Naohiro Okitsu

CFE-1, a Novel Plasmid-Encoded AmpC β-Lactamase with an ampR Gene Originating from Citrobacter freundii

ABSTRACT A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded β-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a β-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 β-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 β-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC β-lactamase, CFE-1, with an ampR gene derived from C. freundii.

Detection of respiratory viruses in nasopharyngeal secretions and middle ear fluid from children with acute otitis media

Conclusions. Our results suggest that various respiratory viruses contribute to the pathogenesis of acute otitis media (AOM). Objective. AOM is one of the most common complications of viral upper respiratory tract infections in children. Recently, the importance of respiratory viruses has been stressed as causative agents of AOM. Subjects and methods. A total of 1092 children ≤10 years old (average age 1.38 years) diagnosed as having AOM between 2002 and 2004 were studied. Bacterial and viral cultures of both nasopharyngeal secretions (NPS) and middle ear fluid (MEF) were performed for all 1092 children. Body temperature, changes of the tympanic membrane, and the number of days from the onset of illness were analyzed. Results. 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.

Characterization of ermB Gene Transposition by Tn1545 and Tn917 in Macrolide-Resistant Streptococcus pneumoniae Isolates

ABSTRACT 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 [MIC90], 128 mg/liter) but susceptibility to rokitamycin (MIC90, 1 mg/liter), with the ermB gene located on Tn1545. Group 2 (65 strains) showed a tendency to high resistance to both antibiotics (MIC90s 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.

Impact of the Seven-valent Pneumococcal Conjugate Vaccine on Acute Otitis Media in Japanese Children: Emergence of Serotype 15A Multidrug-resistant Streptococcus pneumoniae in Middle Ear Fluid Isolates.

Background: Streptococcus pneumoniae is one of the most common bacteria causing acute otitis media (AOM). In Japan, a 7-valent pneumococcal conjugate vaccine (PCV7) was introduced for voluntary vaccination of children in 2010, and it became a recommended vaccination in April 2013. We surveyed the serotypes and antimicrobial susceptibility profile of S. pneumoniae isolates obtained from the middle ear fluid of Japanese children with AOM. Methods: Between April and September 2013, a total of 176 S. pneumoniae isolates were obtained from the middle ear fluid of children aged 0–3 years with AOM. Isolates were collected from various regions of Japan. Minimum inhibitory concentrations were measured by the broth microdilution method. Serotyping was performed by observing the Quellung reaction. Results: Although 45.5% of the strains were susceptible to penicillin G, 42.6% were penicillin-intermediate strains and 11.9% were penicillin-resistant strains. Serotype 19A (27.3%), serotype 15A (14.2%) and serotype 3 (11.9%) showed a high frequency. Although PCV7 types only accounted for 4.5% of all strains, 44.9% were PCV13 types and 55.1% were non-PCV types. Serotype 15A strains were 100% nonsusceptible to penicillin G and all of these strains showed multidrug resistance. Serotype 15A was frequent in children up to 1 year old. Conclusion: After this research was completed, PCV7 was switched to a PCV13 that also contained serotype 3 and serotype 19A. We need to consider the possibility that serotype 15A, which is not included in PCV13, may increase and cause intractable AOM in the future.

Improvement rate of acute otitis media caused by Haemophilus influenzae at 1 week is significantly associated with time to recovery.

ABSTRACT Acute otitis media (AOM) is the most common upper respiratory tract infection in childhood. Children with AOM were enrolled at Tohoku Rosai Hospital between July 2006 and June 2011 if their middle ear fluid cultures after tympanocentesis yielded only Haemophilus influenzae. 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 μg/ml). In the microtiter biofilm assay, the median optical density at 600 nm (OD600) 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.

Detection of Chlamydia trachomatis or Neisseria gonorrhoeae in otorhinolaryngology patients with pharyngeal symptoms

As sexual behaviour has become more diversified, oral sex has become common (especially among younger people) and urogenital micro-organisms, such as Chlamydia trachomatis or Neisseria gonorrhoeae , are also being detected in the pharynx.1– ,5 We performed testing for pharyngeal C trachomatis and N gonorrhoeae in patients with pharyngeal symptoms. Among patients who presented at Tohoku Rosai Hospital otorhinolaryngology outpatient clinic with acute tonsillitis, acute pharyngitis or abnormal pharyngeal sensation syndrome between June 2012 and March 2013, 225 patients gave written informed consent to the collection of specimens and were enrolled …

Binax NOW® Streptococcus pneumoniae test of middle ear fluid for detecting causative pathogens in children with acute otitis media

We investigated rapid diagnosis of acute otitis media, (AOM) with the Binax NOW® Streptococcus pneumoniae test kit. Middle ear fluid specimens were obtained from 38 children with AOM (mean age: 1.1 years). Binax NOW® demonstrated 100% sensitivity and 72% specificity, suggesting it is a useful auxiliary test for AOM.