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Streptococcus pneumoniae serotype 35B, a non-vaccine type, is a major contributor to the increase in pneumococcal infection post-vaccination. We aimed to understand the mechanism of its spread by characterizing 35B. The serotype, type 1 pilus (T1P) positivity, and antimicrobial susceptibility of 319 isolates in 2018-2022 were analysed and compared with those of isolates in 2014-2017 to find the changes. 35B accounted for 40 (12.5%) isolates. T1P positivity was notably higher in 35B (87.5%) than in the other serotypes. To confirm the role of T1P, an adhesion factor, we compared adherence to A549 cells between T1P -positive 35B isolates and their T1P -deficient mutants, showing contribution of T1P to adherence. Penicillin-non-susceptible rate of 35B was 87.5%, and meropenem-resistant 35B rate was 35.0%, which increased from 14.5% of 2014-2017 ( p  = 0.009). Multilocus sequence typing was performed in 35B strains. Prevalence of clonal complex 558, harbouring T1P and exhibiting multidrug non-susceptibility, suggested the advantages of 35B in attachment and survival in the host. The emergence of ST156 isolates, T1P -positive and non-susceptible to β-lactams, has raised concern about expansion in Japan. The increase of serotype 35B in pneumococcal diseases might have occurred due to its predominant colonizing ability after the elimination of the vaccine-serotypes.

Purpose: In this study, we aimed to clarify the risk factors associated with unfavorable outcomes in adults with pneumococcal meningitis (PnM)., Methods: Surveillance was conducted between 2006 and 2016. Adults with PnM (n = 268) were followed up for outcomes within 28 days after admission using the Glasgow Outcome Scale (GOS). After classifying the patients into the unfavorable (GOS1-4) and favorable (GOS5) outcome groups, i) the underlying diseases, ii) biomarkers at admission, and iii) serotype, genotype, and antimicrobial susceptibility for all isolates were compared between both groups., Results: Overall, 58.6% of patients with PnM survived,15.3% died, and 26.1% had sequelae. The number of living days in the GOS1 group was highly heterogeneous. Motor dysfunction, disturbance of consciousness, and hearing loss were the commonest sequelae. Of the underlying diseases identified in 68.9% of the PnM patients, liver and kidney diseases were significantly associated with unfavorable outcomes. Of the biomarkers, creatinine and blood urea nitrogen, followed by platelet and C-reactive protein had the most significant associations with unfavorable outcomes. There was a significant difference in the high protein concentrations in the cerebrospinal fluid between the groups. Serotypes 23F, 6C, 4, 23A, 22F, 10A, and 12F were associated with unfavorable outcomes. These serotypes were not penicillin-resistant isolates possessing three abnormal pbp genes (pbp1a, 2x, and 2b), except for 23F. The expected coverage rate of the pneumococcal conjugate vaccine (PCV) was 50.7% for PCV15 and 72.4% for PCV20., Conclusions: In the introduction of PCV for adults, the risk factors for underlying diseases should be prioritized over age, and serotypes with unfavorable outcomes should be considered., Competing Interests: Declaration of competing interest S. Iwata received grant support from Asahi Kasei Pharma Corporation; and personal fees from Pfizer Japan Inc., Astellas Pharma, Meiji Seika Pharma Co., Ltd., and Gilead Sciences. Inc. K. Ubukata has received payment for lectures and manuscripts from Pfizer Japan Inc., HORIBA, Ltd., and Eiken Chemical Co., Ltd. The other authors declare that they have no conflict of interest., (Copyright © 2023 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Purpose: In Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains., Materials and Methods: Pneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to β-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling., Results: In PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations., Conclusions: NVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines., (Copyright © 2022 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: Invasive pneumococcal disease (IPD) is often fatal, requiring prompt diagnosis and treatment. To evaluate the factors associated with IPD in adults, we retrospectively investigated its characteristics compared to pneumococcal pneumonia without confirmation of invasion (PP)., Methods: Patients >18 years with PP (n = 79) and IPD (n = 53) from whom Streptococcus pneumoniae was isolated were enrolled from two hospitals between 2011 and 2017. Clinical backgrounds, blood test results at admission, initial antimicrobials administered, isolate serotypes, and outcomes were compared between the PP and IPD groups., Results: Patients with IPD exhibited higher mortality (28.3%) than those with PP (2.5%) (p<0.001), regardless of the type of antimicrobials first administered. The majority (80.0%) of fatal cases of IPD were due to vaccine serotypes. Almost all patients with PP (97.4%) and IPD (88.7%) had underlying disease. C-reactive protein (CRP) ≥17.0 mg/dL (odds ratio [OR], 7.1; 95% CI, 2.7-19.0; p<0.001), white blood cell counts <11.0 × 10 3 /μL (OR, 3.2; 95% CI, 1.3-8.4; p = 0.016), and platelet (PLT) counts <16.2 × 10 4 /μL (OR, 2.8; 95% CI, 1.1-7.4; p = 0.036) were significantly more common in IPD. Moreover, 89.5% of cases with both CRP ≥23.8 mg/dL and PLT <18.5 × 10 4 /μL were diagnosed with IPD., Conclusion: Laboratory blood test findings at admission, particularly high CRP and low PLT values, are useful early indicators of IPD in adults. These results could be used to initiate rapid and intensive treatment and improve prognosis., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: Risk factors for death from invasive pneumococcal disease (IPD) have not been clearly established in patients aged under 65 years. We aimed to evaluate contributions of host and bacterial factors to the risk of death from IPD in patients aged under 65 years in Japan., Methods: In this prospective, observational, multicenter cohort study, patients with IPD (n = 581) aged 6-64 years were enrolled between 2010 and 2017. We investigated the role of host and bacterial factors in 28-day mortality., Results: The mortality rate increased from 3.4% to 6.2% in patients aged 6-44 years to 15.5%-19.5% in those aged 45-64 years. Multivariable analysis identified the following risk factors for mortality: age 45-64 years (hazard ratio [HR], 3.4; 95% confidence interval [CI], 1.6-6.8, p = 0.001), bacteremia with unknown focus (HR, 2.0; 95% CI, 1.1-3.7, p = 0.024), meningitis (HR, 2.1; 95% CI, 1.1-4.0, p = 0.019), underlying multiple non-immunocompromising conditions (HR, 2.6; 95% CI, 1.1-7.4, p = 0.023), and immunocompromising conditions related to malignancy (HR, 2.4; 95% CI, 1.0-5.2, p = 0.039). Pneumococcal serotype was not associated with poor outcomes., Conclusions: Host factors, including age of 45-64 years and underlying multiple non-immunocompromising conditions, are important for the prognosis of IPD. Our results will contribute to the development of targeted pneumococcal vaccination strategies in Japan., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: The characteristics of pneumococcal isolates and their associations with outcomes in pediatric meningitis are unclear. This study aimed to clarify serotypes and resistance genotypes of Streptococcus pneumoniae from children with meningitis and evaluate the patient prognoses and backgrounds., Methods: Large-scale surveillance was conducted from 2002 to 2016 through periods I-V. Serotypes and penicillin (PEN) resistance genotypes were analyzed for pneumococcal isolates (n = 459) and cerebrospinal fluid (CSF) samples (n = 25). Furthermore, underlying diseases (n = 251), prognoses (n = 202), and laboratory data were evaluated., Results: The number of meningitis cases decreased drastically after the introduction of 7-valent pneumococcal conjugate vaccine (PCV7) to -53.6% and after switching to PCV13 to -70.2%. In particular, this reduction was apparent at ≤3 years of age. The proportion of the PCV7 serotype decreased sharply from 70.1% before introduction to 2.6% during period V; however, the non-vaccine type increased from 17.5% to 87.2%. The PEN resistance rate (gPRSP) was decreased from approximately 49% to 12.2% during period V. Among cases revealed prognosis, sequelae and mortality rates were 16.3% and 5.4%, respectively. The rate of the patients with underlying diseases was 26.3% and relatively high in ≥6 years. Laboratory data associated with a poor prognosis were low white blood cell count (<12.7 × 10 3 /μL), low platelet count (<28.1 × 10 4 /μL), low CSF-glucose (<36 mg/dL), and high CSF-protein (≥142 mg/dL)., Conclusions: Changes in serotype prevalence warrant continuous monitoring to observe future trends of pneumococcal meningitis, and further developments in multivalent conjugate vaccines are required., Competing Interests: Declaration of competing interest S. Iwata has received grant support from Sumitomo Dainippon Pharma Co., Ltd., Pfizer Japan Inc., Taisho Toyama Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Astellas Pharma Inc., and MSD K. K. K. Ubukata has received payment for lectures from Meiji Seika Pharma Co., Ltd., Pfizer Japan Inc., and HORIBA, Ltd. I. Miyairi has received payments for lectures from Sumitomo Dainippon Pharma Co., Ltd., bioMelieux Japan Ltd., Taisho Toyama Pharmaceutical Co., Ltd., Japan Vaccine Co., Ltd., Sanofi, Tanabe Mitsubishi Pharma Co., Ltd., Abbvie Inc. and Abbott diagnostics. The other authors declare that they have no conflict of interest., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: Streptococcus pneumoniae with a mucoid-type capsule is associated with invasive pneumococcal diseases (IPDs). Despite the introduction of pneumococcal vaccines, IPDs caused by mucoid-type isolates are still prevalent. The present study aimed to characterize mucoid-type S. pneumoniae isolated from IPD patients throughout Japan in 2017 (post-vaccination era)., Methods: A total of 225 mucoid-type isolates were collected. The serotype, antimicrobial susceptibility, and multilocus sequence type of these isolates were determined., Results: The prevalence of IPDs caused by mucoid-type isolates was high in adults, especially in the elderly (≥65 years of age), and prognosis in these patients was significantly poor. Of the mucoid-type isolates, the predominant serotype was serotype 3 (84.4%), and the remaining were serotypes 37 (15.1%) and 8 (0.4%). Antimicrobial susceptibility showed that most mucoid isolates exhibited the penicillin-intermediate resistant S. pneumoniae genotype (gPISP). However, the serotype 3 isolate exhibited the penicillin-resistant S. pneumoniae genotype (gPRSP). This gPRSP isolate was classified into ST166, which is related to serotypes 9 V and 11 strains. Sequence analysis of the capsule-coding regions and its flanking regions indicated that recombination occurred upstream and downstream of the capsule-coding region, suggesting that gPRSP (serotype 9 V/ST166) obtaining the type-3 capsule gene cluster resulted in the emergence of gPRSP (serotype 3/ST166)., Conclusions: Our findings indicated that IPDs caused by mucoid-type S. pneumoniae are still a serious concern and mucoid-type S. pneumoniae with novel phenotype could emerge via capsular switching in response to environmental changes such as introduction of vaccines and improper use of antimicrobial agents., Competing Interests: Declaration of competing interest S. Iwata has received grant support from Sumitomo Dainippon Pharma Co., Ltd., Pfizer Japan Inc., Taisho Toyama Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Astellas Pharma Inc., and MSD K. K. H. Mukae has received grant support from MSD K. K., Astellas Pharma Inc., TAIHO Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co.,Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan K.K., FUJIFILM Pharma Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Toyama Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Toyama Chemical Co.,Ltd., Meiji Seika Pharma Co., Ltd., Novartis Phama Co.,Ltd., Nippon Boehringer Ingelheim Co., Ltd., and Pfizer Japan Inc. T. Ishida has received payment for lectures from MSD K. K. H. Kiyota has received grant support from Taisho Toyama Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Astellas Pharma Inc., Toyama Chemical Co.,Ltd. and Sanofi K.K. K. Ubukata has received payment for lectures from Meiji Seika Pharma Co., Ltd. and Pfizer Japan Inc. The other authors declare that they have no conflict of interest., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: Streptococcus pneumoniae is a commensal bacterium of the human nasopharynx and a major causative pathogen of bacterial diseases worldwide. Pilus of S. pneumoniae is one of the virulence factors which enhance the adhesion to the host epitherial cells in the upper respiratory tract., Methods: We analyzed the serotype distribution and presence of pilus genes, rrgC and sipA, among 785 S. pneumoniae isolates from specimens of patients with invasive or non-invasive disease in a regional Japanese hospital between October 2014 and August 2018. We next performed multilocus sequence typing and penicillin-resistant genotyping for 86 isolates of serotype 35B., Results: Serotype 35B was the most frequent serotype which accounted for 11.0% of total isolates and had pilus genes at high rate (80.2%). Clonal complex (CC) 558 isolates accounted for 77.9% of serotype 35B and were highly positive for rrgC and gPRSP (98.5%). In contrast, all CC2755 isolates (19.8%) were rrgC-negative and gPISP., Conclusions: Our results suggest that CC558 may assist the prevalence of serotype 35B after the introduction of vaccines, as that clone has pili as adhesins in addition to non-susceptibility against penicillin. These results may be useful information for development of optimal preventive strategies. Continuous studies on serotype distribution and virulence factors of S. pneumoniae are necessary., Competing Interests: Declaration of Competing Interest H. Miyazaki was provided a grant for education by Pfizer, USA (grant no. 30049557). K. Ubukata has received consulting fees and honoraria from Meiji Seika Pharma, Japan as well as honoraria from Pfizer, Japan. The other authors declare that there are no other conflicts of interest associated with this study., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

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.

Streptococcus pneumoniae is a main causative agent of serious invasive bacterial infections. However, concurrent infection with invasive pneumococcal disease (IPD) and viral infectious mononucleosis (IM) is rare. We report an infant with serotype 6C infection causing IPD occurring simultaneously with IM. A previously healthy 11-month-old girl referred to our hospital because of fever, leukopenia, and elevated C-reactive protein presented to us with disturbance of consciousness, tachycardia, tachypnea and agranulocytosis. Other findings included tonsillitis with purulent exudates and white spots, bilateral cervical adenopathy, and hepatosplenomegaly. We diagnosed her illness as sepsis and administered a broad-spectrum antibiotic, an antiviral agent, and granulocyte transfusions. After treatment was initiated, fever gradually decreased and general condition improved. IPD was diagnosed based upon isolation of S. pneumoniae of serotype 6C from blood cultures obtained on admission. Concurrently the girl had IM, based upon quantitation of Epstein-Barr viral DNA copies in blood and fluctuating serum antibody titers. Although simultaneous IPD and IM is a rare occurrence, this possibility is important to keep in mind., (Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Invasive pneumococcal disease (IPD) causes considerable morbidity and mortality. We aimed to identify host factors and biomarkers associated with poor outcomes in adult patients with IPD in Japan, which has a rapidly-aging population., Methods: In a large-scale surveillance study of 506 Japanese adults with IPD, we investigated the role of host factors, disease severity, biomarkers based on clinical laboratory data, treatment regimens, and bacterial factors on 28-day mortality., Results: Overall mortality was 24.1%, and the mortality rate increased from 10.0% in patients aged ˂50 years to 33.1% in patients aged ≥80 years. Disease severity also increased 28-day mortality, from 12.5% among patients with bacteraemia without sepsis to 35.0% in patients with severe sepsis and 56.9% with septic shock. The death rate within 48 hours after admission was high at 54.9%. Risk factors for mortality identified by multivariate analysis were as follows: white blood cell (WBC) count <4000 cells/μL (odds ratio [OR], 6.9; 95% confidence interval [CI], 3.7-12.8, p < .001); age ≥80 years (OR, 6.5; 95% CI, 2.0-21.6, p = .002); serum creatinine ≥2.0 mg/dL (OR, 4.5; 95% CI, 2.5-8.1, p < .001); underlying liver disease (OR, 3.5; 95% CI, 1.6-7.8, p = .002); mechanical ventilation (OR, 3.0; 95% CI, 1.7-5.6, p < .001); and lactate dehydrogenase ≥300 IU/L (OR, 2.4; 95% CI, 1.4-4.0, p = .001). Pneumococcal serotype and drug resistance were not associated with poor outcomes., Conclusions: Host factors, disease severity, and biomarkers, especially WBC counts and serum creatinine, were more important determinants of mortality than bacterial factors.

After 7-valent pneumococcal conjugate vaccine (PCV) for children was introduced in Japan in November 2010, we examined changes in Streptococcus pneumoniae serotypes and in genetic antimicrobial drug resistance of isolates from adults with invasive pneumococcal diseases. During April 2010-March 2013, a total of 715 isolates were collected from adults with invasive pneumococcal diseases. Seven-valent PCV serotypes in adults decreased from 43.3% to 23.8%, most noticeably for serotype 6B. Concomitantly, 23-valent pneumococcal polysaccharide vaccine (PPSV23) serotypes decreased from 82.2% to 72.2%; non-PPSV23 serotypes increased from 13.8% to 25.1%. Parallel with serotype changes, genotypic penicillin-resistant S. pneumoniae decreased from 32.4% to 21.1%, and 6 non-PPSV23 serotypes emerged (6D, 15A, 15C, 16F, 23A, and 35B). Respective vaccine coverage rates for 13-valent PCV and PPSV23 differed by disease: 73.9% and 84.3% for patients with pneumonia, 56.4% and 69.2% for patients with bacteremia and sepsis, and 45.7% and 69.3% for patients with meningitis.

An increase in the appearance of nonvaccine serotypes in both children and adults with invasive pneumococcal disease (IPD) after introduction of pneumococcal conjugate vaccine represents a limitation of this vaccine. In this study, we generated three recombinant pneumococcal surface protein A (PspA) proteins comprising PspA families 1 and 2, and we examined the reactivity of antisera raised in mice immunized with a PspA fusion protein in combination with CpG oligonucleotides plus aluminum hydroxide gel. The protective effects of immunization with PspA fusion proteins against pneumococcal challenge by strains with five different PspA clades were also examined in mice. Flow cytometry demonstrated that PspA3+2-induced antiserum showed the greatest binding of PspA-specific IgG to all five challenge strains with different clades. PspA2+4- or PspA2+5-induced antiserum showed the lowest binding of PspA-specific IgG to clade 3. Immunization with PspA3+2 afforded significant protection against pneumococcal challenge by five strains with different clades in mice, but immunization with PspA2+4 or PspA2+5 failed to protect mice from pneumococcal challenge by strains with clades 1 and 3. The binding of PspA-specific IgG in antisera raised by three PspA fusion proteins was examined in 68 clinical isolates from adult patients with IPD. Immunization of mice with PspA3+2-induced antiserum with a high binding capacity for clinical isolates expressing clades 1-4, but not clade 5. Our results suggest that the PspA3+2 vaccine has an advantage over the PspA2+4 or PspA2+5 vaccine in terms of a broad range of cross-reactivity with clinical isolates and cross-protection against pneumococcal challenge in mice., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

From February to December 20XX, penicillin-resistant Streptococcus pneumoniae (PRSP) showing MICs of 16-32 microg/mL to cefotaxime (CTX) and 4-8 microg/mL to meropenem (MEPM) were isolated from 6 patients hospitalized at the general hospital S (2 cases) and hospital A (4 cases), close to the hospital S. Five elderly patients among these six cases came from nursing care facilities or nursing care-related medical facilities. All elderly persons (mean age: 81.7 years) were diagnosed as having pneumonia at the time of admission and the problematic PRSP was isolated from sputum samples collected on admission. Notably, all of these PRSP isolates simultaneously showed high resistance to macrolide agents mediated by an erm (B) gene and to fluoroquinolone agents via mutations in the gyrA and parC genes. Eventually, they were identified as multidrug-resistant S. pneumoniae (MDRSP) with high resistance to many agents. The capsule type of all strains was serotype 19F and multilocus sequence typing (MLST) revealed that they belonged to clonal complex (CC) 7993, which has not been reported before. It was thus concluded that the MDRSP that had spread within the nursing facilities was transmitted to the general hospitals via the elderly inpatients with pneumonia caused by these agents. Although one case finally had a poor outcome, the pneumococcal infection was not the direct trigger of the event. The current ratio of MDRSP is concluded to be very low. However, general hospitals that accept patients for therapeutic purposes from nursing-care facilities have to share epidemiological information in a timely manner with the nursing homes to prevent nosocomial infections.

We aimed to clarify changes in serotypes and genotypes mediating β-lactam and macrolide resistance in Streptococcus pneumoniae isolates from Japanese children who had invasive pneumococcal disease (IPD) after the 7-valent pneumococcal conjugate vaccine (PCV7) was introduced into Japan; 341 participating general hospitals conducted IPD surveillance during April 2010-March 2013. A total of 300 pneumococcal isolates were collected in 2010, 146 in 2011, and 156 in 2012. The proportion of vaccine serotypes in infectious isolates decreased from 73.3% to 54.8% to 14.7% during the 3 years. Among vaccine serotype strains, genotypic penicillin-resistant S. pneumoniae strains also declined each year. Among nonvaccine serotype strains, 19A, 15A, 15B, 15C, and 24 increased in 2012. Increases were noted especially in genotypic penicillin-resistant S. pneumoniae isolates of serotypes 15A and 35B, as well as macrolide resistance mediated by the erm(B) gene in 15A, 15B, 15C, and 24.

In Japan, the heptavalent pneumococcal conjugate vaccine (PCV7) has been introduced on a voluntary basis since February 2010, and official financial support for children under 5 years started in November 2010. The impact of PCV7 on invasive pneumococcal diseases (IPD) in children is unknown. There are 340 medical institutions that actively participated in our surveillance project throughout Japan. We collected 252 strains from patients with IPD in 2006 (pre-PCV7), 280 strains in 2010 (under 10% immunization achieved), and 128 strains in 2011 (50% to 60% immunization). Serotypes and penicillin-resistance genotypes (g) were compared between these years. Multilocus sequence typing was also carried out on these strains. Due to the official promotion, IPD significantly decreased in 2011 (p<0.001). In particular, meningitis and sepsis caused by vaccine type (VT) strains declined (p=0.033, p<0.001). In less than 2 years, among nonvaccine types (NVT), 15A and 22F increased in 2011 (p=0.015, p=0.015). Coverage by PCV7 decreased from 71.8% in 2006 to 51.6% in 2011. Sequence-type diversities accompanied by evolution to gPRSP occurred in both VT and NVT strains. Reduction of IPD caused by VT strains was accomplished, but a rapid increase of NVT raises concern about a future decrease in the efficacy of PCV7.

We sought to identify genotypic resistance classes by real-time PCR in 300 Streptococcus pneumoniae isolates from invasive pneumococcal diseases. Primers and molecular beacon probes were designed for the lytA gene, 3 pbp genes, and the mefA/ermB genes. Targeted sequences of pbp1a, pbp2x, and pbp2b genes in susceptible strain R6 corresponded to those of penicillin G-nonsusceptible strains, including sites within or adjacent to conserved amino acid motifs. If amplification did not occur, the corresponding penicillin-binding protein (PBP) was considered to possess amino acid substitution(s) affecting minimal inhibitory concentrations (MICs) of β-lactam antibiotics. Real-time PCR required 90 min or less. Strains were assigned to six genotypic classes: Genotypic penicillin-susceptible S. pneumoniae (gPSSP) with 3 normal genes (22.3%); genotypic penicillin-intermediate S. pneumoniae (gPISP) (pbp2x) with an abnormal pbp2x gene (25.3%); gPISP (pbp2b) with an abnormal pbp2b gene (7.3%); gPISP (pbp1a+2x) with abnormal pbp1a+2x genes (11.3%); gPISP (pbp2x+2b) with abnormal pbp2x+2b genes (4.7%); or genotypic penicillin-resistant S. pneumoniae (gPRSP) with 3 abnormal PBP genes (29.0%). Sensitivity and specificity of real-time PCR compared with those of conventional PCR were high, 73.7-100% and 97.7-100%, respectively. As for relationships between genotype and β-lactam MICs, 90% of MICs for every resistance class were distributed within three serial dilutions for almost all antibiotics. MICs of each β-lactam antibiotic were estimated with high probability from genotypic patterns. In conclusion, determination of genotypic classes of S. pneumoniae using rapid real-time PCR is useful in selecting effective therapeutic agents for patients with pneumococcal infection.

At least four outbreaks of invasive disease caused by serotype 12F, clonal complex 218 Streptococcus pneumoniae have occurred in the United States over the past two decades. We studied the population structure of this clonal complex using a sample of 203 outbreak and surveillance isolates that were collected over 22 years from 34 US states and eight other countries. Conventional multilocus sequence typing identified five types and distinguished a single outbreak from the others. To improve typing resolution, multilocus boxB sequence typing (MLBT) was developed from 10 variable boxB minisatellite loci. MLBT identified 86 types and distinguished between each of the four outbreaks. Diversity across boxB loci tended to be positively correlated with repeat array size and, overall, best fit the infinite alleles mutation model. Multilocus linkage disequilibrium was strong, but pairwise disequilibrium decreased with the physical distance between loci and was strongest in one large region of the chromosome, indicating recent recombinations. Two major clusters were identified in the sample, and they were differentiated geographically, as western and more easterly US clusters, and temporally, as clusters that predominated before and after the licensure of pneumococcal conjugate vaccines. The diversity and linkage disequilibrium within these two clusters also differed, suggesting different population dynamics. MLBT revealed hidden aspects of the population structure of these hyperinvasive pneumococci, and it may provide a useful adjunct tool for outbreak investigations, surveillance, and population genetics studies of other pneumococcal clonal complexes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We examined associations of serotypes with multilocus sequence typing (MLST) data for 7 housekeeping genes and the genotype concerning penicillin resistance based on penicillin-binding protein (PBP) alterations in Streptococcus pneumoniae isolates from children with meningitis. From throughout Japan, we collected 115 pneumococcal isolates from the cerebrospinal fluid of patients 15 years old or younger from January 2007 to December 2009. We then carried out serotyping, MLST, and genotypic classification. Isolates included 24 serotypes and 52 sequence types (STs) according to MLST, of which 18 were novel. The 4 predominant serotypes included a variety of STs: 14 STs in serotype 6B (n = 24), 2 STs in 19F (n = 17), 6 STs in 23F (n = 14), and 5 STs in 14 (n = 11). Resistance genotypes included 6 types: 44.3% for gPRSP (pbp1a + 2x + 2b), 13.9% for gPISP (pbp1a + 2x), 9.6% for gPISP (pbp2x + 2b), 19.1% for gPISP (pbp2x), 3.5% for gPISP (pbp2b), and 9.6% for gPSSP. Interestingly, the most prevalent serotype of 6B included 7 newly identified STs and a variety of genotypes for resistance. STs in serotypes 23F and 14 were highly diverse, but not in 19F. These results suggest that various genetic elements in S. pneumoniae might be intrinsically susceptible to genetic mutations and recombination, with acceleration of emergence reflecting selection pressures such as antibiotic overuse.

The minimum inhibitory concentrations (MICs) and the mutant prevention concentrations (MPCs) of garenoxacin (GRNX), were compared to those of levofloxacin (LVFX), and moxifloxacin (MFLX) against 78 Streptococcus pneumoniae isolates from otorhinolaryngological infections in Japan during the period January 2007 to June 2007. The MIC and MPC for 90% of the isolates (MIC90 and MPC90) of GRNX were 0.06 and 0.12 microg/mL, respectively, and were the lower values than LVFX and MFLX MIC90s and MPC90s. The ratios of MPC/MIC of GRNX were the lower values than those of LVFX and MFLX.

This is the first report to describe an apparently healthy girl, who developed recurrent occult bacteremia by the same Streptococcus pneumoniae strain, at 11 and 15 months of age. The two separately isolated organisms were demonstrated to have the identical serotype (type 6B), antibiotic susceptibility (intermediately penicillin-resistant), genotypes of penicillin-binding proteins, and patterns of pulse-field gel electrophoresis. The serum levels of anti-type 6B antibodies showed poor responses after both bacteremic episodes, but other immunological workups did not demonstrate any abnormalities. This case indicates that occult bacteremia may recur due to an identical pneumococcal strain in an immunocompetent infant, and that early introduction of pneumococcal conjugate vaccine is necessary in Japan.

Between August 1998 and July 2005, we studied the serotypes and mutation of penicillin-binding protein (PBP) genes of 46 strains of Streptococcus pneumoniae isolated from children with invasive pneumococcal infection in Hokkaido. The clinical diagnosis was pneumonia in 16 cases, occult bacteremia in 15, meningitis in 8, upper respiratory infection in 6, and arthritis in 1. Patients ranged in age from 2 months to 9 years old. Prevalent serotypes were 6B (39.1%), 23F (17.4%) 6A (8.7%), and 19F (8.7%). The serotype coverage rate by the 7-valent pneumococcal vaccine was greater than 70% in children. Mutation of 3 genes (Penicillin-binding S. pneumoniae, PRSP) was detected in 18 isolates, mutation of 10 genes (pbp1a and pbp2x, or pbp2x and pbp2b) in 2, and of pbp2x alone in 15. PRSP was found in serotypes 6A, 6B, and 23F, and the rate of PRSP in 6A, 6B, and 23F was 75.0%, 62.5%, and 55.6%, respectively.

Objectives: Antibiotic susceptibilities, genes mediating beta-lactam and macrolide resistance, and serotypes were analysed for strains of Streptococcus pneumoniae., Methods: A total of 392 strains of S. pneumoniae were isolated from paediatric patients with community-acquired pneumonia between May 2002 and 2004. All strains were classified into six genotype patterns according to the mutations found in the pbp1a, pbp2x and pbp2b genes identified by PCR. These results are represented by adding 'g', indicating genotypic identification., Results: Thirty-nine per cent of the isolates showed mutations in either one or two PBP genes (gPISP, where PISP stands for penicillin-intermediate resistant S. pneumoniae) and 52.3% had mutations in three genes (gPRSP, where PRSP stands for penicillin-resistant S. pneumoniae). The majority of the strains had a macrolide resistance gene: mefA, (30.6%); ermB, (48.5%); or both mefA and ermB, (7.7%). The most frequent serotypes of these strains were: 6B (23.2%), 23F (17.6%), 19F (17.3%), 14 (10.5%) and 6A (8.2%). Serotypes of the seven-valent conjugate vaccine covered 70.9% of all isolates, and 89.8% of gPRSP. Serotypes of the strains with cefotaxime MICs of > or =2 mg/L were almost all of a vaccine type., Conclusions: The results suggest that introduction of conjugate vaccines into infants and children is necessary for the prevention of pneumococcal infections in Japan.

Nasopharyngeal transmission of Streptococcus pneumoniae was evaluated among 23 siblings with acute otitis media (AOM). Restriction fragment length polymorphism revealed that the nasopharyngeal strains were identical between siblings in 12 of 13 clusters of AOM experienced in 11 families. This study demonstrated person-to-person transmission of S. pneumoniae, especially drug-resistant strains, among siblings with AOM.