Correspondence: Atsushi Harimaya, Department of Otolaryngology, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo, Hokkaido 060-8543, Japan. Tel.: +81 11 611 2111, ext. 3491; fax: +81 11 615 5405; e-mail: email@example.com
Alloiococcus otitidis is a recently discovered bacterium frequently associated with otitis media. However, no study is available as to whether A. otitidis has a pathogenic role and induces local immune response in the middle ear as a true pathogen. Whole bacterial sonicate of A. otitidis was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane. Then, Western blot analysis was performed with supernatant of the middle ear effusions from children with A. otitidis-positive otitis media. SDS-PAGE of the bacterial sonicate showed several protein bands, designated A1-A11. Western blot analysis revealed the presence of IgG, secretory IgA, IgG2, and IgM against A. otitidis in the middle ear effusions. Absorption of the specimens with sonicates of other major middle ear pathogens did not alter the reactivity of antibodies against the alloiococcal antigens. The results suggest that specific local immune response against A. otitidis is induced during middle ear infection of the organism as a true pathogen. A5, A6 or A11 is expected to be a main antigenic determinant. This is the first report to show evidence of local antibody response against A. otitidis and to disclose antigenic components of A. otitidis.
However, it has been unclear whether A. otitidis has a pathogenic role in otitis media, and also whether local immune response against the organism is induced in the middle ear cavity or not. In order to clarify these unknown problems, we investigated whether local antibody response is induced in the middle ear cavity during otitis media due to A. otitidis.
Materials and methods
Patient and middle ear effusions
Four A. otitidis-positive specimens were randomly selected from 40 middle ear effusion specimens from children with acute otitis media which had been collected in our previous study (Harimaya et al., 2006b). In all of the specimens, we had already confirmed that any bacteria other than A. otitidis was not detected by neither culture nor PCR which was planned to detect the other major middle ear pathogens (S. pneumoniae, H. influenzae and M. catarrhalis) (Harimaya et al., 2006b). The specimens were from four children with acute otitis media (two males and two females) who ranged in age from 2 to 8 years, with a median age of 6.5 years. Written informed consent was obtained from the parents of all the children. All of the specimens were obtained during myringotomy which was performed as the treatment for acute otitis media. Acute otitis media were diagnosed on signs of inflammation of the tympanic membrane, the presence of middle ear effusion, and symptoms of otalgia, tugging at or rubbing of the ear, fever or irritability.
The external ear canal was disinfected with povidone-iodine before myringotomy. After myringotomy, middle ear effusions were aspirated with Tym-Taps (Xomed-Treace, Jacksonville, FL). The specimens were diluted up to 1 mL by phosphate-buffered saline, and centrifuged at 3000 g for 15 min. The precipitation was used for detection of pathogens as previously reported (Harimaya et al., 2006b), and the supernatant was stored at −80°C until used for immunostaining of Western blot analysis.
Western blot analysis
A strain of A. otitidis (NCFB2890, American Type Culture Collection, Manassas, VA), which was originally isolated from the middle ear of children with otitis media, was used. The bacterium was grown in Brucella broth containing 10% (v/v) horse serum, and the bacterial cells were disrupted by sonication. Then, the whole bacterial sonicate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane in a semidry transfer cassette (Bio-Rad, Hercules, CA). After the transfer, the membrane was blocked in 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h, and cut into strips. Each strip was incubated with a middle ear effusion specimen (diluted 1 : 1000 in TBS-T) for 1 h at room temperature. Because cross-reacting antibodies might be present in the specimens, absorption procedure was performed before use, to investigate antibodies specific for A. otitidis. As the absorption procedure, the middle ear effusions had been pretreated with 1 mg mL−1 proteins of whole bacterial sonicate of other major middle ear pathogens (S. pneumoniae, H. influenzae, M. catarrhalis and Staphylococcus aureus) for 30 min. After washing with TBS-T, the strip was incubated with alkaline phosphatase (AP)-conjugated goat antihuman IgG (Promega Corp, Madison, WI), AP-conjugated goat antihuman IgA (Sigma, Saint Louis, MO), AP-conjugated goat antihuman IgM (Sigma), mouse antihuman secretory component (Sigma), or mouse antihuman IgG2 (Sigma) for 30 min. For the last two, washing with TBS-T and incubation with AP-conjugated goat antimouse IgG (Promega Corp) for 30 min was added. The blot was detected with the color substrate, BCIP/NBT (Promega Corp).
Separation of whole bacterial sonicate by SDS-PAGE
Whole bacterial sonicate of A. otitidis was separated by SDS-PAGE as shown in Fig. 1. Several bands of bacterial components were observed. Clearly visible bands were designated A1 (>107 kDa), A2 (>107 kDa), A3 (100 kDa), A4 (90 kDa), A5 (60–70 kDa), A6 (50–60 kDa), A7 (50 kDa), A8 (40–50 kDa), A9 (40–50 kDa), A10 (35 kDa) and A11 (30 kDa), from high to low molecular weight (Fig. 1).
Detection of immunoglobulins against A. otitidis in middle ear effusions
We firstly investigated the presence of IgG against A. otitidis in middle ear effusions from children with otitis media. Western blot analysis with monoclonal antihuman IgG as secondary antibody showed several bands (Fig. 2a). Mainly, two bands corresponding to A5 and A6 were clearly observed. In addition, other bands corresponding to A1/A2, A4 and A8/A9 were also faintly recognized (Fig. 2a). These bands were similarly detected even after the absorption of the middle ear specimens with bacterial sonicate of the other major pathogens to exclude cross-reacting antibodies. In contrast, these bands were weakened when the specimens had been absorbed with Alloiococcal sonicate (data not shown), showing that the bands were specific for A. otitidis.
We next investigated the presence of secretory IgA against A. otitidis in the middle ear specimens. Western blot analysis with monoclonal antihuman IgA and monoclonal antihuman secretory component, was performed to detect secretory IgA. With antiIgA antibodies, the analysis showed three bands corresponding to A4, A5/A6 and A11 (Fig. 2b). With antisecretory component antibodies, the analysis showed two bands corresponding to A5/A6 and A11 (Fig. 2C). As a result, two bands corresponding to A5/A6 and A11 were recognized by both anti-IgA and antisecretory components (Fig. 2b and c). These results suggest that secretory IgA against A5/A6 or A11 is present in the middle ear cavity.
The presence of IgG2, which is a subtype of IgG and which plays an important role in otitis media (Dhooge et al., 2002), was then investigated. Western blot analysis with antihuman IgG2 antibodies showed three bands corresponding to A4, A5/A6 and A11 (Fig. 2d). The presence of IgM was also investigated. Western blot analysis with antihuman IgM antibodies disclosed a band corresponding to A4 (Fig. 2e).
Although A. otitidis is frequently detected in otitis media cases (Hendolin et al., 1997; Beswick et al., 1999; Leskinen et al., 2002; Harimaya et al., 2006b), it has been unclear whether this organism has enough pathogenic potential to induce otitis media. Even if A. otitidis is frequently detected in middle ear effusions, the bacterium may be one of the normal flora in the middle ear cavity, or it may just be a factor contributing to otitis media in a polymicrobial environment. To clarify this point, we have studied the immunogenicity of A. otitidis and host response against A. otitidis. The organism activates lymphocytes and induces production of cytokines or chemokines, as well as the major middle ear pathogens (Himi et al., 2000; Kita et al., 2000; Tarkkanen et al., 2000; Harimaya et al., 2005). In addition, A. otitidis is a ligand for Toll-like receptor 2 and induces the activation of NF-kappaB (Konishi et al., 2006) or mitogen-activated protein kinases (A. Harimaya et al., submitted for publication). These studies suggest that A. otitidis may have enough immunogenic potential to induce a host immune response, as well as the major pathogens, and also be able to contribute singly to an inflammatory reaction in the middle ear cavity. However, the nature of the antibody response against A. otitidis has not been clarified.
In children with acute otitis media due to middle ear pathogens other than A. otitidis, there are IgG, IgA and IgM antibody against the pathogens in the middle ear (Sloyer et al., 1976; Leinonen et al., 1981; Karjalainen et al., 1990), and IgG is the most frequently detectable immunoglobulin in the middle ear cavity in children with otitis media (Yamanaka & Faden, 1993). Our results showed the presence of IgG against A. otitidis, especially against A5 and A6 proteins in the middle ear cavity of children with acute otitis media. In addition, IgG against A1/A2, A4 and A8/A9 was present. These results suggest that local immune response against A. otitidis is induced in the middle ear cavity during a middle ear infection of this organism.
While a part of IgG or IgM is passively diffused from serum to the middle ear, secretory IgA is locally produced in the middle ear during otitis media and is the main component of the local mucosal immune system in the middle ear cavity (Yamanaka & Faden, 1993). The presence of secretory IgA against a pathogen is evidence of local antibody production in the middle ear cavity, as a result of middle ear infection due to the pathogen (Faden et al., 1989; Yamanaka & Faden, 1993). Therefore, our study suggests that A. otitidis can induce local immune response in the middle ear cavity as a true pathogen. Because secretory IgA is associated with the resolution and prevention of otitis media (Lewis et al., 1980; Marshak et al., 1981), A5/A6 or A11 of the bacterial components may be a candidate for a vaccine antigen.
IgG2 is associated with prevention of otitis media due to the major pathogens (Dhooge et al., 2002), and IgM is also frequently detected in the middle ear cavity of children with otitis media (Faden et al., 1989). Our results showed the presence of IgG2 and IgM against A. otitidis in the middle ear cavity of children with otitis media. Although the role of these antibodies against A. otitidis is not disclosed in this study, our results suggest that they may play some roles in immune response against the organism, as well as in that against other middle ear pathogens (Faden et al., 1989; Dhooge et al., 2002). Further investigation is expected to reveal the immunological roles of these antibodies.
In this study, we showed the presence of antibodies against A. otitidis in the middle ear effusions from otitis media cases due to this organism. Because the number of the specimens in this study was small, further work is expected to establish whether our findings are general. However, our results suggest that any specific local antibody response against A. otitidis is induced. A1/A2, A4, A5, A6, A8/A9 and A11 of bacterial components of A. otitidis seemed to be antigenic determinants of A. otitidis. A5, A6 or A11 in particular is expected to be associated with elimination of this organism and to be a candidate for a vaccine antigen. Our results suggest that this recently discovered organism may be associated as a true pathogen in otitis media, and our results help us to understand the pathogenic role of this organism in the development of otitis media. Based on our findings, it is believed that further investigation into the antigenic determinants will lead to the development of diagnosis, therapy and vaccination for otitis media due to this organism.
We thank Mr Teruo Yashiki and Mr Kiyoshi Sato for their technical assistance. This work was funded by the Sapporo Medical University Foundation for the Promotion of Medical Science.