Pyelonephritis with bacteremia caused by Salmonella Choleraesuis in a Japanese patient with carcinoma of unknown primary origin: A case report.

Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) is a nontyphoidal Salmonella pathogen that causes swine paratyphoids. S. Choleraesuis is a zoonotic pathogen transmitted to humans via contaminated food and causes sepsis. Here, we report a rare case of pyelonephritis caused by S. Choleraesuis in a Japanese patient with a carcinoma of unknown primary origin. On the day of admission, the patient was diagnosed with pyelonephritis associated with ureteral stent obstruction. He had no history of raw pork consumption or gastrointestinal symptoms. Gram-negative rods were isolated from urine and blood cultures, identified as Salmonella enterica subsp. enterica using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The serological typing results were O7: -: 1 and 5; however, the serotypes could not be determined. The isolate was identified as S. Choleraesuis using multilocus sequence typing, nucleotide sequence analysis of the fliC gene, and biochemical examination. Four days after a 14-day course of intravenous piperacillin-tazobactam (9 g/day), the patient showed relapse of the condition. Subsequently, the patient was treated with intravenous ceftriaxone (2 g/day) and oral amoxicillin (1000 mg/day) for 14 days each; recurrence was not observed. This novel case of pyelonephritis with bacteremia was caused by S. Choleraesuis in Japan. Conventional testing methods could not identify the serotypes; however, the case highlights the importance of adopting advanced diagnostic techniques based on molecular biology to ensure accurate pathogen identification.

Bacteriological and molecular characterization of temperature- and CO2-dependent Streptococcus pneumoniae serotype 24F ST162 isolated from Japanese children.

IMPORTANCE: We characterized Streptococcus pneumoniae serotype 24F sequence type (ST) 162 isolated from Japanese children with invasive pneumococcal disease (IPD). Owing to its highly invasive nature, serotype 24F is expected to be isolated from clinically significant cases. Serotype 24F ST162 isolates tested in the present study did not grow at 35°C in ambient air. Therefore, antimicrobial susceptibility testing using the broth microdilution method, which is usually conducted in ambient air, cannot be performed, posing a clinical challenge. Clinical practitioners and laboratory personnel should be aware of the epidemiological, bacteriological, and molecular characteristics of serotype 24F ST162. We believe that our findings can help diagnose and treat IPD caused by serotype 24F ST162, a serotype expected to become problematic in the post-13 valent pneumococcal conjugate vaccine era.

Increase in prevalence of Streptococcus pneumoniae serogroup 24 in children upon introducing 13-valent pneumococcal conjugate vaccine in Japan.

After introducing the 13-valent pneumococcal conjugate vaccine (PCV13) for children, a change in the prevalence of different Streptococcus pneumoniae serotypes that cause invasive pneumococcal diseases (IPDs) has been observed. The prevalence of vaccine serotypes has decreased and that of non-vaccine serotypes has increased. Currently, serogroup 24 has become one of the major non-vaccine serotypes causing IPDs in children in Japan. The aim of this study was to characterize clinical and genomic features of S. pneumoniae serogroup 24 strains isolated from sterile body sites in Japanese children. Serotyping, multi-locus sequence typing and genomic analysis of capsular polysaccharides of 61 strains of serogroup 24 were performed from 2015 to 2021. Among the 61 strains, 36, 23 and two belonged to serotypes 24F, 24B and 24C, respectively. The 24F sequence type (ST) 2572 and 24B ST 2572 were the major serotypes and sequence types observed from 2015 to 2019. By contrast, 24F ST 162 and 24B ST 2754 were the two major serotypes and sequence types observed after 2020. Two strains of serotype 24C were detected for the first time in Japan. Sequence analysis of the abpA gene, which plays a role in the synthesis of capsular polysaccharides in S. pneumoniae , was performed to distinguish different strains of serogroup 24. After the introduction of PCV13 in Japan, serogroup 24 has become one of the most prevalent non-vaccine serotypes causing IPDs in children. This serogroup has not been targeted in the next-generation pneumococcal conjugate vaccines. Therefore, monitoring of S. pneumoniae serogroup 24 that causes IPDs in children is essential.

Extensive Subcutaneous Abscess due to Panton-Valentine Leucocidin-Positive Community-Associated Methicillin-Resistant Staphylococcus aureus in an Infant.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have increased worldwide in people without underlying diseases. CA-MRSA can often cause serious bacterial infections, especially skin and soft tissue infections (SSTI). Here, we describe a case of severe subcutaneous abscess due to Panton-Valentine leucocidin (PVL)-positive CA-MRSA in an infant without underlying diseases. A 4-month-old girl presented with a 4-day history of fever, with extensive redness and swelling of the lumbar region and buttocks. She was diagnosed with extensive subcutaneous abscess of the lumbar region and buttocks. Surgical drainage was performed, and a substantial volume of pus was drained. MRSA was detected in the pus on culture. Antibiotic therapy that covered MRSA was also administered for 3 weeks, and the abscess healed. As it was a severe SSTI due to MRSA, analysis of MRSA revealed PVL-positive MRSA. This patient had no underlying disease or history of antibiotic administration, and as MRSA was present in the nasopharyngeal cavity, it was considered a case of CA-MRSA. Furthermore, the prevalence of PVL-positive CA-MRSA in MRSA isolated from patients with SSTI has also increased in Japan. The Infectious Diseases Society of America recommends surgical intervention and empirical antibiotic therapy for MRSA-complicated SSTI cases in an era of CA-MRSA. Pediatricians must strongly consider the possibility of MRSA in children with severe SSTIs.

Bacillus subtilis variant natto Bacteremia of Gastrointestinal Origin, Japan.

We report a case of bacteremia caused by Bacillus subtilis variant natto after a gastrointestinal perforation in a patient in Japan. Genotypic and phenotypic studies of biotin identified B. subtilis var. natto. This case and 3 others in Japan may have been caused by consuming natto (fermented soybeans).

Clinical and Bacteriological Analysis of Pediatric Pneumococcal Meningitis after 13-Valent Pneumococcal Conjugate Vaccine Introduction in Japan.

Streptococcus pneumoniae is one of the leading causes of meningitis in children. In Japan, since the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13), the number of pneumococcal meningitis due to non-PCV13 serotypes in children has increased. To clarify the clinical outcomes, serotype distributions, and antimicrobial susceptibility of isolated S. pneumoniae strains from pediatric pneumococcal meningitis, we clinically and bacteriologically analyzed 34 cases of pediatric pneumococcal meningitis that were reported after the PCV13 introduction era in Japan. The median age at diagnosis was 1 year (range: 3 months-13 years). Ten (29.4%) patients had underlying diseases. Twenty-nine (85.3%) patients had received at least one dose of any pneumococcal vaccine. Of the 34 patients with pneumococcal meningitis, 6 had sequelae, and 4 died. Nine (26.5%) strains were resistant to penicillin; five (15%) strains to meropenem, with an MIC of 0.5 µg/mL. All strains were susceptible to vancomycin and linezolid. Daptomycin's MIC50 was 0.064 µg/mL and MIC90 was 0.094 µg/mL. Among the tested strains, only four were PCV13 serotypes. Penicillin-resistant S. pneumoniae was isolated from 30.0% of the patients with sequelae and death. Particularly, the proportion of serotype 10A in the sequelae and deceased cases was significantly higher than that in the complete recovery cases. We should carefully monitor the serotype and drug susceptibility of S. pneumoniae strains isolated from patients with meningitis after the PCV13 era and reconsider the treatment strategy to prepare against further drug-resistant pneumococcal strains. IMPORTANCE We analyzed 34 cases of pediatric pneumococcal meningitis that were reported after the 13-valent pneumococcal conjugate vaccine (PCV13) introduction era in Japan. Our study revealed that pneumococcal meningitis in children was mainly caused by non-PCV13 serotypes; all cases with sequelae and death were caused by non-PCV13 serotypes. Moreover, all serotypes of penicillin resistant Streptococcus pneumoniae strains (26.5%; 9/34) were non-PCV13 serotypes. We also analyzed antimicrobial susceptibilities of glycopeptides, linezolid (LZD), and daptomycin (DAP) of isolated S. pneumoniae strains. All tested strains were susceptible to vancomycin, teicoplanin, LZD, and DAP. Especially. DAP demonstrated the best outcome among the tested antibiotics, with MIC90 of 0.094 µg/mL. Pneumococcal meningitis in children continues to persist and is difficult to control with the current conjugate vaccines. Therefore, it is important to monitor the serotype and antimicrobial susceptibility of S. pneumoniae strains isolated from patients with meningitis and accordingly reconsider the treatment strategy.

Epidemiological characteristics in serotype 24 paediatric invasive pneumococcal disease according to an 11-year population-based study in Japan.

After the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13), serotype replacement has occurred in Japan, and serotype 24 has become the most common serotype in paediatric invasive pneumococcal disease (IPD). To understand the characteristics of serotype 24-IPD in Japanese children in the post-PCV13 era, we conducted a retrospective study in children aged ≤15 years from 2010 to 2020 using a database of paediatric IPD surveillance in Chiba prefecture, Japan. We identified a total of 357 IPD cases and collected clinical information on 225 cases (24: 32 cases, non-24: 193 cases). Compared with the non-serotype 24-IPD, serotype 24-IPD was independently related to be <2 years of age [odds ratio (OR) 3.91, 95% confidence interval (CI) 1.47-10.44; P = 0.0064] and bacteremia (OR 2.28, 95% CI 1.01-5.13; P = 0.0475), as a result of the multivariate regression analysis. We also conducted a bacterial analysis, and the isolates of serotype 24-IPD had tendencies of PCG-susceptible (24: 100.0%, non-24: 61.3%; P < 0.0001) and macrolide-resistance (24: 100.0%, non-24: 87.3%; P = 0.0490). Their multilocus sequence typing was mostly ST2572 and the variants, which were unique to Japan. This tendency might have been a result of the progress made in the Japanese PCV13 immunisation programme.

Immunogenicity and safety of routine 13-valent pneumococcal conjugate vaccination outside recommended age range in patients with hematological malignancies and solid tumors.

Hematological malignancy and solid tumor are major risks for invasive pneumococcal disease. Thirteen-valent pneumococcal conjugate vaccine (PCV13) is recommended for immunocompromised patients aged 6 years and older and adults who had not received the vaccine previously. However, vaccination for these individuals is not publicly subsidized in Japan. We measured pneumococcal serotype-specific IgGs (Pn-IgGs) and opsonophagocytic activities (Pn-OPAs) against PCV13 serotypes (1, 3, 5, 6A, 7F, and 19A) in patients with hematological malignancies and solid tumors who were outside the recommended age range for routine vaccination at baseline and at 1 and 6 months after the first dose of PCV13. Pneumococcal serotype-specific memory B cells (Pn-MBCs) against serotype 3 were measured from a portion of the study samples. Thirty-seven patients (30 in the young patient group and 7 in the adult patient group) completed the study. Pn-IgGs were significantly elevated at 1 month post-vaccination and persisted in protection level for 6 months after the first vaccination against all six serotypes measured except serotype 3. Pn-OPAs were significantly elevated and persisted as well against all six serotypes. Pn-MBCs were measured in 10 patients, and 90% of them had at least one detectable Pn-MBC, and 70% of them showed an increased frequency of Pn-MBCs against serotype 3. No serious adverse events were observed up to 1 month after vaccination. PCV13 is thus safe and immunogenic, including against serotype 3, in patients with hematological malignancies and solid tumors outside the recommended age range for routine vaccination.

Genetic system underlying responses of Cryptococcus neoformans to cadmium.

Cryptococcus neoformans, basidiomycetous pathogenic yeast, is basically an environmental fungus and, therefore, challenged by ever changing environments. In this study, we focused on how C. neoformans responds to stress caused by cadmium that is one of high-risk pollutants. By tracking phenotypes of the resistance or sensitivity to cadmium, we undertook forward and reverse genetic studies to identify genes involved in cadmium metabolism in C. neoformans. We found that the main route of Cd2+ influx is through Mn2+ ion transporter, Smf1, which is an ortholog of Nramp (natural resistance-associated macrophage protein 1) of mouse. We found that serotype A strains are generally more resistant to cadmium than serotype D strains and that cadmium resistance of H99, a representative of serotype A strains, was found to be due to a partial defect in SMF1. We found that calcium channel has a subsidiary role for cadmium uptake. We also showed that Pca1 (P-type-ATPase) functions as an extrusion pump for cadmium. We examined the effects of some metals on cadmium toxicity and suggested (i) that Ca2+ and Zn2+ could exert their protective function against Cd2+ via restoring cadmium-inhibited cellular processes and (ii) that Mg2+ and Mn2+ could have antagonistic roles in an unknown Smf1-independent Cd2+ uptake system. We proposed a model for Cd2+-response of C. neoformans, which will serve as a platform for understanding how this organism copes with the toxic metal.

Emergence of Haemophilus influenzae with low susceptibility to quinolones isolated from pediatric patients in Japan.

INTRODUCTION: In 2010, oral fluoroquinolone tosufloxacin (TFX) granules were released as the first oral respiratory quinolone for children in Japan. METHODS: To investigate the recent trend of H. influenzae strains with low susceptibility to quinolones in children, we analyzed the gene sequences of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE of 23 clinical isolates from 15 patients aged <15 years with an MIC of ≥0.5 µg/mL for TFX from 2010 to 2018. RESULTS: Amino acid substitutions were observed in both GyrA and ParC in 13 strains (81%, 13/16), except two strains with a TFX MIC of 0.5 µg/mL with amino acid substitution in only GyrA and one strain with a TFX MIC of 1 µg/mL with no amino acid substitution. Four ST422 strains were observed in 2018, the detection age range was wide (0-7 years), and the residential city was varied. A total of 3/15 patients had a clear history of TFX treatment. CONCLUSIONS: Even for the strain with an MIC of 0.5 µg/mL for TFX, it is highly possible that it harbors a mutation in gyrA, which is the first step toward quinolone resistance, and it may also harbor mutations in both gyrA and parC. Furthermore, several specific sequence type quinolone-resistant H. influenzae strains, particularly ST422, may be widespread among children in Japan. It is necessary to investigate changes in resistance both at the MIC and gene levels. The continuous monitoring of strains and the use of antimicrobial drugs in treatment should be carefully observed.

Population-based study of a free rubella-specific antibody testing and immunization campaign in Chiba city in response to the 2018-2019 nationwide rubella outbreak in Japan.

Japan has not been able to eliminate rubella; as a result, the large rubella epidemic has occurred. Considering the complicated history of the vaccine policy in Japan, some susceptible populations became infected with rubella, resulting in an outbreak. We conducted a large serosurveillance against rubella in Chiba city after initiating free rubella-specific antibody testing and an immunization campaign during 2018-2019. The total number of rubella specific antibody tests that was conducted in the nationwide campaign and Chiba city original campaign was 8277 and 6104, respectively. The proportion of participants with an antibody titer of ≤1:16 using the hemagglutination inhibition (HI) test was higher in those in their 20-30s. On the contrary, the proportion of participants with an antibody titer of <1:8 using the HI test was higher in men in their 40-50s. This discrepancy possibly reflects the complicated history of the vaccine policy. The number of participants in the nationwide immunization campaign in this city was 1517, whereas that in the Chiba city campaign was 3607. The Chiba city campaign was effective against women in their 20-30s (child-bearing generation); however, the nationwide campaign was not sufficiently effective against men in their 40-50s because many workers were did not visit medical facilities to receive the measles-rubella vaccine.

Panton-Valentine leukocidin-positive novel sequence type 5959 community-acquired methicillin-resistant Staphylococcus aureus meningitis complicated by cerebral infarction in a 1-month-old infant.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a pathogen of major importance in pediatric patients. CA-MRSA can cause skin and soft tissue infection in children and young active adults with no predisposing factors, and life-threatening infections such as meningitis or necrotizing pneumonia have been reported. We report here a case of CA-MRSA meningitis complicated by acute left middle cerebral artery (MCA) infarction and necrotizing pneumonia in a previously healthy 1-month-old Vietnamese boy. He was firstly treated with vancomycin, but changed to linezolid because of persistent fever and low vancomycin trough level. He recovered successfully with residual right-sided hemiparesis. The mode of transmission of CA-MRSA and the mechanism of cerebral infarction (thrombotic or embolic) were unknown. The isolate was genotyped as staphylococcal cassette chromosome (SCC) mec type V with a novel sequence type (ST) 5959 harboring the Panton-Valentine leukocidin (PVL) gene. ST 5959 is a double locus variant of ST 59, which is a major PVL-positive CA-MRSA strain isolated in invasive disease in Asian countries. This case report may serve as a warning about the dissemination of PVL-positive CA-MRSA in and around Japan, with the possibility of causing serious life-threatening disease. The potential of linezolid for the treatment of MRSA meningitis as one of the alternative MRSA therapeutic drugs is also discussed.

Bacteriological analysis of Neisseria lactamica isolated from the respiratory tract in Japanese children.

INTRODUCTION: Neisseria lactamica is a commensal bacterium of the upper respiratory tract in humans and is closely related to Neisseria meningitidis. N. lactamica colonization may contribute to preventing N. meningitidis colonization and invasive meningococcal disease. However, the transference of antimicrobial resistance genes from N. lactamica to N. meningitidis has been reported. METHODS: In this study, we aimed to identify N. lactamica using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and performed multilocus sequence typing of seven N. lactamica strains isolated from Japanese children. We also analyzed the antimicrobial susceptibility of these strains and the mutations in their antimicrobial resistance genes (penA, gyrA, and parC). RESULTS: All the N. lactamica strains could be identified using MALDI-TOF MS. All strains were of different sequence types (STs), including five new STs. Five strains had intermediate susceptibility, two were resistant to ampicillin, and all had five out of the five known PBP2 mutations. Six strains were resistant to levofloxacin. Among the quinolone-resistant strains, three had GyrA mutations, and three had both ParC and GyrA mutations. CONCLUSIONS: N. lactamica STs may vary in Japanese children, and penicillin- and quinolone-resistant strains may be prevalent. We should pay attention not only to the drug resistance of N. meningitidis but also to the drug susceptibility of N. lactamica whose drug-resistance genes may transfer to N. meningitidis.

First report on multidrug-resistant non-encapsulated Streptococcus pneumoniae isolated from a patient with pneumonia.

The non-encapsulated Streptococcus pneumoniae (NESp) has emerged and increased in the clinical setting. The majority of NESp strains have been isolated from the nasopharynxes of healthy carriers and from respiratory specimens of patients with otitis media. NESp strains were shown to be more effective than encapsulated counterparts at forming biofilms. Therefore, NESp should become one of the leading causes of emerging refractory respiratory disease after the introduction of pneumococcal conjugate vaccines. We report the first case of multidrug-resistant - including fluoroquinolone-resistant - NESp isolated from the intrabronchial aspirate of a patient with pneumonia. Drug-resistant NESp infections can possibly emerge as a clinical problem and thus the continuous monitoring of NESp infections is of utmost importance.

Two cases of bacterial meningitis due to meropenem-resistant Streptococcus pneumoniae: A threat of serotype 35B, ST 558 lineage.

Although the pneumococcal conjugate vaccine (PCV) has decreased the incidence of invasive pneumococcal disease (IPD) in children, cases of IPD caused by non-PCV serotypes have been increasing. Here, we report two cases of bacterial meningitis caused by meropenem-resistant Streptococcus pneumoniae; in both the cases, 13-valent PCV (PCV13) had been administered. The isolated S. pneumoniae strains were non-PCV13 serotype 35B and resistant to penicillin G, cefotaxime, and meropenem. In addition, multilocus sequence typing (MLST) revealed the sequence type (ST) to be 558. In case 1, a 6-month-old girl recovered without sequelae after antibiotic therapy comprising cefotaxime and vancomycin, whereas in case 2, a 9-month-old boy was treated with an empirical treatment comprising ceftriaxone and vancomycin administration. However, maintaining the blood concentration of vancomycin within the effective range was difficult, due to which the antibiotics were changed to panipenem/betamipron. During the treatment, he presented with seizures, which were effectively controlled with antiepileptic drugs. The rate of incidence of penicillin-susceptible IPD has been substantially increasing after the introduction of PCV. However, an upsurge in IPD cases due to multidrug-resistant (MDR) serotype 35B has been reported in countries where PCV13 was introduced before introducing in Japan. Moreover, an increase in the proportion of MDR serotype 35B and decrease in the susceptibility to broad-spectrum antimicrobials, including meropenem, have been reported. Hence, the number of meningitis cases caused by MDR serotype 35B/ST558 may increase in the future.

Total Synthesis and Antimicrobial Evaluation of 23-Demethyleushearilide and Extensive Antimicrobial Evaluation of All Synthetic Stereoisomers of (16Z,20E)-Eushearilide and (16E,20E)-Eushearilide.

A novel stereoisomer of eushearilide, 23-demethyleushearilide, was synthesized, and the structure-activity relationships of this compound along with known eushearilide stereoisomers were investigated in order to design novel lead compounds for the treatment of fungal infections. It was discovered that all of these congeners, together with the natural product, exhibited a wide range of antimicrobial activity against not only fungi but also against bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).

Identification of Haemophilus influenzae serotype e strains missing the fucK gene in clinical isolates from Japan.

Introduction. Certain nontypeable Haemophilus influenzae cannot be assigned a sequence type (ST) by Multilocus Sequence Typing (MLST) due to the lack of the fucK gene, one of seven MLST loci in H. influenzae, which encodes a fucose-operon enzyme.Aims. To confirm whether the loss of fucK is also found in the encapsulated strains, we analysed clinical isolates of H. influenzae serotype e (Hie).Methodology. We conducted MLST, PFGE, and antimicrobial susceptibility tests of 45 Hie strains; the majority (n=43) were derived from respiratory samples of pediatric patients at Chiba Children's Hospital between 2000 and 2016. The two remaining strains were obtained from the blood of elderly patients with invasive H. influenzae diseases (IHiDs) between 2015 and 2016 at general hospitals. For the fucK-negative strains, PCR analysis for fucose operon was also performed.Results. Four STs (ST18, 122, 621 and 1758) were assigned to 13 strains, and remaining 32 (including one associated with IHiD) were fucK-negative, completely missing the fucose operon. The allelic profiles of six other loci were identical among 31 strains and to that of ST18, 122 and 621, and these strains were genetically closely related. Forty of 45 isolates were ampicillin-sensitive.Conclusions. The loss of fucK was frequently observed in clinical isolates of Hie from children. Moreover, fucK-negative Hie may be the cause of IHiD in adult patients. The majority of Hie, including fucK-negative strains, were shown to be clonally related and were ampicillin sensitive. This represents the first report examining fucK losses in encapsulated H. influenzae.

Clonal spread of serotype 12F ST4846 Streptococcus pneumoniae.

Introduction. In 2016-2017, there was an increase in the number of paediatric invasive pneumococcal disease (IPD) cases caused by Streptococcus pneumoniae serotype 12F in Chiba Prefecture, Japan. Serotype 12F is one of the major causative serotypes of IPD following the introduction of pneumococcal conjugate vaccine 13 (PCV13), and outbreaks of IPD caused by serotype 12F have recently been reported in several countries.Aim. Our goal here was to clarify the relationship among local outbreak strains and the outbreak strains in other countries, and for this we analysed clinical isolates of S. pneumoniae serotype 12F using several genetic identification methods.Methodology. All reported IPD cases caused by serotype 12F were reviewed and bacterial strains were collected and analysed. We also analysed S. pneumoniae serotype 12F strains isolated from other time periods, geographical areas, cases of adult IPD and respiratory specimens as control strains. Multi-locus sequence typing, PFGE and multi-locus variable number tandem repeat analysis (MLVA) were conducted on all isolates.Results. All 26 S. pneumoniae serotype 12F isolates, including control strains, belonged to a single sequence type (ST4846) that was the specific ST in Japan. All tested strains demonstrated five MLVA patterns and two PFGE patterns.Conclusion. We determined that the 2016-2017 outbreak of IPD in Chiba Prefecture was caused by clonally related isolates of serotype 12F. The continuous monitoring of IPD caused by serotype 12F is important for evaluating the impact of re-emerging pneumococcal serotypes following the PCV13 introduction era, and MLVA could be a useful tool for identification of outbreak strains.

Molecular typing, antibiotic susceptibility, and biofilm production in nonencapsulated Streptococcus pneumoniae isolated from children in Japan.

The prevalence of nonencapsulated Streptococcus pneumoniae (NESp) has increased with the introduction of pneumococcal conjugate vaccines in children; however, the bacteriological characteristics of NESp have not been sufficiently clarified. In this study, NESp strains isolated from the nasopharyngeal carriage of children from four nursery schools in Japan were analyzed for molecular type, antibiotic susceptibility, and biofilm productivity. A total of 152 putative S. pneumoniae strains were identified by optochin-susceptibility analysis, of which 21 were not serotypeable by slide agglutination, quellung reaction, or multiplex PCR. Among these 21 strains, three were lytA-negative and, therefore, not S. pneumoniae. The remaining 18 strains were positive for lytA, ply, pspK, and bile solubility and were confirmed as NESp. Therefore, the isolation rate of NESp in the S. pneumoniae strains in this study was 12.0% (18/149). Molecular-typing analyses classified five strains as two existing sequence types (STs; ST7502 and ST7786), and 13 strains formed four novel STs. Horizontal spread was suspected, because strains with the same ST were often isolated from the same nursery school. The NESp isolates were generally susceptible to most antimicrobials, with the exception of macrolides; however, all isolates possessed more than one abnormal penicillin-binding protein gene. Furthermore, NESp strains were more effective than encapsulated counterparts at forming biofilms, which showed obvious differences in morphology. These data indicated that NESp strains should be continuously monitored as emerging respiratory pathogens.