Recombinational exchange of M-fibril and T-pilus genes generates extensive cell surface diversity in the global group A Streptococcus population.

Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as "pilin types." Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

Variation in pneumococcal invasiveness metrics is driven by serotype carriage duration and initial risk of disease.

Streptococcus pneumoniae is an opportunistic pathogen that, while usually carried asymptomatically, can cause severe invasive diseases like meningitis and bacteremic pneumonia. A central goal in S. pneumoniae public health management is to identify which serotypes (immunologically distinct strains) pose the most risk of invasive disease. The most common invasiveness metrics use cross-sectional data (i.e., invasive odds ratios (IOR)), or longitudinal data (i.e., attack rates (AR)). To assess the reliability of these metrics we developed an epidemiological model of carriage and invasive disease. Our mathematical analyses illustrate qualitative failures with the IOR metric (e.g., IOR can decline with increasing invasiveness parameters). Fitting the model to both longitudinal and cross-sectional data, our analysis supports previous work indicating that invasion risk is maximal at or near time of colonization. This pattern of early invasive disease risk leads to substantial (up to 5-fold) biases when estimating underlying differences in invasiveness from IOR metrics, due to the impact of carriage duration on IOR. Together, these results raise serious concerns with the IOR metric as a basis for public health decision-making and lend support for multiple alternate metrics including AR.

Low but Increasing Prevalence of Reduced Beta-lactam Susceptibility Among Invasive Group B Streptococcal Isolates, US Population-Based Surveillance, 1998-2018.

BACKGROUND: Invasive group B Streptococcus (iGBS) isolates with mutations in the pbp2x gene that encodes penicillin binding protein 2x can have reduced beta-lactam susceptibility (RBLS) when susceptible by Clinical and Laboratory Standards Institute (CLSI) criteria. We assessed the emergence and characteristics of RBLS strains in US iGBS isolates. METHODS: We analyzed iGBS isolates from 8 multistate population-based surveillance sites from 1998 to 2018. During 1998-2014, phenotypic antimicrobial susceptibility was determined by broth microdilution; criteria for 6 antibiotics were used to identify RBLS, followed by whole-genome sequencing (WGS). WGS for all isolates was added in 2015; we used phenotypic and genotypic results of >2000 isolates to validate phenotypic RBLS criteria and genotypic predictions. Since 2016, WGS has been used to screen for RBLS with broth microdilution confirmation of predicted RBLS isolates. RESULTS: Of 28 269 iGBS isolates, 28 (0.1%) were nonsusceptible by CLSI criteria; 137 (0.5%) met RBLS criteria. RBLS isolates were detected in all Active Bacterial Core surveillance sites. The RBLS proportion increased, especially since 2013 (odds ratio, 1.17; 95% CI, 1.03-1.32); the proportion that were nonsusceptible remained stable. CONCLUSIONS: The RBSL proportion was low but increasing among US iGBS isolates. Ongoing monitoring is needed to detect emerging threats to prevention and treatment of GBS infections.

Multistate, Population-Based Distributions of Candidate Vaccine Targets, Clonal Complexes, and Resistance Features of Invasive Group B Streptococci Within the United States, 2015-2017.

BACKGROUND: Group B Streptococcus (GBS) is a leading cause of neonatal sepsis and meningitis and an important cause of invasive infections in pregnant and nonpregnant adults. Vaccines targeting capsule polysaccharides and common proteins are under development. METHODS: Using whole genome sequencing, a validated bioinformatics pipeline, and targeted antimicrobial susceptibility testing, we characterized 6340 invasive GBS isolates recovered during 2015-2017 through population-based Active Bacterial Core surveillance (ABCs) in 8 states. RESULTS: Six serotypes accounted for 98.4% of isolates (21.8% Ia, 17.6% V, 17.1% II, 15.6% III, 14.5% Ib, 11.8% IV). Most (94.2%) isolates were in 11 clonal complexes (CCs) comprised of multilocus sequence types identical or closely related to sequence types 1, 8, 12, 17, 19, 22, 23, 28, 88, 452, and 459. Fifty-four isolates (0.87%) had point mutations within pbp2x associated with nonsusceptibility to 1 or more ß-lactam antibiotics. Genes conferring resistance to macrolides and/or lincosamides were found in 56% of isolates; 85.2% of isolates had tetracycline resistance genes. Two isolates carrying vanG were vancomycin nonsusceptible (minimum inhibitory concentration = 2 µg/mL). Nearly all isolates possessed capsule genes, 1-2 of the 3 main pilus gene clusters, and 1 of 4 homologous alpha/Rib family determinants. Presence of the hvgA virulence gene was primarily restricted to serotype III/CC17 isolates (465 isolates), but 8 exceptions (7 IV/CC452 and 1 IV/CC17) were observed. CONCLUSIONS: This first comprehensive, population-based quantitation of strain features in the United States suggests that current vaccine candidates should have good coverage. The ß-lactams remain appropriate for first-line treatment and prophylaxis, but emergence of nonsusceptibility warrants ongoing monitoring.

Visualizing variation within Global Pneumococcal Sequence Clusters (GPSCs) and country population snapshots to contextualize pneumococcal isolates.

Knowledge of pneumococcal lineages, their geographic distribution and antibiotic resistance patterns, can give insights into global pneumococcal disease. We provide interactive bioinformatic outputs to explore such topics, aiming to increase dissemination of genomic insights to the wider community, without the need for specialist training. We prepared 12 country-specific phylogenetic snapshots, and international phylogenetic snapshots of 73 common Global Pneumococcal Sequence Clusters (GPSCs) previously defined using PopPUNK, and present them in Microreact. Gene presence and absence defined using Roary, and recombination profiles derived from Gubbins are presented in Phandango for each GPSC. Temporal phylogenetic signal was assessed for each GPSC using BactDating. We provide examples of how such resources can be used. In our example use of a country-specific phylogenetic snapshot we determined that serotype 14 was observed in nine unrelated genetic backgrounds in South Africa. The international phylogenetic snapshot of GPSC9, in which most serotype 14 isolates from South Africa were observed, highlights that there were three independent sub-clusters represented by South African serotype 14 isolates. We estimated from the GPSC9-dated tree that the sub-clusters were each established in South Africa during the 1980s. We show how recombination plots allowed the identification of a 20 kb recombination spanning the capsular polysaccharide locus within GPSC97. This was consistent with a switch from serotype 6A to 19A estimated to have occured in the 1990s from the GPSC97-dated tree. Plots of gene presence/absence of resistance genes (tet, erm, cat) across the GPSC23 phylogeny were consistent with acquisition of a composite transposon. We estimated from the GPSC23-dated tree that the acquisition occurred between 1953 and 1975. Finally, we demonstrate the assignment of GPSC31 to 17 externally generated pneumococcal serotype 1 assemblies from Utah via Pathogenwatch. Most of the Utah isolates clustered within GPSC31 in a USA-specific clade with the most recent common ancestor estimated between 1958 and 1981. The resources we have provided can be used to explore to data, test hypothesis and generate new hypotheses. The accessible assignment of GPSCs allows others to contextualize their own collections beyond the data presented here.

Genome-wide association analyses of invasive pneumococcal isolates identify a missense bacterial mutation associated with meningitis.

Bacterial mutations predisposing pneumococcus to causing meningitis, a more severe form of invasive pneumococcal disease (IPD), are largely unknown. Knowledge of such mutations may improve our understanding of pathogenesis and inform preventive strategies. Here we report a pneumococcal pbp1b gene mutation (pbp1bA641C causing N214T change in PBP1b transglycosylase domain) that is associated with meningitis in an exploratory cohort of IPD patients (n = 2054, p = 6.8 × 10-6), in an independent confirmatory cohort (n = 2518, p = 2.3 × 10-6), and in a combined analysis (n = 4572, p = 3.0 × 10-10). Patients infected by the pbp1b641C genotype pneumococci show 2.8-fold odds (95% CI 1.7 to 4.8) of meningitis compared to those infected by non-pbp1b641C pneumococci, after controlling for pneumococcal serotype, antibiotic resistance, and patient age. The pbp1bA641C change results in longer time needed for bacterial killing by antibiotic treatment and shows evidence of being under positive selection. Thus, a pneumococcal mutation conferring increased antibiotic tolerance is associated with meningitis among IPD patients.

Epidemiology of Invasive Early-Onset and Late-Onset Group B Streptococcal Disease in the United States, 2006 to 2015: Multistate Laboratory and Population-Based Surveillance.

Importance: Invasive disease owing to group B Streptococcus (GBS) remains an important cause of illness and death among infants younger than 90 days in the United States, despite declines in early-onset disease (EOD; with onset at 0-6 days of life) that are attributed to intrapartum antibiotic prophylaxis (IAP). Maternal vaccines to prevent infant GBS disease are currently under development. Objective: To describe incidence rates, case characteristics, antimicrobial resistance, and serotype distribution of EOD and late-onset disease (LOD; with onset at 7-89 days of life) in the United States from 2006 to 2015 to inform IAP guidelines and vaccine development. Design, Setting, and Participants: This study used active population-based and laboratory-based surveillance for invasive GBS disease conducted through Active Bacterial Core surveillance in selected counties of 10 states across the United States. Residents of Active Bacterial Core surveillance areas who were younger than 90 days and had invasive GBS disease in 2006 to 2015 were included. Data were analyzed from December 2017 to April 2018. Exposures: Group B Streptococcus isolated from a normally sterile site. Main Outcomes and Measures: Early-onset disease and LOD incidence rates and associated GBS serotypes and antimicrobial resistance. Results: The Active Bacterial Core surveillance program identified 1277 cases of EOD and 1387 cases of LOD. From 2006 to 2015, EOD incidence declined significantly from 0.37 to 0.23 per 1000 live births (P < .001), and LOD rates remained stable (mean, 0.31 per 1000 live births). Among the mothers of 1277 infants with EOD, 617 (48.3%) had no indications for IAP and did not receive it, and 278 (21.8%) failed to receive IAP despite having indications. Serotype data were available for 1743 of 1897 patients (91.3%) from 7 sites that collect GBS isolates. Among patients with EOD, serotypes Ia (242 [27.3%]) and III (242 [27.3%]) were most common. Among patients with LOD, serotype III was most common (481 [56.2%]), and this increased from 2006 to 2015 from 0.12 to 0.20 cases per 1000 live births (P < .001). Serotype IV caused 53 cases (6.2%) of EOD and LOD combined. The 6 most common serotypes (Ia, Ib, II, III, IV, and V) caused 881 EOD cases (99.3%) and 853 LOD cases (99.7%). No ß-lactam resistance was identified; 359 isolates (20.8%) tested showed constitutive clindamycin resistance. In 2015, an estimated 840 EOD cases and 1265 LOD cases occurred nationally. Conclusions and Relevance: The rates of LOD among US infants are now higher than EOD rates. Combined with addressing IAP implementation gaps, an effective vaccine covering the most common serotypes might further reduce EOD rates and help prevent LOD, for which there is no current public health intervention.

Molecular Epidemiology, Ecology, and Evolution of Group A Streptococci.

The clinico-epidemiological features of diseases caused by group A streptococci (GAS) is presented through the lens of the ecology, population genetics, and evolution of the organism. The serological targets of three typing schemes (M, T, SOF) are themselves GAS cell surface proteins that have a myriad of virulence functions and a diverse array of structural forms. Horizontal gene transfer expands the GAS antigenic cell surface repertoire by generating numerous combinations of M, T, and SOF antigens. However, horizontal gene transfer of the serotype determinant genes is not unconstrained, and therein lies a genetic organization that may signify adaptations to a narrow ecological niche, such as the primary tissue reservoirs of the human host. Adaptations may be further shaped by selection pressures such as herd immunity. Understanding the molecular evolution of GAS on multiple levels-short, intermediate, and long term-sheds insight on mechanisms of host-pathogen interactions, the emergence and spread of new clones, rational vaccine design, and public health interventions.

Validation of ß-lactam minimum inhibitory concentration predictions for pneumococcal isolates with newly encountered penicillin binding protein (PBP) sequences.

BACKGROUND: Genomic sequence-based deduction of antibiotic minimum inhibitory concentration (MIC) has great potential to enhance the speed and sensitivity of antimicrobial susceptibility testing. We previously developed a penicillin-binding protein (PBP) typing system and two methods (Random Forest (RF) and Mode MIC (MM)) that accurately predicted ß-lactam MICs for pneumococcal isolates carrying a characterized PBP sequence type (phenotypic ß-lactam MICs known for at least one isolate of this PBP type). This study evaluates the prediction performance for previously uncharacterized (new) PBP types and the probability of encountering new PBP types, both of which impact the overall prediction accuracy. RESULTS: The MM and RF methods were used to predict MICs of 4309 previously reported pneumococcal isolates in 2 datasets and the results were compared to the known broth microdilution MICs to 6 ß-lactams. Based on a method that specifically evaluated predictions for new PBP types, the RF results were more accurate than MM results for new PBP types and showed percent essential agreement (MICs agree within ±1 dilution) >97%, percent category agreement (interpretive results agree) >93%, major discrepancy (sensitive isolate predicted as resistant) rate < 1.2%, and very major discrepancy (resistant isolate predicted as sensitive) rate < 1.4% for all 6 ß-lactams. The identification of new PBP types over time was well approximated by a diminishingly increasing curve (Pearson's r = 0.99) and minimally impacted overall MIC prediction performance. CONCLUSIONS: MIC prediction using the RF method could be an accurate alternative of phenotypic susceptibility testing even in the presence of previously uncharacterized PBP types.

Cross-resistance to lincosamides, streptogramins A and pleuromutilins in Streptococcus agalactiae isolates from the USA.

Background: Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of meningitis, sepsis and pneumonia in neonates in the United States. GBS also causes invasive disease in older infants, pregnant women, children and young adults with underlying medical conditions, and older adults. Resistance to lincosamides in the absence of erythromycin resistance is rare in GBS, but has been previously reported in clinical isolates, both on its own or in combination with resistance to streptogramins A and pleuromutilins (L/LSA/LSAP phenotypes). Objectives: To retrospectively screen the Active Bacterial Core surveillance (ABCs) GBS isolate collection for these phenotypes in order to identify the causal genetic determinants and determine whether their frequency is increasing. Methods: Based on MIC data, 65 (0.31%) isolates susceptible to erythromycin (MIC ≤0.25 mg/L) and non-susceptible to clindamycin (MIC ≥0.5 mg/L) were identified among 21 186 GBS isolates. Genomic DNA was extracted and WGS was performed. The presence of 10 genes previously associated with LSA resistance was investigated by read mapping. Results: Forty-nine (75%) isolates carried the lsa (C) gene and expressed the LSAP phenotype, and 12 (18%) carried both the lnu (B) and lsa (E) genes and expressed the LSAP phenotype. The four remaining isolates were negative for all determinants investigated. Conclusions: While the overall observed frequency of these phenotypes among our GBS isolates was quite low (0.31%), this frequency has increased in recent years. To the best of our knowledge, this is the first time the LSAP phenotype has been reported among GBS isolates from the USA.

Genomic Epidemiology of Penicillin-Nonsusceptible Pneumococci with Nonvaccine Serotypes Causing Invasive Disease in the United States.

Conjugate vaccination against seven pneumococcal serotypes (PCV7) reduced disease prevalence due to antibiotic-resistant strains throughout the 2000s. However, diseases caused by resistant nonvaccine type (NVT) strains increased. Some of these emerging strains were derived from vaccine types (VT) that had changed their capsule by recombination. The introduction of a vaccine targeting 13 serotypes (PCV13) in 2010 has led to concern that this scenario will repeat itself. We generated high-quality draft genomes from 265 isolates of NVT pneumococci not susceptible to penicillin (PNSP) in 2009 and compared them with the genomes of 581 isolates from 2012 to 2013 collected by the Active Bacterial Core surveillance (ABCs) of the Centers for Disease Control and Prevention (CDC). Of the seven sequence clusters (SCs) identified, three SCs fell into a single lineage associated with serogroup 23, which had an origin in 1908 as dated by coalescent analysis and included isolates with a divergent 23B capsule locus. Three other SCs represented relatively deep-branching lineages associated with serotypes 35B, 15A, and 15BC. In all cases, the resistant clones originated prior to 2010, indicating that PNSP are at present dominated by descendants of NVT clones present before vaccination. With one exception (15BC/ST3280), these SCs were related to clones identified by the Pneumococcal Molecular Epidemiology Network (PMEN). We conclude that postvaccine diversity in NVT PNSP between 2009 and 2013 was driven mainly by the persistence of preexisting strains rather than through de novo adaptation, with few cases of serotype switching. Future surveillance is essential for documenting the long-term dynamics and resistance of NVT PNSP.

Penicillin-Binding Protein Transpeptidase Signatures for Tracking and Predicting ß-Lactam Resistance Levels in Streptococcus pneumoniae.

UNLABELLED: ß-Lactam antibiotics are the drugs of choice to treat pneumococcal infections. The spread of ß-lactam-resistant pneumococci is a major concern in choosing an effective therapy for patients. Systematically tracking ß-lactam resistance could benefit disease surveillance. Here we developed a classification system in which a pneumococcal isolate is assigned to a "PBP type" based on sequence signatures in the transpeptidase domains (TPDs) of the three critical penicillin-binding proteins (PBPs), PBP1a, PBP2b, and PBP2x. We identified 307 unique PBP types from 2,528 invasive pneumococcal isolates, which had known MICs to six ß-lactams based on broth microdilution. We found that increased ß-lactam MICs strongly correlated with PBP types containing divergent TPD sequences. The PBP type explained 94 to 99% of variation in MICs both before and after accounting for genomic backgrounds defined by multilocus sequence typing, indicating that genomic backgrounds made little independent contribution to ß-lactam MICs at the population level. We further developed and evaluated predictive models of MICs based on PBP type. Compared to microdilution MICs, MICs predicted by PBP type showed essential agreement (MICs agree within 1 dilution) of >98%, category agreement (interpretive results agree) of >94%, a major discrepancy (sensitive isolate predicted as resistant) rate of <3%, and a very major discrepancy (resistant isolate predicted as sensitive) rate of <2% for all six ß-lactams. Thus, the PBP transpeptidase signatures are robust indicators of MICs to different ß-lactam antibiotics in clinical pneumococcal isolates and serve as an accurate alternative to phenotypic susceptibility testing. IMPORTANCE: The human pathogen Streptococcus pneumoniae is a leading cause of morbidity and mortality worldwide. ß-Lactam antibiotics such as penicillin and ceftriaxone are the drugs of choice to treat pneumococcal infections. Some pneumococcal strains have developed ß-lactam resistance through altering their penicillin-binding proteins (PBPs) and have become a major concern in choosing effective patient therapy. To systematically track and predict ß-lactam resistance, we obtained the sequence signatures of PBPs from a large collection of clinical pneumococcal isolates using whole-genome sequencing data and found that these "PBP types" were predictive of resistance levels. Our findings can benefit the current era of strain surveillance when whole-genome sequencing data often lacks detailed resistance information. Using PBP positions that we found are always substituted within highly resistant strains may lead to further refinements. Sequence-based predictions are accurate and may lead to the ability to extract critical resistance information from nonculturable clinical specimens.

A systematic and functional classification of Streptococcus pyogenes that serves as a new tool for molecular typing and vaccine development.

Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development.

Deriving group A Streptococcus typing information from short-read whole-genome sequencing data.

Typing of group A Streptococcus (GAS) is crucial for infection control and epidemiology. While whole-genome sequencing (WGS) is revolutionizing the way that bacterial organisms are typed, it is necessary to provide backward compatibility with currently used typing schemas to facilitate comparisons and understanding of epidemiological trends. Here, we sequenced the genomes of 191 GAS isolates representing 42 different emm types and used bioinformatics tools to derive commonly used GAS typing information directly from the short-read WGS data. We show that emm typing and multilocus sequence typing can be achieved rapidly and efficiently using this approach, which also permits the determination of the presence or absence of genes associated with GAS tissue tropism. We also report on how the WGS data analysis was instrumental in identifying ambiguities present in the commonly used emm type database hosted by the U.S. Centers for Disease Control and Prevention.

Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract.

We performed culture-based and PCR-based tests for pneumococcal identification and serotyping from carriage specimens collected in rural and urban Kenya. Nasopharyngeal specimens from 237 healthy children <5 years old (C-NPs) and combined nasopharyngeal/oropharyngeal specimens from 158 adults (A-NP/OPs, 118 HIV-positive) were assessed using pneumococcal isolation (following broth culture enrichment) with Quellung-based serotyping, real-time lytA-PCR, and conventional multiplexed PCR-serotyping (cmPCR). Culture-based testing from C-NPs, HIV-positive A-NP/OPs, and HIV-negative A-NP/OPs revealed 85.2%, 40.7%, and 12.5% pneumococcal carriage, respectively. In contrast, cmPCR serotypes were found in 93.2%, 98.3%, and 95.0% of these sets, respectively. Two of 16 lytA-negative C-NPs and 26 of 28 lytA-negative A-NP/OPs were cmPCR-positive for 1-10 serotypes (sts) or serogroups (sgs). A-NP/OPs averaged 5.5 cmPCR serotypes/serogroups (5.2 in HIV-positive, 7.1 in HIV-negative) and C-NPs averaged 1.5 cmPCR serotypes/serogroups. cmPCR serotypes/serogroups from lytA-negative A-NP/OPs included st2, st4, sg7F/7A, sg9N/9L, st10A, sg10F/10C/33C, st13, st17F, sg18C/18A/18B/18F, sg22F/22A, and st39. Nine strains of three non-pneumococcal species (S. oralis, S. mitis, and S. parasanguinis) (7 from A-OP, 1 from both A-NP and A-OP, and 1 from C-NP) were each cmPCR-positive for one of 7 serotypes/serogroups (st5, st13, sg15A/15F, sg10F/10C/33C, sg33F/33A/37, sg18C/18A/18B/18F, sg12F/12A/12B/ 44/46) with amplicons revealing 83.6-99.7% sequence identity to pneumococcal references. In total, 150 cmPCR amplicons from carriage specimens were sequenced, including 25 from lytA-negative specimens. Amplicon sequences derived from specimens yielding a pneumococcal isolate with the corresponding serotype were identical or highly conserved (>98.7%) with the reference cmPCR amplicon for the st, while cmPCR amplicons from lytA-negative specimens were generally more divergent. Separate testing of 56 A-OPs and 56 A-NPs revealed that ∼94% of the positive cmPCR results from A-NP/OPs were from OP microbiota. In contrast, A-NPs yielded >2-fold more pneumococcal isolates than A-OPs. Verified and suspected non-pneumococcal cmPCR serotypes/serogroups appeared to be relatively rare in C-NPs and A-NPs compared to A-OPs. Our findings indicate that non-pneumococcal species can confound serotype-specific PCR and other sequence-based assays due to evolutionarily conserved genes most likely involved in biosynthesis of surface polysaccharide structures.

Invasive pneumococcal disease among children with and without sickle cell disease in the United States, 1998 to 2009.

BACKGROUND: Children with sickle cell disease (SCD) are at increased risk of illness and death from invasive pneumococcal disease (IPD). The introduction in 2000 of the 7-valent pneumococcal conjugate vaccine and penicillin prophylaxis for children with SCD has greatly reduced the incidence of IPD in this population. However, a recent report suggested an increase in cases of IPD in children with SCD. METHODS: Using data from Active Bacterial Core surveillance, we analyzed trends in hospitalizations, mortality and serotype among children with SCD compared with other children. We used neonatal screening data to estimate SCD population denominators for each Active Bacterial Core surveillance site. RESULTS: From 1998 to 2009, 3069 cases of IPD occurred among African-American children less than 18 years of age in the Active Bacterial Core surveillance catchment area. Of these, 127 (4.1%) had SCD identified by medical chart review and 185 (6.0%) had 1 or more IPD risk factors, excluding SCD. Rates of IPD among children with SCD declined by 53% (1118 vs. 530 per 100,000) whereas the overall rates among African-American children declined by 74% (54 to 14 per 100,000). For all time periods, children with SCD and IPD were more likely to be hospitalized (84%-92% vs. 31%-56%) and more likely to die (6%-17% vs. 1%-2%) than children with no risk factors. CONCLUSIONS: Although the rate of IPD in children with SCD has dropped dramatically since 7-valent pneumococcal conjugate vaccine introduction, the rate of IPD in children with SCD remains higher than that of the general population of African-American children, pointing to the need for more effective prevention efforts to prevent IPD in children with SCD.

Pneumococcal capsular switching: a historical perspective.

BACKGROUND: Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching. METHODS: Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 were analyzed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events. RESULTS: We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately 19.0 kb to ≥ 58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a. CONCLUSIONS: Capsular switching has been a regular occurrence among pneumococcal populations throughout the past 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicillin-binding protein genes, predated both vaccine introduction and widespread antibiotic use. This type of recombination has likely been an intrinsic feature throughout the history of pneumococcal evolution.

The multidrug-resistant PMEN1 pneumococcus is a paradigm for genetic success.

BACKGROUND: Streptococcus pneumoniae, also called the pneumococcus, is a major bacterial pathogen. Since its introduction in the 1940s, penicillin has been the primary treatment for pneumococcal diseases. Penicillin resistance rapidly increased among pneumococci over the past 30 years, and one particular multidrug-resistant clone, PMEN1, became highly prevalent globally. We studied a collection of 426 pneumococci isolated between 1937 and 2007 to better understand the evolution of penicillin resistance within this species. RESULTS: We discovered that one of the earliest known penicillin-nonsusceptible pneumococci, recovered in 1967 from Australia, was the likely ancestor of PMEN1, since approximately 95% of coding sequences identified within its genome were highly similar to those of PMEN1. The regions of the PMEN1 genome that differed from the ancestor contained genes associated with antibiotic resistance, transmission and virulence. We also revealed that PMEN1 was uniquely promiscuous with its DNA, donating penicillin-resistance genes and sometimes many other genes associated with antibiotic resistance, virulence and cell adherence to many genotypically diverse pneumococci. In particular, we describe two strains in which up to 10% of the PMEN1 genome was acquired in multiple fragments, some as long as 32 kb, distributed around the recipient genomes. This type of directional genetic promiscuity from a single clone to numerous unrelated clones has, to our knowledge, never before been described. CONCLUSIONS: These findings suggest that PMEN1 is a paradigm of genetic success both through its epidemiology and promiscuity. These findings also challenge the existing views about horizontal gene transfer among pneumococci.