ABSTRACT
Staphylococcus aureus is a major pathogen responsible for hospital- and community-acquired infections, with the accessory gene regulator (agr) system playing a central role in virulence control. The ST9 lineage commonly associated with livestock exhibits multidrug resistance but remains understudied in terms of virulence. In this work, we examined nine clinical ST9 methicillin-resistant S. aureus isolates and identified two strains carrying spontaneous frameshift mutations in the agr locus, resulting in the loss of RNAIII and α-toxin expression. These agr-deficient strains exhibited reduced hemolytic activity, enhanced biofilm formation, and attenuated virulence in a Galleria mellonella infection model. These preliminary pieces of evidence suggested a critical role of the agr system in ST9 virulence. Comparative genomic analyses revealed distinct pathogenicity islands in ST9, differing from those in other common clinical lineages. Notably, we identified three novel prophage islands (φST9-A, φST9-B, and φST9-C), with φST9-B carrying the recently characterized tarP virulence gene, suggesting that recombination events within livestock-associated MRSA could contribute to the genetic diversification of ST9. These findings demonstrate the significant virulence potential of the ST9 lineage and the importance of agr-mediated regulation. Moreover, the presence of new prophages highlights the evolutionary adaptability of this lineage. Collectively, our results underscore the clinical threat posed by ST9 MRSA and the necessity for vigilant monitoring to control its spread.
IMPORTANCE
Methicillin-resistant Staphylococcus aureus (MRSA) ST9 commonly associated with livestock remains understudied in terms of virulence mechanisms. This study identifies key insights into ST9 MRSA virulence, focusing on the accessory gene regulator (agr) system, which regulates virulence in S. aureus. We show that agr-deficient ST9 strains exhibit altered virulence phenotypes, including reduced hemolysis and increased biofilm formation. Additionally, our genomic analysis reveals novel prophage islands in ST9, highlighting the lineage’s genetic adaptability and potential for increased virulence. These findings emphasize the need for continued surveillance and targeted strategies to control the spread of ST9 MRSA, with important implications for diagnostic and therapeutic approaches.
KEYWORDS: virulence, LA-MRSA, ST9, agr, prophage
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that can colonize in the hospital, community, and livestock environments, causing a wide range of infections, including pneumonia, septicemia, skin and soft tissue infections, osteomyelitis, and endocarditis (1). MRSA harbors a diverse array of virulence factors, including enzymes, surface-associated proteins, pore-forming toxin, superantigens, phenol-soluble modulins, etc. (2). The virulence of a given lineage can be determined either by the specific regulation of an intrinsic factor, such as the accessory gene regulator (agr), or the acquisition of virulence factors via mobile genetic elements, such as prophage or pathogenicity islands (3).
Sequence type 9 (ST9) has emerged as the most predominant livestock-associated MRSA (LA-MRSA) in Asia since it was first isolated from pig farms in China (4, 5). Jin et al. reported a highly virulent ST9-SCCmecXII MRSA strain causing serious disease in a patient in China (6). Recently, our previous multicenter prospective study uncovered the emergence of ST9 strains in healthcare settings across China and highlighted the multidrug-resistant nature of this lineage (7).
However, the virulence determinants of the ST9 lineage have not been comprehensively characterized. While some studies have attributed ST9 virulence to the presence of immune evasion cluster (IEC) genes, such as scn, chp, and sak (6, 8), the contribution of regulatory systems, such as agr and SaeRS, to the virulence of ST9 has not been investigated. In our previous study (7), we performed the genomic analysis of ST9 strains isolated from patients and identified two strains harboring frameshift mutations in the agr locus. However, whether these mutations contribute to altered virulence phenotypes remains to be elucidated.
In this study, we investigated the contribution of the agr system to the virulence of ST9 lineages by comparing virulence factor expression and virulence phenotypes between agr wild-type and -deficient strains. In addition, the pathogenicity island and novel prophages carried by ST9 lineages have been analyzed to gain a more comprehensive understanding of virulence evolution within this lineage.
MATERIALS AND METHODS
Bacterial strains and growth conditions
Bacterial strains were cultured in tryptic soy broth (TSB) at 37°C with shaking at 200 rpm. The clinical ST9 MRSA strains used in this study were obtained from a previous multicenter prospective study (9), as also described in our previous research (7). The agr-deficient strains were selected for molecular and virulence phenotype experiments, while agr wild-type ST9 strains (N28CSA05 and N29CSA11), which differed by the fewest single nucleotide polymorphism from the mutant strains, were used for comparative analyses. The community-associated MRSA ST72 lineage strain HL1 and its agr-knockout mutant HL1Δagr (10) were used as controls in virulence experiments.
Real-time quantitative reverse transcription-PCR (qRT-PCR)
Overnight bacterial cultures were diluted 1:100 into fresh TSB and incubated at 37°C with shaking for 8 h to reach the stationary phase. Total RNA was extracted using the RNeasy Mini Kit (Qiagen), treated with DNase (TURBO DNA-Free Kit, Ambion), and reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara) following the manufacturer’s instructions. Quantitative PCR was performed using the SYBR Premix Ex Taq PCR Kit (Takara Bio) under PCR conditions of 95°C for 5 min, followed by 45 cycles of 95°C for 5 s, 55°C for 15 s, and 72°C for 15 s using a LightCycler 480 System (Roche). All reactions were conducted in triplicate, with gyrB used as the internal control. Each experiment was independently repeated at least three times (11).
Measurement of α-toxin production
Culture supernatants were collected after 8 h of bacterial growth. Equal volumes of supernatants were loaded onto 15% SDS polyacrylamide gels and electrophoresed at 160 V for 1 h. Proteins were transferred to nitrocellulose membranes using an iBlot Western Blot System (Bio-Rad, America). The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature, followed by overnight incubation with anti-staphylococcal α-toxin mouse serum (1:5,000 dilution; Aladdin) in blocking buffer. After washing three times with washing buffer (Tris-buffered saline, 0.1% Tween-20, pH 7.4), membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:5,000 dilution; Jackson Research) in blocking buffer in the dark for 2 h. Protein bands were visualized using enhanced chemiluminescence detection reagent (BeyoECL Plus, Beyotime, China) according to the manufacturer’s instructions and scanned using a Typhoon Trio+ Variable Mode Imager (GE Healthcare) (10).
Fitness evaluation
Fitness was assessed based on growth curves and maximum optical density (max OD) values (12). Three independent cultures per strain were grown overnight, diluted 1:100 in TSB, and added in triplicate to a flat-bottom honeycomb 100-well plate. The plates were incubated at 37°C with continuous agitation, and OD600 readings were measured every 5 min for 24 h using a Bioscreen C Analyzer (Oy Growth Curves AB Ltd.). Blank wells containing only medium were used to adjust for any changes in OD600 unrelated to microbial growth. The max OD was recorded as the OD600 value at 24 h. Growth curves were constructed using GraphPad Prism 9.
Erythrocyte lysis assay
The hemolytic activity of all tested strains was evaluated as previously described (13). Culture filtrates were collected after 8 h of bacterial growth and serially diluted (1:2 to 1:16) in Dulbecco’s phosphate-buffered saline (PBS). Diluted filtrates were then incubated with 2% (vol/vol) human red blood cells at 37°C for 1 h. Hemolysis was quantified by measuring the optical density at 540 nm using a microplate reader. All assays were performed in triplicate.
Quantitative biofilm assay
Biofilm formation on polystyrene was measured using crystal violet staining, as described by Xie et al. (14). Each strain was cultivated overnight at 37°C, and the cultures were adjusted to a turbidity of 0.5 McFarland standards. A total of 20 µL bacterial suspension and 180 µL TSB medium supplemented with 0.5% glucose were added to a 96-well flat-bottomed microtiter plate in triplicate. After 72 h of incubation at 37°C, the wells were gently washed with PBS three times, stained with 0.1% (w/v) crystal violet (CV) for 10 min, and washed with water. Then, the biofilm-associated CV dye was solubilized with 30% glacial acetic acid for 15 min at room temperature. The OD570 of each well was measured.
Survival assay in Galleria mellonella larvae
Strains were grown to mid-exponential phase, washed once with sterile PBS, then resuspended in PBS at 2 × 108 CFU/mL. Fully matured last-instar G. mellonella larvae (Tianjin huiyude Biotech, Tianjin, China) were weighed (~300 mg), paired, and randomly grouped to ensure consistent quality for each strain across experimental conditions. Each larva was injected in the buttock with 2 × 106 bacterial cells suspended in 10 µL PBS solution and incubated in clean, sterile petri dishes at 37°C. Mortality was recorded over 72 h to calculate survival rates (15).
Genomic DNA extraction and sequencing
Genomic DNA was extracted using the Gentra Puregene Yeast/Bact. Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. DNA concentration was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA), and DNA integrity was confirmed by agarose gel electrophoresis. A minimum of 5 µg of genomic DNA was required for subsequent library preparation. Sequencing libraries were constructed using the SQK-LSK110 Ligation Sequencing Kit in combination with PCR-Free ONT Native Barcode Expansion kits (EXP-NBD104 and EXP-NBD114, Oxford Nanopore Technologies, UK) following the manufacturer’s protocol without additional DNA shearing, thereby enriching for longer reads. Prepared libraries were quantified by Qubit 4.0 Fluorometer (Invitrogen, CA, USA). Sequencing was subsequently performed on the Nanopore MinION sequencing platform (Oxford Nanopore Technologies, UK).
Bioinformatics analysis
Virulence genes were confirmed using ABRicate software (https://github.com/tseemann/abricate) with the Virulence Factor Database as the reference. The agr locus was aligned using ABRicate with a custom reference database constructed from wild-type agr sequences. Hybrid genome assemblies were generated using Unicycler (16), combining second-generation data from a previous study (9) with third-generation data obtained in this study. BLAST was used to compare the ST9 genome with those of other clones to identify pathogenicity islands and discover novel prophage, and Easyfig was used for linear comparison. For strains with only second-generation sequencing data, Mauve and ABRicate were used to confirm the presence of virulence islands by checking gene coverage for integrase and primase enzyme genes coverage. The phylogenetic tree of S. aureus ST9 strains was generated in our previous research (7), and virulence genes/phage islands annotations were performed using the iTOL (https://itol.embl.de/) based on this phylogenetic framework.
Statistical analysis
All statistical analyses were performed using GraphPad Prism software (version 9.0.0; GraphPad Software). For comparisons among multiple groups, either one-way analysis of variance or the non-parametric Kruskal-Wallis test was used depending on data distribution and variance homogeneity. A P-value of <0.05 was considered statistically significant. Data were presented as mean ± standard deviation (SD) unless otherwise stated.
RESULTS
Identification of agr mutations in ST9 MRSA isolates
Nine ST9 isolates were obtained from our previous study (7), all of which were identified as agr type II. Pairwise comparison of the agr gene sequences revealed mutations in two strains: N08CSA36 and N09HSA31. Specifically, strain N08CSA36 harbored a deletion of adenine (A) at position 712 in the agrA gene, while strain N09HSA31 exhibited an insertion of thymine (T) between positions 487 and 488 in the agrC gene (Fig. 1A). Both mutations resulted in frameshift changes that alter the gene reading frame and prevent gene expression.
Fig 1.
(A) Accessory gene regulator (agr) system mutations in N08CSA36 and N09HSA31.1 represent the mutation in N08CSA36, and two represent the mutation in N09HSA31. (B) Molecular structure, signal synthesis, and transduction cascade of the agr quorum sensing system.
To assess the broader prevalence of agr mutations in the ST9 lineage, we further analyzed 206 genomes in our phylogenetic analysis (7). Among them, 37 strains exhibited nucleotide sequence variations in the agr system (not publication). In addition to N08CSA36, five other strains exhibited frameshift mutations in the agrA gene. Similarly, in addition to N09HSA31, three other strains had frameshift mutations in the agrC gene. The remaining 27 strains harbored single nucleotide mutations. Most mutations were located in the sensor histidine kinase AgrC and response regulator protein AgrA, suggesting a potential loss of function (Fig. 1B).
Effect of agr-deficient ST9 strains on virulence factor expression
To determine whether these agr-deficient ST9 strains effectively deactivate the agr regulatory system, we examined the expression of agr-regulated genes using agr wild-type ST9 strains (N28CSA05 and N29CSA11), ST72 MRSA strain HL1, and its agr knockout mutant HL1Δagr as controls.
The transcription levels of agr effectors were tested by RT-PCR in all tested strains. The transcription level of RNAIII was completely abolished in both the agr-deficient ST9 strains and the agr deletion HL1 strain. Furthermore, the RNAIII expression in agr wild-type ST9 strains showed no significant difference from that in HL1 (Fig. 2A).
Fig 2.
Comparison of virulence phenotypes and genotypes between accessory gene regulator (agr)-deficient strains and agr wild-type strains. (A) Expression of the agr system in agr-deficient and agr wild-type strains. The HL1 strain was used for normalization, with gyrB as the internal control gene. The relative expression levels of RNAIII in the agr system were shown. ns indicates no significant difference. (B) Expression of α-toxin in agr-deficient and agr wild-type strains. Lanes 1 and 2 represent N08CSA36 and N09HSA31, respectively, both agr-deficient strains; lanes 3 and 4 represent N28CSA05 and N29CSA11, respectively, both agr wild-type strains; lane 5 represents HL1; and lane 6 represents the HL1 agr knockout strain. (C) Growth curves of agr-deficient and agr wild-type strains in the TSB medium. Different colors represent different strains. (D) Maximum OD600 values. Box plot showing the upper and lower quartiles, median, maximum, and minimum values. Pairwise comparisons of ST9 strains were conducted, and HL1 was compared with its knockout strain. Statistical significance for multiple groups was tested using Šidák’s multiple-comparisons test. **: adjusted P-value < 0.01; ****: adjusted P-value < 0.0001. (E) Biofilm formation ability of agr-deficient and agr wild-type strains at 72 h. Pairwise comparisons of ST9 strains were conducted, and HL1 was compared with its knockout strain. Statistical significance for multiple groups was tested using Šidák’s multiple-comparisons test. *: adjusted P-value < 0.05. (F) Erythrocyte lysis assay of agr-deficient and agr wild-type strains. Dunnett’s multiple-comparisons test was used to compare the hemolytic activity between ST9 agr wild-type strains and HL1 at each dilution. *: adjusted P-value < 0.05; ***: adjusted P-value < 0.001. Significant differences were observed between N29CSA11 and HL1 at dilutions of 1:4 and 1:8. (G) Survival assay of agr-deficient and agr wild-type strains in Galleria mellonella larvae. The x-axis represents time, and the y-axis represents survival rate. Different colors represent different strains.
The expression of α-toxin, regulated by the agr system, was detected via western blot analysis in all strains. Both the agr-deficient ST9 strains and the agr-deletion HL1 strain failed to produce α-toxin but showed expression of surface protein A (Spa), a phenotype consistent with agr inactivation. In contrast, the agr-functional ST9 strains and the HL1 strain produced α-toxin and exhibited weak or no expression of the Spa protein (Fig. 2B).
Phenotypic effects of agr dysfunction on virulence
All tested strains reached the logarithmic phase at the same time in the TSB medium (Fig. 2C), indicating comparable growth rates between the agr-deficient and agr wild-type ST9 strains. However, the final OD600 of the agr-deficient ST9 strains was higher than that of the agr wild-type strain (Fig. 2D), which was similar to the behavior observed in the HL1 strain and its agr deletion mutant.
Hemolysis (lysis of erythrocytes), a crucial virulence determinant of S. aureus, was assessed using an erythrocyte lysis assay. The agr-deficient ST9 strains and the agr deletion HL1 strain completely lost the ability to lyse red blood cells (Fig. 2E). In contrast, the agr wild-type ST9 strains exhibited similar hemolytic ability comparable to HL1. Notably, at 1:4 and 1:8 dilutions, the agr wild-type ST9 strain N29CSA11 showed significantly stronger hemolytic activity than HL1.
Biofilm formation in agr-deficient and agr wild-type ST9 strains was analyzed via crystal violet staining. Strain N08CSA36, which carries a frameshift mutation in agrA, exhibited significantly enhanced biofilm formation compared to agr wild-type strains, particularly N29CSA11 (Fig. 2F). Similarly, the HL1Δagr mutant formed more robust biofilms than HL1. Although N09HSA31 exhibited a slightly higher biofilm formation than the agr wild-type strains, the difference was not statistically significant.
To evaluate the in vivo virulence potential of these strains, we compared the survival rates of G. mellonella larvae following infection with agr-deficient and agr wild-type ST9, as well as HL1 and HL1Δagr. As shown in Fig. 2G, the percentage of surviving larvae was significantly higher following infection with agr-deficient strains N08CSA36, N09HSA31, and HL1Δagr compared to agr wild-type strains N28CSA05, N29CSA11, and HL1 (P < 0.0001). The median survival time was 24 h for agr-deficient ST9 strains and 36 h for agr wild-type ST9 strains. Similarly, the median survival time for HL1 was 15 h, whereas that for HL1Δagr was 36 h, indicating reduced virulence in the absence of a functional agr system.
Virulence factors and pathogenicity islands carried by clinical ST9 MRSA strains
To better understand the virulence potential of the ST9 lineage, we conducted a comparative analysis of virulence genes and pathogenicity islands. As shown in Table 1, ST9 harbored only pathogenicity islands νSa4, νSaα, and νSaβ, while the PVL-containing ΦSa2 found in community-associated MRSA (CA-MRSA) clones ST59, ST1, and ST8 and the immune evasion cluster IEC-containing ΦSa3 found in ST1, ST8, and ST5 were absent in the ST9 clone.
TABLE 1.
Genomic pathogenic islands and phage carried by clinical ST9 straina
| Pathogenic island/phage | N29CSA11 (ST9) |
SA268 (ST59) |
LAC (ST8) |
MW2 (ST1) |
N315 (ST5) |
COL (ST250) |
|---|---|---|---|---|---|---|
| νSa1 | − | + | + | − | − | + |
| νSa2 | − | − | − | − | − | − |
| νSa3 | − | − | − | + | − | − |
| νSa4 | + | − | − | + | + | + |
| νSaα | + | + | + | + | + | + |
| νSaβ (carrying lukDE) | + | + | + | + | + | + |
| ΦSa1 | − | − | − | − | − | − |
| ΦSa2 (carrying PVL) | − | + | + | + | − | − |
| ΦSa3 (carrying IEC) | − | − | + | + | + | − |
+, present; −, absent.
Further analysis of the pathogenicity island structure through comparative genomics revealed that the νSaα structure in clinical ST9 strain N29CSA11 contained an insertion of SaPIbov4 (not publication) consistent with the νSaα structure reported in livestock-derived ST9 strains (8). This SaPIbov4 element carried a truncated vwbp gene and a homolog of scn with 50% sequence identity (not publication), although the function of this gene remains undetermined.
As shown in Fig. 3A, the global virulence gene profiles of ST9 strains were relatively conserved, with most genomes carrying lipase, hyaluronidase, metalloprotease, extracellular adherence protein, and α, β, δ, and γ hemolysins. However, most ST9 strains, except for those in cluster A, lacked the immune evasion cluster, including scn, chp, and sak. Enterotoxins were also rare among ST9 isolates. The νSaα structures in cluster A of the phylogenetic tree and some American strains did not have the SaPIbov4 insertion, whereas most strains isolated from China (cluster D) carried the SaPIbov4 insertion in νSaα, indicating a common origin for this cluster.
Fig 3.
Global distribution of virulence genes in the ST9 lineage and comparison of novel prophage island structures. (A) Global distribution of virulence genes in the ST9 lineage. From left to right, the chart represents the host, region, SCCmec typing, presence of SaPIbov4 integration in νSaα, and the distribution of virulence genes. Different colors represent specific meanings, as indicated in the legend on the left. (B) Structure of prophage φST9-A. (C) Structure of prophage φST9-B. (D) Structure of prophage φST9-C. Red represents the lysogenic region of the prophage island; green represents the DNA replication and metabolism region; blue represents the tail region of the prophage island; and pink represents the lysis region of the prophage island.
Novel prophage in clinical ST9 MRSA strains
To identify novel prophage islands in the ST9 genome, we selected strains from each clinical subclade according to their distribution in the phylogenetic tree, as constructed in our previous study (7), and performed long-read sequencing to obtain complete genomes. Comparative genomics of clinical strains with complete genome sequences identified prophages φST9-A and φST9-B in N08CSA36 (agr-deficient strain) and Prophage φST9-C in N29CSA02.
Prophage φST9-A was inserted between the yfkA gene (encoding an uncharacterized radical SAM protein) and a hypothetical gene. This prophage island contained regions for lysogeny, DNA replication, and metabolism, as well as DNA assembly, head, tail, and lytic regions. No known virulence genes were detected. No prophage island with an identical structure to Prophage φST9-A was found in the NCBI database, but it exhibited similarity to a prophage island in the ST398 genome (Fig. 3B).
Prophage φST9-B was inserted between the sufB gene (encoding an iron-sulfur cluster assembly protein) and a hypothetical gene. This prophage island also contained regions for lysogeny, DNA replication, and metabolism, along with DNA assembly, head, tail, and lytic regions. In the lysogeny region, we identified the gene tarP, a virulence gene discovered in 2018 that has been reported in prophage islands of some ST398 and ST5 strains (17). φST9-B displayed partial structural similarity to prophages from ST398 strain WBG8381 and ST5 strain NL1 (Fig. 3C).
Prophage φST9-C in strain N29CSA02 was inserted into the hlb gene (encoding β-hemolysin), the same insertion site of ΦSa3. No virulence genes were identified in this prophage. However, similar structures have been reported in livestock-associated ST5 strains (18) and in ST6324 (a single-locus variant of ST5) isolated from chickens in China (Fig. 3D).
These novel prophages shared structural similarities with prophages from LA-MRSA lineages, suggesting inter-lineage recombination events in livestock environments. Further analysis of the distribution of these three novel prophage islands in global ST9 strains revealed that these three newly identified prophage islands were exclusively found in a few ST9 strains from China, suggesting that these novel prophage islands are currently locally prevalent (Fig. 4).
Fig 4.
Global prevalence and distribution of the novel prophages in ST9 strains. From inside to outside, each ring represents host, region, SCCmec type, and presence of the three prophages. The specific meanings of different colors are provided in the legend on the left.
DISCUSSION
The accessory gene regulator (agr) system is a well-established quorum-sensing regulator in S. aureus, modulating the expression of numerous virulence factors (19). It upregulates several key secreted virulence proteins, including α-toxin, serine proteases, and lipases, while repressing the expression of surface-associated proteins, such as Spa (20). In our study, two ST9 strains harbored frameshift mutations in the agrA and agrC genes predicted to inactivate the agr system. Functional assays confirmed that these mutations led to a loss of RNAIII transcription, decreased α-toxin production, and impaired hemolytic activity. These phenotypic changes are consistent with agr inactivation and were validated through RT-PCR, western blotting, and erythrocyte lysis assays. Our results suggest that agr dysfunction in the ST9 lineage directly impairs the production of key virulence determinants and alters the virulence profile of these strains. It is important to note, however, that biofilm formation is a complex, multifactorial process influenced by various regulatory pathways. While our results suggest a potential link between agr dysfunction and enhanced biofilm formation in certain ST9 strains, agr is unlikely to be the sole determinant.
Previous studies have shown that agr mutant strains exhibit reduced infectivity in various infection models, including mouse skin infections, acute pneumonia, and rabbit endocarditis (21–23). This underscores the critical role of the agr system and its downstream virulence factors in S. aureus pathogenicity. However, previous studies on the virulence characteristics of the LA-MRSA ST9 lineage have mostly focused on its pathogenicity islands and virulence genes. For example, Yu et al. have proposed that the host shift of ST9 S. aureus from humans to animals involved the loss of IEC genes, such as scn, chp, and sak, which are typically present in human-adapted strains (8). In addition, sporadic cases have reported the presence of IEC-positive ST9 strains (6). However, the role of regulatory systems, such as agr, in the virulence of the ST9 lineage has not been elucidated. Our study demonstrates that agr-deficient ST9 strains lack α-toxin production, resulting in the loss of hemolytic activity and infection potential. These results indicate that, as an epidemic lineage of LA-MRSA, the virulence regulation mechanism of ST9 is similar to other common clinical lineages, with the agr system playing a pivotal role. Furthermore, comparisons with the hypervirulent CA-MRSA clone ST72 (10) revealed that the virulence level of ST9 is comparable to that of ST72, suggesting that ST9 poses a significant clinical threat.
Clinical reports have documented mutations in the agr system that result in regulatory dysfunction most commonly occurring in agrA or agrC, consistent with our findings in the ST9 strains (24). While agr dysfunction reduces bacterial invasiveness, clinical studies have indicated that agr dysfunction in S. aureus is associated with prolonged bacteremia and independently correlates with mortality in critically ill patients (25). Furthermore, agr deficiency has been linked to the downregulation of phenol-soluble modulins, contributing to persistent infections and complicating clinical treatment (26). Additionally, research on bacteria isolated from cystic fibrosis patients found that agr dysfunction could trigger a proinflammatory response (27). Thus, the agr system, as a global regulator in S. aureus, can still influence the progression and prognosis of clinical infections, even after inactivation. The emergence of agr-deficient clinical ST9 strains highlights the importance of monitoring virulence dynamics during infections caused by this lineage.
Prophages and the pathogenicity islands represent critical mobile genetic elements that encode and disseminate virulence determinants in S. aureus (3). The prophage islands in the ST9 genome differ significantly from those in common clinical clones, resulting in a different virulence gene profile. This suggests that ST9 may have distinct origins. Our previous phylogenetic and comparative genomic analyses of antibiotic resistance gene transmission suggested that clinical ST9 strains originated from livestock (7). In this study, we identified several novel prophage islands in clinical ST9 isolates that share structural similarities with those in livestock-associated ST398 and ST5, further supporting the hypothesis that clinical ST9 strains originated from livestock and underwent recombination with other lineages in livestock.
The tarP gene first reported in 2018 was initially found in prophage islands of ST398 and ST5 (17). It encodes a glycosyltransferase that replaces the function of TarS, altering the glycosylation site of wall teichoic acid, the major glycoantigen in S. aureus, thereby enhancing the bacterium’s ability to evade host defenses (17, 28, 29). It has been reported that tarP is also prevalent in other clones, such as CC1 and CC7, in China (30). In this study, we identified tarP in a prophage island of ST9 for the first time, suggesting a broader lineage distribution than previously recognized. The potential impact of tarP on S. aureus virulence and its epidemiological spread warrants further investigation.
This study has several limitations. First, although G. mellonella has been demonstrated to serve as an effective infection model in previous studies (31, 32), the absence of additional mammalian models limits the generalizability of our findings. Second, due to the multidrug-resistant nature of ST9 strains, we were unable to perform genetic complementation or knockout experiments to further elucidate the specific roles of the novel pathogenicity islands, the virulence factor tarP, and the agrA/agrC mutations. Thirdly, since the strains were obtained from a previous multicenter surveillance study, detailed clinical information was not available, preventing us from performing risk factor analyses or correlating agr dysfunction with patient outcomes. These limitations highlight the need for more comprehensive models and functional analyses in future research.
In conclusion, this study demonstrates the occurrence of agr dysfunction in clinical ST9 strains and establishes that agr is crucial for regulating the high virulence potential of the ST9 lineage. Additionally, it reveals that the ST9 lineage has acquired novel pathogenicity islands/virulence factors through genomic exchange in livestock. These findings elucidate the virulence characteristics of the ST9 lineage, underscoring the importance of monitoring its spread in clinical settings.
ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (grant numbers 82172308, 82472284, and 82302572).
During the preparation of this work, the authors used AI (ChatGPT 4) in order to refine the language of the article. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.
Contributor Information
Yan Chen, Email: chenyan@zju.edu.cn.
Lu Sun, Email: lusun@zju.edu.cn.
Min Yue, Zhejiang University College of Animal Sciences, Hangzhou, Zhejiang, China.
DATA AVAILABILITY
The complete genomes of the strains carrying novel prophage islands have been uploaded to the NCBI Genome database with accession numbers CP119571 and CP119341 for chromosomes N08CSA36 and N29CSA02, respectively. The novel prophage island structures have also been deposited in the NCBI GenBank database with accession numbers OQ578993, OQ578994, and OQ578995 for prophages φST9-A, φST9-B, and φST9-C, respectively.
ETHICS APPROVAL
This study was approved by the ethics committees in Sir Run Run Shaw hospital (Ethics account: 20240215-304).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The complete genomes of the strains carrying novel prophage islands have been uploaded to the NCBI Genome database with accession numbers CP119571 and CP119341 for chromosomes N08CSA36 and N29CSA02, respectively. The novel prophage island structures have also been deposited in the NCBI GenBank database with accession numbers OQ578993, OQ578994, and OQ578995 for prophages φST9-A, φST9-B, and φST9-C, respectively.