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Staphylococcus aureus nasal colonization increases the risk of cedar pollinosis

Abstract Background One‐third of the people in Japan are colonized with Staphylococcus aureus (S. aureus) and suffer from virulence factor‐mediated subclinical inflammation of the nares. We investigated whether subclinical inflammation contributed to cedar pollinosis affecting 20 million people annually. Methods The study participants were 814 inhabitants of the A or B prefectures. We compared the colonization rate and population structure of S. aureus, in association with the prevalence of cedar pollinosis, between participants in these two areas. Results A prefecture had twice the annual amount of airborne cedar pollen compared with B. The prevalence of cedar pollinosis was significantly higher in A (23.5%) than in B (13.1%) (p = 0.0004). Moreover, the prevalence of cedar pollinosis was higher in female participants (23.3%) than in male participants (14.7%) (p = 0.003). In addition, the prevalence of cedar pollinosis was higher in S. aureus carriers (24.2%) than in S. aureus noncarriers (17.9%) (p = 0.03). The isolation rate of clonal complex (CC) 508 was higher in the A group (21%) than in the B group (7%) (p = 0.015). Conclusion Nasal colonization of S. aureus is a major risk factor for cedar pollinosis. However, the direct mechanism of this risk is currently unknown.

Staphylococcus aureus superantigens are the strongest T-cell activators and are obtained by horizontal gene transfer under the regulation of restriction modification systems. 11 SauI (type I) is one of the major systems that regulate the acquisition of exogenous virulence genes, and one of the constitutive genes responsible for target specificity, which is hsdS, perfectly correlates with clonal complex (CC). If superantigens are involved in cedar pollinosis, correlation with CC is also expected.
The accessory gene regulator (agr), quorum-sensing system, regulates the expression of staphylococcal superantigens. 12 The agr locus is comprised of a four-gene operon, agrB, agrD, agrC, and agrA, and polymorphisms centering on the former three loci (agr specificity group: referred to as agr subgroup type I through IV) influence quorum-sensing dynamics and result in variant expression of virulence genes in S. aureus. 12,13 In the present study, first we epidemiologically investigated whether the colonization of S. aureus in the nares of inhabitants increases the prevalence of cedar pollinosis in the area. Then, we determined whether there was a correlation between agr type and cedar pollinosis and CC and cedar pollinosis.

| Study setting and design
Based on real-time airborne pollen data released by the Japanese Ministry of the Environment (http://kafun.taiki.go.jp), we selected two areas for our work: A and B prefectures. The former was an underpopulated area located on Shikoku Island, 89% of which consists of mountains. Most of the mountains (94%) are forested, and more than half of the forests (55%) are populated with Japanese cedar or, to a lesser extent, with another pollen producer, Japanese cypress.
Contrarily, the total area occupied by Japanese cedar and cypress forests in B prefecture is equivalent to 7% of that in A prefecture.
We conducted all of the studies in June and July, immediately following the end of the airborne pollen season of 2008.

| Participants and questionnaires
A total of 814 supermarkets' employees in both A (531 employees) and B (283 employees) prefectures participated in this study. We used written questionnaires, together with a verbal explanation of disease symptoms, to obtain data on gender, age, and history of cedar pollinosis (suffering from at least one of the following symptoms seasonally: runny nose, sneezing, blocked nose, itchy nose, itchy eyes, or watery eyes), and the duration of these symptoms. We include only current patients (people who have symptoms currently). Previous patients (people who do not have symptoms but had symptoms previously) were excluded.
Identification of S. aureus was performed using API Staph (Sysmex-bioMérieux, Tokyo, Japan), and detection of femA and B genes was performed using PCR. 11

| Genotyping by MLST and agr subgroup type
We performed multilocus sequence typing (MLST) according to the method described by Enright et al. 14 Sequence types were determined by accessing the website for multilocus sequence typing (http://www.mlst.net/). We performed agr subgroup typing according to the method described by Francois et al., with minor modification. 15 We confirmed the agr subgroup type Ⅳ by using these specific primers: agr4f: 5′-AGCCTATTCCTGTGTGCGACTT-3′, and agr4r: 5′-ACGATAATGCCGTAATAATACCCGT-3′.

| Statistical analysis
We compared the prevalence of pollinosis in various situations employing chi-squared (χ2) test, Student's t-test, and multiple regression analysis. All statistical analyses were conducted using SPSS version 19.0 (Statistical Package for Social Science, Inc., Chicago, IL, USA).

| Ethical disclosure
The study was approved by the ethics committee of our institution, and informed consent to use the data for clinical investigation was obtained from the participants. The investigation was conducted in accordance with the Declaration of Helsinki of 1975.

| Study participants
The participants of this study were 814 employees of 24 super-

| Rate of S. aureus colonization
The rate of S. aureus colonization in A female, B female, A male, and B male was 29.3%, 35.3%, 32.5%, and 34.4% (average: 31.9%), respectively, and a statistically significant difference was not observed between these values. The S. aureus isolation rate from pollinosis participants was 38.4% in A and 40.5% in B (p = 0.815).
3.5 | Association between the population structure of S. aureus and the prevalence of cedar pollinosis Table 2 presents the relationship between CC and prevalence of cedar pollinosis. There were particularly high and low prevalence depending on CC in participants, such as CC 30 (43%) and CC 5 (0%) (p = 0.005), but these were not statistically significant overall, and CC did not increase the incidence of cedar pollinosis. Table 3 presents the relationship between agr type and prevalence of cedar pollinosis. None of the agr types increased the incidence of cedar pollinosis (p = 0.963). Table 4 presents the population structures of 208 S. aureus strains.

| Association between population structure of S. aureus and area
We found genotypic diversity of S. aureus in both areas with more than 18 CCs observed. Except for CC508, which had a higher isolation rate in A (21%) than in B (7%) (p = 0.015), no significant dif-

ference was observed in the composition of CCs between A and B
prefectures. We observed no significant difference in the composition of agr types between A and B prefectures (data not shown).

| DISCUSS ION
In this study, we investigated the prevalence of cedar pollinosis in two areas. Living in the A prefecture was a major risk of cedar pollinosis, compared to those in the B area. The prevalence of cedar pollinosis in the research area was proportional to the amount of cedar pollen.
More than half of the people sensitized serologically by cedar pollen do not develop cedar pollinosis. 3 Some additional stimulation, such as atrophic nasal change caused by hormonal imbalance in perimenopausal women, may contribute to cedar pollinosis progression. 16 In fact, pollinosis was more frequently found in women than men (p = 0.03). Subclinical nasal inflammation caused by S. aureus colonization may contribute to cedar pollinosis progression. 11,17,18 Here, we have reported that colonization of S. aureus in the nares is a risk factor for cedar pollinosis (p = 0.03). We hypothesized that the rates of S. aureus colonization would be higher in A participants or female participants; however, the rates were not significantly different.
We analyzed the association between the population structure of S. aureus and the prevalence of cedar pollinosis. We found no difference in the strength of risk among CCs. It is postulated that among these diverse S. aureus lineages, certain clones, such as CC 30, found in S. aureus carriers are more likely to cause invasive diseases than others. 19 13 However, contrary to our expectations, the present data indicated that all agr strains have approximately the same frequency of cedar pollinosis. We did not observe an agr type that exhibited a special correlation with cedar pollinosis. Taken together, we did not observe different levels of risk between the strains.
We analyzed the association between the population structure of S. aureus and area. The population structure of S. aureus is extremely diverse, [23][24][25] and CC profiles exhibit a geographical feature. structure of S. aureus and the prevalence of cedar pollinosis in more areas. Our goal is to identify S. aureus lineages that actually pose an allergic threat.

ACK N OWLED G M ENTS
We thank Dr. Yoshiaki Suzuki for their critical reading of the manuscript and Mrs. Kumi Kiyooka for her consult to look for participants in this study.

CO N FLI C T O F I NTE R E S T
The authors have stated explicitly that there are no conflicts of interest in connection with this article.