Increasing Resistance in Multiresistant Methicillin-Resistant Staphylococcus aureus Clones Isolated from a Chinese Hospital Over a 5-Year Period

Abstract

Background: The aim was to study the changes in the antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) clones over a 5-year period (from 2000 to 2005) at a representative hospital in Beijing, China. Methods: A total of 100 randomly selected MRSA strains were analyzed using antimicrobial susceptibility testing, pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, SCCmec typing, and PCR for the Panton-Valentine leukocidin virulence factor. Results: Resistance to rifampin greatly increased from 32% (16/50) to 68% (34/50). High-level mupirocin-resistant isolates were found only in 2005, when four were identified. Intermediate susceptibly to quinupristin-dalfopristin increased from 22% (11/50) to 52% (26/50) between 2000 and 2005. The main antimicrobial resistance profiles changed from TC-GM-CI-EM-CM in 2000 to TC-GM-CI-EM-CM-RI in 2005. The main pulsed-field gel electrophoresis type changed from types C, L, and E in 2000 to types J, F, and N, respectively, in 2005. ST239-MRSA-III was the most predominant clone in 2000 and 2005, whereas ST5-MRSA-II was found only in 2005. Conclusions: There were increasing levels of antimicrobial resistance and epidemiological changes in the hospital-associated MRSA strains isolated in this facility between 2000 and 2005.

Introduction

Staphylococcus aureus has become a very important pathogen that causes several diseases, ranging from minor infections of the skin to wound infections, bacteremia, and necrotizing pneumonia. The epidemiological characteristics of S. aureus, especially methicillin-resistant S. aureus (MRSA), are changing rapidly. From the first MRSA strain reported until the present, various hospital-associated MRSA (HA-MRSA) clones have been disseminated worldwide.^3,5,6,12,26 The emergence of new strains of community-associated MRSA in many parts of the world is well documented^15,21 and causes an increasing proportion of healthcare-associated infections.¹³

In China, MRSA is highly endemic in hospitals with mean prevalence rates of >50% in 2005.³³ Two major epidemic MRSA clones with a unique geographic distribution across China, ST239-MRSA-III and ST5-MRSA- II, have been reported recently.²² Multilocus sequence typing for a few isolates over 5 years was completed by us and provided an important clue.³⁴ To investigate the dynamics of MRSA over long time periods, we characterized MRSA isolates from a Chinese hospital collected between 2000 and 2005 to identify changes in antimicrobial resistance, molecular typing, and virulence factors.

Materials and Methods

Isolates

A total of 100 MRSA strains were isolated from several clinical sources including the respiratory tract (n = 76), blood (n = 3), drainage (n = 8), pus (n = 7), wounds (n = 3), and other sources (n = 3) that were randomly selected from Beijing Union Medical Hospital, which has about 1,800 beds. Fifty strains were isolated in 2000, and 50 more were isolated in 2005. All strains were confirmed by PCR analysis of the mecA and nuc genes.⁴

Antimicrobial susceptibility tests

A total of 14 antimicrobial agents were tested, including linezolid, chloramphenicol, tetracycline, gentamicin, vancomycin, ciprofloxacin, erythromycin, clindamycin, rifampin, quinupristin-dalfopristin, teicoplanin, mupirocin, fusidic acid, and trimethoprim. Vancomycin was tested by broth dilution according to Clinical Laboratory Standards Institutes, and the other antimicrobial agents were tested by Etest (AB Biodisk). Clinical Laboratory Standards Institutes breakpoints were used for minimum inhibitory concentration (MIC) interpretation. The resistance breakpoints for mupirocin and fusidic acid were defined as previously described.^9,23

Molecular typing

All isolates were investigated by pulsed-field gel electrophoresis (PFGE), SCCmec typing, and spa typing. Multilocus sequence typing was performed on 29 strains that included representatives of each spa type. Genomic DNA and plasmids were extracted using the DNeasy blood and tissue kit and the QIAprep Spin Miniprep kit, respectively (Qiagen GmbH). Amplifications were performed in a Mycycler™ thermal cycler (Bio-Rad). PFGE was performed using the CHEF-DR III System (Bio-Rad).

PFGE using SmaI was set up according to the American CDC Standardized Protocol for Molecular Subtyping of Listeria monocytogenes by PFGE (www.cdc.gov/PULSENET/protocols.htm). The MRSA suspension incorporated into the agarose block was standardized to have an optical density (OD) of 5.5–6.5, and 2 μl lysostaphin (1 mg/ml) was added to 250 μl of bacterial suspension, which was immediately mixed with an equal volume of 1% low-melting-point agarose. Digital images were analyzed by BioNumerics software (v. 4.6; Bio-Rad) using the Dice coefficient and were represented by unweighted pair group method arithmetic averages (UPGMA) with 1.5% tolerance and 1% optimization settings. A similarity cutoff of 80% and the criterion of a difference of six bands as described by Tenover et al.³⁰ were used to define a cluster. Isolates showing identical or related PFGE patterns were considered to belong to the same clone. Clones were labeled with a capital letter (A, B, C, and so on), and related profiles were indicated by adding a number (A1, A2, B1, B2, and so on).

spa typing, multilocus sequence typing, and SCCmec typing were performed as previously described.^7,8,14,27,35

pvl and mupA genes detection

Panton-Valentine leukocidin (PVL) genes (lukS-PV and lukF-PV) were detected by PCR.²¹ The high-level mupirocin-resistant isolates (based on the Etest method) were detected by PCR for the mupA gene as previously described.¹

Statistical analysis

Statistical analysis was performed with the SPSS software package using chi-square and Fisher's exact tests.

Results

Antimicrobial susceptibility testing

Isolates from the various time periods did not demonstrate resistance or increases in the MIC of vancomycin, teicoplanin, and linezolid. Resistance to rifampin greatly increased from 32% (16/50) to 68% (34/50, p < 0.001). Four high-level mupirocin-resistant isolates were found only in samples from 2005. Intermediate susceptibility rates to quinupristin-dalfopristin increased from 22% (11/50) to 52% (26/50, p < 0.01) from 2000 to 2005. Resistance to ciprofloxacin was universal. Resistance to chloramphenicol, tetracycline, erythromycin, and clindamycin slightly increased from 4% to 6%, 94% to 98%, 98% to 100%, and 98% to 100%, respectively. On the other hand, gentamicin, quinupristin-daltopristin, fusidic acid, and trimethoprim resistance decreased from 100% to 94%, 12% to 8%, 4% to 0%, and 4% to 0%, respectively.

Antibiotic resistance profiles and molecular epidemiology

Overall, 15 PFGE strain types (A through O) and 4 clusters (1 through 4) were identified among the 100 MRSA isolates. Five PFGE types, type (C, J, N, F, and L) were the dominant types, constituting 82% of all isolates. Types C, L, and E were the dominant types from isolates collected in 2000, accounting for 88% (44/50), whereas types J, F, and N were the dominant types in 2005, accounting for 74% (37/50).

Eleven antimicrobial resistant profiles were identified in total. Two predominant profiles (TC-GM-CI-EM-CM-RI and TC-GM-CI-EM-CM) contained 47 and 40 isolates, respectively. The antimicrobial resistance profile TC-GM-CI-EM-CM-RI included 14 isolates in 2000, corresponding to PFGE types E, L, and M, and included 33 isolates in 2005, corresponding to PFGE types A, F, I, J, K, L, and N. The antimicrobial resistance profile TC-GM-CI-EM-CM included 29 isolates in 2000, corresponding to PFGE types C, D, and N, and included 11 isolates in 2005, corresponding to PFGE types C, F, G, and L (Table 1).

Table 1.

Multilocus Sequence Typing, spa, SCCmec, and Pulsed-Field Gel Electrophoresis Types of Methicillin-Resistant Staphylococcus aureus Isolates and Their Antibiotic Resistance Profiles in 2000 and 2005

ST (CC)	spa type	SCCmec	PFGE type	Antibiotic resistance profile	% of isolates in 2000	% of isolates in 2005
5 (5)	t002	II	F	TC-GM-CI-EM-CM	0	16
				CL-TC-GM-CI-EM-CM-MU	0	4
			G	TC-GM-CI-EM-CM	0	2
			H	TC-CI-EM-CM-MU	0	2
				CL-TC-GM-CI-EM-CM-MU	0	2
				TC-CI-EM-CM	0	2
			L1	TC-GM-CI-EM-CM	0	2
72 (8)	t148	Unknown	O	GM-CI-EM-CM	2	0
239 (8)	t1152	III	A	TC-GM-CI-EM-CM-RI	0	2
	t037	III	B	CL-TC-GM-CI-EM-CM-RI-QDA-MU-FU-TR	2	0
			C1	TC-GM-CI-EM-CM	50	2
				TC-GM-CI-EM-CM-FU-TR	2	0
				GM-CI-EM-CM	2	0
			C2	TC-GM-CI-EM-CM	4	0
				GM-CI-EM-CM	2	0
			D	TC-GM-CI-EM-CM	2	0
			E	CL-TC-GM-CI-EM-CM	2	0
		Unknown	E	TC-GM-CI-EM-CM-RI	8	0
	t030	III	F	TC-GM-CI-EM-CM-RI	0	2
			I1	TC-GM-CI-EM-CM-RI	0	6
			I2	TC-GM-CI-EM-CM-RI	0	2
			J1	TC-GM-CI-EM-CM-RI	0	6
			J2	TC-GM-CI-EM-CM-RI	0	22
				TC-CI-EM-CM-RI	0	2
			K	TC-GM-CI-EM-CM-RI	0	2
			L1	TC-GM-CI-EM-CM-RI-QDA	2	0
				TC-GM-CI-EM-CM-RI	16	0
			L2	TC-GM-CI-EM-CM-RI	0	2
			M	TC-GM-CI-EM-CM-RI	4	0
			N1	TC-GM-CI-EM-CM-RI	0	4
			N2	TC-GM-CI-EM-CM	2	0
				TC-GM-CI-EM-CM-RI	0	18

CI, ciprofloxacin; CL, chloramphenicol; CM, clindamycin; EM, erythromycin; FU, fusidic acid; GM, gentamicin; MU, mupirocin; PFGE, pulsed-field gel electrophoresis; QDA, quinupristin/dalfopristin; RI, rifampin; TC, tetracycline; TR, trimethoprim.

Typing all isolates yielded five spa types (Table 1). T037 was the predominant spa type in 2000, accounting for 74% (37/50); and the second most prominent was t030, accounting for 24% (12/50). T030 was the most predominant spa type in 2005, accounting for 66% (33/50); whereas the second was t002, accounting for 30% (15/50, p < 0.001).

Twenty-nine strains that included representatives of each spa type yielded three STs belonging to two clonal complexes (Table 1). ST239 was the predominant ST, which belonged to clonal complex CC8 and constitutes 72.41% of isolates (21/29). The second ST was ST5, which belonged to clonal complex CC5 and constitutes 24.14% of isolates (7/29). ST5 was only found in 2005.

From all isolates, two SCCmec types were identified, namely II and III (Table 1). The most common SCCmec type was type III, which was found in 80 isolates (80/100 or 80%), including 45 isolates from 2000 and 35 isolates (35/50 or 70%) from 2005. The second was type II, which was only found in 15 isolates (15/100 or 15%) from 2005. A total of five isolates (5/100 or 5%) were nontypeable by the multiplex SCCmec typing method.

Detection of pvl and mupA

No isolate was pvl positive. The four high-level mupirocin-resistant isolates were all positive for mupA.

Discussion

MRSA strains associated with community acquisition were not found to have been introduced into this hospital, but there were epidemiological changes in the types of HA-MRSA strains in the facility over the 5 years included in our study, with an associated change in antimicrobial resistance patterns.

The main antimicrobial resistance profile changed from TC-GM-CI-EM-CM in 2000 to TC-GM-CI-EM-CM-RI in 2005. Resistance to rifampin increased from 32% to 68%, and the MIC₅₀ for rifampin increased from 0.008 to 256 μg/ml. Combination therapy with rifampin has been used to treat S. aureus infections^24,29 and has been widely used in China in recent years to treat esophagitis, dysentery (difficult to cure or resistant), skin infections, hordeolum, purulent otitis media, and mycoplasma pneumonia. From our data, we found that an increasing number of resistant isolates are emerging with the wide spread use of rifampin.

The data in this study revealed that resistance to mupirocin increased from 2% to 8%. We found that four high-level-resistant (MupRH) isolates with mupA in 2005,¹¹ which were PFGE types F and H, belonged to the same spa type and STs (t002, ST5-MRSA-II) and belonged to two antimicrobial resistant profiles (CL-TC-GM-CI-EM-CM-MU and TC-CI-EM-CM-MU). One low-level-resistant (MupRL)^2,10 isolate from 2000 was PFGE type B, t037, ST239-MRSA-III, and resistant to 11 antimicrobial drugs (CL-TC-GM-CI-EM-CM-RI-QDA-MU-FU-TR). This result indicated the MupRL isolates and the MupRH isolate were different clones. Beginning in December 2005, mupirocin could be purchased OTC in China. From the experience in other countries,³¹ more attention to it should be paid to this trend. Caution should be taken whenever high-level resistance strains are detected; and control measures should be implemented, because transmissible plasmids have led to outbreaks.²⁰

The other interesting finding was that 10% isolates were resistant to quinupristin-dalfopristin whereas 37% (37/100) of isolates displayed intermediate susceptibility to the drug. The rate of intermediate susceptibility to quinupristin-dalfopristin increased from 22% (11/50) to 52% (26/50) between 2000 and 2005. We are quite curious how this has occurred, because quinupristin-dalfopristin had not been marketed in China until the present. Virginiamycin, another streptogramin A/B combination, has long been used in animal feed as a growth promoter in many countries, including China. This type of use selects for virginiamycin-resistant strains of Enterococcus faecium, which are cross-resistant to quinupristin-dalfopristin¹⁸ and that may pose a risk to public health. As far as we know, staphylococci are generally host-specific, although some exceptions have been noted, such as MRSA from pigs that can colonize humans, as observed in the Netherlands.¹⁶ Thus, whether these isolates resistant to quinupristin-dalfopristin originated from animals needs to be further investigated.

ST5-MRSA-II was a new type found in the hospital in 2005; this strain has also spread widely in European countries and is the predominant MRSA clone in Korea and Japan.¹⁹ Some published data have shown that clonal evolution can occur within a single hospital. In a Mexico City hospital, a local clone (ST30-MRSA-IV) was predominant between 1997 and 2000, but it was completely replaced over a 2-year period by the New York/Japan clone (ST5-MRSA-II).³² Similarly, a study in Spain showed that ST247-MRSA-I was replaced by ST36-MRSA-II between 1998 and 2002.²⁸ The ST5-MRSA-II clone emerged after 2000 and was not previously found in Hong Kong.¹⁷ These results indicate that ST5-MRSA-II has become more common in this Beijing hospital in recent years. Recently, research on the ST5-MRSA clone has provided strong evidence that the geographical spread of MRSA over long distances and across cultural borders is a rare event compared with the frequency with which the staphylococcal cassette chromosome island has been imported.²⁵ Thus, continuous surveillance of MRSA and methicillin-sensitive staphylococcus aureus (MSSA) in hospitals and communities is of great importance for understanding the local epidemiology of MRSA.

Conclusion

Our study showed that there were increasing levels of antimicrobial resistance and epidemiological changes in the HA-MRSA strains collected in the facility between 2000 and 2005.

Footnotes

Acknowledgment

This work was supported by “major infectious diseases such as AIDS and viral hepatitis prevention and control” technology major projects (2008ZX10004-002) from the Ministry of Science and Technology. We thank Beijing Union Medical Hospital for the MRSA isolates supported.

Disclosure Statement

No competing financial interests exist.

References

Anthony

R.M.

, Connor

A.M.

, Power

E.G.

, French

G.L.

1999. Use of the polymerase chain reaction for rapid detection of high-level mupirocin resistance in staphylococci. Eur. J. Clin. Microbiol. Infect. Dis., 18:30–34.

Antonio

, McFerran

, Pallen

M.J.

2002. Mutations affecting the Rossman fold of isoleucyl-tRNA synthetase are correlated with low-level mupirocin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother., 46:438–442.

Aparicio

, Richardson

, Martin

, Vindel

, Marples

R.R.

, Cookson

B.D.

1992. An epidemic methicillin-resistant strain of Staphylococcus aureus in Spain. Epidemiol. Infect., 108:287–298.

Brakstad

O.G.

, Aasbakk

, Maeland

J.A.

1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol., 30:1654–1660.

L.H.

, Severina

E.P.

, Milch

, Thege

M.K.

, Tomasz

1997. Wide geographic distribution of a unique methicillin-resistant Staphylococcus aureus clone in Hungarian hospitals. Clin. Microbiol. Infect., 3:289–296.

Deplano

, Witte

, van Leeuwen

W.J.

, Brun

, Struelens

M.J.

2000. Clonal dissemination of epidemic methicillin-resistant Staphylococcus aureus in Belgium and neighboring countries. Clin. Microbiol. Infect., 6:239–245.

Enright

M.C.

, Day

N.P.

, Davies

C.E.

, Peacock

S.J.

, Spratt

B.G.

2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol., 38:1008–1015.

Feil

E.J.

, Li

B.C.

, Aanensen

D.M.

, Hanage

W.P.

, Spratt

B.G.

2004. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J. Bacteriol., 186:1518–1530.

Finlay

J.E.

, Miller

L.A.

, Poupard

J.A.

1997. Interpretive criteria for testing susceptibility of staphylococci to mupirocin. Antimicrob. Agents Chemother., 41:1137–1139.

10.

Fujimura

, Tokue

, Watanabe

2003. Isoleucyl-tRNA synthetase mutations in Staphylococcus aureus clinical isolates and in vitro selection of low-level mupirocin-resistant strains. Antimicrob. Agents Chemother., 47:3373–3374.

11.

Gilbart

, Perry

C.R.

, Slocombe

1993. High-level mupirocin resistance in Staphylococcus aureus: evidence for two distinct isoleucyl-tRNA synthetases. Antimicrob. Agents Chemother., 37:32–38.

12.

Gomes

A.R.

, Sanches

I.S.

, ires de

S.M.

, Castaneda

, de

L.H.

2001. Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Colombian hospitals: dominance of a single unique multidrug-resistant clone. Microb. Drug Resist., 7:23–32.

13.

Gonzalez

B.E.

, Rueda

A.M.

, Shelburne

S.A.

III , Musher

D.M.

, Hamill

R.J.

, Hulten

K.G.

2006. Community-associated strains of methicillin-resistant Staphylococcus aureus as the cause of healthcare-associated infection. Infect. Control Hosp. Epidemiol., 27:1051–1056.

14.

Harmsen

, Claus

, Witte

, Rothganger

, Claus

, Turnwald

, Vogel

2003. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J. Clin. Microbiol., 41:5442–5448.

15.

Herold

B.C.

, Immergluck

L.C.

, Maranan

M.C.

, Lauderdale

D.S.

, Gaskin

R.E.

, Boyle-Vavra

, Leitch

C.D.

, Daum

R.S.

1998. Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA, 279:593–598.

16.

Huijsdens

X.W.

, van Dijke

B.J.

, Spalburg

, van Santen-Verheuvel

M.G.

, Heck

M.E.

, Pluister

G.N.

, Voss

, Wannet

W.J.

, de Neeling

A.J.

2006. Community-acquired MRSA and pig-farming. Ann. Clin. Microbiol. Antimicrob., 5:26.

17.

, Yung

R.W.

, Ng

T.K.

, Luk

W.K.

, Tse

, Hung

, Enright

, Lyon

D.J.

2005. Contemporary methicillin-resistant Staphylococcus aureus clones in Hong Kong. J. Clin. Microbiol., 43:5069–5073.

18.

Jensen

L.B.

, Hammerum

A.M.

, Aerestrup

F.M.

, van den Bogaard

A.E.

, Stobberingh

E.E.

1998. Occurrence of satA and vgb genes in streptogramin-resistant Enterococcus faecium isolates of animal and human origins in the Netherlands. Antimicrob. Agents Chemother., 42:3330–3331.

19.

K.S.

, Lee

J.Y.

, Suh

J.Y.

, Oh

W.S.

, Peck

K.R.

, Lee

N.Y.

, Song

J.H.

2005. Distribution of major genotypes among methicillin-resistant Staphylococcus aureus clones in Asian countries. J. Clin. Microbiol., 43:421–426.

20.

Leski

T.A.

, Gniadkowski

, Skoczynska

, Stefaniuk

, Trzcinski

, Hryniewicz

1999. Outbreak of mupirocin-resistant staphylococci in a hospital in Warsaw, Poland, due to plasmid transmission and clonal spread of several strains. J. Clin. Microbiol., 37:2781–2788.

21.

Lina

, Piemont

, Godail-Gamot

, Bes

, Peter

M.O.

, Gauduchon

, Vandenesch

, Etienne

1999. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin. Infect. Dis., 29:1128–1132.

22.

Liu

, Wang

, Du

, Shen

, Chen

, Niu

, Ye

, Chen

2009. Molecular evidence for spread of two major methicillin-resistant Staphylococcus aureus clones with a unique geographic distribution in Chinese hospitals. Antimicrob. Agents Chemother., 53:512–518.

23.

MacGowan

A.P.

, Wise

2001. Establishing MIC breakpoints and the interpretation of in vitro susceptibility tests. J. Antimicrob. Chemother., 48,Suppl 1:17–28.

24.

Miller

L.G.

, Quan

, Shay

, Mostafaie

, Bharadwa

, Tan

, Matayoshi

, Cronin

, Tan

, Tagudar

, Bayer

A.S.

2007. A prospective investigation of outcomes after hospital discharge for endemic, community-acquired methicillin-resistant and -susceptible Staphylococcus aureus skin infection. Clin. Infect. Dis., 44:483–492.

25.

Nubel

, Roumagnac

, Feldkamp

, Song

J.H.

, Ko

K.S.

, Huang

Y.C.

, Coombs

, Ip

, Westh

, Skov

, Struelens

M.J.

, Goering

R.V.

, Strommenger

, Weller

, Witte

, Achtman

2008. Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus. Proc. Natl. Acad. Sci. U. S. A., 105:14130–14135.

26.

Oliveira

, Santos-Sanches

, Mato

, Tamayo

, Ribeiro

, Costa

, de

L.H.

1998. Virtually all methicillin-resistant Staphylococcus aureus (MRSA) infections in the largest Portuguese teaching hospital are caused by two internationally spread multiresistant strains: the “Iberian” and the “Brazilian”. clones of MRSA. Clin. Microbiol. Infect., 4:373–384.

27.

Oliveira

D.C.

, de

L.H.

2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother., 46:2155–2161.

28.

Perez-Roth

, Lorenzo-Diaz

, Batista

, Moreno

, Mendez-Alvarez

2004. Tracking methicillin-resistant Staphylococcus aureus clones during a 5-year period (1998 to 2002) in a Spanish hospital. J. Clin. Microbiol., 42:4649–4656.

29.

Schwalm

J.D.

, El-Helou

, Lee

C.H.

2004. Clinical outcome with oral linezolid and rifampin following recurrent methicillin-resistant Staphylococcus aureus bacteremia despite prolonged vancomycin treatment. Can. J. Infect. Dis., 15:97–100.

30.

Tenover

F.C.

, Arbeit

R.D.

, Goering

R.V.

, Mickelsen

P.A.

, Murray

B.E.

, Persing

D.H.

, Swaminathan

1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol., 33:2233–2239.

31.

Upton

, Lang

, Heffernan

2003. Mupirocin and Staphylococcus aureus: a recent paradigm of emerging antibiotic resistance. J. Antimicrob. Chemother., 51:613–617.

32.

Velazquez-Meza

M.E.

, ires de

S.M.

, Echaniz-Aviles

, Solorzano-Santos

, Miranda-Novales

, Silva-Sanchez

, de

L.H.

2004. Surveillance of methicillin-resistant Staphylococcus aureus in a pediatric hospital in Mexico City during a 7-year period (1997 to 2003): clonal evolution and impact of infection control. J. Clin. Microbiol., 42:3877–3880.

33.

Wang

, Liu

, Sun

, Xu

, Xie

, Chen

2008. In vitro activity of ceftobiprole, linezolid, tigecycline, and 23 other antimicrobial agents against Staphylococcus aureus isolates in China. Diagn. Microbiol. Infect. Dis., 62:226–229.

34.

Xiaomei

, Yixin

, Lihua

, Jianzhong

2009. Multilocus sequence typing of methicillin-resistant Staphylococcus aureus. Chin. J. Prev. Med., 2:137–140.

35.

Zhang

, McClure

J.A.

, Elsayed

, Louie

, Conly

J.M.

2005. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol., 43:5026–5033.