Diagnostic pitfalls in Staphylococcus spp. surveillance: lessons from a multinational university student cohort

Background: Staphylococcus aureus is a leading cause of severe and hard-to-treat human infections, particularly when resistant to cefoxitin due to the presence of the mecA gene [1]. However, diagnostic challenges arise from the misidentification of S. aureus and related species when relying on classical identification methods (mannitol fermentation; coagulase production), as well as from the detection of strains carrying the mecA gene but phenotypically susceptible to cefoxitin - known as "stealth" strains [2]. Objective: Building upon a previous collection of S. aureus from healthy students’ nares [3], we aimed to expand this collection with new samples, assess the occurrence of “stealth” isolates, and further investigate cases of incongruent identification. Methods: Nasal swab samples (n=557) from 507 students (median-23-years; 9 countries) attending a large university (Porto district) were collected between March 2022 and November 2024. They were inoculated onto mannitol-salt agar and, in parallel, enriched in brain-heart-broth with 6.5% NaCl further plated onto ChromID® MRSA-SMART. Isolates deriving from mannitol-salt (only fermenting colonies) and chromogenic (all typical colonies) agar media were stored for species identification (MALDI-TOF MS), cefoxitin-susceptibility (disk-diffusion), and mecA gene screening (PCR). Results: Staphylococcus aureus was identified in 46% (256/557; 6 countries) of cases. Other Staphylococcus species included S. haemolyticus (n=5), S. capitis (n=3), S. warneri (n=3), S. saprophyticus (n=1), S. simulans (n=1), and S. ureilyticus (n=1). These isolates expressed variable coagulase production (7 positive, 7 negative). On another hand, a non-fermenting S. aureus was detected (cromogenic medium). Ten (1.8%) students were colonized with methicillin-resistant staphylococci species carrying mecA including S. aureus (n=6), S. haemolyticus (n=3), S. ureilyticus (n=1). The mecA gene was also detected in 4/16 (25%; 3 Portuguese, 1 Italian) S. aureus susceptible to cefoxitin, the so-called “stealth” strains. Screening is ongoing in more isolates. Conclusions: Our study highlights the importance of integrating both phenotypic and genotypic methods for Staphylococcus accurate identification. Furthermore, the detection of stealth strains in healthy students underscores the need for robust community-based screening, as S. aureus carriage may be underestimated. Future studies will unveil whether these strains are capable of reversion to resistance.