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.

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.

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.

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.

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.

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.

Microbial aetiology of community-acquired pneumonia in hospitalised adults: A prospective study utilising comprehensive molecular testing.

OBJECTIVES: This study aimed to describe the microbial aetiology of community-acquired pneumonia (CAP) in adults admitted to a tertiary care hospital and assess the impact of syndromic polymerase chain reaction (PCR) panels on pathogen detection. METHODS: Conducted at Haukeland University Hospital, Norway, from September 2020 to April 2023, this prospective study enrolled adults with suspected CAP. We analysed lower respiratory tract samples using both standard-of-care tests and the BIOFIRE® FILMARRAY® Pneumonia Plus Panel (FAP plus). The added value of FAP Plus in enhancing the detection of clinically relevant pathogens, alongside standard-of-care diagnostics, was assessed. RESULTS: Of the 3238 patients screened, 640 met the inclusion criteria, with 384 confirmed to have CAP at discharge. In these patients, pathogens with proven or probable clinical significance were identified in 312 (81.3%) patients. Haemophilus influenzae was the most prevalent pathogen, found in 118 patients (30.7%), followed by SARS-CoV-2 in 74 (19.3%), and Streptococcus pneumoniae in 64 (16.7%). Respiratory viruses were detected in 186 (48.4%) patients. The use of FAP plus improved the pathogen detection rate from 62.8% with standard-of-care methods to 81.3%. CONCLUSIONS: Pathogens were identified in 81% of CAP patients, with Haemophilus influenzae and respiratory viruses being the most frequently detected pathogens. The addition of the FAP plus panel, markedly improved pathogen detection rates compared to standard-of-care diagnostics alone.

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.

Metagenomic next-generation sequencing of nasopharyngeal microbiota in COVID-19 patients with different disease severities.

Throughout the COVID-19 pandemic, extensive research has been conducted on SARS-COV-2 to elucidate its genome, prognosis, and possible treatments. However, few looked at the microbial markers that could be explored in infected patients and that could predict possible disease severity. The aim of this study is to compare the nasopharyngeal microbiota of healthy subjects, moderate, under medication, and recovered SARS-COV-2 patients. In 2020, 38 nasopharyngeal swabs were collected from 6 healthy subjects, 14 moderates, 10 under medication and 8 recovered SARS-COV-2 patients at the Prince Mohammed Bin Abdulaziz Hospital Riyadh. Metatranscriptomic sequencing was performed using Minion Oxford nanopore sequencing. No significant difference in alpha as well as beta diversity was observed among all four categories. Nevertheless, we have found that Streptococcus spp including Streptococcus pneumoniae and Streptococcus thermophilus were among the top 15 most abundant species detected in COVID-19 patients but not in healthy subjects. The genus Staphylococcus was found to be associated with COVID-19 patients compared to healthy subjects. Furthermore, the abundance of Leptotrichia was significantly higher in healthy subjects compared to recovered patients. Corynebacterium on the other hand, was associated with under-medication patients. Taken together, our study revealed no differences in the overall microbial composition between healthy subjects and COVID-19 patients. Significant differences were seen only at specific taxonomic level. Future studies should explore the nasopharyngeal microbiota between controls and COVID-19 patients while controlling for confounders including age, gender, and comorbidities; since these latter could affect the results and accordingly the interpretation.IMPORTANCEIn this work, no significant difference in the microbial diversity was seen between healthy subjects and COVID-19 patients. Changes in specific taxa including Leptotrichia, Staphylococcus, and Corynebacterium were only observed. Leptotrichia was significantly higher in healthy subjects, whereas Staphylococcus and Corynebacterium were mostly associated with COVID-19, and specifically with under-medication SARS-COV-2 patients, respectively. Although the COVID-19 pandemic has ended, the SARS-COV-2 virus is continuously evolving and the emergence of new variants causing more severe disease should be always kept in mind. Microbial markers in SARS-COV-2 infected patients can be useful in the early suspicion of the disease, predicting clinical outcomes, framing hospital and intensive care unit admission as well as, risk stratification. Data on which microbial marker to tackle is still controversial and more work is needed, hence the importance of this study.

Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min-1, and 63,218 min-1 mM-1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci.

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)

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.

Identification and genetic engineering of pneumococcal capsule-like polysaccharides in commensal oral streptococci.

Capsular polysaccharides (CPS) in Streptococcus pneumoniae are pivotal for bacterial virulence and present extensive diversity. While oral streptococci show pronounced antigenicity toward pneumococcal capsule-specific sera, insights into evolution of capsule diversity remain limited. This study reports a pneumococcal CPS-like genetic locus in Streptococcus parasanguinis, a predominant oral Streptococcus. The discovered locus comprises 15 genes, mirroring high similarity to those from the Wzy-dependent CPS locus of S. pneumoniae. Notably, S. parasanguinis elicited a reaction with pneumococcal 19B antiserum. Through nuclear magnetic resonance analysis, we ascertained that its CPS structure matches the chemical composition of the pneumococcal 19B capsule. By introducing the glucosyltransferase gene cps19cS from a pneumococcal serotype 19C, we successfully transformed S. parasanguinis antigenicity from 19B to 19C. Furthermore, substituting serotype-specific genes, cpsI and cpsJ, with their counterparts from pneumococcal serotype 19A and 19F enabled S. parasanguinis to generate 19A- and 19F-specific CPS, respectively. These findings underscore that S. parasanguinis harbors a versatile 19B-like CPS adaptable to other serotypes. Remarkably, after deleting the locus's initial gene, cpsE, responsible for sugar transfer, we noted halted CPS production, elongated bacterial chains, and diminished biofilm formation. A similar phenotype emerged with the removal of the distinct gene cpsZ, which encodes a putative autolysin. These data highlight the importance of S. parasanguinis CPS for biofilm formation and propose a potential shared ancestry of its CPS locus with S. pneumoniae. IMPORTANCE: Diverse capsules from Streptococcus pneumoniae are vital for bacterial virulence and pathogenesis. Oral streptococci show strong responses to a wide range of pneumococcal capsule-specific sera. Yet, the evolution of this capsule diversity in relation to microbe-host interactions remains underexplored. Our research delves into the connection between commensal oral streptococcal and pneumococcal capsules, highlighting the potential for gene transfer and evolution of various capsule types. Understanding the genetic and evolutionary factors that drive capsule diversity in S. pneumoniae and its related oral species is essential for the development of effective pneumococcal vaccines. The present findings provide fresh perspectives on the cross-reactivity between commensal streptococci and S. pneumoniae, its influence on bacteria-host interactions, and the development of new strategies to manage and prevent pneumococcal illnesses by targeting and modulating commensal streptococci.

RNA cis-regulators are important for Streptococcus pneumoniae in vivo success.

Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and a rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogen Streptococcus pneumoniae TIGR4. By evaluating native locus mutants of each regulator that result in constitutively active or repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with either constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display exceptional sensitivity to mis-regulation or constitutive gene repression in both planktonic culture and in vivo environments. Thus, despite lack of obvious phenotypes associated with constitutive gene expression in vitro, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is substantial in vivo, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms.

Serotype distribution and antimicrobial resistance of pediatric Streptococcus pneumoniae isolated from inpatients and outpatients at Beijing Children's Hospital.

BACKGROUND: Understanding the epidemiology of Streptococcus pneumoniae (S. pneumoniae) isolates is important for pneumonia treatment and prevention. This research aimed to explore the epidemiological characteristics of S. pneumoniae isolated from pediatric inpatients and outpatients during the same period. METHODS: S. pneumoniae were isolated from unsterile samples of inpatients and outpatients younger than five years old between March 2013 and February 2014. The serotypes were determined using diagnostic pneumococcal antisera. The resistance of each strain to 13 antibiotics was tested using either the E-test or the disc diffusion method. The Sequence Types (STs) were analyzed via Multilocus Sequence Typing (MLST). RESULTS: The dominant serotypes obtained from inpatients were 19F (32.9 %), 19A (20.7 %), 23F (10.7 %), 6A (10.0 %), and 14 (8.6 %), while those from outpatients were 19F (13.6 %), 23F (12.9 %), 6A (10.0 %), 6B (10.0 %), and 19A (7.9 %). The coverage rates of 13-valent Pneumococcal Conjugate Vaccine (PCV) formulations were high in both groups. The nonsusceptibility to penicillin, cefuroxime, imipenem, erythromycin, and trimethoprim-sulfamethoxazole among the inpatient isolates was 7.1 %, 92.8 %, 65.7 %, 100 %, and 85.0 %, respectively, while that among the outpatient isolates was 0.7 %, 50.0 %, 38.6 %, 96.4 %, and 65.7 %, respectively. There were 45 and 81 STs detected from the pneumococci isolated from inpatients and outpatients, respectively. CC271 was common among both inpatients and outpatients (43.6 % and 14.3 %). CONCLUSIONS: Pneumococcal vaccine-related serotypes are prevalent among both inpatients and outpatients, especially among inpatients, who exhibit more severe antibiotic resistance. Therefore, universal immunization with PCV13 would decrease the hospitalization rate due to S. pneumoniae and the antibiotic resistance rate of S. pneumoniae.

Prevalence of linezolid resistance in Streptococcus pneumoniae isolates: a systematic review and meta-analysis.

Aim: This study aimed to understand the current level of linezolid (LNZ) resistance in Streptococcus pneumoniae isolates reported over the past 10 years. Material & methods: An electronic search was conducted for the following keywords: ((Streptococcus pneumoniae [title/abstract]) OR (Pneumococcus [title/abstract]) OR (Pneumococci [title/abstract]) AND (linezolid [title/abstract]) OR (Zyvox [title/abstract])) OR (Zyvoxid [title/abstract])). Result: Out of all the studies, 80 had a cross-sectional design, while 11 followed a cohort approach. The prevalence of LNZ resistance among S. pneumoniae isolates ranged from 0% to 4.86%. Discussion: Urgent, high-powered, randomized, controlled trials with participants from endemic regions are needed to gain a comprehensive understanding of the impact on and significance of LNZ treatment to patients.

Utility of quantitative loop mediated isothermal amplification (qLAMP) assay for the diagnosis of invasive pneumococcal disease (IPD).

PURPOSE: Quantitative LAMP (qLAMP) assay is one of the recent and emerging diagnostic tests for infectious diseases. Only a few studies exist comparing this assay with quantitative real-time PCR (qPCR) for the diagnosis of invasive pneumococcal disease (IPD). AIM: To compare the diagnostic performance of qLAMP assay with qPCR targeting autolysin gene for the diagnosis of invasive pneumococcal disease. METHODS: Ninety six blood samples and 73 CSF samples from patients clinically suspected with community acquired pneumonia and acute meningitis were tested by qPCR and qLAMP assays using previously published primers and protocols. The qPCR was considered as the gold standard test and the diagnostic performance was assessed by calculating sensitivity, specificity, positive and negative predictive values, and kappa coefficient for the level of agreement between the tests. Chi-squared/Fisher exact test was used to compare categorical variables (positive/negative). RESULTS: Thirty two blood samples and 22 CSF samples were positive by qPCR while 24 and 20 samples were positive by qLAMP assay respectively. The sensitivity of qLAMP assay was only 86.4% and 75% when tested on CSF and blood samples respectively. However, the qLAMP assay was in substantial to almost perfect agreement when compared with qPCR. The results were statistically significant in both sample types (P < 0.001). CONCLUSIONS: The performance of qLAMP assay can vary based on the specimen type. It has very high specificity and had substantial to almost perfect agreement, and thus may be an alternative to qPCR for the diagnosis of IPD.

Multiplex PCR for respiratory bacteria in acute care.

The purpose of the study was to evaluate the clinical utility of multiplex PCR for detecting bacterial respiratory pathogens in nasopharyngeal samples. Acutely ill adults in the emergency department with respiratory infection symptoms, fever, chest pain or poor general condition were enrolled for this cohort study. Samples were stored at -70 °C until being analysed with multiplex PCR for seven respiratory bacteria. Of the 912 patients enrolled, those with positive bacterial samples (n = 130, 14%) were significantly younger than those with a negative finding (55.5 years vs 62.2 years, p < 0.001), and their mean C-reactive protein (CRP) concentration was higher (110 mg/L vs 59 mg/L, p < 0.0001). Patients with a positive respiratory bacterial finding had a higher probability of pneumonia (35% vs 13%, p < 0.001) and a higher likelihood of receiving a prescription for antibiotics than those with a negative finding (79% vs 59%, p < 0.0001). Positive detection of Streptococcus pneumoniae was associated with a 4.5-fold risk of pneumonia in a multivariate model and detection of an atypical respiratory pathogen with a 9-fold risk. Bacterial PCR performed on nasopharyngeal samples appeared to offer a valuable addition to the diagnostics of infections in adults in acute care.

The Moraxella catarrhalis AdhC-FghA system is important for formaldehyde detoxification and protection against pulmonary clearance.

Multidrug-resistant clinical isolates of Moraxella catarrhalis have emerged, increasing the demand for the identification of new treatment and prevention strategies. A thorough understanding of how M. catarrhalis can establish an infection and respond to different stressors encountered in the host is crucial for new drug-target identification. Formaldehyde is a highly cytotoxic compound that can be produced endogenously as a by-product of metabolism and exogenously from environmental sources. Pathways responsible for formaldehyde detoxification are thus essential and are found in all domains of life. The current work investigated the role of the system consisting of the S-hydroxymethyl alcohol dehydrogenase (AdhC), a Zn-dependent class III alcohol dehydrogenase, and the S-formyl glutathione hydrolase (FghA) in the formaldehyde detoxification process in M. catarrhalis. Bioinformatics showed that the components of the system are conserved across the species and are highly similar to those of Streptococcus pneumoniae, which share the same biological niche. Isogenic mutants were constructed to study the function of the system in M. catarrhalis. A single fghA knockout mutant did not confer sensitivity to formaldehyde, while the adhC-fghA double mutant is formaldehyde-sensitive. In addition, both mutants were significantly cleared in a murine pulmonary model of infection as compared to the wild type, demonstrating the system's importance for this pathogen's virulence. The respective phenotypes were reversed upon the genetic complementation of the mutants. To date, this is the first study investigating the role of the AdhC-FghA system in formaldehyde detoxification and pathogenesis of M. catarrhalis.