Skin levels of toxic shock syndrome toxin-1 predict the severity of atopic dermatitis

To the Editor:

The skin of most patients with atopic dermatitis (AD) is colonized or infected with Staphylococcus aureus that produces superantigen exotoxins, including toxic shock syndrome toxin-1 (TSST-1).¹ Skin colonization by TSST-1–producing S aureus is associated with AD severity and chronicity.^2,3 However, production of TSST-1 by bacterial isolates in vitro may not reflect its presence on the skin.

To investigate whether presence of TSST-1 on the skin is associated with AD severity, we studied 45 patients: 5 with almost clear skin, 27 with mild AD, and 13 with moderate AD according to Eczema Area and Severity Index (EASI) score. S aureus was cultured from lesional skin of 14 patients, nonlesional skin of 7 patients, and both lesional and nonlesional skin of 13 patients, for a cumulative positivity of 34 of 45 samples (76%). Consistent with the results of a recent meta-analysis,⁴ the lesional skin swabs contained more S aureus colony-forming units (CFUs) than the nonlesional skin swabs (944 ± 526 vs 195 ± 188 [P = .01]). TSST-1 and/or staphylococcal enterotoxin B (SEB) cross-reactive superantigens (SEB, staphylococcal enterotoxin C, and staphylococcal enterotoxin [SE]-like U) were detected in skin swab washes from 27 of the samples 34 patients with culturable S aureus in their skin (skin S aureus–positive), but in none of the samples from the 11 patients without culturable S aureus in their skin (skin S aureus–negative). TSST-1 was detected in 12 of the 34 skin samples of the S aureus–positive patients (range 0.3-4.15 mg/mL; mean ± SD = 1.4 ± 0.5 mg/mL), and SEB cross-reactive superantigens were detected in the samples of 25 of the S aureus–positive patients (range 1.0-18.0 mg/mL; mean ± SD = 3.6 ± 1.3 mg/mL), with 10 patients having both superantigens. Of the 34 patients whose skin was positive for S aureus, 7 had neither superantigen (Fig 1, A). The presence of TSST-1 in skin swab washings was associated with significantly higher EASI scores than in the samples without TSST-1 (8.64 ± 4.25 vs 4.36 ± 1.19 [P = .027]) (Fig 1, B). There was no difference in EASI scores between patients with SEB cross-reactive superantigens in their skin swab washings and patients without such superantigens (6.83 ± 2.41 vs 5.08 ± 2.69 [P = .44]) (Fig 1, B). These results indicate that the presence of TSST-1 in skin swab washings of patients with AD is selectively associated with more severe disease.

FIG 1.

Association of TSST-1 in skin swab washings with disease severity in patients with AD and S aureus cultured from their skin. A, Presence of TSST-1 and/or SEB cross-reactive superantigens in skin swab washings from the skin of 34 S aureus–positive patients with AD. B, EASI scores of the skin S aureus–positive patients with AD with or without TSST-1 (left) or SEB cross-reactive superantigens (right) in skin swab washings Red dots represent the 10 patients whose skin washings were positive for both TSST-1 and SEB cross-reactive superantigens. Columns and bars represent means ± SEMs. *P < .05. ns, Not significant by the Student t test.

TSST-1 interferes with keratinocyte proliferation, delays healing of skin from infections, and is the superantigen most capable of traversing mechanical barriers and mucosal surfaces.^5-7 We examined whether S aureus and its products can be detected is the sera of patients with AD and whether TSST-1 in skin washings predicts their presence in the serum. No S aureus could be cultured from the sera of the 45 patients studied. Quantitative PCR analysis revealed the presence of DNA encoding the S aureus structural gene femA in the sera of 28 of 45 of the patients (62%), including 7 who lacked culturable S aureus in their skin. This may be an underestimate because the PCR assay may not detect small fragments of degraded S aureus DNA. The number of femA copies in sera of patients with AD was 194 ± 5/mL (range 6-1000/mL). Altogether, 41 of 45 patients with AD were positive for S aureus by skin culture and/or by the presence of femA in their blood, for an overall yield of 91% for S aureus presence when both methodologies were used versus 78% when lesional and nonlesional skin were cultured (Fig 2, A). Thus, serum measurement of femA level in combination with skin culture greatly increases the rate of detection of S aureus in patients with AD. Whether healthy S aureus carriers have femA needs investigation.

FIG 2.

Association of S aureus DNA in serum with TSST-1 in skin swab washings from patients with AD and S aureus cultured from the skin. A, Frequency of serum positivity for S aureus femA gene and culturable S aureus from lesional (L) and/or nonlesional (NL) skin alone or in combination in 45 patients with AD. B, Frequency of detection of DNA encoding superantigen genes in sera from the 28 patients with AD who were serum-positive for S aureus femA. C, TSST-1 and SEB cross-reactive (SEB-XR) superantigens in skin swab washings from with AD and culturable S aureus in their skin.

There were no differences in EASI scores between patients with femA in their serum (serum femA–positive) and those with no serum femA (serum femA–negative) (6.3 ± 2.2 vs 5.0 ± 1.7 [P = .42]) or between patients with S aureus–positive, serum femA–positive skin and patients with S aureus–positive, serum femA–negative skin (7.0 ± 2.6 vs 4.8 ± 2.1 [P = .25]). There was no significant difference between serum femA–positive patients and serum femA–negative patients as to S aureus skin load (1155 ± 730 vs 884 ± 950 CFUs in lesional skin [P = .67]; 85 ± 59 vs 26 ± 27 CFUs in nonlesional skin, [P = .15]). There was no correlation between the amounts of TSST-1 in skin swabs and EASI score or in the amounts of serum femA DNA.

The genes for TSST-1, staphylococcal enterotoxin A and the 6-membered enterotoxin gene cluster (which encodes staphylococcal enterotoxin G, and SE-like I, SE-like M, SE-like N, SE-like O, and SE-like U) were detected in all 28 S aureus–positive, femA-positive sera, whereas DNA encoding SE-like X was detected in 27 of these sera (Fig 2, B). This strongly suggests the presence of the S aureus clonal group USA200.⁸ Only 6 of the 28 femA-positive sera were positive for DNA encoding SE-like Q, a superantigen expressed by many (albeit not all) S aureus USA200 strains. This suggests subclonal variation in strains carried by different patients. Superantigen genes were not detected in the 17 sera negative for femA. Neither TSST-1 nor SEB cross-reactive superantigen proteins were detected in the 45 sera, but they were readily detected in culture supernatants of the S aureus USA200 strain MN8. Mass spectrometry failed to detect S aureus–derived peptides in 3 femA-positive sera tested.

TSST-1 and/or SEB-cross reactive superantigens were detected in the skin washings from only skin S aureus–positive patients; they were present in 95% of the skin samples from S aureus–positive, serum femA–positive patients versus in 54% of the skin samples from S aureus–positive, serum femA–negative patients (P = .007) (Fig 2, C), suggesting that the presence of S aureus superantigens in the skin and S aureus DNA is determined in tandem. All 12 patients with TSST-1 in their skin washings had S aureus DNA (femA) in their sera. Further, TSST-1 in skin washings was detected only among the 21 S aureus–positive, serum femA–positive patients, and it was present in 12 (57%) of them (Fig 2, C). Of the 25 patients with SEB cross-reactive superantigens in their skin washings, 10 also had TSST-1 in those skin washings. Of the 15 with SEB cross-reactive superantigens, but not TSST-1, in their skin washings, only 8 (53%) had S aureus DNA in their serum (Fig 2, C). Thus, the presence of TSST-1, but not SEB cross-reactive superantigens, in skin predicts the presence of circulating S aureus DNA in patients with AD. The fact that not all patients with superantigen-encoding DNA in their serum had superantigen in their skin swabs could be due to degradation of superantigens or to their presence in the sampled skin in quantities below the level of detection. Alternatively, sites other than the sampled ones may be source of the serum DNA.

In summary, TSST-1 in skin washings is a biomarker for more severe AD, and it predicts the presence of circulating S aureus DNA, including TSST-1 DNA, in patients with AD. Bacterial DNA binds to Toll-like receptor 9.⁹ Whether activation of Toll-like receptor 9–expressing B cells and plasmacytoid dendritic cells by circulating S aureus DNA contributes to AD severity requires investigation.

Abbreviations used

AD

Atopic dermatitis

CFU

Colony-forming unit

EASI

Eczema Area and Severity Index

SE

Staphylococcal enterotoxin

SEB

Staphylococcal enterotoxin B

TSST-1

Toxic shock syndrome toxin-1