Changing characteristics of S. aureus bacteremia caused by PVL-negative, MRSA strain over 11 years

Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) has emerged as an important cause of infection. We conducted a longitudinal study to evaluate changes in clinical and microbiological characteristics as well as outcomes of sequence type (ST) 72 MRSA bacteremia. We reviewed adult patients enrolled in a prospective cohort with ST72 MRSA bacteremia from August 2008 to December 2018 at Asan Medical Center, Seoul, South Korea. Changes in clinical characteristics, outcomes, and microbiological characteristics of patients over time were evaluated. Generalized linear and linear regression models were used to evaluate changes. Of the 1,760 isolates, 915 (62%) were MRSA bacteremia and 292 (31.9%) were ST72 MRSA. During the study period, the relative risk (RR) of MRSA bacteremia decreased annually by 3.7%; however, among MRSA bacteremia, RR of ST72 MRSA increased annually by 8.5%. Vancomycin minimum inhibitory concentration (MIC) decreased over the study period. Metastatic infection, persistent bacteremia, and recurrence of bacteremia within 12 weeks decreased significantly. There were no significant changes in 30-d and 12-week mortality. Antibiotic susceptibility of ST72 MRSA was evaluated, and the resistance rate to erythromycin decreased significantly. ST72 MRSA incidence increased annually; its vancomycin MIC and erythromycin resistance rate decreased over the 11 years.

Microbiology analysis. The first bloodstream isolates from each patient were used for microbiological and molecular assessments. All isolates were confirmed as MRSA by polymerase chain reaction detection of the mecA gene, and multi-locus sequence typing was performed as described elsewhere 18 . The vancomycin minimum inhibitory concentrations (MICs) were determined using a broth microdilution method based on the manufacturer's protocol. Antimicrobial susceptibilities were tested by standard techniques according to the Clinical and Laboratory Standards Institute.
The staphylococcal cassette chromosome mec (SCCmec) type, agr functionality, and spa sequence typing were identified using previously described methods 19,20 . The agr disfunction was determined by analyzing δ-hemolysin activity as described previously 21 . The assignment of spa type was performed using BIONUMERICS (APPLIED MATHS).
Statistical analysis. All variables for each year's clinical characteristics, agr functionality, SCCmec type, and spa type were summarized, and annual changes were analyzed using a generalized linear model. Continuous variables were analyzed using a linear regression model. We compared patients in the first 3 years (2008-2010) and in the final 3 years (2016-2018). Categorical variables were compared using the Chi-squared test or Fisher's exact test, as appropriate. Continuous variables were compared using Student's t test and the Mann-Whitney U-test. A two-tailed p value less than 0.05 was considered significant. All statistical analyses were performed using SPSS software, version 21.0 (IBM Corp., Armonk, NY, USA). Ethics approval. This study was approved by the hospital ethics committee.

Results
Annual change in clinical characteristics and outcomes of ST72 MRSA. From July 2008 to December 2018, there were 1,760 episodes of SAB, and 915 (62%) were MRSA bacteremia. Among the 915 MRSA isolates, 292 (31.9%) were ST72 MRSA. During the study period, the relative risk (RR) of MRSA bacteremia decreased annually by 3.7% (p < 0.01) and among MRSA bacteremia, the RR of ST72 MRSA increased annually by 8.5% (p < 0.01) (Fig. 1a). Locations of acquisition of ST72 MRSA over an 11-year period are shown in Fig. 1b (Fig. 2a).
Annual changes in microbiological characteristics and antibiotic susceptibility of ST72 MRSA. The mean vancomycin MIC was 1.08 mg/L, and this decreased annually by 0.01 (R 2 = 0.025; p = 0.01).  (Fig. 3). The proportions of agr dysfunction, SCCmec type, and spa type did not differ over the study period. Antibiotic susceptibility tests were conducted on 10 antibiotics. There was no change in the resistance rate to most antibiotics, but that to erythromycin decreased significantly during the study period (RR 0.89; p < 0.01).  Table 2. There were no significant differences in the location of acquisition, underlying disease, and characteristics of infection between the two groups. The patients in the first 3 years were more likely to have metastatic infection (    Annual changes in clinical characteristics and outcomes of patients with community-onset vs. hospital-acquired infection. We analyzed patients with community-onset and HA infection (Tables 3   and 4). Metastatic infection (RR 0.89; p = 0.04) and recurrent bacteremia (RR 0.64; p = 0.01) decreased in patients

Discussion
In this study, we evaluated the clinical, microbiological, and genotypic changes in ST72 MRSA bacteremia over 11 years. The RR of MRSA bacteremia decreased and among these, the RR of ST72 MRSA increased during the study period. S. aureus pneumonia increased. Metastatic infection, persistent bacteremia, and recurrent bacteremia decreased during the study period. The rate of erythromycin resistance decreased significantly over the 11 years. PVL-negative ST72 MRSA is a major CA-MRSA strain in South Korea and is an important cause of nosocomial infection. The incidence of infections caused by ST72 MRSA is increasing in both community and healthcare settings. Kim et al. 6 23 reported that ST72-SCCmecIV was associated with reduced mortality compared with ST5-SCCmecII. In this study, 30-d and 12-week mortality did not differ over the study period. CVC-related infection, bone and joint infection, and infective endocarditis have been suggested as risk factors for bacteremia complication [26][27][28] . In the present study, there were no changes in CVC-related infection, bone and joint infection, and infective endocarditis, but the incidence of bacteremia complications, such as metastatic infection, persistent bacteremia, and recurrent bacteremia, decreased. In subgroup analysis, metastatic infection and recurrent bacteremia only decreased in community-onset infection. These results suggest changes in community-onset MRSA, and further research is warranted.
A high vancomycin MIC is associated with worse clinical outcomes and treatment failure 29-31 . Rybak et al. 32 reported an increase in the vancomycin MIC of S. aureus from 1986 to 2007. The cell wall-active antibiotics inhibit bacterial growth by inhibiting peptidoglycan biosynthesis 33 . Molecular events of bacteria occur after treatment with cell wall-active antibiotics 34,35 . S. aureus activates a protective cell wall stress stimulons in response to the inhibition of cell envelope damage or cell wall synthesis caused by several antibiotics 34,36,37 . These factors probably increase the vancomycin MIC. In this study, the vancomycin MIC decreased over the 11-year study period, and the change was significant in HA infection. This may be due to the difference in the study period or study lesions. Additionally, there may be changes in microbiological and genomic factors of S. aureus. Further studies are necessary to assess changes in the microbiological and genomic factors of S. aureus that are associated with vancomycin MIC.
In Korea, resistance rates to clindamycin, erythromycin, ciprofloxacin, and gentamicin were lower in ST72-SCCmecIV MRSA than in ST5-SCCmecII MRSA strain 6,9,23 . The resistance rate of ST72 MRSA to erythromycin was from 35.4% to 60.0% 9,23 . In this study, the erythromycin resistance rate of ST72 MRSA decreased over the 11-year study period significantly. The resistance rate of ST72 MRSA in the final 3 years was significantly lower than ST72 MRSA in the first 3 years (15.9% vs. 42.0%, p < 0.01). In subgroup analysis, the erythromycin resistance rate of ST72 MRSA decreased only in community-onset infection. Several mechanisms cause erythromycin resistance to S. aureus; the presence of multicomponent macrolide efflux pumps (msrA, msrB) and enzymatic modification (EreA, EreB) of the antibiotics by enzymes [38][39][40][41] . In addition, macrolide resistance genes are present in erythromycin-resistant S. aureus 42 . Reduced use of macrolides or altered resistance genes of ST72 MRSA may contribute to decreased erythromycin resistance. Further studies are needed regarding changes in the mechanisms of erythromycin resistance. www.nature.com/scientificreports/     www.nature.com/scientificreports/ This study has several limitations. First, this was a single-center study and our findings cannot be generalized to South Korea. Second, we evaluated ST72 MRSA types only, but analysis with other MRSA types, such as ST5 and ST239, may be helpful for understanding changes in S. aureus in Korea. However, this was the first large-scale study to analyze longitudinal changes in ST72 MRSA in Korea. This study will benefit future studies in their analysis of changes in S. aureus in community and nosocomial environments.
In conclusion, ST72 MRSA is an important pathogen in both community and nosocomial settings and its incidence increased over the 11-year study period. Bacteremia complication, vancomycin MIC, and resistance rate to erythromycin decreased, and these results suggest changes in the characteristics of ST72 MRSA. Further studies, including genome-wide studies, are needed to understand the reasons for changes of ST72 MRSA.

Data availability
The data of this study are available by contacting the corresponding author upon reasonable request.