A 10-year retrospective study of methicillin-resistant Staphylococcus aureus from burn wound infection in southeast China from 2013 to 2022

Background Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most commonly encountered pathogens among burn patients incurring substantial morbidity and mortality. To investigate the epidemiology and features of MRSA in burn wound infections, we conducted a 10-year retrospective study on MRSA isolated from burn patients with burn wound infections from southeast China from 2013 to 2022. Methods One hundred MRSA isolates (10 isolates each year) from burn wound infection among burn patients from 2013 to 2022 were randomly selected and enrolled. In addition to the clinical data of the 100 burn patients, MRSA isolates were characterized by antimicrobial susceptibility testing, detection of toxin genes, and molecular typing. Results The median time from the onset of burns and admission to MRSA detected was 13 and 5 days, respectively. No MRSA isolate was found resistant to quinupristin/dalfopristin, linezolid, and vancomycin. Toxin gene seg was found most frequently (90%) followed by sea (70%) and eta (64%). CC8 (74%), ST239 (70%), and SCCmec III (72%) were the most common CC, ST, and SCCmec types, respectively. Conclusion ST239-III (70%) was the predominant clone found in MRSA from burn wound infection among burn patients in southeast China. ST239-III was less found from 2018 to 2022. A higher diversity of MRSA clones was observed in these recent 5 years than that from 2013 to 2017.


Introduction
Millions of people get burned across the world every year.Burns are one of the most devastating injuries leading to high mortality and morbidity (Mater et al., 2020;Savetamal, 2021).Secondary to the loss of the skin barrier and suppression of the immune system, burn patients are more likely to develop invasive infections, leading to serious complications and even death.As the fourth most common type of trauma across the world, the World Health Organization (WHO) has estimated that ∼11 million burn patients require medical attention and result in ∼265,000 deaths each year (Mater et al., 2020).In China, approximately 5,000-10,000 people per million suffer burn injuries, and 10% of them require medical intervention (Chen et al., 2018).
Staphylococcus aureus is a serious public health concern and an important clinical pathogen that can cause a variety of infections in the skin and soft tissue, respiratory tract, bloodstream, and catheter (Jiang et al., 2018;Mahmoudi et al., 2019).S. aureus, particularly methicillin-resistant S. aureus (MRSA), remains a leading cause of gram-positive burn wound infections worldwide and has been one of the major contributors to increase the morbidity and mortality rates (Branski et al., 2009).Researchers analyzed 29 articles on multidrug-resistant bacteria outbreaks in burn units, and they found that one of the most frequent bacteria was MRSA (Girerd-Genessay et al., 2016).MRSA burn wound infection can potentially cause a fatal sequence of burn wound sepsis, invasive infection, septicemia, multiple organ failure, and even death (Kalligeros et al., 2019).Moreover, MRSA has been the second most common pathogen only to Pseudomonas aeruginosa among burn patients with bacteremia as reported (Kalligeros et al., 2019).
To learn more about the characteristics of MRSA from burn wound infections, we carried out a 10-year retrospective study of MRSA isolated from burn patients with burn wound infections from 2013 to 2022 in Ruijin Hospital in Shanghai, hosting mostly burn patients from southern China.In addition, we would also explore the dynamics of the epidemiology of MRSA in burn wound infections over these 10 years.

Study design
This study was carried out in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, a tertiary teaching hospital in Shanghai, with more than 3,000 beds serving patients from all over China.Department of Burn is one of the best burn units in China providing medical care to burn victims across the country, mainly from southeast China.
Clinical diagnosis of burn wound infections relies on the appearance of the wound, laboratory abnormalities, and wound cultures (D'Abbondanza and Shahrokhi, 2021).Changes in the appearance of the burn wound, including conversion of partialthickness to full-thickness injury, loss of previously viable skin grafts, rapid cellulitis expansion of healthy tissue surrounding the injury, rapid eschar separation, or tissue necrosis, may indicate an acute infection.This typically manifests as some combination of pain, purulence, edema, malodor, or discoloration of the skin graft or donor site.Systemic alterations and laboratory abnormalities may help to diagnose burn wound infections.Leukocytosis (white blood cell count of >10,000 cells/mm 3 ) and/or multiple protein biomarkers including acute-phase reactants (Creactive protein/erythrocyte sedimentation rate and sedimentation rate), anticoagulant factors, cytokines, and tissue injury biomarkers (serum lactate) may or may not be associated with burn wound infections.Surface wound swabs and cultures may help identify the predominant pathogen and can be used as surveillance if there is any clinical concern about changes in the burn wound (Church et al., 2006;D'Abbondanza and Shahrokhi, 2021;Ladhani et al., 2021).Diagnosis of burn wound infections can be complicated and requires comprehensive consideration.The burn patients with burn wound infections enrolled in this study were diagnosed by MRSA-positive cultures of wound swabs combined with clinical signs and symptoms and/or laboratory results mentioned above.
This study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, and the review committee removed the requirement of informed consent for this retrospective study, which just focused on bacteria and did not involve patient interventions.

MRSA isolates
From January 2013 to December 2022, 410 non-repetitive MRSA isolates were collected from cultures of wound swabs from burn patients with burn wound infections in Ruijin Hospital, and 100 MRSA isolates were randomly selected and enrolled (10 isolates each year) using the random number generation function of Microsoft Office Excel 2016 (Microsoft Corporation, Redmond, WA, USA).The initial species were identified by MALDI-TOF mass spectrometry (bioMérieux, Marcy-l'Étoile, France).

Antimicrobial susceptibility testing
The broth microdilution method was used for antimicrobial susceptibility testing in accordance with the guidelines of the Clinical and Laboratory Standards Institute issued in 2023 (CLSI 2023) (Institute CaLS., 2023).Antibiotics selected and tested are presented in Table 2. S. aureus ATCC29213 strain was included as a control strain for the antimicrobial susceptibility testing.

Detection of toxin genes
Thirteen significant toxin genes were detected by polymerase chain reaction (PCR) containing lukS/F-PV (encoding Panton-Valentine leukocidin), tst (encoding toxic shock syndrome toxin 1), eta and etb (encoding exfoliative toxin A and B), and sea-see and seg-sej (encoding staphylococcal enterotoxins SEA-SEE and SEG-SEJ) as described previously (Gu et al., 2020).

Molecular typing
Multilocus sequence typing (MLST), spa typing, SCCmec typing, and agr typing were performed by PCR and/or sequencing as described previously (Gu et al., 2016(Gu et al., , 2020)).mecA detection was performed by PCR to confirm the MRSA strains.

Statistical analysis
The t-test, chi-square test, or Fisher's exact test was performed for statistical analysis as appropriate, and a two-sided P < 0.05 was considered statistically significant.In this study, statistical analysis was performed using the SAS 8.2 software package (SAS Institute Inc., Cary, NC, USA).

Clinical data
From January 2013 to December 2022, the median age of burn patients with MRSA burn wound infection was 37 years (range: 10 months−89 years; interquartile range: 15-54 years), and the sex distribution (male/female) was 75/25% (P < 0.0001).The median time from the onset of burns to receiving medical attention was 1 day (range: 0.5 h−90 days); the median time from admission to detection of MRSA was 5 days (range: 1-54 days), and the median time from the onset of burns to MRSA detected was 13 days (range: 1-98 days).Burn by direct exposure to flame accounted for 45%, and scalding accounted for 50%.Furthermore, the incidence of burn by direct exposure to flame was higher in 2013-2017 (P = 0.0090), and scalding was higher in 2018-2022 (P = 0.0164).More information about the burn patients with MRSA burn wound infection is presented in Table 1.

Antimicrobial resistance
All MRSA isolates in this study have been mecAconfirmed by PCR.No isolate was found resistant to quinupristin/dalfopristin, linezolid, and vancomycin.In addition to cefoxitin, benzylpenicillin, and oxacillin, all the MRSA isolates were resistant to tigecycline as well.The resistance rates of MRSA isolates to gentamicin, ciprofloxacin, levofloxacin, moxifloxacin, tetracycline, and rifampin were significantly higher in 2013-2017 (P < 0.05), as shown in Table 2.However, inducible clindamycin resistance was higher in 2018-2022 (P = 0.0166).

Toxin genes
The seg was found most frequently (90%) followed by sea (70%) and eta (64%), as shown in Table 3.The eta and sea were found more frequently among the MRSA isolates in 2013-2017 (P < 0.0001 and P = 0.0001, respectively).However, seb was observed more frequently among the MRSA isolates in 2018-2022 (P = 0.0196).The toxin gene etb has not been discovered in this study.
Our study is a 10-year retrospective of MRSA from burn wound infections from 2013 to 2022, and we split it into two groups by year as 2013-2017 and 2018-2022 to find out how MRSA might change over time.As shown in Table 1, in the last 5 years in 2018-2022, the incidence of burn by direct exposure to flame was lower and instead the incidence of burn by scalding was higher.More notably, topical antibiotics such as silver sulfadiazine/sulfamylon and mupirocin were more frequently used in 2018-2022, which can prevent infections in burn patients.The microbial load on the open burn wounds and the risk of infections could be greatly reduced by the wide application of effective topical antimicrobials.A series of studies have demonstrated the role of topical antimicrobials in reducing morbidity and mortality among burn patients with severe burn injuries (partial-thickness or full-thickness skin involvement), especially before early excision (Church et al., 2006).
Several studies have shown that partial antibiotic resistance rates of S. aureus and MRSA have declined in recent years (Murray et al., 2009;Jiang et al., 2018;Mahmoudi et al., 2019).In our study, the resistance rates of MRSA to gentamicin, ciprofloxacin, tetracycline, rifampin, and other series antibiotics from 2018 to 2022 were significantly lower than that in 2013-2017.However, inducible clindamycin resistance was higher in 2013-2017.It is still unclear what accounts for this trend of MRSA resistance, or if it is simply due to chance.One research studied vancomycin susceptibility trends of MRSA isolated from burn wounds and found that the proportion of MRSA isolates exhibiting higher vancomycin MICs increased significantly (Zorgani et al., 2015).In our study, the 75% vancomycin MIC value of MRSA isolates was 2 µg/ml; the 21% vancomycin MIC value was 1 µg/ml, and only 4% vancomycin MIC value was ≤0.5 µg/ml.Those high MIC values indicate that vancomycin heteroresistance might emerge, although relevant information was not available.However, there is an urgent need to implement infection control measures to prevent the spread of MRSA, especially in burn patients.
In addition to the high positive rates of enterotoxin genes such as seh (90%) and sea (70%), exfoliative toxin A (ETA) gene eta was also frequently detected accounting for 64%, which was much higher than the S. aureus isolates we studied before from bloodstream infections, skin and soft tissue infections, and colonization (Gu et al., 2015(Gu et al., , 2016(Gu et al., , 2020;;Zhang et al., 2015;He et al., 2021).Staphylococcal exfoliative toxins are responsible for staphylococcal scalded skin syndrome, which is characterized by dehydration, detachment of superficial skin layers, and secondary infections (Ahmad-Mansour et al., 2021).The prevalence of ETA in MRSA and methicillin-susceptible (MSSA) strains does not differ considerably as reported; nevertheless, 4% of MSSA strains carry the eta or etb gene, while approximately 10% of MRSA strains possess the eta gene, according to a recent study (Ahmad-Mansour et al., 2021).However, resistant strains such as MRSA with eta-positive may pose a problem nowadays or in future, especially among burn patients.CC8 (74%) was found as the dominant clonal complex in MRSA burn wound infection among burn patients, and ST239-III was the most common clone with 70%.CC8 or ST239-III is a predominant healthcare-associated MRSA (HA-MRSA) clone across the world including America, Europe, Africa, Middle East, and Asia (Lakhundi and Zhang, 2018).As to burn infections, a study conducted at Jiangxi Burn Center in China reported that SCCmecIII-CC239-t030 was the most common clone (Chen et al., 2018).ST239-SCCmec III/t037 has emerged as the major MRSA clone in burn patients in Iran as reported in 2017 (Goudarzi et al., 2017).It is interesting that ST239-III was less found in recent 5 years in 2018-2022 (46 vs. 24, P < 0.0001), and ST59-I was more found in 2018-2022 (0 vs. 8, P = 0.0099) in our study.The diversity of MRSA clones from 2018 to 2022 was obviously greater than that in 2013-2017 (4 vs. 14, P = 0.0004), as shown in Table 5.It might suggest that besides the predominant clone ST239-III, other MRSA clones should also be watched and monitored among burn patients.In this study, livestockassociated MRSA (LA-MRSA) clone CC398-V occurred in 2017, 2018, 2020, 2021, and 2022 has emerged as a cause of hospital outbreaks, and more discoveries in recent years might indicate its greater ability to spread among patients in hospital along with time.ST398 has been reported as one of the predominant MRSA clones with ST239 in hospitals in India (Patil et al., 2023), and MRSA CC398 has been described in human colonization and infections over these years including our previous studies (Ballhausen et al., 2017;Gu et al., 2020;He et al., 2021;Silva et al., 2022).
MRSA colonization upon admission and during hospitalization among burn patients cannot be ignored, and it could result in multiclonal MRSA outbreaks in the burn units if decolonization protocols are not implemented (Kalligeros et al., 2019;Kim et al., 2019).The implementation of universal decolonization with intranasal mupirocin was effective as reported, and the prevalence of HA-MRSA in burn centers was significantly decreased after the application of the decolonization protocol (Johnson et al., 2016;Kim et al., 2019).Burn patients colonized or infected with MRSA are more likely to be a main reservoir transfer to others; a comprehensive concept to control the spread of all multidrug-resistant pathogens including MRSA is deeply needed in burn units, perhaps temporarily shut down, supplemented by intensive cleaning, are effective measures to stop the transmission events (Baier et al., 2018).
TABLE Clinical data of burn patients with MRSA burn wound infection from to .
TABLE Prevalence of toxin genes among MRSA isolated from burn wound infection from to .
uninfected burn patients.It has been reported that 42-65% of deaths in burn patients can be attributed to infections over the past decades (D'Abbondanza and Shahrokhi, 2021).The bacteria responsible for infection appear in chronological order, changing with the time since the initial burn happened.So far, one of the most common pathogens involved in burn wound infections remains S. aureus ( TABLE Distribution of MRSA clones from burn wound infection from to .