This study was conducted at the bacteriology unit of CHU Angré.

This study analyzed stored clinical isolates from routine inpatient and outpatient samples collected at the Angré University Hospital between January 2022 and February 2024. All unique, non-duplicate isolates phenotypically identified as Staphylococcus aureus in the microbiology laboratory were eligible. Identification was confirmed by PCR targeting the nuc gene (and the NRPS marker, when applicable).

The exclusion criteria were screening/colonization specimens, environmental isolates, duplicates (per repository records), uncertain identification, and obvious contaminants. Isolates were preserved either as deep nutrient agar stabs at room temperature or at –80˚C in Brain Heart Infusion (BHI) broth supplemented with 10% glycerol.

Since 2020, the laboratory has archived approximately 100 S. aureus isolates per year (with more pronounced losses of viability in 2020). For this study, inclusion was restricted to 2022-2024 because deep nutrient agar preservation from these years was better maintained, yielding higher viability and fewer losses at subculture. Isolates were selected consecutively, without replacement, following the chronological order of archival entry until 200 unique clinical isolates were reached (100 MRSA and 100 MSSA). One isolate per patient/clinical episode was selected. If an isolate was non-viable on subculture or its record was entirely missing, the next one in chronological order was included. Selection was not conditioned by ward, and the distribution of wards among the included isolates broadly mirrored that of the available archives, indicating no notable ward-related bias. A 1:1 (MRSA/MSSA) scheme was retained, consistent with the proportions observed in the laboratory database, where methicillin-resistant and methicillin-susceptible isolates were relatively balanced and available during 2022-2024. The isolates originated from diverse clinical specimens, including wound swabs, blood, pus, urine, and puncture/aspiration fluids (ascitic and pleural).

The collected strains were grown on nutritive and Chapman agar and incubated at 37˚C for 24 h. Preliminary identification of the strains was performed using Gram staining and catalase, DNase, and coagulase tests, supplemented by the VITEK® 2 automated system (bioMérieux, Marcy-l’Étoile, France). Preliminary identification was confirmed by molecular identification targeting the nuc gene (species-specific) and NRPS molecular marker [10] [11] by conventional PCR. Reference strains S. aureus ATCC 43300 (MRSA) and ATCC 25923 (MSSA) were used as controls. Primary culture was performed on nutrient agar and Chapman agar and incubated at 37˚C for 24 h. Initial identification relied on Gram staining and catalase, DNase, and coagulase tests, complemented by the VITEK® 2 automated system (bioMérieux, Marcy-l’Étoile, France). The identification of S. aureus was confirmed by conventional PCR targeting the nuc gene (species-specific) and the NRPS molecular marker [10] [11] . Reference strains S. aureus ATCC 43300 (MRSA) and ATCC 25923 (MSSA) were used as controls.

Severity was operationalized as a binary outcome (severe versus non-severe). An episode was classified as severe if at least one of the following criteria was met: admission or transfer to the ICU or critical care; documented Staphylococcus aureus bacteremia or sepsis, defined as explicit mention in records, a positive blood culture associated with initiation of antistaphylococcal therapy, or at least two concordant positive blood cultures (excluding single positive bottles judged as contaminants or not treated and followed by a negative control); evidence of a deep or invasive infection such as pneumonia, endocarditis, osteoarticular infection, deep abscess, necrotizing fasciitis, meningitis, or pyelonephritis; requirement for drainage or surgery related to infection, including abscess evacuation, debridement, or hardware placement or revision; adverse outcomes such as death during the episode or explicit documentation of septic state, severe sepsis, or septic shock; or persistent fever, defined as either explicit mention in records or temperature ≥ 38˚C recorded on at least two occasions over 72 h despite antimicrobial therapy. Episodes that did not meet any of these criteria were classified as non-severe.

Models were adjusted for covariates reliably available in the Laboratory Information System (LIS): methicillin-resistance status (MRSA/MSSA), specimen site (grouped as blood vs. other), and an age proxy based on the ward of care, with two categories: pediatrics/neonatology and adult services (internal medicine/geriatrics, surgery/traumatology, emergency/ICU, pulmonology, and other wards). This proxy was chosen because the ward is exhaustively recorded and closely reflects the age distribution at our institution. Information on immune status and prior antibiotic exposure was not captured consistently enough for valid adjustment and was, therefore, excluded.

Methicillin resistance was assessed using two approaches.

Agar diffusion method: A bacterial suspension equivalent to 0.5 McFarland was spread on a Mueller-Hinton agar and a cefoxitin disc (30 μg) was applied. After incubation at 37˚C for 18 - 24 h, the inhibition diameters were measured and interpreted according to the recommendations of the Antibiogram Committee of the French Society of Microbiology (CA-SFM, 2024). A diameter ≤ 22 mm was interpreted as indicative of an MRSA strain, while a diameter ≥ 22 mm indicated an MSSA strain [12] .

Automated method: In parallel, a few isolates were analyzed using the VITEK® system (bioMérieux, Marcy-l’Étoile, France), which provides a sensitivity profile, including methicillin resistance detection.

To evaluate the virulence potential of the Staphylococcus aureus isolates, conventional PCR targeted the PVL locus (lukS-PV/lukF-PV) and the tsst1 gene. PVL detection used the primer set (luk-PV-1/luk-PV-2); tsst1 was amplified with the corresponding tsst1 primer pair. Primer details and expected amplicon sizes are provided in Table 1 .

Genomic DNA was extracted from fresh cultures grown on nutrient agar using the Biofact ® DNA/RNA Amplification Kit (Biofact, South Korea) according to the manufacturer’s instructions. DNA extracts were quantified by spectrophotometry using the A260/A280 ratio and stored at −20˚C for polymerase chain reaction (PCR).

The pvl and tsst1 genes were detected using conventional PCR. Amplifications were run on a CFX96 thermocycler (Bio-Rad) using the HOT FIREPol® Master Mix 5× (Solis BioDyne, Tartu, Estonia), a ready-to-use mixture containing DNA polymerase, dNTPs, Mg²⁺ and reaction buffer. Each reaction was set up in a final volume of 22 µL containing 4 µL of HOT FIREPol® Master Mix 5×, 0.75 µL of each primer (10 µM), 3 µL of template DNA, and 13.5 µL of nuclease-free water.

Thermal cycling comprised an initial denaturation step, followed by cycles of denaturation, annealing, and extension, and a final extension for each target ( Table 2 ). PCR products were separated on 1.5% agarose gels containing SYBR Safe® (Invitrogen®) at 100 V for 30 min, and bands were interpreted according to their expected sizes.

Data were entered and analysed using Excel and R. Associations between variables were assessed using the chi-squared test, and a p-value < 0.05 was considered statistically significant.

This study used isolates stored from routine diagnostic samples collected at the Angré University Hospital. No data were collected that could be used to determine the patients’ personal data, and all information was processed in an anonymized form. In accordance with current regulations, formal ethical approval was not required.

A total of 200 isolates were included, with equal representation of MRSA and MSSA strains. Table 3 summarizes the general characteristics of these isolates.

Continued

*Subset computed only for isolates with available age data (n = 110).

The 200 clinical Staphylococcus aureus isolates were evenly split between MRSA and MSSA, with most originating from adult wards. Blood was the predominant specimen type, and severe presentations were relatively uncommon (16% of cases). Age data were available for just over half of the isolates; within this subset, cases more often involved patients aged < 15 years than ≥15 years. Overall, pvl (72%) and tsst1 (60.5%) were frequent, underscoring the high burden of virulence determinants in this collection.

Detection of virulence genes by PCR ( Figure 1 ) revealed a high prevalence of pvl and tsst1 genes.

The distribution of the combined patterns was as follows: 54% of isolates (n = 108) were pvl⁺/tsst1⁺, 21% (n = 42) pvl⁺/tsst1⁻, 14% (n = 28) pvl⁻/tsst1⁺, and 11% (n = 22) pvl⁻/tsst1⁻.

The isolates were grouped into four clinical categories: pediatrics/neonatology, adult medicine, surgery/traumatology, and critical care (including ICU, pulmonology, and other high-acuity units). Pediatrics/neonatology contributed 38.0% of isolates (76/200), of which 40.8% were pvl-positive (31/76) and 48.7% were tsst1-positive (tsst1⁺) (37/76). Adult medicine accounted for 26.5% (53/200), with 81.1% pvl-positive (pvl⁺) (43/53) and 47.2% tsst1-positive (25/53). Surgery/traumatology represented 23.5% (47/200), with 89.4% pvl-positive (42/47) and 74.5% tsst1-positive (35/47). Critical care comprised 12.0% (24/200), and all isolates in this group were double positive (pvl⁺/tsst1⁺, 24/24).

Isolates were classified into two categories based on the suspected clinical severity of infection at the time of collection, according to the information available on the accompanying sheets ( Table 3 ).

PVL positivity was higher in severe than in non-severe episodes (90.09% [30/32] vs. 30.77% [64/137]; p = 0.0002). It was also more frequent in adults than in pediatric patients (88.9% [48/54] vs. 40.78% [31/76]; p = 0.002). No significant differences were observed in pvl rates between specimen types (blood vs. other: 71.2% [99/139] vs. 82.0% [50/61]; p = 0.117). pvl co-occurrence with tsst1 did not differ meaningfully (pvl⁺ among tsst1⁺ vs. tsst1⁻: 76.1% [70/92] vs. 68.2% [30/44]; p = 0.406).

For tsst1, no significant associations were found with severity (59.1% [19/32] vs. 45.5% [77/168]; p = 0.180), methicillin resistance (71.0% [49/69] vs. 64.2% [43/67]; p = 0.465), ward (adults vs. pediatrics: 64.8% [35/54] vs. 69.5% [57/82]; p = 0.580), or specimen type (67.0% [93/139] vs. 68.9% [42/61]; p = 0.870).

The pvl gene was detected in 75% of MRSA (75/100) and 69% of MSSA (69/100) cases, with no significant difference between the groups (p = 0.345). The tsst1 gene was found in 64% of MRSA (64/100) and 57% of MSSA (57/100) and was not significant (p = 0.311). Combined pvl/tsst1 profiles are summarized separately for MRSA and MSSA ( Figure 2 ).

Distribution of virulence gene profiles (pvl and tsst1) among Staphylococcus aureus isolates according to methicillin resistance (MRSA vs. MSSA).

Four profiles were identified: doubly positive (pvl⁺/tsst-1⁺), pvl⁺/tsst-1⁻, pvl⁻/tsst-1⁺ and doubly negative (pvl⁻/tsst-1⁻). The pvl⁺/tsst-1⁺ profile was prevalent in MRSA (64%) and MSSA (50%) isolates, respectively. The pvl⁺/tsst-1⁻ profile was more common in MSSA (23%) than in MRSA (16%), whereas pvl⁻/tsst1⁺ accounted for 12% of MRSA and 16% of MSSA. The pvl⁻/tsst-1⁻ profile was the least represented, especially in MRSA (8%) compared with MSSA (11%).

This study is part of local efforts to molecularly characterize isolates within the Staphylococcus aureus complex, highlighting the distribution of pvl and tsst1 virulence genes in stored strains. We observed a high prevalence (pvl: 72.0%; tsst1: 60.5%), in agreement with several studies from sub-Saharan Africa, where high frequencies of pvl have been reported, for example, in Ghana (75%) [15] and The Gambia (77%) [6] , while exceeding the values observed in Senegal (47%) and in several countries included in the multicenter study by Breurec et al. We observed high prevalence (pvl: 72.0%; tsst1: 60.5%), in agreement with several studies from sub-Saharan Africa where high frequencies of pvl have been reported, for example in Ghana (75% ) and The Gambia (77% ), while exceeding the values observed in Senegal (47%) and in several countries included in the multicentre study by Breurec & al. (2011) [16] . Conversely, in Europe and Asia, pvl frequencies are often lower (≤30%), with marked local variability (Greece: 19% pvl⁺ among S. aureus; China: 20%) [17] [18] ). These discrepancies may reflect, on the one hand, the composition of our sample (inpatients and outpatients), which indicates co-circulation of community and nosocomial strains, and, on the other hand, irregular access to care in our setting, which may favour the spread of strains carrying virulence genes. They also fit within heterogeneous epidemiological contexts, as highlighted by Shallcross et al. They also fit within heterogeneous epidemiological contexts, as highlighted by Shallcross et al. (2013) [19] . In addition, the circulation of mobile genetic elements (prophages, pathogenicity islands, and plasmids) can facilitate the acquisition and sometimes co-occurrence of virulence determinants [20] [21] . In fact, 54% of our isolates were pvl⁺/tsst1⁺, an unusual and rare profile internationally but already described in Pakistan [22] , suggesting that some local lineages may combine these factors.

Distribution by clinical ward showed an over-representation of pvl⁺/tsst1⁺ profiles in critical care units, where this profile reached 100% of isolates, as well as high rates in surgery/trauma wards (89.3%). This concentration in high-risk areas is consistent with the role of S. aureus toxins in severe forms (deep abscesses, necrotizing pneumonia, complicated infections), while recognizing that clinical virulence results from a set of bacterial and host factors [2] [4] . In adult medicine, the pvl gene was present in 81.1% of isolates, indicating substantial circulation of virulent strains outside surgical departments, whereas tsst1 was less frequent (47.1%), possibly reflecting greater lineage diversity or lower selective pressure for this gene in that context. In pediatrics and neonatology, which accounted for most samples, the rates were more moderate for PVL (40%) and tsst1 (48.6%); the relatively high prevalence of tsst1 in this young population with fragile immune systems raises questions about superantigen-specific interactions, even without proven toxic shock [5] . Overall, the critical care, surgery, and adult medicine departments concentrated pvl⁺/tsst1⁺ profiles, reinforcing the hypothesis that particularly virulent strains contribute to severe S. aureus infections [23] [24] , whereas the more balanced distribution observed in pediatrics suggests greater genetic diversity of strains and variability in pathogenic potential according to department and patient profile.

Our results revealed that a significant proportion of Staphylococcus aureus strains carrying the pvl gene were present among both susceptible (MSSA) and resistant (MRSA) isolates. This contrasts with data reported in Abidjan by Kacou-N’Douba et al. (2011), where, although pvl was present in more than 60% of samples, no MRSA strain carried pvl [3] . The current co-circulation of pvl⁺ MRSA observed in our study may reflect local epidemiological evolution, possibly related to the introduction and/or selection of more virulent clones. This aligns with patterns documented elsewhere, including the introduction of pvl⁺ isolates into hospitals with subsequent local dissemination [25] , and the establishment of pvl⁺ community lineages in West Africa [26] . Recent molecular epidemiology studies from the region support this observation: in Ghana, a multicenter whole-genome sequencing study reported a high pvl prevalence (65% overall, 84% in MRSA), largely dominated by ST152 [27] , while in Nigeria, pvl genes were detected in 12.5% of isolates from soft-tissue infections, in both MRSA and MSSA, with recurrent infection independently associated with pvl carriage [28] .

In our cohort, pvl and tsst1 remained common regardless of MSSA/MRSA status; although they were slightly higher in MRSA, the differences were not statistically significant. This lack of a formal association suggests that, in our context, the dissemination of these virulence genes does not directly depend on methicillin resistance. Internationally, the link between pvl and resistance is heterogeneous and varies by region. In Nigeria, pvl is mainly carried by MSSA, although pvl⁺ MRSA are also present [29] . In Gaza (Palestine), pvl was detected in both groups at comparable frequencies (MRSA, 30.5%; MSSA, 28.2%) [30] .

The differences between studies can be explained, at least in part, by local epidemiological factors and the circulation of lineages combining resistance and virulence. Without typing (spa/MLST) in our study, we remain cautious and do not propose clonal attribution. As a hypothesis supported by African literature, a recent synthesis reported a continent-wide increase in the distribution of ST1, ST22, and ST152 clones [26] , the latter of which is frequently pvl⁺ [31] . This hypothesis is further reinforced by the Ghanaian findings, where ST152 accounted for the majority of pvl-positive MRSA, suggesting the regional expansion of this epidemic lineage [27] . The absence of differences across specimen types suggests that pvl functions mainly as a general virulence determinant, acting across multiple infection sites, and is more closely linked to the clonal background than to the site of isolation [19] .

Clinically, we observed a significant association between pvl and disease severity. This finding is consistent with reports linking pvl to deep-seated skin and soft tissue infections (dSSTIs) and necrotizing pneumonia [13] [32] [33] . However, pvl-positive isolates are also found in mild infections, underscoring that severity depends on multiple host- and site-related factors [1] [19] . The higher pvl frequency in adults than in pediatric patients should be interpreted with caution, since the ward (adults vs. pediatrics) was used as a proxy for age due to missing data. This approach may lead to misclassification, which usually attenuates, rather than generates, associations. Therefore, the observed signal is plausible; however, confirmation using actual age data is required. In contrast, tsst1 was not associated with disease severity in the present study population. This aligns with the notion that tsst1 is mainly a marker of a specific syndrome (toxic shock) rather than a consistent predictor of severity across all S. aureus infections [24] [34] . Finally, the co-occurrence of pvl and tsst1 alone is not sufficient to establish a systematic link with methicillin resistance or severity outside the targeted syndromes.

This study had some limitations. First, the isolates were obtained from a single hospital, which limits the generalizability of the findings to the entire country. Second, some clinical data linked to the isolates were incomplete, constraining the assessment of risk factors (prior antibiotic exposure, immune status, and comprehensive resistance profiles). Finally, our analysis focused on two major virulence genes (pvl and tsst1) without in-depth molecular typing namely MLST (multilocus sequence typing of seven housekeeping genes to assign sequence types and clonal complexes), spa typing (sequencing the polymorphic X region of the spa gene to derive t-types), and SCCmec characterization (defining the staphylococcal cassette chromosome mec that carries mecA/mecC and ccr genes) which would have refined the characterization of circulating lineages and their epidemiological potential.

Despite these constraints, the high prevalence of virulence determinants suggests an elevated risk of invasive disease and severe complications in the future. In settings with limited diagnostic and therapeutic resources, the circulation of virulent and potentially methicillin-resistant strains poses major challenges for patient care and safety.

From a public health perspective in Côte d’Ivoire, these findings highlight the need for strengthened microbiological surveillance of S. aureus that integrates both resistance and virulence markers. Priorities include reinforcing hospital laboratory capacity (basic molecular biology), establishing a small sentinel network to monitor MRSA and virulence genes with regular reporting, incorporating S. aureus indicators into infection prevention programs, and providing targeted training on early diagnosis, isolation, and antimicrobial stewardship. Finally, a One Health approach should be considered, taking into account community reservoirs and possible zoonotic sources of infection.