Comparison of genotypes and enterotoxin genes between Staphylococcus aureus isolates from blood and nasal colonizers in a Korean hospital.

In this study, we investigated the genetic background of 70 Staphylococcus aureus isolates (36 methicillin-resistant S. aureus [MRSA] and 34 methicillin-susceptible S. aureus [MSSA]) obtained from blood at a Korean tertiary-care hospital, using spa typing, multilocus sequence typing, and SCCmec typing. In addition, the prevalence of enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, and sek), tst, and pvl genes among the samples was assessed via polymerase chain reaction, and the results were compared with those of 95 isolates of S. aureus obtained from nasal swabs. All MRSA isolates from blood, except one, belonged to three major clones: sequence type (ST)5-MRSA-II, ST72-MRSA-II (or IVA), and ST239-MRSA-III, among which ST5-MRSA-II was the predominant clone. The prevalence of enterotoxin genes in the S. aureus isolates obtained from blood differed significantly from those from the nasal swabs for the sea, seb, sec, and seh gene. In particular, the seb and sec genes were detected exclusively in the MRSA isolates of ST5 or spa-CC002, thereby suggesting the co-adaptation of virulence genes with the genetic background and their contribution to biological fitness.

INTRODUCTION

Staphylococcus aureus, which normally colonizes the anterior nares, can induce a variety of infections, ranging from superficial lesions to toxic shock syndrome and severe systemic infections. Since the first report in 1961 in U.K., the emergence and dissemination of methicillin-resistant S. aureus (MRSA) has become a subject of great global concern (1). In Korea, MRSA is responsible for more than 60% of S. aureus infections in hospitals, and constitutes a continuing threat to public health (2-4). Clinical MRSA isolates from Korean hospitals generally are identified as the two predominant clones, ST5-MRSA-II or ST239-MRSA-III (5-7). However, the majority of studies have focused on any clinical MRSA strains, without regard to their disease characteristics. That is to say, there are few studies reporting the molecular characteristics of MRSA and methicillin-susceptible S. aureus (MSSA) causing bacteremia in Korea (8).

Virulence determinants may have been crucial to the evolution of contemporary epidemic strains of S. aureus (9, 10). So far, a variety of virulence genes have been identified in S. aureus. They include staphylococcal enterotoxins (SEs), staphylococcal exofoliative toxins (ETs), toxic shock syndrome toxin 1 (TSST-1), and the Panton-Valentine leukocidin (PVL). Among these, the SEs are emetic toxins, and have been implicated in food poisoning in humans. TSST-1, which is also a member of the superantigenic toxin family, has been associated with several acute or chronic human diseases, including toxic shock syndrome and, possibly, sudden infant death syndrome and Kawasaki syndrome (11). PVL is known to be associated with tissue necrosis, and is frequently found in community-associated MRSA (CA-MRSA) (12). It has been demonstrated that the horizontal transfer of these virulence genes is one of the causes of the emergence of new virulent MRSA strains (9). Recently, the prevalence of enterotoxin genes in S. aureus isolates associated with staphylococcal food poisoning in Korea was investigated (13).

Detailed knowledge of genotype and virulence gene content may be a prerequisite for understanding of the genetic basis of prevailing clones of bacterial pathogens (9). Genetic backgrounds and the prevalence of enterotoxins may be different between disease-causing and colonizing strains. Thus, we have attempted to characterize the molecular characteristics of S. aureus isolated from blood in a Korean hospital, and have compared them with isolates of S. aureus from nasal swab specimens. In addition, we have evaluated the prevalence of virulence genes among S. aureus isolates obtained from blood and nasal specimens.

MATERIALS AND METHODS

Bacterial isolates

Seventy isolates of S. aureus obtained from blood were consecutively collected at a tertiary-care hospital (Samsung Medical Center) in Seoul, Korea over a five-month period (January to May) in 2006. In addition, 95 S. aureus isolates from nasal swabs of children attending an outpatient clinic in a tertiary-care hospital (14) were also included in this study. These isolates were collected from the same hospital over a similar period (December 2005 to February 2006) with the S. aureus isolates from blood. All of the isolates were identified using a Staphaurex Plus Kit (Murex Diagnostics Ltd., Dartford, U.K.), which were confirmed by molecular typing methods such as multilocus sequence typing (MLST) and spa typing.

Antimicrobial susceptibility testing

In vitro antimicrobial susceptibility testing was performed by a broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) (15). The minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined: oxacillin, penicillin, gentamicin, ciprofloxacin, clindamycin, erythromycin, rifampin, vancomycin, teicoplanin, tetracycline, and trimethoprim-sulfamethoxazole. Susceptibility interpretive criteria used were those established in the CLSI standard M100-S16 (15). S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Escherichia coli ATCC 25922 were employed as control strains.

Genotyping

In order to determine the genotype of S. aureus isolates, spa typing was initially conducted as previously described (16-18). With the BURP algorithm (Ridom GmbH), spa types were clustered into spa clonal complex (spa-CC) (16, 19). MLST was performed for 28 S. aureus isolates from blood, which were representative of each spa type, in accordance with a previously described method (20) with the exception of a primer pair amplifying the yqiL gene (forward, 5'-TAT TAG CAG CAT ACA GGA C-3'; reverse, 5'-CAC CTT TAC GTT GAG GAA TC-3'). The staphylococcal chromosomal casette mec (SCCmec) types of all MRSA isolates were determined by the method of multiplex polymerase chain reaction (PCR) (21).

Virulence gene detection

The detection of toxin genes (sea, seb, sec, sed, see, seg, seh, sei, sek, and tst) was achieved via the method previously described by Jarraud et al. (10). PCR was performed independently for each toxin gene. The Panton-Valentine leukocidin (pvl) gene was detected using the primers previously described by Lina et al. (22).

Statistical analysis

Fisher's exact t-test and chi-square test were utilized to determine the significance of differences in resistance, where appropriate.

RESULTS

Antimicrobial resistances

Among the 70 S. aureus isolates from blood, 36 isolates (51.4%) were found to be methicillin-resistant and 34 isolates (48.6%) were methicillin-susceptible. More than 80% of MRSA isolates were resistant to gentamicin, ciprofloxacin, clindamycin, and erythromycin. In addition, tetracycline resistance rate among MRSA isolates was 72.2%. However, less than 20% of MSSA isolates proved resistant to those antimicrobials except penicillin (Table 1). No S. aureus isolates were found to be resistant to vancomycin or teicoplanin.

Table 1

Antimicrobial resistance of S. aureus isolates from blood

MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus.

Among 95 S. aureus isolates from nasal specimens, 18 were methicillin-resistant (18.9%). Among them, 44 (46.3%), 18 (18.9%), 11 (11.6%), 10 (10.5%), and 3 isolates (3.2%) were resistant to erythromycin, gentamicin, tetracycline, clindamycin, and ciprofloxacin, respectively (14).

Genotypes of S. aureus isolates from blood

Thirty-six MRSA isolates from blood evidenced 10 spa types from three spa-CCs (Table 2). Among the 36 MRSA isolates, 24 isolates (66.7%) belonged to spa-CC002, which share the same DMGMK motif. As five representative isolates were identified as sequence type (ST)5 in MLST analysis and all but one were identified as SCCmec type II, the MRSA isolates of spa-CC002, may represent ST5-MRSA-II. Five MRSA isolates belonged to spa-CC037, all of which harbor the WGKAOMQ motif. All five isolates were identified as SCCmec type III, and two representative isolates evidenced ST239 and a single locus variant (SLV) of ST239. Thus, this group may represent ST239-MRSA-III and its allies. Seven MRSA isolates were associated with spa-CC084, which evidence the ST72. Each of the two MRSA isolates of spa type UJGGMDMGGM were identified as SCCmec types II or IVA, and two isolates of spa type UJGFGMDMGGM were identified as SCCmec type IVA. A remaining isolate of spa-CC084 showed spa type UJGFMB represents ST188-MRSA-IIIA.

Table 2

Genotypes of 36 MRSA and 34 MSSA isolates from blood

*MLST was performed on representative isolates for each spa type (20); †NT, non-typeable (21); ‡Alleles and ST that were not found in the MLST website (http://saureus.mlst.net) were designated 'new'.

MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; MLST, multilocus sequence typing.

The 34 MSSA isolates were differentiated into 18 spa types, which could be further classified into four spa-CCs and a singleton (Table 2). Nine MSSA isolates belonged to spa-CC037 (26.5%), which harbored six spa types. We performed MLST for each MSSA isolate of the six spa types of spa-CC037. Among them, five showed ST30 or its SLV, and one showed ST239. Twelve MSSA isolates belonged to spa-CC084, and six MSSA isolates belonged to spa-CC008. Each two MSSA isolates of spa-CC084 represented ST188 and ST513, which shared the same alleles in three loci: glpF, gmk, and pta. MSSA isolates of the spa-CC008 were consistent with ST8 and its double locus variant, ST630. Only one MSSA isolate showed a spa type in spa-CC002, TJMBMDMGMK, which is the most prevalent clone among MRSA isolates.

Prevalence of enterotoxin genes and pvl gene

Among the 70 S. aureus isolates obtained from blood, 50 of the isolates (71.4%) harbored at least one enterotoxin gene (Table 3, 4). The most prevalent enterotoxin gene was seg (58.6%), followed by the sei (55.7%), tst (44.3%), and sec (34.3%) genes. The see and pvl genes were not detected in any of the S. aureus isolates obtained from blood. MRSA and MSSA isolates from blood showed different prevalences of enterotoxin genes. Whereas the majority of MRSA isolates from blood contained sec (66.7%), seg (86.1%), sei (83.3%), and tst (72.2%) genes, less than 30% of the MSSA isolates from blood were positive for those genes. With regard solely to the sea gene, the MSSA isolates showed significantly higher prevalence than was observed in the MRSA isolates (P=0.041).

Table 3

Prevalence of enterotoxin genes and pvl gene

MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus.

Table 4

Combination of enterotoxin genes in S. aureus from blood and nasal specimens

Among the 95 S. aureus isolates obtained from nasal specimens, 78 isolates (82.1%) were positive for at least one enterotoxin gene. The most prevalent enterotoxin gene in S. aureus isolates from nasal specimens was sei (70.5%), followed by seg (61.1%), tst (52.6%), and sea (47.4%) genes. As compared with the S. aureus isolated from blood, S. aureus isolates from nasal specimens showed significantly higher prevalence of sea and seh genes (P=0.041 and 0.001, respectively) and significantly lower prevalence of seb and sec genes (P=0.031 and <0.001, respectively) (Table 3). No seb, sed, see, and pvl genes were detected in the S. aureus isolates from nasal specimens. By way of contrast with the S. aureus isolates from blood, no significant differences in prevalence were detected between MRSA and MSSA isolates.

Among S. aureus isolates from blood, the most prevalent combination of enterotoxin genes was a combination of sec-seg-sei-tst (21 isolates, 30.0%), followed by combinations of seg-sei (6 isolates, 8.6%) and sea-sek (6 isolates, 8.6%) (Table 4). However, only one isolate from the nasal specimens showed combination of sec-seg-sei-tst. The most prevalent combinations of enterotoxin genes in S. aureus isolates from nasal specimens were sea-seg-sei-tst (12 isolates, 12.6%), sea-seg-seh-sei-tst (11 isolates, 11.6%), and seg-sei (10 isolates, 10.5%) (Table 4).

Distribution of enterotoxin genes by genotypes

In this analysis, we included all S. aureus isolates obtained from blood and nasal specimens (Table 5). Among the 29 isolates of spa-CC002, most contained sec (89.7%), seg (93.1%), sei (89.7%), and tst (89.7%) genes. Overall, 22 isolates of the spa-CC002 (75.9%) showed a combination of sec, seg, sei, and tst genes. It is worthy of note that the sec gene is found only in S. aureus isolates of spa-CC002. Only one isolate (3.5%) of spa-CC002 harbored no enterotoxin genes.

Table 5

Distribution of enterotoxin genes by spa clonal complexes (spa-CC)

Among the 48 isolates of spa-CC037, sea, seg, sei, and tst genes were found in more than 50%. The distribution of enterotoxin genes in S. aureus isolates differed with respect to their sources. Whereas the sea and sek genes were detected in 92.9% and 42.9% of the isolates from blood, they were positive for 61.8% and 3.0% of the isolates from nasal specimens, respectively. However, S. aureus isolates from blood contained seh and sei genes at rates of 7.2% and 50.0%, but isolates from nasal specimens contained those genes at rates of 26.5% and 91.2%, respectively. Four isolates (8.3%) were found to be negative for all enterotoxin genes. Isolates of spa-CC008 harbored few enterotoxin genes.

DISCUSSION

According to the results of genotypic analysis using spa typing and MLST, MRSA strains from blood have different genetic backgrounds from MSSA strains. Whereas the majority of MRSA strains were limited to three spa groups, the MSSA strains were dispersed into a variety of groups. In this study, ST5-MRSA-II corresponding to spa-CC002 was the most prevalent MRSA clone that caused bacteremia, a result which was consistent with the result of previous study (23), in which clinical isolates from all types of specimens were included. However, only one MSSA isolate belonged to ST5. spa-CC002 or ST5 was not a principal clone in the S. aureus isolates obtained from nasal specimens (4 out of 95 isolates, 4.2%) (14). spa-CC084, which corresponds to CC72 in MLST, was the second most prevalent genotype in MRSA strains from blood, but was not detected in MSSA strains. This clone was the most prevalent in the MRSA isolates from nasal specimens (9 out of 18 isolates, 50.0%) (14). spa-CC084 was the most prevalent clone in the MSSA strains obtained from blood, and was also frequently detected in the MRSA strains. In addition, it was also the most prevalent in S. aureus isolates obtained from nasal specimens (34 out of 95 isolates, 35.8%) (14). However, it was not detected in MRSA isolates obtained from nasal specimens, that is, all S. aureus isolates of spa-CC037 obtained from nasal specimens were found to be methicillin-susceptible.

Notably, spa-CC037 of MSSA may differ from that of MRSA. The MRSA isolates of spa-CC037 corresponded to ST239 or its single locus variants, which were previously shown (23). However, only some strains of MSSA of spa-CC037 (spa type, WGKAOMQ) might belong to ST239, and the other six MSSA strains of spa-CC037 belonged to ST30 or its single locus variants. Although clones of ST239 and ST30 share the same allele at only one locus of arcC, ST239 is believed to emerge via the incorporation of a large portion of chromosome of ST30 into that of ST8 (24). In this study, spa types of ST8 (YHGFMBQBLO and YGFCBQBLO; spa-CC008) were quite different from those of ST239 (WGKAOMQ and WGKAKAOMQ; spa-CC037). The emergence and molecular evolution of ST8, ST30, and ST239 should be investigated further, because they constitute the prevalent epidemic clones of MRSA worldwide.

The prevalence of enterotoxin genes in S. aureus isolates from blood differed significantly from those obtained from nasal specimens with regard to the sea, seb, sec, and seh genes; whereas the seb and sec genes were present at higher levels in the blood isolates, the sea and seh genes were more frequently detected in the isolates from nasal specimens. Cha et al. (13) previously reported the prevalence of virulence genes in S. aureus isolates associated with staphylococcal food poisoning in Korea. In their study, the sea gene was detected in the majority of isolates (91.9%). In their study, the isolates with the sea gene were distributed in diverse clones, but were primarily detected in ST1, ST30, and ST59. That is, S. aureus strains that cause food poisoning are different clones from those that cause bacteremia or nasal colonization, according to MLST. The seh gene was also detected frequently in food poisoning-associated S. aureus isolates (198/332 isolates, 59.6%) (13). The seh gene was only infrequently detected in clinical isolates of S. aureus from Jordan (25). As the nasal carriage of S. aureus strains can be a cause of the contamination of manually handled food and transmission among humans, the relatively high presence of sea and seh genes in S. aureus isolates from nasal specimens constitutes a continuing matter of concern (26).

The tst gene encodes for TSST-1, which is associated with staphylococcal toxic shock syndrome (TSS) and is considered to be the cause of nearly all cases of menstrual TSS, and of at least 50% of nonmenstrual cases (11). As a whole, there was not significant difference of prevalence of tst gene between blood and nasal isolates. It is particularly noteworthy that the tst gene was highly prevalent in MRSA isolates from blood (72.2%). However, it was not significant as compared with MRSA isolates from nasal carriage (50.0%). Contrast to MRSA isolates, MSSA isolates from nasal carriage showed higher prevalence of tst gene than those from blood (53.3% vs. 14.7%). As has been reported by Chini et al. (27), the tst gene is considered to coexist with the enterotoxin gene cluster (egc), which includes seg and sei. However, among 97 isolates harboring both the seg and sei genes, 70 isolates (72.2%) were positive for the tst gene in our study.

The sec gene was frequently detected in S. aureus isolates from blood rather than from nasal specimens, which is a significant finding. The sec gene has been detected in only one isolate among food poisoning-associated isolates (13). This virulence gene was present exclusively in the MRSA isolates of a particular clone, spa-CC002 corresponding to ST5 in this study. A previous study in Korea reported that sec gene was detected mostly in clinical MRSA isolates with SCCmec type II, which may be correlated with ST5 in Korea. This clone, the most prevalent one in Korean hospitals (6, 7, 13, 23, 28), is characterized by the sec-seg-sei-tst gene combination. In general, prevalent S. aureus clones tended to harbor more virulence genes than minor clones. It has been reported that successful S. aureus clones usually harbor the seg and sei genes (29). This suggests that repertoires of virulence genes may be coadapted to the genetic background, and that they may contribute to the biological fitness of the lineages into certain environments (9, 29). It is currently known that only several epidemic MRSA clones (such as CC5, CC8, CC22, CC30, and CC45) have spread worldwide (30, 31).

Despite the general relationships existing between genetic backgrounds and repertoires of virulence genes, there is some evidence to suggest considerable transmission of virulence genes on a background of clonality (10). Variations in virulence genes within a certain clone are suggestive of a common horizontal transfer of such genes. For example, the sea, seb, and sed genes existed solely in some isolates of spa-CC002. In addition, the sec, seg, sei, and tst genes existed in the majority of isolates of spa-CC002, but two (seg) or three (sec, sei, and tst) isolates did not harbor them.

In short, S. aureus isolates from blood have genotypes different from those obtained from nasal specimens. In addition, the genetic background of MRSA isolates that cause bacteremia was different from those of the MSSA isolates. S. aureus isolates from blood showed different prevalences in virulence genes from those obtained from nasal specimens. Some prevalent clones have characteristic virulence gene repertoires. The possession of virulence genes may affect the biological fitness of S. aureus, in addition to their pathogenesis, as suggested by van Belkum et al. (29). However, S. aureus clones causing bacteremia may differ from those causing nasal colonizer (7, 14, 32). The clonal difference of S. aureus isolates between two groups could be due to demographic differences and may indicate the different way of acquisitions of virulence genes. Thus, it should also be mentioned that this study has the limitation that the population of nasal colonizer is different from bacteremic patients.