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J Clin Microbiol. Sep 2008; 46(9): 2842–2847.
Published online Jul 9, 2008. doi:  10.1128/JCM.00521-08
PMCID: PMC2546764

Molecular Epidemiology of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Global Clinical Trials[down-pointing small open triangle]

Abstract

Determining the genetic characteristics of Staphylococcus aureus is important for better understanding of the global and dynamic epidemiology of this organism as we witness the emergence and spread of virulent and antibiotic-resistant clones. We genotyped 292 S. aureus isolates (105 methicillin resistant and 187 methicillin susceptible) using a combination of pulsed-field gel electrophoresis, multilocus sequence typing, and SCCmec typing. In addition, S. aureus isolates were tested for the presence of the Panton-Valentine leukocidin (PVL) genes. Isolates were recovered from patients with uncomplicated skin infections in 10 different countries during five phase III global clinical trials of retapamulin, a new topical antibiotic agent. The most common methicillin-resistant clone had multilocus sequence type 8, pulsed-field type USA300, and SCCmec type IV and possessed the PVL genes. This clone was isolated exclusively in the United States. The most common PVL-positive, methicillin-susceptible clone had multilocus sequence type 121 and pulsed-field type USA1200. This clone was found primarily in South Africa and the Russian Federation. Other clones were found at lower frequencies and were limited in their geographic distribution. Overall, considerable genetic diversity was observed within multilocus sequence type clonal complexes and pulsed-field types.

Staphylococcus aureus is a longstanding subject of epidemiological investigation. In recent years, such studies have typically involved genotyping via a variety of molecular techniques, including pulsed-field gel electrophoresis (PFGE) (27), multilocus sequence typing (MLST) (9), and spa typing (25). In addition, researchers have used the presence or absence of resistance and virulence elements to characterize isolates. Because of its potential for pathogenicity, methicillin-resistant S. aureus (MRSA) has received the most attention, and molecular epidemiological analyses have been instrumental in establishing characteristic profiles for health care-associated (HA) as well as community-associated (CA) strains. For example, the well-known association of type IV staphylococcal cassette chromosome mec (SCCmec) and the genes encoding Panton-Valentine leukocidin (PVL) with CA-MRSA (7) has been useful not only for strain identification but also for documenting the changing epidemiological MRSA landscape, with the most common North American CA-MRSA clone, USA300, rapidly moving into health care settings (12).

A variety of studies have shown S. aureus, and especially MRSA, to be highly clonal relative to many other species of bacterial pathogens (10, 21). Thus, the majority of MRSA isolates associated with an outbreak typically belong to one or a few different clonal types. In comparison, methicillin-susceptible S. aureus (MSSA) isolates are often more genetically variable and have commonly been the subject of more general surveillance studies (14) but relatively few studies with molecular typing. Considering the global and dynamic nature of MRSA in HA and CA infections, continued surveillance is important for clearer understanding of the epidemiology of these organisms.

Here we report the molecular characterization of S. aureus isolates obtained from uncomplicated skin infections during five phase III global clinical trials of retapamulin, a new topical antibiotic agent. To our knowledge, this report represents the largest global data set of MRSA and MSSA isolates in which each isolate was subjected to MLST, PFGE, SCCmec (MRSA only), and PVL analyses. The molecular characterization and geographic distribution of these isolates are compared to those from previous reports of CA-MRSA, HA-MRSA, and MSSA.

MATERIALS AND METHODS

Bacterial isolates.

Skin specimens were obtained at the baseline visit for all patients enrolled in five global clinical trials conducted in 2004 to 2005 to evaluate the safety and efficacy of retapamulin, a new topical antibiotic agent for use in the treatment of secondarily infected traumatic lesions (SITL), secondarily infected dermatitis (SID), and impetigo. All isolates were collected in the community setting. A description of the isolates included in this study is shown in Table Table1.1. A total of 1,442 S. aureus isolates were recovered at baseline in these clinical trials. One hundred eight (7.5% of all S. aureus) isolates were determined to be MRSA by the clinical trial central laboratory, and all but three discrepant isolates (i.e., methicillin susceptible upon repeat testing by GlaxoSmithKline) were included for genotyping in this investigation. A total of 1,334 MSSA isolates (92.5% of all S. aureus isolates) were recovered from baseline in all five clinical trials. Of those, a subset of 977, which included all of the MSSA isolates from the SITL and SID clinical trials, were tested to determine the presence of the PVL genes and mecA (see below). Genotyping was performed on 118 of 121 PVL-positive MSSA isolates (excluding 3 discrepant cultures that were methicillin resistant upon repeat testing by GlaxoSmithKline) and an additional 69 PVL-negative MSSA isolates. The latter were selected to be representative of the entire set of PVL-negative MSSA isolates in terms of geographic distribution, clinical response, and testing arm. All isolates were recovered in 2004 to 2005 from investigator sites in North America, Europe, and other countries.

TABLE 1.
Overview of S. aureus isolates recovered from patients during five clinical trials in 2004 to 2005

PVL, mecA, and SCCmec testing.

Genomic DNA was obtained by boiling colonies in 50 μl of filter-sterilized water for 15 min. Triplex PCR for detecting mecA, lukS-PV-lukF-PV, and rplC (positive control) was performed in 25-μl reaction volumes with the following components: 1× PCR buffer, 1.5 mM MgCl2, 200 μM each deoxynucleoside triphosphate, 1.25 U Taq polymerase, 1 μl DNA template, and 200 nM each primer. Previously established multiplex PCR technology was utilized to determine the SCCmec type of each MRSA isolate (20, 21). Isolates belonging to atypical amplicon patterns were further characterized using previously established methodologies (20). The multiplex PCR for characterizing SCCmec types was performed as described above but with 3 mM MgCl2. Using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA), the cycle program for multiplex PCR was predenaturation for 10 min at 95°C; 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 2 postextension cycles for 7 min at 72°C.

MLST.

MLST was performed according to the protocol of Enright et al. (9), with some modifications of the PCR conditions. Chromosomal DNA was extracted using the DNeasy tissue kit (Qiagen Inc., Valencia, CA) according to the manufacturer's specifications. PCR was carried out with reaction volumes of 48.7 μl containing 130 ng of chromosomal DNA, 15 nmol each of forward and reverse gene-specific primers, and 45 μl of PCR Supermix High Fidelity (Invitrogen, Carlsbad, CA). The PCR was performed with an initial 5-min denaturation at 94°C; 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 70°C for 90 s; and a final extension step of 72°C for 5 min. The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA) by following the manufacturer's instructions. Direct sequencing of purified PCR products was performed with a BigDye terminator cycle sequencing kit and a model 3730XL Genetic Analyzer (both from Applied Biosystems, Foster City, CA). Multiple sequencing primers were used to ensure the accuracy of sequencing results from both strands. Contigs were assembled using Sequencher, version 4.5 (Gene Codes, Ann Arbor, MI); homologous DNA sequence alignments were generated by ClustalX 1.83 (29); and sequences were analyzed and manually edited in GeneDoc 2.6 (19). Allelic profiles and sequence types were determined using the public MLST database (www.mlst.net), and clonal complex designations were made using eBURST, version 3.

Primers.

The primers used to determine the presence of mecA and the SCCmec type have been described by Oliveira et al. (21) and Okuma et al. (20). The primers used to determine the presence of the PVL genes have been described by Lina et al. (15). All other primers used for PCR and sequencing in this study are listed in Table Table22.

TABLE 2.
Sequences of primers used in PCR and sequencing

PFGE.

For each isolate, DNA was prepared by in situ lysis of cells encased in agarose plugs and digested with SmaI as described by Goering (11). PFGE was performed using a CHEF DR III system (Bio-Rad, Hercules, CA) at 6 V/cm, 14°C, and a 120° included angle, with switching from 5 to 15 s for 10 h, followed by switching from 15 to 60 s for 13 h (18). Images of ethidium bromide-stained gels were archived using a Bio-Rad GelDoc 1000 system. PFGE profiles were analyzed using BioNumerics, version 4.01 (Applied Maths, Kortrijk, Belgium). Isolates were given specific strain designations based on comparison (i.e., at least 80% similarity) with published pulsed-field patterns (e.g., the CDC national database) and other molecular characterization as noted above.

RESULTS

Table Table33 shows the PVL status, SCCmec type, clonal complex (CC), sequence type (ST), and pulsed-field type (PFT) by geographic location for the 105 MRSA isolates recovered in this study. Tables Tables44 and and55 summarize MSSA PFT, CC, and ST characteristics by geographic distribution for PVL-positive and -negative isolates, respectively. The results concerning significant staphylococcal strain types are as follows.

TABLE 3.
SCCmec type, PFGE, and MLST results for PVL-positive and PVL-negative MRSA isolates by countrya
TABLE 4.
PFGE and MLST results by country for PVL-positive MSSA isolatesa
TABLE 5.
PFGE and MLST results by country for PVL-negative MSSA isolatesa

USA300 and USA500.

Of the 105 MRSA isolates, 53 (50%) had PFT USA300, SCCmec IV, and CC8 (Table (Table3).3). Of these, 49 (92%) were PVL positive and were collected in the United States (Table (Table3).3). The overwhelming majority of these were CC8:ST8 and PFT USA300-0114, the most common strain of CA-MRSA in the United States (8, 28) Four PFT USA300 MRSA isolates were PVL negative, had a mixture of CC8 STs, and were collected in Costa Rica and India (one and three isolates, respectively). Interestingly, 10 of the MSSA isolates tested also had PFT USA300 and CC8:ST8 (Tables (Tables44 and and5).5). Of these, five were PVL positive and came from the United States (Table (Table4),4), while the five PVL-negative isolates came from a variety of locations, including the United States (n = 1), South Africa (n = 2), Peru (n = 1), and Poland (n = 1) (Table (Table55).

A small number of PFT USA500 MRSA isolates, related to PFT USA300 (e.g., CC8, SCCmec IV) but HA and typically PVL negative, were found in Germany (n = 3) and South Africa (n = 2) (Table (Table3).3). Two CC8 MSSA isolates from South Africa also exhibited PFTs similar to USA500 (Table (Table55).

USA400.

Two MRSA isolates, one PVL positive and the other PVL negative, had PFT USA400, SCCmec IV, and CC1:ST1 (Table (Table3),3), a pattern similar to that of MW2, the first CA-MRSA strain reported in the United States (3, 26). Fourteen MSSA isolates had PFT USA400 and CC1:ST1 or CC1:SLV1 (Tables (Tables44 and and5).5). These were a mixture of predominantly PVL-negative isolates originating in India (n = 6), South Africa (n = 5), the United States (n = 2), and Germany (n = 1) (Tables (Tables44 and and55).

Other CA-MRSA strains.

In Europe the characteristic CA-MRSA strain associated with major outbreaks has CC80:ST80 and SCCmec IV and is PVL positive (13, 31). However, only one ST80 isolate, a PVL-positive MSSA strain from South Africa, was found in our data set (Table (Table4).4). CA-MRSA USA1000 and USA1100 (SCCmec IV, ST59 and ST30, respectively) have also been described (23, 31). However, the only ST59 isolates recovered in these studies were a PVL-negative, SCCmec type IIIa MRSA isolate from India (Table (Table3),3), an ST59 PVL-negative MSSA isolate from the United States, and an ST59 PVL-negative MSSA isolate from the United Kingdom (Table (Table5),5), all with unique PFTs. While no PFT USA1100 MRSA isolates were identified in this study, six PVL-positive, CC30:ST30 (or SLV30) MSSA isolates related to this PFT were recovered from India (n = 3), Germany (n = 1), Russia (n = 1), and Canada (n = 1) (Table (Table44).

EMRSA-15.

Two PVL-positive MRSA isolates from India were similar to the CC22:ST22, SCCmecIV epidemic MRSA-15 (EMRSA-15) strain, which has been well documented in European HA outbreaks (2) (Table (Table3).3). In addition, 23 PVL-positive MSSA isolates from South Africa (n = 16) and India (n = 7) exhibited PFTs related to EMRSA-15 (Table (Table44).

USA200.

In both the United States and elsewhere, the CC30:ST36, SCCmec II, USA200 strain (also known as EMRSA-16) is a well-documented cause of HA infection (5, 6). However, only one PVL-negative USA200 MRSA isolate, from South Africa, was found in our data set (Table (Table3).3). Interestingly, an additional 12 PVL-positive and -negative MSSA isolates of this strain type were recovered from the United States (n = 5), Germany (n = 3), South Africa (n = 2), Costa Rica (n = 1), and India (n = 1) (Tables (Tables44 and and55).

Other HA-MRSA strains.

MRSA strains classified as USA100 and USA800 are well known as causes of HA infection. Both have CC5:ST5 with related pulsed-field patterns but differ in the SCCmec type (II versus IV, respectively) (16). Within our MRSA data set (Table (Table3),3), seven isolates from the United States (n = 5) and Germany (n = 2) had PFT USA100 (also termed the New York/Japan clone in some reports [5]). PFT USA800 MRSA isolates were recovered from the United States (n = 4), Costa Rica (n = 5), Peru (n = 1), and France (n = 1). While more difficult to differentiate in the absence of SCCmec, a total of seven PVL-positive and -negative CC5:ST5 MSSA isolates with PFT 100/800 were isolated in the United States (n = 2), Germany (n = 3), India (n = 1), and South Africa (n = 1) (Tables (Tables44 and and55).

The Brazilian MRSA clone has been characterized as CC8:ST239 with SCCmec type IIIa (1). Among the 50 PVL-negative MRSA isolates analyzed in our study, 12 had CC8:ST239 or related (i.e., ST241 or SLV241) sequence types with PFTs categorized as “unique” but with some similarity (<80%) to the Brazilian pattern (Table (Table3).3). All were recovered in India and harbored SCCmec type III or IIIa. An additional ST239 MRSA isolate with a unique SCCmec type was recovered in Russia.

Additional MSSA strains of interest.

In addition to those noted above, within the 118 PVL-positive MSSA isolates, the most common strain type (n = 42) was CC121:ST121 or CC121:SLV121 and PFT USA1200 (Table (Table4).4). These were recovered primarily in South Africa (n = 25) and Russia (n = 11). Six additional USA1200 isolates were seen within the PVL-negative MSSA set from the United States (n = 3), Peru (n = 1), South Africa (n = 1), and Germany (n = 1) (Table (Table5).5). The second most frequent strain (n = 23), found exclusively among PVL-positive MSSA isolates, exhibited a PFT related to EMRSA-15 and had CC22:ST22, -SLV22, or -DLV22. Of these isolates, 16 were recovered in South Africa and 7 in India. The remaining PVL-positive MSSA isolates were distributed at a low frequency among a variety of strain types (Table (Table4).4). Among the 69 PVL-negative MSSA isolates, no specific strain type was represented by more than 9 isolates (i.e., USA200 and USA400) (Table (Table5).5). Twenty of these 69 isolates had unique PFTs with a variety of CC:STs.

Clonality of S. aureus.

Isolates of S. aureus that differ in methicillin resistance and the presence of PVL genes also differ in their levels of clonality. Among 55 PVL-positive MRSA isolates, 84% had ST8 and PFT USA300 (Table (Table3).3). No other clone was present at a frequency higher than 5%. Among 50 PVL-negative MRSA isolates, the two most frequent clones (CC8:ST239 with a unique PFT and CC5:ST5 with PFT USA800) accounted for 40% of the isolates recovered. Two other clones (CC8 PFT USA300 and CC5 PFT USA100) accounted for an additional 22%. Among PVL-positive MSSA isolates, two clones (CC22 PFT EMRSA-15 and CC121 PFT USA1200) accounted for 55% of isolates (Table (Table4).4). The remaining strain types each represented ≤8% of isolates. The PVL-negative MSSA isolates were more diverse, with the two most frequent clones (CC30:ST30 USA 200 and CC45:ST45 USA600) accounting for only 20% and two other clones (CC8:ST8 USA300 and CC1:ST1 USA400) accounting for another 16% of isolates (Table (Table55).

DISCUSSION

In this study, 292 S. aureus isolates from patients with uncomplicated skin infections in 10 countries were characterized by use of multiple molecular techniques. Our results concur with earlier reports (4, 28, 30) but add new and important findings on the epidemiology of both MRSA and MSSA. Within the population examined, PVL-positive MRSA strains such as USA300 are still predominantly found in the United States compared with the other countries included in the study. Also, in a number of instances, genetic backgrounds associated with MRSA outbreaks were found in MSSA isolates. Finally, there was considerable diversity in terms of STs (but not necessarily CCs) within known PFTs.

While the major PVL-positive SCCmec type IV CA-MRSA strains initially appeared regionally specific (31), recent findings have shown some CA-MRSA strains to have spread globally (30). The results of this study suggest that, as of 2004 to 2005, the frequency of different PVL-positive genotypes normally associated with CA-MRSA is still relatively low, at least with respect to S. aureus strains causing skin infections in the countries included in these clinical trials. The notable exception is CC8 USA300 in the United States. It should be noted that not a single MRSA isolate of the major European CC80:ST80 CA-MRSA clone (and only one such MSSA isolate) was recovered, despite the inclusion of several European countries in the clinical trials.

Another interesting finding is the number of MSSA genotypes associated with MRSA outbreak strains. Some strains, such as ST30 MSSA, are known to be ancestral forms of HA-USA200 and CA-USA1100 MRSA strains (24). However, other strains, such as USA400, have been studied primarily as MRSA (3, 26), although the data in some cases may reflect the increased attention to the genetic characterization of MRSA in contrast to MSSA isolates. MRSA strains are believed to have arisen via multiple acquisitions of SCCmec, and they may gain and lose resistance and virulence determinants over time (17, 22). Thus, the relationship between MRSA and MSSA strains with similar genetic backgrounds may reflect both the intermittent acquisition of SCCmec within existing MSSA populations and the persistence of MSSA strains along with their related MRSA strains in a given environment.

It is noteworthy that the epidemiological conclusions of this report are limited to the countries from which patients were enrolled during the study period (2004 to 2005). For example, while ST30 MRSA (i.e., USA1100, South West Pacific clone) has been found in at least 14 countries (30), only 2 of those countries, the United Kingdom and The Netherlands, were included in this study; therefore, the lack of these isolates in our data set is not surprising. Also, since these countries differ in their overall levels of MRSA, the genotypes of MRSA isolates observed in this study will be representative mainly of locations where MRSA is more common. In addition, the fact that we observed no CC80:ST80 CA-MRSA isolates despite the inclusion of several European countries suggests that the global frequencies of epidemic clones of S. aureus may be generally in flux.

It has been noted previously that some STs are associated with multiple PFTs (13). The present study confirms this and adds some new insights. First, we observed several novel PFTs in this study. Many are associated with STs that are already associated with known PFTs. For example, ST8 is associated with PFTs USA300 and USA500, but this study shows ST8 to also be associated with at least one novel PFT. Furthermore, we have provided evidence that multiple STs within the same CC are often found within the same PFT. For example, PFT USA900 contained isolates with four different STs within CC15.

We observed different levels of clonality in different populations of S. aureus. PVL-positive MRSA isolates were primarily represented by a single dominant strain, while the subset of PVL-negative MSSA isolates tested was composed of multiple strains that were present at a low frequency. These differences could reflect different ages and rates of epidemic spread of these populations.

A potentially disturbing finding is the large numbers of PVL-positive MSSA isolates collected in South Africa and India. While the frequency of PVL-positive MSSA is generally considered to be low around the world, this study, in agreement with other recent work (4), provides evidence that some regions may have exceptional levels of PVL-positive MSSA isolates. This underscores the importance of continued surveillance of MSSA strains such as CC121 USA1200, which may be important both as causes of infection and as potential reservoirs of resistance and virulence factors that can be acquired by MRSA strains.

In summary, by use of a combination of molecular techniques, we have presented a view of the molecular epidemiology of MRSA and MSSA causing uncomplicated skin infections. MRSA and MSSA strains shared the same genetic backgrounds with high frequency, and there was considerable diversity within both CCs and PFTs. The single most frequently observed MRSA clone had ST8, PFT USA300, and SCCmec type IV and was PVL positive.

Acknowledgments

We thank the GlaxoSmithKline U.S.-based sequencing facility, under the management of Ganesh Sathe, for its efforts on this project.

Footnotes

[down-pointing small open triangle]Published ahead of print on 9 July 2008.

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