Effects of Pneumococcal Conjugate Vaccine on Genotypic Penicillin Resistance and Serotype Changes, Japan, 2010-2017.

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.

Genome Evolution to Penicillin Resistance in Serotype 3 Streptococcus pneumoniae by Capsular Switching.

Streptococcus pneumoniae isolates of serotype 3 were collected from cases of invasive pneumococcal disease (n = 124) throughout Japan between April 2010 and March 2013. A penicillin-resistant S. pneumoniae (PRSP) isolate from an adult patient, strain KK0981 of serotype 3, was identified among these strains. Whole-genome analysis characterized this PRSP as a recombinant strain derived from PRSP of serotype 23F with the cps locus (20.3 kb) replaced by that of a penicillin-susceptible strain of serotype 3.

Detection of pathogens by real-time PCR in adult patients with acute exacerbation of bronchial asthma.

BACKGROUND: Respiratory tract infection is a major cause of acute exacerbation of bronchial asthma (AEBA). Although recent findings suggest that common bacteria are causally associated with AEBA, a comprehensive epidemiologic analysis of infectious pathogens including common/atypical bacteria and viruses in AEBA has not been performed. Accordingly, we attempted to detect pathogens during AEBA by using real-time polymerase chain reaction (PCR) in comparison to conventional methods. METHODS: We prospectively enroled adult patients with AEBA from August 2012 to March 2014. Infectious pathogens collected in nasopharyngeal swab and sputum samples were examined in each patient by conventional methods and real-time PCR, which can detect 6 bacterial and 11 viral pathogens. The causal association of these pathogens with AEBA severity and their frequency of monthly distribution were also examined. RESULTS: Among the 64 enroled patients, infectious pathogens were detected in 49 patients (76.6%) using real-time PCR and in 14 patients (21.9%) using conventional methods (p < 0.001). Real-time PCR detected bacteria in 29 patients (45.3%) and respiratory viruses in 28 patients (43.8%). Haemophilus influenzae was the most frequently detected microorganism (26.6%), followed by rhinovirus (15.6%). Influenza virus was the significant pathogen associated with severe AEBA. Moreover, AEBA occurred most frequently during November to January. CONCLUSIONS: Real-time PCR was more useful than conventional methods to detect infectious pathogens in patients with AEBA. Accurate detection of pathogens with real-time PCR may enable the selection of appropriate anti-bacterial/viral agents as a part of the treatment for AEBA.

Molecular Characterization of Invasive Streptococcus dysgalactiae subsp. equisimilis, Japan.

We collected ß-hemolytic streptococci (1,611 isolates) from patients with invasive streptococcal infections in Japan during April 2010-March 2013. Streptococcus dysgalactiae subsp. equisimilis (SDSE) was most common (n = 693); 99% of patients with SDSE infections were elderly (mean age 75 years, SD ±15 years). We aimed to clarify molecular and epidemiologic characteristics of SDSE isolates and features of patient infections. Bacteremia with no identified focus of origin and cellulitis were the most prevalent manifestations; otherwise, clinical manifestations resembled those of S. pyogenes infections. Clinical manifestations also differed by patient's age. SDSE isolates were classified into 34 emm types; stG6792 was most prevalent (27.1%), followed by stG485 and stG245. Mortality rates did not differ according to emm types. Multilocus sequence typing identified 46 sequence types and 12 novel types. Types possessing macrolide- and quinolone-resistance genes were 18.4% and 2.6%, respectively; none showed ß-lactam resistance. Among aging populations, invasive SDSE infections are an increasing risk.

Serotype Changes and Drug Resistance in Invasive Pneumococcal Diseases in Adults after Vaccinations in Children, Japan, 2010-2013.

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.

Direct identification of Streptococcus agalactiae and capsular type by real-time PCR in vaginal swabs from pregnant women.

Most group B streptococcus (GBS) infections in newborns are with capsular type Ia, Ib, or III. To prevent these infections more effectively, we developed a real-time PCR method to simultaneously detect GBS species and identify these 3 capsular types in vaginal swab samples from women at 36-39 weeks of gestation. DNA to be detected included those of the dltS gene (encoding a histidine kinase specific to GBS) and cps genes encoding capsular types. PCR sensitivity was 10 CFU/well for a 33-35 threshold cycle. Results were obtained within 2 h. Direct PCR results were compared with results obtained from cultures. Samples numbering 1226 underwent PCR between September 2008 and August 2012. GBS positivity rates by direct PCR and after routine culture were 15.7% (n = 192) and 12.6% (n = 154), respectively. Sensitivity and specificity of direct PCR relative to culture were 96.1% and 95.9%. Of GBS positive samples identified by PCR, capsular types determined directly by real-time PCR were Ia (n = 24), Ib (n = 32), and III (n = 26). Real-time PCR using our designed cycling probe is a practical, highly sensitive method for identification of GBS in pregnant carriers, allowing use of prophylactic intrapartum antibiotics in time to cover the possibility of unexpected premature birth.

Changes in capsule and drug resistance of Pneumococci after introduction of PCV7, Japan, 2010-2013.

We aimed to clarify changes in serotypes and genotypes mediating ß-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.

[Genetic analysis of multidrug-resistant Streptococcus pneumoniae including meropenem resistance that was isolated from elderly residents with pneumonia in nursing-care facilities].

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.

Longitudinal surveillance of Haemophilus influenzae isolates from pediatric patients with meningitis throughout Japan, 2000-2011.

In Japan, ß-lactamase-nonproducing, ampicillin-resistant organisms have been evident among Haemophilus influenzae type b (Hib) isolates since 2000, when no appropriate vaccine had been approved. We therefore performed molecular analysis of agents causing H. influenzae meningitis nationwide over the following 10 years. Some 285 institutions have participated in surveillance since 2000. The capsular type and resistance genes of 1,353 isolates and 23 cerebrospinal fluid samples from pediatric patients with meningitis we had received from 2000 to 2011 were analyzed by polymerase chain reaction. Blood and spinal fluid test results obtained when patients were admitted were examined for correlation with outcomes. Hib was found in 98.9 % of isolates. We received more than 100 Hib isolates per year until vaccination began in December 2008, when these isolates decreased, especially since establishment of a special fund to promote vaccination in November 2010. Decreased incidence among infants 7 months to 2 years old has been particularly notable. However, the rate of ampicillin-resistant organisms has increased to more than 60 % of all isolates since 2009. We received 587 replies to a questionnaire concerning outcomes, indicating 2 % mortality and 17.7 % serious morbidity. Age of 6 months or younger and presence of disseminated intravascular coagulation at admission were related to an unfavorable outcome (p < 0.05), but ampicillin resistance was not. Combination therapy with third-generation cephem and carbapenem agents was used initially for 72 % of patients. Routine immunization can prevent Hib meningitis in children.

Antibiotic susceptibility in relation to genotype of Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae responsible for community-acquired pneumonia in children.

Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae are the main pathogens causing community-acquired pneumonia (CAP). We identified S. pneumoniae (n = 241), H. influenzae (n = 123), and M. pneumoniae (n = 54) as causative pathogens from clinical findings and blood tests from pediatric CAP patients (n = 903) between April 2008 and April 2009. Identification of genes mediating antimicrobial resistance by real-time PCR was performed for all isolates of these three pathogens, as was antibiotic susceptibility testing using an agar dilution method or broth microdilution method. The genotypic (g) resistance rate was 47.7 % for penicillin-resistant S. pneumoniae (gPRSP) possessing abnormal pbp1a, pbp2x, and pbp2b genes, 62.6 % for ß-lactamase-nonproducing, ampicillin-resistant (gBLNAR) H. influenzae possessing the amino acid substitutions Ser385Thr and Asn526Lys, and 44.4 % for macrolide-resistant M. pneumoniae (gMRMP) possessing a mutation of A2063G, A2064G, or C2617A. Serotype 6B (20.3 %) predominated in S. pneumoniae, followed by 19F (15.4 %), 14 (14.5 %), 23F (12.0 %), 19A (6.2 %), and 6C (5.4 %). Coverage for the isolates by heptavalent pneumococcal conjugate vaccine (PCV7) and PCV13, respectively, was calculated as 68.5 and 80.9 %. A small number of H. influenzae were identified as type b (6.5 %), type e (0.8 %), or type f (0.8 %); all others were nontypeable. Proper use of antibiotics based on information about resistance in CAP pathogens is required to control rapid increases in resistance. Epidemiological surveillance of pediatric patients also is needed to assess the effectiveness of PCV7 and Hib vaccines after their introduction in Japan.

Molecular epidemiological analyses of Clostridioides difficile isolates in a university hospital in Japan.

Background: We performed molecular epidemiological analyses of Clostridioides difficile isolates in a university hospital in Japan to reveal the risk of C. difficile infection. Methods: Cultured isolates from 919 stool samples from 869 patients obtained from July 2015 to August 2016 were subjected to toxin gene detection, ribotyping, multilocus sequence typing, antimicrobial susceptibility testing, and quantitative real-time polymerase chain reaction testing for C. difficile toxin gene expression. Results: Of the 919 stool samples from 869 patients, C. difficile was isolated from 153 samples (16.6%), of which 49 (32%) and 104 (68%) were from patients with and without C. difficile infection, respectively. Analyses showed genetic diversity, with ST8 and ST17 strains of healthcare-associated infections, some of which caused C. difficile infections. There was no significant difference in the transcription levels of C. difficile toxin genes between isolates from patients with and without C. difficile infection. Conclusions: Major Japanese clonal strains, ST8 and ST17, have been in the hospital environment for a long time and cause healthcare-associated C. difficile infections. The C. difficile toxin genes were transcribed in the isolates from both patients with and without C. difficile infection but were no significant relationship with the development of C. difficile infection.

Rapid effectiveness of minocycline or doxycycline against macrolide-resistant Mycoplasma pneumoniae infection in a 2011 outbreak among Japanese children.

BACKGROUND: Mycoplasma pneumoniae is a major pathogen causing community-acquired pneumonia in children and young adults. Outbreaks typically occur at intervals of several years. In 2011, a widespread outbreak was associated with macrolide-resistant M. pneumoniae (MRMP) in Japanese children, often those of school age. METHODS: Two hundred fifty-eight children were diagnosed with M. pneumoniae-associated pneumonia based on chest radiography, real-time polymerase chain reaction (PCR), and antibody titers between January and December 2011. Mycoplasma pneumoniae cultures obtained from nasopharyngeal samples using appropriate broth were subjected to real-time PCR, by which decreases in M. pneumoniae in patients treated with minocycline (MIN), doxycycline (DOX), or tosufloxacin (TFX) were calculated. Mutations of the 23S ribosomal RNA gene that confer high resistance to macrolides in M. pneumoniae were identified by DNA sequencing. RESULTS: Among 202 M. pneumoniae isolates from M. pneumoniae-associated pneumonia patients, 176 (87.1%) were MRMP. Macrolide-resistant M. pneumoniae infection was significantly related to school age (P < .01) and initial administration of macrolides (P < .01). Minocycline or DOX (n = 125) or TFX or levofloxacin (n = 15) was used for definitive treatment of MRMP patients. Minocycline or DOX was significantly more effective than TFX (P ≤ .05) in achieving defervescence within 24 hours and in decreasing numbers of M. pneumoniae DNA copies 3 days after initiation. CONCLUSIONS: 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.

A practical approach estimating etiologic agents using real-time PCR in pediatric inpatients with community-acquired pneumonia.

To evaluate pathogens in pediatric inpatients with community-acquired pneumonia (CAP), an Acute Respiratory Diseases Study Group organized by ten Japanese medical institutions devised a rapid, reliable process based on real-time PCR results in nasopharyngeal swab samples plus admission blood test results. From April 2008 to April 2009, we enrolled 903 children with CAP based on chest radiographs and clinical findings who were hospitalized within 5 days of onset. Comprehensive real-time PCR was used to detect 6 bacteria and 11 respiratory viruses. The swab specimens also were used for bacterial cultures. After initial determination of presence or absence of viral and mycoplasmal infections, significant bacterial contributions were defined by bacterial identification, clinical efficacy of antimicrobial agent, and reference to blood test results. Children were stratified by age: below 1 year, 1 year, 2-5 years, or at least 6 years old. Among patients studied, 34.4 % were diagnosed with viral infection; 21.8 %, bacterial infection; 17.5 %, viral/bacterial co-infection; 5.9 %, mycoplasmal infection; 0.3 %, mycoplasmal/bacterial co-infection; and 1.7 %, viral/mycoplasmal co-infection. The remaining 18.4 % had unknown pathogens. Purely viral infection was suggested mainly in infants younger than 1 year; mycoplasmal infection typically occurred in children at least 6 years old. Our results suggest usefulness of real-time PCR for nasopharyngeal samples together with blood tests in estimating etiologic agents in clinical settings.

[Morphological changes in penicillin-resistant Streptococcus pneumoniae and beta-lactamase-nonproducing, ampicillin-resistant Haemophilus influenzae after exposure to oral antibacterial agents].

Morphological changes in penicillin-resistant Streptococcus pneumoniae (PRSP) and beta-lactamase-nonproducing, ampicillin-resistant Haemophilus influenzae (BLNAR) after exposure to oral antibacterial agents could be observed over time under a phase-contrast microscope. Morphological changes in BLNAR were also observed using a scanning electron microscope. The organisms used in this study were ME19F strain identified as genotypic(g) gPRSP (serotype: 19F) and JPH002 strain identified as gBLNAR (serotype: b). The antibacterial agents used were amoxicillin (AMPC), cefditoren (CDTR), tebipenem (TBPM), and tosufloxacin (TFLX). The concentration of each antibacterial agent to which the bacteria were exposed was set at the blood level one hour after Cmax when administered to children at the usual dose. Bacteriolysis of gPRSP cells started after exposure of only 20minutes to TBPM, and 90% of the cells were lysed within 2 hours. A high bactericidal action of TBPM on gPRSP was supported by these findings. When gBLNAR was exposed to AMPC and TBPM, lysis from spheroplasts and cells with vacuoles were sometimes observed. In contrast, after gBLNAR was exposed to CDTR, lysis occurred after marked filamentation in the cells, but after exposure to TFLX, cells deduced to be killed after mild filamentation without lysis. Time-dependent morphological changes that reflect the differences in bactericidal activity and PBP affinity among beta-lactams provide beneficial information to select antibacterial agents.

Expression profiles of hsa-miR-148a-3p and hsa-miR-125b-5p in human breast milk and infant formulae.

BACKGROUND: Milk-derived microRNAs (miRNAs), including hsa-miR-148a-3p (miR-148a) and hsa-miR-125b-5p (miR-125b), have been shown to be beneficial to the gastrointestinal function in infants. Here, we investigated their expression during lactation in humans and determined whether the infant formulae available in Japan contain these miRNAs. METHODS: Healthy Japanese women (n = 16) who gave birth vaginally or by cesarean section at the Teine Keijinkai Hospital between 1 September 2020, and 31 April 2021 were included in this study. Breast milk was collected by nurses on days 4 or 5 after delivery (hereinafter, transition milk) and on day 30 of postpartum (hereinafter, mature milk). The levels of miR-148a and miR-125b in breastmilk and six commercially available infant formulae were compared and evaluated using quantitative reverse transcription-polymerase chain reaction. RESULTS: In all participants, the miR-148a level in mature breastmilk was significantly lower than that in the transition milk. The changes in miR-125b expression during lactation showed similar trends to the changes in miR-148a expression. The miR-148a and miR-125b levels in all analyzed infant formulae were lower than 1/500th and 1/100th of those in mature breastmilk, respectively. CONCLUSIONS: The levels of both miR-148a and miR-125b in human breast milk decreased on day 30 postpartum compared with those in the transition milk. Additionally, the expression of these miRNAs in infant formulae available in Japan was very low. Further studies with larger populations are required to understand precisely the lactational changes in the expression of miR148a and miR-125b in breast milk.

Direct detection by real-time PCR of ftsI gene mutations affecting MICs of ß-lactam agents for Haemophilus influenzae isolates from meningitis.

One resistance mechanism of Haemophilus influenzae to ampicillin involves decreased affinity of penicillin-binding protein (PBP) 3 for ß-lactam antibiotics reflecting amino acid substitutions in PBP3 encoded by the ftsI gene. Three amino acid substitutions, Ser385Thr, Arg517His, and Asn526Lys, are especially responsible for ß-lactam resistance. We constructed a new real-time polymerase chain reaction (PCR) to directly detect these substitutions in addition to 16S ribosomal RNA (rRNA), cap, and bla(TEM) genes. Our real-time PCR was evaluated using 206 clinical H. influenzae strains isolated from pediatric patients with meningitis. Relative sensitivities and specificities of real-time PCR were 90.5-100% and 96.3-100% for all resistance classes compared with our previously reported conventional PCR. In addition, real-time PCR shortened time required from 3 h by conventional PCR to 1.5 h. When correlations between combinations of amino acid substitutions in the ftsI gene detected by real-time PCR and minimum inhibitory concentrations (MICs) of ß-lactam antibiotics were evaluated, MIC(90)s of ampicillin for ß-lactamase-nonproducing ampicillin-intermediate-resistant strains with Asn526Lys, ß-lactamase-nonproducing, ampicillin-resistant strains with Ser385Thr, and ß-lactamase-nonproducing ampicillin-resistant strains with both Asn526Lys and Ser385Thr, respectively, were two, four, and eight times higher than those for sensitive strains. Similarly, MIC(90)s of cephalosporins for these strains, respectively, were two, 16-32, and 16-32 times higher than those for sensitive strains. Thus, real-time PCR can guide antibiotic use.

Molecular epidemiologic characteristics of Streptococcus pneumoniae isolates from children with meningitis in Japan from 2007 through 2009.

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.

In vitro activity of tebipenem, a new oral carbapenem antibiotic, against beta-lactamase-nonproducing, ampicillin-resistant Haemophilus influenzae.

In vitro activity of tebipenem, a new oral carbapenem antibiotic, against clinical Haemophilus influenzae isolates was compared with those of 8 reference agents. Isolates were classified into 6 resistance classes after PCR identification of beta-lactamase genes and ftsI gene mutations. For all isolates, the minimal concentration at which 90% of isolates were inhibited was lower for tebipenem than for the reference oral antibiotics, except for cefditoren. Tebipenem also showed excellent bactericidal activity against beta-lactamase-nonproducing, ampicillin-resistant isolates.

Clinical aspects of invasive infection with Streptococcus dysgalactiae subsp. equisimilis in elderly patients.

The number of patients with severe invasive infections (mainly exhibiting bacteremia) with Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been increasing worldwide. We herein report the clinical aspects of invasive infections (cellulitis, pneumonia, and urosepsis) occurring with SDSE in 13 elderly patients (mean age 84 years, range 69-99 years) diagnosed at a hospital for elderly individuals during the period January 2005-June 2009. Ten subjects had underlying diseases, including neurologic disorders, diabetes mellitus, and others. Eleven patients presented to the hospital emergency department, and the most common symptom was high fever or respiratory distress. Primary care and emergency department doctors treating elderly patients with high fever should keep in mind invasive SDSE infection as a differential diagnosis, especially when an elderly person has underlying illnesses. To detect SDSE in elderly subjects, blood cultures should be obtained before the administration of antimicrobials because, as we found, the patients' symptoms were limited.

Diverse mutations in the ftsI gene in ampicillin-resistant Haemophilus influenzae isolates from pediatric patients with acute otitis media.

To clarify molecular changes in beta-lactamase-nonproducing, ampicillin-resistant (BLNAR) Haemophilus influenzae, which is increasing in pediatric patients with acute otitis media (AOM) in Japan, we identified amino acid (aa) substitutions in penicillin-binding protein 3 for the BLNAR strains. Of 191 H. influenzae strains isolated from middle ear fluid of pediatric AOM patients between October 2005 and March 2008, BLNAR strains determined by PCR accounted for 49.2%. Of the BLNAR strains, 91.5% possessed 4 aa substitutions: Met377Ile, Ser385Thr, Leu389Phe, and either Asn526Lys or Arg517His. Additionally, the emergence of BLNAR strains possessing a new aa substitution of Val329Ala in the conserved aa motif of Ser327-Thr-Val-Lys, or Val511Ala adjacent to the conserved aa motif of Lys512-Thr-Gly, was noted. Transformation of the ftsI gene into the Rd reference strain (ATCC 51907) demonstrated that these two aa substitutions reduced susceptibility to amoxicillin more than to cephalosporins. Pulsed-field gel electrophoretic profiles of BLNAR strains were highly diverse. These results suggested that inadequate antibiotic use may increase BLNAR strains by selecting mutations in the ftsI gene and that such use may have favored the new aa substitutions.