methicillin-resistant staphylococcus aureus in spain: molecular 1

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Methicillin-Resistant Staphylococcus aureus in Spain: molecular 1 epidemiology and utility of different typing methods 2 3 Ana Vindel, 1 Oscar Cuevas, 2 Emilia Cercenado, 2 Carmen Marcos, 1 Verónica 4 Bautista, 1 Carol Castellares, 2 Pilar Trincado, 1 Teresa Boquete, 1 Maria Pérez- 5 Vázquez, 1 Mercedes Marín, 2 Emilio Bouza 2 6 and the Spanish Group for the Study of Staphylococcus 7 1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 8 Madrid, Spain 9 2 Servicio de Microbiología, Hospital General Universitario “Gregorio Marañón,” 10 Madrid, Spain 11 12 13 Running title: Methicillin-resistant Staphylococcus aureus in Spain: molecular 14 epidemiology 15 16 Key words: Methicillin-resistant Staphylococcus aureus. PFGE. SCCmec. agr. 17 spa-typing. BURP. PVL. MLST. 18 19 Word count: 3647 20 21 22 Corresponding author: Emilia Cercenado 23 Servicio de Microbiología, Hospital General Universitario “Gregorio Marañón,” 24 C/ Dr. Esquerdo, 46 25 28007 Madrid, Spain 26 Phone: +34-91-586-8459 27 Fax: +34-91-504-4906 28 E-mail: [email protected] 29 30 31 This study was presented in part at the 47 th Interscience Conference on 32 Antimicrobial Agents and Chemotherapy, Chicago, Illinois, USA, 2007 (abstract 33 C2-148). 34 35 36 Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. J. Clin. Microbiol. doi:10.1128/JCM.01579-08 JCM Accepts, published online ahead of print on 1 April 2009 on February 3, 2018 by guest http://jcm.asm.org/ Downloaded from

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Page 1: Methicillin-Resistant Staphylococcus aureus in Spain: molecular 1

Methicillin-Resistant Staphylococcus aureus in Spain: molecular 1

epidemiology and utility of different typing methods 2

3

Ana Vindel,1 Oscar Cuevas,2 Emilia Cercenado,2 Carmen Marcos,1 Verónica 4

Bautista,1 Carol Castellares,2 Pilar Trincado,1 Teresa Boquete,1 Maria Pérez-5

Vázquez,1 Mercedes Marín,2 Emilio Bouza2 6

and the Spanish Group for the Study of Staphylococcus 7

1Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 8

Madrid, Spain 9 2Servicio de Microbiología, Hospital General Universitario “Gregorio Marañón,” 10

Madrid, Spain 11

12

13

Running title: Methicillin-resistant Staphylococcus aureus in Spain: molecular 14

epidemiology 15

16

Key words: Methicillin-resistant Staphylococcus aureus. PFGE. SCCmec. agr. 17

spa-typing. BURP. PVL. MLST. 18

19

Word count: 3647 20

21

22

Corresponding author: Emilia Cercenado 23

Servicio de Microbiología, Hospital General Universitario “Gregorio Marañón,” 24

C/ Dr. Esquerdo, 46 25

28007 Madrid, Spain 26

Phone: +34-91-586-8459 27

Fax: +34-91-504-4906 28

E-mail: [email protected] 29

30

31

This study was presented in part at the 47th Interscience Conference on 32

Antimicrobial Agents and Chemotherapy, Chicago, Illinois, USA, 2007 (abstract 33

C2-148). 34

35

36

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01579-08 JCM Accepts, published online ahead of print on 1 April 2009

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ABSTRACT 1

2

In a point-prevalence study performed in 145 Spanish hospitals in 2006, 3

we collected 463 isolates of Staphylococcus aureus in a single day. Of these, 4

135 (29.2%) were resistant to methicillin (MRSA). Susceptibility testing was 5

performed by a miocrodilution method, and mecA was detected by PCR. The 6

isolates were analyzed using pulsed-field gel electrophoresis (PFGE) after SmaI 7

digestion, SCCmec typing, agr typing, spa typing with BURP analysis, and 8

MLST. The 135 MRSA isolates showed resistance to ciprofloxacin (93.3%), 9

tobramycin (72.6%), gentamicin (20.0%), erythromycin (66.7%), and 10

clindamycin (39.3%). Among the isolates resistant to erythromycin, 27.4% 11

showed the M-phenotype. All of the isolates were susceptible to glycopeptides. 12

Twelve resistance patterns were found, of which four accounted for 65% of the 13

isolates. PFGE revealed 36 different patterns, with 13 major clones (including 14

two predominant clones with variable antibiotypes that accounted for 52.5% of 15

the MRSA isolates) and 23 sporadic profiles. Two genotypes were observed for 16

the first time in Spain: SCCmec IV, accounting for 87.4% of the isolates (70.1% 17

were type IVa, 23.9% type IVc, 0.9% type IVd, and 5.1% type IVh); and 18

SCCmec I and SCCmec II, accounting for 6.6% and 5.2% of isolates, 19

respectively, with one non-typeable isolate. Only one of the isolates produced 20

the Panton-Valentine leukocidin. The isolates presented agr type 2 (82.2%), 21

type 1 (14.8%) and type 3 (3.0%). spa typing revealed 32 different types, the 22

predominant ones being t067 (48.9%) and t002 (14.8%), as well as BURP 23

CC067 (78%). The clone ST125-MRSA-IV was the most prevalent throughout 24

Spain. In our experience, PFGE, spa typing, SCCmec typing and MLST 25

presented a good correlation for the majority of the MRSA strains; we suggest 26

the use of the spa typing and PFGE typing for epidemiological surveillance, 27

since this combination is useful for both long-term and short-term studies. 28

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INTRODUCTION 1

2

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of 3

hospital-acquired infections worldwide (5, 25). The appearance of MRSA in the 4

community, and the potential risk of it entering hospitals are also a matter of 5

concern (29, 44). Moreover, the increasing prevalence of multidrug-resistant 6

MRSA and the emergence of isolates with intermediate or high-level 7

vancomycin resistance emphasize the importance of infection control measures 8

(2, 49, 50). Although MRSA isolation rates have been increasing throughout the 9

world for the last few decades and in some areas the figures reach >50%, there 10

are considerable variations in the prevalence of MRSA according to geographic 11

area (3, 18, 21, 39, 44). In Spain, the prevalence of MRSA increased from 1.5% 12

in 1986 to 29.2% in 2006, although it seems to have stabilized (13). Despite the 13

worldwide increase in isolation rates, only a limited number of clones of MRSA 14

have spread in most countries (20). 15

Historically, the dissemination of epidemic clones such as EMRSA-15, 16

EMRSA-16, the Iberian clone, and the Brazilian clone, as well as the high 17

incidence of the community-acquired MRSA USA300 clone has led to increased 18

use of molecular typing methods (11, 38, 42, 47, 53). 19

In recent years, a variety of molecular techniques have been used for 20

MRSA typing. Of these, SmaI macrorestriction is the gold standard for analysis 21

of local epidemiology in the short term, spa typing in combination with BURP 22

(based upon repeat pattern) analysis has become a frontline tool for routine 23

epidemiological typing, and MLST (multilocus sequence typing)/SCCmec typing 24

is the reference method for defining MRSA clones (10, 34, 37, 46). 25

The aim of the present study was to determine which clones are 26

circulating in Spain and whether the strains have spread between hospitals, by 27

analyzing a representative sample of isolates collected in a point-prevalence 28

study. Isolates were grouped using PFGE and spa typing and assigned to 29

MRSA clones on the basis of MLST/SCCmec typing. The congruence between 30

the different grouping methods was assessed. 31

32

33

34

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MATERIALS AND METHODS 1

2

Bacterial isolates. A point-prevalence study involving 145 Spanish hospitals on 3

a single day in 2006 yielded a total of 463 clinical isolates of S. aureus. Full 4

details of the study design and identification of the isolates have been published 5

previously (13, 14). Of the total number of isolates, 135 were MRSA. 6

Susceptibility testing. Antimicrobial susceptibility testing was performed using 7

the MicroScan automated broth microdilution method and the Pos Combo 23S 8

panel (MicroScan, Siemens, Sacramento, California, USA) following the 9

manufacturer’s guidelines. MIC breakpoints were determined following the 10

Clinical and Laboratory Standards Institute (CLSI) recommendations (9). Full 11

details of antimicrobial susceptibility testing have been published elsewhere (13, 12

14). 13

Methicillin-resistance detection. The mecA gene was detected by PCR as 14

described by Geha et al (24). 15

Pulsed-field gel electrophoresis (PFGE). All 135 MRSA isolates were 16

genotyped by PFGE after SmaI digestion of chromosomal DNA, prepared using 17

a modification of the protocol described by Cookson et al (10). This technique 18

has been fully described previously (12). Analysis of the gels was performed 19

according to the criteria of Tenover et al (48), and a dendrogram was 20

constructed with Molecular Analyst Software (Bio-Rad) using the Dice 21

correlation coefficient (28) and the unweighted pair-group method with 22

averages, with a tolerance position of 0.8%. According to our previous studies 23

(12, 53), each PFGE type was assigned the letter E, followed by a number that 24

correlated with the date of isolation, while each subtype was assigned the letter 25

of the main genotype to which it was closest. Sporadic strains were indicated in 26

each case. In our previous studies (12, 53), PFGE type E1 corresponded to 27

ST247-MRSA-I, types E3 and E10 corresponded to ST146-MRSA-IV, types E6, 28

E9, E15, E16, and E17 corresponded to ST228-MRSA-IV, types E7, E8, E11 29

corresponded to ST125-MRSA-IV, and type E12 corresponded to ST36-MRSA-30

II (53). PFGE type A was a community-acquired genotype that corresponded to 31

ST8-MRSA-IV (6). 32

Multiplex PCR SCCmec typing. SCCmec types were determined using a 33

multiplex PCR strategy that generated a specific amplification pattern for each 34

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SCCmec structural type according to the method described by Oliveira et al 1

(40). Additional typing of isolates was performed by two different PCR methods 2

in order to determine the SCCmec IV subtypes IVb, IVc, IVd, and IVh (37), and 3

to detect SCCmec V (56). 4

Detection of Panton-Valentine leukocidin (PVL) genes. The PVL genes 5

(lukS-PV and lukF-PV) were detected by PCR following the method described 6

by Lina et al (32). S. aureus ATCC 49775 (a PVL-positive strain) was used as a 7

positive amplification control. 8

Determination of accessory gene regulator (agr) types. A scheme of two 9

PCR reactions based on the method described by Shopsin et al (45) was used 10

for the determination of specific agr groups. 11

spa typing and BURP analysis. The polymorphic X region of the protein A 12

gene (spa) was amplified from all MRSA isolates as previously described (27, 13

46). By application of the BURP algorithm implemented by the software, spa 14

types with more than five repeats were clustered into different groups, with the 15

calculated cost between members of a group being less than or equal to 6. The 16

spa type was assigned using Ridom StaphType software (36). 17

MLST. Several representative strains from each type and subtype of PFGE 18

were selected for determination of the sequence type (ST). None of the 19

sporadic PFGE genotypes were typed by this method. MLST typing was 20

performed following the method described by Enright et al (16). Allelic profiles 21

and ST types were assigned using the MLST database (http://www.mlst.net). 22

23

RESULTS 24

25

Resistance patterns. Of the 135 MRSA isolates studied, 93.3% were resistant 26

to ciprofloxacin, 72.6% to tobramycin, 20% to gentamicin, 66.7% to 27

erythromycin, and 39.3% to clindamycin. Of the isolates resistant to 28

erythromycin, 27.4% showed the M-phenotype. All the isolates were susceptible 29

to vancomycin (MIC ≤2 mg/L) and to teicoplanin (MIC ≤8 mg/L). Full details of 30

the susceptibility of the isolates have been published elsewhere (13). Table I 31

shows the different resistance patterns of the MRSA strains. Twelve different 32

patterns were found, of which four accounted for 65% of the isolates. Seven 33

isolates (5.2%) were only resistant to oxacillin. Multiresistance to one, two, 34

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three, four, and five additional antibiotics, was observed in 6.7%, 25.1%, 29.6%, 1

20.8%, and 12.6% of the MRSA isolates, respectively. 2

3

Pulsed-field gel electrophoresis (PFGE). Genotyping by PFGE of the 135 4

MRSA isolates grouped 112 into 13 genotypes (E7, E8, E10, E11, E12, E13, 5

E15, E16, E17, E18, E19, E20, and A). Two genotypes (E19 and E20) were 6

observed for the first time in Spain. Genotypes E7 (with subtypes E7a, and E7b; 7

28.1%) and E8 (with subtypes E8a, and E8b; 24.4%) predominated, and 8

together accounted for 52.5% of the isolates. The remaining 23 isolates 9

belonged to 23 sporadic profiles that were each represented by a single isolate. 10

The Figure shows the genetic relationships between the 36 PFGE patterns 11

identified. 12

13

SCCmec types. The distribution of the different SCCmec types among the 14

different genotypes is shown in Table 2. SCCmec IV accounted for 86.7% of the 15

isolates (117), with 70.1% of these carrying SCCmec IVa, 23.9% carrying 16

SCCmec IVc, 0.9% carrying SCCmec IVd, and 5.1% carrying SCCmec IVh. 17

SCCmec I was identified in 10 isolates (7.4%), and SCCmec II was found in 7 18

isolates (5.2%). One isolate could not be typed by any of the methods used in 19

this study for SCCmec typing. 20

SCCmec type IVa (present in 60.8% of all MRSA isolates) was included 21

in seven major genotypes (E7, E8, E10, E16, E19, E20, and A) and in eight 22

sporadic isolates. SCCmec type IVc was in four major genotypes (E7, E8, E10, 23

and E11), and in seven sporadic isolates. SCCmec IVh was in genotype E13 24

(ST22-MRSA-IV). SCCmec I was present in genotypes E15, E17, and E18 25

(ST228-MRSA-I), and SCCmec II in genotype E12 (ST36-MRSA-II). 26

27

agr types. Table 2 shows the distribution of the agr types and their correlation 28

with the genotypes observed by PFGE and by SCCmec typing. The most 29

frequent agr type was type 2 (82.2%), which grouped strains belonging to 9 30

major clones (E7, E8, E10, E11, E15, E16, E17, E18, and E20) and to 14 31

sporadic MRSA strains. agr type 1 was present in 14.8% of the strains 32

belonging to 3 major clones (E-13, E-19, and A) and to 8 sporadic MRSA 33

strains. Only 3.0% of MRSA strains harboured agr type 3; these strains 34

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belonged to the E12 clone (3 strains) and to 1 sporadic MRSA isolate. None of 1

the MRSA strains presented agr type 4. 2

3

pvl genes. Only one isolate presented the PVL. The MRSA strain showed 4

PFGE genotype A (community-acquired) and was also resistant to erythromycin 5

(see Table 2). The origin of the isolate was a wound infection from a child. 6

7

spa types. The different spa types observed are shown in Table 3. Among the 8

135 MRSA strains, 32 different spa types were identified: 21 were represented 9

by a single strain, and 7 were new spa types not described previously. spa type 10

t067 was the most frequent (48.9% of the isolates), followed by spa type t002 11

(14.8%). By application of the BURP algorithm, the MRSA strains were 12

clustered into 3 groups: CC067 (77.7%), CC211 (6.0%), and CC012 (3.7%). 13

The only PVL-positive isolate belonged to spa type t008 (spa CC211). Two spa 14

types were considered “non-founder”, 5 were singletons (one of which, t032, 15

grouped 6 MRSA strains), and two were excluded (3%). 16

17

Correlation between the different molecular typing methods. The 18

correlations between the BURP group (spa CC), spa type, PFGE genotype, agr 19

type, and BURST group MLST type and SCCmec type are shown in Table 2 20

and the Figure. Both PFGE and spa typing showed 100% typeability and 21

excellent reproducibility, although PFGE showed more discriminatory power 22

than spa typing. MRSA strains belonging to different epidemic PFGE genotypes 23

(E7, E8, E10, E11, E15, E16, and E20) presented either spa type t067 (43.0%) 24

or type t002 (11.9%), and both were grouped in CC067 and in MLST CC5 25

(ST125 and ST228). The six isolates designated as E13 by PFGE belonged to 26

spa type t032 and were grouped as singletons, corresponding to clone ST22-27

MRSA IVh. On the other hand, 12 MRSA strains considered sporadic isolates 28

on the basis of their PFGE pattern belonged to spa types t067 (8 strains, 5.9%) 29

and t002 (4 strains, 2.9%). In addition we observed discrepancies between both 30

methods in the classification of five MRSA isolates. Three isolates belonging to 31

genotype E7, one isolate belonging to genotype E8, and another isolate 32

belonging to genotype E12 presented different spa types: t012 and t021 33

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(CC012, ST30-MRSA IV), three t084 singletons (ST582-MRSA IV) and two t166 1

singletons (ST36-MRSA II and ST125-MRSA IV) (Figure). 2

In general we observed a high concordance between the MLST clonal 3

complexes and the BURP groups (Figure). 4

Isolates belonging to MLST CC5 (ST125 with PFGE genotypes E7, E8, 5

E11, and E20; ST146 with the genotype E10; and ST228 with the genotypes 6

E15, E16, E17, and E18) were grouped in BURP CC067 (with the exception of 7

the five isolates described above). MLST CC8 (ST8 with PFGE genotypes E19, 8

and A) presented the spa type t008, belonging to BURP group CC211. CC22 9

(ST22 with the genotype E13, related to the clone EMRSA-15) presented spa 10

type t032, which appeared in this analysis as a singleton. Finally, MLST CC30 11

(ST36, genotype E12, related to clone EMRSA-16) presented spa types t012 12

and t018, grouped in BURP CC012 and spa type t166 (singleton). 13

14

DISCUSSION 15

16

MRSA is among the most frequently identified antimicrobial drug-17

resistant pathogens worldwide and has evolved in relatively few lineages. It has 18

been demonstrated that some lineages are ecologically highly successful and 19

that most isolates belong to pandemic clones (17). The present study revealed 20

that >90% of the isolates were multiresistant and that >30% were resistant to at 21

least four antimicrobial agents. In 2002, the predominant (23.9%) pattern in 22

MRSA in Spain involved resistance to ciprofloxacin, erythromycin, clindamycin, 23

gentamicin, and tobramycin (12). In the present study, performed 4 years later 24

using the same methodology, we observed a significant decrease in this 25

multiresistance pattern (P=0.018), representing only 12.6%. In contrast, 26

multiresistance to ciprofloxacin, erythromycin, and tobramycin has significantly 27

increased from 8.2% in 2002 (12) to 17.0% in 2006 (P=0.042). These results 28

indicate that strains are becoming more susceptible and that the M-phenotype 29

of resistance to macrolides and the presence of the ant4’ gene that confers 30

resistance to tobramycin but not to gentamicin are becoming more prevalent. Of 31

the 135 MRSA isolates, only one was community-acquired and presented the 32

M-phenotype of resistance to macrolides. 33

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We characterized the MRSA isolates by using different molecular typing 1

tools. After analysis of the data, we found interesting clinical and 2

epidemiological findings. First, the spa-type t067 (ST5-MRSA-IV) was dominant 3

among the Spanish MRSA isolates, a situation not described in other countries. 4

Second, we found a high clonality of the MRSA isolates obtained in this 5

nationwide prevalence study which demonstrates that most isolates belong to 6

pandemic clones; and third, we found that PFGE, spa typing, SCCmec typing, 7

and MLST presented a good correlation for most MRSA strains. 8

The PFGE analysis revealed 36 different genotypes that included two 9

predominant clones (E7 and E8) and one community-acquired clone (profile A). 10

PFGE is known to be a highly discriminatory and valuable technique for typing 11

of S. aureus (10), and has been used by the Spanish Reference Laboratory for 12

staphylococci for local investigation and national surveillance of MRSA since 13

1996 (53). It has been argued that the stability of PFGE may be insufficient for 14

its application to long-term epidemiological studies due to the high degrees of 15

genetic variation that have been observed among pandemic clones with a long 16

evolutionary history (4). However, we have already reported that the 17

predominant clones in Spain did not experiment significant changes from 1996 18

(53) to 2002 (12). Moreover, in the present study (2006), we identified the same 19

predominant clones as well as two new clones: E19 (ST8) and E20 (ST125), 20

the latter being closely related to the predominant E7 and E8 clones. These 21

genotypes belong to ST125, which continues to be responsible for more than 22

half of the nosocomial MRSA infections in Spain (59.3%), although it is unusual 23

in the rest of Europe (23, 42). Our results validate the use of PFGE for long-24

term nationwide epidemiological studies, although we consider that this 25

technique presents difficulties in interlaboratory reliability. Nevertheless, 26

multicenter studies using PFGE are now possible due to standardization of 27

electrophoresis conditions (8, 10) and normalization and analysis software (15). 28

The most frequent SCCmec type found was SCCmec IV, which was 29

present in 86.7% of isolates. Its presence in the predominant clones, in the 30

majority clones, in the sporadic isolates and in the community-acquired clone 31

suggests a great degree of promiscuity and successful persistence (43). Since 32

SCCmec IV is currently one of the most frequent nosocomial SCCmec types 33

found in several countries (1, 22, 33, 44, 47), the antimicrobial resistance 34

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patterns of isolates presenting this type varied considerably. In our study, 97.5% 1

and 78.6% of MRSA isolates with multiresistance to three or four antimicrobials, 2

respectively, showed this type. The permanence of this type of SCCmec in 3

hospitals over long periods of time has probably favoured its multiresistance 4

due to antibiotic pressure. In our study, most type IV strains belonged to 5

subtype IVa (70.1%), followed by subtype IVc (23.9%). In one study performed 6

in the Unites States (1), subtype IVa was identified in 87.1% of MRSA isolates 7

and subtype IVd in 5.7%. In a Japanese study, type IV SCCmec strains were 8

also the most frequent, comprising 53.6% of all strains, and the frequencies of 9

type IVc and type IVd were 38.1% and 10.3%, respectively (33). 10

SCCmec types I and II, historically associated with multiresistance (more 11

than 3 antimicrobials), were very uncommon in this study, and in certain cases 12

were associated with sporadic isolates. 13

Characterization of SCCmec was not 100% typeable in our study and 14

showed weak discriminatory power. Even when we used three different typing 15

schemes, one isolate was non-typeable (sporadic isolate). In addition, the 16

elevated number of isolates harbouring type IV limited the discriminatory power 17

of this technique. Although this type can be differentiated in many subtypes, a 18

second multiplex PCR is necessary increasing the cost of the determinations 19

(37). Since new alleles are frequently described, an ever-increasing number of 20

primers will be necessary in order to discriminate between different subtypes (7, 21

30). 22

Only one of the 135 MRSA isolates was PVL-positive, and corresponded 23

to a community-acquired wound infection in a child. This isolate presented the 24

characteristics most frequently described among PVL-positive MRSA in Spain, 25

including PFGE profile A, SCCmec IVa, ST8, and spa type t008 (CC008) (6). 26

The finding of only one PVL-positive isolate could be due to the characteristics 27

of our study: a point-prevalence study performed on a single day in 145 Spanish 28

hospitals (13). However, we have previously published a higher prevalence of 29

PVL-positive MRSA isolates which is consistent with the increased prevalence 30

of PVL-positive MRSA in Europe (52, 54). 31

Concerning the agr types, one previous study indicated that PFGE, 32

MLST, and spa genotypes are so strongly correlated with agr types that the 33

former can be used to predict the latter indistinctly, and that no MLST, spa, or 34

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PFGE pattern occurs in more than one agr group (55). In our study, most 1

isolates presented agr type 2 (82.2%), although strains belonging to BURP 2

group CC067 presented agr groups 2 or 1 indistinctly, and strains belonging to 3

BURP group CC012 presented agr groups 2 or 3 indistinctly. The same study 4

cited above (55) also indicates that in certain cases, strains belonging to the 5

same MLST type can present different agr groups. These observations need to 6

be confirmed by additional data, although inter-strain recombination and intra-7

strain rearrangements would be important sources of variation that could 8

explain these observations (43). In our study, in all cases there were 9

unequivocal correlations between MLST and agr type. We have not found in the 10

literature any studies analyzing the agr type in a large series of MRSA isolates. 11

Recently, a method based on the sequence of the protein A gene (spa 12

typing) represents a marked improvement in the typing of MRSA. It is 13

reproducible, easy to use, and the availability of a central database 14

(http://spa.ridom.de) enables comparisons to be made with data obtained in 15

different laboratories and countries. Several studies have demonstrated that it is 16

applicable to both local and global epidemiological studies (31, 35). The 17

application of this method in Spain revealed that two spa types (t067 and t002) 18

were dominant (63.7% of all MRSA strains). 19

The high frequency of t067 (48.9%) in Spain contrasted with the relatively 20

low frequency of t067 (0.97%) found in other European countries (Austria, 21

Denmark, Finland, Germany, The Netherlands, Norway, Sweden, and 22

Switzerland). 23

In the case of spa type t002, the global frequency found was 5.79% 24

(Austria, Belgium, Canada, China, Croatia, Cyprus, Denmark, Estonia, Finland, 25

France, Germany, Hungary, Iceland, Israel, Italy, Japan, Lebanon, The 26

Netherlands, Norway, Romania, Sweden, Switzerland, Taiwan, United 27

Kingdom, and the United States), whereas this frequency was higher in Spain 28

(14.8%). The high frequency of t067 and t002 in Spanish hospitals limits the 29

usefulness of spa typing for local investigation and makes it necessary to 30

differentiate these frequent strains by PFGE. 31

Concerning the correlation between the different typing methods used in 32

our study, SCCmec types encompassed multiple MLST types, spa types, MLST 33

CCs, and spa CCs, a fact that has been observed elsewhere (41, 51). 34

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When we compared the PFGE genotypes using spa typing, the 1

predominant clones (E7 and E8) presented a variety of spa types, although 2

most belonged to the same BURP group (CC067). However, we observed 3

discordant results (group violations) between the spa type assignment and the 4

profile obtained by PFGE, as described in other studies (26, 43). In addition, we 5

encountered MRSA isolates belonging to different profiles—E7, E8 and E11 6

(ST125), and E10 (ST146)—sharing the same spa type (t002 and t067). These 7

discrepancies have also been described by Hallin et al (26), suggesting that 8

they could be due to intergenomic recombination. An elevated number of 9

sporadic isolates, as defined by their PFGE profiles, harboured the same spa 10

type as the predominant clones. This could be due to the high discriminatory 11

power of PFGE. These different profiles could reflect the occurrence of genetic 12

events (19). A recent report suggests that the combination of PFGE and spa 13

typing for epidemiological surveillance studies makes it possible to maintain the 14

discriminatory power and typeability needed in short-term and long-term studies 15

(19). 16

The application of MLST is especially useful for long-term 17

epidemiological studies due to the low mutation rate of the seven housekeeping 18

genes analysed by the method (16). However, we consider that spa typing and 19

PFGE typing for epidemiological surveillance is the most useful technique for 20

both long-term and short-term studies. In our study, this combination of typing 21

techniques predicted the MLST CCs, except for the five isolates included as 22

group violations. Other studies have also suggested that this combination 23

reasonably predicts the MLST CCs (19). 24

In summary, this study demonstrates that strains of MRSA in Spain have 25

become significantly more susceptible to gentamicin and clindamycin than 26

previous years, and that there is persistence of the ST125-MRSA-IV clone, 27

which includes the previously described predominant clones E7 and E8, and the 28

new closely related clone, E20. The use of spa typing in this study allowed us to 29

detect two predominant types (t067 and t002) in Spain that are very uncommon 30

in other countries. In our experience, PFGE, spa typing, SCCmec typing, and 31

MLST presented a good correlation for most MRSA strains. Due to the high 32

percentage of spa types t067 and t002 (CC067 BURP), we consider that spa 33

typing should be combined with PFGE to provide the necessary discriminatory 34

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power and typeability for local and long-term epidemiological studies, as well as 1

the possibility of inter-laboratory comparisons. The combination of PFGE and 2

the assignment of BURP CC could predict eBURST CC without performing 3

MLST in a larger number of MRSA strains. 4

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ACKNOLEDGMENTS 1

2

Supported by Ministerio de Sanidad y Consumo, Instituto de Salud 3

Carlos III - FEDER, Spanish Network for the Research in Infectious Diseases 4

(REIPI RD06/0008). 5

We are grateful to Wyeth-Farma for financing the transport of samples 6

and the laboratory material used in this study. We thank Dr. Ito for the 7

Staphylococcus aureus control strains subtyping SCCmec IV. 8

We would like to thank to Thomas O’Boyle for his help in the translation 9

of the manuscript. 10

The members of the Spanish Group for the Study of Staphylococcus and 11

the staff of the microbiology services of all participating hospitals are as follows: 12

Andalucía: (Hospital de Torrecárdenas, Almería). Adolfo Sicilia, Armando 13

Reyes; (Hospital Universitario Puerta del Mar, Cádiz). Juan García Herruzo, 14

Pilar Marín; (Hospital Punta de Europa, Algeciras, Cádiz). Inés Ruiz; (Hospital 15

de Jerez de La Frontera, Cádiz). Luis Calbo, José Luis de Francisco Ramírez; 16

(Hospital de La Línea de La Concepción, Cádiz). Eustaquio Arrimadas, Antonio 17

Sánchez Porto; (Hospital Universitario Reina Sofía, Córdoba). Manuel Casal, 18

Manuel Causse del Río; (Hospital Infanta Margarita, Cabra, Córdoba). Carlos 19

Plata, Rocío Tejero; (Hospital Universitario Virgen de las Nieves, Granada). 20

Manuel de la Rosa, Mª Fe Bautista; (Hospital Universitario San Cecilio, 21

Granada). Carmen Maroto, Trinidad Escobar; (Hospital Juan Ramón Jiménez, 22

Huelva). Lucía Pascual, José Saavedra; (Hospital Universitario de Jaén). 23

Inocente Cuesta, Inmaculada Carazo; (Hospital Regional Carlos Haya, 24

Málaga). Juan Hernández Molina; (Hospital Virgen de la Victoria, Málaga). 25

Alfonso Pinedo, Mª Victoria García, Mar Gallardo; (Hospital Virgen Macarena, 26

Sevilla). Álvaro Pascual, Marina de Cueto, Evelio Perea; (Hospital Universitario 27

Virgen del Rocío, Sevilla). Javier Aznar, María de Toro Crespo; (Hospital 28

Nuestra Señora de Valme, Dos Hermanas, Sevilla). Estrella Martín Mazuelos, 29

José Luis García López; Aragón: (Hospital General San Jorge, Huésca). Miguel 30

Ferrero; (Hospital Obispo Polanco, Teruel). Pilar Chocarro; (Hospital de 31

Alcañiz, Teruel). Carmen Navarro, Luis Torres; (Hospital Nuestra Señora de 32

Gracia, Zaragoza). Cristina Miñana; (Hospital Miguel Servet, Zaragoza). Mª 33

José Revillo, Antonio Rezusta; (Hospital Clínico Universitario Lozano Blesa, 34

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Zaragoza). Carmen Rubio, Estrella Durán; Asturias: (Hospital Monte Naranco, 1

Oviedo, Asturias). Fernando Vázquez, Carlos Aranaz; (Hospital Central de 2

Asturias, Oviedo, Asturias). Mª Jesús Santos, Ana Fleites, Marta Lantero; 3

(Instituto Nacional de Silicosis, Oviedo, Asturias). Isabel Sánchez Folgueras; 4

(Fundación Hospital de Jove, Gijón, Asturias). Elisa Hidalgo; (Hospital de 5

Cabueñes, Gijón, Asturias). Luis Otero, Mª Dolores Miguel; (Hospital San 6

Agustín, Avilés, Asturias). Pilar Prendes, Mª Carmen Galarraga, Gema Sierra; 7

(Hospital Comarcal de Jarrio, Asturias). Pedro de la Iglesia; (Hospital Carmen y 8

Severo Ochoa, Cangas de Narcea, Asturias). José Francisco Ordás, Lucía 9

Barreiro; Baleares: (Hospital Son Dureta, Palma de Mallorca). José Luis Pérez, 10

Nuria Borrell, Antonio Oliver; (Hospital Can Misses, Ibiza); Javier Sánchez 11

Gómez, Adoración Hurtado; Canarias: (Hospital Nuestra Señora de La 12

Candelaria, Santa Cruz de Tenerife). Nínive Batista, Isabel Gutiérrez González; 13

(Hospital Universitario de Canarias, La Laguna, Santa Cruz de Tenerife). 14

Antonio Sierra, Mª Antonia Miguel Gómez; (Hospital Universitario Insular de 15

Gran Canaria, Las Palmas de Gran Canaria). Antonio Manuel Martín, Araceli 16

Hernández; Cantabria: (Hospital Marqués de Valdecilla, Santander). Luis 17

Martínez, Ana Belén Campo, Cecilia García de la Fuente; (Hospital Comarcal 18

de Laredo, Cantabria). Purificación Mellado; Castilla-La Mancha: (Complejo 19

Hospitalario Universitario de Albacete, Albacete). Mª Dolores Crespo, Elena 20

Escribano, Juan José Palomar; (Hospital General de Ciudad Real). Mª Dolores 21

Romero, Sonia Solís; (Hospital General La Mancha-Centro, Alcázar de San 22

Juan, Ciudad Real). Rafael Carranza; (Hospital Virgen de la Luz, Cuenca). 23

Carmen Martínez Medina, Germán Seseña; (Hospital Universitario de 24

Guadalajara). Julia Bisquert; Daniel Tena; (Hospital Virgen de la Salud, 25

Toledo). Luis Díaz Pierna, Mª Victoria Martino; (Hospital Nacional de 26

Parapléjicos, Toledo). Ana Mª Leturia; Castilla-León: (Hospital Nuestra Señora 27

de Sonsoles, Ávila). Rosario Ibáñez, Rafael Sánchez Arroyo; (Hospital General 28

Yagüe, Burgos). Isidro Pozas, Eva Ojeda, Gregoria Mejías; (Hospital Provincial 29

Divino Vallés, Burgos). Carmen Lizondo; Mª Dolores Badía; (Hospital Comarcal 30

Santiago Apóstol, Miranda de Ebro, Burgos). Carmen Gimeno; (Hospital Santos 31

Reyes, Aranda de Duero, Burgos); Sonia Junquera; (Hospital de León). Felipe 32

Cachón, Mª Isabel Fernández Natal; (Hospital del Bierzo, Ponferrada, León). 33

Carlos Fuster; (Complejo Hospitalario Río Carrión, Palencia). Elena Álvarez 34

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Alonso, Mª Antonia García Castro; (Hospital Clínico de Salamanca). José Elías 1

García Sánchez, José Ángel García Rodríguez, Marta Fernández Vázquez; 2

(Hospital General de Segovia). Santiago García Carbajosa, Pablo Carrero, 3

Susana Hernando Real; (Complejo Hospitalario de Soria). Ángel Campos, Ana 4

Beltrán, Carmen Aldea; (Hospital Universitario Río Hortega, Valladolid). Pilar 5

Pérez Pascual, Armando Alberte; (Hospital Universitario de Valladolid). Antonio 6

Rodríguez Torres, Miguel Ángel Bratos; (Hospital de Medina del Campo, 7

Valladolid). Belén Lorenzo; (Hospital Virgen de la Concha, Zamora). Mª Fe 8

Brezmes, Luis López Urrutia; Cataluña: (Hospital Vall d’Hebron, Barcelona). 9

Guillem Prats; (Hospital Clinic i Provincial de Barcelona). Mª Teresa Jiménez de 10

Anta, Francesc Marco; (Hospital de la Santa Creu i Sant Pau, Barcelona). Pere 11

Coll, Beatriz Mirelis; (Hospital Comarcal Alt Penedès, Barcelona). Anna 12

Vilamala, Carmen San José; (Hospital Universitario de Bellvitge, Hospitalet de 13

Llobregat, Barcelona). Rogelio Martín, Fe Tubau; (Laboratorio de Referencia de 14

Cataluña-Hospital del Mar, Hospitalet Llobregat, Barcelona). Margarita Salvadó, 15

Araceli González; (Corporación Sanitaria del Parc Taulí, Sabadell, Barcelona). 16

Dionisia Fontanals, Dolors Mariscal; (Hospital Mutua de Terrassa, Barcelona). 17

Josep Lite; (Residencia Sant Camil, Sant Pere de Ribes, Barcelona). Francisca 18

Corcoy, Roser Angrill; (Hospital San Joan de Deu, Manresa, Barcelona). 19

Monserrat Morta, Dolors Estivill; (Hospital Provincial de Santa Caterina, 20

Girona). Mª. Luisa Urcuola; (Hospital Universitario Doctor Josep Trueta, 21

Girona). Jordi de Batlle Surroca, Montserrat Motje; (Hospital Universitari Arnau 22

de Vilanova, Lleida). Antoni Nogues, Mercé García; (Hospital Universitario Joan 23

XXIII, Tarragona). Josep Mª Santamaría, Frederic Gómez, J. Tapiol; Ceuta: 24

(Hospital Cruz Roja de Ceuta). José López Barba; Extremadura: (Hospital 25

Universitario Infanta Cristina, Badajoz). Javier Blanco; (Hospital San Pedro de 26

Alcántara, Cáceres). Pilar Teno; Jesús Viñuelas; Galicia: (Complejo 27

Hospitalario Universitario Juan Canalejo, La Coruña). Rosa Villanueva, Ana 28

Fernández González; (Hospital General Juan Cardona, El Ferrol, La Coruña). 29

Manuel Rodríguez Jove; (Complejo Hospitalario Arquitecto Marcide-Novoa 30

Santos, El Ferrol, La Coruña). Andrés Agulla, María Rodríguez Mayo; (Hospital 31

Clínico Universitario de Santiago de Compostela, La Coruña). Prof. Benito 32

Regueiro, Fernanda Pardo; (Complejo Hospitalario Xeral-Calde, Lugo). Pilar 33

Alonso, Amparo Coira; (Complejo Hospitalario de Ourense). Gloria Esteban, 34

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Begoña Fernández Pérez; (Complejo Hospitalario de Pontevedra); Marta 1

García Campello, Patricia Álvarez García; (Complejo Hospitalario Xeral-Cies, 2

Vigo, Pontevedra). Teresa González del Blanco, Isabel Otero; (Hospital do 3

Meixoeiro, Vigo, Pontevedra). Julio Torres, Francisco José Vasallo; (Policlínico 4

de Vigo-POVISA, Pontevedra) Joaquina Sevillano, Irene Rodríguez Conde; La 5

Rioja: (Hospital San Millan, Logroño). Luis Borque, Inés Olarte, Estibaliz 6

Ugalde; Madrid: (Hospital General Universitario Gregorio Marañón, Madrid). 7

Emilio Bouza, Emilia Cercenado, Oscar Cuevas; (Hospital La Paz, Madrid). 8

Avelino Gutiérrez, Adela García Perea; (Hospital Universitario Doce de 9

Octubre, Madrid). Joaquín Rodríguez Otero, Fernando Chaves; (Fundación 10

Jiménez Díaz, Madrid). Ignacio Gadea, Ricardo Fernández Roblas; (Hospital 11

Central de la Defensa, Madrid). Francisco Hervás, Mª Victoria Buezas, Paloma 12

Sánchez Santana; (Hospital Clínico San Carlos, Madrid). Juan José Picazo, 13

Paloma Merino; (Hospital Ramón y Cajal, Madrid). Fernando Baquero, Mª 14

Isabel Morosini; (Hospital Santa Cristina, Madrid). Carmen Pazos; (Hospital 15

Carlos III, Madrid). Margarita Baquero, Ana Mª Enríquez; (Hospital Puerta de 16

Hierro, Madrid). Diego Dámaso, Isabel Sánchez Romero; (Hospital Universitario 17

de la Princesa, Madrid). Manuel López Brea, Teresa Alarcón; (Hospital de la 18

Cruz Roja, Madrid). Rosario Cortés, Mª Victoria Portús; (Hospital de 19

Cantoblanco, Madrid). Amalia de Urmeneta; (Hospital Príncipe de Asturias, 20

Alcalá de Henares Madrid). María Beltrán, Rosa González; (Hospital General 21

de Móstoles, Madrid). José Luis Gómez Garcés, Liliana Vargas; (Hospital de 22

Madrid, Madrid). Almudena Burillo, Esteban Aznar; (Hospital de Madrid, 23

Montepríncipe, Madrid). Almudena Burillo, Aida Sánchez; (Hospital de Madrid, 24

Torrelodones, Madrid). Almudena Burillo, Alicia Rico; (Hospital Severo Ochoa, 25

Leganés, Madrid). Isabel Wilhelmi, Concepción Gómez Criado; (Fundación 26

Hospital de Alcorcón, Madrid). Alberto Delgado, José Valverde; (Hospital 27

Universitario de Getafe, Madrid). Juan Ignacio Alós, Margarita Sánchez 28

Concheiro; Melilla: (Hospital Comarcal de Melilla). Mª Isabel Galán; (Hospital 29

Militar Pages, Melilla). Juan Antonio Tur Sánchez; Murcia: (Hospital Morales 30

Meseguer, Murcia). Cristóbal Ramírez, Carmen Guerrero; (Hospital 31

Universitario Virgen de la Arrixaca, El Palmar, Murcia). Manuel Segovia, 32

Genoveva Yagüe, Ana Menasalvas; Navarra: (Hospital Virgen del Camino, 33

Pamplona). Víctor Martínez de Artola, Xavier Beristain; (Hospital San Juan de 34

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Dios, Pamplona). Coro Pina, Ana Fontaneda; (Hospital de Navarra, Pamplona). 1

Inés Dorronsoro, José Javier García Irure; (Clínica Universitaria de Navarra, 2

Pamplona). José Leiva, Silvia Hernáez; País Vasco: (Hospital Donostia, San 3

Sebastián, Guipuzkoa). Emilio Pérez Trallero, José Mª García Arenzana; 4

(Policlínica Gipuzkoa, San Sebastián, Guipuzkoa). José Antonio Jiménez 5

Alfaro, Borja Redondo; (Hospital de Zumárraga, Guipuzkoa). Lourdes Arteche, 6

Ana Iturzaeta, Aitziber Aguinaga; (Hospital de Basurto, Bilbao Bizkaia). Ramón 7

Cisterna, Karmele Ibarra; (Hospital de Santa Marina, Bilbao, Bizkaia). Felicitas 8

Calvo; (Hospital San Eloy, Baracaldo, Bizkaia). Ignacio Corral, Luis Elorduy, 9

Inés Martínez Rienda; (Hospital de Galdakao, Bizcaia). Mª José López de 10

Goikoetxea; (Hospital Txagorritxu, Vitória). Lourdes Michaus, A. Perales; 11

(Hospital Santiago Apóstol, Vitória). Alicia Labora, Andrés Canut; Valencia: 12

(Hospital General Universitario de Alicante, Alicante). Joaquín Plazas. Mariano 13

Andreu López; (Hospital de San Juan, Alicante). Victoria Ortiz de la Tabla, 14

Alfredo Zorraquino; (Hospital de la Vega Baja, Orihuela, Alicante). Nieves 15

Gonzalo; (Hospital de Elda, Alicante). Victoria Sánchez, Encarna Fuentes; 16

(Hospital General Universitario de Elche, Alicante). Gloria Royo, Pilar López; 17

(Hospital General de Castellón). Rosario Moreno, Alfonso García del Busto; 18

(Hospital La Plana, Villareal, Castellón). Alberto Yagüe; (Hospital Universitario 19

La Fe, Valencia). Manuel Gobernado, José Luis López Hontangas; (Hospital 20

Clínico Universitario de Valencia). Juan García de Lomas, Nuria Tormo, Tomás 21

García Lozano; (Instituto Valenciano de Oncología, Valencia). Joaquín 22

Máiquez; (Hospital Peset, Valencia). José Miguel Nogueira, Manuel Canós; 23

(Consorcio Hospitalario General Universitario de Valencia). Mª Rosario Llucián; 24

(Hospital Arnau de Vilanova, Valencia). Ana Mª Lloret, Montserrat Bosque; 25

(Hospital de la Ribera, Alzira, Valencia). Antonio Guerrero, Javier Colomina; 26

(Hospital de Requena, Valencia). José Mª García Aguayo; (Hospital Francisco 27

de Borja, Gandía, Valencia). Luis Andreu Miguel, Mª Carmen Alonso Jiménez; 28

(Hospital Lluís Alcanyis, Xátiva, Valencia). Rosa Mª Ferreruela; (Hospital 29

General Básico de la Defensa, Quart Poblet, Valencia). José Luis Hernández 30

Tintorer, Ignacio Vírseda; (Hospital de Sagunto, Valencia). Josep Prat Fornells, 31

Rosa Escoms; (Hospital Moliner, Serra, Valencia). Salvador Giner. 32

33

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41

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Table 1. Resistance phenotypes of MRSA 1

2

3

Profile No. of isolates (%)

Only oxacillin-resistant 7 (5.2)

ERY 1 (0.8)

CIP 8 (5.9)

TOB+CIP 26 (19.2)

ERY+CIP 7 (5.2)

ERY+CLI 1 (0.7)

ERY+TOB+CIP 23 (17.0)

ERY+CIP+CLI 13 (9.6)

GEN+TOB+CIP 4 (3.0)

ERY+CIP+CLI+TOB 22 (16.3)

ERY+CIP+TOB+GEN 6 (4.5)

ERY+CIP+TOB+GEN+CLI 17 (12.6)

Abbreviations: CIP, ciprofloxacin; CLI, clindamycin; ERY, erythromycin; GEN, 4

gentamicin; TOB, tobramycin. 5

6

7

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Table 2. Correlation between the different molecular typing methods 1

2

SCCmec type (No.) agr type (No.)

PFGE (No.)

CC BURST

spa CC BURP (No.) I II IVa IVc IVd IVh

1 (20) E13 (6) CC22 Singletons (6) - - - - - 6 E19 (5) CC8 CC211 (5) - - 4 - 1 - A (1) CC8 CC211 (1) - - 1 - - - Sporadics (8) CC067 (3) - - 3 - - - CC211 (2) 1 - - 1 - - Non-founder (1) - - - 1 - - Singleton (1) - - - 1 - - Excluded (1) - - 1 - - - 2 (111) E7 (38) CC5 CC067 (35) - - 30 5 - - CC012 (1) - - - 1 - - Singletons (2) - - 2 - - - E8 (33) CC5 CC067 (32) - - 21 11 - - CC012 (1) - - 1 - - - E10 (9) CC5 CC067 (9) - - 6 3 - - E11 (1) CC5 CC067 (1) - - - 1 - - E15 (3) CC5 CC067 (3) 3 - - - - - E16 (1) CC5 CC067 (1) - - 1 - - - E17 (3) CC5 CC067 (1) 1 - - - - - Excluded (2) 2 - - - - - E 18 (1) CC5 Excluded (1) 1 E 20 (8) CC5 CC067 (8) - - 8 - - - Sporadics (14) CC067 (12) 1 4 3 4 - - CC012 (1) - - 1 - - - Non-founder (1) 1 - - - - - 3 (4) E12 (3) CC30 CC012 (2) - 2 - - - - Singleton (1) - 1 - - - - Sporadics (1) Singleton (1) Not determined

Total (%)

10 (7.4)

7 (5.2)

82 (60.8)

28 (20.7)

1 (0.8)

6 (4.4)

3

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Table 3. Distribution of spa types and BURP groups (spa CC) 1

2

3

spa CC (BURP)

No. (%) spa

type* No. of isolates

CC067 105 (77.7) t067 66 t002 20 t001 1 t010 2 t045 2 t105 1 t109 3 t837 1 t1683 1 t1954 1 t2220 1 t3734 1 t3739 1 t3746 1 t3748 2 t3753 1 CC211 8 (6.0) t008 6 t051 1 t211 1 CC012 5 (3.7) t012 2 t018 1 t021 1 t037 1 Singletons 11 (8.1) t032 6 t084 1 t127 1 t166 2 t3742 1 Non-founder 2 (1.5) t108 1 t1197 1 Excluded 4 (3.0) t059 1

t3744 3

*The most frequent spa types are marked in bold 4

5

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Figure 1. Dendrogram showing the genetic relationships between the 135 1

MRSA isolates and correlations between the different typing 2

methods 3

4

5

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1

2

3

4

5

6

7

8

9

MLST

CC ST PFGE

(No. of isolates) spa TYPE

(No. of isolates) BURP group

Sporadic (1) t002 (1) Sporadic (1) t002 (1) Sporadic (1) t067 (1)

CC067

t105 (1)

t002 (5) 5 146 E10 (9)

t067 (3)

CC067

Sporadic (1) t002 (1) Sporadic (1) t002 (1)

CC067

t109 (1) CC067 5 228 E17 (3)

t3744 (2) Excluded

Sporadic (1) t067 (1) Sporadic (1) t067 (1) Sporadic (1) t067 (1) Sporadic (1) t1683 (1)

CC067

E8b (1) t002 (1)

t837 (1)

t1954 (1)

t2220 (1)

t3746 (1)

CC067

t012 (1) CC012

t045 (1)

t002 (8)

t067 (17)

5 125 E8a (32)

t3739 (1)

CC067

Sporadic (1) t067 (1) CC067 Sporadic (1) t3742 (1) Singleton Sporadic (1) t127 (1) Singleton

t010 (2)

t067 (12) CC067

t166 (1) Singleton

t3753 (1) CC067

t021 (1) CC012

5 125 E7a (19)

t002 (2) CC067

Sporadic (1) t067 (1)

Sporadic (1) t067 (1) CC067

t3748 (2) CC067

t084 (1) Singleton 5 125 E7b (19)

t067 (16) CC067

t3734 (1) 5 125 E20 (8)

t067 (7) CC067

5 125 E11 (1) t067 (1) CC067

Sporadic (1) t067 (1) CC067

5 228 E18 (1) t3744 (1) Excluded

t067 (1)

5 228 E15 (3) t109 (2)

CC067

22 22 E13 (6) t032 (6) Singleton

Sporadic (1) t108 (1) Non-founder

Sporadic (1) t037 (1) CC012

t012 (1)

t018 (1) CC012

30 36 E12 (3)

t166 (1) Singleton

5 228 E16 (1) t067 (1) CC067

Sporadic (1) t211 (1) CC211

8 8 E19 (5) t008 (5) CC211

Sporadic (1) t051 (1) CC211

Sporadic (1) t1197 (1) Non-founder

8 8 A (1) t008 (1) CC211

Sporadic (1) t059 (1) Excluded Sporadic (1) t001 (1)

Sporadic (1) t045 (1) CC067

“Group violations” are marked in grey

100

9590

85

807570

65

60

555045

40

35

3025

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