Geographical migration and fitness dynamics of Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading cause of pneumonia and meningitis worldwide. Many different serotypes co-circulate endemically in any one location1,2. The extent and mechanisms of spread and vaccine-driven changes in fitness and antimicrobial resistance remain largely unquantified. Here using geolocated genome sequences from South Africa (n = 6,910, collected from 2000 to 2014), we developed models to reconstruct spread, pairing detailed human mobility data and genomic data. Separately, we estimated the population-level changes in fitness of strains that are included (vaccine type (VT)) and not included (non-vaccine type (NVT)) in pneumococcal conjugate vaccines, first implemented in South Africa in 2009. Differences in strain fitness between those that are and are not resistant to penicillin were also evaluated. We found that pneumococci only become homogenously mixed across South Africa after 50 years of transmission, with the slow spread driven by the focal nature of human mobility. Furthermore, in the years following vaccine implementation, the relative fitness of NVT compared with VT strains increased (relative risk of 1.68; 95% confidence interval of 1.59-1.77), with an increasing proportion of these NVT strains becoming resistant to penicillin. Our findings point to highly entrenched, slow transmission and indicate that initial vaccine-linked decreases in antimicrobial resistance may be transient.

Novel pneumococcal capsule type 33E results from the inactivation of glycosyltransferase WciE in vaccine type 33F.

The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anticapsule antibodies; however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [→5)-ß-D-Galf2Ac-(1→3)-ß-D-Galp-(1→3)-α-D-Galp-(1→3)-ß-D-Galf-(1→3)-ß-D-Glcp-(1→], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS did not significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F.

Discovery and Characterization of Pneumococcal Serogroup 36 Capsule Subtypes, Serotypes 36A and 36B.

Streptococcus pneumoniae can produce a wide breadth of antigenically diverse capsule types, a fact that poses a looming threat to the success of vaccines that target pneumococcal polysaccharide (PS) capsule. Yet, many pneumococcal capsule types remain undiscovered and/or uncharacterized. Prior sequence analysis of pneumococcal capsule synthesis (cps) loci suggested the existence of capsule subtypes among isolates identified as "serotype 36" according to conventional capsule typing methods. We discovered these subtypes represent two antigenically similar but distinguishable pneumococcal capsule serotypes, 36A and 36B. Biochemical analysis of their capsule PS structure reveals that both have the shared repeat unit backbone [→5)-α-d-Galf-(1→1)-d-Rib-ol-(5→P→6)-ß-d-ManpNAc-(1→4)-ß-d-Glcp-(1→] with two branching structures. Both serotypes have a ß-d-Galp branch to Ribitol. Serotypes 36A and 36B differ by the presence of a α-d-Glcp-(1→3)-ß-d-ManpNAc or α-d-Galp-(1→3)-ß-d-ManpNAc branch, respectively. Comparison of the phylogenetically distant serogroup 9 and 36 cps loci, which all encode this distinguishing glycosidic bond, revealed that the incorporation of Glcp (in types 9N and 36A) versus Galp (in types 9A, 9V, 9L, and 36B) is associated with the identity of four amino acids in the cps-encoded glycosyltransferase WcjA. Identifying functional determinants of cps-encoded enzymes and their impact on capsule PS structure is key to improving the resolution and reliability of sequencing-based capsule typing methods and discovering novel capsule variants indistinguishable by conventional serotyping methods.

Comparison of next generation technologies and bioinformatics pipelines for capsular typing of Streptococcus pneumoniae.

Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. In silico serotyping from WGS has not yet been applied to long-read sequences produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F (n = 17; 14.3%), 14 (n = 10; 8.4%), and 15B/C (n = 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.

RCandy: an R package for visualizing homologous recombinations in bacterial genomes.

SUMMARY: Homologous recombination is an important evolutionary process in bacteria and other prokaryotes, which increases genomic sequence diversity and can facilitate adaptation. Several methods and tools have been developed to detect genomic regions recently affected by recombination. Exploration and visualization of such recombination events can reveal valuable biological insights, but it remains challenging. Here, we present RCandy, a platform-independent R package for rapid, simple and flexible visualization of recombination events in bacterial genomes. AVAILABILITY AND IMPLEMENTATION: RCandy is an R package freely available for use under the MIT license. It is platform-independent and has been tested on Windows, Linux and MacOSX. The source code comes together with a detailed vignette available on GitHub at https://github.com/ChrispinChaguza/RCandy. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Endocarditis Caused by Nontypeable Streptococcus pneumoniae.

The Streptococcus pneumoniae capsule is regarded as indispensable in bacteremia. We report an infant with a ventricular septal defect and infective endocarditis caused by nontypeable S. pneumoniae. In-depth investigation confirmed a deficient capsule yet favored pneumococcal fitness for causing infective endocarditis, rather than a host immune disorder, as the cause of infective endocarditis in this case.

Fast and flexible bacterial genomic epidemiology with PopPUNK.

The routine use of genomics for disease surveillance provides the opportunity for high-resolution bacterial epidemiology. Current whole-genome clustering and multilocus typing approaches do not fully exploit core and accessory genomic variation, and they cannot both automatically identify, and subsequently expand, clusters of significantly similar isolates in large data sets spanning entire species. Here, we describe PopPUNK (Population Partitioning Using Nucleotide K -mers), a software implementing scalable and expandable annotation- and alignment-free methods for population analysis and clustering. Variable-length k-mer comparisons are used to distinguish isolates' divergence in shared sequence and gene content, which we demonstrate to be accurate over multiple orders of magnitude using data from both simulations and genomic collections representing 10 taxonomically widespread species. Connections between closely related isolates of the same strain are robustly identified, despite interspecies variation in the pairwise distance distributions that reflects species' diverse evolutionary patterns. PopPUNK can process 103-104 genomes in a single batch, with minimal memory use and runtimes up to 200-fold faster than existing model-based methods. Clusters of strains remain consistent as new batches of genomes are added, which is achieved without needing to reanalyze all genomes de novo. This facilitates real-time surveillance with consistent cluster naming between studies and allows for outbreak detection using hundreds of genomes in minutes. Interactive visualization and online publication is streamlined through the automatic output of results to multiple platforms. PopPUNK has been designed as a flexible platform that addresses important issues with currently used whole-genome clustering and typing methods, and has potential uses across bacterial genetics and public health research.

A Nationwide Outbreak of Invasive Pneumococcal Disease in Israel Caused by Streptococcus pneumoniae Serotype 2.

BACKGROUND: Invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae serotype 2 (Sp2) is infrequent. Large-scale outbreaks were not been reported following pneumococcal conjugate vaccine (PCV) implementation. We describe a Sp2 IPD outbreak in Israel, in the PCV13 era, with focus on Sp2 population structure and evolutionary dynamics. METHODS: The data were derived from a population-based, nationwide active surveillance of IPD since 2009. PCV7/PCV13 vaccines were introduced in July 2009 and November 2010, respectively. Sp2 isolates were tested for antimicrobial susceptibility, multilocus sequence typing, and whole-genome sequencing (WGS) analysis. RESULTS: Overall, 170 Sp2 IPD cases were identified during 2009-2019; Sp2 increased in 2015 and caused 6% of IPD during 2015-2019, a 7-fold increase compared with 2009-2014. The outbreak was caused by a previously unreported molecular type (ST-13578), initially observed in Israel in 2014. This clone caused 88% of Sp2 during 2015-2019. ST-13578 is a single-locus variant of ST-1504, previously reported globally including in Israel. WGS analysis confirmed clonality among the ST-13578 population. Single-nucleotide polymorphism-dense regions support a hypothesis that the ST-13578 outbreak clone evolved from ST-1504 by recombination. All tested strains were penicillin-susceptible (minimum inhibitory concentration <0.06 µg/mL). The ST-13578 clone was identified almost exclusively (99%) in the Jewish population and was mainly distributed in 3 of 7 Israeli districts. The outbreak is still ongoing, although it began declining in 2017. CONCLUSIONS: To the best of our knowledge, this is the first widespread Sp2 outbreak since PCV13 introduction worldwide, caused by the emerging ST-13578 clone.

Nontoxic electron collimators.

PURPOSE: The goal of this work was to develop and test nontoxic electron collimation technologies for clinical use. METHODS: Two novel technologies were investigated: tungsten-silicone composite and 3D printed electron cutouts. Transmission, dose uniformity, and profiles were measured for the tungsten-silicone. Surface dose, relative dose output, and field size were measured for the 3D printed cutouts and compared with the standard cerrobend cutouts in current clinical use. Quality assurance tests including mass measurements, Megavoltage (MV) imaging, and drop testing were developed for the 3D printed cutouts as a guide to safe clinical implementation. RESULTS: Dose profiles of the flexible tungsten-silicone skin shields had an 80-20 penumbra values of 2-3 mm compared to 7-8 mm for cerrobend. In MV transmission image measurements of the tungsten-silicone, 80% of the pixels had a transmission value within 2% of the mean. An ∼90% reduction in electron intensity was measured for 6 MeV and a 6.4 mm thickness of tungsten-silicone and 12.7 mm thickness for 16 MeV. The maximum difference in 3D printed cutout versus cerrobend output, surface dose, and full width at half-maximum (FWHM) was 1.7%, 1.2%, and 1.5%, respectively, for the 10 cm × 10 cm cutouts. CONCLUSIONS: Both flexible tungsten-silicone and 3D printed cutouts were found to be feasible for clinical use. The flexible tungsten-silicone was of adequate density, flexibility, and uniformity to serve as skin shields for electron therapy. The 3D printed cutouts were dosimetrically equivalent to standard cerrobend cutouts and were robust enough for handling in the clinical environment.

A mosaic tetracycline resistance gene tet(S/M) detected in an MDR pneumococcal CC230 lineage that underwent capsular switching in South Africa.

OBJECTIVES: We reported tet(S/M) in Streptococcus pneumoniae and investigated its temporal spread in relation to nationwide clinical interventions. METHODS: We whole-genome sequenced 12 254 pneumococcal isolates from 29 countries on an Illumina HiSeq sequencer. Serotype, multilocus ST and antibiotic resistance were inferred from genomes. An SNP tree was built using Gubbins. Temporal spread was reconstructed using a birth-death model. RESULTS: We identified tet(S/M) in 131 pneumococcal isolates and none carried other known tet genes. Tetracycline susceptibility testing results were available for 121 tet(S/M)-positive isolates and all were resistant. A majority (74%) of tet(S/M)-positive isolates were from South Africa and caused invasive diseases among young children (59% HIV positive, where HIV status was available). All but two tet(S/M)-positive isolates belonged to clonal complex (CC) 230. A global phylogeny of CC230 (n=389) revealed that tet(S/M)-positive isolates formed a sublineage predicted to exhibit resistance to penicillin, co-trimoxazole, erythromycin and tetracycline. The birth-death model detected an unrecognized outbreak of this sublineage in South Africa between 2000 and 2004 with expected secondary infections (effective reproductive number, R) of ∼2.5. R declined to ∼1.0 in 2005 and <1.0 in 2012. The declining epidemic could be related to improved access to ART in 2004 and introduction of pneumococcal conjugate vaccine (PCV) in 2009. Capsular switching from vaccine serotype 14 to non-vaccine serotype 23A was observed within the sublineage. CONCLUSIONS: The prevalence of tet(S/M) in pneumococci was low and its dissemination was due to an unrecognized outbreak of CC230 in South Africa. Capsular switching in this MDR sublineage highlighted its potential to continue to cause disease in the post-PCV13 era.

Global Distribution of Invasive Serotype 35D Streptococcus pneumoniae Isolates following Introduction of 13-Valent Pneumococcal Conjugate Vaccine.

A newly recognized pneumococcal serotype, 35D, which differs from the 35B polysaccharide in structure and serology by not binding to factor serum 35a, was recently reported. The genetic basis for this distinctive serology is due to the presence of an inactivating mutation in wciG, which encodes an O-acetyltransferase responsible for O-acetylation of a galactofuranose. Here, we assessed the genomic data of a worldwide pneumococcal collection to identify serotype 35D isolates and understand their geographical distribution, genetic background, and invasiveness potential. Of 21,980 pneumococcal isolates, 444 were originally typed as serotype 35B by PneumoCaT. Analysis of the wciG gene revealed 23 isolates from carriage (n = 4) and disease (n = 19) with partial or complete loss-of-function mutations, including mutations resulting in premature stop codons (n = 22) and an in-frame mutation (n = 1). These were selected for further analysis. The putative 35D isolates were geographically widespread, and 65.2% (15/23) of them was recovered after the introduction of pneumococcal conjugate vaccine 13 (PCV13). Compared with serotype 35B isolates, putative serotype 35D isolates have higher invasive disease potentials based on odds ratios (OR) (11.58; 95% confidence interval[CI], 1.42 to 94.19 versus 0.61; 95% CI, 0.40 to 0.92) and a higher prevalence of macrolide resistance mediated by mefA (26.1% versus 7.6%; P = 0.009). Using the Quellung reaction, 50% (10/20) of viable isolates were identified as serotype 35D, 25% (5/20) as serotype 35B, and 25% (5/20) as a mixture of 35B/35D. The discrepancy between phenotype and genotype requires further investigation. These findings illustrated a global distribution of an invasive serotype, 35D, among young children post-PCV13 introduction and underlined the invasive potential conferred by the loss of O-acetylation in the pneumococcal capsule.

Photodynamic effect of meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins on HaCaT keratinocytes.

Sixteen porphyrins, including neutral, anionic and cationic meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins were herein evaluated in terms of their photosensitizing properties against HaCaT keratinocytes. After an initial screening, the cationic porphyrins were studied in more details, by both determining their log POW and performing PDT assays in lower porphyrin concentrations. Porphyrins presenting two or more adjacent positively charged groups, directly linked to the macrocycle meso positions, appeared to be the most effective photosensitizers. The present study also included the dicationic 5,10-diphenyl-15,20-di(1-methylpyridinium-4-yl)porphyrin (14b), which has previously shown promising results on a psoriasis-like in vivo model. Overall results indicated that the beneficial effect related to porphyrins on psoriasis can be related to the decreasing of keratinocyte viability. Furthermore, some of the cationic porphyrins studied appeared as candidates to be utilized as photosensitizers for psoriasis treatment.

Comparison of gene-by-gene and genome-wide short nucleotide sequence-based approaches to define the global population structure of Streptococcus pneumoniae.

Defining the population structure of a pathogen is a key part of epidemiology, as genomically related isolates are likely to share key clinical features such as antimicrobial resistance profiles and invasiveness. Multiple different methods are currently used to cluster together closely related genomes, potentially leading to inconsistency between studies. Here, we use a global dataset of 26 306 Streptococcus pneumoniae genomes to compare four clustering methods: gene-by-gene seven-locus MLST, core genome MLST (cgMLST)-based hierarchical clustering (HierCC) assignments, life identification number (LIN) barcoding and k-mer-based PopPUNK clustering (known as GPSCs in this species). We compare the clustering results with phylogenetic and pan-genome analyses to assess their relationship with genome diversity and evolution, as we would expect a good clustering method to form a single monophyletic cluster that has high within-cluster similarity of genomic content. We show that the four methods are generally able to accurately reflect the population structure based on these metrics and that the methods were broadly consistent with each other. We investigated further to study the discrepancies in clusters. The greatest concordance was seen between LIN barcoding and HierCC (adjusted mutual information score=0.950), which was expected given that both methods utilize cgMLST, but have different methods for defining an individual cluster and different core genome schema. However, the existence of differences between the two methods shows that the selection of a core genome schema can introduce inconsistencies between studies. GPSC and HierCC assignments were also highly concordant (AMI=0.946), showing that k-mer-based methods which use the whole genome and do not require the careful selection of a core genome schema are just as effective at representing the population structure. Additionally, where there were differences in clustering between these methods, this could be explained by differences in the accessory genome that were not identified in cgMLST. We conclude that for S. pneumoniae, standardized and stable nomenclature is important as the number of genomes available expands. Furthermore, the research community should transition away from seven-locus MLST, whilst cgMLST, GPSC and LIN assignments should be used more widely. However, to allow for easy comparison between studies and to make previous literature relevant, the reporting of multiple clustering names should be standardized within the research.

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.

Non-pharmaceutical interventions for COVID-19 transiently reduced pneumococcal and Haemophilus influenzae carriage in a cross-sectional pediatric cohort in Southampton, UK.

The Southampton pneumococcal carriage study of children under 5 years old continued during the coronavirus disease 2019 (COVID-19) pandemic. Here, we present data from October 2018 to March 2023 describing prevalence of pneumococci and other pathobionts during the winter seasons before, during, and after the introduction of non-pharmaceutical interventions (NPIs) to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Nasopharyngeal swabs were collected from children attending outpatient clinics at a secondary care hospital and community healthcare sites. Pre-NPIs, in 2019/2020, the carriage prevalence of pneumococci at the hospital site was 32% (n = 161 positive/499 participants). During NPIs, this fell to 19% (n = 12/64), although based on fewer participants compared to previous years due to COVID-19 restrictions on health-care attendance. In 2021/2022, after NPIs had eased, prevalence rebounded to 33% (n = 15/46) [compared to NPIs period, χ2 (1, N = 110) =2.78, P = 0.09]. Carriage prevalence at community healthcare sites fell significantly from 27% (n = 127/470) in 2019/2020 to 19% during the NPI period (n = 44/228) in 2020/2021 [χ2 (1, N = 698) =4.95, P = 0.026]. No rebound was observed in 2021/2022 [19% (n = 56/288)]. However, in a multivariate logistic regression model, neither site had a significantly lower carriage prevalence during the NPI period compared to the post NPI period. A reduction in serotype diversity was observed in 2020/2021. Carriage of Haemophilus influenzae was particularly affected by NPIs with a significant reduction observed. In conclusion, among children under 5 years of age, transient, modest, and statistically non-significant alterations in carriage of both Streptococcus pneumoniae and H. influenzae were associated with SARS-CoV-2 NPIs.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) continues to be a major contributor to global morbidity and mortality. Using our long-running pediatric study, we examined changes in pneumococcal carriage prevalence in nearly 3,000 children under the age of 5 years between the winters of 2018/2019 and 2022/2023. This period coincided with the severe acute respiratory syndrome coronavirus 2 pandemic and, in particular, the implementation of national strategies to limit disease transmission in the UK. We observed a transient reduction of both Streptococcus pneumoniae and Haemophilus influenzae in these populations during this period of non-pharmaceutical interventions. This aligned with the reduction in invasive pneumococcal disease seen in the UK and is therefore a likely contributor to this phenomenon.

Essential role of proline synthesis and the one-carbon metabolism pathways for systemic virulence of Streptococcus pneumoniae.

Virulence screens have indicated potential roles during Streptococcus pneumoniae infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect S. pneumoniae virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δfhs and ΔproABC mutants. S. pneumoniae capsular serotype 6B BHN418 Δfhs and ΔproABC mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 ΔproABC strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of proABC was strain specific, as the D39 ΔproABC strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δfhs and ΔproABC strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δfhs had an impaired stringent response, and when cultured in sera, BHN418 Δfhs and ΔproABC were under increased oxidative stress and had altered lipid profiles. Loss of proABC also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the S. pneumoniae metabolic functions affected by S. pneumoniae one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae. We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential.

Streptococcus pneumoniae serotype 33G: genetic, serological, and structural analysis of a new capsule type.

IMPORTANCE: Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen with the greatest burden of disease in Asia and Africa. The pneumococcal capsular polysaccharide has biological relevance as a major virulence factor as well as public health importance as it is the target for currently licensed vaccines. These vaccines have limited valency, covering up to 23 of the >100 known capsular types (serotypes) with higher valency vaccines in development. Here, we have characterized a new pneumococcal serotype, which we have named 33G. We detected serotype 33G in nasopharyngeal swabs (n = 20) from children and adults hospitalized with pneumonia, as well as healthy children in Mongolia. We show that the genetic, serological, and biochemical properties of 33G differ from existing serotypes, satisfying the criteria to be designated as a new serotype. Future studies should focus on the geographical distribution of 33G and any changes in prevalence following vaccine introduction.

Pneumococcal carriage and changes in serotype distribution post- PCV13 introduction in children in Matiari, Pakistan.

BACKGROUND: In early 2021, the 10-valent Pneumococcal conjugate vaccine (PCV10) was replaced with 13-valent (PCV13) by the federal directorate of immunization (FDI), Pakistan. We assessed the impact of a higher valent vaccine, PCV13, on the serotype distribution of nasopharyngeal carriage in rural Pakistan. METHODS: Children <2 years were randomly selected from two rural union councils of Matiari, Sindh in Pakistan between September-October,2022. Clinical, sociodemographic and vaccination histories were recorded. Nasopharyngeal swabs were collected and processed at Infectious Disease Research Laboratory, Aga Khan University, Karachi. Whole genome sequencing was performed on the culture positive isolates. RESULTS: Of the 200 children enrolled, pneumococcus was detected in 140(70 %) isolates. Majority of age-eligible children (60.1 %,110/183) received 3 PCV13 doses. PCV10 carriage declined from 13.2 %(78/590) in 2017/18 to 7.2 % (10/140) in 2022, additional PCV13 serotypes (3, 6A/6C and 19A) decreased from 18.5 %(109/590) to 11.4 %(16/140) while non-PCV13 serotypes increased from 68.3 %(403/590) to 81.4 %(114/140). There were 88.5 %(n = 124), 80.7 %(n = 113), 55.0 %(n = 77), and 46.0 %(n = 65) isolates predicted to be resistant to cotrimoxazole, penicillin(meningitis cut-off), tetracycline, and erythromycin respectively. CONCLUSION: Replacing PCV10 with PCV13 rapidly decreased prevalence of PCV13 carriage among vaccinated children in Matiari, Pakistan. Vaccine-driven selection pressure may have been responsible for the increase of non-PCV13 serotypes.

Impact of pneumococcal conjugate vaccines on invasive pneumococcal disease-causing lineages among South African children.

Invasive pneumococcal disease (IPD) due to non-vaccine serotypes after the introduction of pneumococcal conjugate vaccines (PCV) remains a global concern. This study used pathogen genomics to evaluate changes in invasive pneumococcal lineages before, during and after vaccine introduction in South Africa. We included genomes (N = 3104) of IPD isolates from individuals aged <18 years (2005-20), spanning four periods: pre-PCV, PCV7, early-PCV13, and late-PCV13. Significant incidence reductions occurred among vaccine-type lineages in the late-PCV13 period compared to the pre-PCV period. However, some vaccine-type lineages continued to cause invasive disease and showed increasing effective population size trends in the post-PCV era. A significant increase in lineage diversity was observed from the PCV7 period to the early-PCV13 period (Simpson's diversity index: 0.954, 95% confidence interval 0.948-0.961 vs 0.965, 0.962-0.969) supporting intervention-driven population structure perturbation. Increases in the prevalence of penicillin, erythromycin, and multidrug resistance were observed among non-vaccine serotypes in the late-PCV13 period compared to the pre-PCV period. In this work we highlight the importance of continued genomic surveillance to monitor disease-causing lineages post vaccination to support policy-making and future vaccine designs and considerations.

Pan-pathogen deep sequencing of nosocomial bacterial pathogens in Italy in spring 2020: a prospective cohort study.

BACKGROUND: Nosocomial infections pose a considerable risk to patients who are susceptible, and this is particularly acute in intensive care units when hospital-associated bacteria are endemic. During the first wave of the COVID-19 pandemic, the surge of patients presented a significant obstacle to the effectiveness of infection control measures. We aimed to assess the risks and extent of nosocomial pathogen transmission under a high patient burden by designing a novel bacterial pan-pathogen deep-sequencing approach that could be integrated with standard clinical surveillance and diagnostics workflows. METHODS: We did a prospective cohort study in a region of northern Italy that was severely affected by the first wave of the COVID-19 pandemic. Inpatients on both ordinary and intensive care unit (ICU) wards at the San Matteo hospital, Pavia were sampled on multiple occasions to identify bacterial pathogens from respiratory, nasal, and rectal samples. Diagnostic samples collected between April 7 and May 10, 2020 were cultured on six different selective media designed to enrich for Acinetobacter baumannii, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae, and DNA from each plate with positive growth was deep sequenced en masse. We used mSWEEP and mGEMS to bin sequencing reads by sequence cluster for each species, followed by mapping with snippy to generate high quality alignments. Antimicrobial resistance genes were detected by use of ARIBA and CARD. Estimates of hospital transmission were obtained from pairwise bacterial single nucleotide polymorphism distances, partitioned by within-patient and between-patient samples. Finally, we compared the accuracy of our binned Acinetobacter baumannii genomes with those obtained by single colony whole-genome sequencing of isolates from the same hospital. FINDINGS: We recruited patients from March 1 to May 7, 2020. The pathogen population among the patients was large and diverse, with 2148 species detections overall among the 2418 sequenced samples from the 256 patients. In total, 55 sequence clusters from key pathogen species were detected at least five times. The antimicrobial resistance gene prevalence was correspondingly high, with key carbapenemase and extended spectrum ß-lactamase genes detected in at least 50 (40%) of 125 patients in ICUs. Using high-resolution mapping to infer transmission, we established that hospital transmission was likely to be a significant mode of acquisition for each of the pathogen species. Finally, comparison with single colony Acinetobacter baumannii genomes showed that the resolution offered by deep sequencing was equivalent to single-colony sequencing, with the additional benefit of detection of co-colonisation of highly similar strains. INTERPRETATION: Our study shows that a culture-based deep-sequencing approach is a possible route towards improving future pathogen surveillance and infection control at hospitals. Future studies should be designed to directly compare the accuracy, cost, and feasibility of culture-based deep sequencing with single colony whole-genome sequencing on a range of bacterial species. FUNDING: Wellcome Trust, European Research Council, Academy of Finland Flagship program, Trond Mohn Foundation, and Research Council of Norway.