Shin Takei

Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells.

OBJECTIVES The aim of this study is to examine the internalization of nontypeable Haemophilus influenzae (NTHi) into human epithelial cells. METHODS Bactericidal assay was applied to examine the effects of antibiotics against cell-adherent NTHi using HEp-2 cells. A trans-well chamber assay was applied to examine the internalization and penetration of NTHi using Detroit562 cells. RESULTS The adherence of NTHi to HEp-2 cells was noted after 2h of incubation. Azithromycin had a strong bactericidal effect against both cell-associated and non-adherent NTHi, while ceftriaxone did not show bactericidal effects on NTHi adhered to the HEp-2 cells. Three (60.0%) out of five NTHi isolates from the nasopharynx of children with intractable acute otitis media (AOM) internalized into and subsequently penetrated through the epithelial cells at various degrees. Azithromycin had a strong bactericidal effect against the cell-internalized NTHi, while ceftriaxone was bactericidal only against extracellular NTHi. CONCLUSION The potential of NTHi as the intracellular pathogen may contribute to the persistent existence of this pathogen that result in the prolonged and intractable clinical course of AOM. Azithromycin may be a therapeutically significant antibiotic for patients with prolonged respiratory tract infections due to NTHi.

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.

Haemophilus influenzae and Haemophilus haemolyticus in tonsillar cultures of adults with acute pharyngotonsillitis

OBJECTIVE The aim of this study was to evaluate the clinical implication of Haemophilus haemolyticus, one of the closest relative of Haemophilus influenzae, on acute pharyngotonsillitis. METHODS We applied polymerase chain reaction (PCR) for 16S ribosomal DNA (rDNA) and IgA protease gene (iga) to distinguish H. haemolyticus and H. influenzae. RESULTS Among the 199 Haemophilus spp. isolated from 214 patients with acute pharyngotonsillitis, 52 (24.3%) H. influenzae strains and 23 (10.7%) H. haemolyticus strains were identified by polymerase chain reaction (PCR) for 16S rDNA and IgA protease gene (iga). All H. haemolyticus strains showed hemolysis on horse blood agar and there were no other Haemophilus spp., nonhemolytic H. haemolyticus and H. influenzae variant strains that had absent iga gene. H. hemolyticus showed close genetic relationship with H. influenzae evaluated by pulsed field gel electrophoresis (PFGE). The cases of acute pharyngotonsillitis showing WBC=7000/mm(3) or CRP=8 mg/dl were frequently found among cases with H. influenzae rather than cases with H. haemolyticus. CONCLUSION H. haemolyticus is a pharyngeal commensal that is isolated frequently from adults with acute pharyngotonsillitis.

Evaluation of a rapid immunochromatographic ODK-0901 test for detection of pneumococcal antigen in middle ear fluids and nasopharyngeal secretions.

Since the incidence of penicillin-resistant Streptococcus pneumoniae has been increasing at an astonishing rate throughout the world, the need for accurate and rapid identification of pneumococci has become increasingly important to determine the appropriate antimicrobial treatment. We have evaluated an immunochromatographic test (ODK-0901) that detects pneumococcal antigens using 264 middle ear fluids (MEFs) and 268 nasopharyngeal secretions (NPSs). A sample was defined to contain S. pneumoniae when optochin and bile sensitive alpha hemolytic streptococcal colonies were isolated by culture. The sensitivity and specificity of the ODK-0901 test were 81.4% and 80.5%, respectively, for MEFs from patients with acute otitis media (AOM). In addition, the sensitivity and specificity were 75.2% and 88.8%, respectively, for NPSs from patients with acute rhinosinusitis. The ODK-0901 test may provide a rapid and highly sensitive evaluation of the presence of S. pneumoniae and thus may be a promising method of identifying pneumococci in MEFs and NPSs.

PspA Family Distribution, Antimicrobial Resistance and Serotype of Streptococcus pneumoniae Isolated from Upper Respiratory Tract Infections in Japan

Background The protection against pneumococcal infections provided by currently available pneumococcal polysaccharide conjugate vaccines are restricted to the limited number of the serotypes included in the vaccine. In the present study, we evaluated the distribution of the pneumococcal capsular type and surface protein A (PspA) family of pneumococcal isolates from upper respiratory tract infections in Japan. Methods A total of 251 S. pneumoniae isolates from patients seeking treatment for upper respiratory tract infections were characterized for PspA family, antibiotic resistance and capsular type. Results Among the 251 pneumococci studied, the majority (49.4%) was identified as belonging to PspA family 2, while most of the remaining isolates (44.6%) belonged to family 1. There were no significant differences between the distributions of PspA1 versus PspA2 isolates based on the age or gender of the patient, source of the isolates or the isolates’ susceptibilities to penicillin G. In contrast, the frequency of the mefA gene presence and of serotypes 15B and 19F were statistically more common among PspA2 strains. Conclusion The vast majority of pneumococci isolated from the middle ear fluids, nasal discharges/sinus aspirates or pharyngeal secretions represented PspA families 1 and 2. Capsular serotypes were generally not exclusively associated with certain PspA families, although some capsular types showed a much higher proportion of either PspA1 or PspA2. A PspA-containing vaccine would potentially provide high coverage against pneumococcal infectious diseases because it would be cross-protective versus invasive disease with the majority of pneumococci infecting children and adults.

Distribution of fibronectin-binding protein genes ( prtF1 and prtF2 ) and streptococcal pyrogenic exotoxin genes ( spe ) among Streptococcus pyogenes in Japan

Two hundred and seventy-two strains of Streptococcus pyogenes isolated from patients with invasive and noninvasive infections in Japan were evaluated for the prevalence of fibronectin-binding protein genes (prtF1 and prtF2). The possible associations of the genes with streptococcal pyrogenic exotoxin genes, macrolide resistance genes, and emm types were also evaluated. Overall, about 50% of S. pyogenes isolates carried fibronectin-binding protein genes. The prevalence of the prtF1 gene was significantly higher among isolates from noninvasive infections (71.4%) than among isolates from invasive infections (30.8%; P = 0.0037). Strains possessing both the prtF1 and prtF2 genes were more likely to be isolates from noninvasive infections than isolates from invasive infections (50.6% vs 15.4%; P = 0.019). S. pyogenes isolates with streptococcus pyrogenic exotoxin genes (speA and speZ) were more common among isolates without fibronectin-binding protein genes. The speC gene was more frequently identified among isolates with fibronectin-binding protein genes (P = 0.05). Strains belonging to emm75 or emm12 types more frequently harbored macrolide resistance genes than other emm types (P = 0.0094 and P = 0.043, respectively). Strains carrying more than one repeat at the RD2 region of the prtF1 gene and the FBRD region of the prtF2 gene were more prevalent among strains with macrolide resistance genes than among strains negative for macrolide resistance genes. These genes (i.e., the prtF1, prtF2, and spe genes) may enable host-bacteria interaction, and internalization in the host cell, but may not enable infection complications such as invasive diseases.

Minimal biofilm eradication concentration of antimicrobial agents against nontypeable Haemophilus influenzae isolated from middle ear fluids of intractable acute otitis media

Nontypeable Haemophilus influenzae (NTHi) makes the clinical course of acute otitis media (AOM) intractable by forming a biofilm that may hamper the clearance of the bacteria from middle ear cavity. In this study, we evaluated the minimum biofilm eradication concentration (MBEC) of antimicrobial agents against biofilm-forming NTHi strains. Twelve NTHi strains isolated from middle ear fluids of Japanese children with intractable AOM before antimicrobial treatment were evaluated for MBEC of fluoroquinolones in comparison with those of β-lactams and macrolides. AMPC and CDTR required much higher concentration, i.e., high MBECs, to suppress the biofilm formation of NTHi. In contrast, fluoroquinolones followed by macrolides showed lower MBECs. MBEC would be a good parameter to infer the efficacies of antimicrobials against NTHi in biofilm.

Mucoepidermoid Carcinoma in the Nasopharynx.

We report a case of mucoepidermoid carcinoma in the nasopharynx. A woman, aged 59, who suffered from chronic otitis media with effusion was referred to our department due to a mass lesion on the Rosenmullers fossa. The mass lesion was pathologically diagnosed as a mucoepidermoid carcinoma. CT and MRI scans showed a well bordered round shadow which was limited to the orifice of the left eustachian tube, without invasion of the skull base.As the patient did not agree to surgical treatment, we selected radiotherapy using a gamma knife for treatment of the nasopharyngeal tumor. The tumor responded well to gamma knife treatment and disappeared with 30Gy irradiation (Edge dose). The patient has shown no recurrence of the tumor for a year and 6 months after treatment. Gamma knife radiotherapy may be one potential treatment strategy for intractable lesions of the head and neck such as mucoepidermoid carcinoma in the nasopharynx.