Molecular Characterization of Staphylococcus aureus from Surgical Site Infections in Orthopedic Patients in an Orthopedic Trauma Clinical Medical Center in Shanghai

Abstract

Background:

Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of surgical site infections (SSIs). The aim of our study was to characterize molecularly S. aureus isolates from SSIs in orthopedic patients in Shanghai, China.

Methods:

Eighty-two S. aureus isolates (46 methicillin-susceptible Staphylococcus aureus [MSSA] and 36 MRSA) were collected from SSIs in orthopedic patients. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and a variety of clinically important toxin genes were detected. The sequence type, spa type, and agr group were determined to analyze the genotypes of all the isolates collected. In addition, MRSA isolates were characterized by staphylococcal cassette chromosome mec (SCCmec) type as well.

Results:

The strains showed susceptibility to antibiotics such as teicoplanin, minocycline, quinupristin-dalfopristin, linezolid, mupirocin, and vancomycin. Ten pvl-positive isolates (three MRSA and seven MSSA) were found among all isolates. Eight community-associated MRSA (CA-MRSA) isolates were found, six of which belonged to ST59-MRSA-IV but most MRSA isolates (20/36, 55.6%) belonged to ST239-MRSA-III-t030/t037 with a wide range of antibiotic resistance. By contrast, MSSA isolates were more diverse in both molecular characterizations and virulence factors, with eight MSSA isolates harboring more than six toxin genes detected.

Conclusions:

ST239-MRSA-III-t030/t037 was the epidemic clone, and healthcare-associated MRSA (HA-MRSA) strains might be the major pathogen causing S. aureus SSIs in orthopedic patients.

Surgical site infections (SSIs) are defined as infections occurred within 30 d after surgery or 1 y after surgery in patients receiving implants that affect the incision or deep tissue at the operation site, even deep to the bone or organs, causing bacteremia or sepsis and leading to severe and complex problems [1]. Although perioperative antibiotic prophylaxis has reduced the incidence of SSIs [2], it still has a high occurrence rate worldwide and has been the third most common nosocomial infection [3]. Staphylococcus aureus is an important pathogen in SSIs, especially after orthopedic surgery [4].

The S. aureus metagenome contains a number of toxin genes such as pvl (encoding Panton-Valentine leukocidin), tst (encoding toxic shock syndrome toxin-1), eta and etb (encoding exfoliative toxin A and B), and enterotoxins genes, which are mostly controlled by the agr locus [5]. They may play an important role in the pathogenesis of S. aureus infections. Currently, increasingly widespread antibiotic resistance is a challenge to the therapy of patients with S. aureus infections. Methicillin-resistant S. aureus (MRSA) is widespread and exhibits multi-drug resistance frequently to several substances belonging to different antibiotic classes such as aminoglycosides, macrolides, lincosamides, tetracyclines, trimethoprim, and sulfonamides [6,7]. Furthermore, as an increasing number of vancomycin-resistant S. aureus (VRSA) isolates has been reported recently [8], the developing resistance to antibiotics has been a serious threat to the therapy of S. aureus infections. Therefore, the presence of toxins together with the developing resistance to antibiotics leads to major challenges in preventing and treating infections in surgical patients [9].

Typing of S. aureus is an essential component of an effective surveillance system to describe epidemiologic trends and implement infection control strategies. Typing methods contain multi-locus sequence typing (MLST), Staphylococcus protein A gene (spa) typing, staphylococcal cassette chromosome mec (SCCmec) typing, and accessory gene regulator (agr) typing [5,10 –13]. These typing methods have been adopted widely and their results have a good degree of concordance and can be compared internationally. Pulsed-field gel electrophoresis (PFGE) is a method used widely for typing S. aureus especially in investigations of outbreak or local molecular epidemiology, however, it is labor-intensive and difficult to standardize among laboratories. As far as we know, there is a limited number of publications on molecular characterization of S. aureus from SSIs in orthopedic patients worldwide, none of which is from Shanghai. Therefore, our study aimed to investigate the epidemiology and genetic diversity of S. aureus isolates from SSIs in orthopedic patients as well as the antibiotic resistance pattern and toxin gene profile.

Patients and Methods

Bacterial isolates

The Orthopedics Trauma Clinical Medical Center of Shanghai Jiao Tong University Affiliated Sixth People's Hospital is the largest orthopedic center in east China with 550 beds. It served 340,655 outpatients in 2011 and 388,630 outpatients in 2012. There were 35,560 orthopedic trauma operations in 2011 and 38,596 in 2012, accounting for 60%–70% of trauma operations in the Shanghai area. In addition to Shanghai, the orthopedic patients come to this hospital from all provinces in China, especially east China.

According to computerized discharge records, during the period January 2011 to December 2012, 328 organisms were recovered from 649 SSIs samples from orthopedic patients; approximately 25% were S. aureus. All 82 S. aureus isolates were saved and enrolled in this study. Surgical procedures performed included osteosynthesis of fractures, total knee and hip replacements, lumbar spine surgery, amputation, bone graft, and other orthopedic operations.

This study was approved by Ruijin Hospital Ethics Committee (Shanghai Jiao Tong University School of Medicine). The Review Board did not require informed consent for this retrospective study because it focused mainly on bacteria without interventions involving patients.

Identification and antimicrobial susceptibility testing

Eighty-two S. aureus isolates were identified by a combination of phenotypic tests as described previously [14]. The agar dilution and broth dilution methods were performed to detect the minimum inhibitory concentrations (MIC) of vancomycin and oxacillin, respectively, whereas resistance or susceptibility to penicillin, cefoxitin, gentamicin, kanamycin, tobramycin, fosfomycin-trometamol, erythromycin, tetracycline, teicoplanin, minocycline, ciprofloxacin, clindamycin, sulfamethoxazole-trimethoprim, chloramphenicol, rifampicin, quinupristin-dalfopristin, linezolid, fusidic acid, and mupirocin were measured by disk diffusion method [15]; the disk interpretive criteria for fosfomycin-trometamol and fusidic acid were used from the studies by Lu et al. [16] and Skov et al. [17]. A 5-mcg mupirocin disk was used to determine susceptibility to mupiricin. A 200-mcg mupirocin disk test would be performed to detect high-level mupirocin resistance if there was any resistance to 5 mcg mupirocin. The penicillin disk diffusion zone edge test was performed for β-lactamase detection, and inducible clindamycin resistance was tested by the D-test. Staphylococcus aureus ATCC25923 and ATCC29213 (American Type Culture Collection, Rockville, MD) were used as quality control strains for the disk diffusion test and MIC detection, respectively.

Detection of molecular characteristics

Deoxyribonucleic acid was extracted by the simplified alkaline-lysis method [14]. The typing methods including MLST, spa typing, and agr typing were performed as described previously [18]. Methicillin-resistant S. aureus isolates were checked by the detection of the mecA gene, and the SCCmec types of MRSA were determined by the method described previously [12].

Using polymerase chain reaction (PCR), we detected a variety of clinically important toxin genes, including sea-see and seg-sej (encoding staphylococcal enterotoxins SEA-SEE and SEG-SEJ); eta and etb (encoding exfoliative toxin A and B); pvl (encoding Panton-Valentine leukocidin); tst (encoding toxic shock syndrome toxin-1); and hla, hlb, hld, hlg, hlg-2 (encoding α-, β-, δ-, γ-, and γ variant hemolysins) as described previously [5].

Statistical analysis

Data were analyzed using the χ² or Fisher exact test as appropriate with a two-sided p value <0.05 used for statistical significance. All statistical analysis was conducted by SAS 8.2 (SAS Institute Inc., Cary, NC).

Results

Clinical data

A total of 82 S. aureus isolates from SSIs in patients undergoing orthopedic surgery were collected in this study. The mean age was 41 y (range, 7–78 y). The gender distribution was 62 males (75.6%) and 20 females (24.4%). The mean and median time between the surgery and infection was 3 d and 1 d, respectively (range, 0–16 d). As to the distribution of SSIs according to type of orthopedic surgery, osteosynthesis of fractures was the most common procedure (n=45; 54.9%), followed by total knee and hip replacements (n=8; 9.8%), bone graft (n=7; 8.5%), lumbar spine surgery (n=5; 6.1%), amputation (n=3, 3.7%) and other (n=14, 17.1%). In addition, 35 of the 82 patients underwent implant surgery.

Antimicrobial susceptibility testing

Thirty-six (43.9%) of 82 S. aureus isolates were MRSA in this study as determined by the broth dilution method of oxacillin and PCR confirmation of mecA gene presence. We did not find any isolate that was resistant to teicoplanin, minocycline, quinupristin-dalfopristin, linezolid, mupirocin, or vancomycin. The resistance rates for the 82 S. aureus strains to sulfamethoxazole-trimethoprim, chloramphenicol, fusidic acid, and rifampicin were 6.1%, 7.3%, 2.4%, and 24.4%, respectively, and the rates determined for the other antibiotics were all above 30% overall. Methicillin-susceptible Staphylococcus aureus (MSSA) isolates showed a low resistance rate to all the agents except penicillin and fosfomycin-trometamol. However, MRSA isolates showed a high resistance rate to almost all antibiotics as presented in Table 1. Twelve isolates were found inducible resistant to clindamycin based on the D-test. Six MSSA isolates that were found susceptible to penicillin were β-lactamase–negative according to the penicillin zone-edge test. We did not detect any high-level mupirocin resistance.

Table 1.

The Antibiotic Resistance Rate of Eighty-Two Staphylococcus aureus Isolates from Surgical Site Infections

	Resistance rate (%)
Antibiotic	Overall	MSSA	MRSA
Penicillin	92.7	87.0	100
Oxacillin	43.9	0	100
Gentamicin	40.2	13.0	75.0
Kanamycin	45.1	15.2	83.3
Tobramycin	47.6	23.9	77.8
Fosfomycin-trometamol	64.6	56.5	75.0
Erythromycin	65.9	52.2	83.3
Tetracycline	40.2	17.4	69.4
Teicoplanin	0	0	0
Minocycline	0	0	0
Ciprofloxacin	40.2	13.0	75.0
Clindamycin^a	47.6	19.6	83.3
Sulfamethoxazole-trimethoprim	6.1	0	13.9
Chloramphenicol	7.3	2.2	13.9
Rifampicin	24.4	6.5	47.2
Quinupristin-dalfopristin	0	0	0
Linezolid	0	0	0
Fusidic acid	2.4	0	5.6
Mupirocin	0	0	0
Vancomycin	0	0	0

Twelve isolates (11 MSSA and 1 MRSA) inducible resistance to clindamycin.

MSSA=methicillin-susceptible Staphylococcus aureus; MRSA=methicillin-resistant Staphylococcus aureus.

Virulence factors

Eighteen toxin genes were detected in this study, and the prevalence of these toxin genes is presented in Table 2. Four of five hemolysin genes (hla, hlb, hlg, and hlg-2) were frequently found positive. The hla gene was positive in all 82 S. aureus isolates. The sea, seg, and sei were the most prevalent enterotoxin genes (36.6%, 22.0%, and 22.0%, respectively), whereas sed and sej were found once in the same MSSA isolate. Ten isolates (seven MSSA and three MRSA) and three isolates (one MSSA and two MRSA) harbored pvl and tst, respectively, but no isolate that was both pvl- and tst-positive was found. see, eta, etb, and hld were not detected among any the collected isolates. Furthermore, MRSA is more likely to carry the sea gene (24 versus six, p<0.0001), however, MSSA tends to carry the seg and sei genes (15 versus three, p=0.0084; 15 versus three, p=0.0084, respectively) according to the statistical analysis.

Table 2.

Prevalence of Toxin Genes among Eighty-Two Staphylococcus aureus Isolates from Surgical Site Infections

	Positive isolates		No. distributing in
Toxin gene	No.	%	MSSA (n=46)	MRSA (n=36)	p
sea	30	36.6	6	24	<0.0001
seb	11	13.4	6	5	0.9112
sec	4	4.9	2	2	0.8011
sed	1	1.2	1	0	0.3734
see	0	0	0	0	—
seg	18	22.0	15	3	0.0084
seh	2	2.4	2	0	0.2053
sei	18	22.0	15	3	0.0084
sej	1	1.2	1	0	0.3734
eta	0	0	0	0	—
etb	0	0	0	0	—
pvl	10	12.2	7	3	0.3445
tst	3	3.7	1	2	0.4183
hla	82	100	46	36	—
hlb	37	45.1	18	19	0.2178
hld	0	0	0	0	—
hlg	74	90.2	41	33	0.7009
hlg-2	72	87.8	40	32	0.7907

sea-see and seg-sej, gene encoding staphylococcal enterotoxins SEA-SEE and SEG-SEJ; eta and etb, gene encoding exfoliative toxin A and B; pvl, gene encoding Panton-Valentine leukocidin; tst, gene encoding toxic shock syndrome toxin-1; hla, hlb, hld, hlg and hlg-2, gene encoding α-, β-, δ-, γ-, and γ variant hemolysins.

p value, two-sided p value calculated by the χ² or Fisher exact test as appropriate.

MSSA=methicillin-susceptible Staphylococcus aureus; MRSA=methicillin-resistant Staphylococcus aureus.

Molecular characteristics and toxin genes

Among the 82 S. aureus isolates, we found 34 different spa types and 19 sequence types (STs). As mentioned before, 36 MRSA isolates were observed in the survey. Four SCCmec types (II–V) occurred among the MRSA isolates, and the most common SCCmec type was SCCmecIII, which was found in 23 isolates (63.9%); two MRSA isolates could not be SCCmec typed. The most frequent spa-type and ST among MRSA isolates were t030/t037 and ST239 (55.6% and 63.9%, respectively) and they were also the most common types among all isolates (24.4% and 30.5%, respectively). Therefore, the most common clone among MRSA clones from SSIs in patients undergoing orthopedic surgery was ST239-MRSA-III-t030/t037 as presented in Table 3. This was followed by ST59-MRSA-IV as well as ST5-MRSA-II. The 46 MSSA isolates showed a large diversity in spa types and STs (25 and 19 types, respectively). ST1281-t164, ST188-t189, and ST7-t091 were slightly more frequent than other types, which were found in five (10.9%), five (10.9%), and four (8.7%) isolates, respectively. Eight MSSA isolates harboring six or more toxin genes were detected with a diversity of genotypes.

Table 3.

Molecular Characterization and Antibiotic Resistance Pattern of Eighty-Two Staphylococcus aureus Isolates from Surgical Site Infections

Clone (n)	agr group	Antibiotic resistance pattern	Toxin gene profiles	No. of isolates
ST239- SCCmecIII-t030 (16)	I	P-OX-CX-CN-K-TOB-E-CIP-CC-RA	hla-hlb-hlg-hlg2	1
	I	P-OX-CX-CN-K-TOB-E-TE-CIP-CC-RA	sea-hla-hlb-hlg-hlg2	1
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC	sea-hla-hlg-hlg2	1
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-RA	hla-hlb-hlg-hlg2	2
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-RA	sea-hla-hlb-hlg-hlg2	2
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-RA	sea-hla-hlg	1
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-RA	sea-hla-hlg-hlg2	4
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-RA-FD	sea-hla-hlg-hlg2	1
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-RA-FD	sea-hla-hlg	1
	I	P-OX-CX-CN-K-TOB-FOT-TE-CIP-RA	sea-hla-hlb-hlg-hlg2	1
	I	P-OX-CX-CN-K-TOB-FOT-TE-CIP-RA	sea-hla-hlg-hlg2	1
ST239- SCCmecIII-t037 (4)	I	P-OX-CX-CN-K-TOB-E-CIP-CC	sea-hla-hlg-hlg2	1
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-SXT	sea-hla-hlg	1
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-SXT-CL	sea-hla-hlb-hlg-hlg2	1
	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-SXT-CL	sea-hla-hlg-hlg2	1
ST239- SCCmecIII-t298 (2)	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-SXT	sea-hla-hlg-hlg2	2
ST239- SCCmecIII-t459 (1)	I	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-RA	sea-hla-hlg-hlg2	1
ST5- SCCmecII-t002 (2)	II	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC	seg-sei-hla-hlb-hlg-hlg2	1
	II	P-OX-CX-CN-K-TOB-FOT-E-TE-CIP-CC-CL	sec-seg-sei-tst-hla-hlg-hlg2	1
ST5- SCCmecII-t010 (1)	II	P-OX-CX-K-TOB-FOT-E-TE-CIP-CC	sec-seg-sei-tst-hla-hlb-hlg-hlg2	1
ST59- SCCmecII-t163 (1)	I	P-OX-CX-FOT	sea-hla-hlb-hlg-hlg2	1
ST59- SCCmecIV-t172 (1)	I	P-OX-CX-FOT-RA	sea-seb-hla-hlb	1
ST59- SCCmecIV-t437 (2)	I	P-OX-CX-K-E-CC	seb-hla-hlb-hlg-hlg2	2
ST59- SCCmecIV-t441 (2)	I	P-OX-CX-CN-K-TOB-E-CC-CL	pvl-sea-seb-hla-hlb-hlg2	1
	I	P-OX-CX-FOT-E-CC-CL	pvl-sea-seb-hla-hlb-hlg2	1
ST88- SCCmecIV-t2310 (1)	III	P-OX-CX	hla-hlb-hlg-hlg2	1
ST88- SCCmecNT-t12147 (1)	III	P-OX-CX-FOT-E-CC	pvl-hla-hlg-hlg2	1
ST7- SCCmecNT-t12146 (1)	I	P-OX-CX-CN-K-TOB-E-TE-CIP-CC	hla-hlb-hlg-hlg2	1
ST630- SCCmecV-t5554 (1)	I	P-OX-CX	hla-hlb-hlg-hlg2	1
ST1281-t164(5)	I	P-FOT-E	seg-sei-hla-hlg-hlg2	3
	I	P-FOT-E	seg-sei-hla-hlb-hlg-hlg2	1
	I	P-FOT	seg-sei-hla-hlg-hlg2	1
ST188-t189 (5)	I	P	seb-hla-hlg-hlg2	1
	I	P	hla-hlb-hlg-hlg2	1
	I	FOT	seb-hla-hlg-hlg2	1
	I	P-FOT	hla-hlg	1
	I	P-FOT	hla-hlg-hlg2	1
ST7-t091 (4)	I	P-TOB	hla-hlb-hlg-hlg2	1
	I	P-TOB-TE	hla-hlb-hlg-hlg2	1
	I	P-TOB-FOT-TE	hla-hlg-hlg2	1
	I	P-TOB-FOT-E-TE	hla-hlg-hlg2	1
ST7-t796 (2)	I	P-CN-K-TOB-FOT-E-CC	hla-hlg-hlg2	2
ST7-t6284 (1)	I	P-TOB-TE	hla-hlg-hlg2	1
ST15-t084 (3)	II	P	hla-hlg-hlg2	1
	II	P-FOT-E	hla-hlg-hlg2	1
	II	P-FOT-E-CC	hla-hlg-hlg2	1
ST1-t127 (2)	III	P	seh-hla-hlb-hlg-hlg2	1
	III	P-CN-K-TOB-FOT-E-CC	sea-seb-seh-hla-hlg-hlg2	1
ST217-t309 (2)	I	None	pvl-seg-sei-hla	1
	I	P-FOT-E	pvl-seg-sei-hla	1
ST239-t030 (2)	I	P-CN-K-TOB-FOT-E-TE-CIP-CC-RA	sea-hla-hlb-hlg-hlg2	2
ST6-t701 (2)	I	P-E	sea-hla-hlb-hlg-hlg2	1
	I	P-FOT	sea-hla-hlb-hlg-hlg2	1
ST630-t377 (2)	I	P-CIP	hla-hlb-hlg-hlg2	1
	I	P-TE-CIP	hla-hlb-hlg-hlg2	1
ST630-t12148 (1)	I	P-E-TE-CIP-CC	hla-hlb-hlg-hlg2	1
ST1301-t12145 (1)	IV	E	pvl-sea-seg-sei-hla-hlg-hlg2	1
ST1281-t377(1)	I	P-FOT-E-CC	hla-hlb-hlg-hlg2	1
ST20-t164 (1)	I	P	seg-sei-hla-hlg-hlg2	1
ST2315-t11687 (1)	II	P-FOT-CIP	sec-seg-sei-hla-hlg-hlg2	1
ST25-t078 (1)	I	P-FOT-E	pvl-seb-seg-sei-hla-hlb-hlg-hlg2	1
ST398-t1255(1)	I	P-FOT-E	pvl-hla	1
ST398-t034 (1)	I	FOT-E-RA	pvl-hla	1
ST398-t571 (1)	I	P-FOT-E	hla	1
ST5-t954 (1)	II	P-E	seg-sei-hla-hlg-hlg2	1
ST5-t002 (1)	II	P-CN-K-TOB-FOT-E	sec-sed-seg-sei-sej-hla-hlg-hlg2	1
ST5-t570 (1)	II	P	seg-sei-tst-hla-hlb-hlg-hlg2	1
ST59-t437 (1)	I	P-K-E-CC-CL	seb-hla-hlb-hlg-hlg2	1
ST59-t163 (1)	I	None	seb-hla-hlb-hlg-hlg2	1
ST88-t2310 (1)	III	None	pvl-hla-hlg-hlg2	1
ST965-t062 (1)	II	P	seg-sei-hla-hlb-hlg-hlg2	1

Clone, ST-SCCmec-spa-type; ST, sequence type by multi-locus sequence typing; SCCmec, staphylococcal cassette chromosome mec; spa, Staphylococcus protein A gene; agr, accessory gene regulator; NT, not-typeable.

All the drugs tested in our study were P-OX-CX-CN-K-TOB-FOT-E-TE-TEC-MH-CIP-CC-SXT-CL-RA-QD-LZD-FD-MUP-VAN. P, penicillin (10 units); OX, oxacillin (tested by broth dilution method); CX, cefoxitin (30 mcg); CN, gentamicin (10 mcg); K, kanamycin (30 mcg); TOB, tobramycin (10 mcg); FOT, fosfomycin-trometamol (200 mcg); E, erythromycin (15 mcg); TE, tetracycline (30 mcg); TEC, teicoplanin (30 mcg); MH, minocycline (30 mcg); CIP, ciprofloxacin (5 mcg); CC, clindamycin (2 mcg); SXT, sulfamethoxazole-trimethoprim (25 mcg); CL, chloramphenicol (30 mcg); RA, rifampicin (5 mcg); QD, quinupristin-dalfopristin (15 mcg); LZD, linezolid (30 mcg); FD, fusidic acid (10 mcg); MUP, mupirocin (5 mcg); VAN, vancomycin (tested by agar dilution method).

pvl, gene encoding Panton-Valentine leukocidin; sea-see and seg-sej, gene encoding staphylococcal enterotoxins SEA-SEE and SEG-SEJ; eta and etb, gene encoding exfoliative toxin A and B; tst, gene encoding toxic shock syndrome toxin 1; hla, hlb, hld, hlg, and hlg-2, gene encoding α-, β-, δ-, γ-, and γ variant hemolysins.

Three MSSA isolates were susceptible to all tested drugs and two carried pvl (ST88-t2310 and ST217-t309, respectively). The other five pvl-positive MSSA isolates were ST217-t309, ST398-t1255, ST398-t034, ST25-t078, and ST1301-t12145. Also, three pvl-positive MRSA isolates were ST59-MRSA-IV-t441 (two isolates) and ST88-MRSA-NT-t12147 (1 isolate), and the former two MRSA isolates were both sea- and seb-positive. In addition, three tst-positive isolates that were distributed in two MRSA isolates and one MSSA isolate all belonged to ST5 (agrII). The only MSSA isolate that was both sed- and sej-positive was ST5-t002 (agrII). Specifically, sea and seb were found in SCCmecIII and SCCmecIV MRSA isolates, however, sec, seg, and sei were all found in SCCmecII MRSA isolates. Grouping of agr allele indicated that agr I to IV was detected in 65 (79.3%), 11 (13.4%), 5 (6.1%), and 1 (1.2%) isolates, respectively, and agrI appeared much more frequently both in MRSA and MSSA isolates than the other three agr groups.

Discussion

Staphylococcus aureus is the most common cause of SSIs and is the primary pathogen in 20% of SSIs as reported [19,20]. Moreover, S. aureus is the main causative agents in orthopedic surgery, representing more than 40% of all concerned microorganisms, leading to substantial morbidity and mortality of SSIs [20]. Recently, the percentage of MRSA among S. aureus SSIs isolates in China was 30%–40% [6,21] and the percentage of MRSA in this study was 43.9%. Minocycline, trimethoprim-sulfamethoxazole, rifampicin, linezolid, quinupristin-dalfopristin, and vancomycin can be used for treating MRSA SSIs empirically according to Infectious Diseases Society of America guidelines with the result of our antimicrobial susceptibility testing [22]. However, treatment still differs for the infections caused by MSSA and MRSA and different surgeons may treat in different ways. Furthermore, it is known that vancomycin, linezolid, and teicoplanin are used widely for treating MRSA SSIs; however, in recent studies, the susceptibility rate of teicoplanin seemed to be reduced in China [6,21]. Hence, minocycline, trimethoprim-sulfamethoxazole, chloramphenicol, quinupristin-dalfopristin, fusidic acid, and mupirocin may be other good choices for the therapy of MRSA SSIs. However, it should be noted that treatment with chloramphenicol and quinupristin-dalfopristin can have serious associated toxicities and mupirocin is a topical agent.

The mecA gene was detected in the 36 MRSA isolates; SCCmecIII was the major SCCmec type among 36 MRSA isolates (n=23; 63.9%), followed by SCCmecIV (n=7; 19.4%), SCCmecII (n=3; 8.3%), and SCCmecV (n=1; 2.8%). The main genotype among SCCmecIV strains was ST59, whereas ST5 and ST239 were the main genotype among SCCmecII and SCCmecIII strains, respectively. Health care-associated MRSA (HA-MRSA) strains are usually associated with SCCmecI, II, or III, however, community-associated MRSA (CA-MRSA) are associated with SCCmecIV or V. Because of the unclear boundary between hospital and community in China and the limitation of clinical data, we distinguished HA-MRSA and CA-MRSA according to the SCCmec type. Thus, HA-MRSA strains (26/36, 72.2%) were the major pathogen causing SSIs among MRSA isolates in this survey. The number of CA-MRSA isolates was eight, two (25%) of which were pvl-positive. Panton-Valentine leukociclin is considered a strong cytotoxic factor in S. aureus infections, and highly expressed in invasive CA-MRSA infections as reported recently [23]. In spite of the pvl-positive isolate that was not SCCmec typed (ST88-MRSA-NT-t12147), the other two pvl-positive MRSA isolates were both ST59-MRSA-IV-t441, belonging to CA-MRSA. Although ST59 is not a pandemic clone of pvl-positive CA-MRSA such as ST30 and ST80, it has also been found in the United States and China [13,24,25] and we found two ST59 pvl-positive CA-MRSA as presented previously. ST88 pvl-positive MRSA is relatively less common but has been reported in China, Bangladesh, Belgium, and Nigeria [13,25] and we found two ST88 pvl-positive isolates (one MRSA and one MSSA) in our study. The other six pvl-positive MSSA exhibit diverse genotypes.

The most common clone found in our study was ST239-MRSA-III-t030/t037. This might imply that ST239-MRSA-III-t030/t037 is an epidemic clone among patients with SSIs. Similarly, the common clone in blood stream infection caused by S. aureus is the same clone in China [18], but pneumonia and skin and soft tissue infection (SSTI) tend to be caused by CA-MRSA such as ST59-MRSA-IV [26,27]. To our knowledge, this is the first investigation of molecular characterization of S. aureus isolates from orthopedic SSIs in Shanghai. Unfortunately, we have not found similar investigations of whether there are other epidemic clones among orthopedic patients with SSIs in other cities or countries. As reported by Aamot et al. [9], who studied S. aureus deep incisional SSIs in orthopedic patients, it is different that almost all the S. aureus isolates collected were MSSA, and the molecular characteristics and toxin genes are also quite different from the MSSA of our study. Another report on S. aureus bone and joint infections also shows a major difference from ours as well as the clone of pvl-positive or tst-positive isolates [28]. It might imply that the S. aureus orthopedic SSIs we studied differ greatly from the deeper infections or there is a difference between countries, and MSSA isolates always expressed a great diversity in both molecular characterizations and virulence factors.

However, the Brazilian or Hungarian clone (ST239) of MRSA is a highly transmissible, multi-antibiotic– and antiseptic-resistant variant of MRSA that was recently reported as prevalent in Asia, America, and Europe [29 –32]. ST239-MRSA-III is still a common epidemic clone in many other S. aureus infections such as blood stream infection and invasive infections [18,33]. The less common ST of MRSA in our study was ST59, which spread in Asia in recent years as a pvl-positive MRSA clone and might be linked to health care settings as reported [34]. Therefore, hospital infection control is still important in preventing MRSA SSIs.

Some researchers have demonstrated that the S. aureus strains causing staphylococcal enterotoxin (SE)-mediated diseases always belonged to agr group I or II, whereas agr group III and agr group IV strains are mostly involved in toxic shock syndrome toxin-1 (TSST-1)–mediated diseases and generalized exfoliative syndrome, respectively [5]. However, our results do not confirm this. For example, among the five agrIII isolates of our study, we found three were enterotoxin gene-positive, but tst gene of all five isolates was not detected. The eta and etb genes of the only agr IV isolate were negative, but pvl, sea, seg, and sei genes were positive. We believe that if one or more virulence factors were more common than others, it may be because it contributes to the infection or is there by chance because it is present in a common clone. However, we have not found any outstanding relation between toxin genes and S. aureus clones or antibiotic resistance pattern except pvl as presented in Table 3.

Footnotes

Acknowledgments

We thank Jie Li, Shu-Zhen Xiao, and Sheng-Yuan Zhao (Ruijin Hospital, Shanghai Jiao Tong University School of Medicine) for the kind help with this work. We also thank Xiao-Kui Guo (Department of Microbiology and Parasitology, Shanghai Jiao Tong University School of Medicine) for the support for the molecular experiments.

This study was supported by the research special fund for public welfare industry of health (No. 201002021) and scientific research project of independent innovation of Shanghai Putuo district health system (PKW11204). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Disclosure Statement

Li-Zhong Han has received grant support from Pfizer and BioMerieux. All other authors report no potential conflicts of interest.

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