A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded ␤-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a ␤-lactamase could be transferred by either conjugation or transformation with plasmid pKU601 into E. coli ML4947. The substrate and inhibition profiles of this enzyme resembled those of the AmpC ␤-lactamase. The resistance gene of pKU601, which was cloned and expressed in E. coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with the ampC gene of Citrobacter freundii GC3. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 99.0% identical to the ampR gene from C. freundii GC3. In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding the ampR-ampC genes in C. freundii were identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26 composite transposon); these results confirm the evidence of the translocation of a ␤-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC ␤-lactamase, CFE-1, with an ampR gene derived from C. freundii.

Respiratory viruses were detected in 360 of 1092 NPS specimens, including 157 isolates of respiratory syncytial virus and 88 of influenza virus. Among 1092 MEF specimens, 102 were virus-positive, including 43 for respiratory syncytial virus and 29 for influenza virus. In 75 children, respiratory viruses were only detected in MEF. The viral detection rate was higher in children with fever at an early stage of their illness. The tympanic membrane changes associated with viral infection tended to be less severe, while changes were more severe in cases with bacterial infection, especially co-infection with bacteria and viruses.

In Streptococcus pneumoniae, the ermB gene is carried by transposons, such as Tn917 and Tn1545. This study investigated the relationship between macrolide resistance and the presence of the ermB gene on Tn917 or Tn1545 in 84 Japanese pneumococcal isolates. Macrolide-resistant strains were classified into two groups as follows. Group 1 (19 strains) showed a tendency to high resistance to erythromycin (MIC at which 50% of isolates are inhibited, 4 mg/liter; MIC at which 90% of isolates are inhibited [MIC 90 ], 128 mg/liter) but susceptibility to rokitamycin (MIC 90 , 1 mg/liter), with the ermB gene located on Tn1545. Group 2 (65 strains) showed a tendency to high resistance to both antibiotics (MIC 90 s for both erythromycin and rokitamycin, >128 mg/liter), with the ermB gene located on Tn917. There were no strains with constitutive macrolide resistance in either group. All of the strains in group 2 had a deletion in the promoter region of ermB and an insertion of the TAAA motif in the leader peptide. The results of pulsed-field gel electrophoresis and serogrouping showed that Tn1545 spread clonally while Tn917 spread both horizontally and clonally. In conclusion, in Japanese macrolide-resistant S. pneumoniae isolates, the ermB gene is carried and spread primarily by Tn917.

The susceptibilities of the isolates to ampicillin were determined, and microtiter biofilm assays and invasion assays using BEAS-2B cells were performed. The association between these bacterial characteristics and clinical relapses of AOM and treatment failures was evaluated. Seventy-four children (39 boys and 35 girls) with a median age of 1 year (interquartile range [IQR], 0.25 to 2 years) were enrolled. Among 74 H. influenzae isolates, 37 showed intermediate resistance or resistance to ampicillin (MIC, >2 g/ml). In the microtiter biofilm assay, the median optical density at 600 nm (OD 600 ) was 0.68 (IQR, 0.24 to 1.02), and 70 isolates formed biofilms. The median invasion rate was 15% (IQR, 0 to 10%), and 46 isolates invaded BEAS-2B cells. Relapses and treatment failures occurred in 19 and 6 children, respectively. There was no significant difference in the invasion rates between patients with and those without relapses or treatment failures. Also, there was no significant association between biofilm formation and relapse or treatment failure. The improvements in the severity scores after 1 week were significantly associated with the recovery time (P < 0.0001). We did not identify any significant association between relapse or treatment failure and bacterial factors. AOM has a multifactorial etiology, and this may explain why we could not find a significant association. An improvement in the severity score after 1 week of treatment may be a useful predictor of the outcome of AOM.A cute otitis media (AOM) is the most common disease of the upper respiratory tract in childhood, and treatment of AOM is the most frequent reason that children in the United States take antibiotics (1). In Japan, the incidence of AOM has increased recently, and many children now need hospitalization to receive intravenous antibiotics for the treatment of intractable AOM with persistent purulent otorrhea (2).We previously reported that Haemophilus influenzae can form a biofilm both in vitro (3) and in vivo (4). Bacterial biofilms are recognized as having an important role in various human infections, and the bacteria in a biofilm are more resistant to antibiotic therapy than are planktonic microorganisms, suggesting that biofilms might play an important role in the pathogenesis and chronicity of otitis media (3). In addition, an investigation of the mechanism of airway epithelium invasion by H. influenzae revealed that the bacteria are internalized by the adenoid cells of children (5). We also previously demonstrated that H. influenzae isolated from clinical samples can invade and destroy human bronchial epithelial cells (BEAS-2B cells) (6), suggesting that such activity might delay the resolution of AOM. However, the association between biofilm formation or invasion of bronchial epithelial cells and the clinical course and outcome of AOM due to H. influenzae has been unclear.Accordingly, we measured the biofilm formation and invasion of bronchial epithelial cells by H. influenzae isolated from children with AOM, and we evaluated the ...