Long-term evolution of Streptococcus mitis and Streptococcus pneumoniae leads to higher genetic diversity within rather than between human populations.

Evaluation of the apportionment of genetic diversity of human bacterial commensals within and between human populations is an important step in the characterization of their evolutionary potential. Recent studies showed a correlation between the genomic diversity of human commensal strains and that of their host, but the strength of this correlation and of the geographic structure among human populations is a matter of debate. Here, we studied the genomic diversity and evolution of the phylogenetically related oro-nasopharyngeal healthy-carriage Streptococcus mitis and Streptococcus pneumoniae, whose lifestyles range from stricter commensalism to high pathogenic potential. A total of 119 S. mitis genomes showed higher within- and among-host variation than 810 S. pneumoniae genomes in European, East Asian and African populations. Summary statistics of the site-frequency spectrum for synonymous and non-synonymous variation and ABC modelling showed this difference to be due to higher ancestral bacterial population effective size (Ne) in S. mitis, whose genomic variation has been maintained close to mutation-drift equilibrium across (at least many) generations, whereas S. pneumoniae has been expanding from a smaller ancestral bacterial population. Strikingly, both species show limited differentiation among human populations. As genetic differentiation is inversely proportional to the product of effective population size and migration rate (Nem), we argue that large Ne have led to similar differentiation patterns, even if m is very low for S. mitis. We conclude that more diversity within than among human populations and limited population differentiation must be common features of the human microbiome due to large Ne.

Environmental and genetic regulation of Streptococcus pneumoniae galactose catabolic pathways.

Efficient utilization of nutrients is crucial for microbial survival and virulence. The same nutrient may be utilized by multiple catabolic pathways, indicating that the physical and chemical environments for induction as well as their functional roles may differ. Here, we study the tagatose and Leloir pathways for galactose catabolism of the human pathogen Streptococcus pneumoniae. We show that galactose utilization potentiates pneumococcal virulence, the induction of galactose catabolic pathways is influenced differentially by the concentration of galactose and temperature, and sialic acid downregulates galactose catabolism. Furthermore, the genetic regulation and in vivo induction of each pathway differ, and both galactose catabolic pathways can be turned off with a galactose analogue in a substrate-specific manner, indicating that galactose catabolic pathways can be potential drug targets.

Evaluation of a multiplex-qPCR for paediatric pleural empyema-An observational study in hospitalised children.

Pleural empyema is a serious complication of pneumonia in children. Negative bacterial cultures commonly impede optimal antibiotic therapy. To improve bacterial identification, we developed a molecular assay and evaluated its performance compared with bacterial culture. Our multiplex-quantitative PCR to detect Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Haemophilus influenzae was assessed using bacterial genomic DNA and laboratory-prepared samples (n = 267). To evaluate clinical performance, we conducted the Molecular Assessment of Thoracic Empyema (MATE) observational study, enrolling children hospitalised with empyema. Pleural fluids were tested by bacterial culture and multiplex-qPCR, and performance determined using a study gold standard. We determined clinical sensitivity and time-to-organism-identification to assess the potential of the multiplex-qPCR to reduce the duration of empiric untargeted antibiotic therapy. Using spiked samples, the multiplex-qPCR demonstrated 213/215 (99.1%) sensitivity and 52/52 (100%) specificity for all organisms. During May 2019-March 2023, 100 children were enrolled in the MATE study; median age was 3.9 years (IQR 2-5.6). A bacterial pathogen was identified in 90/100 (90%) specimens by multiplex-qPCR, and 24/100 (24%) by bacterial culture (P <0.001). Multiplex-qPCR identified a bacterial cause in 68/76 (90%) culture-negative specimens. S. pneumoniae was the most common pathogen, identified in 67/100 (67%) specimens. We estimate our multiplex-qPCR would have reduced the duration of untargeted antibiotic therapy in 61% of cases by a median 20 days (IQR 17.5-23, range 1-55). Multiplex-qPCR significantly increased pathogen detection compared with culture and may allow for reducing the duration of untargeted antibiotic therapy.

Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts.

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS-11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A260/280. In case DNA was pure (A260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A260/280 values > 2.0. The A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S. pneumoniae, and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.

Effect of erythromycin residuals in food on the development of resistance in Streptococcus pneumoniae: an in vivo study in Galleria mellonella.

Background: The use of antimicrobials to treat food animals may result in antimicrobial residues in foodstuffs of animal origin. The European Medicines Association (EMA) and World Health Organization (WHO) define safe antimicrobial concentrations in food based on acceptable daily intakes (ADIs). It is unknown if ADI doses of antimicrobials in food could influence the antimicrobial susceptibility of human-associated bacteria. Objectives: This aim of this study was to evaluate if the consumption of ADI doses of erythromycin could select for erythromycin resistance in a Galleria mellonella model of Streptococcus pneumoniae infection. Methods: A chronic model of S. pneumoniae infection in G. mellonella larvae was used for the experiment. Inoculation of larvae with S. pneumoniae was followed by injections of erythromycin ADI doses (0.0875 and 0.012 µg/ml according to EMA and WHO, respectively). Isolation of S. pneumoniae colonies was then performed on selective agar plates. Minimum inhibitory concentrations (MICs) of resistant colonies were measured, and whole genome sequencing (WGS) was performed followed by variant calling to determine the genetic modifications. Results: Exposure to single doses of both EMA and WHO ADI doses of erythromycin resulted in the emergence of erythromycin resistance in S. pneumoniae. Emergent resistance to erythromycin was associated with a mutation in rplA, which codes for the L1 ribosomal protein and has been linked to macrolide resistance in previous studies. Conclusion: In our in vivo model, even single doses of erythromycin that are classified as acceptable by the WHO and EMA induced significant increases in erythromycin MICs in S. pneumoniae. These results suggest the need to include the induction of antimicrobial resistance (AMR) as a significant criterion for determining ADIs.

Estimating geographical spread of Streptococcus pneumoniae within Israel using genomic data.

Understanding how pathogens spread across geographical space is fundamental for control measures such as vaccination. Streptococcus pneumoniae (the pneumococcus) is a respiratory bacterium responsible for a large proportion of infectious disease morbidity and mortality globally. Even in the post-vaccination era, the rates of invasive pneumococcal disease (IPD) remain stable in most countries, including Israel. To understand the geographical spread of the pneumococcus in Israel, we analysed 1174 pneumococcal genomes from patients with IPD across multiple regions. We included the evolutionary distance between pairs of isolates inferred using whole-genome data within a relative risk (RR) ratio framework to capture the geographical structure of S. pneumoniae. While we could not find geographical structure at the overall lineage level, the extra granularity provided by whole-genome sequence data showed that it takes approximately 5 years for invasive pneumococcal isolates to become fully mixed across the country.This article contains data hosted by Microreact.

Expansion of pneumococcal serotype 23F and 14 lineages with genotypic changes in capsule polysaccharide locus and virulence gene profiles post introduction of pneumococcal conjugate vaccine in Blantyre, Malawi.

Since the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in Malawi in 2011, there has been persistent carriage of vaccine serotype (VT) Streptococcus pneumoniae, despite high vaccine coverage. To determine if there has been a genetic change within the VT capsule polysaccharide (cps) loci since the vaccine's introduction, we compared 1022 whole-genome-sequenced VT isolates from 1998 to 2019. We identified the clonal expansion of a multidrug-resistant, penicillin non-susceptible serotype 23F GPSC14-ST2059 lineage, a serotype 14 GPSC9-ST782 lineage and a novel serotype 14 sequence type GPSC9-ST18728 lineage. Serotype 23F GPSC14-ST2059 had an I253T mutation within the capsule oligosaccharide repeat unit polymerase Wzy protein, which is predicted in silico to alter the protein pocket cavity. Moreover, serotype 23F GPSC14-ST2059 had SNPs in the DNA binding sites for the cps transcriptional repressors CspR and SpxR. Serotype 14 GPSC9-ST782 harbours a non-truncated version of the large repetitive protein (Lrp), containing a Cna protein B-type domain which is also present in proteins associated with infection and colonisation. These emergent lineages also harboured genes associated with antibiotic resistance, and the promotion of colonisation and infection which were absent in other lineages of the same serotype. Together these data suggest that in addition to serotype replacement, modifications of the capsule locus associated with changes in virulence factor expression and antibiotic resistance may promote vaccine escape. In summary, the study highlights that the persistence of vaccine serotype carriage despite high vaccine coverage in Malawi may be partly caused by expansion of VT lineages post-PCV13 rollout.

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae.

Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

A novel biosensing strategy for identification of three important bacteria causing meningitis.

Bacterial meningitis is an acute infection which requires rapid diagnosis and treatment due to the high mortality and serious consequences of the disease. The purpose of this study was to design a homemade multiplex PCR and a novel fluorescence biosensor on chip (FBC) to detect three important agents of meningitis including Streptococcus pneumoniae (S. pneumoniae), Neisseria meningitidis (N. meningitidis), and Haemophilus influenzae (H. influenzae). The homemade multiplex PCR can diagnose three bacterial species simultaneously. Fabrication of FBC was carried out based on the deposition of lead nanoparticles on a quartz slide using the thermal evaporation method. Then, the SH-Cap Probe/Target ssDNA /FAM-Rep probe was loaded on lead film. The evaluation of the fluorescence reaction when the probes bind to the target ssDNA was assessed by a Cytation 5 Cell Imaging Multimode Reader Bio-Tek. The limit of detections (LOD) in homemade PCR and FBC to identify S. pneumoniae were 119 × 102 CFU/mL (0.27 ng/µL) and 380 CFU/mL (9 pg/µL), respectively. The LODs of homemade PCR and FBC for detection of N. meningitidis were 4.49 CFU/mL (1.1 pg/µL) and 13 × 103 CFU/mL (30 pg/µL), respectively. Our results confirmed the LODs of homemade PCR and FBC in detection of H. influenzae were 15.1 CFU/mL (30 fg/µL) and 41 × 102 CFU/mL (90 pg/ µL), respectively. Both techniques had appropriate sensitivity and specificity in detection of S. pneumoniae, N. meningitidis and H. influenzae.

Biological puzzles solved by using Streptococcus pneumoniae: a historical review of the pneumococcal studies that have impacted medicine and shaped molecular bacteriology.

The major human pathogen Streptococcus pneumoniae has been the subject of intensive clinical and basic scientific study for over 140 years. In multiple instances, these efforts have resulted in major breakthroughs in our understanding of basic biological principles as well as fundamental tenets of bacterial pathogenesis, immunology, vaccinology, and genetics. Discoveries made with S. pneumoniae have led to multiple major public health victories that have saved the lives of millions. Studies on S. pneumoniae continue today, where this bacterium is being used to dissect the impact of the host on disease processes, as a powerful cell biology model, and to better understand the consequence of human actions on commensal bacteria at the population level. Herein we review the major findings, i.e., puzzle pieces, made with S. pneumoniae and how, over the years, they have come together to shape our understanding of this bacterium's biology and the practice of medicine and modern molecular biology.

Transcriptional regulation of TacL-mediated lipoteichoic acids biosynthesis by ComE during competence impacts pneumococcal transformation.

Competence development is essential for bacterial transformation since it enables bacteria to take up free DNA from the surrounding environment. The regulation of teichoic acid biosynthesis is tightly controlled during pneumococcal competence; however, the mechanism governing this regulation and its impact on transformation remains poorly understood. We demonstrated that a defect in lipoteichoic acid ligase (TacL)-mediated lipoteichoic acids (LTAs) biosynthesis was associated with impaired pneumococcal transformation. Using a fragment of tacL regulatory probe as bait in a DNA pulldown assay, we successfully identified several regulatory proteins, including ComE. Electrophoretic mobility shift assays revealed that phosphomimetic ComE, but not wild-type ComE, exhibited specific binding to the probe. DNase I footprinting assays revealed the specific binding sequences encompassing around 30 base pairs located 31 base pairs upstream from the start codon of tacL. Expression of tacL was found to be upregulated in the ΔcomE strain, and the addition of exogenous competence-stimulating peptide repressed the tacL transcription in the wild-type strain but not the ΔcomE mutant, indicating that ComE exerted a negative regulatory effect on the transcription of tacL. Mutation in the JH2 region of tacL upstream regulatory sequence led to increased LTAs abundance and displayed higher transformation efficiency. Collectively, our work identified the regulatory mechanisms that control LTAs biosynthesis during competence and thereby unveiled a repression mechanism underlying pneumococcal transformation.

Posibilities for use of whole genome sequencing (WGS) for the analysis of Streptococcus pneumoniae isolates.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive coccus causing both non-invasive and invasive infectious diseases. Pneumococcal diseases are vaccine preventable. Invasive pneumococcal diseases (IPD) meeting the international case definition are reported nationally and internationally and are subject to surveillance programmes in many countries, including the Czech Republic. An important part of IPD surveillance is the monitoring of causative serotypes and their frequency over time and in relation to ongoing vaccination programmes. In the world and in the Czech Republic, whole genome sequencing (WGS) is increasingly used for pneumococci, which allows for serotyping from sequencing data, precise analysis of their genetic relationships, and the study of genes present in their genome. Whole-genome sequencing enables the generation of reliable and internationally comparable data that can be easily shared. Sequencing data are analysed using bioinformatics tools that require knowledge in the field of natural sciences with an emphasis on genetics and expertise in bioinformatics. This publication presents some options for pneumococcal analysis, i.e., serotyping, multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), whole genome MLST (wgMLST), single nucleotide polymorphism (SNP) analysis, assignment to Global Pneumococcal Sequence Cluster (GPSC), and identification of virulence genes and antibiotic resistance genes. The WGS strategies and applications for Europe and WGS implementation in practice are presented. WGS analysis of pneumococci allows for improved IPD surveillance, thanks to molecular serotyping, more detailed typing, generation of internationally comparable data, and improved evaluation of the effectiveness of vaccination programmes.

AmiA and AliA peptide ligands, found in Klebsiella pneumoniae, are imported into pneumococci and alter the transcriptome.

Klebsiella pneumoniae releases the peptides AKTIKITQTR and FNEMQPIVDRQ, which bind the pneumococcal proteins AmiA and AliA respectively, two substrate-binding proteins of the ABC transporter Ami-AliA/AliB oligopeptide permease. Exposure to these peptides alters pneumococcal phenotypes such as growth. Using a mutant in which a permease domain of the transporter was disrupted, by growth analysis and epifluorescence microscopy, we confirmed peptide uptake via the Ami permease and intracellular location in the pneumococcus. By RNA-sequencing we found that the peptides modulated expression of genes involved in metabolism, as pathways affected were mostly associated with energy or synthesis and transport of amino acids. Both peptides downregulated expression of genes involved in branched-chain amino acid metabolism and the Ami permease; and upregulated fatty acid biosynthesis genes but differed in their regulation of genes involved in purine and pyrimidine biosynthesis. The transcriptomic changes are consistent with growth suppression by peptide treatment. The peptides inhibited growth of pneumococcal isolates of serotypes 3, 8, 9N, 12F and 19A, currently prevalent in Switzerland, and caused no detectable toxic effect to primary human airway epithelial cells. We conclude that pneumococci take up K. pneumoniae peptides from the environment via binding and transport through the Ami permease. This changes gene expression resulting in altered phenotypes, particularly reduced growth.

Respiratory Syncytial Virus and Influenza Infections in Children in Ulaanbaatar, Mongolia, 2015-2021.

BACKGROUND: Data available for RSV and influenza infections among children < 2 years in Mongolia are limited. We present data from four districts of Ulaanbaatar from April 2015 to June 2021. METHODS: This study was nested in an enhanced surveillance project evaluating pneumococcal conjugate vaccine (PCV13) impact on the incidence of hospitalized lower respiratory tract infections (LRTIs). Our study was restricted to children aged < 2 years with arterial O2 saturation < 93% and children with radiological pneumonia. Nasopharyngeal (NP) swabs collected at admission were tested for RSV and influenza using qRT-PCR. NP swabs of all patients with radiological pneumonia and of a subset of randomly selected NP swabs were tested for S. pneumoniae (S.p.) by qPCR and for serotypes by culture and DNA microarray. RESULTS: Among 5705 patients, 2113 (37.0%) and 386 (6.8%) had RSV and influenza infections, respectively. Children aged 2-6 months had a higher percentage of very severe RSV infection compared to those older than 6 months (42.2% versus 31.4%, p-value Fisher's exact = 0.001). S.p. carriage was detected in 1073/2281 (47.0%) patients. Among S.p. carriage cases, 363/1073 (33.8%) had S.p. and RSV codetection, and 82/1073 (7.6%) had S.p. and influenza codetection. S.p. codetection with RSV/influenza was not associated with more severe LRTIs, compared to only RSV/influenza cases. CONCLUSION: In Mongolia, RSV is an important pathogen causing more severe LRTI in children under 6 months of age. Codetection of RSV or influenza virus and S.p. was not associated with increased severity.

Novel method for production and purification of untagged pneumococcal surface protein A from clade 1.

Streptococcus pneumoniae can cause diseases with high mortality and morbidity. The licensed vaccines are based on capsular polysaccharides and induce antibodies with low cross reactivity, leading to restricted coverage of serotypes. For surpassing this limitation, new pneumococcal vaccines are needed for induction of broader protection. One important candidate is the pneumococcal surface protein A (PspA), which can be classified in 6 clades and 3 families. We have reported an efficient process for production and purification of untagged recombinant PspA from clade 4 (PspA4Pro). We now aim to obtain a highly pure recombinant PspA from clade 1 (PspA1) to be included, together with PspA4Pro, in a vaccine formulation to broaden response against pneumococci. The vector pET28a-pspA1 was constructed and used to transform Escherichia coli BL21(DE3) strain. One clone with high production of PspA1 was selected and adapted to high-density fermentation (HDF) medium. After biomass production in 6 L HDF using a bioreactor, the purification was defined after testing 3 protocols. During the batch bioreactor cultivation, plasmid stability remained above 90% and acetate formation was not detected. The final protein purification process included treatment with a cationic detergent after lysis, anion exchange chromatography, cryoprecipitation, cation exchange chromatography, and multimodal chromatography. The final purification process showed PspA1 purity of 93% with low endotoxin content and an overall recovery above 20%. The novel established process can be easily scaled-up and proved to be efficient to obtain a highly pure untagged PspA1 for inclusion in vaccine formulations. KEY POINTS: • Purification strategy for recombinant PspA1 from Streptococcus pneumoniae • Downstream processing for untagged protein antigens, the case of PspA1 • Purification strategy for PspA variants relies on buried amino acids in their sequences.

In silico exploration of phenolics as modulators of penicillin binding protein (PBP) 2× of Streptococcus pneumoniae.

Infections caused by multidrug-resistant Streptococcus pneumoniae remain the leading cause of pneumonia-related deaths in children < 5 years globally, and mutations in penicillin-binding protein (PBP) 2 × have been identified as the major cause of resistance in the organism to beta-lactams. Thus, the development of new modulators with enhanced binding of PBP2x is highly encouraged. In this study, phenolics, due to their reported antibacterial activities, were screened against the active site of PBP2x using structure-based pharmacophore and molecular docking techniques, and the ability of the top-hit phenolics to inhibit the active and allosteric sites of PBP2x was refined through 120 ns molecular dynamic simulation. Except for gallocatechin gallate and lysidicichin, respectively, at the active and allosteric sites of PBP2x, the top-hit phenolics had higher negative binding free energy (ΔGbind) than amoxicillin [active site (- 19.23 kcal/mol), allosteric site (- 33.75 kcal/mol)]. Although silicristin had the best broad-spectrum effects at the active (- 38.41 kcal/mol) and allosteric (- 50.54 kcal/mol) sites of PBP2x, the high thermodynamic entropy (4.90 Å) of the resulting complex might suggest the need for its possible structural refinement for enhanced potency. Interestingly, silicristin had a predicted synthetic feasibility score of < 5 and quantum calculations using the DFT B3LYP/6-31G+ (dp) revealed that silicristin is less stable and more reactive than amoxicillin. These findings point to the possible benefits of the top-hit phenolics, and most especially silicristin, in the direct and synergistic treatment of infections caused by S. pneumoniae. Accordingly, silicristin is currently the subject of further confirmatory in vitro research.

Invasive pneumococcal diseases in Chinese children: a multicentre hospital-based active surveillance from 2019 to 2021.

This study aimed to provide data for the clinical features of invasive pneumococcal disease (IPD) and the molecular characteristics of Streptococcus pneumoniae isolates from paediatric patients in China. We conducted a multi-centre prospective study for IPD in 19 hospitals across China from January 2019 to December 2021. Data of demographic characteristics, risk factors for IPD, death, and disability was collected and analysed. Serotypes, antibiotic susceptibility, and multi-locus sequence typing (MLST) of pneumococcal isolates were also detected. A total of 478 IPD cases and 355 pneumococcal isolates were enrolled. Among the patients, 260 were male, and the median age was 35 months (interquartile range, 12-46 months). Septicaemia (37.7%), meningitis (32.4%), and pneumonia (27.8%) were common disease types, and 46 (9.6%) patients died from IPD. Thirty-four serotypes were detected, 19F (24.2%), 14 (17.7%), 23F (14.9%), 6B (10.4%) and 19A (9.6%) were common serotypes. Pneumococcal isolates were highly resistant to macrolides (98.3%), tetracycline (94.1%), and trimethoprim/sulfamethoxazole (70.7%). Non-sensitive rates of penicillin were 6.2% and 83.3% in non-meningitis and meningitis isolates. 19F-ST271, 19A-ST320 and 14-ST876 showed high resistance to antibiotics. This multi-centre study reports the clinical features of IPD and demonstrates serotype distribution and antibiotic resistance of pneumococcal isolates in Chinese children. There exists the potential to reduce IPD by improved uptake of pneumococcal vaccination, and continued surveillance is warranted.

Epidemiología molecular de la colonización nasofaríngea neumocócica en niños de Sevilla, tras la implementación del programa de vacunación con VNC13 en Andalucía (España) / Molecular epidemiology of pneumococcal carriage in children from Seville, following implementation of the PCV13 immunization program in Andalusia, Spain

Introducción: El programa de vacunación universal con la vacuna antineumocócica conjugada 13-valente (VNC13) se implantó en Andalucía en diciembre de 2016. Métodos: Estudio transversal de colonización nasofaríngea por Streptococcus pneumoniae. Se seleccionó a 397 niños sanos en centros de atención primaria de Sevilla durante los periodos 1/4/2018-28/2/2020 y 1/11/2021-28/2/2022 (periodo VNC13). Se utilizó una colección histórica de un estudio de colonización desarrollado en niños sanos y con infección respiratoria superior entre el 1/01/2006 y el 30/06/2008 (periodo VNC7) para comparar las distribuciones de serotipos/genotipos y las tasas de resistencias antibióticas. Resultados: Un total de 76 (19%) niños estaban colonizados con S. pneumoniae en el periodo VNC13 y se dispuso de 154 aislamientos del periodo VNC7. La colonización por serotipos incluidos en VNC13 disminuyó significativamente entre los periodos VNC13 y VNC7 (11 vs. 38%; p=0,0001); los serotipos 19F (8%), 3 (1%) y 6B (1%) fueron los únicos serotipos vacunales circulantes. Los serotipos 15B/C y 11A fueron los serotipos no VNC13 más prevalentes durante el periodo VNC13 (14% y 11%, respectivamente); este último se incrementó de forma significativa entre periodos de tiempo (p=0,04). El serotipo 11A solo se asoció en el periodo VNC13 con variantes resistentes a la ampicilina del clon Spain9V-ST156 (ST6521 y genéticamente relacionado ST14698), no detectados en el periodo anterior. Conclusiones: Hubo una circulación muy residual de los serotipos vacunales durante el periodo VNC13, con excepción del serotipo19F. El serotipo 11A se incrementó de forma significativa entre los periodos VNC13 y VNC7 por expansión clonal del genotipo resistente a la ampicilina ST6521.(AU)

Background: The 13-valent pneumococcal conjugate vaccine (PCV13) universal vaccination program was introduced in December 2016 in Andalusia. Methods: A cross-sectional study was conducted on the molecular epidemiology of pneumococcal nasopharyngeal colonization. A total of 397 healthy children were recruited from primary healthcare centres in Seville for the periods 1/4/2018 to 28/2/2020 and 1/11/2021 to 28/2/2022 (PCV13 period). Data from a previous carriage study conducted among healthy and sick children from 1/01/2006 to 30/06/2008 (PCV7 period) were used for comparison of serotype/genotype distributions and antibiotic resistance rates. Results: Overall, 76 (19%) children were colonized with S. pneumoniae during the PCV13 period and there were information available from 154 isolates collected during the PCV7 period. Colonization with PCV13 serotypes declined significantly in the PCV13 period compared with historical controls (11 vs. 38%, P=0.0001), being serotypes 19F (8%), 3 (1%) and 6B (1%) the only circulating vaccine types. Serotypes 15B/C and 11A were the most frequently identified non-PCV13 serotypes during the PCV13 period (14% and 11%, respectively); the later one increased significantly between time periods (P=0.04). Serotype 11A was exclusively associated in the PCV13 period with ampicillin-resistant variants of the Spain9V-ST156 clone (ST6521 and genetically related ST14698), not detected in the preceding period. Conclusions: There was a residual circulation of vaccine types following PCV13 introduction, apart from serotype 19F. Serotype 11A increased between PCV13 and PCV7 periods due to emergence and clonal expansion of ampicillin-resistant genotype ST6521.(AU)

Progresión temporal de la distribución de los serotipos de Streptococcus pneumoniae productores de enfermedad neumocócica invasiva en Galicia (España) y su relación con la resistencia a antibióticos (periodo 2011-2021) / Temporal progression of the distribution of Streptococcus pneumoniae serotypes causing invasive pneumococcal disease in Galicia (Spain) and its relationship with resistance to antibiotics (period 2011-2021)

Introducción: Streptococcus pneumoniae causa enfermedades graves en la población susceptible. La vacuna neumocócica conjugada (PCV) 13-valente (PCV13) se incluyó en el calendario infantil en 2011. Este estudio analiza la evolución de los serotipos de neumococo y de sus resistencias tras la PCV13. Métodos: Se incluyeron los neumococos serotipados en Galicia en 2011-2021. Se estudió la sensibilidad antibiótica siguiendo criterios EUCAST. Se analizaron los datos en 3 subperíodos: inicial (2011-2013), medio (2014-2017) y final (2018-2021). Se calcularon las prevalencias de los serotipos y el porcentaje de resistencia a los antibióticos más representativos. Resultados: Se incluyeron 2.869 aislados. Inicialmente el 42,7% presentaba tipos capsulares incluidos en la PCV13, frente al 15,4% al final. Los incluidos en la PCV20 y no en la PCV13 y PCV15 fueron el 12,5% inicialmente y el 41,3% al final. El 26,4% de los serotipos a lo largo del estudio no estaban incluidos en ninguna vacuna. La prevalencia del serotipo 8 se multiplicó casi por 8 y la del 12F se triplicó. El serotipo 19A fue el más resistente inicialmente. La resistencia de los serotipos 11A y 15A aumentó a lo largo del estudio. Conclusiones: La introducción de la PCV13 en la población infantil determinó un cambio en los serotipos de neumococo hacia los incluidos en la PCV20 y los no incluidos en ninguna vacuna. El serotipo 19A inicialmente fue el más resistente, y el 15A, no incluido en ninguna vacuna, merece un especial seguimiento. El serotipo 8, que fue el que más se incrementó, no mostró resistencia destacable.(AU)

Introduction: Streptococcus pneumoniae causes serious diseases in the susceptible population. The 13-valent pneumococci conjugate vaccine (PCV13) was included in the children's calendar in 2011. The objective of the study was to analyze the evolution of pneumococcal serotypes and their resistance after PCV13. Methods: This study included the pneumococci serotyped in Galicia in 2011-2021. Antibiotic susceptibility was analyzed following EUCAST criteria. The data was analyzed in 3 sub-periods: initial (2011-2013), middle (2014-2017) and final (2018-2021). The prevalence of serotypes and their percentage of resistance to the most representative antibiotics were calculated. Results: A total of 2.869 isolates were included. Initially, 42.7% isolates presented capsular types included in PCV13, compared to 15.4% at the end. Those included in PCV20 and not in PCV13 and PCV15 were 12.5% at baseline and 41.3% at the end; 26.4% of the isolates throughout the study had serotypes not included in any vaccine. The prevalence of serotype 8 multiplied almost by 8 and that of 12F tripled. The 19A serotype was initially the most resistant, while the resistance of serotypes 11A and 15A increased throughout the study. Conclusions: The introduction of PCV13 in the pediatric population determined a change in pneumococcal serotypes towards those included in PCV20 and those not included in any vaccine. Serotype 19A was initially the most resistant and the 15A, not included in any vaccine, deserves special follow-up. Serotype 8, which increased the most, did not show remarkable resistance.(AU)

High-resolution genomics identifies pneumococcal diversity and persistence of vaccine types in children with community-acquired pneumonia in the UK and Ireland.

BACKGROUND: Streptococcus pneumoniae is a global cause of community-acquired pneumonia (CAP) and invasive disease in children. The CAP-IT trial (grant No. 13/88/11; https://www.capitstudy.org.uk/ ) collected nasopharyngeal swabs from children discharged from hospitals with clinically diagnosed CAP, and found no differences in pneumococci susceptibility between higher and lower antibiotic doses and shorter and longer durations of oral amoxicillin treatment. Here, we studied in-depth the genomic epidemiology of pneumococcal (vaccine) serotypes and their antibiotic resistance profiles. METHODS: Three-hundred and ninety pneumococci cultured from 1132 nasopharyngeal swabs from 718 children were whole-genome sequenced (Illumina) and tested for susceptibility to penicillin and amoxicillin. Genome heterogeneity analysis was performed using long-read sequenced isolates (PacBio, n = 10) and publicly available sequences. RESULTS: Among 390 unique pneumococcal isolates, serotypes 15B/C, 11 A, 15 A and 23B1 were most prevalent (n = 145, 37.2%). PCV13 serotypes 3, 19A, and 19F were also identified (n = 25, 6.4%). STs associated with 19A and 19F demonstrated high genome variability, in contrast to serotype 3 (n = 13, 3.3%) that remained highly stable over a 20-year period. Non-susceptibility to penicillin (n = 61, 15.6%) and amoxicillin (n = 10, 2.6%) was low among the pneumococci analysed here and was independent of treatment dosage and duration. However, all 23B1 isolates (n = 27, 6.9%) were penicillin non-susceptible. This serotype was also identified in ST177, which is historically associated with the PCV13 serotype 19F and penicillin susceptibility, indicating a potential capsule-switch event. CONCLUSIONS: Our data suggest that amoxicillin use does not drive pneumococcal serotype prevalence among children in the UK, and prompts consideration of PCVs with additional serotype coverage that are likely to further decrease CAP in this target population. Genotype 23B1 represents the convergence of a non-vaccine genotype with penicillin non-susceptibility and might provide a persistence strategy for ST types historically associated with vaccine serotypes. This highlights the need for continued genomic surveillance.