Molecular and Phenotypic Characterization Revealed High Prevalence of Multidrug-Resistant Methicillin-Susceptible Staphylococcus aureus in Chongqing,Southwestern China

Abstract

Methicillin-susceptible Staphylococcus aureus (MSSA) accounts for ∼40% of staphylococcal infections in China. However, the molecular characterization of MSSA is not well described. In this study, 124 MSSA strains collected in 2013 from a comprehensive teaching hospital in Chongqing, Southwestern China, were subjected to antibiotics susceptibility testing and molecular typing, including multilocus sequence typing, staphylococcal protein A (spa) gene typing, accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) typing, Panton–Valentine leukocidin (pvl) gene detection, and antibiotic-resistant gene detection. MSSA strains exhibited high genetic heterogeneity. A total of 10 PFGE groups, 26 sequence types, and 47 spa types were identified. Type I (62.9%) was the most frequent agr type, followed by type II (15.3%), type IV (11.3%), and type III (10.5%). The prevalence of pvl genes was 27.4% (34/124). Notably, 44.4% (55/124) of MSSA strains were multidrug resistance (MDR), and MDR isolates were mostly resistant to penicillin, erythromycin, and clindamycin. The resistance gene blaZ was present in 84.7% of strains, ermC was present in 85.5% of strains, ermA was present in 28.2% of strains, tetK was present in 16.1% of strains, tetM was present in 6.5% of strains, and aacA-aphD was present in 2.6% of strains. These data demonstrated the high prevalence of MDR MSSA in Chongqing, thereby indicating the need to control MSSA infection.

Introduction

Staphylococcus aureus is a major human pathogen that can cause various infections, ranging from benign superficial skin eruptions to life-threatening bacteremia, endocarditis, pneumonia, and toxic shock syndrome.¹ Understanding the genotypic characteristics of S. aureus clones helps control the spread of S. aureus. Several molecular typing methods, including pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal protein A (spa) gene typing, and accessory gene regulator (agr) typing, have been applied to the evolution and epidemiology surveys of S. aureus.^2–5 The number of infections caused by methicillin-resistant S. aureus (MRSA) has rapidly increased since the late 1980s; a molecular typing survey of MRSA revealed that several major clones, including ST1, ST5, ST8, ST59, and ST239, cause most of the MRSA infections worldwide.⁶ However, several infections are caused by methicillin-susceptible S. aureus (MSSA); ∼40% of staphylococcal infections in China were caused by MSSA,^7–9 but little is known about the molecular characteristics of MSSA.

MSSA isolates usually exhibit high genetic diversity without clear geographical or temporal clustering. Grundmann et al.¹⁰ indicated the widespread diversity of MSSA strains without a predominant geographic pattern in Europe, thereby contradicting the clonal clustering of MRSA isolates. Miko et al.¹¹ reported that several prevalent MSSA strains (spa t002 and spa t008) were analogous to the predominant MRSA clones. The primary MSSA clones also differed among studies in China. The prevalent MSSA clones, such as ST1, ST5, ST6, ST7, ST25, ST59, ST88, ST188, ST398, and ST2155, have been identified in China.^8,12,13 Liu et al.⁸ reported ST188-t189-agr I (20.6%) as the predominant MSSA clone in central–southern China. Another study by Chen et al.¹² indicated that ST398-t571 is the most frequent MSSA clone; ST5-t002 and ST59-t437 coexist in MRSA and MSSA. MSSA reportedly exhibits high prevalence of several virulence genes, such as those encoding for Panton–Valentine leukocidin (pvl), hemolysins, toxic shock syndrome toxin, and staphylococcal enterotoxin. The prevalence of virulence factors among MSSA isolates was significantly higher than that among MRSA isolates.⁸ Furthermore, MSSA is more frequently associated with bacteremia, endocarditis, and sepsis than MRSA, thereby indicating a possible “reversal of roles” between the organisms with the two resistance patterns.¹⁴ The association of antimicrobial resistance profiles (ARPs) in MSSA strains and their molecular characteristics may be useful for the clinical treatment of MSSA infections.

MSSA causes a large burden of disease in nosocomial and community-associated infections, but the characteristics of MSSA strains in Southwestern China remain poorly understood. In this study, we utilized molecular typing methods combined with antibiotic susceptibility testing and polymerase chain reaction (PCR)-based profiling of pvl and resistance genes to characterize a collection of MSSA isolates from a comprehensive teaching hospital in Chongqing for the molecular characterization and genotype/phenotype correlation of MSSA strains in Southwestern China.

Materials and Methods

Bacterial isolates

A total of 124 MSSA strains were collected between January 2013 and December 2013 from a comprehensive teaching hospital in Chongqing, China (Southwest Hospital Affiliated to The Third Military Medical University). This hospital in Southwestern China is a large hospital (2,900 beds) that annually handles ∼3.2 million outpatients. In 2013, 319 isolates of S. aureus were cultured and kept frozen at −80°C in tryptic soy broth supplemented with 40% glycerol. The genomic DNA was extracted as described by Unal et al.,¹⁵ and 124 strains (38.9%) were characterized as MSSA by determining their femB and mecA genes as previously described.¹⁶ These MSSA isolates were obtained from individual hospitalized patients in the Departments of Pediatrics (n = 38, 30.7%), Respiratory Medicine (n = 18, 14.5%), Emergency (n = 18, 14.5%), Burn Medicine (n = 11, 8.9%), Joint Surgery (n = 11, 8.9%), Oncology (n = 6, 4.8%), Geriatric Medicine (n = 4, 3.2%), Neurology (n = 4, 3.2%), and other departments (Dermatology, Urology, and Endocrinology; n = 14, 11.3%). The isolates were derived from diverse clinical specimens, including respiratory tract secretions (n = 35, 28.2%), blood (n = 28, 22.6%), abscesses or wounds (n = 27, 21.8%), and other fluids (cerebrospinal fluid, joint fluid, and abdominal fluid; n = 34, 27.4%).

The study was approved by the Laboratory Animal Welfare and Ethics Committees of the Third Military Medical University, Chongqing, China. Given that the data were collected and interpreted anonymously, the need for written informed consent from the participants was waived by the university ethics committees.

Molecular typing experiments

Multilocus sequence typing

Seven housekeeping genes (arcC, aroE, glp, gmk, pta, tpi, and yqiL) of all the MSSA strains of interest were amplified and sequenced as described.¹⁷ The allelic profiles and sequence types (STs) were assigned according to the MLST Website (http://saureus.mlst.net).

spa typing

A variable number of 21 to 27 bp repeats were contained in the X region of the spa gene in S. aureus; the 24 bp repeat is the most common. The entire X region of each MSSA isolate was amplified by PCR as described by Shopsin et al.¹⁸ The PCR products were sequenced and analyzed, and each isolate was assigned a specific spa type based on the spa database Website (www.ridom.de/spaserver).

agr typing

agr allele types (I–IV) were determined by multiplex PCR using the agr group-specific primers as described by Gilot et al.¹⁹ The PCR products of 441, 575, 323, and 659 bp represent the agr types I, II, III, and IV, respectively.

PFGE typing

DNA extraction and SmaI restriction were performed as previously described. ²⁰ The SmaI-digested DNA fragments of the MRSA strain N315 served as PFGE standard molecular sizes and were included in each gel. The PFGE patterns were analyzed with BioNumerics version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium) according to the unweighted pair-group matching analysis clustering algorithm. The strains with 90% identity were categorized to the same group.

pvl gene detection

The pvl genes of all isolates were detected by PCR with primers pvl-1 and pvl-2 as previously described.²¹

Antimicrobial susceptibility testing

Antimicrobial susceptibility was determined by the broth dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI).²² The tested antimicrobial agents included oxacillin (OXA), levofloxacin (LEV), penicillin (PEN), erythromycin (ERY), clindamycin (CLI), rifampicin (RIF), ciprofloxacin (CIP), gentamicin (GEN), tetracycline (TET), trimethoprim–sulfamethoxazole (SXT), linezolid (LZD), teicoplanin (TEC), and vancomycin (VAN). CLSI breakpoints were used for minimum inhibitory concentration (MIC) interpretation.

Resistance gene detection

The genetic determinants for macrolide and lincosamide resistance (ermA and ermC genes), TET resistance (tetK and tetM genes), aminoglycoside resistance (aacA-aphD gene), and β-lactams resistance (blaZ gene) were detected by PCR according to previously described protocols.^23,24

Definitions

According to the diversity of the epidemic MSSA strains, the clone comprising >5% of the isolates was considered the major prevalent clone. An isolate was considered to have multidrug resistance (MDR) when the isolate was resistant to at least one agent in three or more antimicrobial categories.²⁵

Results

Molecular typing of MSSA isolates

The molecular characteristics of all MSSA isolates are shown in Table 1. The MSSA strains were genetically diverse. A total of 26 STs were identified for the 124 MSSA isolates. The most frequent STs were ST22 (11.3%, 14/124), ST121 (10.5%, 13/124), ST15 (10.5%, 13/124), ST7 (9.7%, 12/124), ST398 (6.5%, 8/124), and ST188 (5.6%%, 7/124), which accounted for 54.1% of the tested MSSA strains. A total of 47 spa types were determined with the major prevalent types of t11413 (9.7%, 12/124), t571 (8.9%, 11/124), t7164 (8.1%, 10/124), and t002 (6.5%, 8/124), thereby accounting for 33.2% of the MSSA strains. Other spa types, such as t030 (4.8%, 6/124), t189 (4.0%, 5/124), t2592 (4.0%, 5/124), and t2663 (3.2%, 4/124), were also identified.

Table 1.

Relationships Between agr Types and Other Molecular Types in MSSA Isolates

agr type (no.)	MLST (no.)	spa type (no.)	pvl+ (no.)
Type I(78)	ST22(14)	t11413(11), t284(1), t571(2)	10
	ST7(12)	t7164(6), t2663(4), t6193(2)	1
	ST398(8)	t571(4), t034(1), t030(1), t2874(1), t7160(1)	2
	ST188(7)	t189(5), t030(1), t8914(1)	0
	ST239(6)	t037(2), t030(1), t1202(1), t3297(1), t7164(1)	1
	ST25(5)	t078(2), t349(1), t5231(1), t9506(1)	1
	ST59(4)	t163(2), t172(1), t437(1)	0
	ST943(4)	t7164(3), t2617(1)	0
	ST464(3)	t3297(3)	0
	ST630(3)	t377(3)	0
	ST97(2)	t267(1), t359(1)	1
	ST121(2)	t030(2)	1
	ST217(2)	t11413(1), t571(1)	0
	ST6(1)	t701(1)	0
	ST15(1)	t11518(1)	0
	ST20(1)	New(1)	0
	ST617(1)	t1081(1)	0
	ST1281(1)	t164(1)	0
	ST1920(1)	t030(1)	0
Type II(19)	ST15(12)	t2325(3), t571(4), t002(2), t693(1), t10029(1), t11518(1)	0
Type III (13)	ST5(6)	t002(5), t062(1)	1
	ST59(1)	t002(1)	0
	ST88(6)	t2592(5), t1376(1)	4
	ST1(3)	t127(2), t114(1)	2
	ST1920(2)	t5356(2)	0
	ST938(1)	t318(1)	1
	ST30(1)	t1130(1)	1
Type IV(14)	ST121(11)	t159(3), t284(3), t435(2), t850(1), t2086(1), t2092(1)	8
	ST45(2)	t1081(2)	0
	ST946(1)	New(1)	0

agr, accessory gene regulator; MLST, multilocus sequence typing; MSSA, methicillin-susceptible Staphylococcus aureus; pvl, Panton–Valentine leukocidin; spa, staphylococcal protein A.

For agr typing, agr type I was identified as the most prevalent, accounting for 62.9% (78/124) of the MSSA isolates, followed by type II (15.3%, 19/124), type IV (11.3%, 14/124), and type III (10.5%, 13/124). The agr type II isolates were assigned to ST15 (63.2%, 12/19), ST5 (31.6%, 6/19), and ST59 (5.2%, 1/19), whereas among the 14 agr type IV isolates, 11 belonged to ST121 (78.6%, 11/14). The presence of pvl genes was 27.4% (34/124). ST22 (71.4%, 10/14), ST121 (69.2%, 9/13), ST88 (66.7%, 4/6), and ST1 (66.7%, 2/3) MSSA strains seemed to frequently carry pvl genes (Table 1). All 124 MSSA strains were analyzed by PFGE, whereas 8 ST398 strains were nontypable probably because of DNA methylation.²⁶ Among the 10 PFGE groups (A to J) identified, groups A (26.7%, 31/116), B (26.7%, 31/116), and D (24.1%, 28/116) accounted for 77.6% of the typable MSSA isolates (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/mdr). Most ST121 strains belonged to PFGE group D (92.3%, 12/13), most ST7 MSSA strains belonged to PFGE group A (91.7%, 11/12), and most ST15 MSSA strains belonged to PFGE group B (91.7%, 11/12). Most agr IV isolates belonged to PFGE group D (85.7%, 12/14).

Antimicrobial susceptibility determination

Antibiotic susceptibility testing showed that 77.4% (96/124) of the MSSA isolates were resistant to PEN, 55.6% (69/124) isolates were resistant to ERY, 47.6% (59/124) isolates were resistant to CLI, 26.6% (33/124) isolates were resistant to SXT, 16.9% (21/124) isolates were resistant to TET, 7.3% (9/124) isolates were resistant to GEN, 4.8% (6/124) isolates were resistant to CIP, and 2.4% (3/124) isolates were resistant to LEV. All tested MSSA isolates were susceptible to RIF, OXA, VAN, LZD, and TEC.

MDR MSSA isolates and resistance gene carriage

Based on the definitions, 44.4% (55/124) of the tested MSSAs were MDR isolates. Among these MDR strains, 65.5% (36/55) were resistant to three categories of antimicrobials, 30.9% (17/55) were resistant to four categories of antimicrobials, and 3.6% (2/55) were resistant to seven tested antimicrobial agents (six categories) (Table 2). For MSSA STs, all 4 ST943 strains were MDR with varied ARPs; 9 out of 13 ST121 strains (69.2%) were MDR, and most of which (88.9%, 8/9) were resistant to PEN, ERY, and CLI. Three ST398 MDR isolates were resistant to at least four categories of antimicrobial agents (Table 2). Approximately 67.9% (19/28) of PFGE group D strains were MDR MSSAs, including 1 strain (ST45-t1081) that was resistant to 7 tested antimicrobial agents.

Table 2.

ARPs of MDR Isolates

MDR isolates resistant to (no.)	ARPs of MDR isolates (no.)	MLST (no.)	PFGE type (no.)
Three antibiotics (36)	PEN, ERY, CLI (31)	ST121(8), ST22(6), ST7(4), ST88(3), ST15(2), ST217(2), ST5(2), ST1(1), ST188(1), ST630(1), ST943(1)	D(15), A(9), B(3), E(2), C(1), J(1)
	PEN, ERY, SXT (2)	ST15(1), ST25(1)	B(2)
	PEN, ERY, TET (1)	ST15(1)	B(1)
	PEN, SXT, TET (1)	ST7(1)	A(1)
	ERY, SXT, TET (1)	ST943(1)	B(1)
Four antibiotics (8)	PEN, ERY, CLI, TET (3)	ST7(1), ST30(1), ST121(1)	A(1), D(2)
	PEN, ERY, CLI, SXT (3)	ST59(1), ST88(1), ST398(1)	B(1), J(1), nontypable(1)
	PEN, ERY, CLI, CIP (1)	ST630(1)	B(1)
	PEN, ERY, TET, GEN (1)	ST45(1)	D(1)
Five antibiotics (6)	PEN, ERY, CLI, SXT, TET (3)	ST943(2), ST15(1)	A(2), B(1)
	PEN, ERY, CLI, SXT, GEN (3)	ST5(1), ST7(1), ST1920(1)	A(1), C(2)
Six antibiotics (3)	PEN, ERY, CLI, SXT, CIP, TET (2)	ST398(2)	Non-typable(2)
	PEN, ERY, CLI, SXT, TET, GEN (1)	ST5(1)	B(1)
Seven antibiotics (2)	PEN, ERY, CLI, SXT, CIP, LEV, GEN (2)	ST45(1), ST617(1)	J(1), D(1)

ARP, antimicrobial resistance profile; CIP, ciprofloxacin; CLI, clindamycin; ERY, erythromycin; GEN, gentamicin; LEV, levofloxacin; MDR, multidrug resistance; PEN, penicillin; PFGE, pulsed-field gel electrophoresis; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline.

The resistance genes carried by MSSA strains were detected. The results demonstrated that 84.7% (105/124) of the MSSA strains harbored blaZ, which is a major determinant for PEN resistance. The genetic determinants for macrolide and lincosamide resistance (ermA and ermC genes) were detected in this study, whereas the ermB gene was not detected because hospital strains seldom carry ermB.²⁷ The ermC gene is primarily responsible for ERY and CLI resistance; this gene was carried by 85.5% (106/124) MSSA strains. Moreover, 28.2% (35/124) MSSA isolates carried the ermA gene. Lina et al.²⁸ indicated that ermC is predominant in MSSA isolates, mainly those with an inducible expression. No association was observed between the phenotypic ERY resistance and the presence of erm gene, as indicated in a previous study.²⁹ The resistance to TET in S. aureus is either based on the widely disseminated tetM gene that encodes for modification of the ribosome or mediated by the tetK gene-encoded efflux.²⁴ The tetK and tetM genes were, respectively, carried by 16.1% (20/124) and 6.5% (8/124) of the MSSA isolates. S. aureus can carry a bifunctional enzyme-coding gene, aacA-aphD, for cross-resistance to clinically used aminoglycosides, such as GEN,²⁴ and we detected that 22.6% (28/124) of the MSSA strains harbored the aacA-aphD gene.

Discussion

MSSA is genetically defined as the absence of the mecA gene for an alternative PEN-binding protein 2a, with the reduced affinity for β-lactams and the presence of an S. aureus-specific gene, such as femB; phenotypically, MSSA is indicated by an OXA MIC of ≤2 mg/L. In this study, MSSA isolates from Chongqing were observed to be genetically diversified, with six major STs (ST22, ST121, ST15, ST7, ST398, and ST188) and four major spa types (t11413, t571, t7164, and t002). Several of these types (ST7, ST398, ST188, and t571) were previously characterized as the most dominant molecular types in other cities in China.^12,13,30 The high diversity of MSSA isolates may be attributed to the temporal or geographic dynamics of endemic and epidemic S. aureus clones.^10,31 ST22 has been established as one of the major international MLST of MRSA.³² However, ST22 has been detected in MSSA strains from Beijing, China,³⁰ and other countries.¹⁷ We proved that ST22 strains (11.3%, 14/124) are the most common group among MSSA isolates in Chongqing. Further investigation of the roles of ST22 isolates with a large collection of MSSA isolates is proposed. Previously, ST121 has been characterized as a hypervirulent clone and is seldom reported in China.^33,34 Notably, ST121 has become a major prevalent MSSA clone (10.5%, 13/124) in Chongqing, Southwestern China. The agr locus belongs to the core variable genome and is linked to the STs of MSSA strains.³⁰ In Chongqing, agr I was the most common agr type in MSSA isolates and was often linked to ST22, ST7, ST398, and ST188, whereas agr II was usually associated with ST15; agr III was mainly related to ST88, but ST121 accounted for most of the agr IV strains. Our findings are consistent with those of other studies.^30,35

PVL is linked to skin and soft tissue infections caused by S. aureus; the protein may be carried by MRSA and MSSA, most often by the so-called community-associated MRSA strains.³⁶ In Chongqing, PVL-positive MSSA is fairly common, with a positive rate of 27.4% (34/124), which is similar to that presented in several recent studies.^30,37 The main pvl-positive MSSA clones were ST121-agr IV (72.7%, 8/11), ST22-agr I (71.4%, 10/14), and ST88-agr III (66.7%, 4/6), thereby providing evidence that these clones may be associated with abscess formation and tissue necrosis.

Since the emergence of MRSA in the late 1980s, the antibiotic resistance of MSSA has been largely neglected. MSSA is reportedly susceptible to most antibiotic agents, except PEN, in Europe and the United States.^11,38,39 The high prevalence of MDR MSSA isolates (44.4%, 55/124) in Chongqing was detected in this study. This rate was higher than that reported by a previous study, where 22.6% (36/159) of the MSSA isolates collected in Beijing were simultaneously resistant to more than three antimicrobial classes.³⁰ The discrepancy between phenotype and genotype of the MSSA isolates was observed, such that 77.4% (96/124) of the MSSA isolates were resistant to PEN, however, 84.7% (105/124) of the MSSA strains harbored blaZ; 85.5% (106/124) of the MSSA strains carried ermC, but only 55.6% (69/124) of the isolates were resistant to ERY and 47.6% (59/124) of the isolates were resistant to CLI. In addition, 22.6% (28/124) of the MSSA isolates carried aacA-aphD, whereas only 7.3% (9/124) of the strains were resistant to GEN. Similar phenomena were observed in a previous study.²⁹ Sekiguchi et al.⁴⁰ proposed that a mutation in the coding or promoter region of the drug resistance gene in several MSSA isolates might contribute to the discordance between phenotype and genotype. More than half of MDR MSSA strains (65.5%, 36/55) showed resistance to three categories of antimicrobials, but MSSA strains were detected in Chongqing, even those showing resistance to six classes of antimicrobial agents. Therefore, infections caused by MDR MSSA strains need to be controlled in China.

In conclusion, this study reveals the high prevalence of MDR MSSA in Chongqing, Southwestern China. Although MRSA is a primary problem for S. aureus infections, MSSA still comprises a large proportion of infections in China probably because of its high virulence and growing resistance. MSSA strains are genetically diverse and commonly carry pvl genes. The MDR MSSA strains are mostly resistant to PEN, ERY, and CLI (89.1%, 49/55) with high levels of the corresponding resistant genes, such as blaZ and ermC. Therefore, efforts to fight infections caused by MSSA in China should be intensified.

Footnotes

Acknowledgments

This study was supported by a grant from the National Natural Scientific foundation of China (No. 81471993) and the New Drug Development Project of China (2012ZX09103301-038).

Disclosure Statement

No competing financial interests exist.

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