The affinity of [ 3 H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of ␤-lactamase-negative ampicillin (AMP)-resistant (BLNAR)Haemophilus influenzae for which the AMP MIC was >1.0 g/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the ftsI gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of the dacB gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the ftsI gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-ThrGly (KTG) motif, was substituted for Arg-517 in group I strains (n ‫؍‬ 9), and Lys-526 was substituted for Asn-526 in group II strains (n ‫؍‬ 12). In group III strains (n ‫؍‬ 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of ␤-lactams for H. influenzae transformants into which the ftsI gene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for ␤-lactams. There was no clear relationship between 7-bp deletions in the dacB gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in ␤-lactam resistance, these data indicate that mutations in the ftsI gene are the most important for development of resistance to ␤-lactams in BLNAR strains.Haemophilus influenzae is one of the important pathogens causing respiratory tract infection (RTI), acute otitis media (AOM), pneumonia, and purulent meningitis. Ampicillin (AMP) resistance in this organism is due to two well-known mechanisms. One is resistance mediated by the production of TEM-1 (20) and ROB-1 (13) ␤-lactamases, and the other is decreasing affinity of AMP for penicillin-binding proteins (PBPs) involved in peptidoglycan synthesis (14)(15)(16)19). Strains with resistance due to the latter mechanism are termed ␤-lactamase-negative AMP-resistant (BLNAR) H. influenzae.In surveillance studies conducted in the United States the incidence of ␤-lactamase-producing AMP-resistant (BLPAR) H. influenzae has gradually increased (6, 7, 10) and accounted for 36.4% of all isolates (5) in 1994 and 1995. In contrast, BLNAR isolates remain extremely uncommon in the United States.In Japan, according to nation wide surveillance studies conducted in 1997 and 1998, the proportion of clinical isolates supposed to be BLNAR has rapidly increased to 28.8% in parallel with the increased prevalence of penicillin (PEN)-resistant Streptcoccus pneumoniae. The characteristic of resistant H. influenzae isolates is that the AMP MIC is Ն1 g/ml, whereas the MIC for susce...

Among 380 Mycoplasma pneumoniae isolates from 3,678 pediatric patients with community-acquired pneumonia, 50 macrolide-resistant strains had an A2063G transition in domain V of the 23S rRNA, whereas 5 had an A2064G transition. These resistant strains increased rapidly from April 2002 to December 2006.For Mycoplasma pneumoniae, a major etiologic agent of lower respiratory tract infections acquired in the community, 14-membered ring macrolides (ML) generally are recognized as first-choice agents. In Japan, ML-resistant (ML r ) M. pneumoniae possessing a 23S rRNA mutation first was isolated from pediatric patients with community-acquired pneumonia (CAP) and bronchitis as reported in 2001 by Okazaki et al. (4). Patient symptoms appeared to be prolonged when isolates showed ML resistance (5).We subjected M. pneumoniae isolated from pediatric patients with CAP between 2002 and 2006 to susceptibility evaluation for eight agents, including ML. In strains showing ML resistance, the 23S rRNA gene was analyzed.Between April 2002 and December 2006, 3,678 clinical samples were sent to our laboratory from pediatricians affiliated with 10 institutions participating in the Acute Respiratory Diseases Study Group. All samples originating from pediatric patients diagnosed with pneumonia according to clinical symptoms and chest X-ray images were collected after informed consent was given by the patients and/or their parents or guardians.Immediately after receipt, the samples were suspended in 1.5 ml of pleuropneumonia-like organism (PPLO) broth (Difco, Detroit, MI). DNA then was extracted by using Extragen II (Tosoh, Tokyo, Japan) according to the manufacturer's protocol. Real-time PCR to detect M. pneumoniae was performed as described previously (2) using the extracted DNA. Culture of M. pneumoniae was carried out for PCR-positive samples using PPLO broth according to previously described methods (6).The MICs of eight agents for M. pneumoniae isolates were determined with microdilution methods using PPLO broth.These agents were erythromycin, clarithromycin, azithromycin, josamycin, rokitamycin, telithromycin, minocycline, and levofloxacin. M. pneumoniae M129 strain was used as a control.The full length of the 23S rRNA gene was sequenced by methods described previously (3) in 55 M. pneumoniae strains showing ML resistance.For patients with adequate clinical information, clinical courses of CAP caused by ML r M. pneumoniae (n ϭ 53) were compared to those of CAP with ML-susceptible (ML s ) M. pneumoniae (n ϭ 58). Variables compared included (i) the number of days from initiation of ML treatment until defervescence to 37°C and (ii) whether or not initial treatment with ML was changed later to another agent. Body temperature that exceeded 38°C at least once daily was defined as ongoing fever. Table 1 Table 2 shows the MIC range, MIC 50 , and MIC 90 for eight agents according to the presence or absence of a mutation of the 23S rRNA gene in the 380 M. pneumoniae isolates; 50 strains had an A2063G transition in domain V, and 5 strains had...

Macrolides are inappropriate as first-choice agents against MRMP in terms of shortening the clinical course and decreasing M. pneumoniae. Control and prevention of MRMP outbreaks in children require early decreases in M. pneumoniae as well as improvement of clinical findings.

Clinical isolates of Haemophilus influenzae from Japan (n = 296) and the United States (n = 100) were tested by the microdilution method for susceptibility in vitro to 10 beta-lactam antibiotics and molecular mechanisms of beta-lactam resistance. For all isolates, PCR was used to identify six elements, including beta-lactamase-producing ampicillin (AMP)-resistance (BLPAR) and beta-lactamase-nonproducing AMP-resistance (BLNAR) genes as follows: (1) TEM-1 type beta-lactamase gene, (2) ROB-1 type beta-lactamase gene, (3) part of normal ftsI gene encoding PBP3, which is involved in septal peptidoglycan synthesis, (4) a portion of the ftsI gene possessing some amino acid substitutions commonly detected in BLNAR strains, (5) p6 gene encoding P6 membrane proteins specific to H. influenzae, and (6) serotype b capsule gene. In Japanese and U.S. isolates, respective prevalences of each resistance class in Japan and the United States were 55.1% and 46% for beta-lactamase-nonproducing, AMP-susceptible (BLNAS); 3.0% and 26% for the TEM-1 type beta-lactamase gene; 0% and 10% for the ROB-1 type; 26.4% and 13% for low-BLNAR with a low degree of AMP resistance; and 13.2% and 0% for BLNAR strains. A few remaining isolates were beta-lactamase-producing strains with a mutation in the ftsI gene. MICs of all beta-lactam agents against low-BLNAR strains were 2-8 times higher than against BLNAS. MICs of cephalosporin antibiotics against BLNAR strains were 16-32 times higher than against BLNAS. The rank order of beta-lactam MIC90 values against BLNAR strains was piperacillin = ceftriaxone = cefditoren (0.25 microg/ml), meropenem (0.5), cefotaxime (1), AMP = cefpodoxime (8), cefdinir (16), amoxicillin (16), and cefaclor (64). Serotype b isolates were few in both countries (2.4% in Japan, 3% in the United States). Differences in proportions of respective AMP-resistant genes in H. influenzae isolates between the two countries might reflect differences in antibiotic agents ordinarily given to outpatients with community-acquired bacterial infections.

A total of 395 Haemophilus influenzae strains from 226 Japanese institutions participating in the Nationwide Surveillance Study Group for Bacterial Meningitis were received from 1999 to 2002. All strains were analyzed by PCR to identify the resistance genes, and their susceptibilities to ␤-lactam agents were determined. Of these strains, 29.1% were ␤-lactamase nonproducing and ampicillin (AMP) susceptible (BLNAS) and lacked all resistance genes; 15.4% were ␤-lactamase producing and AMP resistant and had the bla TEM-1 gene; 30.6% were ␤-lactamase nonproducing and AMP resistant (low-BLNAR) and had a Lys-526 or His-517 amino acid substitution in ftsI encoding PBP 3; 13.9% were ␤-lactamase nonproducing and AMP resistant (BLNAR) and had an additional substitution of Thr-385 in ftsI; 9.1% were amoxicillin-clavulanic acid resistant (BLPACR I) and had the bla TEM-1 gene and a Lys-526 or His-517 amino acid substitution in ftsI; and 1.8% showed resistance similar to that of the BLPACR I group (BLPACR II) but had bla TEM-1 gene and ftsI substitutions, as was the case for the BLNAR strains. All but three strains were serotype b. The prevalence of BLNAR strains has increased rapidly: 0% in 1999, 5.8% in 2000, 14.1% in 2001, and 21.3% in 2002. The MICs at which 90% of BLNAR isolates were inhibited were as follows: AMP, 16 g/ml; cefotaxime, 1 g/ml; ceftriaxone, 0.25 g/ml; and meropenem, 0.5 g/ml. All of these values were higher than those for the BLNAS counterpart strains. The relatively wide distributions of the ␤-lactam MICs for BLNAR strains presumably reflect variations in ftsI gene mutations. Pulsed-field gel electrophoresis suggested the rapid spread of specific H. influenzae type b strains throughout Japan. Expedited vaccination, rapid identification, and judicious antibiotic use could slow their spread.Penicillin-intermediate Streptococcus pneumoniae and penicillin-resistant S. pneumoniae isolates from patients with respiratory tract infections (RTIs) have emerged and increased in number in Japan since 1988 (1). In contrast to the phenotypes of S. pneumoniae isolates from the United States (15), strains with an abnormal PBP 2X and strains for which penicillin G MICs at which 50% of isolates are inhibited (MIC 50 s) were 0.06 g/ml and cefotaxime (CTX) MIC 50 s were 0.125 to 0.5 g/ml accounted for 20% of all strains collected throughout Japan between 1998 and 2000 (22). The high prevalence of these resistant organisms is attributed to the frequent use of oral and intravenous cephem antibiotic agents in Japan (15,22). A similar situation is now evident among Haemophilus influenzae isolates from patients with RTIs, in which a rapid increase in the number of ␤-lactamase-nonproducing, ampicillin (AMP)-resistant (BLNAR) H. influenzae strains has been detected (11,23). Resistance in BLNAR strains results from mutations in the ftsI gene encoding PBP 3, which mediates septal peptidoglycan synthesis (24). Amino acid substitutions identified at three positions in the ftsI gene are very important for resistance phenotypes: (i) Asn-...

We have developed a real-time reverse transcription-PCR (RT-PCR) method to detect 13 respiratory viruses: influenza virus A and B; respiratory syncytial virus (RSV) subgroup A and B; parainfluenza virus (PIV) 1, 2, and 3; adenovirus; rhinovirus (RV); enterovirus; coronavirus (OC43); human metapneumovirus (hMPV); and human bocavirus (HBoV). The new method for detection of these viruses was applied simultaneously with real-time PCR for the detection of six bacterial pathogens in clinical samples from 1700 pediatric patients with community-acquired pneumonia (CAP). Of all the patients, 32.5% were suspected to have single bacterial infections; 1.9%, multiple bacterial infections; 15.2%, coinfections of bacteria and viruses; 25.8%, single viral infections; and 2.1%, multiple viral infections. In the remaining 22.6%, the etiology was unknown. The breakdown of suspected causative pathogens was as follows: 24.4% were Streptococcus pneumoniae, 14.8% were Mycoplasma pneumoniae, 11.3% were Haemophilus influenzae, and 1.4% were Chlamydophila pneumoniae. The breakdown of viruses was as follows: 14.5% were RV, 9.4% were RSV, 7.4% were hMPV, 7.2% were PIV, and 2.9% were HBoV. The new method will contribute to advances in the accuracy of diagnosis and should also result in the appropriate use of antimicrobials.

To clarify year-to-year changes in capsular serotypes, resistance genotypes, and multilocus sequence types of Streptococcus pneumoniae, we compared isolates collected from patients with invasive pneumococcal disease before and after introductions of 7- and 13-valent pneumococcal conjugate vaccines (PCV7 and PVC13, respectively). From April 2010 through March 2017, we collected 2,856 isolates from children and adults throughout Japan. Proportions of PCV13 serotypes among children decreased from 89.0% in fiscal year 2010 to 12.1% in fiscal year 2016 and among adults from 74.1% to 36.2%. Although nonvaccine serotypes increased after introduction of PCV13, genotypic penicillin resistance decreased from 54.3% in 2010 to 11.2% in 2016 among children and from 32.4% to 15.5% among adults. However, genotypic penicillin resistance emerged in 9 nonvaccine serotypes, but not 15A and 35B. Multilocus sequence typing suggested that resistant strains among nonvaccine serotypes may have evolved from clonal complexes 156 and 81. A more broadly effective vaccine is needed.

This infection is an increasing threat to aging populations.