Outbreak Reports: A Foodborne Outbreak Associated with ST59-spa t441-SCCmec IVa Methicillin-resistant Staphylococcus aureus Producing Enterotoxins A and B — Puyang City, Henan Province, China, September 2024

Introduction: On September 16, 2024, the Puyang City CDC received a report of a suspected foodborne disease outbreak involving 14 individuals who developed nausea, vomiting, and diarrhea following attendance at a hotel banquet. Upon notification, the District CDC immediately deployed a specialized investigation team to characterize the epidemiological features of the outbreak, identify the causative pathogen, assess potential transmission risks, and implement effective control and prevention measures.

Methods: We integrated comprehensive on-site epidemiological investigations, clinical symptom analyses, and laboratory diagnostics to isolate and identify pathogenic agents from retained food samples, environmental specimens, and anal swabs collected from affected cases. The recovered isolates underwent enterotoxin-virulence-gene profiling, antimicrobial-susceptibility testing, and phylogenetic analyses. Additionally, we characterized the architecture of the enterotoxin-A-linked pathogenicity island vSaβ.

Results: A total of 4 S. aureus strains were successfully isolated from 22 leftover food samples, 2 environmental swabs, and 2 patient anal swabs. Contaminated donkey and goose meat was identified as the outbreak source. All isolates harbored sea and seb enterotoxin genes, exhibited PEN-OXA-ERY-CLI resistance patterns, and were identified as clonal ST59-spa t441-SCCmec IVa CA-MRSA strains. Phylogenetic analysis positioned the outbreak strains within the Asia-Pacific clade, distinguishing them from the North American ST59 sublineage. Comprehensive analysis of the sea-associated virulence island vSaβ identified a novel structural arrangement containing a type A IEC cluster (sea-sak-chp-scn).

Conclusions: The detection of foodborne ST59 CA-MRSA clones in this outbreak underscores the prevalence and transmission risks associated with this hypervirulent lineage. These findings emphasize the critical need to strengthen surveillance measures for CA-MRSA among food industry workers.

On September 16, 2024, a district CDC in Puyang City received notification of a suspected foodborne disease outbreak involving 14 individuals who developed nausea, vomiting, and diarrhea following attendance at a hotel banquet. Upon receiving this report, the District CDC immediately deployed a specialized investigation team to characterize the epidemiological features of the outbreak, identify the causative pathogen, evaluate potential transmission risks, and implement comprehensive control and prevention measures.

At 19:50 on September 16, 2024, the district CDC received notification from the Puyang City Health Commission regarding a suspected foodborne illness outbreak that had occurred during a hotel banquet at 12:00 that day. Investigation revealed that 70 individuals attended the banquet, of whom 14 subsequently developed illness. Among the affected individuals, 2 patients required hospitalization, 10 received outpatient treatment (2 with intravenous infusions and 8 with oral medication), and 2 with mild symptoms required no treatment. All patients exhibited symptom onset before 23:00 on September 16, 2024. The estimated incubation period was 4 hours, with detailed case onset times presented inSupplementary Table S1 and Figure S1. The affected population comprised 10 males and 4 females, with ages ranging from 6 to 55 years; notably, 71.4% of cases occurred in individuals aged 7–14 years. A total of 26 samples (Supplementary Table S2) were collected for laboratory analysis, including 22 leftover food samples, 2 environmental samples, and 2 anal swabs from patients, all submitted to Puyang CDC on September 17.

Rapid screening via multiplex fluorescence polymerase chain reaction (PCR) identified S. aureus-specific nucleic acids in 4 samples (Supplementary Table S2): 2 food samples and 2 anal swabs from patients (patient 1 and patient 5). Following the guidelines of GB 4789.10-2016 (National Food Safety Standards, Food Microbiology Inspection, Staphylococcus aureus Test) (1) and WS/T 80-1996 (Diagnostic Criteria and Principles of Management for Food Poisoning of Staphylococcus aureus) (2), these 4 samples underwent enrichment culture, strain isolation, Gram staining, and plasma coagulase testing. The suspected strains were confirmed as S. aureus through MALDI-TOF MS (Supplementary Table S2). Four S. aureus strains were isolated from the 4 positive samples. Fluorescent PCR assay identified the sea and seb enterotoxin genes in all 4 strains. Additionally, enzyme-linked immunosorbent assay (ELISA) detected the secreted forms of enterotoxins A and B in the culture medium (BHI) of these strains. Antimicrobial susceptibility testing using VITEK 2 Compact revealed that all 4 strains exhibited resistance to penicillin (PEN), oxacillin (OXA), erythromycin (ERY), and clindamycin (CLI). The results are presented in Figure 1A.

Figure 1. 

Distribution of antimicrobial resistance genes and virulence factors in four ST59 S. aureus isolates. (A) Heat maps display antibiotic susceptibility profiles across all strains; (B) Antimicrobial resistance gene distributions across all strains.

Note: White blocks indicate antibiotic susceptibility or gene absence, while colored blocks represent antibiotic resistance or gene presence.

Abbreviation: PEN=penicillin; OXA=oxacillin; GEN=gentamicin; Cipro=Ciprofloxacin; LEV=levofloxacin; Moxi=moxifloxacin; ERY=erythromycin; CLI=clindamycin; Q/D=quinupristin/dalfopristin; LZD=linezolid; VAN=vancomycin; TET=tetracycline; TGC=tigecycline; NIT=nitrofurantoin; RIF=rifampin; SXT=trimethoprim/sulfamethoxazole.

Whole-genome sequencing was conducted on 4 isolates: 3 strains underwent next-generation sequencing (NGS) analysis, while 1 strain (DC54089) was characterized using third-generation sequencing (TGS). The DC54089 genome comprised 3,444,506 bp (GC content: 36.05%) and contained 1 circular chromosome (harboring the sea and seb genes) along with 2 circular plasmids, collectively encoding 3,104 coding DNA sequences (CDS). All 4 strains shared identical origins and were confirmed as ST59-spa t441-SCCmec IVa clones, exhibiting matching antibiotic resistance and virulence gene profiles. Multiple resistance genes were detected, including mecA, ermB, blaZ, and APH(3')-IIIa (Figure 1B). Additionally, virulence genes encoding hemolysins, capsule synthesis proteins, adhesion factors, Panton–Valentine Leukocidin (PVL) toxin, exoenzymes, enterotoxins (sea, seb, seq, sek), and immune evasion cluster (IEC) components (scn, sak, and chp) were identified (Figure 2), demonstrating substantial pathogenic potential.

Figure 2. 

Virulence gene profiles and genetic variations across the four outbreak strains.

Note: White blocks denote gene absence, while colored blocks indicate gene presence. The horizontal color bar (left to right) represents genes associated with hemolysins, capsule formation, adhesion factors, PVL, secretion systems, exoenzymes, enterotoxins, and additional virulence determinants.

Abbreviation: PVL=panton-valentine leucocidin.

Core genome single nucleotide polymorphisms (SNPs) were identified across the 4 strains, and phylogenetic analysis was performed on the genomic sequences of these outbreak strains alongside 103 S. aureus ST59 reference strains using Snippy (version 4.6.0, Melbourne, Australia) (3). The phylogenetic tree (Figure 3) revealed that all outbreak strains isolated from food matrices clustered together with those from human samples within the same evolutionary branch (Clade China-II-I). Moreover, the staphylococcal food poisoning (SFP) strains from Henan Province formed a distinct branch (Clade Henan MRSA). Core-genome multilocus sequence typing (cgMLST) analysis demonstrated that strain DC54087 clustered tightly with the other 3 strains (DC54086, DC54088, and DC54089), differing by only a single allelic locus (≤10 alleles). These findings collectively confirm that the 4 outbreak strains originated from a common source. Detailed analysis of the sea-associated virulence island vSaβ revealed structural divergence compared to 6 reference ST59 SCCmec variants (Figure 4). Collinearity analysis using Easyfig (version 2.2.5, Brisbane, Australia) identified a type A IEC gene cluster (sea-scn-sak-chp) with partial deletions in the ΦSa3 gene. Two copies of scn and distinct rearrangements of the chp and scn loci revealed a novel gene sequence within the vSaβ region. Based on clinical symptoms, epidemiological evidence, laboratory findings, and genomic analysis, this outbreak was determined to be a foodborne illness caused by ST59-spa t441-SCCmec IVa MRSA producing enterotoxins A and B.

Figure 3. 

Phylogenetic reconstruction of the ST59 lineages.

Note: A total of 107 ST59 sequences (4 from this study and 103 from the NCBI database) were analyzed. The Henan MRSA ST59 isolates are shown in red. The reference genome is the completely sequenced ST59 MRSA isolate M013 (NCBI BioSample accession: CP003166). The filled squares indicate the presence of individual genes, while the empty squares indicate their absence, as specified at the top of each column. The clade ranges, sources, spa types, and SCCmec types of 107 strains have been presented on the left side.

Abbreviation: MRSA=methicillin-resistant Staphylococcus aureus; SCCmec types=Staphylococcal cassette chromosome mec types.

Figure 4. 

Comparison of genomic island (vSaβ) among ST59 isolates with different SCCmec types.

Note: Arrows and arrowheads represent open reading frames (ORFs) and the direction of transcription. The nucleotide similarities in the structure are indicated by grey shading. No. (4) represents the Henan representative strain DC54089.

Following these findings, the District CDC implemented comprehensive public health interventions: 1) conducted thorough disinfection of cold dish storage areas, cutting boards, and knives; 2) ensured safe disposal of all remaining food items; 3) mandated immediate self-inspection protocols within the affected hotel; 4) provided enhanced food safety training for all employees, emphasizing proper food processing techniques, rigorous hand hygiene practices, and effective disinfection protocols.

Staphylococcus aureus represents a clinically significant pathogen frequently implicated in foodborne intoxication events. This organism produces over 29 distinct staphylococcal enterotoxins (SEs) and staphylococcal enterotoxin-like (SEl) toxins. Among these, SEA, SEB, SEC, SED, and SEE constitute the most prevalent enterotoxins, collectively accounting for approximately 95% of SFP outbreaks globally (4).

The SFP outbreak documented in Puyang City resulted from contamination of donkey and goose meat (served as cold mixed dishes) with S. aureus enterotoxins A and B. This determination was substantiated through comprehensive epidemiological evidence, clinical symptom analysis, and laboratory findings. Multiple lines of evidence supported this conclusion: 1) all affected individuals shared exposure to a single hotel banquet; 2) symptom onset occurred synchronously within a 12-hour window (Supplementary Figure S1), consistent with the characteristic incubation period for SFP; 3) patients presented with uniform clinical manifestations, predominantly nausea and vomiting with or without diarrhea, which are pathognomonic for SFP; 4) multiplex fluorescence PCR and bacterial culture successfully identified S. aureus in specimens from two patients and two food samples. The four isolated strains demonstrated the capacity to produce enterotoxins A and B. Phylogenetic analysis of these SFP strain genomes (Clade Henan MRSA) alongside 103 ST59 sequences revealed that the outbreak strains clustered within Clade China-II-I, a lineage predominantly comprising strains of food origin. The two contaminated food items were identified as chilled donkey meat and chilled Cantonese-style roast goose. However, the absence of specimens from kitchen staff or the processing environment precluded definitive identification of the contamination source.

Currently, ST6, ST7, ST943, ST5, ST2315, ST15, ST59, and ST7591 represent the predominant causative strains of SFP in China, with most demonstrating methicillin sensitivity (5–8). Our investigation identified the ST59-t441 SEA/SEB-positive MRSA strain as the causative agent, marking the first documented SFP outbreak in Henan Province. This finding aligns with retrospective analyses demonstrating that ST59 constitutes the primary MRSA type responsible for food poisoning outbreaks across multiple Chinese cities, including Shijiazhuang, Suzhou, and locations throughout Sichuan Province (6–8). Furthermore, ST59 has emerged as the most prevalent CA-MRSA strain isolated from Eastern Asian communities and has established dominance in healthcare settings over the past two decades (9).

Phylogenetic analysis demonstrated that the SFP outbreak strains exhibited distant genetic relationships with North American sub-lineages while clustering closely with ST59 MRSA strains isolated from food and human samples throughout China, confirming their classification within the Asia-Pacific clone. Within the phylogenetic tree, most Chinese ST59 strains carried seb, whereas only strains within Clade China-II-I and North American sub-lineages harbored both sea and seb genes (Figure 3). All ST59-t441 MRSA isolates detected in this investigation carried the characteristic human IEC genes scn, sak, and chp, indicating their potential association with human hosts. Unfortunately, the absence of specimens from kitchen staff prevented determination of the outbreak source.

ST59 strains commonly exhibit elevated prevalence of the seb, seq, and sek genes. In this outbreak, the ST59-t441 MRSA strains harbored both sea and seb genes and actively secreted enterotoxins A and B in vitro. The synergistic action of these dual toxins likely accounts for the severe clinical manifestations and subsequent hospitalization observed in several patients. Structural analysis of the sea-associated virulence island vSaβ identified a previously unreported configuration featuring a type A IEC cluster (sea-sak-chp-scn). Consistent with earlier findings, genetic environment analysis indicated that the ST59-t441 MRSA strains from this outbreak lacked the characteristic νSaβ structure due to staphylococcal prophage ΦSA3 insertion (10). Within these ST59-t441 MRSA strains, certain ΦSA3-related genes were absent, while two copies of the scn gene were detected. The functional implications of these genomic alterations on strain pathogenicity and horizontal sea transfer warrant additional investigation.

The detection of foodborne ST59 CA-MRSA clones in this outbreak underscores both the widespread distribution and transmission potential of this hypervirulent lineage. To support comprehensive risk assessment efforts, we recommend strengthening surveillance protocols for CA-MRSA among healthy populations, with a particular emphasis on food industry workers.