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J Clin Microbiol. Oct 2005; 43(10): 5150–5157.
PMCID: PMC1248511

Comparison of Genetic Backgrounds of Methicillin-Resistant and -Susceptible Staphylococcus aureus Isolates from Portuguese Hospitals and the Community


In order to understand the origins of the dominant methicillin-resistant Staphylococcus aureus (MRSA) clones in Portuguese hospitals, we compared the genetic backgrounds of nosocomial MRSA with methicillin-susceptible S. aureus (MSSA) isolates from the same hospitals (n = 155) and from the community (n = 157) where they were located. Pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, and agr type analysis revealed that the genetic backgrounds correspondent to the dominant MRSA clones in Portuguese hospitals during the last 15 years (Iberian ST247, Brazilian ST239, and EMRSA-15 ST22) were scarcely or not found among the present MSSA collection. The four major MSSA clones encountered (A-ST30, B-ST34, C-ST5, and H-ST45) correspond, or are very similar, to the background of other international MRSA pandemic clones, i.e., EMRSA-16, New York/Japan, Pediatric, and Berlin clones. However, with the exception of the Pediatric clone, none of these MRSA clones has been detected in Portugal. Our findings suggest the three major MRSA clones identified in Portuguese hospitals have not originated from the introduction of SCCmec into dominant MSSA backgrounds present in the Portuguese nosocomial or community environment but were probably imported from abroad. In contrast, the MRSA Pediatric clone might have originated in our country by the acquisition of SCCmec type IV into MSSA clone C. Furthermore, we provide evidence that the introduction of SCCmec into sensitive clones is most likely a relatively infrequent event that seems to depend not exclusively on the presence of a successful MSSA lineage.

Staphylococcus aureus is a major human pathogen capable of causing a wide range of infections of different severity, such as skin abscesses and wound infections, osteomyelitis, endocarditis, pneumonia, meningitis, bacteremia, and toxic shock syndrome. Gradually, S. aureus has evolved resistance to all classes of antibiotics (36). Methicillin-resistant S. aureus (MRSA) strains have emerged by acquisition of a mobile genetic element called staphylococcal chromosomal cassette (SCC) mec, which carries the mecA gene. Five different types of SCCmec (types I to V), which differ in size and structure, have been described for S. aureus (11, 12, 17).

It is now clear that methicillin resistance has emerged by the introduction of SCCmec into at least five phylogenetically distinct successful methicillin-susceptible S. aureus (MSSA) lineages (5, 28) resulting in a relatively small number of pandemic MRSA clones spread worldwide, namely the Iberian (ST247-SCCmec IA), Brazilian (ST239-III), New York/Japan (ST5-II), Pediatric (ST5-IV), EMRSA-16 (ST36-II), EMRSA-15 (ST22-IV), and Berlin (ST45-IV) clones (1, 7, 27). On the other hand, there is evidence that resistance has been transferred to S. aureus on more than five occasions, as some lineages have acquired different structural types of SCCmec (7, 28).

The nosocomial prevalence of MRSA in Portugal was estimated as close to 50% between 1993 and 1997 (19-21) and 47.5% in 2003 (J. Melo-Cristino, personal communication). The European Antimicrobial Resistance Surveillance System reported an MRSA prevalence in Portugal in blood isolates of around 39% in 2002, which is one of the highest in Europe (33). During the last 15 years there were successive waves of dominant MRSA clones in Portuguese hospitals: (i) in 1992 and 1993, the Iberian clone replaced the Portuguese clone (ST239-III variant) widely spread in the country in the mid-1980s and early 1990s; (ii) in 1994 and 1995, the Brazilian clone was introduced, and its representation has rapidly increased since then; and (iii) in 2001, clone EMRSA-15 emerged and is nowadays becoming the dominant clone in the hospital setting (1). In addition, the Pediatric clone was described for the first time in a pediatric hospital in Lisbon in coexistence with the Iberian clone between 1992 and 1996 (31).

Although reports of community-acquired MRSA (CA-MRSA) are increasing worldwide, the single study evaluating the prevalence of MRSA among the Portuguese community revealed a low carriage rate (<1%) (30). In that study, the two out of seven CA-MRSA isolates not healthcare associated produced enterotoxins A and B and were both ST82.

In order to understand the origins of the nosocomial major MRSA clones present in Portuguese hospitals, we compared the genetic background of MRSA collected in different periods from three hospitals located in Lisbon and Oporto with MSSA isolates from those hospitals and from the community where they were located.


Bacterial strains.

A total of 312 MSSA isolates divided into nosocomial (n = 155) and community (n = 157) collections were included in the present study.

Nosocomial isolates were collected from single patients at three Portuguese hospitals: Hospital Geral de Santo António (49 HSA isolates) and Instituto Português de Oncologia do Porto (39 IPOP isolates), both located in Oporto, and Hospital de São José (53 HSJ isolates) located in Lisbon. The three nosocomial subcollections included invasive and carriage isolates that were recovered in different periods corresponding to the successive waves of dominant MRSA clones in Portuguese hospitals, namely, 1992-1993 (introduction of the Iberian clone), 1995 (introduction of the Brazilian clone), and 2001 (emergence of clone EMRSA-15). HSA isolates were collected between December 1992 and February 1993, the HSJ isolates were collected between September and November 1995 (49 isolates), and one isolate in July 1996 and three isolates between January and February 1997 and the IPOP isolates were collected between January and December 2001. Also included in the nosocomial collection were 14 carriage isolates obtained between June and September 2003 from health care workers at Hospital São Francisco Xavier (Lisbon) (BOH isolates).

Community isolates were obtained from different subcollections. One collection originated from a community-based study on the prevalence of S. aureus colonizing healthy young Portuguese populations including children attending day care centers and draftees (30). From 1,331 children aged 0 to 5 years old attending 16 day care centers located in different geographical areas of Lisbon, nasopharyngeal samples were obtained between February and March 1996 and 1997. S. aureus was isolated from 210 children, from which 64 MSSA (DCC isolates) representing several day care centers were randomly selected. From 823 draftees, males and females aged 17 to 22 years old, nasal swab cultures were obtained between May and June 1996 and between October and November 1997. S. aureus was isolated from 280 draftees, out of which 77 were originally from Lisbon or Oporto and were all included in the present study (draftees isolates). Finally, 16 isolates were obtained from nasal, pharyngeal, or nasopharyngeal swabs of children and adult patients who attended an outpatient clinic in Oporto, Instituto Nacional Ricardo Jorge (RJ isolates) (1992 and 1993).

DNA isolation, detection of mecA and PVL genes, and determination of agr type.

Chromosomal DNA was extracted by incubating cells grown overnight in a solid medium in 20 μl of TE 1× (10 mM Tris, 1 mM EDTA, pH 8) with lysostaphin at 0.5 mg/ml for the lysis step for 30 min, followed by a denaturation step of 15 min at 95°C. The mixture was harvested at 13,000 rpm for 5 min, and 2 μl of the supernatant was used as DNA template in the PCRs. The presence of the mecA and Panton-Valentine leukocidin (PVL) genes was determined by PCR as described previously (16, 23). The accessory gene regulator (agr) group was determined by a multiplex PCR according to the methods of Jarraud et al. (13).

Molecular typing.

Pulsed-field gel electrophoresis (PFGE) was performed as described by Chung et al. (3) on all 312 isolates. The resulting band patterns were analyzed by visual inspection followed by the analysis with the Bionumerics software (version 4.0; Applied Maths, Ghent, Belgium) for relatedness evaluation. Dendrograms were generated from similarity matrixes calculated with the Jaccard coefficient, and patterns were clustered by unweighted pair group method with averages using an optimization of 0.25% and a tolerance of 1.0%.

Nosocomial IPOP and HSA isolates, community DCC isolates, and all remaining isolates that presented a PFGE pattern not observed among the previous collections were further characterized by spa typing (14, 32) (total, 171 isolates). As done previously (14, 32), spa types with similar repeat profiles were grouped together as part of a same lineage (spa lineage), which was identified in the present study by capital letters. Multilocus sequence typing (MLST) was performed in representatives of each PFGE type/spa lineage as described previously (6), with the exception that primer arcCF2 (5′-CCT TTA TTT GAT TCA CCA GCG-3′) (4) was used. For spa typing and MLST, PCR products were purified with a Wizard PCR Preps purification system (Promega, Madison, WI) and used as templates for sequencing of both strands at Macrogen, Seoul, South Korea. MLST alleles and sequence types (STs) were identified using the MLST database (http://www.mlst.net) hosted by Imperial College.


Genetic diversity.

A total of 20 different PFGE patterns were found among the 312 MSSA isolates. Further characterization by spa typing distributed the isolates into 72 spa types. Isolates showing related spa types could be grouped in 19 spa lineages showing congruence between clustering by PFGE and spa typing, with the exception of spa lineages B and P, which were associated with two PFGE patterns each, B and Q and P and L, respectively. PFGE analysis versus spa typing analysis is depicted in Fig. Fig.1.1. MLST performed on representatives of each PFGE pattern and/or spa lineage identified 20 STs, confirming the existence of 20 distinct clones among the 312 MSSA isolates. Application of the eBURST algorithm to the 20 STs recognized 17 groups, which were defined as clusters of closely related STs in which a single difference in the allelic profile was tolerated and therefore considered to belong to 17 clonal complexes (CCs) (8). Figure Figure22 illustrates the molecular characterization of strains representing the 20 clonal types defined by PFGE, spa typing, and MLST as well as the CC assignment generated by eBURST. Interestingly, some clones belonging to different CCs presented a higher PFGE similarity than clones included in the same CC. For instance, clone PFGE L-ST188 showed a PFGE similarity of 63.2% with clone PFGE Y-ST106 but only 55.3% with clone PFGE M-ST573 (Fig. (Fig.2).2). These discrepancies may be due to the different spectra of changes detected by the two typing methods. PFGE examines both nucleotide changes that are in specific restriction sites and changes that involve large insertions or deletions of DNA in the whole genome, while MLST detects nucleotide changes only within the seven amplified housekeeping gene fragments. Moreover, to complicate the interpretation, bands of the same size are assumed to be identical in PFGE and unrelated fragments that are indistinguishable by size can occur by chance, especially as the genetic distance between strains increases. Despite the fact that in one case in the present study the PFGE and allelic profile similarities were not connected, it did not constitute an issue for typing, since the isolates were sufficiently unrelated and classified into different clones.

FIG. 1.FIG. 1.
PFGE analysis versus spa type analysis of MSSA strains. Distribution of the 312 MSSA isolates into 20 PFGE types is shown. The area of each circle is proportional to the number of isolates included in the PFGE type, and white and gray areas correspond ...
FIG. 2.
Molecular characterization of MSSA strains and comparison with MRSA pandemic clones. Shown from left to right are (i) a dendrogram indicating the estimated relationships of PFGE types based on Bionumerics analysis, including representatives of seven international ...

Despite the high genetic variability, over half (61%) of the isolates belonged to four major clonal types (represented by more than 10% of the isolates) and three CCs: clone A (PFGE type A, spa type 33 or related, ST30, CC30), clone B (PFGE type B, spa type 497 or related, ST34, CC30), clone C (PFGE type C, spa type 2 or related, ST5, CC5), and clone H (PFGE type H, spa type 136 or related, ST45, CC45).

Hospital versus community MSSA isolates.

Sixteen out of 20 clones, including the four major clones, A, B, C, and H, were present in both nosocomial and community settings (Fig. (Fig.1).1). However, some of the major clones were clearly represented by different proportions: clone C was overrepresented in the hospital setting (chi homogeneous square test, P = 0.039), and clone B contained proportionally more isolates from the community (chi homogeneous square test, P < 0.0001).

Among the 155 nosocomial isolates, four major clones could be identified, clones A (n = 34, 22%), C (n = 22, 14%), and H and K (n = 17 each, 11%), whereas clone B (n = 49, 32%) followed by clone A (n = 37, 24%) embraced 86 out of the 157 (55%) community isolates (Fig. (Fig.3).3). Clone K was characterized by PFGE type K, spa type 21 or related, and ST15. The clonal distribution among the nosocomial isolates varied according to the hospital: clone A was predominant in both hospitals in Oporto, while clones C and K were the principal clones in hospital HSJ located in Lisbon. A relatively large number of genetic backgrounds (n = 7) was found among the 14 isolates recovered from health care workers from another hospital in Lisbon, and the major clones included only three isolates each.

FIG. 3.
Clonal type distribution. Numbers underlined represent the major clonal types in each collection or subcollection. Minor clones (clones harboring a maximum of 5% of all isolates) were classified as “other” clonal types and include STs ...

Although the community isolates belonged to two major clones, the different populations were not evenly distributed: children attending day care centers more often carried strains from clone B (n = 32, 50%) in comparison with draftees (n = 14, 18%) (chi homogeneous square test, P = 0.001). No significant difference was found in the clonal distribution of community isolates from Oporto versus Lisbon (data not shown). The isolates recovered from patients attending the outpatient clinic in Oporto (RJ isolates) were distributed into several clonal types, as happened with the nosocomial BOH isolates, rather than showing a significant major clone, probably due to the reduced number of isolates representing these subcollections.

A considerable number of minor clones, each harboring a maximum of 5% of all isolates, was found among both the hospital and community isolates. Minor clones reached 30 and 21% of all hospital and community isolates, respectively (Fig. (Fig.33).

MSSA versus MRSA background.

Comparing the genetic backgrounds of the major MSSA clones (A, B, C, and H) with international pandemic MRSA lineages, including the main MRSA clones present in Portugal during the last 15 years, revealed that clones A and B correspond to slight variations (MLST single and double locus variants, respectively) of the genetic background of MRSA clone EMRSA-16 (ST36, SCCmec type II), clone C corresponds to the background of the New York/Japan (ST5-II) and Pediatric (ST5-IV) clones, and clone H corresponds to the background of the Berlin clone (ST45-IV). In addition, two minor MSSA clones, J (6% of all isolates) and T (one isolate), are related to two additional widely spread MRSA clones. Clone J (PFGE J, spa type 1 or related, ST8) corresponds to a single and a double locus variant of the Brazilian (ST239-III) and Iberian (ST247-IA) clones, respectively, and clone T corresponds to clone EMRSA-15 (ST22-IV). On the other hand, eBURST analysis showed that 67% of all isolates belonged to one of the five major CCs described for MRSA, i.e., CC30, CC5, CC45, CC8, and CC22 (Fig. (Fig.1,1, right panel).

A dendrogram representing the clustering of the 20 MSSA PFGE types together with representatives of the seven major international MRSA clones (Iberian, Brazilian, New York/Japan, Pediatric, EMRSA-15, EMRSA-16, and Berlin) confirmed the relatedness between MSSA found in the present study and MRSA pandemic clones (Fig. (Fig.22).

mecA and PVL gene amplification and agr specificity group.

The presence of the mecA gene was determined by PCR amplification, and only mecA-negative isolates were included in the present study. We have assessed the prevalence of the PVL gene in isolates representing all clonal types from both the community and hospital populations. Two of 86 isolates tested were PVL-positive MSSA strains. These two isolates belonged to clone F (PFGE F, spa type 441, ST121). Interestingly, among the 17 strains belonging to clone F (9 from the hospital and 8 from the community), only 2 isolates harbored the PVL gene. These two isolates were found in the community among carriage isolates from a child attending a day care center and an Air Force draftee.

The determination of the agr specificity group for representatives of the 20 MSSA clones revealed that half belonged to agr type 1. The remaining clones belonged to agr type 2 (seven clones) and type 3 (three clones) (Fig. (Fig.2).2). Although agr type 3 included three clonal types only, it was associated with the major clones A and B as well as clone G and therefore harbored 44% of all MSSA isolates. Interestingly, the three clonal types belonging to CC1 showed three different agr types (Fig. (Fig.22).


The incidence of MRSA in Portuguese hospitals is one of the highest in Europe. We previously drew a temporal scheme for the evolution of MRSA clonal types in Portuguese hospitals (1). In the present study, the population structure of Portuguese MSSA, isolated from clinical and nonclinical isolates, has been determined. Among the nosocomial isolates four subcollections were used, corresponding to different hospitals and periods. The community subcollections were obtained from the geographical area served by the hospitals from children attending day care centers (<5 years) and Air Force draftees (17 to 22 years) with nasal carriage of S. aureus and from a small group of patients attending an outpatient clinic.

PFGE, spa typing, and MLST analysis distributed the 312 isolates into 20 clonal types. A search in the MLST database (http://www.mlst.net/) revealed that 15 out of the 20 STs identified in the present study are exclusively or mostly associated with MSSA isolates, evidencing a high genetic diversity among the MSSA population, as found by others (2, 6). Despite the high diversity, eBURST, a computer algorithm used to solve population structures based on MLST data (8), grouped 67% of the MSSA isolates, including clinical and carriage isolates, into four major clonal complexes (CC30, CC5, CC45, and CC8) corresponding to four of the five major MRSA clusters spread worldwide (7, 28, 29, 34). On the other hand, the main STs found in the present study have been found in other countries showing MSSA geographical spread. These observations support the evidence that the same main MSSA lineages seem to be disseminated in different regions of the world and that pandemic MRSA originated by the introduction of SCCmec into these most successful lineages. The fact that clinical and carriage strains as well as MSSA and MRSA isolates fall into the same main clusters is in agreement with Melles et al., who proposed that essentially any S. aureus genotype carried by humans can transform into a life-threatening human pathogen (18).

In comparison with the scenario observed for nosocomial Portuguese MRSA clones, the representation of the major MSSA clones in Portuguese hospitals appears to be more stable over time. Whether the slight differences in the clonal distribution between hospitals are due to the different sampling moments or to the different geographic location of the hospitals is currently unclear. Comparison of the genetic backgrounds of the MSSA clones found in the present study with the backgrounds of the major international pandemic MRSA lineages revealed some overlapping. The major MSSA clones, A-ST30, B-ST34, C-ST5, and H-ST45, correspond or show a high degree of similarity in PFGE, spa type, and MLST to EMRSA-16 (ST36), the New York/Japan or Pediatric clones (ST5), and the Berlin clone (ST45).

However, to the best of our knowledge, none of these clones with the exception of the Pediatric MRSA has been reported among nosocomial Portuguese MRSA isolates recovered between 1990 and 2001. The Pediatric MRSA clone was first described in a pediatric Portuguese hospital (31) and could have originated in our country by the introduction of a variant of SCCmec type IV (26) into MSSA clone C. We cannot rule out the hypothesis that the remaining main MSSA clones will not become major MRSA clones in Portugal in the future.

Interestingly, the genetic backgrounds corresponding to the three dominant MRSA clones in Portuguese hospitals during the last 15 years (Iberian-ST247, Brazilian-ST239, and EMRSA-15-ST22) or their ancestral genotypes were not detected or scarcely found among the present MSSA collection, clones J-ST8 (ancestral of ST239 and ST247) and T-ST22. Moreover, in the present collection the prevalence of the MSSA clone J increased between 1992 and 2001, which may indicate that most MSSA isolates belonging to this clone were probably isolated after the introduction of the Iberian clone (1992 to 1993) in Portuguese hospitals. These observations suggest the Iberian, Brazilian, and EMRSA-15 MRSA clones have not originated from the introduction of SCCmec into dominant MSSA backgrounds present in the Portuguese nosocomial or community environment but were probably imported from abroad.

The fact that there is no congruence between the genetic backgrounds of the major MSSA and MRSA clones within a population indicates that the presence of a successful MSSA lineage is not the sole factor necessary for SCCmec acquisition or for the success of an MRSA clone. Moreover, the introduction of SCCmec into sensitive clones seems to be a relatively infrequent event compared to the geographical spread of MRSA clones.

CA-MRSA isolates are reported to differ genetically from hospital-acquired MRSA (24). In contrast, community-acquired MSSA isolates are no different than hospital-acquired MSSA, as reported by Enright et al. (6). In the present study, clone A was overrepresented in the hospital setting, whereas clone B was the main clone in the community collection. Nevertheless, clone B was only predominant among isolates collected from children attending day care centers (50%). The different age categories may be an explanation for the observed difference. Moreover, confined environments such as day care centers, where children have close contacts, facilitate the easy spread of bacteria and therefore a high clonal expansion (15). Nevertheless, we cannot exclude the hypothesis that the restricted number of isolates is not a confounding factor.

PFGE and MLST analysis indicated distinct genetic backgrounds for CA-MRSA associated with each geographic origin, namely ST80 in Europe, STs 1, 8, and 59 in the United States, and ST30 in Oceania (34). Although in the present study we have not detected any MSSA sharing the background of the major European CA-MRSA clone, ST80, we detected MSSA isolates of STs 1, 8, and 30. ST1 and ST8 represented minor clones, but ST30, which was initially described among CA-MRSA in Australia and recently reported to have spread in the community in Europe both in The Netherlands and in Latvia (22, 35), corresponded to the main clone found in the present study. In addition, the background of the only two true CA-MRSA strains described so far in Portugal (30) was not found among the MSSA nasal carriage population in our work that in part overlaps the S. aureus isolates from the Sa-Leão et al. study (30). Apparently, as happened with the major hospital-acquired MRSA, we anticipate CA-MRSA ST82 was probably imported from abroad.

PVL is a toxin associated with S. aureus strains causing severe skin infections (16) and with highly virulent necrotizing pneumonia in young patients (9). We assessed the presence of PVL genes in representatives of each MSSA clonal type. The two single PVL-positive isolates belonged to clone F and were recovered in the community from one child and one draftee. The reason why only 2 out of the 17 clone F isolates harbored PVL may be due to the fact that PVL determinants are carried by different temperate phages (25) and therefore should have integrated independently in the two isolates with background F.

As a further contribution to the genotypic characterization of the MSSA isolates, we determined the agr sequence types for representatives of each MSSA clone. The four successive major MRSA clones present in Portuguese hospitals during the last 15 years belonged to the same agr group 1 (10). However, the major MSSA and MRSA clones coexisting in the same hospital do not belong to the same agr type. It would be interesting to clarify the agr-associated interference between MSSA and MRSA. In addition, the fact that the three clonal types belonging to CC1 showed different agr types is intriguing and needs further investigation, since previous studies have linked the genetic background of S. aureus isolates to the agr type (10, 13).

In summary, the major hospital-acquired MRSA clones as well as the rare cases of CA-MRSA in Portugal are not related to the MSSA circulating in the nosocomial or the community setting in the country, which provides supportive evidence that SCCmec moves rarely and that pandemic MRSA spreads independently of the local MSSA clones.


This work was supported by project FCG 61052 from Fundação Calouste Gulbenkian, Portugal, awarded to H. de Lencastre. T. Conceição and C. Simas were supported by grants 13/09/04 CB and 3/01/02 CB from IBET, Portugal.

We thank Barry N. Kreiswirth and Steve Naidich for the new spa assignments (spa types 577 to 582, 603, 609 to 614, and 620).

This publication made use of the Multi Locus Sequence Typing website (http://www.mlst.net), which is hosted at Imperial College, London, United Kingdom.


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