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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.

The core Mga (multiple gene activator) regulon of group A Streptococcus (GAS) contains genes encoding proteins involved in adhesion and immune evasion. While all GAS genomes contain genes for Mga and C5a peptidase, the intervening genes encoding M and M-like proteins vary between strains. The genetic make-up of the Mga regulon of GAS was characterized by utilizing a collection of 1,688 GAS genomes that are representative of the global GAS population. Sequence variations were examined with multiple alignments, and the expression of all core Mga regulon genes was examined by quantitative reverse transcription-PCR in a representative strain collection. In 85.2% of the sampled genomes, the Mga locus contained genes encoding Mga, Mrp, M, Enn, and C5a peptidase proteins. These isolates account for 53% of global infections. Only 9.1% of genomes did not contain either an mrp or an enn gene. The pairwise identity within Enn (68.6%) and Mrp (83.2%) protein sequences was higher than within M proteins (44.7%). Gene expression varied between strains tested, but high expression was recorded for all genes in at least one strain. Previous nomenclature issues were clarified with molecular gene definitions. Our findings support a shift in focus in the GAS research field to further consider the role of Mrp and Enn in virulence and vaccine development.IMPORTANCE While the GAS M protein has been the leading vaccine target for decades, the bacteria encode many other virulence factors of interest for vaccine development. In this work, we show that emm-like genes are encoded in a remarkable majority of GAS genomes and expressed at a level similar to that for the emm gene. In collaboration with the U.S. Centers for Disease Control, we developed molecular definitions of the different emm and emm-like gene families. This clarification should abrogate mistyping of strains, especially in the area of whole-genome typing. We have also updated the emm-typing collection by removing emm-like gene se

OBJECTIVES: PCR-based typing of the emm gene Streptococcus pyogenes often results in the amplification of multiple bands. This has resulted in the misclassification of strains into types based on non-emm gene sequences. We aimed to improve the specificity of the emm typing PCR reaction using a primer called CDC3, the sequence for which has been previously used to identify emm genes in silico. METHODS: The proposed primer CDC3 was validated in silico from a global database of 1688 GAS genomes and in vitro with 32 isolates. PCR reactions were performed on genomic DNA from each isolate, using the published CDC1 forward primer with the CDC2 reverse primer or the new CDC3 reverse primer. The products were examined by gel electrophoresis, and representative PCR products were sequenced. RESULTS: In 1688 S. pyogenes genomes, the previous CDC2 reverse primer annealed in silico in 1671 emm genes and also in 2109 non emm genes in close proximity, whereas the new CDC3 primer annealed in 1669 emm genes only. The remaining 19 genes without a CDC3 binding site were chimeric emm genes. The PCR pair CDC1+CDC3 produced a single band at appropriate molecular weight in all 32 isolates tested, while the CDC1+CDC2 pair produced more than one band in 13 of 32 isolates (40%). CONCLUSIONS: The new CDC3 primer is more specific for emm genes than the previous CDC2 primer and represents a simple solution to reduce the potential for mistyping S. pyogenes strains.

Background: Invasive pneumococcal disease remains an important health priority owing to increasing disease incidence caused by pneumococci expressing non-vaccine serotypes. We previously defined 621 Global Pneumococcal Sequence Clusters (GPSCs) by analysing 20 027 pneumococcal isolates collected worldwide and from previously published genomic data. In this study, we aimed to investigate the pneumococcal lineages behind the predominant serotypes, the mechanism of serotype replacement in disease, as well as the major pneumococcal lineages contributing to invasive pneumococcal disease in the post-vaccine era and their antibiotic resistant traits. Methods: We whole-genome sequenced 3233 invasive pneumococcal disease isolates from laboratory-based surveillance programmes in Hong Kong (n=78), Israel (n=701), Malawi (n=226), South Africa (n=1351), The Gambia (n=203), and the USA (n=674). The genomes represented pneumococci from before and after pneumococcal conjugate vaccine (PCV) introductions and were from children younger than 3 years. We identified predominant serotypes by prevalence and their major contributing lineages in each country, and assessed any serotype replacement by comparing the incidence rate between the pre-PCV and PCV periods for Israel, South Africa, and the USA. We defined the status of a lineage as vaccine-type GPSC (≥50% 13-valent PCV [PCV13] serotypes) or non-vaccine-type GPSC (>50% non-PCV13 serotypes) on the basis of its initial serotype composition detected in the earliest vaccine period to measure their individual contribution toward serotype replacement in each country. Major pneumococcal lineages in the PCV period were identified by pooled incidence rate using a random effects model. Findings: The five most prevalent serotypes in the PCV13 period varied between countries, with only serotypes 5, 12F, 15B/C, 19A, 33F, and 35B/D common to two or more countries. The five most prevalent serotypes in the PCV13 period varied between countries, with

Community-acquired pneumonia (CAP) is a major cause of morbidity in children. This study estimated the proportion of children with pneumococcal CAP among children hospitalised with CAP in Belgium and describes the causative serotype distribution after implementation of the 7-valent pneumococcal conjugate vaccine. Children 0-14 years hospitalised with X-ray-confirmed CAP were prospectively enrolled in a multicentre observational study. Acute and convalescent blood samples were collected. Pneumococcal aetiology was assessed by conventional methods (blood or pleural fluid cultures with Quellung reaction capsular typing or polymerase chain reaction [PCR] in pleural fluid), and recently developed methods (real-time PCR in blood and World Health Organization-validated serotype-specific serology). A total of 561 children were enrolled. Pneumococcal aetiology was assessed by conventional methods in 539, serology in 171, and real-time PCR in blood in 154. Pneumococcal aetiology was identified in 12.2% (66/539) of the children by conventional methods alone but in 73.9% by the combination of conventional and recently developed methods. The pneumococcal detection rate adjusted for the whole study population was 61.7%. Serotypes 1 (42.3%), 5 (16.0%), and 7F(7A) (12.8%) were predominant. In conclusion, Streptococcus pneumoniae remains the predominant bacteria in children hospitalised for CAP in Belgium after implementation of 7-valent pneumococcal conjugate vaccine, with non-vaccine-serotypes accounting for the majority of cases. The use of recently developed methods improves diagnosis of pneumococcal aetiology.

BACKGROUND: Pneumococcal disease is a significant cause of morbidity and mortality in young children in Nepal, and currently available pneumococcal conjugate vaccines offer moderate coverage of invasive disease isolates. METHODS: A prevalence study of children aged 1.5 to 24 months in urban and rural Nepal was conducted. In the urban group, nasopharyngeal swabs (NPS) were transported using silica desiccant packages (SDP) with delayed processing (2 weeks), or skim-milk-tryptone-glucose-glycerin (STGG) with immediate processing (within 8 hours). Pneumococcal nasopharyngeal carriage prevalence, serogroup/type distribution and isolate genotypes (as defined by multilocus sequence typing) were determined. RESULTS: 1101 children were enrolled into the study: 574 in the urban group and 527 in the rural group. Overall carriage prevalence based on culture from specimens transported and stored in STGG was 58.7% (337/574), compared to 40.9% (235/574) in SDP. There was concordance of detection of pneumococcus in 67% of samples. Using the SDP method, pneumococcal carriage prevalence was higher in the rural population (69.2%; 364/526) compared to the urban population (40.9%; 235/574). Serogroup/type distribution varied with geographical location. Over half of the genotypes identified in both the urban and rural pneumococcal populations were novel. CONCLUSION: The combination of delayed culture and transport using SDP underestimates the prevalence of pneumococcal carriage; however, in remote areas, this method could still provide a useful estimate of carriage prevalence and serogroup/type distribution. Vaccine impact is unpredictable in a setting with novel genotypes and limited serotype coverage as described here. Consequently, continued surveillance of pneumococcal isolates from carriage and disease in Nepali children following the planned introduction of pneumococcal conjugate vaccines introduction will be essential.

BACKGROUND: Indigenous children in Australia and Alaska have very high rates of chronic suppurative lung disease (CSLD)/bronchiectasis. Antibiotics, including frequent or long-term azithromycin in Australia and short-term beta-lactam therapy in both countries, are often prescribed to treat these patients. In the Bronchiectasis Observational Study we examined over several years the nasopharyngeal carriage and antibiotic resistance of respiratory bacteria in these two PCV7-vaccinated populations. METHODS: Indigenous children aged 0.5-8.9 years with CSLD/bronchiectasis from remote Australia (n = 79) and Alaska (n = 41) were enrolled in a prospective cohort study during 2004-8. At scheduled study visits until 2010 antibiotic use in the preceding 2-weeks was recorded and nasopharyngeal swabs collected for culture and antimicrobial susceptibility testing. Analysis of respiratory bacterial carriage and antibiotic resistance was by baseline and final swabs, and total swabs by year. RESULTS: Streptococcus pneumoniae carriage changed little over time. In contrast, carriage of Haemophilus influenzae declined and Staphylococcus aureus increased (from 0% in 2005-6 to 23% in 2010 in Alaskan children); these changes were associated with increasing age. Moraxella catarrhalis carriage declined significantly in Australian, but not Alaskan, children (from 64% in 2004-6 to 11% in 2010). While beta-lactam antibiotic use was similar in the two cohorts, Australian children received more azithromycin. Macrolide resistance was significantly higher in Australian compared to Alaskan children, while H. influenzae beta-lactam resistance was higher in Alaskan children. Azithromycin use coincided significantly with reduced carriage of S. pneumoniae, H. influenzae and M. catarrhalis, but increased carriage of S. aureus and macrolide-resistant strains of S. pneumoniae and S. aureus (proportion of carriers and all swabs), in a 'cumulative dose-response' relationship. CONCLUSIONS: Over time, similar

Bacterial isolates are often transported between laboratories for research and diagnostic purposes. Silica desiccant packets (SDPs), which are inexpensive and do not require freezing, were evaluated for storage and recovery of bacterial isolates. Conditions such as inoculum size, swab type and temperature of storage were investigated using ten Streptococcus pneumoniae isolates. The optimized protocol was then tested using 49 additional S. pneumoniae isolates representing 40 serogroups. Overall, S. pneumoniae growth was considered satisfactory (>100 colony forming units) for 98/109 (89.9%) and 20/20 (100%) swabs after 14 days at room temperature or 28 days at 4° C, respectively. Storage in SDPs did not impact on the ability of S. pneumoniae isolates to be subsequently serotyped. When the survival of nine other clinically relevant bacterial species was tested, seven were viable after 28 days at room temperature, the exceptions being Neisseria gonorrhoeae and Haemophilus influenzae. SDPs are suitable for transport and short-term storage of bacterial species including S. pneumoniae.

emm sequence typing is the most widely used method for defining group A streptococcal (GAS) strains, and has been applied to isolates in all regions of the world. We did a systematic review of the global distribution of GAS emm types. 102 articles and reports were included (38 081 isolates). Epidemiological data from high-income countries were predominant, with sparse data from low-income countries. The epidemiology of GAS disease in Africa and the Pacific region seems to be different from that in other regions, particularly high-income countries. In Africa and the Pacific, there were no dominant emm types, a higher diversity of emm types, and many of the common emm types in other parts of the world were less common (including emm 1, 4, 6, and 12). Our data have implications for the development of GAS vaccines. On the basis of the available data, the current formulation of the experimental multivalent emm vaccine would provide good coverage in high-income countries, particularly USA, Canada, and Europe, but poor coverage in Africa and the Pacific, and only average coverage in Asia and the Middle East.

Serum opacity factor (SOF) is a bifunctional cell surface protein expressed by 40-50% of group A streptococcal (GAS) strains comprised of a C-terminal domain that binds fibronectin and an N-terminal domain that mediates opacification of mammalian sera. The sof gene was recently discovered to be co-transcribed in a two-gene operon with a gene encoding another fibronectin-binding protein, sfbX. We compared the ability of a SOF(+) wild-type (WT) serotype M49 GAS strain and isogenic mutants lacking SOF or SfbX to invade cultured HEp-2 human pharyngeal epithelial cells. Elimination of SOF led to a significant decrease in HEp-2 intracellular invasion while loss of SfbX had minimal effect. The hypoinvasive phenotype of the SOF(-) mutant could be restored upon complementation with the sof gene on a plasmid vector, and heterologous expression of sof49 in M1 GAS or Lactococcus lactis conferred marked increases in HEp-2 cell invasion. Studies using a mutant sof49 gene lacking the fibronectinbinding domain indicated that the N-terminal opacification domain of SOF contributes to HEp-2 invasion independent of the C-terminal fibronectin binding domain, findings corroborated by observations that a purified SOF N-terminal peptide could promote latex bead adherence to HEp- 2 cells and inhibit GAS invasion of HEp-2 cells in a dose-dependent manner. Finally, the first in vivo studies to employ a single gene allelic replacement mutant of SOF demonstrate that this protein contributes to GAS virulence in a murine model of necrotizing skin infection.