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.

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.

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.

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.

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.

Identification of pathogens by comprehensive real-time PCR versus conventional methods in community-acquired pneumonia in Japanese adults.

BACKGROUND: Community-acquired pneumonia (CAP) has high morbidity and mortality. Unfortunately, the pathogen detection rate using conventional culture methods is relatively low. We compared comprehensive real-time polymerase chain reaction (real-time PCR) analysis of nasopharyngeal swab specimens (NPS) and sputum samples against conventional methods for ability to detect causative pathogens of CAP. METHODS: We prospectively enrolled adult CAP patients, including those with prior antibiotic use, from December 2012 to May 2014. For each patient, causative pathogens were investigated conventionally and by real-time PCR that can identify 6 bacterial and 11 viral pathogens. RESULTS: Patients numbered 92 (mean age, 63 years; 59 male), including 30 (33%) with prior antibiotic use. Considering all patients, identification of causative pathogens by real-time PCR was significantly more frequent than by conventional methods in all patients (72% vs. 57%, p = 0.018). In patients with prior antibiotic use, identification rates also differed significantly (PCR, 77%; conventional, 50%; p = 0.027). Mixed infections were more frequent according to real-time PCR than conventional methods (26% vs. 4%, p < 0.001). By the real-time PCR, Streptococcus pneumoniae was most frequently identified (38%) as a causative pathogen, followed by Haemophilus influenzae (37%) and Mycoplasma pneumoniae (5%). PCR also identified viral pathogens (21%), with sensitivity enhanced by simultaneous examination of both NPS and sputum samples rather than only NPS samples. CONCLUSIONS: Real-time PCR of NPS and sputum samples could better identify bacterial and viral pathogens in CAP than conventional methods, both overall and in patients with prior antibiotic treatment.

Host Factors and Biomarkers Associated with Poor Outcomes in Adults with Invasive Pneumococcal Disease.

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.

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.

Pathogens in COPD exacerbations identified by comprehensive real-time PCR plus older methods.

Respiratory infection is a major cause of exacerbation in chronic obstructive pulmonary disease (COPD). Infectious contributions to exacerbations remain incompletely described. We therefore analyzed respiratory tract samples by comprehensive real-time polymerase chain reaction (PCR) in combination with conventional methods. We evaluated multiple risk factors for prolonged hospitalization to manage COPD exacerbations, including infectious agents. Over 19 months, we prospectively studied 46 patients with 50 COPD exacerbations, collecting nasopharyngeal swab and sputum samples from each. We carried out real-time PCR designed to detect six bacterial species and eleven viruses, together with conventional procedures, including sputum culture. Infectious etiologies of COPD exacerbations were identified in 44 of 50 exacerbations (88%). Infections were viral in 17 of 50 exacerbations (34%). COPD exacerbations caused by Gram-negative bacilli, including enteric and nonfermenting organisms, were significantly associated with prolonged hospitalization for COPD exacerbations. Our results support the use of a combination of real-time PCR and conventional methods for determining both infectious etiologies and risk of extended hospitalization.

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.

[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.

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.

Prevalence of macrolide resistance among group A streptococci isolated from pharyngotonsillitis.

A GAS Surveillance Study Group was organized to analyze group A streptococci (GAS) isolated from patients with acute pharyngotonsillitis, with participating pediatricians submitting swab samples between April and October 2012. Molecular analysis of emm typing and multilocus sequence typing (MLST), as well as antimicrobial susceptibility testing, were carried out for 363 GAS isolates. Strains belonging to emm1 were most prevalent (25.6%), followed in turn by emm12 (23.7%), emm28 (16.3%), and emm89 (15.3%). In emm1, 87.2% of strains, typed as ST28 or ST661, showed macrolide (ML) resistance mediated by the mef(A) gene. In emm12 (ST36 and ST465), 64.3% of strains were resistant to MLs because of mef(A) or erm(B), as was also true for 81.7% of emm28 (ST52). In emm89 (ST101 and ST646), however, such resistance was not seen. Due to the high levels of ML resistance observed, GAS isolates from individuals with penicillin allergies need to be isolated and their antimicrobial susceptibility tested, rather than automatically giving the patient a ML.

Associations of macrolide and fluoroquinolone resistance with molecular typing in Streptococcus pyogenes from invasive infections, 2010-2012.

In this study, the relationship between emm type and antibiotic resistance in 283 invasive group A streptococcal strains collected during surveillance from 2010 to 2012 was analysed. Strains were characterised by emm typing, multilocus sequence typing and antimicrobial susceptibility. Resistance rates for macrolide antibiotics conferred by mef(A), erm(A) or erm(B) were high (54.4%). The most prevalent (40.3%) macrolide resistance mediated by mef(A) was present in 92.2% of emm1 strains. It was found that 53.3% of emm12 strains and 91.7% of emm28 strains had erm(A) or erm(B) genes. Intermediate resistance to fluoroquinolones caused by a mutation in parC was found in 14.1% of strains. Furthermore, three strains (1.1%) with high resistance caused by mutations in both parC and gyrA were detected. Fluoroquinolone resistance was present in various emm types.

Rapid decrease of 7-valent conjugate vaccine coverage for invasive pneumococcal diseases in pediatric patients in Japan.

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.

Late-onset invasive group B Streptococcal infection with serotype VIII in a neonate having congenital biliary atresia.

A female newborn was admitted to our department 15 days after birth for insufficient sucking and jaundice. The patient's blood and urine cultures were both positive for group B streptococcal (GBS) infection. A maternal vaginal sample at 35 weeks' gestation was negative for GBS in culture-based microbiologic screening. The patient recovered shortly after receiving systemic antibiotic therapy. On the basis of clinical evidence of white stool and progressive jaundice, we suspected that the newborn had complications related to congenital biliary atresia (CBA); surgery was performed. Isolates from the mother's vaginal sample obtained when the patient was 25 days old, along with neonatal blood, revealed identical patterns (serotype VIII and sequence type 1) of GBS capsular and multilocus sequence typing, suggestive of maternal transmission. Molecular epidemiologic examination may be useful to clarify the transmission route and etiology; culture-based microbiologic screening appears to have limitations for detecting the route of transmission.

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.