Kazuma Yamauchi

Genotyping of Streptococcus pneumoniae and Haemophilus influenzae Isolated from Paired Middle Ear Fluid and Nasopharynx by Pulsed-Field Gel Electrophoresis

Twenty-eight isolates of Streptococcus pneumoniae and 30 isolates of Haemophilus influenzae from paired nasopharynx and middle ear fluids of 21 children with acute otitis media (AOM) were evaluated to determine genotypes by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE). Among the 28 isolates of S. pneumonaie, 21 isolates (75.0%) possessed mutations in the pbp1a,pbp2x, and pbp2b genes, and 7 isolates (25%) had mutations in the pbp2x gene. Nineteen isolates (67.9%) expressed the mefE gene, and 5 isolates (17.9%) possessed the ermB gene. Among the 30 isolates of H. influenzae, 5 isolates (16.7%) had mutations in pbp3 genes, 3 isolates (10.0%) produced β-lactamase, and 2 (6.7%) isolates possessed mutations both in the pbp3 gene and the β-lactamase gene. Ten out of the 14 pairs (71.4%) of the restriction fragment patterns of S. pneumoniae from paired nasopharynx and middle ear fluids were indistinguishable following PFGE analysis. The same patterns were identified among 5 children of unrelated families. The restriction fragment patterns of H. influenzae isolated by PFGE were also indistinguishable in 13 out of the 15 pairs (86.7%) of nasopharynx and middle ear fluids. The genetic similarity between nasopharyngeal and middle ear isolates suggests that the causative bacteria migrate from the nasopharynx into the middle ear cavity via the Eustachian tube. Some resistant strains might be prevalent. In children with AOM, the nasopharynx could have been colonized by a virulent strain of bacteria that replaced the benign, commensal bacteria and then progressed to the middle ear, where they caused AOM.

Determination of pneumococcal serotypes/genotypes in nasopharyngeal secretions of otitis media children by multiplex PCR.

The appropriate clinical applications of pneumococcal polysaccharide vaccines against recent increases in antimicrobial resistant Streptococcus pneumoniae (S. pneumoniae) urgently require accurate analytical methodologies for determining and characterizing the serotypes. The results of current immunological determinations of serotypes with anti-capsular polysaccharide-specific sera are difficult to interpret in terms of quellung changes of the pneumococci. In this study, we applied the multiplex PCR technique for the rapid identification of pneumococci and simultaneous rapid determinations of their serotypes and genotypes that directly correlated with antimicrobial susceptibilities from nasopharyngeal secretions (NPS). Serogroups 6, 19F and 23F were the predominant capsular types of S. pnuemoniae in the NPS samples. Strains of serotypes 19F and 23F frequently had mutations in pbp1a, pbp2x and pbp2b and expressed ermB and mefA; they also were mostly resistant to both penicillin G (PCG) and clarithromycin (CAM). Two NPS samples contained the strain of serotype 19F together with the strain of serotype 23F, although only the strain of serotype 19F was identified by a conventional bacterial culture. Pneumococci were identified in six NPS samples and their serotypes determined by the multiplex PCR, while a conventional bacterial culture failed to identify the pathogens. Our findings suggest that PCR-based serotyping and genotyping can provide an accurate and rapid distribution of pneumococcal serotypes and antimicrobial resistance. The relatively minor populations in the nasopharynx may be determined using molecular techniques.

Increase of Macrolide‐Resistant Streptococcus pneumoniae‐Expressing mefE or ermB Gene in the Nasopharynx among Children with Otitis Media

Objective: To evaluate prevalence of macrolide resistant strains and the genotypes of the resistance among Streptococcus pneumoniae isolated from the nasopharynx of children with otitis media.

Protection against systemic fatal pneumococcal infection by maternal intranasal immunization with pneumococcal surface protein A (PspA)

Streptococcus pneumoniae is a leading causative pathogen responsible for various types of bacterial infectious diseases in children. The aim of this study was to evaluate the protection conferred against fatal pneumococcal infections during infancy by maternal intranasal immunization with pneumococcal surface protein A (PspA). Four-week-old female BALB/c mice were immunized with PspA mixed with, or without, cholera toxin B (CTB) intranasally twice a week for 3 weeks. After the final immunization, they were mated with male mice to obtain offspring. Offspring at 10 days old were intraperitoneally inoculated with a pneumococcus strain, TIGR4, serotype 4. After the infections their survival periods were monitored. Anti-PspA-specific IgG antibody was induced in sera and breast milk at birth and maintained for 14 days during nursing periods in the PspA-immunized mother mice. At birth, offspring delivered from PspA-immunized mother mice had levels of anti-PspA-specific IgG antibody in sera same to those in their mothers on the day of birth. The survival times to death of offspring delivered from PspA-immunized mother mice after systemic fatal pneumococcal infections were significantly extended compared to those of controls. These findings suggest that maternal intranasal immunization with PspA could be an attractive procedure to employ against pneumococcal infections in early childhood.

High Prevalence of Streptococcus pneumoniae with Mutations in pbp1a, pbp2x, and pbp2b Genes of Penicillin-Binding Proteins in the Nasopharynx in Children in Japan

Objective: To evaluate the resistances of Streptococcus pneumoniae to β-lactams developed by stepwise alterations in high-molecular-weight penicillin-binding proteins (PBPs) with a reduced binding affinity of β-lactams. Among the numerous mutations in pbp genes that alter the affinity for β-lactams, the decreased affinity of PBP1A, 2X and 2B is especially important in the development of resistances to β-lactams. Study Design: Retrospective review. Methods:In this study, we investigated the mutations in pbp1a, pbp2x, and pbp2b genes evaluated by polymerase chain reaction (PCR) in 866 pneumococcal isolates collected from the nasopharynx of Japanese children with acute otitis media. Results: 210 strains (24.3%) exhibited no mutations in the three pbp genes. 333 strains (38.5%) had mutations in the three pbp genes, 78 (9.0%) in two pbp genes, whereas 245 (28.3%) displayed mutations in only one pbp gene. Among the 656 strains with mutations in pbp genes, 620 (94.5%) strains had mutations in pbp2x. The annual prevalence of antimicrobial-resistant S. pneumoniae showed a gradual increase in strains with mutations in the three pbp genes and a parallel decrease in strains without mutations. Conclusions: PCR-based genotyping can characterize the antimicrobial resistances in pneumococci along with minimal inhibitory concentrations (MICs). Physicians should pay attention to the recent increase in antimicrobial-resistant S. pneumoniae when treating pediatric acute otitis media.

Activation of NF-κB via Endosomal Toll-Like Receptor 7 (TLR7) or TLR9 Suppresses Murine Herpesvirus 68 Reactivation

ABSTRACT In order to understand and possibly treat B-cell malignancies associated with latent gammaherpesvirus infection, it is vital to understand the factors that control the balance between the two transcriptional states of gammaherpesviruses: latency and lytic replication. We used murine gammaherpesvirus 68 (MHV 68) as a model system to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts reactivation from latency in vitro and establishment of latent infection in vivo. We found that treatment with TLR7 ligand R848 or TLR9 ligand CpG oligodeoxynucleotide (ODN) suppresses reactivation of MHV 68 in vitro. These suppressive effects correlated with the ability to activate cellular transcription factor NF-κB. Downregulation of TLR9 by RNA interference in vitro led to a reduction of nuclear levels of NF-κB p65 and consequently to an increase of spontaneous reactivation in cells latently infected with MHV 68, indicating that the TLR9 pathway suppresses spontaneous reactivation events. In vivo, sustained stimulation of TLR7 by repeated R848 treatment led to an increased frequency of infected splenocytes compared to mock-treated control results. Frequencies of infected splenic B cells in tlr7 −/− or tlr9 −/− mice after establishment of latency did not differ from those seen with their wild-type counterparts. Nevertheless, MHV 68-infected B cells from tlr9 −/− mice showed a higher frequency of reactivation than B cells from wild-type or tlr7 −/− mice in ex vivo reactivation assays. Thus, we show a suppressive effect of TLR7 or TLR9 triggering on MHV 68 reactivation that correlates with NF-κB activation and that the mere presence of a functional TLR9 signaling pathway contributes to dampen lytic gammaherpesvirus reactivation in infected cells. IMPORTANCE A hallmark of gammaherpesviruses is their establishment of latency in B cells that is reversible through lytic reactivation. Latency can result in B-cell malignancies. Activation of the innate immune system is thought to contribute to controlling the switch between the transcriptional states of latency and reactivation. Nevertheless, the mechanisms involved are not clear. Here, we show that engagement of Toll-like receptor 7 (TLR7) and TLR9 suppresses reactivation of murine gammaherpesvirus MHV 68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected cells. TLR7 and TLR9 are innate immunity sensors of nucleic acids localized in endosomes. Additionally, we demonstrate that impairment of TLR9 signaling in latently infected B cells leads to increased reactivation. Thus, activated endosomal TLR7 and TLR9 pathways play an important role in promoting establishment of latent gammaherpesvirus infection. Counteracting signaling of these pathways allows reactivation and could represent treatment targets in gammaherpesvirus-associated malignancies.

Evaluation of mutations in penicillin binding protein-3 gene of non-typeable Haemophilus influenzae isolated from the nasopharynx of children with acute otitis media

Conclusion Younger children tend to harbor more resistant strains because they are exposed to these pathogens more often through contacts with siblings or attendance at day-care centers and are frequently treated with antibiotics. The high prevalence of BLNAR strains should be taken into account in the treatment of AOM in young children. Objective Non-β-lactamase-producing ampicillin-resistant (BLNAR) strains with mutations in penicillin-binding protein (PBP) genes of Haemophilus influenzae have been prevalent recently among younger children. Material and methods We investigated mutations in the ftsI gene encoding PBP-3 of H. influenzae isolated from the nasopharynx of children with acute otitis media (AOM) using polymerase chain reaction (PCR). Results Strains containing the bla gene (β-lactamase-producing ampicillin-resistant) were identified in 4.7% of cases. Strains with mutations in the ftsI gene (BLNAR) were identified in 23.3% of cases. Strains without mutations in the ftsI gene and that did not contain the bla gene (non-β-lactamase-producing ampicillin-susceptible) were identified in 70.7% of cases. Strains with both expression of the bla gene and mutations in the ftsI gene (β-lactamase-producing amoxicillin–clavulanate-resistant) were identified in 1.3% of cases. The MICs of ampicillin against the strains evaluated in this study were 0.5–2.0 μg/ml. Cefditoren-pivoxil had the lowest MIC90 against the strains (0.06 μg/ml). Strains with mutations in the ftsI gene (BLNAR) were broadly identified among young children.

Genetic characteristics of Haemophilus influenzae and Streptococcus pneumoniae isolated from children with conjunctivitis-otitis media syndrome

Acute conjunctivitis is the most common ocular disorders among children and frequently concomitant with acute otitis media (AOM) as conjunctivitis-otitis syndrome. In this study, we evaluated prevalence of causative pathogens and PCR-based genotypes of Haemophilus influenzae and Streptococcus pneumoniae among children with conjunctivitis-otitis media syndrome. Nontypeable H. influenzae (NTHi) is identified most often at 61.8% in conjunctiva exudates followed by S. pneumoniae at 28.2% and Moraxella catarrhalis at 19.1%. Genetic β-lactamase nonproducing ampicillin resistant (gBLNAR) strains of NTHi and genetic penicillin resistant S. pneumoniae (gPRSP) were identified at 72.1% and at 74.2% among conjunctiva isolates by polymerase chain reaction (PCR), respectively. Pneumococcal strains having either ermB or mefE genes were identified at 93.5% among conjunctiva isolates. The restriction fragment of patterns of 89.7% pairs of H. influenzae isolates and 100% pairs of pneumococcal isolates from conjunctiva exudates, middle ear fluids (MEFs) and nasopharyngeal swabs were identical. In contrast to the previous reports, most prevalent strains from conjunctivitis-otitis media syndrome was BLNAR H. influenzae in this study. The causative pathogen responsible for acute conjunctivitis will be originated from the nasopharynx. In the absence of MEFs one can possibly rely on the nasopharyngeal culture to guide an appropriate treatment.

Primary laryngeal cryptococcosis resembling laryngeal carcinoma

A case of an 82-year-old female with primary laryngeal cryptococcosis who had undergone long-term corticosteroid therapy for chronic obstructive pulmonary disease and rheumatoid arthritis is reported. She complained hoarseness with swallowing pain and irritability of the larynx for over a month. Endoscopic examination revealed a white, exudative irregular region on right arytenoid that mimicked a laryngeal carcinoma. Histological examination showed pseudoepitheliomatous hyperplasia and severe submucosal inflammation with ovoid budding yeasts by Grocotts stain. A serological study indicated a high titer of cryptococcal antigen. After treating with oral fluconazole for 3 months, her primary lesion of larynx turned to be clear. We implicate a long-term use of steroids as the significant risk factor in developing cryptococcosis of the larynx.

Activation of NFκB via Endosomal Toll-like Receptors 7 or 9 Contributes to Limiting Murine Herpes Virus 68 Reactivation

ABSTRACT In order to understand and possibly treat B-cell malignancies associated with latent gammaherpesvirus infection, it is vital to understand the factors that control the balance between the two transcriptional states of gammaherpesviruses: latency and lytic replication. We used murine gammaherpesvirus 68 (MHV 68) as a model system to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts reactivation from latency in vitro and establishment of latent infection in vivo. We found that treatment with TLR7 ligand R848 or TLR9 ligand CpG oligodeoxynucleotide (ODN) suppresses reactivation of MHV 68 in vitro. These suppressive effects correlated with the ability to activate cellular transcription factor NF-κB. Downregulation of TLR9 by RNA interference in vitro led to a reduction of nuclear levels of NF-κB p65 and consequently to an increase of spontaneous reactivation in cells latently infected with MHV 68, indicating that the TLR9 pathway suppresses spontaneous reactivation events. In vivo, sustained stimulation of TLR7 by repeated R848 treatment led to an increased frequency of infected splenocytes compared to mock-treated control results. Frequencies of infected splenic B cells in tlr7 −/− or tlr9 −/− mice after establishment of latency did not differ from those seen with their wild-type counterparts. Nevertheless, MHV 68-infected B cells from tlr9 −/− mice showed a higher frequency of reactivation than B cells from wild-type or tlr7 −/− mice in ex vivo reactivation assays. Thus, we show a suppressive effect of TLR7 or TLR9 triggering on MHV 68 reactivation that correlates with NF-κB activation and that the mere presence of a functional TLR9 signaling pathway contributes to dampen lytic gammaherpesvirus reactivation in infected cells. IMPORTANCE A hallmark of gammaherpesviruses is their establishment of latency in B cells that is reversible through lytic reactivation. Latency can result in B-cell malignancies. Activation of the innate immune system is thought to contribute to controlling the switch between the transcriptional states of latency and reactivation. Nevertheless, the mechanisms involved are not clear. Here, we show that engagement of Toll-like receptor 7 (TLR7) and TLR9 suppresses reactivation of murine gammaherpesvirus MHV 68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected cells. TLR7 and TLR9 are innate immunity sensors of nucleic acids localized in endosomes. Additionally, we demonstrate that impairment of TLR9 signaling in latently infected B cells leads to increased reactivation. Thus, activated endosomal TLR7 and TLR9 pathways play an important role in promoting establishment of latent gammaherpesvirus infection. Counteracting signaling of these pathways allows reactivation and could represent treatment targets in gammaherpesvirus-associated malignancies.