In this study, we investigated the impact of bacterial colonization and common infections on naturally occurring antibody levels in humans, with a specific focus on anti-αGal antibodies, using S. aureus as the model organism. Our findings suggest that nasal colonization by S. aureus is negatively associated with plasma concentrations of anti-αGal antibodies, whereas skin abscesses, primarily caused by S. aureus, are followed by increased levels of these antibodies.

TRIFMA was chosen over ELISA for antibody measurements due to its higher sensitivity, broader dynamic range, and improved signal stability [17, 36,37,38]. By utilizing lanthanide chelates as fluorescent labels, TRIFMA reduces background noise and enhances detection limits, making it particularly suitable for measuring low-abundance antibodies and detecting subtle changes over time. Additionally, TRIFMA offers a more stable readout with lower variability, ensuring greater reproducibility in our analyses.

The previously reported reactivity of anti-αGal antibodies with S. aureus [20] does not necessarily indicate that this organism expresses or displays αGal antigens. The αGal epitope is exceptionally rare in bacteria [39,40,41] and has not been detected in selected S. aureus strains [41]. A more plausible explanation for this reactivity is the synergistic polyreactivity exhibited by anti-αGal antibodies [1]. These antibodies consist of multiple clones with distinct specificities beyond their shared αGal reactivity. Through synergistic interactions, these clones achieve broad-spectrum antigen reactivity [1]. In pilot experiments, we have observed minimal binding to well-characterized mutants of the Reynolds strain [42] expressing capsule serotypes 5 or 8, suggesting that these capsular polysaccharides are not significant antigens for these antibodies (data not shown).

Our a priori hypothesis was that nasal S. aureus colonization would be associated with higher anti-αGal antibody levels due to increased exposure to the bacterium. However, contrary to this expectation, we found that nasal S. aureus colonization was associated with 35% lower plasma levels of anti-αGal antibodies compared to non-colonized individuals. Although this unexpected result could represent a type I statistical error, it appears robust given its consistency across stratified analyses. One potential explanation is that S. aureus colonization may actively reduce plasma anti-αGal antibody levels, either by inducing partial immune tolerance or by displacing other microorganisms that more strongly stimulate these antibodies. Since anti-αGal antibodies are thought to contribute to antimicrobial defense [1] and correlate with broader pathogen-reactive antibody levels [11, 12], another possibility is that higher levels of these antibodies confer protection against S. aureus colonization, or reflect underlying mucosal immunity. However, the cross-sectional design of this substudy precludes causal inference, and these interpretations should be considered speculative.

We observed that a single skin abscess event led to a 30% increase in plasma anti-αGal antibody levels within 187 days post-infection. The 187-day cutoff was chosen for statistical reasons, as it represents the median follow-up time, ensuring balanced group sizes and minimizing the impact of extreme values. Although not selected based on specific clinical considerations, it provides a standardized approach for assessing antibody responses post-abscess. Future research may explore optimal follow-up timing in relation to clinical outcomes. The 30% increase in plasma anti-αGal antibody levels may seem modest, but it is noteworthy given that only a small subset (in the order of 1%) of anti-αGal antibody clones is expected to exhibit polyreactivity toward a reactive but non-αGal-presenting pathogen targeted by these antibodies [1]. For such a small subset to account for the observed increase in total anti-αGal antibody levels, its concentration must increase disproportionately. Specifically, if this subset originally constituted 1% of the total anti-αGal antibodies, then a 30-fold expansion of this subset would be required to drive a 30% increase in the overall antibody pool. This calculation, detailed in Supplementary Table 4, highlights the potential magnitude of expansion required for a polyreactive subset to significantly influence total anti-αGal levels.

No statistically significant increase in antibody levels was observed in samples collected after 187 days, suggesting that the rise in anti-αGal antibodies is transient following a single exposure and subsequently declines. Additionally, non-skin abscesses, such as anal, oral, and deep neck abscesses, did not correlate with increased anti-αGal antibody levels. S. aureus is more rarely involved in such abscesses [26, 27, 43,44,45]. The pathogens causing these non-skin abscesses may lack reactivity with anti-αGal antibodies, which could explain why these infections did not result in elevated antibody levels. A limitation of this substudy is the lack of access to the actual pathogenic organisms, which prevented us from directly examining their nature or their reactivity with anti-αGal antibodies. Thus, we cannot rule out the possibility that pathogens other than S. aureus may have contributed to the induction of anti-αGal antibodies.

Our study also demonstrated that patients with recurrent skin abscesses had higher plasma levels of anti-αGal antibodies compared with controls, further suggesting that skin abscesses significantly influence anti-αGal antibody levels. Limitations in this substudy include its cross-sectional design, lack of case-control matching, and the small sample size for the stratified analysis of B-antigen-expressing individuals, with only two cases available for analysis. Conversely, our substudy of single abscess events in healthy individuals was more robust, utilizing paired samples taken before and after the abscess event. This design is unique and provides valuable insight. A limitation to that substudy is the reliance on registry-based abscess diagnoses without validation, though the registry used is considered highly reliable [33].

While this study focuses on bacterial exposures as drivers of naturally occurring antibodies, specifically IgG anti-αGal antibodies, we acknowledge that specific host-related factors may be of particular importance for the production of some naturally occurring antibodies, but not necessarily others. In the context of skin abscesses, S. aureus, and anti-αGal antibodies, atopy may be particularly relevant. Atopic individuals are at increased risk of S. aureus skin infections [46] and may also be more prone to producing IgE anti-αGal antibodies [47], which could be important in future investigations.

In conclusion, this study suggests that common infections help shape the repertoire of naturally occurring antibodies in humans. The observation of lower anti-αGal antibody levels in S. aureus nasal carriers might imply an association between reduced systemic anti-αGal levels and colonization. Further studies are warranted to clarify this relationship and to better understand the role of anti-αGal antibodies in mucosal immunity.