Antibiotic susceptibility and molecular characterization based on whole-genome sequencing of 
Staphylococcus aureus is a gram-positive opportunistic pathogen that primarily colonizes the skin and mucous membranes, affecting approximately 30% of healthy adults . It is a major cause of skin and soft tissue infections, especially in already colonized individuals . Recent studies have shown that S. aureus is the leading cause of bacterial death in 135 countries and is the bacterium responsible for the greatest number of deaths among people older than 15 years worldwide . Although most local skin infections are self-limiting, they sometimes serve as entry points for S. aureus to enter deeper tissues and the bloodstream . S. aureus is one of the major pathogenic bacteria causing bloodstream infections, and invasive S. aureus infection poses a major medical burden and is associated with high mortality (19.1%), a high recurrence rate (9.3%), and residual functional impairment (34%) . Even with appropriate treatment, invasive S. aureus infections are associated with high mortality, high recurrence rates, and lasting damage in more than one-third of survivors . S. aureus is also notorious for multidrug antimicrobial resistance (AMR) , and the emergence and spread of methicillin-resistant S. aureus (MRSA) has placed a heavy burden on health care systems . MRSA isolates produce an altered penicillin-binding protein (PBP 2a or PBP 2′) that has reduced affinity for penicillin, encoded by the mecA gene, which is carried on a genome segment called the staphylococcal cassette chromosome mec (SCC mec ) on mobile genetic elements (MGE) . Epidemiological studies in the early 2000s reported an increasing burden of invasive MRSA infections in both healthy and chronically ill children . Since then, community-acquired (CA)-MRSA has become an established pathogen worldwide . There is considerable geographic variation in CA- S. aureus disease and the clonal types that cause disease, but recent epidemiological reports indicate that the national burden of CA-MRSA infection in adults and children is declining, whereas rates of invasive methicillin-sensitive S. aureus (MSSA) infection are relatively unchanged . Because of the dynamic changes in the prevalent S. aureus clonotypes, molecular characterization of S. aureus isolates within a specific region is important for the control of infectious diseases . A variety of detection methods, such as multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), SCC mec typing, and determination of polymorphisms in the X region encoding protein A ( spa typing), have been used to study the molecular characteristics of S. aureus . Recently, many studies have utilized whole-genome sequencing (WGS) for phylogenetic analysis and epidemiological investigations of S. aureus . WGS can be used to conduct a comprehensive analysis of the molecular characteristics of S. aureus and provide a deeper understanding of specific clonal groups . In this study, a total of 108 S. aureus isolates were collected from patients with invasive infections at West China Second University Hospital from January 2018 to July 2023, and all the isolates were subjected to WGS. We performed 15 antibiotic resistance phenotype tests. Afterward, different isolates were typed by ST, spa , CCs and SCC mec , and the carriage of virulence genes and resistance genes was analyzed. All the isolates were comprehensively characterized with the aim of providing useful information on the prevalence and spread of invasive S. aureus infections in women and children. 

Invasive infection-derived S. aureus isolates This study was conducted from January 2018 to July 2023 at West China Second University Hospital, Sichuan University, which is one of the largest specialty hospitals for children and women in China and is the National Regional Medical Center of Southwest China. The hospital’s clinical laboratories have been accredited by the College of American Pathologists (CAP) and the China National Accreditation Service for Conformity Assessment (CNAS) under the ISO15189 accreditation standard. The enrolled subjects were patients from Southwest China who presented with invasive S. aureus infection and were admitted to this hospital. The participant eligibility criteria included the following: (1) patients who had clinical specimens (blood, marrow, cerebrospinal fluid (CSF) or pleural effusion) from which S. aureus was isolated and cultured; and (2) patients who were diagnosed with sepsis, meningococcal disease or pleurisy according to the International Classification of Diseases 11th Revision (ICD-11). Specimens were collected by specialized sample collection personnel, nurses or physicians following the standard operating procedure (SOP) of ISO/TS 20658:2017-Medical Laboratories-Requirements for collection, transport, receipt, and handing of samples. Blood and sterile body fluid samples that were contaminated were excluded. Isolate identification and antimicrobial susceptibility testing Patient samples were cultured in vials via the BD BACTECTM FX system (BD Medical Technology, NJ, USA) and then subcultured onto Columbia Agar + 5% sheep blood plates (Autobio, Zhengzhou, China), after which the plates were incubated at 35 °C for 24–48 h in a 5% carbon dioxide (CO 2 ) environment. All the isolates were identified via matrix-assisted laser desorption ionization time‒of‒flight mass spectrometry (MALDI-TOF MS; Vitek MS system; bioMerieux, Rhône, France), and the results were confirmed via PubMLST whole-genome signature sequence alignment. Antimicrobial susceptibility testing was performed with Vitek AST-GP67 cards (bioMerieux, Rhône, France) following the manufacturer’s instructions. of the following 15 antibiotics were tested: rifampin (RIF), nitrofurantoin (NIT), linezolid (LNZ), vancomycin (VAN), quinupristin-dalfopristin (QD), tigecycline (TGC), moxifloxacin (MFX), ciprofloxacin (CIP), gentamicin (GEN), tetracycline (TCY), sulfamethoxazole/trimethoprim (SXT), oxacillin (OXA), clindamycin (CLI), erythromycin (ERY) and penicillin (PEN). S. aureus ATCC25923 was used as a quality control strain, and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines . Genome sequencing The genomic DNA of the S. aureus isolates was extracted via a QIAamp DNA Minikit (Qiagen, Hilden, Germany), and a NEBNext ® Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, NEB, United States) was then used to generate sequencing libraries. The Illumina NovaSeq PE150 platform at Beijing Novogene Biotechnology Co., Ltd. (Illumina, San Diego, CA, USA) was used to sequence the entire genome of S. aureus , with a coverage rate of approximately 200×. Assembly and annotation of sequencing data SPAdes software (v3.15.5) was used to assemble the sequencing data, and different K-mers (21, 33, 55, 77) were selected for assembly to obtain the best assembly results . The assembled data were subjected to further processing with the Seqkit tool (v2.5.1) ( https://github.com/shenwei356/seqkit ) to filter out fragments < 200 bp in length. All assembled genomes were annotated for genes via Prokka software (v1.14.6) ( https://github.com/tseemann/prokka ). Genomic bioinformatic analysis The assembled genomic data were subjected to molecular typing, including MLST, spa typing, clonal complex (CC) typing and SCC mec typing. ST typing and CC typing were performed using mlst software (v2.19.0)  and PubMLST database ( https://pubmlst.org/organisms/staphylococcus-aureus ). CCs here were defined as STs that matched the central genotype (ST) at 4 or more loci unless they more closely matched another central genotype, and CC59 included ST59, ST338, ST8788 and ST8789. spa and SCCmec were typed using the Center for Genomic Epidemiology website ( https://cge.food.dtu.dk/services/spaTyper ) and SCC mec Finder, respectively . Furthermore, SCCmec XI-XV were aligned and verified by reference sequence . ABRicate software (v1.0.1) (Seemann T, Abricate, GitHub https://github.com/tseemann/abricate ) was used to search for virulence factor genes and antibiotic resistance genes in the VFDB and NCBI databases based on FASTA files, respectively , and the positive sequences were verified in the annotated genome (.gff). Since the coa template sequences are not fully registered in the VFDB database, we constructed a staphylocoagulase (SC) alignment dataset based on ABRicate (Supplementary material coa.fa). The fasta files of type I-X SC serotype template sequences referred in NCBI database  (the GenBank numbers from I to X are AB158549.1, BA000018.3, CP000253.1, AB158550.1, AB158551.1, AB158552.1, NC_003923.1, AB158553.1, AB158554.1 and AB158555.1 respectively). The general feature format (.gff) files generated by Prokka annotation were used to create multiFASTA alignments of core genes (> 99%) using Roary (v3.13.0) (Figure ) . Snp-sites (v2.5.1, https://github.com/andrewjpage/snp-sites ) was then used to remove duplicate sites in the multiFASTA alignment. The pruned aln file was used to construct a maximum likelihood tree based on the alignment generated by RAxML (v8.2.12, https://github.com/stamatak/standard-RAxML ) using the GTRGAMMA method . S. aureus MW2, Newman, MRSA252 and JP80 were used as references, and their GenBank numbers on the NCBI website are BA000033.2, AP009351.1, BX571856.1 and AP017922.1, respectively. Finally, RAxML trees, AMR distributions and virulence factor genes were managed and annotated using iTOL  (v6, https://itol.embl.de ). Statistical analysis Data were assessed for significant differences using Statistical Package for the Social Sciences (SPSS) software (v25.0; Chicago, IL, USA). The Chi-square test, Fisher’s exact test, and T test were applied in this study. When comparing multiple groups, based on the chi-square test, the Bonferroni method was used to test whether the differences between multiple groups were statistically significant. A P value < 0.05 was considered to indicate statistical significance. 

S. aureus isolates This study was conducted from January 2018 to July 2023 at West China Second University Hospital, Sichuan University, which is one of the largest specialty hospitals for children and women in China and is the National Regional Medical Center of Southwest China. The hospital’s clinical laboratories have been accredited by the College of American Pathologists (CAP) and the China National Accreditation Service for Conformity Assessment (CNAS) under the ISO15189 accreditation standard. The enrolled subjects were patients from Southwest China who presented with invasive S. aureus infection and were admitted to this hospital. The participant eligibility criteria included the following: (1) patients who had clinical specimens (blood, marrow, cerebrospinal fluid (CSF) or pleural effusion) from which S. aureus was isolated and cultured; and (2) patients who were diagnosed with sepsis, meningococcal disease or pleurisy according to the International Classification of Diseases 11th Revision (ICD-11). Specimens were collected by specialized sample collection personnel, nurses or physicians following the standard operating procedure (SOP) of ISO/TS 20658:2017-Medical Laboratories-Requirements for collection, transport, receipt, and handing of samples. Blood and sterile body fluid samples that were contaminated were excluded. 

Patient samples were cultured in vials via the BD BACTECTM FX system (BD Medical Technology, NJ, USA) and then subcultured onto Columbia Agar + 5% sheep blood plates (Autobio, Zhengzhou, China), after which the plates were incubated at 35 °C for 24–48 h in a 5% carbon dioxide (CO 2 ) environment. All the isolates were identified via matrix-assisted laser desorption ionization time‒of‒flight mass spectrometry (MALDI-TOF MS; Vitek MS system; bioMerieux, Rhône, France), and the results were confirmed via PubMLST whole-genome signature sequence alignment. Antimicrobial susceptibility testing was performed with Vitek AST-GP67 cards (bioMerieux, Rhône, France) following the manufacturer’s instructions. of the following 15 antibiotics were tested: rifampin (RIF), nitrofurantoin (NIT), linezolid (LNZ), vancomycin (VAN), quinupristin-dalfopristin (QD), tigecycline (TGC), moxifloxacin (MFX), ciprofloxacin (CIP), gentamicin (GEN), tetracycline (TCY), sulfamethoxazole/trimethoprim (SXT), oxacillin (OXA), clindamycin (CLI), erythromycin (ERY) and penicillin (PEN). S. aureus ATCC25923 was used as a quality control strain, and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines . 

The genomic DNA of the S. aureus isolates was extracted via a QIAamp DNA Minikit (Qiagen, Hilden, Germany), and a NEBNext ® Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, NEB, United States) was then used to generate sequencing libraries. The Illumina NovaSeq PE150 platform at Beijing Novogene Biotechnology Co., Ltd. (Illumina, San Diego, CA, USA) was used to sequence the entire genome of S. aureus , with a coverage rate of approximately 200×. 

SPAdes software (v3.15.5) was used to assemble the sequencing data, and different K-mers (21, 33, 55, 77) were selected for assembly to obtain the best assembly results . The assembled data were subjected to further processing with the Seqkit tool (v2.5.1) ( https://github.com/shenwei356/seqkit ) to filter out fragments < 200 bp in length. All assembled genomes were annotated for genes via Prokka software (v1.14.6) ( https://github.com/tseemann/prokka ). 

The assembled genomic data were subjected to molecular typing, including MLST, spa typing, clonal complex (CC) typing and SCC mec typing. ST typing and CC typing were performed using mlst software (v2.19.0)  and PubMLST database ( https://pubmlst.org/organisms/staphylococcus-aureus ). CCs here were defined as STs that matched the central genotype (ST) at 4 or more loci unless they more closely matched another central genotype, and CC59 included ST59, ST338, ST8788 and ST8789. spa and SCCmec were typed using the Center for Genomic Epidemiology website ( https://cge.food.dtu.dk/services/spaTyper ) and SCC mec Finder, respectively . Furthermore, SCCmec XI-XV were aligned and verified by reference sequence . ABRicate software (v1.0.1) (Seemann T, Abricate, GitHub https://github.com/tseemann/abricate ) was used to search for virulence factor genes and antibiotic resistance genes in the VFDB and NCBI databases based on FASTA files, respectively , and the positive sequences were verified in the annotated genome (.gff). Since the coa template sequences are not fully registered in the VFDB database, we constructed a staphylocoagulase (SC) alignment dataset based on ABRicate (Supplementary material coa.fa). The fasta files of type I-X SC serotype template sequences referred in NCBI database  (the GenBank numbers from I to X are AB158549.1, BA000018.3, CP000253.1, AB158550.1, AB158551.1, AB158552.1, NC_003923.1, AB158553.1, AB158554.1 and AB158555.1 respectively). The general feature format (.gff) files generated by Prokka annotation were used to create multiFASTA alignments of core genes (> 99%) using Roary (v3.13.0) (Figure ) . Snp-sites (v2.5.1, https://github.com/andrewjpage/snp-sites ) was then used to remove duplicate sites in the multiFASTA alignment. The pruned aln file was used to construct a maximum likelihood tree based on the alignment generated by RAxML (v8.2.12, https://github.com/stamatak/standard-RAxML ) using the GTRGAMMA method . S. aureus MW2, Newman, MRSA252 and JP80 were used as references, and their GenBank numbers on the NCBI website are BA000033.2, AP009351.1, BX571856.1 and AP017922.1, respectively. Finally, RAxML trees, AMR distributions and virulence factor genes were managed and annotated using iTOL  (v6, https://itol.embl.de ). 

Data were assessed for significant differences using Statistical Package for the Social Sciences (SPSS) software (v25.0; Chicago, IL, USA). The Chi-square test, Fisher’s exact test, and T test were applied in this study. When comparing multiple groups, based on the chi-square test, the Bonferroni method was used to test whether the differences between multiple groups were statistically significant. A P value < 0.05 was considered to indicate statistical significance. 

Clinical characteristics of S. aureus isolates A total of 108 nonrepetitive invasive S. aureus isolates were collected in West China Second Hospital/National Regional Medical Center (Southwest China) from January 2018 to July 2023. The detailed clinical information and characteristics of the isolates are summarized in Table . Among the individuals from whom the isolates were derived, 43.52% (47/108) were male and 56.48% (61/108) were female. Their ages ranged from 0 to 68 years old, and the median age (P25-P75) was 9.5 (0.44-13) years old. The hosts of all isolates had sepsis, of which 19.44% (20/108) also had osteoarthritis, 18.52% (20/108) also had pneumonia, 14.81% (16/108) also had tumors, 11.11% (12/108) also had skin infection, and so on. The specimen types were blood (95.37%, 103/108), cerebrospinal fluid (2.78%, 3/108) and pleural effusion (1.85%, 2/108). Molecular typing of S. aureus isolates MLST analysis revealed that there were thirty-two different STs among the 108 isolates, including 8 novel STs (ST8772, ST8786, ST8787, ST8788, ST8789, ST8790, ST8791 and ST8792) and 7 novel alleles ( pta [1041], pta [1042], aroE [1210], tpi [956], tpi [957], yqiL [1143], and arcC [1010]). We annotated all novel ST-type isolates in red font on the phylogenetic tree (Fig. 2). Among the isolates, the prevalent STs were ST22 (17.59%, 19/108), ST59 (15.74%, 17/108), ST5 (7.41%, 8/108), ST188 (7.41%, 8/108) 108) and ST398 (7.41%, 8/108) (Fig. A). In this study, the 32 different STs were divided into 10 CCs and 8 singletons. Excluding the untyped singletons (43.51%, 47/108), the prevalent CCs were CC59 (21.30%, 23/108), CC22 (19.44%, 21/108), CC1 (12.96%, 14/108), and CC5 (12.04%, 13/108) (Fig. B). A total of 45 spa types were detected among 108 S. aureus isolates, and 1 isolate of S. aureus did not have a specific spa type. Among them, t437 and t309 were the most prevalent types, both accounting for 14.81% (16/108) of the isolates (Fig. C). Interestingly, all t002 (5.56%, 6/108) S. aureus isolates belonged to ST5 and CC5, all t189 (7.41%, 8/108) isolates belonged to CC1, and all t437 and t441isolates belonged to CC59. All the above molecular characteristics were marked and annotated on the phylogenetic tree, and the results are shown in Fig. . Comparison of molecular typing between MRSA and MSSA According to previous studies , OXA-resistant (MIC ≥ 4 µg/mL) S. aureus isolates were defined as MRSA; we thus divided the 108 isolates into two major groups, MRSA ( n = 29) and MSSA ( n = 79). The main ST type composing the MRSA group was ST59 (48.28%, 14/29), and the main ST types composing the MSSA group were ST22 (22.78%, 18/79), ST5 (10.13%, 8/79) and ST188 (10.13%, 8/79). The mecA gene was detected in 29 MRSA isolates, 28 isolates were classified as specific SCC mec types, which were divided into two categories: IV (62.07%, 18/29) and V (34.48%, 10/29) (Fig. D). ST59 accounted for more than half of SCC mec IV (66.67%, 12/18) isolates, and spa t437 accounted for 44.83% (13/29) of all MRSA isolates. Among the 29 MRSA isolates tested, CC59-IV-t437 (34.48%) and CC59-V-t437 (13.79%) were the main lineages, and among the 79 MSSA isolates, CC22-t309 (18.89%), CC1-t189 (10.13%), and CC5-t002 (7.60%) were the main lineages (Fig. E and F). Antimicrobial susceptibility of S. aureus The antibiotic susceptibility test results of the isolates are shown in Fig. . All isolates were susceptible to RIF, NIT, LNZ, VAN, QD, and TGC. The rate of resistance to PEN among the isolates was the highest, at 91.67% (99/108), and all the MRSA isolates were resistant. The rates of resistance to erythromycin (ERY) and clindamycin (CLI) were the next highest, both at 65.74% (71/108). The rates of resistance to MFX, CIP, GEN, TCY, SXT and OXA among the isolates were low, at 2.78% (3/108), 4.63% (5/108), 4.63% (5/108), 12.96% (14/108), 13.89% (15/108) and 26.85% (29/108), respectively (Fig. A). We divided the isolates into MRSA and MSSA isolates according to their susceptibility to eight other antibiotics except OXA. The chi-square test results showed that the resistance rates of MRSA to TCY (31.03% vs. 6.33%, p = 0.001), ERY (82.76% vs. 59.49%, p = 0.044), and CLI (82.76% vs. 59.49%, p = 0.024) were significantly higher than those of MSSA to these antibiotics ( p < 0.05) (Fig. B). Comparison of antimicrobial resistance phenotypes of different molecular lineages of MRSA The 29 MRSA isolates were divided into four groups: CC59-IV-t437 (34.38%, 10/29), CC59-V-t437 (13.79, 4/29), CC59-IV-t441 (10.35%, 3/29) and others (41.38%, 12/29) to compare the antibiotic resistance phenotypes. The test analysis revealed that the resistance rates of CC59-IV-t437, CC59-V-t437 and CC59-IV-t441 to CLI and ERY were 100%, which was significantly higher than that of MRSA of other molecular lineages (Fig. ). Comparison of antimicrobial resistance phenotypes of different molecular lineages of MSSA The 79 MSSA isolates were divided into four groups: CC22-t309 (18.99%, 15/79), CC1-t189 (10.13%, 8/79), CC5-t002 (7.59%, 6/79) and others (63.29%, 50/79) to compare the antibiotic resistance phenotypes. The test analysis revealed that the resistance rate of CC5-t002 to GEN, SXT and CLI was significantly higher than that of other molecular lineages of MSSA (Fig. ). Carriage of S. aureus virulence factor genes and antibiotic resistance genes To better understand the molecular characteristics of the 108 isolates of S. aureus , we analyzed the carriage of 81 virulence factor genes and 27 antibiotic resistance genes. The results are shown in the heatmap in Fig. . All of these isolates carried the gene spa and coa , which encodes staphylococcal protein A and SC, respectively. And the most common SC serotypes in MRSA and MSSA were type VII (79.31%) and type II (37.97%), respectively (Table ). coa and vWbp act as cofactors to promote the formation of blood clots. More than 99% of the isolates carried the genes hla , hlb , and hld that express hemolysins, the icaA , icaB , icaC , and icaD genes that regulate biofilm formation, sspA , sspB , sspC , geh , and lip that encode secreted enzymes, isdA , isdB , isdC , isdD , isdE , isdF , and strB that are related to nutritional metabolism, ebp/map and sdrC that are related to adhesion, and adsA , sbi , and capB-G that are related to immune modulation. An antibiotic resistance-related gene carried by all isolates was tet (38) , but not all isolates were resistant to tetracycline. The gene carriage of each clinical isolate is shown in Table . Comparison of virulence factor genes and antibiotic resistance genes in MRSA and MSSA of different molecular lineages We also analyzed the virulence factor genes and antibiotic resistance gene profile of different molecular lineages in MRSA and MSSA (Table -S6). In MRSA, the aminoglycoside antibiotic-related genes ant (6)-Ia , aph (2’’)-Ii , the β-lactam antibiotic gene blaPC1 , and the trifluoperazine-related dfrE gene were different in CC59-IV-t437, CC59-V-t437, CC59-IV-t441, others. In MRSA, the aminoglycoside antibiotic-related genes ant (6)-Ia , aph (2’’)-Ii , β-lactam antibiotic gene blaPC1 and trifluocorticoid-related dfrE gene were different in CC59-IV-t437, CC59-V-t437, CC59-IV-t441 and others. The carrying rates of aph (2’’)-Ii and dfrE in CC59-V-t437 (50% and 50%) were significantly higher than those in the other three groups. In MSSA, the gene mph(C) associated with macrolide resistance and the gene msr(A) associated with drug efflux pumps were different in CC22-t309, CC1-t189, CC5-t002 and others, and the carrying rate of CC5-t002 (16.67% and 16.67%) was significantly higher than that of the other three groups. 

S. aureus isolates A total of 108 nonrepetitive invasive S. aureus isolates were collected in West China Second Hospital/National Regional Medical Center (Southwest China) from January 2018 to July 2023. The detailed clinical information and characteristics of the isolates are summarized in Table . Among the individuals from whom the isolates were derived, 43.52% (47/108) were male and 56.48% (61/108) were female. Their ages ranged from 0 to 68 years old, and the median age (P25-P75) was 9.5 (0.44-13) years old. The hosts of all isolates had sepsis, of which 19.44% (20/108) also had osteoarthritis, 18.52% (20/108) also had pneumonia, 14.81% (16/108) also had tumors, 11.11% (12/108) also had skin infection, and so on. The specimen types were blood (95.37%, 103/108), cerebrospinal fluid (2.78%, 3/108) and pleural effusion (1.85%, 2/108). 

S. aureus isolates MLST analysis revealed that there were thirty-two different STs among the 108 isolates, including 8 novel STs (ST8772, ST8786, ST8787, ST8788, ST8789, ST8790, ST8791 and ST8792) and 7 novel alleles ( pta [1041], pta [1042], aroE [1210], tpi [956], tpi [957], yqiL [1143], and arcC [1010]). We annotated all novel ST-type isolates in red font on the phylogenetic tree (Fig. 2). Among the isolates, the prevalent STs were ST22 (17.59%, 19/108), ST59 (15.74%, 17/108), ST5 (7.41%, 8/108), ST188 (7.41%, 8/108) 108) and ST398 (7.41%, 8/108) (Fig. A). In this study, the 32 different STs were divided into 10 CCs and 8 singletons. Excluding the untyped singletons (43.51%, 47/108), the prevalent CCs were CC59 (21.30%, 23/108), CC22 (19.44%, 21/108), CC1 (12.96%, 14/108), and CC5 (12.04%, 13/108) (Fig. B). A total of 45 spa types were detected among 108 S. aureus isolates, and 1 isolate of S. aureus did not have a specific spa type. Among them, t437 and t309 were the most prevalent types, both accounting for 14.81% (16/108) of the isolates (Fig. C). Interestingly, all t002 (5.56%, 6/108) S. aureus isolates belonged to ST5 and CC5, all t189 (7.41%, 8/108) isolates belonged to CC1, and all t437 and t441isolates belonged to CC59. All the above molecular characteristics were marked and annotated on the phylogenetic tree, and the results are shown in Fig. . 

According to previous studies , OXA-resistant (MIC ≥ 4 µg/mL) S. aureus isolates were defined as MRSA; we thus divided the 108 isolates into two major groups, MRSA ( n = 29) and MSSA ( n = 79). The main ST type composing the MRSA group was ST59 (48.28%, 14/29), and the main ST types composing the MSSA group were ST22 (22.78%, 18/79), ST5 (10.13%, 8/79) and ST188 (10.13%, 8/79). The mecA gene was detected in 29 MRSA isolates, 28 isolates were classified as specific SCC mec types, which were divided into two categories: IV (62.07%, 18/29) and V (34.48%, 10/29) (Fig. D). ST59 accounted for more than half of SCC mec IV (66.67%, 12/18) isolates, and spa t437 accounted for 44.83% (13/29) of all MRSA isolates. Among the 29 MRSA isolates tested, CC59-IV-t437 (34.48%) and CC59-V-t437 (13.79%) were the main lineages, and among the 79 MSSA isolates, CC22-t309 (18.89%), CC1-t189 (10.13%), and CC5-t002 (7.60%) were the main lineages (Fig. E and F). 

S. aureus The antibiotic susceptibility test results of the isolates are shown in Fig. . All isolates were susceptible to RIF, NIT, LNZ, VAN, QD, and TGC. The rate of resistance to PEN among the isolates was the highest, at 91.67% (99/108), and all the MRSA isolates were resistant. The rates of resistance to erythromycin (ERY) and clindamycin (CLI) were the next highest, both at 65.74% (71/108). The rates of resistance to MFX, CIP, GEN, TCY, SXT and OXA among the isolates were low, at 2.78% (3/108), 4.63% (5/108), 4.63% (5/108), 12.96% (14/108), 13.89% (15/108) and 26.85% (29/108), respectively (Fig. A). We divided the isolates into MRSA and MSSA isolates according to their susceptibility to eight other antibiotics except OXA. The chi-square test results showed that the resistance rates of MRSA to TCY (31.03% vs. 6.33%, p = 0.001), ERY (82.76% vs. 59.49%, p = 0.044), and CLI (82.76% vs. 59.49%, p = 0.024) were significantly higher than those of MSSA to these antibiotics ( p < 0.05) (Fig. B). 

The 29 MRSA isolates were divided into four groups: CC59-IV-t437 (34.38%, 10/29), CC59-V-t437 (13.79, 4/29), CC59-IV-t441 (10.35%, 3/29) and others (41.38%, 12/29) to compare the antibiotic resistance phenotypes. The test analysis revealed that the resistance rates of CC59-IV-t437, CC59-V-t437 and CC59-IV-t441 to CLI and ERY were 100%, which was significantly higher than that of MRSA of other molecular lineages (Fig. ). 

The 79 MSSA isolates were divided into four groups: CC22-t309 (18.99%, 15/79), CC1-t189 (10.13%, 8/79), CC5-t002 (7.59%, 6/79) and others (63.29%, 50/79) to compare the antibiotic resistance phenotypes. The test analysis revealed that the resistance rate of CC5-t002 to GEN, SXT and CLI was significantly higher than that of other molecular lineages of MSSA (Fig. ). 

S. aureus virulence factor genes and antibiotic resistance genes To better understand the molecular characteristics of the 108 isolates of S. aureus , we analyzed the carriage of 81 virulence factor genes and 27 antibiotic resistance genes. The results are shown in the heatmap in Fig. . All of these isolates carried the gene spa and coa , which encodes staphylococcal protein A and SC, respectively. And the most common SC serotypes in MRSA and MSSA were type VII (79.31%) and type II (37.97%), respectively (Table ). coa and vWbp act as cofactors to promote the formation of blood clots. More than 99% of the isolates carried the genes hla , hlb , and hld that express hemolysins, the icaA , icaB , icaC , and icaD genes that regulate biofilm formation, sspA , sspB , sspC , geh , and lip that encode secreted enzymes, isdA , isdB , isdC , isdD , isdE , isdF , and strB that are related to nutritional metabolism, ebp/map and sdrC that are related to adhesion, and adsA , sbi , and capB-G that are related to immune modulation. An antibiotic resistance-related gene carried by all isolates was tet (38) , but not all isolates were resistant to tetracycline. The gene carriage of each clinical isolate is shown in Table . 

We also analyzed the virulence factor genes and antibiotic resistance gene profile of different molecular lineages in MRSA and MSSA (Table -S6). In MRSA, the aminoglycoside antibiotic-related genes ant (6)-Ia , aph (2’’)-Ii , the β-lactam antibiotic gene blaPC1 , and the trifluoperazine-related dfrE gene were different in CC59-IV-t437, CC59-V-t437, CC59-IV-t441, others. In MRSA, the aminoglycoside antibiotic-related genes ant (6)-Ia , aph (2’’)-Ii , β-lactam antibiotic gene blaPC1 and trifluocorticoid-related dfrE gene were different in CC59-IV-t437, CC59-V-t437, CC59-IV-t441 and others. The carrying rates of aph (2’’)-Ii and dfrE in CC59-V-t437 (50% and 50%) were significantly higher than those in the other three groups. In MSSA, the gene mph(C) associated with macrolide resistance and the gene msr(A) associated with drug efflux pumps were different in CC22-t309, CC1-t189, CC5-t002 and others, and the carrying rate of CC5-t002 (16.67% and 16.67%) was significantly higher than that of the other three groups. 

S. aureus is one of the most common pathogenic bacteria that causes infections in hospitalized patients. The high mortality and recurrence rate of invasive S. aureus infection has placed a burden on the global medical care system. S. aureus is also notorious for its ability to acquire and carry multiple antibiotic resistance (AMR) , with methicillin-susceptible S. aureus (MSSA) evolving into methicillin-resistant S. aureus (MRSA) after acquisition of staphylococcal cassette chromosome mec (SCC mec ) elements or gene mutations . The global spread of MRSA has placed a heavy burden on healthcare resources . Phenotypic and genomic analyzes of S. aureus in specific regions are important for monitoring bacterial prevalence characteristics and developing prevention and control strategies . MRSA infections are of great concern because of their high morbidity and mortality and the resistance of the offending isolates to all available penicillins and most other β-lactams (except ceftaroline and ceftobiprole) . Fortunately, the prevalence of MRSA in this study was reduced (26.85%), which is similar to the results of a recent study in Tianjin (34.83%) . The prevalence of MRSA in this study (26.85%) was much lower than the prevalence in China in 2010–2011 (47.5%)  and lower than the prevalence in Latin America in 2011–2014 (45.4%) . It is reassuring to note that MRSA prevalence has declined in most cities in China since 2011, similar to the results of recent studies in Finland . In addition, we found that oxacillin resistance in this study was exhibited by all MRSA isolates, which was mediated by mecA , and no mecA homolog mecC was detected, which is consistent with most recent studies . Although historical research data show that the prevalent strains of S. aureus vary in different regions and periods, CC59 has begun to dominate the epidemic in various regions around the world in recent years. According to research by Wang et al., ST5 has been a competitor to ST239 in Chinese hospitals since 2013 , and now ST239 has lost its dominance and been replaced by the common clone ST59 . ST59 is the most successful and persistent CA-MRSA clone in the Asia-Pacific region, displaying tremendous overall phylogenetic diversity . Our study identified ST59/CC59 (include CC59-IV-t437, CC59-V-t437 and CC59-IV-t441) as the major clonal complex of MRSA prevalence in the region. This result is consistent with the finding by Yu et al. that CC59-ST59 and CC5-ST5/ST764 are the key lineages responsible for the development of methicillin resistance in S. aureus in the Chinese clinical setting . CC59 is also the most prevalent MRSA in Taiwan, and CC59 in Taiwan and mainland China may have the same genetic background . Similar epidemic clonal have been documented in the United States, Malaysia, Singapore, Hungary, Portugal, the Czech Republic, and several Latin American countries in recent decades , suggesting that this appears to be a widespread phenomenon. In addition, among the identified SCC mec , type IV accounted for 62.07% of MRSA isolates, indicating that this cassette was frequently transferred. This may be because type IV SCC mec has an advantage due to its small size and simple genetic composition . MSSA clones prevalent in hospitals have been reported, such as ST1, ST7, ST15, ST22, ST59, and ST398 . ST398 is the most common MSSA lineage in China, with a prevalence of 5.5–26.6% , and the prevalence of ST398-MSSA in Hainan Province increased significantly from 5.5% (2013 to 2014) to 18.4% (2018 to 2019) . Previous reports have shown that ST188 has strong biofilm formation and adhesion capabilities and remains the most common MSSA clone in childhood infections in China . This study found that the ST22/CC22-t309(18.99%), ST188/CC1-t189(10.13%) and ST5/CC5-t002(7.59%) lineages were dominant in MSSA in this region. Notably, ST22(24.06%,19/79) became the most prevalent molecular type in the region. Recently, the hypervirulent S. aureus ST22 has also attracted the attention of many scholars, the study by Yu et al. specifically focused on ST22 S. aureus . The study by Baldan et al. showed that ST22 has a significantly greater ability to invade A549 cells than ST228 . The prevalence of ST22 S. aureus in this area deserves further exploration of its epidemic trend, because its high virulence poses a greater threat to infection in women and children. In addition, in general, the number of spa types and MLST in the MSSA group was greater than that in the MRSA group, indicating that MSSA isolates were more diverse than MRSA isolates and that MSSA isolates could not be ignored. The rates of resistance of the isolates in this study to PEN, ERY, CLI, OXA, SXT, TCY, GEN, CIP, and MFX were 91.67%, 65.74%, 65.74%, 25.93%, 1389%, 12.96%, 4.63%, 4.63% and 2.78%, respectively, which are similar to those derived from the results of the Chinese Antibiotic Resistance Surveillance System in the first half of 2023. The rate of resistance to PEN was higher than that in a recent University of Hartford study on nasal S. aureus isolates (74%) , indicates that PEN is no longer suitable for treating S. aureus infections in this area. The rate of resistance to ERY in this study was much higher than that in Athens (12.2%) , the United States (41%), Russia (2%), and Australia (1%) ( http://resistancemap.cddep.org/AntibioticResistance.php ), which maybe be because macrolides were once heavily used in China, because they are safe to use in children, and because they are exempt from skin testing before antibiotic treatment . In this study, the rate of S. aureus resistance to CLI and SXT was also much higher than that at Athens Children’s Hospital (8.10% and 0.00%, respectively), which may be due to the different clinical antibiotic usage habits in China and Europe . Our studies showed that the antibiotic resistance phenotypes differ between MRSA and MSSA and their respective lineages. Except for SXT, for which MSSA had a higher resistance rate than MRSA, the rates of resistance of MSSA to TCY, ERY and CLI were lower than those of MRSA, which is also consistent with the data from the Chinese Antibiotic Resistance Surveillance System. In MRSA, the resistance rate of CC59 clone group to ERY and CLI was 100%, which was higher than other molecular types. In MSSA, the resistance rate of CC5 clone group to SXT, CLI, ERY, PEN was 100%, and the resistance rate to GEN, SXT, CLI was higher than other types of MSSA, and there were significant differences after testing. Notably, the proportion of SXT is very low in both the entire MRSA group and the CC59 clone complex, which accounts for the largest proportion, which may have a suggestive effect on the treatment of invasive MRSA infections. However, SXT can impair tetrahydrofolate production, leading to DNA damage, induction of stress responses, and accumulation of reactive oxygen species (ROS), and care should be taken with the dosage used . In addition, the CC5 clone group in MSSA has a 100% resistance rate to multiple antibiotics, which deserves people’s attention. Virulence factors play an important role in the pathogenesis of S. aureus infection . According to existing studies, S. aureus pathogenicity largely depends on the carriage of a series of virulence factors related to exotoxins, biofilms, secreted enzymes, adhesion, immune modulation, and nutritional metabolism . Among the 81 virulence factor genes we analyzed, common virulence factors such as spa and hla were expressed in almost all isolates, which is similar to the results of most studies in mainland China , which confirmed that these are the most common virulence factors of S. aureus and that their distribution does not differ among different regions. Notably, the frequency of pvl in this study was 33.33%, which was higher than that in mainland China . The high prevalence of pvl in invasive infections in women and children deserves attention. Immune evasion cluster (IEC) genes specifically bind to compounds of the human innate immune system to protect bacteria from the human innate immune system, which prolongs the duration of bacterial infection . Our study found that the prevalence of key members of the IEC, scn , chp , and sak , was 98.15%, 75.00%, and 82.40%, respectively, and they may be key factors that lead to prolonged bacterial infection and progression to invasive infection. This study is the first to apply WGS technology to explore the molecular biological characteristics of invasive S. aureus isolated from children and women in Southwest China. Children’s immune system development is immature, and women have some specific infection routes, such as the pelvic cavity and birth canal. Moreover, S. aureus can also be transmitted vertically from mother to child. Therefore, in children’s and women’s specialty hospitals, the biological characteristics of S. aureus may differ from those in other hospital populations, and relevant data analysis and theoretical support are needed for specific patient groups. In recent years, researchers have begun to pay more attention to the epidemic characteristics of S. aureus in children . In addition, all of the 108 isolates enrolled in this study were invasive clinical pathogens isolated from patients’ blood, cerebrospinal fluid, and pleural effusion, which were critically ill patients and different from other studies of S. aureus isolated from the environment, food and ordinary patients. However, our study also has the following limitations. (1) There is a lack of strain monitoring in community (maternal and child) medical institutions, which is due to insufficient detection capabilities at the community level and the fact that pathogenic isolation cannot be achieved. (2) The number of isolates in this study is relatively limited (108 isolates), which is due to our focused on critically ill patients with invasive infections, and the limited sample size may affected our ability to explore the specific characteristics among different molecular lineages. (3) Core gene analysis rather than pangenome analysis was used in the bioinformatics part, which is due to the library construction and sequencing technology used in this study, and the degree of information mining was relatively inadequate. 

In summary, this study found that the most common MRSA and MSSA molecular lineages in women and children in Southwest China were CC59-IV-t437 and CC22-t309, and CC5 in MSSA was highly resistant to multiple antibiotics, which deserves attention. This study aims to provide valuable information for the prevention and treatment of invasive S.aureus diseases. 

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2 Supplementary Material 3 Supplementary Material 4 

