resistant to non-beta-lactam antimicrobials as shown in Table 2. The FA-resistant isolates exhibited more than 50% of resistance rates to erythromycin (n = 44), clindamycin (n = 36), trimethoprim-sulfamethoxazole (n = 36) and ciprofloxacin (n = 35), but less than 50% of resistance rates to others including gentamicin (n = 27), rifampin (n = 26) and tetracycline (n = 13). All 55 isolates mentioned above were susceptible to vancomycin and linezolid.

Table 1 Distribution of FA MIC Levels in S. epidermidis Isolates

Table 2 Prevalence of Antibiotic Resistance in FA-Resistant S. epidermidis

Detection of FA Resistance Determinants

Among 55. S. epidermidis isolates with FA resistance, 47 (85.5%) and 4 (7.3%) were positive for fusB and fusC, respectively. However, no resistance genes were detected in four strains.

Molecular Characteristics of S. epidermidis Clinical Isolates with FA Resistance

PCR was used to amplify arcC, aroE, gtr, mutS, pyrR, tpi and yqiL genes from 55. Strains of FA resistant Staphylococcus epidermidis. The molecular characteristics of the 55 FA-resistant S. epidermidis isolates are listed in Figure 1. A total of 14 STs were identified among the 55. S. epidermidis isolates with FA resistance, among which ST2 was the most prevalent, accounting for 43.7% (24/55), followed by ST130 (9.1%, 5/55), ST64 (7.3%, 4/55), ST59 (5.5%, 3/55), ST210 (3.6%, 2/55) and ST23 (3.6%, 2/55). Other STs was only found one isolate each, such as ST125. The STs for five isolates were not identified. In the study, ST2 was the most prevalent so that the comparison of drug resistance between ST2 and non-ST2 strains is presented in Table 3. The resistance profiles of 24 ST2 S. epidermidis isolates were similar that they were resistance to penicillin, ciprofloxacin, levofloxacin, gentamicin and trimethoprim-sulfamethoxazole, however, susceptibility to vancomycin, linezolid and tetracycline.

Table 3 The Comparison of Drug Resistance Between ST2 Type and Non-ST2 in FA-Resistant S. epidermidis

Figure 1 Results of PFGE and molecular characteristics of FA-resistant S. epidermidis isolates.

According to the 448 ST models in the MLST database, the eBURST software analysis was performed, and the 55 clinical isolates in this study were analyzed by eBURST algorithm. Most of the isolates belonged to SCCmec III (n = 18), while 4, 3, 3 and 1 isolates belonged to SCCmec IV a, SCCmec IVd, SCCmec Vand SCCmec I, respectively. Intriguingly, half of the 24 ST2 strains were SCC mec III, and only one strain was SCCmec V. It was found that most of the strains in the clinical isolates belonged to CC2, accounting for 43.7% (24/55), while 15 and 8 strains belonged to CC5 and CC247, respectively. The founder of the clinical isolates found in this study was ST2, which was consistent with the founder found in the analysis of the S. epidermidis global database, and ST5 has the largest number in the subgroup founder, which was the single-locus variant (SLV) of ST2 (Figure 2).

Figure 2 The value represents the ST type name, the blue point is the CC type Founder, the yellow dot is Subgroup founder, the black dot is all other ST types, and the dot size indicates the number of CC types. The solid line between the two points indicates that there is only one housekeeping gene difference between the two ST types.

Results of Pulsed Field Gel Electrophoresis (PFGE)

According to the band analysis of pulsed field gel electrophoresis, the clones with more than 80% similarity were divided into 20 types (A-T) (Figure 1), showing considerable diversity. Seventeen isolates including 12 ST2 isolates (70.6%, 12/17) belonged to PFGE Q type, indicating clonal dissemination of S. epidermidis isolates with FA resistance. Of 12 ST2 isolates with same PFGE type isolated from blood (6), catheter (5) and sputum (1) mainly distributed in ICU and comprehensive disease area. Seven isolates including 3 ST130, 2 ST2, 1 ST248 and 1 ST210 isolates belonged to PFGE type E. These 7 isolates with same PFGE type were isolated from catheter (3), urine (2), blood (1) and peritoneal dialysate (1), respectively.

Discussion

Staphylococcus epidermidis, as a conditional pathogen, often causes a series of infections in people with low immunity or in infants and the elderly. To understand the origin of FA-resistant S. epidermidis and monitor them from different sources, analysis of molecular characteristics was performed in our study.

Chen et al found that the majority of FA-resistant S. epidermidis isolates were also resistant to methicillin.²² Our result was consistent with the above study that Methicillin-resistant Staphylococcus epidermidis was the predominates. McLaws et al reported a high prevalence rate (46%, 23/50) of resistance to fusidic acid in S. epidermidis clinical isolates.¹² Twenty-five percent of methicillin-resistant CNS and 15% of methicillin susceptible CNS strains isolated from blood cultures of septicemic patients in Turkey were resistant to FA.²³ Compared with the reports mentioned above, the prevalence of FA in the present study was relatively low.

Two major FA resistance mechanisms were reported, the alteration of the drug target site caused by the 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, which have been reported in S. aureus.⁴ In Staphylococci, high-level FA resistance is usually associated with mutations in fusA, while low-level resistance is generally caused by plasmid-mediated resistance genes including fusB, fusC and fusD.²⁴ In the present study, 47 FA-resistant S. epidermidis clinical isolates were positive for fusB, while only four were positive for fusC. Colonized Staphylococci on skin may be reservoir for fusidic acid resistance genes.²⁵ The monitor for presence of fusidic acid resistance genes among S. epidermidis should be helpful for preventing the dissemination of FA-resistant S. epidermidis.

So far, a total of 448 ST types of S. epidermidis have been found worldwide. The main ST types of FA-resistant S. epidermidis in the drug resistance spectrum were ST2, ST130, ST64 and ST59. Studies had shown that ST2 was the main ST type of clinical isolates of S. epidermidis in the reports in the United States, Germany and other countries.^26,27 In our study, ST2 was also found to be the most prevalent clone among S. epidermidis isolates with FA resistance, which was consistent with the main ST type of S. epidermidis found in other countries. In the present study, 24 ST2 MRSE with FA resistance isolated from blood, catheter, sputum, urine and ascites distributed among 9 wards, indicating that ST2 S. epidermidis clone with FA resistance has disseminated in this hospital. The drug resistance rate of ST2 to gentamicin, ciprofloxacin, rifampicin, levofloxacin and moxifloxacin was more than 5 times higher than that of non-ST2. A report from China showed that 91.7% (297/324) of S. epidermidis from the community and hospital environments belonged to CC2.¹⁵ CC2 comprises 74% of the S. epidermidis isolates from 17 national centers between 1996 and 2001.¹⁴ The majority (62/71; 87.3%) of S. epidermidis clinical isolates from US hospitals belonged to CC2.²⁸ We speculate that acquiring resistance to FA, as well as multi-resistance to other antimicrobial agents, contributes to the spread of S. epidermidis ST2 clone. ST23 was found among linezolid-resistant S. epidermidis isolates.^29,30 In the present study, ST23 was identified in 2 S. epidermidis isolates with resistance to FA, which was first found among S. epidermidis isolates isolated from China. Although ST125 exist in online database (http://sepidermidis.mlst.net/), the literatures about S. epidermidis ST125 isolates was not found. The present study first reported ST125 among S. epidermidis clinical isolates. Taken together, the dissemination of S. epidermidis ST2 clone with FA resistance can cause trouble in controlling S. epidermidis infections.

In addition, although the results of PFGE for the 55 strains showed considerable diversity (20 PFGE types), 17 isolates including 12 ST2 isolates belonged to PFGE Q type, indicating clonal dissemination of S. epidermidis isolates with FA resistance has occurred. Clonal dissemination was the main reason for the cross-medical structure, cross-regional and even transnational global epidemic of S. epidermidis. Therefore, the etiological methods of tracking and monitoring strains should clarify the homology and clonal dissemination of S. epidermidis strains timely and accurately at the type, subtype and molecular level, so as to effectively control the nosocomial infection of Staphylococcus epidermidis and the cloning and dissemination of bacterial drug resistance genes.