Impact of anaerobic and oligotrophic conditions on survival of Alloiococcus otitidis, implicated as a cause of otitis media.

The survival of Alloiococcus otitidis (NCFB2890) with different nutritional supplements, including brain-heart infusion broth (BHI), phosphate-buffered saline (PBS), distilled water (DW), and middle ear effusion (MEE), as well as various atmospheres (aerobic, microaerobic, anaerobic), was compared using cultures, LIVE/DEAD staining, and transmission electron microscopy. The bacterial morphological traits and viability were maintained in BHI and MEE under aerobic conditions but were rapidly lost in PBS and DW. In contrast, anaerobic conditions did not support viability at all. Thus, the bacteria critically required an aerobic atmosphere for its survival as well as the appropriate nutrients, implying that culture of this pathogen from clinical specimens would become more difficult through oxygen depletion depending on a slight change in the middle ear atmosphere.

Thymic stromal lymphopoietin enhances tight-junction barrier function of human nasal epithelial cells.

Epithelial-derived thymic stromal lymphopoietin (TSLP) triggers dendritic cell (DC)-mediated Th2-type inflammatory responses and is a master switch for allergic inflammatory diseases. In the present study, the expression and induction of TSLP and the effects of TSLP on the tight-junctional barrier of human nasal epithelial cells (HNECs) have been investigated in order to elucidate the role of TSLP in allergic rhinitis. We have found high expression of TSLP in the epithelium from patients with allergic rhinitis with recruitment and infiltration of DCs. In vitro, TSLP is significantly produced in HNECs after treatment with a toll-like receptor 2 (TLR2) ligand, Pam(3)Cys-Ser-(Lys)(4), and a mixture of interleukin-1beta and tumor necrosis factor-alpha. Treatment with TSLP rapidly enhances the barrier function of cultured HNECs, together with an increase of tight-junction proteins claudin-1, -4, -7, and occludin. The nasal-epithelial-derived TSLP thus not only activates DCs but also preserves the epithelial barrier via the upregulation of tight-junction proteins, thereby regulating antigen sensitization during the early stage of allergic rhinitis.

Preliminary study of proinflammatory cytokines and chemokines in the middle ear of acute otitis media due to Alloiococcus otitidis.

Alloiococcus otitidis is a newly discovered organism frequently detected in otitis media. However, the association of the organism with the development of otitis media has not been disclosed in detail yet. In the middle ear, proinflammatory cytokines and chemokines are released in association with infection by pathogens, and these cytokines contribute to the induction of an inflammatory reaction. To investigate the profile of inflammation-related cytokines in the acute phase of A. otitidis infection, we analyzed the release of proinflammatory cytokines and chemokines in middle ear effusions of acute otitis media due to A. otitidis, in comparison with acute otitis media due to the well-known Gram-positive middle ear pathogen Streptococcus pneumoniae. The amounts of proinflammatory cytokines (IL-8, IL-1beta, IL-6, TNF-alpha) and CXC chemokines (IP-10, I-TAC) were significantly increased in the A. otitidis group as well as in the S. pneumoniae group. Various inflammation-related cytokines/chemokines were induced in the A. otitidis-infected middle ear, and the profile of cytokines was very similar to that in S. pneumoniae infection. This preliminary study suggests that A. otitidis has the potential to induce these cytokines, contributing to the development of an inflammatory reaction in the middle ear cavity in a similar manner to S. pneumoniae.

Remarkably high prevalence of fts I gene mutations in Haemophilus influenzae isolates from upper respiratory tract infections in children of the Sapporo district, Japan.

Recently, the frequency of isolation of beta-lactamase-negative ampicillin resistant (BLNAR) strains of Haemophilus influenzae in Japanese children has been increasing rapidly. Drug resistance in BLNAR strains is associated with mutations of the fts I gene, which encodes penicillin-binding protein 3. In the otolaryngological field, only a few reports have been available concerning fts I gene mutations in BLNAR. We investigated the prevalence of fts I gene mutations, by polymerase chain reaction (PCR) genotyping, in H. influenzae isolates from the upper respiratory tracts of children in the Sapporo district, Japan. When the isolates were classified according to PCR genotyping, 34 (44.2%) of 77 isolates were beta-lactamase-negative ampicillin-sensitive (g-BLNAS), 8 (10.4%) were g-low-BLNAR, 30 (39.0%) were g-high-BLNAR, 2 (2.6%) were beta-lactamase-positive ampicillin-resistant (g-BLPAR), and 3 (3.9%) were beta-lactamase-positive ampicillin/clavulanic acid-resistant (g-high-BLPACR). Mutations in the fts I gene were generally parallel to ampicillin susceptibility, and were frequently observed in children who were 7 years or younger. Of the beta-lactams tested, cefditoren showed the strongest inhibition of H. influenzae isolates, and it inhibited g-BLNAR and g-BLPACR. This study revealed a remarkably high prevalence of fts I gene mutations (g-BLNAR and g-BLPACR) in our district. Furthermore, a regional difference in the prevalence of fts I gene mutations was observed even at the district level.

Protein kinase C enhances tight junction barrier function of human nasal epithelial cells in primary culture by transcriptional regulation.

The epithelium of upper respiratory tissues such as human nasal mucosa forms a continuous barrier via tight junctions, which is thought to be regulated in part through a protein kinase C (PKC) signaling pathway. To investigate the mechanisms of the regulation of PKC-mediated tight junction barrier function of human nasal epithelium in detail, primary human nasal epithelial cells were treated with the PKC activator 12-O-tetradecanoylophorbol-13-acetate (TPA). In primary human nasal epithelial cells, treatment with TPA led not only to activation of phosphorylation of PKC, myristoylated alanine-rich C kinase substrate, and mitogen-activated protein kinase but also expression of novel PKC-delta, PKC-theta, and PKC-epsilon. Treatment with TPA increased transepithelial electrical resistance, with tight junction barrier function more than 4-fold that of the control, together with up-regulation of tight junction proteins, occludin, zona occludens (ZO)-1, ZO-2 and claudin-1 at the transcriptional level. Furthermore, it affected the subcellular localization of the tight junction proteins and the numbers of tight junction strands. The up-regulation of barrier function and tight junction proteins was prevented by a pan-PKC inhibitor, and the inhibitors of PKC-delta and PKC-theta but not PKC-epsilon. In primary human nasal epithelial cells, transcriptional factors GATA-3 and -6 were detected by reverse transcription-polymerase chain reaction. The knockdown of GATA-3 using RNA interference resulted in inhibition of up-regulation of ZO-1 and ZO-2 by treatment with TPA. These results suggest that TPA-induced PKC signaling enhances the barrier function of human nasal epithelial cells via transcriptional up-regulation of tight junction proteins, and the mechanisms may contribute to a drug delivery system.

High prevalence of erythromycin resistance and macrolide-resistance genes, mefA and ermB, in Streptococcus pneumoniae isolates from the upper respiratory tracts of children in the Sapporo district, Japan.

Our previous study demonstrated that the frequency of penicillin-resistant Streptococcus pneumoniae (PRSP) was lower in our district than in districts in other Japanese studies. In this study, we investigated the prevalence of erythromycin resistance. The susceptibility to erythromycin and the distribution of the macrolide-resistance genes, mefA and ermB, were examined in S. pneumoniae isolates from the upper respiratory tracts of children in four cities in the Sapporo district, Hokkaido prefecture, Japan. Of the 156 isolates, 27 (17.3%) were erythromycin-sensitive, 6 (3.9%) were erythromycin-intermediately resistant, and 123 (78.9%) were erythromycin-resistant. Fifty-nine (37.8%) had the mefA gene, 89 (57.1%) had the ermB gene, and 129 (82.7%) had the mefA and/or the ermB gene. The ermB-positive isolates tended to show high resistance to erythromycin. Erythromycin-resistant isolates and the macrolide-resistance genes were often present in infants or younger children. The frequency of erythromycin-resistant isolates in the four cities was very high, ranging from 76.3% to 83. 3%, as high as the national average. Although erythromycin-resistant isolates generally tend to show cross-resistance to penicillin, the frequency of PRSP was very low in this study, as compared with other Japanese studies. Erythromycin resistance was frequently recognized not only in PRSP but also in penicillin-sensitive S. pneumoniae (PSSP) as well. In Japan, erythromycin resistance may have already become widespread, even in local areas where penicillin resistance is not especially prevalent.

Changes of gap and tight junctions during differentiation of human nasal epithelial cells using primary human nasal epithelial cells and primary human nasal fibroblast cells in a noncontact coculture system.

The epithelium of upper respiratory tissues such as nasal mucosa forms a continuous barrier to a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the intercellular junctions, referred to as gap and tight junctions. However, changes of gap and tight junctions during differentiation of human nasal epithelial (HNE) cells are still unclear. In the present study, to investigate changes of gap and tight junctions during differentiation of HNE cells in vitro, we used primary human HNE cells cocultured with primary human nasal fibroblast (HNF) cells in a noncontact system. In HNE cells cocultured with HNF cells for 2 weeks, numerous elongated cilia-like structures were observed compared to those without HNF cells. In the coculture, downregulation of Cx26 and upregulation of Cx30.3 and Cx31 were observed together with extensive gap junctional intercellular communication. Furthermore, expression of the tight junction proteins claudin-1, claudin-4, occludin and ZO-2 was increased. These results suggest that switching in expression of connexins and induction of tight junction proteins may be closely associated with differentiation of HNE cells in vitro and that differentiation of HNE cells requires unknown soluble factors secreted from HNF cells.

Interleukin-8 induction via NF-kappaB, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways in human peripheral blood mononuclear cells by Alloiococcus otitidis.

Alloiococcus otitidis is a newly recognized species of gram-positive bacteria frequently associated with otitis media. Although immunostimulating activity of this organism has been investigated, little is known about the signaling pathways of chemokine/cytokine induction by this organism. We investigated the role of NF-kappaB and mitogen-activated protein (MAP) kinases in chemokine interleukin-8 (IL-8) production by human peripheral blood mononuclear cells (PBMCs) after stimulation with A. otitidis. The organism could induce in vitro IL-8 production in human PBMCs. I kappa B alpha, NF-kappaB, p38 MAP kinase, p44/42 MAP kinase (ERK1/2) became phosphorylated in PBMCs after stimulation with A. otitidis. And, inhibitors of NF-kappaB (caffeic acid phenylethyl ester), p38 (SB 203580), or ERK1/2 (PD 98059) significantly reduced IL-8 induction by the organism. These results were similar to findings in IL-8 induction by Streptococcus pneumoniae, another gram-positive major middle ear pathogen. Our preliminary study suggests that multiple pathways including NF-kappaB, p38, and ERK1/2 were simultaneously activated, and were associated with IL-8 induction by A. otitidis in human PBMCs.

Colonization and turnover of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in otitis-prone children.

Recurrent otitis media are frequently intractable during childhood. It is unclear whether recurrent otitis media is caused by etiological bacteria colonization or by new infections. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were isolated from the nasopharynx of 7 otitisprone and 2 non-prone children with recurrent otitis media. Plural bacterial species and strains were found in all children while affected by otitis media. The same strain was repeatedly isolated from all otitisprone children even after administration of antibiotics but was not from the non-prone children. Antibiotic susceptibility did not differ significantly among the same repeatedly isolated strains. This pilot study suggests that the etiological bacteria tend to colonize and is hard to eliminate in otitis-prone children.

Evidence of local antibody response against Alloiococcus otitidis in the middle ear cavity of children with otitis media.

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.

Alloiococcus otitidis is a ligand for collectins and Toll-like receptor 2, and its phagocytosis is enhanced by collectins.

Alloiococcus otitidis has been found to be associated with otitis media with effusion. In this study we investigated whether TLR2 and collectins, surfactant protein A (SP-A) and mannose-binding lectin (MBL), interacted with A. otitidis. Both SP-A and MBL bound to A. otitidis in a Ca(2+)-dependent manner. A. otitidis induced IL-8 secretion from U937 cells and NF-kappaB activation in TLR2-transfected HEK293 cells. However, the cells transfected with the mutant TLR2(P681H) did not respond to A. otitidis. In addition, A. otitidis co-sedimented a recombinant soluble form of the extracellular TLR2 domain, indicating direct binding of the bacterium to TLR2. SP-A and MBL augmented the phagocytosis of A. otitidis by J774A.1 cells. The collectin-stimulated phagocytosis of A. otitidis was significantly attenuated when fucoidan and polyinosinic acid were co-incubated. Immunoblotting analysis revealed that MBL was present in the middle ear effusion from patients with otitis media. These results demonstrate that A. otitidis is a ligand for the collectins and TLR2, and that the collectins enhance the phagocytosis of A. otitidis by macrophages, suggesting important roles of the collectins and TLR2 in the innate immunity of the middle ear against A. otitidis infection.

High incidence of Alloiococcus otitidis in children with otitis media, despite treatment with antibiotics.

Acute otitis media (AOM) and otitis media with effusion (OME) are common diseases in childhood. Alloiococcus otitidis is a newly recognized species of gram-positive bacterium which was recently discovered as a pathogen associated with OME. Although some studies show that A. otitidis is frequently detected in children with OME, no study is available concerning the clinical efficiency of antibiotics against this organism. The prevalence of A. otitidis in 116 middle ear effusion specimens from 36 AOM and 52 OME patients was examined by culture and PCR. In addition, the prevalence of the bacterium was retrospectively investigated in relation to antibiotic use. A. otitidis was detected in 20 (50%) AOM and 47 (61%) OME specimens. The organism was the most frequent bacterium in AOM as well as in OME and was highly detected even in patients who had been treated with antibiotics, such as beta-lactams or erythromycin. The incidence of A. otitidis in our study was higher than that in Western countries, and our results suggest that drug-resistant strains of A. otitidis may be frequently spread in Japanese children. Our study suggests that antibiotics such as beta-lactams or erythromycin may not be sufficiently effective to eliminate this organism. Further investigation is expected to reveal the clinical role of the organism in otitis media.

High frequency of Alloiococcus otitidis in the nasopharynx and in the middle ear cavity of otitis-prone children.

OBJECTIVE: Colonization of middle ear pathogens is facilitated in the nasopharynx of otitis-prone children, and is associated with the development of otitis media. Recently, a new species of bacterium, Alloiococcus otitidis, is considered as one of the major middle ear pathogens. However, as far as we know, no study has been reported concerning the prevalence of A. otitidis in the nasopharynx of otitis-prone children. And, no study has been conducted on the association of A. otitidis in the nasopharynx with otitis media. METHODS: The frequency of A. otitidis in 83 middle ear effusions (MEE) and 56 nasopharyngeal swab (NPS) specimens from 56 children with otitis media was investigated by culture and polymerase chain reaction (PCR). RESULTS: A. otitidis was detected in 24 (28.9%) of MEE and in 6 (10.7%) of NPS specimens. When the frequency was investigated in relation to proneness to otitis media, A. otitidis was detected in 16 (64%) of 25 MEE and in 5 (29.4%) of 17 NPS specimens from otitis-prone children, whereas it was detected in 8 (13.8%) of 58 MEE and in 1 (2.6%) of 39 NPS specimens from non-otitis-prone children. The frequency of A. otitidis in both NPS and MEE specimens was significantly higher in otitis-prone than in non-otitis-prone children. CONCLUSIONS: Our results suggest that colonization of A. otitidis is facilitated in the nasopharynx of otitis-prone children. And, nasopharyngeal colonization of A. otitidis may be associated with the frequency of otitis media.

Alterations of pbp1a, pbp2b, and pbp2x in Streptococcus pneumoniae isolates from children with otolaryngological infectious disease in the Sapporo district of Japan.

Evaluation of beta-lactam susceptibility and polymerase chain reaction (PCR)-based genotyping of penicillin-binding proteins (PBP) 1A, 2B, and 2X were performed for Streptococcus pneumoniae isolates from children with otolaryngological infectious disease in the Sapporo district, Hokkaido Prefecture, Japan. Of 174 S. pneumoniae isolates, 14 (8%) were penicillin-resistant S. pneumoniae (PRSP), 87 (50%) were penicillin-intermediately-resistant, and 73 (42%) were penicillin-sensitive. Seventy-six (44%) had alterations in all of the three genes examined (pbp1a, pbp2b, and pbp2x), 81 (47%) had alterations in one or two of the genes, and 17 (10%) had no alterations. Isolates with alterations in all three genes showed low susceptibility to penicillin, while, in contrast, isolates with no alteration showed relatively high susceptibility to penicillin. Similar relationships were observed for other beta-lactams. The prevalence of PRSP in our study ranged from 5% to 12.8% (average, 8%), and there was much variation in the prevalence of PBP gene alterations among the cities. The results suggest that local differences in patterns of PBP gene alterations can be observed even at the district level. PCR-based genotyping of PBP genes is rapid, convenient, and useful to investigate genetic susceptibility to beta-lactams. Further, not only nationwide or prefectural surveys but also local surveillance at the district level is important for determining antimicrobial susceptibility status in daily practice.

Difference in cytokine production and cell activation between adenoidal lymphocytes and peripheral blood lymphocytes of children with otitis media.

We evaluated the immunological potential of adenoidal lymphocytes from children with recurrent otitis media. Interleukin-4 release and CD69 expression were lower in adenoidal lymphocytes than in peripheral blood lymphocytes (PBL). Our results suggest that there may be a difference between the immunological potential of adenoidal lymphocytes and that of PBL in children with otitis.

Induction of CD69 expression and Th1 cytokines release from human peripheral blood lymphocytes after in vitro stimulation with Alloiococcus otitidis and three middle ear pathogens.

Alloiococcus otitidis is a recently discovered pathogen of otitis media. However, only a limited number of studies are available about the pathogenic and immunological role of A. otitidis. The aim of this study was to investigate the activation and the cytokine production of human peripheral blood lymphocytes at the early immune response after stimulation with A. otitidis. After stimulation of whole human peripheral blood lymphocytes for 18 h with whole killed A. otitidis or the three major middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), the expression of CD69 and the production of cytokines were analyzed. The expression of CD69 on T cells and B cells was dose-dependently enhanced after stimulation with A. otitidis. The release of interleukin (IL)-12 was induced after stimulation with A. otitidis, whereas the release of IL-4 was not induced after stimulation with A. otitidis. In addition, the release of interferon (IFN)-gamma was induced after stimulation with A. otitidis. Although the release of IFN-gamma started within 18 h after stimulation with A. otitidis, intracellular production of IFN-gamma was not observed in either CD4+ T cells or CD8+ T cells within 18 h upon stimulation. The patterns of CD69 expression and T helper-type 1 (Th1)-promoting cytokines production were similarly shown when human peripheral blood lymphocytes were stimulated with the other three major pathogens. Our results suggest that A. otitidis has sufficient immunogenic potential to modulate a host immune response, like the other three major middle ear pathogens, and also suggest that the immunogenicity of A. otitidis is very similar, at the early immune response, to that of the three major middle ear pathogens.