Antimicrobial Resistance and Molecular Epidemiology of Staphylococcus aureus Causing Bloodstream Infections at Ruijin Hospital in Shanghai from 2013 to 2018

Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA) is an important issue associated with significant morbidity and mortality and well known as a predominant pathogen causing bloodstream infection (BSIs) globally. To estimate the antibiotic resistance and molecular characteristics of S. aureus causing BSIs in Shanghai, 120 S. aureus isolates (20 isolates each year) from the patients with S. aureus BSIs from 2013 to 2018 were randomly selected and enrolled in this study. Fifty-three (44.2%) MRSA isolates were determined, and no isolate was found resistant to vancomycin, daptomycin, synercid, linezolid and ceftaroline. The toxin genes tst, sec, seg and sei were found more frequently among MRSA isolates compared with MSSA isolates (all P < 0.0001). Twenty-nine sequence types (STs) were identified, and ST5 (23.3%) was the most common ST, followed by ST398 (11.7%) and ST764 (10.0%). SCCmec II (73.6%) was the most frequent SCCmec type among MRSA isolates. The dominant clonal complexes (CCs) were CC5 (ST5, ST764, ST965 and ST3066; 36.7%) and the livestock-associated clone CC398 (ST398, 11.7%). MRSA-CC5 was the predominant CC among MRSA isolates (37/53, 69.8%), and CC5-II MRSA was found in 34 isolates accounting for 91.9% (34/37) among CC5 MRSA isolates. In addition, all 29 tst-positive MRSA isolates were CC5-MRSA as well. Our study provided the properties and genotypes of S. aureus causing BSIs at Ruijin Hospital in Shanghai from 2013 to 2018, and might suggest of value clues for the further study insights into pathogenic mechanisms intrinsically referring to the development of human-adapted S. aureus clones and their diffusions.

Toxin genes detection. A total of 13 significant toxin genes clinically including lukS/F-PV, tst, eta, etb, sea-see and seg-sej were detected on all 120 S. aureus isolates in this study by polymerase chain reaction (PCR) as described previously 13 . lukS/F-PV encodes Panton-Valentine leukocidin; tst encodes toxic shock syndrome toxin 1; eta and etb encodes exfoliative toxin A and B; sea-see and seg-sej encodes staphylococcal enterotoxins SEA-SEE and SEG-SEJ. Molecular typing. Multilocus sequence typing (MLST), spa typing and agr typing were performed on all 120 S. aureus isolates according to the guidelines on the websites (https://pubmlst.org/, http://spa.ridom.de/ index.shtml) and other published documents and researches 13,14 . mecA detection was performed on all 120 S. aureus isolates as well to confirm the existence of MRSA. SCCmec types of MRSA were determined by the previous method as described 15 . The gene blaZ, which produces beta-lactamase and inactivates penicillin by hydrolyzing the beta-lactam ring, was detected on all 120 S. aureus isolates 16 . Statistical analysis. The chi-square or Fisher's exact test was used for statistical analysis as appropriate, and a two-sided P value of <0.05 was considered for statistical significance. All statistical analysis in this study was conducted by the software package SAS 8.2 (SAS Institute Inc., Cary, NC, USA).

Results
clinical data. From January 2013 to December 2018, the median age of patients with SAB in this study was 59 years (range: 7 months-97 years; interquartile range: 44-69 years), and the sex distribution (male/ female) was 67.5%/32.5%. The mortality of patients with SAB was 25.8%, while 15 (12.5%) patients were transferred with unknown outcomes.
The incidence of S. aureus accounting for BSIs was 7.4% at Ruijin Hospital in Shanghai from August 2015 to December 2018, for the system transferring in 2015 and data missing as described in materials and methods.
Antimicrobial resistance. Fifty-three (44.2%) S. aureus isolates were confirmed as MRSA in this study, and all 53 MRSA isolates were mecA-positive. We did not discover any isolate resistant to vancomycin, daptomycin, synercid, linezolid and ceftaroline. No isolate was found showing a reduced vancomycin susceptibility or intermediate to vancomycin among the 120 S. aureus isolates in this study (supplementary information). The resistance rates of antibiotics tested for overall 120 S. aureus isolates from 2013 to 2018 were presented in Table 1. All 53 MRSA isolates detected in the study were resistant to penicillin, and 51 (51/67, 76.1%) MSSA isolates were resistant to penicillin. Sixty-eight (56.7%) S. aureus isolates including 17 MRSA and 51 MSSA isolates were detected blaZ-positive, and all 51 MSSA isolates resistant to penicillin were blaZ-positive as well. Forty-six (46/53, 86.8%) MRSA isolates were observed showing multi-drug resistance in this study. Virulence factors. The toxin genes etb and see were not discovered among all S. aureus isolates in this study.
The seg was found most frequently among the toxin genes screened, occurring in 60 isolates (50.0%) as presented in Table 2. The tst, sec, seg and sei were found more frequently among MRSA isolates compared with MSSA isolates (all P < 0.0001). However, sed was observed more frequently among MSSA isolates (P = 0.0254), and sed was detected only among eight MSSA isolates as shown in Table 2. Besides, sej was found only among four MSSA isolates, but there was no significant difference statistically between MSSA and MRSA isolates (P = 0.1946).

Discussion
S. aureus or MRSA is an important pathogen and frequent cause of invasive infections as well as bloodstream infections around the world. In this study, SAB accounted for 7.4% of BSIs cases in Shanghai from 2013 to 2018, much lower than that in a regional burn center in Jiangxi province in China, which is 25.9% from 2012 to 2016 8 . Coincidentally, in the United States, the incidence of MRSA BSIs in hospitals and communities dropped off 74% and 40% respectively from 2005 to 2016 17 . According to the latest data provided by the European Antimicrobial Resistance Surveillance Network (EARSNet) (2013-2016), more than a third of countries with low and high MRSA prevalence have reported significantly decreasing trends among bloodstream infections, and the population-weighted average MRSA BSIs percentage has dropped from 18.1% in 2013 to 13.7% in 2016 18 . The exact reasons for this specific decline among MRSA BSIs are not fully understood. However, in despite of these positive developments, S. aureus or MRSA still remains a priority for public health in Europe, with 10 of 30 countries reporting prevalence rates of MRSA > 25%, including Greece 19 .
The mortality rate of S. aureus BSIs in Shanghai was 25.8% from 2013 to 2018 in this study, similar to that reported in other published researches of adult patient with S. aureus BSIs (around 20-30%) in other countries 3,5,20 . However, it was much higher than that among infants with S. aureus BSIs both in Europe and USA which have shown overall mortality of 6.4-16% 7,21,22 . The prevalence of methicillin resistance among S. aureus isolates in Hong Kong has risen to >50% while the mortality rates of MRSA bloodstream infections are close to one-third as reported 23 . The proportion of MRSA among S. aureus BSIs in Shanghai was 44.2% in this study. Nevertheless, methicillin resistance is always significantly associated with higher mortality as well as comorbid conditions, intensive care unit admission, and prior exposure to antibiotics 24 . In addition, chronic lung disease, previous hospitalization and older patients (>79 years) are related with increased mortality as well 23 . The mortality in MSSA infections significantly declined and the average time to anti-staphylococcal therapy in MSSA infections decreased even though the mortality in MRSA infections was unchanged 25 . The declining mortality in MSSA infections might be related to the reduction in the duration of targeted therapy. These results emphasize the potential for rapid diagnostics and early optimization of treatment to impact outcomes in MSSA bacteremia. Nevertheless, it was revealed that the treatment failure rate of complicated MRSA bloodstream infections was as high as 40% 26 . MRSA might still need more attention when the patient was determined with MRSA bloodstream infections.
CC5 (S. aureus: 44/120, 36.7%; MRSA: 37/53, 69.8%) was the dominant clone among patients with SAB at Ruijin Hospital in Shanghai from 2013 to 2018 in this study, slightly different from our previous research conducted in Shanghai from 2009 to 2011 that CC8 (ST239) might be more common among patient with SAB before 27 . However, shifts may be occurring in the molecular epidemiology of S. aureus and MRSA among patients with bloodstream infections. As reported from a single-center surveillance in Greece, the widespread HA-MRSA clone ST239-III gradually decreased with the increase of the isolation frequency of these two CCs: HA-MRSA CC5, mostly belonging to ST5-II; and community-associated MRSA (CA-MRSA) CC80, mainly represented by ST80-IV-t044 19 . In this study, CC5-II MRSA was found in 34 isolates accounting for 91.9% (34/37) among CC5 MRSA isolates, suggesting that CC5 MRSA or CC5-II MRSA might be more frequent occurring among patients with MRSA BSIs in Shanghai. It has been suggested that CC5 has become an increasingly frequent cause of MRSA infections as well as bacteremia acquired by hospitalized patients from some other reports and that healthcare exposure is no longer a discriminator of USA100 vs USA300 infections [28][29][30] . An analysis of SAB conducted in  www.nature.com/scientificreports www.nature.com/scientificreports/ Spain over 15 years (2002-2017) revealed that CC5 was the most prevalent CC and the proportion of CC5 among healthcare-associated S. aureus isolates was higher than that among community-associated S. aureus isolates, and CC5 was much more associated with methicillin resistance 4 . Simultaneously, CC5 MRSA was also prevalent in bloodstream infections in nine Latin American countries except in Colombia and Ecuador 29 . Clonal substitution appears to be a common phenomenon, and continuous surveillance is essential to identify molecular epidemiological changes in S. aureus and MRSA.