Pneumococcal Strains Research Articles

On the issue of the etiology of community-acquired pneumonia in children

According to global statistics, the number of cases of community-acquired pneumonia in children exceeds 150 million per year, and despite the introduction of vaccinations, this figure has not significantly decreased. Respiratory viruses, particularly respiratory syncytial virus, are recognized as the main cause of community-acquired pneumonia among preschool children during non-epidemic periods. Pneumococci also continue to play a significant role among bacterial pathogens. There is a significant increase in the prevalence of M. pneumoniae in the etiological structure of community-acquired pneumonia in recent years. Modern clinical guidelines for the use of antibiotics recommend taking into account patient age, vaccination status, and data on circulating pneumococcal strains when prescribing treatment. The COVID-19 pandemic and its subsequent relaxation of strict restrictions in the last two years have led to changes in the structure of infectious diseases, including the causes of lower respiratory tract infections.

Prediction of post-PCV13 pneumococcal evolution using invasive disease data enhanced by inverse-invasiveness weighting.

After introducing pneumococcal conjugate vaccines (PCVs), serotype replacement occurred in Streptococcus pneumoniae. Predicting which pneumococcal strains will become common in carriage after vaccination can enhance vaccine design, public health interventions, and understanding of pneumococcal evolution. Invasive pneumococcal isolates were collected during 1998-2018 by the Active Bacterial Core surveillance (ABCs). Carriage data from Massachusetts (MA) and Southwest United States were used to calculate weights. Using pre-vaccine data, serotype-specific inverse-invasiveness weights were defined as the ratio of the proportion of the serotype in carriage to the proportion in invasive data. Genomic data were processed under bioinformatic pipelines to define genetically similar sequence clusters (i.e., strains), and accessory genes (COGs) present in 5-95% of isolates. Weights were applied to adjust observed strain proportions and COG frequencies. The negative frequency-dependent selection (NFDS) model predicted strain proportions by calculating the post-vaccine strain composition in the weighted invasive disease population that would best match pre-vaccine COG frequencies. Inverse-invasiveness weighting increased the correlation of COG frequencies between invasive and carriage data in linear or logit scale for pre-vaccine, post-PCV7, and post-PCV13; and between different epochs in the invasive data. Weighting the invasive data significantly improved the NFDS model's accuracy in predicting strain proportions in the carriage population in the post-PCV13 epoch, with the adjusted R2 increasing from 0.254 before weighting to 0.545 after weighting. The weighting system adjusted invasive disease data to better represent the pneumococcal carriage population, allowing the NFDS mechanism to predict strain proportions in carriage in the post-PCV13 epoch. Our methods enrich the value of genomic sequences from invasive disease surveillance.IMPORTANCEStreptococcus pneumoniae, a common colonizer in the human nasopharynx, can cause invasive diseases including pneumonia, bacteremia, and meningitis mostly in children under 5 years or older adults. The PCV7 was introduced in 2000 in the United States within the pediatric population to prevent disease and reduce deaths, followed by PCV13 in 2010, PCV15 in 2022, and PCV20 in 2023. After the removal of vaccine serotypes, the prevalence of carriage remained stable as the vacated pediatric ecological niche was filled with certain non-vaccine serotypes. Predicting which pneumococcal clones, and which serotypes, will be most successful in colonization after vaccination can enhance vaccine design and public health interventions, while also improving our understanding of pneumococcal evolution. While carriage data, which are collected from the pneumococcal population that is competing to colonize and transmit, are most directly relevant to evolutionary studies, invasive disease data are often more plentiful. Previously, evolutionary models based on negative frequency-dependent selection (NFDS) on the accessory genome were shown to predict which non-vaccine strains and serotypes were most successful in colonization following the introduction of PCV7. Here, we show that an inverse-invasiveness weighting system applied to invasive disease surveillance data allows the NFDS model to predict strain proportions in the projected carriage population in the post-PCV13/pre-PCV15 and pre-PCV20 epoch. The significance of our research lies in using a sample of invasive disease surveillance data to extend the use of NFDS as an evolutionary mechanism to predict post-PCV13 population dynamics. This has shown that we can correct for biased sampling that arises from differences in virulence and can enrich the value of genomic data from disease surveillance and advance our understanding of how NFDS impacts carriage population dynamics after both PCV7 and PCV13 vaccination.

A 3’UTR-derived small RNA represses pneumolysin synthesis and facilitates pneumococcal brain invasion

Pneumolysin (Ply) of Streptococcus pneumoniae (pneumococcus) at relatively high and low levels facilitates pneumococcal invasion into the lung and brain, respectively; however, the regulatory mechanisms of Ply expression are poorly understood. Here, we find that a small RNA plyT, processed from the 3’UTR of the ply operon, is expressed higher in anaerobically- than in statically-cultured pneumococcus D39. Using bioinformatic, biochemical and genetic approaches, we reveal that PlyT inhibits Ply synthesis and hemolytic activities by pairing with an RBS-embedded intergenic region of the ply operon. The RNA-binding protein SPD_1558 facilitates the pairing. Importantly, PlyT inhibition of Ply synthesis is stronger in anaerobic culture and leads to lower Ply abundance. Deletion of plyT decreases the number of pneumococci in the infected mouse brain and reduces the virulence, demonstrating that PlyT-regulated lower Ply in oxygen-void microenvironments, such as the blood, is important for pneumococcus to cross the blood-brain barrier and invade the brain. PlyT-mediated repression of Ply synthesis at anoxic niches is also verified in pneumococcal serotype 4 and 14 strains; moreover, the ply operon with a 3’UTR-embedded plyT, and the pairing sequences of IGR and plyT are highly conserved among pneumococcal strains, implying PlyT-regulated Ply synthesis might be widely employed by pneumococcus.

A nisin-inducible chromosomal gene expression system based on ICE Tn5253 of Streptococcus pneumoniae, transferable among streptococci and enterococci

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.

Cytokine secretion by in vitro cultures of lung epithelial cells, differentiated macrophages and differentiated dendritic cells incubated with pneumococci and pneumococcal extracellular vesicles.

Streptococcus pneumoniae is an important human pathogen that can colonize the respiratory tract of healthy individuals. The respiratory tract mucosa is thus the first barrier for this pathogen. In this study, we have tested three models of the respiratory epithelium with immune cells: (i) monolayer of A549 human lung epithelial cells, (ii) A549 + macrophages differentiated from the human monocytic THP-1 cell line (dMφ) and (iii) A549 + dMφ + dendritic cells differentiated from THP-1 (dDC) using a two-chamber system. Pneumococcal strains Rx1 (non-encapsulated) and BHN418 (serotype 6B) were incubated with the cells and secretion of IL-6, IL-8, IL-1β, TNF-α and IL-10 was evaluated. Overall, the models using co-cultures of A549 + dMφ and A549 + dMφ + dDC elicited higher levels of pro-inflammatory cytokines and the non-encapsulated strain elicited an earlier cytokine response. BHN418 pspA (pneumococcal surface protein A) and pspC (pneumococcal surface protein C) knockouts elicited similar cytokine secretion in the co-culture models, whereas BHN18 ply (pneumolysin) knockout induced much lower levels. The results are in accordance with the activation of the inflammasome by Ply. Finally, we evaluated pneumococcal extracellular vesicles (pEVs) in the co-culture models and observed secretion of pro-inflammatory cytokines in the absence of cytotoxicity. Since pEVs are being studied as vaccine candidate against pneumococcal infections, the co-cultures of A549 + dMφ and A549 + dMφ + dDC are simple models that could be used to evaluate pEV vaccine batches.

Ultrastructural, metabolic and genetic characteristics of determinants facilitating the acquisition of macrolide resistance by Streptococcus pneumoniae

AimsTo investigate the molecular events associated with acquiring macrolide resistance genes [mefE/mel (Mega) or ermB] in Streptococcus pneumoniae (Spn) during nasopharyngeal colonization. Methods and resultsGenomic analysis of 128 macrolide-resistant Spn isolates revealed recombination events in genes of the conjugation apparatus, or the competence system, in strains carrying Tn916-related elements. Studies using confocal and electron microscopy demonstrated that during the transfer of Tn916-related elements in nasopharyngeal cell biofilms, pneumococcal strains formed clusters facilitating their acquisition of resistance determinants at a high recombination frequency (rF). Remarkably, these aggregates comprise both encapsulated and nonencapsulated pneumococci that span extracellular and intracellular compartments. rF assessments showed similar rates regardless Mega was associated with large integrative and conjugative elements (ICEs) (>23 kb) or not (∼5.4 kb). The rF for Mega Class IV(c) insertion region (∼53 kb) was three orders of magnitude higher than the transformation of the capsule locus. Metabolomics studies of the microenvironment created by colonization of human nasopharyngeal cells revealed a link between the acquisition of ICEs and the pathways involving nicotinic acid and sucrose. ConclusionsPneumococcal clusters, both extracellular and intracellular, facilitate macrolide resistance acquisition, and ICEs were acquired at a higher frequency than the capsule locus. Metabolic changes could serve as intervention targets.

Abstract We063: CD73 EXPRESSION ON NEUTROPHILS PROTECTS AGAINST CARDIOMYOCYTE DAMAGE DURING INVASIVE STREPTOCOCCUS PNEUMONIAE INFECTION

In recent years, Streptococcus pneumoniae (pneumococcus)-induced cardiac events have emerged as one of the most serious and life-threatening infection outcomes of invasive pneumococcal disease (IPD), leading to major adverse cardiac events (MACE) with high mortality rates. S. pneumoniae has the ability to invade myocardium and damage cardiomyocytes through the release of bacterial factors and formation of bacterial-filled cardiac microlesions. We previously found that polymorphonuclear cells (PMNs) or neutrophils are crucial for host defense against S. pneumoniae lung infection and that extracellular adenosine (EAD) production, by the exonucleosidase CD73, controlled the anti-bacterial functions of these cells. The objective of this study was to explore the role of PMNs and CD73 in cardiac damage during invasive pneumococcal infection. Upon intra-peritoneal ( i.p .) injection with TIGR4, an invasive pneumococcal strain, C57BL/6 mice showed an increased influx of PMNs into the cardiac tissue as determined by flow cytometry. However, the increased PMN numbers failed to contain bacterial burden in the heart and showed a positive correlation with serum levels of the cardiac damage marker Troponin-1 as determined through ELISA. Depletion of PMNs prior infection reduced pneumococcal burden in the heart and lowered the Troponin-1 levels suggesting that PMNs induce cardiac damage during infection. While exploring the mechanisms underlying the detrimental PMN response, we found that the late stage of IPD was associated with reduced CD73 expression on PMNs. The role of CD73 in regulating cardiac damage was tested in vivo using CD73 -/- mice which had significantly higher bacterial burden and cardiac damage compared to wild-type mice despite similar PMN numbers. The role of CD73 expression on PMNs was also tested ex vivo using the HL-1 cardiomyocyte cell line which showed increased LDH release upon S. pneumoniae infection in presence of CD73 -/- PMNs, a phenotype reversed by supplementation of exogenous adenosine. Similar iincreased damage in presence of CD73 -/- PMNs was seen with C57BL/6 Mouse Primary Cardiac Microvascular Endothelial Cells which lead to increased migration of beads across endothelium. Our findings suggest that dysregulation of CD73 on PMNs during invasive pneumococcal infection leads to an inability to control bacterial numbers and increased cardiac damage, and that the EAD-pathway can be a potential pharmacological target to limit disease severity.

The Crisis of Macrolide Resistance in Pneumococci in Latin America.

Macrolide antibiotics are recommended for the treatment of pneumococcal pneumonia and invasive pneumococcal disease (IPD). Prior to 2000, ∼10% of Streptococcus pneumoniae strains isolated from IPD cases in Latin American countries were resistant to macrolides. The mechanism of resistance to macrolides was associated mainly with the efflux pump known as the macrolide efflux genetic assembly, since most pneumococcal strains carried the mef(A/E) gene, whereas <6% strains carried both the methylase gene ermB and mef(A/E). In the first decade of this century, a significant increase in the prevalence of macrolide resistance was observed in pneumococcal strains in both Mexico and Peru. Approximately 30% of S. pneumoniae strains in these countries were already resistant to erythromycin, while the prevalence in Colombia, Argentina, and Brazil remained below 10%. During the last decade, we have been experiencing a worrisome increase in pneumococcal strains carrying resistance to macrolides, with a prevalence of up to 80% for resistance to erythromycin. The mechanism for disseminating macrolide resistance has evolved. Currently, more than 55% of invasive S. pneumoniae macrolide-resistant strains carry both the ermB and the mef(A/E)/mel genes. Lessons learned from the current macrolide resistance crisis in Latin America can inform interventions in other regions.

Epidemiological and bacterial profile ofchildhood meningitis in Tunisia.

The worldwide burden of disease of bacterial meningitis remains high, despite the decreasing incidence following introduction of routine vaccination campaigns.The aim of our study was to evaluate the epidemiological and bacteriological profile of paediatric bacterial meningitis (BM) in Tunisian children, during the period 2003-2019, following the implementation of Haemophilus influenzae type b (Hib) vaccine (April 2011) and before 10-valent pneumoccocal conjugate vaccine (PCV10) introduction to the childhood immunization program.All bacteriologically confirmed cases of BM admitted to children's hospital of Tunis were recorded (January 2003 to April 2019). Serogroups of Neisseria meningitidis (Nm) and serotypes of Streptococcus pneumoniae (Sp) and H. influenzae (Hi) and antibiotic resistance were determined using conventional and molecular methods.Among 388 cases, the most frequent species were Sp (51.3%), followed by Nm (27.5%) and Hi(16.8%). We observed a significant decrease in Hi BM rate during the conjugated Hib vaccine use period (P < 0.0001). The main pneumococcal serotypes were 14, 19F, 6B, 23F and 19A and the serotype coverage of PCV10, PCV13, PCV15 and PCV20 was 71.3 and 78.8%, 79.4 and 81.9% respectively. Themost frequent Nm serogroup was B (83.1%). Most Hi strains were of serotype b (86.9%). High levels of resistance were found: Sp and Nm to penicillin (respectively 60.1 and 80%) and Hi to ampicillin (42.6%). All meningococcal and Hi isolates were susceptible to third-generation cephalosporins and 7.2% of pneumococcal strains had decreased susceptibility to these antibiotics.The Hib conjugate vaccine decreased the rate of BM. Sp dominated the aetiology of BM in children in Tunisia. Conjugate vaccines introducing decreases not only BM cases but also antimicrobial resistance.

Bacterial surface lipoproteins mediate epithelial microinvasion by Streptococcus pneumoniae.

Streptococcus pneumoniae, a common colonizer of the upper respiratory tract, invades nasopharyngeal epithelial cells without causing disease in healthy participants of controlled human infection studies. We hypothesized that surface expression of pneumococcal lipoproteins, recognized by the innate immune receptor TLR2, mediates epithelial microinvasion. Mutation of lgt in serotype 4 (TIGR4) and serotype 6B (BHN418) pneumococcal strains abolishes the ability of the mutants to activate TLR2 signaling. Loss of lgt also led to the concomitant decrease in interferon signaling triggered by the bacterium. However, only BHN418 lgt::cm but not TIGR4 lgt::cm was significantly attenuated in epithelial adherence and microinvasion compared to their respective wild-type strains. To test the hypothesis that differential lipoprotein repertoires in TIGR4 and BHN418 lead to the intraspecies variation in epithelial microinvasion, we employed a motif-based genome analysis and identified an additional 525 a.a. lipoprotein (pneumococcal accessory lipoprotein A; palA) encoded by BHN418 that is absent in TIGR4. The gene encoding palA sits within a putative genetic island present in ~10% of global pneumococcal isolates. While palA was enriched in the carriage and otitis media pneumococcal strains, neither mutation nor overexpression of the gene encoding this lipoprotein significantly changed microinvasion patterns. In conclusion, mutation of lgt attenuates epithelial inflammatory responses during pneumococcal-epithelial interactions, with intraspecies variation in the effect on microinvasion. Differential lipoprotein repertoires encoded by the different strains do not explain these differences in microinvasion. Rather, we postulate that post-translational modifications of lipoproteins may account for the differences in microinvasion.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is an important mucosal pathogen, estimated to cause over 500,000 deaths annually. Nasopharyngeal colonization is considered a necessary prerequisite for disease, yet many people are transiently and asymptomatically colonized by pneumococci without becoming unwell. It is therefore important to better understand how the colonization process is controlled at the epithelial surface. Controlled human infection studies revealed the presence of pneumococci within the epithelium of healthy volunteers (microinvasion). In this study, we focused on the regulation of epithelial microinvasion by pneumococcal lipoproteins. We found that pneumococcal lipoproteins induce epithelial inflammation but that differing lipoprotein repertoires do not significantly impact the magnitude of microinvasion. Targeting mucosal innate immunity and epithelial microinvasion alongside the induction of an adaptive immune response may be effective in preventing pneumococcal colonization and disease.

Exploring factors shaping antibiotic resistance patterns in Streptococcus pneumoniae during the 2020 COVID-19 pandemic.

Non-pharmaceutical interventions implemented to block SARS-CoV-2 transmission in early 2020 led to global reductions in the incidence of invasive pneumococcal disease (IPD). By contrast, most European countries reported an increase in antibiotic resistance among invasive Streptococcus pneumoniae isolates from 2019 to 2020, while an increasing number of studies reported stable pneumococcal carriage prevalence over the same period. To disentangle the impacts of the COVID-19 pandemic on pneumococcal epidemiology in the community setting, we propose a mathematical model formalizing simultaneous transmission of SARS-CoV-2 and antibiotic-sensitive and -resistant strains of S. pneumoniae. To test hypotheses underlying these trends five mechanisms were built into the model and examined: (1) a population-wide reduction of antibiotic prescriptions in the community, (2) lockdown effect on pneumococcal transmission, (3) a reduced risk of developing an IPD due to the absence of common respiratory viruses, (4) community azithromycin use in COVID-19 infected individuals, (5) and a longer carriage duration of antibiotic-resistant pneumococcal strains. Among 31 possible pandemic scenarios involving mechanisms individually or in combination, model simulations surprisingly identified only two scenarios that reproduced the reported trends in the general population. They included factors (1), (3), and (4). These scenarios replicated a nearly 50% reduction in annual IPD, and an increase in antibiotic resistance from 20% to 22%, all while maintaining a relatively stable pneumococcal carriage. Exploring further, higher SARS-CoV-2 R0 values and synergistic within-host virus-bacteria interaction mechanisms could have additionally contributed to the observed antibiotic resistance increase. Our work demonstrates the utility of the mathematical modeling approach in unraveling the complex effects of the COVID-19 pandemic responses on AMR dynamics.

Liposome-based dry powder vaccine immunization targeting the lungs induces broad protection against pneumococcus

Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.

A clade of Streptococcus pneumoniae clonal complex 320 with increased tolerance to β-lactam antibiotics in a Chinese metropolitan city

ObjectivesWe characterized the population structure and features of clinical Streptococcus pneumoniae isolates associated with invasive pneumococcal disease (IPD) from 2009 to 2017 in a Chinese metropolitan city using a whole-genome sequencing approach. MethodsSeventy-nine pneumococcal strains, including 60 serogroup-19 strains from children enduring IPD from a paediatric hospital in Shenzhen, were subjected to whole-genome sequencing. Population structure was characterized through phylogenetic analysis, sequence typing, serotyping, virulence factor, and antimicrobial drug resistance (AMR) gene profiling, combining the publicly available related WGS data. Clinical demography and antibiotic susceptibility profiles were compared among different populations to emphasize the higher-risk populations. Genetic regions associated with AMR gene mobilization were identified through comparative genomics. ResultsThese IPD strains mainly belonged to clonal complex 320 (CC320) and were composed of serotypes 19A and 19F. In addition to sporadic possible importation-related isolates (ST320), we identified an independent clade, CC320_SZpop (ST271), that predominantly circulated in Shenzhen and possibly expanded its range. Clinical features and antibiotic susceptibility analysis revealed that CC320_SZpop might manifest much higher pathogenicity and tolerance to β-lactams. Specific virulence factors in Shenzhen isolates of CC320_SZpop were identified. Furthermore, an ca. 40 kb hotspot genomic region enduring frequent recombination was identified, possibly associated with the divergence of S. pneumoniae strains. ConclusionA novel pneumococcal clade, CC320_SZpop, circulating in Shenzhen and other regions in China, possibly under expansion, was found and deserves more study and surveillance. Our study also emphasizes the importance of continuous genomic surveillance of clinical S. pneumoniae isolates, especially IPD isolates.

Strain features of pneumococcal isolates in the pre- and post-PCV10 era in Pakistan.

Pakistan is amongst the four countries with the highest number of pneumococcal deaths. While the PCV10 vaccine was introduced in Pakistan in October 2012, data regarding the impact of the vaccine on the population dynamics of Streptococcus pneumoniae in Pakistan remain obscure. Using whole genome sequencing of 190 isolates (nasopharyngeal carriage=75, disease=113, unknown sites=2) collected between 2002 and 2020, this study presents characteristics of pneumococcal strains in Pakistan in the pre- and post-vaccine era. The isolates were characterized on the basis of serotype distribution, genetic lineages (or Global Pneumococcal Sequence Cluster, GPSC) and antibiotic resistance. A high level of diversity in serotype and genetic lineages of pneumococci was observed in Pakistan. Among 190 isolates, we identified 54 serotypes, 67 GPSCs and 116 sequence types (STs) including 23 new STs. The most prevalent GPSCs and their associated serotypes in nasopharyngeal carriage were GPSC54 (expressing serotype 9V), GPSC5 (15A and 7B, and serogroup 24), GPSC25 (15B/15C), GPSC67 (18C) and GPSC376 (6A and 6D). Similarly, among 113 disease-causing isolates, the most prevalent GPSC/serotype combinations were GPSC2 (serotype 1), GPSC10 (serotypes 14, 10A, 19A and 19F), GPSC43 (serotypes 13, 11A, 23B, 35A and 9V), GPSC67 (serotypes 18A and 18C) and GPSC642 (serotype 11A). Of the 190 isolates, the highest levels of resistance were observed against penicillin (58.9 %, n=122), erythromycin (29.5 %, n=56), clindamycin (13.2 %, n=25), co-trimoxazole (94.2 %, n=179) and tetracycline/doxycycline (53.2 %, n=101). A higher proportion of disease-causing isolates were multidrug resistant as compared to carriage isolates (54 % vs 25 %). Our data suggest limited coverage of PCV10 in nasopharyngeal (21.6 %, 16/74) as well as disease-causing (38.1 %, 16/42) isolates among children ≤5 years old; however, higher valent vaccine PCV13 would increase the coverage rates to 33.8 % in nasopharyngeal and 54.8 % in disease-causing isolates, whereas PCV24/25 would offer the highest coverage rates. Owing to the diversity of serotypes observed during the post-vaccine period, the suggested inclusion of serotype in future vaccine formulations will require investigations with larger data sets with an extended temporal window. This article contains data hosted by Microreact.

Robust Immune Response and Protection against Lethal Pneumococcal Challenge with a Recombinant BCG-PspA-PdT Prime/Boost Scheme Administered to Neonatal Mice.

Pneumococcal diseases are an important public health problem, with high mortality rates in young children. Although conjugated pneumococcal vaccines offer high protection against invasive pneumococcal diseases, this is restricted to vaccine serotypes, leading to serotype replacement. Furthermore, the current vaccines do not protect neonates. Therefore, several protein-based pneumococcal vaccines have been studied over the last few decades. Our group established a recombinant BCG expressing rPspA-PdT as a prime/rPspA-PdT boost strategy, which protected adult mice against lethal intranasal pneumococcal challenge. Here, we immunized groups of neonate C57/Bl6 mice (6-10) (at 5 days) with rBCG PspA-PdT and a boost with rPspA-PdT (at 12 days). Controls were saline or each antigen alone. The prime/boost strategy promoted an IgG1 to IgG2c isotype shift compared to protein alone. Furthermore, there was an increase in specific memory cells (T and B lymphocytes) and higher cytokine production (IFN-γ, IL-17, TNF-α, IL-10, and IL-6). Immunization with rBCG PspA-PdT/rPspA-PdT showed 100% protection against pulmonary challenge with the WU2 pneumococcal strain; two doses of rPspA-PdT showed non-significant protection in the neonates. These results demonstrate that a prime/boost strategy using rBCG PspA-PdT/rPspA-PdT is effective in protecting neonates against lethal pneumococcal infection via the induction of strong antibody and cytokine responses.

Molecular characteristics and antimicrobial resistance of invasive pneumococcal isolates from children in the post-13-valent pneumococcal conjugate vaccine era in Shenzhen, China

This study aimed to evaluate the molecular epidemiology and antimicrobial resistance of invasive pneumococcal isolates from children in Shenzhen, China, in the early stage of the pneumococcal 13-valent conjugated vaccine (PCV-13) era from 2018 to 2020. Invasive pneumococcal strains were isolated from hospitalized children with invasive pneumococcal diseases (IPDs) from January 2018 to December 2020. The serotype identification, multilocus sequence typing (MLST), and antibiotic susceptibility tests were performed on all culture-confirmed strains. Sixty-four invasive strains were isolated mainly from blood (70.3%). Prevalent serotypes were 23F (28.1%), 14 (18.8%), 19F (15.6%), 6A/B (14.1%), and 19A (12.5%), with a serotype coverage rate of 96.9% for PCV13. The most common sequence types (STs) were ST876 (17.1%), ST271 (10.9%), and ST320 (7.8%). Half of the strains were grouped in clonal complexes (CCs): CC271 (21.9%), CC876 (20.3%), and CC90 (14.1%). Meningitis isolates showed a higher resistance rate (90.9% and 45.5%) to penicillin and ceftriaxone than the rate (3.8% and 9.4%) of non-meningitis isolates. The resistance rates for penicillin (oral), cefuroxime, and erythromycin were 53.13%, 73.4%, and 96.9%, respectively. The dual ermB and mefA genotype was found in 81.3% of erythromycin-resistant strains. The elevated minimum inhibitory concentration (MIC) of β-lactam antibiotics and dual-genotype macrolide resistance were related mainly to three major serotype-CC combinations: 19F-CC271, 19A-CC271, and 14-CC876. Invasive pneumococcus with elevated MICs of β-lactams and increased dual ermB and mefA genotype macrolide resistance were alarming. Expanded PCV13 vaccination is expected to reduce the burden of paediatric IPD and to combat antibiotic-resistant pneumococcus in Shenzhen.

Prevalence and resistance characteristics of multidrug-resistant Streptococcus pneumoniae isolated from the respiratory tracts of hospitalized children in Shenzhen, China.

PCV13 introduction in China has led to a significant reduction of vaccine serotype Streptococcus pneumoniae. However, non-vaccine serotypes with highly resistance and invasiveness were often reported in the post-pneumococcal conjugate vaccine era and there was regional differences. A total of 669 S. pneumoniae strains were collected from the respiratory tracts of hospitalized children at Shenzhen Children's Hospital in 2021 and 2022. Antimicrobial resistance (AMR) characteristics were assessed through antibiotic susceptibility testing performed with the VITEK 2 compact system. AMR genes and single nucleotide polymorphisms (SNPs) in pbp1a, pbp2b, and pbp2x were identified via analysis of whole genome sequencing data. Statistical examination of the data was conducted employing chi-square and Fisher's exact tests. We found that non-vaccine serotypes strains had accounted for 46.6% of all the pneumococcal isolated strains. The most common non-vaccine serotype is 23A, with a prevalence rate of 8.9%, followed by 15A (6.6%), 6E (5.7%), 34 (3.2%), and 15B (2.9%). The multidrug resistance rates (MDR) of vaccine serotypes were 19F (99.36%), 19A (100%), 23F (98.08%), 6B (100%), and 6C (100%). Meanwhile, the MDR of non-vaccine serotypes were 15B (100.00%), 6E (100%), 15C (100%), 34 (95.24%), and 23A (98.31%). Resistance rates of 6E to more than six antibiotic classes reached 89.47%, which is similar to 19F (83.33%) and 19A (90%). Unique resistance profiles were also identified for non-vaccine serotypes, including significantly higher resistance to chloramphenicol in 6E, 15B, and 15C than in 19F and 19A. Furthermore, through genome sequencing, we revealed strong correlation of cat-TC with chloramphenicol resistance, patA/patB with tetracycline resistance, ermB and pmrA with erythromycin resistance. The introduction of PCV13 into China from 2017 has led to a shift in the dominant composition of pneumococcal strains. There has been a notable rise and spread of multidrug-resistant non-vaccine serotypes among children. Specifically, the non-vaccine serotype 6E, which was not widely reported in China previously, has emerged. To comprehend the resistance mechanisms, it is crucial to further investigate the molecular and genetic characteristics of these non-vaccine serotypes.

Clinical features relating to pneumococcal colony phase variation in hospitalized adults with pneumonia.

Background. Streptococcus pneumoniae is a major causative bacteria of pneumonia and invasive pneumococcal disease (IPD); however, the mechanisms underlying its severity and invasion remain to be defined. Pneumococcal colonies exhibit opaque and transparent opacity phase variations, which have been associated with invasive infections and nasal colonization, respectively, in animal studies. This study evaluated the relationship between the opacity of pneumococcal colonies and the clinical presentation of pneumococcal pneumonia.Methods. This retrospective study included adult patients hospitalized with pneumococcal pneumonia between 2012 and 2019 at four tertiary medical institutions. Pneumococcal strains from lower respiratory tract specimens were determined for their serotypes and microscopic colony opacity, and the association between the opacity phase and the severity of pneumonia was evaluated. Serotypes 3 and 37 with mucoid colony phenotypes were excluded from the study because their colony morphologies were clearly different.Results. A total of 92 patients were included. Most patients were older adults (median age: 72 years) and males (67 %), and 59 % had community-acquired pneumonia. Of the 92 patients, 41 (45 %), 12 (13 %), and 39 (42 %) patients had opaque, transparent, and mixed variants in their pneumococcal colony, respectively. The opaque and non-opaque pneumococcal variants had no statistically significant difference in patient backgrounds. Although the pneumonia severity index score did not differ between the opaque and non-opaque groups, the rate of bacteremia was significantly higher in the opaque group than in the non-opaque group. Serotype distribution was similar between the groups.Conclusions. Opaque pneumococcal variants may cause pneumonia and invasive diseases in humans. This study could help elucidate IPD, and opacity assessment may serve as a predictor for IPD.

Fusion of PspA to detoxified pneumolysin enhances pneumococcal vaccine coverage

Despite the implementation of conjugate vaccines in several countries, S. pneumoniae continues to pose a great burden worldwide, causing around 1 million annual deaths. Pneumococcal proteins have long been investigated as serotype-independent vaccines against this pathogen, with promising results. However, it is a consensus that one antigen alone will not be sufficient to provide long-term protection with wide coverage. Amongst the most well studied pneumococcal proteins are PspA and pneumolysin (Ply), two major virulence factors required by the bacterium for successful invasion of host tissues. PspA is highly immunogenic and protective, but it is structurally variable; pneumolysin is conserved among different pneumococci, but it is toxic to the host. To overcome these limitations, N-terminal PspA fragments have been genetically fused to non-toxic pneumolysin derivatives (PlD) to create PspA_PlD chimeras. Mouse immunization with these fusions confers protection against pneumococcal strains expressing heterologous PspAs, which correlates with antibody-induced complement C3 deposition on the surface of multiple pneumococcal strains. Analysis of mutant strains lacking PspA or Pneumolysin shows that both proteins contribute to the antibody-mediated enhancement in complement deposition induced by the fusion. These results expand previous data evaluating PspA_PlD and demonstrate that the fusion combines the protective traits of both proteins, inducing antibodies that efficiently promote complement deposition on multiple strains and cross-protection.

Carbon source-dependent capsule thickness regulation in Streptococcus pneumoniae.

The polysaccharide capsule of Streptococcus pneumoniae plays a major role in virulence, adherence to epithelial cells, and overall survival of the bacterium in the human host. Galactose, mannose, and N-acetylglucosamine (GlcNAc) are likely to be relevant for metabolization in the nasopharynx, while glucose is the primary carbon source in the blood. In this study, we aim to further the understanding of the influence of carbon sources on pneumococcal growth, capsule biosynthesis, and subsequent adherence potential. We tested the growth behavior of clinical wild-type and capsule knockout S. pneumoniae strains, using galactose, GlcNAc, mannose, and glucose as carbon source for growth. We measured capsule thickness and quantified capsule precursors by fluorescein isothiocyanate (FITC)-dextran exclusion assays and 31P-nuclear magnetic resonance measurements, respectively. We also performed epithelial adherence assays using Detroit 562 cells and performed a transcriptome analysis (RNA sequencing). We observed a reduced growth in galactose, mannose, and GlcNAc compared to growth in glucose and found capsular size reductions in mannose and GlcNAc compared to galactose and glucose. Additionally, capsular precursor measurements of uridine diphosphate-(UDP)-glucose and UDP-galactose showed less accumulation of precursors in GlcNAc or mannose than in glucose and galactose, indicating a possible link with the received capsular thickness measurements. Epithelial adherence assays showed an increase in adherence potential for a pneumococcal strain, when grown in mannose compared to glucose. Finally, transcriptome analysis of four clinical isolates revealed not only strain specific but also common carbon source-specific gene expression. Our findings may indicate a careful adaption of the lifestyle of S. pneumoniae according to the monosaccharides encountered in the respective human niche.