Masaki Suzumoto

Household Transmission of Streptococcus pneumoniae among Siblings with Acute Otitis Media

ABSTRACT Nasopharyngeal transmission of Streptococcus pneumoniae was evaluated among 23 siblings with acute otitis media (AOM). Restriction fragment length polymorphism revealed that the nasopharyngeal strains were identical between siblings in 12 of 13 clusters of AOM experienced in 11 families. This study demonstrated person-to-person transmission of S. pneumoniae, especially drug-resistant strains, among siblings with AOM.

Intranasal immunization with recombinant outer membrane protein P6 induces specific immune responses against nontypeable Haemophilus influenzae

OBJECTIVE Nontypeable Haemophilus influenzae (NTHi) is one of the leading causative pathogens for otitis media. The outer membrane protein P6 of NTHi is highly conserved among the strains and is an attractive candidate for a preventive vaccine. However, for the production of a relatively small amount P6 containing lipopolysaccharides, the development of a recombinant version of this protein is required. This study was designed to investigate the specific mucosal immunity induced by intranasal immunization of recombinant P6 (rP6) with cholera toxin (CT). METHODS BALB/c mice were immunized with of rP6 (30 microg) and CT (2 microg) intranasally every 2 days for 2 weeks. Anti-rP6 specific IgG, IgA and IgM antibodies and the subclass of anti-rP6 specific IgG antibody were determined by enzyme linked immunosorbent assay (ELISA). Anti-rP6 specific IgA in nasopharyngeal washings were also determined by ELISA. Nasopharyngeal clearance of inoculated NTHi after the intranasal immunization were assessed. All statistical differences between the two groups were assessed by ANOVA parametric test. RESULTS Intranasal immunization with rP6 and CT evoked rP6-specific mucosal IgA immune response as well as the systemic IgG immune response against rP6 and enhanced nasopharyngeal clearance of inoculated live NTHi. CONCLUSION These results indicate the good immunogenicities of rP6 to induce specific immune responses against NTHi. Intranasal immunization with rP6 will be an effective approach to protect infections of NTHi.

Antimicrobial resistance of Haemophilus influenzae isolated from the nasopharynx of Japanese children with acute otitis media

Conclusion. A high prevalence of penicillin-binding protein gene-mutated (PGM) strains of Haemophilus influenzae should be taken into account when treating otitis media in children. Objective. To evaluate the prevalence of β-lactamase-non-producing ampicillin-resistant strains of H. influenzae with mutations in the ftsI gene encoding penicillin-binding protein 3 (PBP3) among children with otitis media. Material and methods. A total of 644 nasopharyngeal isolates of H. influenzae were collected from pediatric acute otitis media patients with or without otitis media with effusion at the clinics of the Department of Otolaryngology—Head and Neck Surgery, Wakayama Medical University Hospital and 6 affiliated hospitals in Wakayama Prefecture between January 1999 and December 2003. MICs to ampicillin (AMP), cefdinir (CFD), cefaclor (CCL), cefpodoxime (CPD) and cefcapene (CFPN) were determined by a microbroth dilution method according to the recommendations of the National Committee for Clinical Laboratory Standards. Types of mutations in the PBP3 gene (ftsI) were evaluated by means of a polymerase chain reaction (PCR)-based genotyping method. The β-lactamase gene (bla) was also identified by means of PCR. Results. β-lactamase-producing (BLP) strains having the bla gene were identified in 16 isolates (2.5%). PGM strains were identified in 279 isolates (43.3%). There were 242 PGM1-non-BLP strains (37.6%) with mutations in the variable mutated locus of ftsI, 35 PGM2-non-BLP strains (5.4%) with mutations in the highly mutated locus of ftsI and 2 BLP-PGM strains (0.3%) with mutations in ftsI that produced β-lactamase. BLP-non-PGM strains producing β-lactamase without mutations in ftsI were identified in 14 isolates (2.2%). MICs of PGM1-non-BLP strains to AMP were 0.5–2.0 µg/ml. The MIC90 of CDN to the PGM1-non-BLP strains was the lowest (0.06 µg/ml). The proportion of PGM1-non-BLP strains increased rapidly during 1999–2002 and then decreased in 2003. In contrast, the proportion of PGM2-non-BLP strains increased in 2003.

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.

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.

A scoring system for management of acute pharyngo-tonsillitis in adults

Abstract Objectives The aim of this study was to develop and evaluate a scoring system for the management of acute pharyngo-tonsillitis. Methods We conducted a prospective study between May 2004 and June 2005. Patients with acute pharyngo-tonsillitis were evaluated for causative pathogens and were assessed clinical symptoms and pharyngo-tonsillar finding by a clinical scoring system. Results A total 214 adult patients were enrolled in this study. Streptococcus pyogenes were identified at 13.6%. Thirty-one viruses were also identified by PCR. They were adenovirus (4.8%), influenza virus (1.0%), RS virus (6.3%), and human metapneumovirus (2.9%). Numbers of total white blood cells and levels of C-reactive protein showed a significant positive correlation with clinical scores (p <0.001) and were also higher in cases with S. pyogenes. The clinical scores rapidly improved after the antimicrobial treatments in moderate cases and severe cases. Conclusion The current study strongly suggested that the clinical scoring system reflected disease severity well and would be very useful for evaluating clinical course and decision making for the antimicrobial treatment of acute pharyngo-tonisllitis.

Nasopharyngeal Carriage of Drug-resistant Streptococcus pneumoniae in Children with Acute Otitis Media Evaluated by Polymerase Chain Reaction-based Genotyping of Penicillin-binding Proteins

A polymerase chain reaction (PCR)-based genotyping of the penicillin-binding protein (PBP) genes pbp1a , pbp2x and pbp2b was used to characterize Streptococcus pneumoniae isolated from the nasopharynx of children with acute otitis media (AOM). Mutations were observed in pbp1a , pbp2x and pbp2b genes in 36.5% of the strains. Decreased susceptibility to g -lactam antibiotics was closely associated with the frequency of mutations in the three PBP genes. Of penicillin-intermediately-resistant S. pneumoniae strains, 54.5% appeared to be genetically similar to penicillin-resistant S. pneumoniae strains. Of penicillin-susceptible S. pneumoniae strains, 33.3% had mutations in the pbp2x gene and showed relatively high MICs to cephalosporins. Strains with mutations in the three PBP genes were often isolated from children h 2 years old. Evaluation of mutations in PBP genes using PCR will prove useful for studying the epidemiology of antibiotic resistance.

Functions of tonsils in the mucosal immune system of the upper respiratory tract using a novel animal model, Suncus murinus

Conclusion. The human palatine tonsils and the nasopharyngeal tonsil were considered the defense mechanism against ingested or inhaled foreign pathogens. The current findings suggest that the tubal tonsils possess abilities of active transportation of foreign antigens, and will act as inductive and effector sites in the mucosal immune system. Our results also indicated a significant difference in roles of immune responses among individual tonsillar organs, suggesting functional sub-compartmentalization. Objectives. To address the function of tonsils in inducing local immune responses, we evaluated the antigen uptake of tubal tonsils and the induction of specific immune responses in a small laboratory animal with both tubal and palatine tonsils, i.e. Suncus murinus. Materials and methods. S. murinus were injected with 2×106 CFU of FITC-labeled Staphylococcus aureus via the right tympanic cavity. The distribution of the FITC-labeled S. aureus was examined under a fluorescent microscope. S. murinus were also immunized with 100 µg of ovalbumin (OVA) mixed with 2 µg of cholera toxin (CT) via the right external ear meatus every 2 days for 2 weeks. One week after the final immunization, sera, pairs of tubal and palatine tonsils, and the neck lymph nodes were obtained to evaluate the induction of specific immune responses. Results. The FITC-labeled S. aureus particles were detected in tubal tonsils and also in cervical lymph nodes. Total IgA-producing cells and OVA-specific antibody-producing cells were identified in the immunized tubal tonsils. Trans-external ear meatus immunization of tubal tonsils also evoked systemic antibody responses.