Abstract
We describe the genetic and microbiological characteristics of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates with agr dysfunction from a tertiary-care hospital in Korea. Of these, ST5-SCCmec type II-agr group II MRSA isolates, which are known to be prevalent in hospital-acquired infections in Korea, were the most abundant, because of the clonal spread of a specific agr-defective lineage. This finding suggests that the loss of agr function may confer a potential advantage in a hospital setting. Clonal spread of a specific defective-agr strain was not observed among community-associated MRSA or methicillin-susceptible S. aureus clones, regardless of community or hospital acquisition of infection. agr-defective clones, including ST5 and ST239 MRSA, were enriched for heteroresistant vancomycin-intermediate S. aureus.
TEXT
The accessory gene regulator (agr) is a global regulator that controls staphylococcal virulence factors and other accessory gene functions (1). Genetically engineered mutants of Staphylococcus aureus with defective agr have substantially reduced virulence (2). Notwithstanding the importance of agr for staphylococcal virulence, several reports suggest that agr dysfunction confers potential advantages in a health care setting and a survival advantage under vancomycin selection pressure (3, 4). agr dysfunction has also been associated with persistent bacteremia (5). In this study, we compare the prevalence of agr dysfunction within and between methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) populations and describe the genetic and microbiological characteristics of agr-defective strains in Korea.
Between August 2008 and September 2011, 639 nonduplicate, consecutive S. aureus isolates were prospectively collected from patients with bacteremia at the Asan Medical Center, a 2,700-bed tertiary-care teaching hospital in Seoul, South Korea. Of 639 S. aureus isolates, 407 (64%) were MRSA and 232 (36%) were MSSA. We used δ-hemolysin activity to determine agr functionality as described previously (6) because agr dysfunction results in a defect in δ-hemolysin production. The agr genotype was determined by using a rapid, PCR-based assay that has been described previously (7). Multilocus sequence type (MLST) determination and staphylococcal cassette chromosome mec (SCCmec) typing were performed as previously described (8, 9).
One hundred twenty-three (30%) agr group I, 276 (68%) agr group II, and 3 (1%) agr group III MRSA isolates were studied. The most frequent SCCmec type was II (282 isolates, 70%) followed by IV (97 isolates, 24%). Among the MRSA isolates, the most frequent sequence types (STs) were ST5 (276, 70%) and ST72 (96, 24%). The most common MRSA clone was ST5-SCCmec type II-agr group II (274/407 [67%]), followed by ST72-SCCmec type IV-agr group I (93/407 [23%]). One hundred thirty-three (57%) agr group I, 32 (14%) agr group II, 52 (22%) agr group III, and 6 (3%) agr group IV MSSA isolates were studied. Twenty-seven different STs were found among the MSSA isolates. The major STs were ST188 (46 isolates, 20%), ST72 (29, 13%), ST1 (27, 12%), ST30 (25, 11%), ST6 (19, 8%), and ST5 (16, 7%). Similar to the MRSA isolates, each ST in the MSSA isolates had a particular agr genotype.
Table 1 shows the differences in genotypic characteristics between S. aureus bloodstream isolates with functional agr and isolates with dysfunctional agr. Of 407 MRSA isolates, 302 (74%) had dysfunctional agr (nonhemolytic strains) and 105 (26%) had functional agr (hemolytic strains). Strikingly, nearly all of the MRSA isolates with agr dysfunction were ST5 (270/302 [89%]) and nearly all of the ST5 isolates had agr dysfunction (270/276 [98%]), raising the possibility of clonal spread. In contrast to the results for the MRSA isolates, only 13% (29/232) of the MSSA isolates had agr dysfunction and no predominant STs were found in MSSA isolates with agr dysfunction.
Table 1.
Genotypic characteristics of S. aureus bloodstream isolates stratified by agr functionality
| Genotype | No. (%) of isolates |
P valuea | |
|---|---|---|---|
| Dysfunctional agr | Functional agr | ||
| MRSA (n = 407) | 302 | 105 | |
| agr genotype | <0.001 | ||
| I | 29 (9.6) | 94 (89.5) | |
| II | 270 (89.4) | 6 (5.7) | |
| III | 2 (0.7) | 1 (1.0) | |
| ND | 1 (0.3) | 4 (3.8) | |
| SCCmec type | <0.001 | ||
| I | 2 (0.7) | 3 (2.9) | |
| II | 274 (90.7) | 8 (7.6) | |
| III | 15 (5.0) | 2 (1.9) | |
| IV | 10 (3.3) | 87 (82.9) | |
| NDb | 1 (0.3) | 5 (4.8) | |
| MLST | <0.001 | ||
| ST1 | 2 (1.9) | ||
| ST5 | 270 (89.4) | 6 (5.7) | |
| ST72 | 10 (3.3) | 86 (81.9) | |
| ST239 | 17 (5.6) | 1 (1.0) | |
| Otherc | 5 (1.7) | 10 (9.5) | |
| MSSA (n = 232) | 29 | 203 | |
| agr genotype | 0.024 | ||
| I | 10 (34.5) | 123 (60.6) | |
| II | 6 (20.7) | 26 (12.8) | |
| III | 10 (34.5) | 42 (20.7) | |
| IV | 6 (2.9) | ||
| ND | 3 (10.3) | 6 (2.9) | |
| MLST | 0.030 | ||
| ST1 | 3 (10.3) | 24 (11.8) | |
| ST5 | 5 (17.2) | 11 (5.4) | |
| ST30 | 7 (24.1) | 18 (8.9) | |
| ST72 | 3 (10.3) | 26 (12.8) | |
| ST188 | 4 (13.8) | 42 (20.7) | |
| Otherd | 7 (24.1) | 82 (40.4) | |
P values comparing the values for the two groups were determined with a two-sided Fisher exact test.
ND, not determined.
The other MLSTs were ST254 (2 isolates), ST291 (1 isolates), and ST89 (2 isolates) in the dysfunctional-agr group and ST101 (2 isolates), ST188 (1 isolate), ST199 (1 isolate), ST254 (2 isolates), ST6 (1 isolate), ST8 (2 isolates), and ND (1 isolate) in the functional-agr group.
The other MLSTs were ST6 (1 isolate), ST8 (1 isolate), ST59 (1 isolate), ST101 (1 isolate), ST378 (1 isolate), ST1153 (1 isolate), and ST1156 (1 isolate) in the dysfunctional-agr group and ST6 (18 isolates), ST8 (2 isolates), ST15 (11 isolates), ST25 (1 isolate), ST45 (1 isolate), ST59 (3 isolates), ST96 (1 isolate), ST97 (7 isolates), ST101 (3 isolates), ST121 (7 isolates), ST217 (1 isolate), ST513 (10 isolates), ST573 (1 isolate), ST580 (1 isolate), ST587 (3 isolates), ST630 (9 isolates), ST883 (1 isolate), ST1821 (1 isolate), and ST2238 (1 isolate) in the functional-agr group.
The analysis of combined genotypes of S. aureus isolates according to agr functionality showed that whereas the ST5-SCCmec type II-agr group II genotype was the most prevalent among MRSA isolates with agr dysfunction, the ST72-SCCmec type IV-agr group I genotype predominated among MRSA isolates with functional agr (Table 2). In addition, nearly all of the MRSA isolates (15/16) with the ST239-SCCmec type III-agr group I genotype had agr dysfunction. Among the MSSA isolates, predominant clones were not found in either the dysfunctional-agr or the functional-agr group. However, one-third of the MSSA isolates with ST5 agr group II and ST30 agr group III genotype had agr dysfunction. In Korea, ST5-SCCmec type II-agr group II and ST239-SCCmec type III-agr group I MRSA strains account for the majority of hospital-acquired MRSA infections (10, 11). The major types in community-associated MRSA infections are ST72-SCCmec type IV-agr group I and ST1-SCCmec type IV-agr group III (11, 12). Our results suggest that ST5-SCCmec type II-agr group II and ST239-SCCmec type III-agr group I clones with agr dysfunction were clonally spread in the hospital.
Table 2.
Distribution of combined genotypes in S. aureus bloodstream isolates stratified by agr functionality
| Genotype | No. of isolates |
|
|---|---|---|
| Dysfunctional agr | Functional agr | |
| MRSA (n = 407) | 302 | 105 |
| ST5-SCCmec II-agr II | 270 | 4 |
| ST72-SCCmec IV-agr I | 10 | 83 |
| ST89-SCCmec II-agr III | 2 | |
| ST239-SCCmec III-agr I | 15 | 1 |
| ST239-SCCmec II-agr I | 2 | |
| ST254-SCCmec I-agr I | 1 | 2 |
| Other | 2 | 15 |
| MSSA (n = 232) | 29 | 203 |
| ST1-agr III | 2 | 24 |
| ST5-agr II | 5 | 11 |
| ST6-agr I | 18 | |
| ST30-agr III | 7 | 17 |
| ST72-agr I | 3 | 25 |
| ST188-agr I | 4 | 41 |
| Other | 8 | 67 |
Sequencing of the 3′ end of agrA was performed as described previously (13). Among 270 ST5-SCCmec type II-agr group II isolates with agr dysfunction, 238 (88%) had a nine-adenine mutation (referred to as agrA-9A), 14 (5%) had an agrA-8A mutation, and 18 (7%) had wild-type agrA-7A. This suggests that there was clonal spread of at least two ST5 lineages. The Mu3 and Mu50 strains of S. aureus have this agrA-9A mutation without any other mutations in the agr locus (14). Given that both of these strains are nonhemolytic (agr dysfunctional), the agrA-9A mutation may be important for agr functionality. The agrA-8A mutation was known as a cause of agr dysfunction (6, 13). In our ST5 MRSA isolates, the agrA-9A or agrA-8A mutation would be a main cause of agr dysfunction. All ST239-SCCmec type III-agr group I isolates with agr dysfunction had wild-type agrA-7A. Most of agr-defective MSSA isolates (25/29 [83%]) had agrA-7A. agr dysfunction in these isolates would result from various mutations in other regions of the agr locus (3). Shopsin et al. (15) reported agr functional analysis with S. aureus isolates recovered from colonized individuals and suggested that isolates with agr dysfunction were capable of transmission. They also suggested that although agr dysfunction is adaptive for survival in the infected host, it appears to confer only a short-term survival advantage (3). However, in ST5-SCCmec type II-agr group II and ST239-SCCmec type III-agr group I MRSA, agr dysfunction may confer long-term advantages in transmission and survival in the hospital environment. Since there were no case clusters or outbreaks of these clones in our hospital during the study period, this suggests that those were endemic. The predominance of the ST5-SCCmec type II-agr group II clone among MRSA isolates with agr dysfunction needs to be further verified in a multicenter study in Korea.
Previous studies suggested that agr dysfunction in MRSA is associated with attenuated vancomycin activity, vancomycin heteroresistance, and an increased proclivity toward vancomycin intermediate resistance (4, 14, 16). To investigate the relationships between agr functionality and vancomycin resistance, we estimated the vancomycin susceptibility by using the Etest (AB Biodisk, Solna, Sweden) and the heteroresistant vancomycin-intermediate S. aureus (hVISA) phenotype by using population analysis profiling of MRSA isolates as previously described (17). An isolate was identified as hVISA if the ratio of the AUC of the test isolate to that of the reference strain (Mu3; ATCC 700698) was ≥0.9. The vancomycin MICs for MRSA isolates ranged from 0.5 to 3 μg/ml. Of 407 MRSA isolates, 127 (31%) were hVISA. The most common STs of hVISA isolates were ST5 (72%), followed by ST72 (13%) and ST239 (12%). Most of the hVISA isolates (107/127, 84%) were agr defective. The hVISA phenotype was significantly more common in isolates with dysfunctional agr than in those with functional agr (107/302 [35%] versus 20/105 [19%], P < 0.002) (Table 3). However, agr dysfunction was not significantly associated with high vancomycin MICs. Among 107 hVISA isolates with agr dysfunction, 91 (85%) were ST5-SCCmec type II-agr group II, 12 (11%) were ST239-SCCmec type III-agr group I, 2 (2%) were ST239-SCCmec type II-agr group I, 1 (1%) was ST254-SCCmec type I-agr group I, and 1 (1%) was ST72-SCCmec type IV-agr group I. The higher prevalence of hVISA in the present study (31%) than in previous studies (overall, 1.3%) (18) could be explained by the clonal spread of specific agr-defective strains (ST5 and ST239 clones) with the hVISA phenotype.
Table 3.
Microbiological characteristics and outcomes of S. aureus bacteremia stratified by agr functionality
| Genotype | No. (%) of isolates |
P value | |
|---|---|---|---|
| Dysfunctional agr | Functional agr | ||
| MRSA (n = 407) | 302 | 105 | |
| Vancomycin MIC (Etest) results: | 0.068 | ||
| ≤1 | 97 (32.1) | 21 (20.0) | |
| 1.5 | 135 (44.7) | 57 (54.3) | |
| 2 | 62 (20.5) | 26 (24.8) | |
| 3 | 8 (2.6) | 1 (1.0) | |
| hVISA phenotype | 107 (35.4) | 20 (19.0) | 0.002 |
| Persistent bacteremia | 79 (26.2) | 19 (18.1) | 0.096 |
| MSSA (n = 232) | 29 | 203 | |
| Persistent bacteremia | 14 (6.9) | 0.226 | |
We also investigated the association of agr dysfunction with persistent bacteremia (Table 3). Persistent bacteremia was defined as bacteremia for ≥7 days while the patient was receiving appropriate antibiotic therapy (5, 19) and was compared with nonpersistent bacteremia with regard to agr functionality. Of 407 MRSA isolates, 98 (24%) caused persistent bacteremia. Of 232 MSSA isolates, 14 (6%) caused persistent bacteremia. Among the MRSA isolates, agr dysfunction was associated with a trend toward persistent bacteremia (P = 0.096). However, agr dysfunction was not significantly associated with persistent bacteremia among the MSSA isolates (P = 0.226).
In conclusion, we showed that agr dysfunction was common among the nosocomial MRSA isolates in our institution because of the clonal spread of specific clones with agr dysfunction, ST5-SCCmec type II-agr group II and ST239-SCCmec type III-agr group I. This suggests that agr dysfunction may confer a particular advantage on these clones in hospital settings. In addition, agr dysfunction was significantly associated with hVISA.
ACKNOWLEDGMENTS
This research was supported by a Bio R&D program through the National Research Foundation of Korea, which is funded by the Ministry of Education, Science and Technology (grants 2009-0091639, 2009-0091640, and 2009-0091641).
Footnotes
Published ahead of print 17 December 2012
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