Serotype Distribution, Antimicrobial Susceptibility, Multilocus Sequencing Type and Virulence of Invasive Streptococcus pneumoniae in China: A Six-Year Multicenter Study

Background:Streptococcus pneumoniae is an important human pathogen that can cause severe invasive pneumococcal diseases (IPDs). The aim of this multicenter study was to investigate the serotype and sequence type (ST) distribution, antimicrobial susceptibility, and virulence of S. pneumoniae strains causing IPD in China.Methods: A total of 300 invasive S. pneumoniae isolates were included in this study. The serotype, ST, and antimicrobial susceptibility of the strains, were determined by the Quellung reaction, multi-locus sequence typing (MLST) and broth microdilution method, respectively. The virulence level of the strains in the most prevalent serotypes was evaluated by a mouse sepsis model, and the expression level of well-known virulence genes was measured by RT-PCR.Results: The most common serotypes in this study were 23F, 19A, 19F, 3, and 14. The serotype coverages of PCV7, PCV10, PCV13, and PPV23 vaccines on the strain collection were 42.3, 45.3, 73.3 and 79.3%, respectively. The most common STs were ST320, ST81, ST271, ST876, and ST3173. All strains were susceptible to ertapenem, levofloxacin, moxifloxacin, linezolid, and vancomycin, but a very high proportion (>95%) was resistant to macrolides and clindamycin. Based on the oral, meningitis and non-meningitis breakpoints, penicillin non-susceptible Streptococcus pneumoniae (PNSP) accounted for 67.7, 67.7 and 4.3% of the isolates, respectively. Serotype 3 strains were characterized by high virulence levels and low antimicrobial-resistance rates, while strains of serotypes 23F, 19F, 19A, and 14, exhibited low virulence and high resistance rates to antibiotics. Capsular polysaccharide and non-capsular virulence factors were collectively responsible for the virulence diversity of S. pneumoniae strains.Conclusion: Our study provides a comprehensive insight into the epidemiology and virulence diversity of S. pneumoniae strains causing IPD in China.

Antibiotic Resistance and Molecular Biological Characteristics of Non-13-Valent-Pneumococcal Conjugate Vaccine Serogroup 15 Streptococcus pneumoniae Isolated From Children in China

Background: The isolation rate of serogroup 15 Streptococcus pneumoniae has been increasing since developing countries began administering the 13-valent pneumococcal conjugate vaccine.Methods: We detected the antibiotic resistance and molecular characteristics of 126 serogroup 15 S. pneumoniae strains isolated from children in China. Serotypes were determined via the Quellung reaction. Antibiotic resistance was tested using the E-test or disc diffusion method. Sequence types were assigned via multilocus sequence typing. Data were analyzed using WHONET 5.6 software.Results: The frequencies of S. pneumoniae serotypes 15A, 15B, 15C, and 15F were 29.37, 40.48, 28.57, and 1.59%, respectively. Continuous-monitoring data from Beijing showed that the annual isolation rates of serogroup 15 S. pneumoniae were 7.64, 7.17, 2.58, 4.35, 3.85, 7.41, and 10.53%, respectively, from 2013 to 2019. All 126 serogroup 15 strains were susceptible to vancomycin and ceftriaxone. The non-susceptibility rate to penicillin was 78.57%. All strains were resistant to erythromycin with high minimum inhibitory concentrations (MICs). The multidrug resistance rate was 78.57%. The most common clonal complexes were CC3397, CC6011, CC10088, CC9785, and ST8589.Conclusion: Serogroup 15 S. pneumoniae is common among children in China, and these strains should be continuously monitored.

Age-Dependent Serotype-Associated Case-Fatality Rate in Invasive Pneumococcal Disease in the Autonomous Community of Madrid between 2007 and 2020

The aim of this study was to investigate the serotype-associated fatality rate in cases of invasive pneumococcal disease (IPD) in the Spanish region of Madrid between 2007 and 2020. Serotyping was performed by Pneumotest Latex and the Quellung reaction using commercial antisera. Case-fatality rate was estimated as the ratio between the number of deaths at hospital discharge and the number of cases attributable to each serotype. To evaluate the association measures, the odds ratios with a 95% confidence interval were calculated. Twenty five pneumococcal serotypes were associated to mortality and comprised 87.8% of the total number of isolates characterized. Serotypes 8, 3, 19A, 1, 7F, 22F, 12F, and 11A were the most prevalent (≥3% each). Serotypes 31, 11A, and 19F were significantly associated to high case-fatality rates (>20% each). The lower significantly associated case-fatality rate (<10% each) was found in serotypes 5, 1, 12B, 7F, 12F, 8, 33, and 10A. The serotypes with higher mortality levels (≥0.04 per 100,000 population) were 11A (fatality 24.0%), 3 (fatality 18.7%), 19A (fatality 12.5%), and 8 (fatality 7.2%). Serotype 3 was worrisome because it is associated with important fatality levels combined with very high incidence and mortality rates. Serotype 11A also showed a high fatality with marked incidence and mortality levels. Some few frequent serotypes as 31, 19F, and 15A despite its high fatality had low levels of mortality. By contrast other serotypes as 8 showing low fatality had high mortality ranges because it shows a wide extended distribution. Finally, common serotypes, such as 1 and 5, presented small mortality length, due to their low case-fatality rates.

PCR and Culture Analysis of Streptococcus pneumoniae Nasopharyngeal Carriage in Healthy Children

Invasive Streptococcus pneumoniae disease is preceded by asymptomatic nasopharyngeal carriage. Measuring carriage in healthy populations provides data on what serotypes are present in communities, which is of interest in the era of polyvalent pneumococcal conjugate vaccines. Nasopharyngeal swabs from a survey of 682 and 800 healthy children in 2016 and 2018, respectively, were analyzed by culture and Quellung reaction to determine rates of carriage and serotypes. All swabs from 2016 and 300 randomly selected swabs from 2018 were then analyzed using real-time semi-quantitative PCR (qPCR) to detect S. pneumoniae gene targets lytA, piaA, and SP2020 and determine serotype. There were 71 (10.4%) and 68 (8.5%) culture positive samples in 2016 and 2018, respectively. All of these were also positive by qPCR except one that was equivocal. In total, 46.0% of 2016 swabs were positive by qPCR. In 2018, results from the selected sample extrapolated to the complete sample showed 49.0% positive by qPCR. PCV13 serotypes were detected in 29.3% and 21.7% of S. pneumoniae qPCR positive samples from 2016 and 2018, respectively; compared with only 8.4% and 6.0% PCV13 serotypes detected by Quellung reaction in culture positive samples. Compared with culture, qPCR detected S. pneumoniae more frequently. Further, qPCR serotyping detected PCV13 serotypes in a larger proportion of samples than culture and Quellung reaction did, showing that, despite established universal childhood PCV13 immunization, vaccine serotypes can still be detected in a large proportion of young children.

Multiple clonal types of nonvaccine serotypes constitute β-lactams nonsusceptible pneumococcus isolates from adult pneumonia patients in Japan

Background and objective The global spread of antimicrobial-nonsusceptible Streptococcus pneumoniae is a major concern. Molecular epidemiology of those strains in relation to vaccine serotype remains to be explored in Japan. This study aimed to elucidate the distribution of molecular types with the serotypes and antimicrobial susceptibility of pneumococcus strains isolated from adult pneumonia patients. Methods We enrolled adult pneumonia patients from four sites in Japan between September 2011 and August 2014. S. pneumoniae isolates from sputum and blood were analyzed for serotyping by the Quellung reaction and for antimicrobial susceptibility by the agar dilution method and e-test and for multilocus sequence typing. Results In total, 204 isolates were analyzed from 200 patients with a median age of 72.5 years, of whom 55 (27.5%) patients had received a 23-valent pneumococcal polysaccharide vaccine (PPSV23). We detected 41 clonal complexes (CCs) and 62 sequence types (STs), including 10 new STs: CC/ST 180 of serotype 3 was the most common followed by CC/ST 236 of serotype 19F and CC/ST 99 of serotype 11A; 144 (70.6%) isolates were PPSV23 serotypes; 40 (19.6%) and 121 (59.3%) isolates were b-lactam nonsusceptible (bNS) and multidrug-nonsusceptible strains, respectively. Among the bNS strains, 18 (45%) were nonvaccine serotypes, and 4 CCs (CC236, CC63, CC242, and CC558) comprised of 62.5% of them.Conclusion Multiple CCs of bNS strains, including nonvaccine serotype are spreading. It is crucial to monitor the antimicrobial susceptibility, serotypes, and molecular types of pneumococci to predict the effectiveness of vaccines in preventing pneumonia by bNS pneumococci strains.

Serotype Distribution and Characteristics of the Minimum Inhibitory Concentrations of Streptococcus pneumoniae Isolated from Pediatric Patients in Kunming, China

Streptococcus pneumoniae (S. pneumoniae) is the main conditional pathogen of acute respiratory infection in infants, children, and older adults worldwide. It was great significant to identify the epidemic characteristics of serotypes and antibiotic susceptibility for the prevention and treatment of S. pneumoniae diseases. This research assessed the serotype distribution and the minimum inhibitory concentrations (MICs) of S. pneumoniae isolated from pediatric patients to provide information on the epidemiology and antibiotic resistance of S. pneumoniae in Kunming, China. A total of 140 S. pneumoniae isolates were collected from pediatric patients at Kunming Children’s Hospital from January 2016 to October 2017. Serotype identification was done by Quellung reaction and multiplex polymerase chain reaction. MICs were determined by E-test. 140 isolates distributed in 13 types of serotypes. The top-three prevalent serotypes were 19F, 19A, and 6B. The immunization coverage rate of 13-valent pneumococcal conjugate vaccine (PCV) was relatively higher and should be introduced into the vaccination program in the region. MIC50 of penicillin, ceftriaxone, and levofloxacin was 1 μg/mL. MIC50 for meropenem and vancomycin was 0.38 μg/mL. MIC90 of penicillin, ceftriaxone, and levofloxacin was 1.5 μg/mL and that of meropenem and vancomycin was 0.5 μg/mL. The MIC90 of erythromycin was > 256 μg/mL. In summary, S. pneumoniae had low resistance rates to penicillin, ceftriaxone, levofloxacin, vancomycin, and meropenem, and these antibiotics could be the first-line agents for children with pneumococcal infections in Kunming.

Etiology, antibiotic susceptibility and prognostic factors of pediatric community-acquired sepsis in Addis Ababa, Ethiopia

Introduction: There is a scarcity of data on pediatric community-acquired sepsis (CAS) in Ethiopia. We sought to determine the etiology, role of Streptococcus pneumoniae, antibiotic susceptibility pattern, and prognostic factors in children with CAS in Addis Ababa, Ethiopia. Methodology: A prospective cross-sectional study of 101 children aged 0-15 years with suspected CAS was performed at two major hospitals in Addis Ababa, Ethiopia. Blood culture, antibiotic susceptibility testing, amplification of the autolysin (lytA) gene and typing S. pneumoniae by sequencing and Quellung reaction were performed. Data were analyzed using descriptive statistics and logistic regression. Results: The prevalence of culture-positive CAS was 18.81% (19/101). S. pneumoniae (21.1%) (Serotypes 19A (n = 2), 33C and 12F) and Klebsiella pneumoniae (21.1%) were the most common causes of CAS. Half of K. pneumoniae isolates were resistant to gentamicin and ceftriaxone. The most common antibiotics used for treatment were a combination of ampicillin with gentamicin (47.5%). The presence of lower respiratory tract infections (LRTIs) in the preceding 3 months was an independent predictor associated with culture-proven sepsis (adjusted odds ratio (AOR), 7.02; 95% confidence interval (CI), 1.42 - 34.64; P = 0.02). The case-fatality rate was 11.9% (12/101). Presence of underlying comorbidity (AOR, 6.8; 95% CI, 1.59-28.7; P = 0.009) was an independent predictor of mortality. Conclusions: S. pneumoniae and K. pneumoniae were the major causes of CAS and there was a substantial level of antibiotic resistance. Presence of LRTIs in the preceding 3 months was a predictor of culture-proven CAS whereas underlying comorbidity was a predictor of mortality.

Post-Vaccination Streptococcus pneumoniae Carriage and Virulence Gene Distribution among Children Less Than Five Years of Age, Cape Coast, Ghana

In 2012, Ghana introduced PCV13 into its childhood immunization program. To monitor the pneumococcus after PCV13 vaccination, we analyzed serotypes, antibiotic resistance, and virulence genes of pneumococcal carriage isolates among children under five years of age. We obtained nasopharyngeal swabs from 513 children from kindergartens and immunization centers in Cape Coast, Ghana. Pneumococcal serotypes were determined by multiplex-PCR and Quellung reaction. Antibiotic resistance and virulence genes prevalence were determined by disc diffusion and PCR respectively. Overall, carriage prevalence was 29.4% and PCV13 coverage was 38.4%. Over 60% of the isolates were non-PCV13 serotypes and serotype 23B was the most prevalent. One isolate showed full resistance to penicillin, while 35% showed intermediate resistance. Resistance to erythromycin and clindamycin remained low, while susceptibility to ceftriaxone, levofloxacin and vancomycin remained high. Penicillin resistance was associated with PCV13 serotypes. Forty-three (28.5%) strains were multidrug-resistant. Virulence genes pavB, pcpA, psrP, pilus-1, and pilus-2 were detected in 100%, 87%, 62.9%, 11.9%, and 6.6% of the strains, respectively. The pilus islets were associated with PCV13 and multidrug-resistant serotypes. PCV13 vaccination had impacted on pneumococcal carriage with a significant increase in non-PCV13 serotypes and lower penicillin resistance. Including PcpA and PsrP in pneumococcal protein-based vaccines could be beneficial to Ghanaian children.

How nasopharyngeal pneumococcal carriage evolved during and after a PCV13-to-PCV10 vaccination programme switch in Belgium, 2016 to 2018

Background The current carriage study was set up to reinforce surveillance during/after the PCV13-to-PCVC10 switch in Belgium. Aim This observational study monitored carriage of Streptococcus pneumoniae (Sp) serotypes, particularly those no longer covered (3, 6A, 19A), as well as Haemophilus influenzae (Hi), because PCV10 contains the non-typeable Hi protein D. Methods A total of 2,615 nasopharyngeal swabs from children (6–30 months old) attending day care were collected in three periods over 2016–2018. Children’s demographic and clinical characteristics and vaccination status were obtained through a questionnaire. Sp and Hi were identified by culture and PCR. Pneumococcal strains were tested for antimicrobial (non-)susceptibility by disc diffusion and serotyped by Quellung-reaction (Quellung-reaction and PCR for serotypes 3, 6A, 19A). Results The carriage prevalence of Sp (> 75%) remained stable over the successive periods but that of Hi increased (87.4%, 664 Hi-carriers/760 in 2016 vs 93.9%, 895/953 in 2017–2018). The proportion of non-PCV13 vaccine serotypes decreased (94.6%, 438 isolates/463 in 2016 vs 89.7%, 599/668 in 2017–2018) while that of PCV13-non-PCV10 vaccine serotypes (3 + 6A + 19A) increased (0.9%, 4 isolates/463 in 2016 vs 7.8%, 52/668 in 2017–2018), with serotype 19A most frequently identified (87.9%, 58/66 isolates). Non-susceptibility of pneumococci against any of the tested antibiotics was stable over the study period (> 44%). Conclusions During and after the PCV13-to-PCV10 vaccine switch, the proportion of non-PCV13 serotypes decreased, mainly due to a serotype 19A carriage prevalence increase. These results complement invasive pneumococcal disease surveillance data, providing further basis for pneumococcal vaccination programme policy making.

Characterization of Streptococcus pneumoniae strains isolated from patients with meningitis before the introduction of pneumococcal conjugate vaccine in Serbia

Introduction: Pneumococcal meningitis is a serious disease which affects mostly children ≤ 2 years, adults ≥ 65 years and immunodeficient patients. The introduction of pneumococcal conjugate vaccine (PCV) into immunization programs worldwide has led to a significant decrease in the incidence of invasive pneumococcal disease, reduction of antibiotic resistance and changes in the distribution of pneumococcal serotypes. In 2018, PCV10 was introduced into the National Immunization Program in Serbia. Aim: The aim of this study was to analyze the serotype distribution and antibiotic susceptibility of pneumococcal strains isolated from meningitis cases in the pre-vaccinal period (2009-2018) in Serbia. Material and methods: Meningeal isolates were sent into the National Reference Laboratory for streptococci between January 2009 and December 2018 for serotyping and antimicrobial susceptibility testing (AST). Serotyping was performed by Quellung reaction, while AST was performed using disk diffusion method and E-test. Results: A total of 199 strains were analyzed and 32 different serotypes have been identified. Among the 55 (27.64%) strains from children ≤16 years, 17 different serotypes were detected of which 19F, 14, 6A, and 6B were the most common. Regarding the 144 (72.36%) adult isolates, 30 different serotypes were present, the most common being 3, 19F, 14, 23F, 6A and 6B. The coverage of pediatric serotypes was 61.82% for PCV10 and 78.18% for PCV13. Statistically significant number of isolates showed resistance to: penicillin (53.26%), erythromycin (45.73%), clindamycin (40.20%), trimethoprim-sulfamethoxazole (34.17%) and tetracycline (34.17%). The isolates from children were more resistant to beta-lactams and macrolides (p < 0.05). Conclusion: During the pre-vaccinal period in Serbia, vaccinal serotypes dominated over non-vaccinal serotypes. Resistance is intermediate to high in the dominant serotypes of children and low in the most common adult serotype 3. Taking into consideration the possible changes in the pneumococcal population in the future, continued monitoring of post-vaccine serotype and resistance trends are essential.