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.

Corynebacterium Species Inhibit Streptococcus pneumoniae Colonization and Infection of the Mouse Airway

The stability and composition of the airway microbiome is an important determinant of respiratory health. Some airway bacteria are considered to be beneficial due to their potential to impede the acquisition and persistence of opportunistic bacterial pathogens such as Streptococcus pneumoniae. Among such organisms, the presence of Corynebacterium species correlates with reduced S. pneumoniae in both adults and children, in whom Corynebacterium abundance is predictive of S. pneumoniae infection risk. Previously, Corynebacterium accolens was shown to express a lipase which cleaves host lipids, resulting in the production of fatty acids that inhibit growth of S. pneumoniae in vitro. However, it was unclear whether this mechanism contributes to Corynebacterium-S. pneumoniae interactions in vivo. To address this question, we developed a mouse model for Corynebacterium colonization in which colonization with either C. accolens or another species, Corynebacterium amycolatum, significantly reduced S. pneumoniae acquisition in the upper airway and infection in the lung. Moreover, the lungs of co-infected mice had reduced pro-inflammatory cytokines and inflammatory myeloid cells, indicating resolution of infection-associated inflammation. The inhibitory effect of C. accolens on S. pneumoniae in vivo was mediated by lipase-dependent and independent effects, indicating that both this and other bacterial factors contribute to Corynebacterium-mediated protection in the airway. We also identified a previously uncharacterized bacterial lipase in C. amycolatum that is required for inhibition of S. pneumoniae growth in vitro. Together, these findings demonstrate the protective potential of airway Corynebacterium species and establish a new model for investigating the impact of commensal microbiota, such as Corynebacterium, on maintaining respiratory health.

WhyD tailors surface polymers to prevent bacteriolysis and direct cell elongation in Streptococcus pneumoniae

Penicillin and related antibiotics disrupt cell wall synthesis in bacteria and induce lysis by misactivating cell wall hydrolases called autolysins. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae (Sp), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked lipoteichoic acids. Because LytA binds to these polymers, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation and lysis. We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active PG synthesis. Our results support a model in which the WTA tailoring activity of WhyD directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.

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.

Immunobiological Properties of Antigenic Preparations Streptococcus pneumoniae and their Mixtures

Relevance. Type-specific immunity does not protect against infection with other pneumococcal serotypes. The phenomenon of the change of serotypes dominating the population of Streptococcus pneumoniae is known, in part due to the intensive recombination process and the phenomenon of «capsule switching». Therefore, the development of a serotype-independent pneumococcal vaccine is an important global public health priority. Ams. Investigation of immunobiological properties of candidate components of a future vaccine with serotype-independent activity. Materials and methods. For immunization of mice, preparations of the capsular polysaccharide of pneumococcus serotype 3 (CPS) were used; protein-containing fraction (PCF) obtained from an aqueous extract of S. pneumoniae 6B cells; recombinant pneumolysin (Ply); mixtures of drugs (CPS + Plу; CPS + PCF; PCF + Plу); conjugate vaccine Prevnar 13 (manufactured by PFIZER Inc. USA). Mice were immunized intraperitoneally, 2 times with an interval of 14 days. Intact mice were used as a control group. To assess the humoral immune IgG response, the method of solid-phase ELISA was used. Phagocytic activity was studied at 7, 14, 21 and 28 days after the second immunization. The cytokine level was determined in the blood sera of mice after the second immunization 2, 4, 8, and 24 hours later on a NovoCyte flow cytometer (ACEA Biosciences, USA) using the MACSPIex CytoKine 10 Kit mouse (Miltenyi Biotec Inc., USA) according to the manufacturer's instructions. Results. Immunization of mice with Ply as well as mixtures with CPS and PCF caused a significant increase in the level of antibodies to Ply. It was found that there was no apparent decrease in the level of antigen-specific antibodies when antigens were administered in combination with others. Pneumolysin, used alone or in combination with PCF and CPS, induces the production of antiinflammatory cytokines IL-4, IL-10, and IL-5 detected throughout the study. This is confirmed by a study of the opsonophagocytic activity of neutrophils from immunized CPS + Ply, Ply + PCF and Ply mice; a significant increase in the number of eosinophils is observed in their blood due to the stimulation of their production of IL-5. Conclusions. As a result of the studies, it was shown that Ply, used alone or in combination with CPS and PCF, has the highest immunogenicity: it stimulates a significant increase in the level of specific antibodies, stimulates Th-2, and induces the production of anti-inflammatory cytokines.

Intra-Species Interactions in Streptococcus pneumoniae Biofilms

Streptococcus pneumoniae is a human pathogen responsible for high morbidity and mortality worldwide. Disease is incidental and is preceded by asymptomatic nasopharyngeal colonization in the form of biofilms. Simultaneous colonization by multiple pneumococcal strains is frequent but remains poorly characterized. Previous studies, using mostly laboratory strains, showed that pneumococcal strains can reciprocally affect each other’s colonization ability. Here, we aimed at developing a strategy to investigate pneumococcal intra-species interactions occurring in biofilms. A 72h abiotic biofilm model mimicking long-term colonization was applied to study eight pneumococcal strains encompassing 6 capsular types and 7 multilocus sequence types. Strains were labeled with GFP or RFP, generating two fluorescent variants for each. Intra-species interactions were evaluated in dual-strain biofilms (1:1 ratio) using flow cytometry. Confocal microscopy was used to image representative biofilms. Twenty-eight dual-strain combinations were tested. Interactions of commensalism, competition, amensalism and neutralism were identified. The outcome of an interaction was independent of the capsular and sequence type of the strains involved. Confocal imaging of biofilms confirmed the positive, negative and neutral effects that pneumococci can exert on each other. In conclusion, we developed an experimental approach that successfully discriminates pneumococcal strains growing in mixed biofilms, which enables the identification of intra-species interactions. Several types of interactions occur among pneumococci. These observations are a starting point to study the mechanisms underlying those interactions.

Increase in Penicillin and multidrug resistance in Streptococcus pneumoniae (1993-2016): report from a tertiary care hospital laboratory, Pakistan

Background: Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. With the emergence of penicillin-resistant S. pneumoniae (PRSP), treatment has become challenging. The Clinical and Laboratory Standards Institute (CLSI) in 2008 revised its guidelines for S. pneumoniae and recommended separate penicillin breakpoints for meningeal and non-meningeal strains. Similar to penicillin’s, resistance to other classes of antibiotics has emerged globally. Objective: The objective of this study is to determine the trend of resistance to antimicrobials in S. pneumoniae infections and the impact of new CLSI guidelines on penicillin susceptibility among meningeal isolates. Methodology: Twenty-four years (1993-2016) data from S. pneumoniae isolates and their antimicrobial susceptibility was retrieved from the computerized database. Data was divided into two groups for analysis, pre-2008 and post 2008. Results: Penicillin resistance remained unchanged in non-meningeal isolates during both study periods. A significant rise in penicillin resistance in meningeal isolates was observed in the second period 2008-2016 (2.9% vs 36.2%). High resistance rates were observed for co-trimoxazole, tetracycline and erythromycin. Increased trend of multi-drug resistant (MDR) strains were also noted, from 11% in 1999 to 36% in 2016. Conclusion: The emergence of MDR strains is evident from our dataset. It seems like the rise in PRSP in meningeal isolates is due to revised CLSI guidelines. Overall low resistance to penicillin in non-meningeal isolates and no resistance to ceftriaxone is encouraging and will assist in drafting local guidelines. Cautious use of antimicrobials are essential to reduce further emergence of antimicrobial resistance in indigenous isolates.

Pandemic Influenza Infection Promotes Streptococcus pneumoniae Infiltration, Necrotic Damage, and Proteomic Remodeling in the Heart

Adverse cardiac events are a common complication of viral and bacterial pneumonia. For over a century, it has been recognized that influenza infection promotes severe forms of pulmonary disease mainly caused by the bacterium Streptococcus pneumoniae .

A novel pneumococcal surface protein K of nonencapsulated Streptococcus pneumoniae promotes transmission among littermates in an infant mouse model with influenza A virus co-infection

We established an infant mouse model for colonization and transmission by nonencapsulated Streptococcus pneumoniae (NESp) strains to gain important information about its virulence among children. Invasive pneumococcal diseases have decreased dramatically since the worldwide introduction of pneumococcal capsular polysaccharide vaccines. Increasing prevalence of non-vaccine serotypes including NESp has been highlighted as a challenge in treatment strategy, but the virulence of NESp is not well understood. Protective strategy against NESp colonization and transmission between children require particularly urgent evaluation. NESp lacks capsules, a major virulent factor of pneumococci, but can cause a variety of infections in children and older people. PspK, a specific surface protein of NESp, is a key factor in establishing nasal colonization. In our infant mouse model for colonization and transmission by NESp strains, NESp could establish stable nasal colonization at the same level as encapsulated serotype 6A in infant mice, and could be transmitted between littermates. Transmission was promoted by NESp surface virulence factor PspK and influenza virus co-infection. However, PspK-deletion mutants lost the ability to colonize and transmit to new hosts. Promotion of NESp transmission by influenza was due to increased susceptibility of the new hosts. PspK was a key factor not only in establishment of nasal colonization, but also in transmission to new hosts. PspK may be targeted as a new candidate vaccine for NESp infection in children.