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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 (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.

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.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.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.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.

Primary malignant lymphoma of the thyroid gland is not a rare disease that we come across in our hospital. Here we report a rare case of adenomatous goiter infiltrated by malignant lymphoma. A 42-year-old woman was admitted to our hospital because of slow-growing anterior neck tumor and rapid-growing axillary tumor. The right anterior neck tumor was 12 cm in diameter and the right axillary tumor was 7 cm in diameter. Fine needle aspiration cytology revealed that these two tumors were class V and malignant lymphoma was suspected. We performed an open biopsy of the neck tumor and histopathological examination revealed that diffuse large B-cell lymphoma co-existed with adenomatous goiter. Eight courses of CHOP were conducted for the patient and CR was ob-tained in her axillary tumor but her neck tumor was NC. We performed surgical resection of the neck tumor and histopathological examination revealed the pathological CR of malignant lymphoma.

Measles virus was isolated from the middle ear fluid (MEF) of two infant cases of acute otitis media (AOM) associated with measles. This is the first report on the isolation of measles virus from the MEF in patients with AOM, and possibility of the measles virus as a causative agent of AOM was suggested.

Summary Objective: Nasopharynx is thought to be a very important site as becterial reservoir for acute otitis media (AOM). In this study, we investigated on the homogeneity of nasopharyngeal microflora at the different location of nasopharynx of children with AOM. Methods: Thirty nasopharyngeal samples of 15 children with AOM, two samples harvested from both nostrils of each child, were cultured and analyzed by patterns of antibiotic susceptibility and pulsed-field gel electrophoresis analysis (PFGE). Results: A total of 30 nasopharyngeal samples were cultured and 19 isolates of Streptococcus pneumoniae from 10 children (66.7%), 8 isolates of Haemophilus influenzae from 4 children (26.7%), and 12 isolates of Moraxella catarrhalis from 7 children (46.7%) were obtained. In all children except three, the nasopharyngeal microflora at right and left orifice of the eustachian tubes showed no obvious differences in the bacterial species and quantities. Furthermore, in children with the same species of were cultured from right and left orifice of the eustachian tubes at the same time, all nine couples of S. pneumoniae isolates, four couples of H. influenzae isolates, and five couples of M. catarrhalis isolates showed about the same susceptibility and PFGE patterns. Conclusion: These results suggest that the microflora at the different location of nasopharynx of children with AOM is almost homogeneous, irrespective of the clinical signs.

A clinical isolate ofEscherichia colifrom 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 intoE. coliML4947. 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 inE. coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with theampCgene ofCitrobacter freundiiGC3. DNA sequence analysis also identified a gene upstream ofampCwhose sequence was 99.0% identical to theampRgene fromC. freundiiGC3. In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding theampR-ampCgenes inC. freundiiwere identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26composite 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 anampRgene derived fromC. freundii.

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 …

An enzyme-linked immunosorbent assay specific to outer membrane protein P6 (P6-ELISA) was applied for detecting Haemophilus influenzae in middle ear fluids (MEFs) from acute otitis media (AOM) patients and in nasopharyngeal secretions (NPSs) from acute rhinosinusitis patients. P6-ELISA had a sensitivity of 83.3% for MEFs and 71.5% for NPSs and a specificity of 85.6% for MEFs and 92.5% for NPSs, respectively. Real-time PCR exhibited significant differences in the number of ompP1 gene copies among samples determined by P6-ELISA to be positive and negative for H. influenzae. However, because the P6-ELISA test has the reactivity in Haemophilus species include two commensals H. haemolyticus and H. parainfluenzae, it is thus a weak method in order to detect only NTHi correctly. Consequently, diagnosis using the P6-ELISA should be based on an overall evaluation, including the results of other related examinations and clinical symptoms to prevent misleading conclusions in clinical setting.

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.

We examined antibacterial activities of 4 kinds of macrolides (MLs), erythromycin (EM), clarithromycin (CAM), azithromycin (AZM) and rokitamycin (RKM), against 4 bacterial species of clinical strains isolated in 2004. Bacterial isolates used were 51 strains of methicillin-susceptible Staphylococcus aureus (MSSA), 20 of Streptococcus pyogenes, 68 of Streptococcus agalactiae, and 120 of Streptococcus pneumoniae. Macrolide resistance genes, ermB and mefE, in macrolide-resistant S. pyogenes and S. agalactiae, and all of pneumococci were analyzed by PCR. Antimicrobial activities against macrolide-susceptible MSSA of EM and CAM, were more potent than those of RKM. By contrast, against S. pneumoniae, RKM was more effective than EM, CAM and AZM. Against S. pyogenes and S. agalactiae, 4 antibiotics showed similar antimicrobial activities. Twelve, 1 and 2 strains of MSSA, S. pyogenes and S. agalactiae, respectively, were resistant to EM, CAM and AZM, whereas RKM was active to almost, but not quite, of them. Among 120 strains of S. pneumoniae, 76 (63.3%) were resistant to EM (MIC;or = 0.5 microg/mL), and 23, 15 and 28 strains were highly resistant (MIC;128 microg/mL) to EM, CAM and AZM, respectively. By contrast, for RKM, there were far fewer resistant strains, and there was no highly resistant strain. PCR analyses of macrolide-resistant genes revealed that 1 resistant strain of S. pyogenes and 2 of S. agalactiae carried mefE and ermB, respectively. In the case of S. pneumoniae, 59, 19 and 5 strains, respectively, carried ermB, mefE and both ermB and mefe. We also studied about bactericidal activities and postantibiotic effects (PAE) of MLs using macrolide-susceptible, and ermB- and mefE-carrying S. pneumoniae, and observed morphological alterations of the strains treated with the drugs by a scanning electron microscope. It was demonstrated that RKM had superior bactericidal activities and PAE than other 3 drugs, and potent destructive effects to all of 3 strains.

Summary Objective Acute otitis media (AOM) is one of the most common complications of viral respiratory tract infections in children, but the role of each virus is still to be elucidated. We analyzed AOM associated with infection by cytomegalovirus (CMV), which is known as one of the major causes of viral respiratory tract infection. Methods Four hundred and ninety-five children (292 boys and 203 girls) diagnosed as having AOM in 2002 were studied. All of the children were under 6 years old, with the average age being 1.31 ± 1.36 years. Bacterial and viral culture of both nasopharyngeal secretions (NPS) and middle ear fluid (MEF) was performed in all 495 children. The levels of glutamyl pyruvic transaminase (GPT) and the serum IgM antibody for CMV were measured. CMV infection was defined on the basis of isolation of this virus by culture and/or positive anti-CMV IgM antibody. NPS and MEF specimens of the subjects diagnosed as having CMV infection were tested for the virus by nested PCR. Results Twelve of the 495 children were found to have CMV infection. They included 6 boys and 6 girls aged from 3 to 25 months, with the average age being 11 ± 7 months. Among 10 children in whom CMV infection was diagnosed by viral culture, CMV was isolated from NPS alone in nine cases and from both NPS and MEF in one case. Nested PCR was performed in all 12 subjects diagnosed as having CMV infection, and all NPS samples were positive, as were 8 MEF samples. We obtained serum samples from 205 children under 2 years of age, including 9 with CMV infection. The mean serum GPT level of 124 children in whom no viruses were detected was 20.7 ± 14.4 IU/L. While, the serum GPT levels of 9 children with CMV infection ranged from 10 to 280 IU/L with the average titer being 78.4 ± 81.9 IU/L, and the GPT levels of the children with CMV infection were significantly higher than those of the children in whom no viruses were detected ( p Conclusion Our results suggested that CMV is a causative pathogen of AOM, and that CMV infection should be suspected in patients with AOM and liver dysfunction.

In Streptococcus pneumoniae , the ermB gene is carried by transposons, such as Tn 917 and Tn 1545 . This study investigated the relationship between macrolide resistance and the presence of the ermB gene on Tn 917 or Tn 1545 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 Tn 1545 . 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 Tn 917 . 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 Tn 1545 spread clonally while Tn 917 spread both horizontally and clonally. In conclusion, in Japanese macrolide-resistant S. pneumoniae isolates, the ermB gene is carried and spread primarily by Tn 917 .

Identification of pathogens in childhood community-acquired pneumonia (CAP) is not easy. However, it is believed that nasopharyngeal colonization of pathogenic bacteria leads to childhood CAP, so the etiology is inferred by the isolates obtained from nasopharynx of children with CAP. Among the pathogens of childhood CAP, Streptococcus pneumoniae (SP) is the most important agent and macrolides resistant SP (MRSP) is increasingly reported. We investigated the characterization of the mechanism of macrolide resistance in isolates of MRSP by the presence of the ermB gene or the mefA gene and clindamycin (CLDM) resistance. In addition, we also assessed the efficacy of azithromycin (AZM) in children with CAP who were isolated MRSP from nasopharynx. During a 6 month period between January and June in 2002, children with CAP who were treated with a 3 day regimen of AZM and isolated SP from nasopharynx were enrolled. Clinical outcome was based on assessment of fever on the fourth day of treatment. MIC measurements were obtained by broth microdilution and interpreted according to NCCLS criteria. 53 patients were enrolled and MRSP were isolated in 41 children. Of 41 MRSP isolates, 25 isolates were identified CLDM resistance. The AZM MIC90 of CLDM resistant MRSP isolates was 128 microg/ml. On the other hand, that of CLDM sensitive MRSP isolates was 8 microg/ml. However, AZM was effective in 20 children isolated CLDM resistant MRSP and 15 out of 16 children isolated CLDM sensitive MRSP. On this background, despite high rates of MRSP in Japan, AZM continues to be clinically effective for the treatment of childhood CAP.

Summary Objective: Nasopharyngeal microflora contains some beta-lactamase-producing microorganisms. In this study, we investigated in vitro on the indirect pathogenicities of Haemophilus parainfluenzae ( H. parainfluenzae ) and Moraxella catarrhalis ( M. catarrhalis ) against the antipneumococcul activities of some beta-lactams. Methods: We compared the antimicrobial and bactericidal activities of beta-lactams against penicillin-susceptible Streptococcus pneumoniae (PSSP) with or without presence of the enzymes of two species of beta-lactamase-producing microorganisms, H. parainfluenzae and M. catarrhalis . Results: When adding the enzymes extracted from these two beta-lactamase-producing microorganisms in equivalent amounts of 10 7 CFU/spot, the minimum inhibitory concentrations of amoxicillin (AMPC) and cefaclor (CCL) increased to >64 μg/mL. Even third-generation cephalosporins, such as cefditren (CDTR) and ceftriaxone (CTRX) showed marked increases with the enzyme of M. catarrhalis . In time-kill kinetics, same phenomenon was observed in mixed culture indicating the indirect pathogenicities of distinct bacteria, not extracted enzymes, on the cidal activities of beta-lactams against PSSP. Clavulanic acid (CVA)/AMPC, faropenem (FRPM), and imipenem (IPM) were not affected by these beta-lactamase-producing strains with respect to their activities against PSSP. However, these two beta-lactamase-producing strains and their enzymes did not show any significant influence on the antipneumococcul activities of beta-lactams, until the number of bacterial cells reached >10 8 CFU/mL. Conclusion: Our results suggest that these two species of beta-lactamase-producing microorganisms in the nasopharyngeal microflora may act as indirect pathogens on the antipneumococcul activities of beta-lactams with reflecting their substrate profiles, but this is dependent on sufficient amounts of enzyme for their influence as indirect pathogens.

Sir, Ketolides are a new class of semisynthetic macrolide derivatives that show excellent activity against Streptococcus pneumoniae, even against erythromycin-resistant isolates. Previous investigators have not found any telithromycin-resistant isolates among S. pneumoniae with the constitutive macrolide–lincosamide–streptogramin B (cMLSB) phenotype, even though staphylococci or Streptococcus pyogenes with the cMLSB phenotype can develop resistance to this drug.1–3 The lack of induction of methylase production by this drug is one reason for such a difference.4 Recently, Hamilton-Miller & Shah5 reported that resistance to the ketolide cethromycin (formerly ABT-773) could be induced in S. pneumoniae with the cMLSB phenotype by erythromycin or other related antibiotics. In this study, we examined whether 55 isolates of erythromycin-resistant S. pneumoniae (MIC ≥ 1 mg/L) carrying the mef(A) and/or erm(B) genes could develop resistance to telithromycin, a ketolide with different substitutions from cethromycin (ABT-773), when exposed to erythromycin. Fifty-five clinical isolates of erythromycin-resistant S. pneumoniae (obtained from the sputum of patients with lower respiratory tract infections between 1998 and 2000) were used. These isolates were identified by their sensitivity to optochin and the bile solubility test, and by PCR amplification of the lytA gene. S. pneumoniae ATCC 6305 was used as the quality control strain. Reference samples of the following antimicrobial agents of known potency were kindly supplied in powder form by the indicated manufacturers: erythromycin (Shionogi Pharmaceutical Co., Osaka, Japan), clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan), azithromycin (Pfizer Laboratories, Groton, CT, USA), rokitamycin (Asahi Kasei, Tokyo, Japan), telithromycin (Nippon HoechstMarion-Roussel, Tokyo, Japan) and clindamycin (Upjohn, Tokyo, Japan). All isolates were tested for susceptibility to the six antibiotics listed above at concentrations between 0.015 and 128 mg/L. MICs were determined by the two-fold agar dilution method using susceptibility test agar (Mueller–Hinton agar medium; Eiken Chemicals, Tokyo, Japan) with 8% Strepto Haemo supplement (SHS; Eiken Chemicals). DNA was obtained as previously reported, and the presence of macrolide resistance genes was investigated by PCR using primers and amplification conditions that have been described previously.6 Induction of telithromycin resistance was examined by the following two methods. (i) Agar dilution method: induction of telithromycin resistance was evaluated by comparing the MICs for telithromycin in the presence and absence of a subinhibitory concentration of erythromycin (0.1 mg/L). Two susceptibility agar plates were prepared for each isolate: one contained only telithromycin and the other contained telithromycin plus erythromycin. The MIC was determined in accordance with the susceptibility test. (ii) Disc diffusion method: A bacterial suspension (2 mL of ∼108 cfu/mL) was inoculated onto 10 mL of susceptibility test agar containing 8% SHS and spread over the surface. After excess suspension was removed, paper discs (8 mm diameter high discs; Tokyo Roshi Kaisha, Tokyo, Japan) containing erythromycin or rokitamycin at 20 μg/disc or telithromycin at 5 μg/disc were placed on the surface of the agar plate. Then the plates were incubated overnight at 35°C and induction of telithromycin resistance was assessed from the shape of the zone of inhibition around the telithromycin disc nearest either the erythromycin or the rokitamycin disc. Fifteen isolates carrying only the mef(A) gene had the M phenotype. Among the other 40 isolates, 25 carried the erm(B) gene, and 15 had both the mef(A) and erm(B) genes. All 40 isolates showed a high

Emergence of bacterial resistance has rendered ineffective a number of previously valuable antibiotic treatments and now threatens the effectiveness of others. beta-Lactam resistance is no longer predominantly a hospital-treated problem; it has now become an important issue in community medicine. More than 100-beta-lactamases have been identified and classified according to their structure, substrate specificity, and whether they are chromosomal or plasmid-mediated. beta-Lactamase production is rare among Gram-positive pathogens, important exceptions being Staphylococcus aureus and Enterococcus faecalis. By contrast, many Gram-negative pathogens are beta-lactamase-positive; inducible and/or hyper-productive strains are particularly challenging in the clinical setting. Surveillance programs have shown that, in general, beta-lactam resistance is on the increase, and that the plasmid-mediated beta-lactamase have developed rapidly over past decade such as ESBLs and carbapenemases.

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.