The Promiscuous Pneumococcus---Evolution and Biology

Competitive interactions between bacteria play an important role in shaping microbial communities on host surfaces. To survive antagonistic interactions in the nasopharynx, Streptococcus pneumoniae has developed offensive and defensive tactics that involve the production of lytic peptides called pneumocins and their associated immunity proteins. The production of pneumocins and immunity proteins is controlled by the extracellular accumulation of the signal peptide, BlpC. The locus encoding these proteins is characterized by significant allelic heterogeneity, in particular, in the genes encoding BlpC, pneumocin peptides and immunity proteins. This diversity reflects the impact of selective pressure from competing strains. Variability in immunity proteins provides a defensive strategy by allowing individual bacteria to avoid pneumocin mediated lysis. Because immunity tends to be restricted to specific pneumocins, defensive immunity can be offset by altering the repertoire of secreted peptides. Finally, production of distinct BlpC types restricts the broadcast of the “on” signal to only similarly responsive members of the population. This prevents the stimulation of neighboring strains that may be capable of producing a protective response. In addition to providing a direct competitive advantage during colonization, pneumocin secretion may also provide a means for genetic adaptation. Cross talk between the regulatory systems controlling pneumocin production and pneumococcal competence suggests that coordinate expression of the machinery required for horizontal gene transfer with the production of lytic peptides provides an adaptive advantage to pneumocin producing strains by increasing the pool of available genetic material.

Improving our knowledge of genetic transformation affecting the capsule biosynthesis locus is critical for better understanding of how pneumococci evolve in response to the introduction of vaccines targeting the bacteria's extracellular polysaccharides. Import of genetic polymorphisms at the polysaccharide locus has been observed in previous studies, but to what extent recombination can drive the evolution of serotypes remains not well understood. To study this process we examine the evolution within closely related, epidemiologically common serogroups 6, 19 and 23 from over 3,000 pneumococcal isolates collected in a refugee camp in Mae La, Thailand. To identify a clonal phylogeny for each of those serogroups we identify dense clusters of polymorphisms on each branch of a phylogenetic tree and assume them to be recombinations. By removing them, we arrive at clonal phylogenies of the studied serogroups, with recombination and mutation events mapped on each tree. This picture shows a strong impact of recombination on the emergence of capsular diversity, particularly in serogroup 6. Interestingly, it suggests that some serotypes have emerged multiple times, ie against independent clonal backgrounds, a scenario likely driven by the selective pressure of the immune system. Comparison of the inferred recombination sequences with the sequences of all 92 serotype sequences suggests other serogroups and nonpneumococcal species as likely sources of genetic diversity. Overall, this study suggests that the adaptive potential of recombination in Streptococcus pneumoniae may be even greater than currently assumed.

GENETIC RECOMBINATION AND NASOPHARYNGEAL CARRIAGE DYNAMICS IN PNEUMOCOCCAL SEROTYPE-1 STRAINS FROM AFRICA
C. Chaguza 1 , J.E. Cornick 1 , C. Chewapreecha 2 , F. Yalcin 2 , M. Senghore 3 , S. Govindpersad 4 , A. Von Gottberg 4 , J. Collard 5  Background and Aims: Streptococcus pneumoniae serotype 1 ranks among the most prevalent invasive pneumococcal serotypes worldwide, particularly in the developing countries. Although carriage is seen as a prerequisite for both invasive disease and genetic recombination, serotype 1 is rarely detected in the nasopharyngeal carriage. The rarity in detection during colonisation drove the assumption that it rarely undergoes recombination since it occurs exclusively in carriage. We aimed to test the hypothesis that recombination does not occur in serotype 1 strains and to establish its association to the geographical origin of the strains and its consequence to nasopharyngeal carriage. Methods: We analysed 448 Illumina sequenced genomic-DNA libraries collected through the Pneumococcal African Genomics (PAGe) Consortium using a novel approach for detecting recombinant DNA fragments by statistical evaluation of polymorphism density in aligned sequences. Results: Almost 23965 segregating (polymorphic) sites were identified and ≥80% of these emerging through >300 genetic recombination exchanges. Genes encoding antimicrobial resistance targets were also frequently involved in recombination. Furthermore, we observed a high geographical clustering, clonal population structure and variation in the levels of recombination between geographically distinct strains. Conclusion: The presence of both ancestral and recent genetic recombination events suggests its role in the evolution of serotype 1 strains. The findings further suggest that the rarity in the nasopharyngeal carriage could explain the observed slow acquisitions of DNA as a consequence of limited exposures. Variation in the levels of recombination is driven by the differences in selective pressures in different locales.

ISPPD-0282
The Promiscuous Pneumococcus---Evolution and Biology Background and Aims: The differing propensity of pneumococcal strains to establish carriage is of importance in transmission and disease. We compared two strains (6B and 23F) with previously similar carriage rates in human challenge experiments and found reduced carriage of the 23F strain. Methods: 60 healthy adults were intranasally inoculated with type 6B and 60 adults with type 23F Streptococcus pneumoniae in dose ranging studies. Carriage rates detected by nasal wash and microbiological culture were compared. The strains were also compared in vitro for colony morphology, adherence to cultured nasopharyngeal epithelial cells, complement deposition and opsonophagocytic killing. The whole genome sequences of both strains were also compared. Results: There was markedly greater colonisation of human volunteers by type 6B compared to 23F (p<0.0001). The 6B strain showed greater adherence than 23F using nasopharyngeal epithelial cells (p=0.03) and was less susceptible to complement deposition (p<0.001). There was no difference in opsonophagocytic killing (p=0.1). Two genes, pcpA and aliB, were unique to 6B while the 23F strain had an amiC frameshift mutation. The amiC mutation was demonstrated to drastically reduce carriage in a mouse model. Conclusion: Dramatic differences in carriage rates were associated with differences in protein expression particularly amiC, pcpA and aliB. Murine models and previous human studies suggest that amiC is the most significant of these factors. Background: Invasive pneumococcal disease is a major cause of morbidity and mortality, presenting invasive infections such as lobar pneumonia, bacteremia and meningitis. The increasing incidence of penicillin resistance in Stretococcus pneumoniae is a growing concern. The evolution of this species of bacteria has enabled it to develop resistance to many other antibiotics. Aim: In this study, the Affymetrix Genechip microarray was employed to investigate the role of a wide range of genes involved in the development of penicillin resistance. Methods: Strains with different susceptibilities to penicillin was extracted before and after been exposed to penicillin stress. The respective cDNA was hybridized onto gene chip expression arrays masked with probes representing the known genome of S. pneumoniae TIGR4. Results: Genes encoding choline binding proteins (CBPs) showed significant levels of expression. cbp C gene showed an increased mRNA expression level in the penicillin resistant strain with a 4.96 fold, whereas the cbp genes A, D, J, G and F showed decreased expression levels in the penicillin resistant strain upon penicillin stress. No significant change was observed in the expression levels of cbps A, C, J, G and F in the penicillin sensitive strain. This suggests that the decreased expression of these genes in the resistant strain reduces its capacity to undergo cell separation and autolysis, hence failing to lyse and become tolerant. Conclusion: Resistance to penicillin might be due to impaired autolytic pathway which may have been modified with various adaptive regulatory mechanisms within the bacterial cell. Streptococcus pneumoniae can invade host immune system with the help of virulence factors that interact directly with host cellular receptors. One such virulence factor is Pneumococcal surface protein C (PspC). Whole genome sequence analyses of various clinical isolates revealed the presence of two different pspC genes in few strains, pspC1 encoding a choline binding version and pspC2 encoding a cell wall anchored LPXTG protein. Interestingly, PspC2 displayed an overall low sequence homology to various PspC proteins which are known to interact with human Factor H (FH). Further investigations showed that despite of lacking the conserved hexapeptide motif known to bind FH, PspC2 binds to human FH through its amino terminal domain. Unlike other members of the PspC protein family, PspC2 utilizes a novel sequence of amino acids for this interaction. Here we defined a novel FH binding motif utilized by PspC2 to interact with human FH. Further structural and biochemical investigations will provide more insights into the molecular basis of PspC2-FH interaction and its role in pneumococcal disease. Background and Aims: The psaBCAD operon of Streptococcus pneumoniae encodes an ABC-Mn 2+ -permease complex (psaBCA) and thiol peroxidase (tpxD), which may be co-transcribed. We have recently shown that TpxD expression is modulated in accordance with H 2 O 2 concentrations, and that TpxD plays a role in the precise control of H 2 O 2 levels. This study aimed to show that TpxD mediates pneumococal responses to H 2 O 2 . Methods: Wild-type D39, D39ΔtpxD and D39ΔpsaR were used. TpxD expression was determined by real-time PCR (RT-PCR) and immunoblotting. Global gene expression was checked by microarray analysis. Detection of reversibly oxidized bacterial cysteine residues was done by using biotinylated iodoacetamide followed by immunoblotting.

THIOL PEROXIDASE MEDIATES STREPTOCOCCUS PNEUMONIAE RESPONSES TO OXIDATIVE STRESS
Results: Challenging D39 with H 2 O 2 resulted in psaBCA down-regulation while tpxD expression was up-regulated. However, in ΔtpxD and ΔpsaR-mutants, H 2 O 2 had no effect on psaBCA expression, implying that both TpxD and PsaR are involved in the effect of H 2 O 2 on the psa operon. Microarray analysis revealed that in D39 challenged with H 2 O 2 , 148 genes were differentially expressed, of which 16 were transcriptional regulators. Conversely, H 2 O 2 had only marginal effect on the transcriptome of D39ΔtpxD-mutant, indicating that TpxD mediates the pneumococcal response to H 2 O 2 . Furthermore, the level of reversibly oxidized cysteine residues in protein extracts of H 2 O 2challenged bacteria was higher compared to untreated bacteria, while in the ΔtpxD-mutant there was almost no effect. Conclusions: Our data suggest that TpxD exhibits two functions associated with H 2 O 2 reduction: (i) It transmits oxidative signals to upstream effectors, such as transcription factors; (ii) It provides antioxidant defense by direct reduction of H 2 O 2 .

ISPPD-0123
The Promiscuous Pneumococcus---Evolution and Biology Background: Colonization and persistence in the human nasopharynx are prerequisites for Streptococcus pneumoniae (Sp) disease and carriage, which normally occurs during early childhood. Studies related to the mechanism of persistence have revealed a number of colonization and virulence factors as well as regulators implicated in nasopharyngeal colonization and pathogenesis. Expression of genes encoding these factors has never been studied in the human nasopharynx. Therefore, this study developed the technology to detect Sp genes in human nasopharyngeal samples and quantified mRNA transcripts of genes shown to be important for persistence and virulence. Methods: Nasopharyngeal samples were collected from healthy children <3 years of age in Peru. DNA and total RNA were extracted with commercial kits (Qiagen). A panel of 21 primers that amplified Sp sequences were designed, in silico analyzed, and their specificity was validated against 20 related strains present in the human nasopharynx. Results: Primers were utilized in conventional PCR, quantitative (q)PCR reactions to find that 100% of samples contained the genes ply, lytC, pavA, lytA, comD, codY and mgrA, whereas the following set of genes were present in >90% of samples including nanA, nanB, pspA, and rrgB. Gene expression studies, (q)RT-PCR, of these 11 targets revealed that lytC, lytA and nanB are highly expressed whereas all others, except rrgB, have a moderate to low level of expression. Conclusion: This is the first study to evaluate expression of virulence, colonization and regulator related genes in the nasopharynx of healthy children and highlights the importance of lytC in nasopharyngeal persistence.

POPULATION DYNAMICS OF STREPTOCOCCUS PNEUMONIAE VACCINE SEROTYPES ON HUMAN PHARYNGEAL CELLS
J.E. Vidal 1 , F. Sakai 1 , J. Concepción-Acevedo 2 , J.R. Shak 1 , S. Chochua 1 , K.P. Klugman 1 1 Hubert Department of Global Health, Emory University, Atlanta, USA; 2 Department of Biology, Emory University, Atlanta, USA Background: Streptococcus pneumoniae (Sp) persists in the human nasopharynx forming biofilms. Nearly half children carry at least 2 different serotypes. On abiotic surfaces, biofilms are regulated by the quorum sensing system LuxS/AI-2. However, virtually nothing is known about biofilms grown on human cells. Methods: Our well established biofilm bioreactor (biBio), with human pharyngeal cells, was utilized to simulate the microenvironment Sp finds in the respiratory epithelium. biBio was infected with a mixture of two vaccine types (2SsBs). The structure and population dynamics of 2SsBs were studied. Results: Biofilms formed on human cells maintained their population, average of ~5.6x10 8 CFU of biofilm cells/ cm 2 , 24 h post-inoculation. Planktonic/invasive Sp cells coming off the biofilm structure remained stable (~1.98x10 6 CFU/ml). Populations of D39ΔluxS, D39ΔcomC or D39Δply were significantly lower than wt biofilms. Transcriptional upregulation of important genes was observed in biofilms grown on human cells (i.e. lytA, psaA, pspA and ply). Ply was located on the cell wall and the biofilm matrix. Mixtures of two vaccine serotypes (similar or different CSPpherotype, e.g. 4, 6B, 19A, 19F or 23F) were inoculated into the biBio to find out three population dynamics: 1) biomass of both vaccine types remained similar, 2) population of a serotype predominated (fitness advantage) or 3) persistence of a serotype eradicated the other strain. Confocal microscopy studies revealed fused 2SsBs structures made by a mixture of specific serotypes. Conclusion: Population dynamics of Sp vaccine types is fine-tuned by specific mechanisms allowing, or not, persistence of Sp strains on human pharyngeal cells.

STREPTOCOCCUS PNEUMONIAE SEROTYPE 1'S ESCAPE FROM DETECTION IN CARRIAGE: A GENETIC MECHANISM?
M. Alaerts 1 , E. Heinsbroek 2 , A. Chirambo 1 , A. Phiri 3 , N. Background: Streptococcus pneumoniae Serotype 1 causes a high proportion of invasive pneumococcal disease (IPD), particularly in developing countries, yet it is rarely detected in healthy carriers. This may occur because it is carried for very short periods of time and transmission rates are high or alternatively, because it goes undetected in carriage. We hypothesized that Serotype 1 has the capacity to switch between a non-encapsulated carriage state colonizing the nasopharynx and an encapsulated virulent state causing IPD, through genetic recombination. Deletion and re-insertion of the functional portion of the Serotype 1 cps locus through homologous recombination involving the flanking IS1167 sequences could provide such a switch mechanism. This deletion was recently detected in 4 nontypeable isolates from Native American communities. Methods: Monthly nasopharyngeal swabs were taken from each member of households participating in a longitudinal carriage study in Karonga, Malawi. Households with at least one Serotype 1 carriage event were recruited to this study. Molecular methods were used to test for the presence of the Serotype 1 deletion variant in all pneumococci positive swabs. Results: Five households were selected with a total of 112 swabs available for testing. Pneumococcal growth was obtained from 65 swabs. Six swabs were Serotype 1 positive. The deletion variant was not detected. Conclusion: We did not detect the Serotype 1 cps deletion variant in this pilot study. We plan a larger study which includes nasopharyngeal aspirates and will use DNA extracted directly from samples without a culturing step to increase sensitivity. Background and Aims: The aim of the present study was to find out the prevalence of various serogroups/types of Streptococcus pneumoniae (SP) isolated from clinical samples. Methods: A total of 544 strains of S. pneumoniae isolated from patients aged 1 mo-60 yr attending the outpatient Department of KSCH and Smt .S.K. Hospital, New Delhi, India during 1988-1997 were biochemically characterized using standard procedures. The serotyping of the strains was carried out using Quellung reaction using type specific pneumococcal antisera. Results: S. pneumoniae strains (86.4%) were found to be typable and they constituted 18 serogroups/types. The percentage of SP strains isolated in children (<13yr) and adults/adolescents (>13yr) from CSF and blood were 31.3 and 13.2 and 12.1 and 20.2 respectively. The other samples that yielded SP were pus (17.5%), Sputum/ bronchial aspirate/gastric aspirate (17.1%), peritoneal fluid (3.5%), conjunctival swab (2.4%), and ear swab (1.8%). The most common serogroups/types encountered in the present study were type 1 (32.3%), 3 (16.5%), 19 (10.3%) followed by 5 (8.0%), 15 (7.6%), 6 (6.8%), 7 (6.5%), 27 (5.1%), 13 (4.6%), 23 (3.8%), 9 (2.3%), 14 (2.0%), 18 ,12, 29, 37 and 39 (1.1%) each and 21 (0.6%). 10 out of 18 serogroups/types isolated in the present study are included in the 23valent pneumococcal vaccine. All the strains were found to be sensitive to Penicillin. Conclusion: The study emphasizes the need for continued epidemiological surveillance of pneumococcal infections as it helps to monitor regularly the prevalent serogroups/types of S. pneumoniae circulating in the community. In the future for the development of a safe successful polyvalent vaccine suitable for Indian conditions should include locally prevalent serogroups/types known to cause invasive infections in children and adults.

Microbiology, Lady Hardinge Medical College and Associated Hospitals, Delhi, India
Background and Aims: The aim of the present study was to find out the distribution of Streptococcus pneumoniae serotypes causing eye infections. The antimicrobial susceptibility pattern of the strains was carried out to find out the prevalence of drug resistance among the commonly used antibiotics. Methods: A total of 176 strains of S. pneumoniae isolated from patients aged 1m-60 yr attending the outpatient Department of Eye of KSCH and Smt .S.K. Hospital, New Delhi, India during 1981-1995 were biochemically characterized using standard procedures. The serotyping of the strains was carried out using Quellung reaction using type specific pneumococcal antisera. The antibiotic susceptibility pattern of these strains was determined by disk diffusion method. Results: S. pneumoniae strains (96.6%) were found to be typable and they constituted 16 serogroups/types. The most common serotype was 3 (19.6%) followed by 12 (15.3%), 6 (13.6%), 33 (11.4%), 28 (8.0%), 4.5% each of 19 and 46, 45 (3.4%) and 2.8% each of 11, 13, 23, 40 (2.3%), 18, 29 (,1.7%), 1.1% each of 8 and 21. The eye infections caused by these strains were dachryocystitis ( 47.2%), conjunctivitis (45.5%), endopthalmitis (4.0%) and lacrymal gland infection (3.4%). All the strains were sensitive to penicillin, ampicillin, cloxacillin chloramphenicol, erythromycin, co-trimoxazole, gentamicin and streptomycin. There were 2 strains, 1 each of serotype 3 (endopthalmitis) and 12 (conjunctivitis) resistant to tetracycline.  Susceptibility of S. pneumoniae to penicillin, chloramphenicol, tetracycline and erythromycin were 82%, 96%, 59% and 92% respectively. Those of GBS were 100%, 99%, 91% and 85% respectively. Conclusion: Our preliminary data shows a negative association between S. pneumoniae and GBS in Gambian infants. This may have important clinical implications for species replacement post pneumococcal vaccination. Background and Aims: Pneumococcal conjugated vaccines (PCV) have been registered, but not reimbursed in Serbia. The aim of this study was to determine serotypes and PCV coverage of invasive Streptococcus pneumoniae in Serbia. Methods: A total of 78 invasive S. pneumoniae isolates (25 from children; 53 from adults) were collected throughout country during the period January 2012-October 2013. The serotypes, antimicrobial susceptibility and macrolide resistance genes were determined using Quellung reaction, disk diffusion test and PCR, respectively. Results: Ten serotypes accounted for 79.5% of the isolates (3, 4, 14, 7F, 19F, 23F, 22F, 9V, 9N and 8), among which the most prevailing was serotype 3 (20.5%). The most common serotypes among children ≤5 years were: 19F (15.8%) and 23F (15.8%), while serotype 3 predominated among adults ≥65 years (27.8%). The serotype coverage of 7-valent, 10-valent, and 13-valent PCV for isolates originated from children <5years was: 52.6%, 68.4% and 84.2%, respectively. Penicillin non-susceptibility was strongly associated with serotypes 14, 23F and 19F. Penicillin and cefotaxime/ceftriaxone resistance for meningitis strains were 44% and 18.5%, while for non-meningitis isolates were 11% and 4.9%, respectively. Among 17 macrolide resistant isolates (21.8%), 64.7% harbored ermB gene and 35.3% mefA. Co-resistance to erythromycin and penicillin was 9% of strains. All isolates were fully susceptible to vancomycin, linezolid, fluoroquinolones, telithromycin and rifampicin. Conclusion: The most common serotypes causing invasive pneumococcal diseases in Serbia were: 3, 4, 14, 7F, 19F and 23F. PCV-7, PCV-10 and PCV-13 vaccine coverage in children ≤5 years were 52.6%, 68.4% and 78.9%, respectively. Background: Pneumococcal serotypes and sequence types (ST) are closely associated. The introduction of the pneumococcal conjugate vaccine (PCV) should select against STs that are associated with vaccine serotypes. Serotype 6B is included in PCV10 that was introduced in Kenya in 2011. We examined the change in proportions of serogroup 6 serotypes before and after vaccine introduction, and investigated the diversity among serogroup 6 isolates from Kilifi. Methods: Invasive and carriage serogroup 6 isolates from hospital (Kilifi District Hospital) and community samples from children and adults collected between 1994 and 2013 were investigated. We serotyped, by Quellung and PCR, 345 isolates and derived whole genome sequences, using the Illumina technology, for 57 isolates collected before 2008. Results: The proportion of serotype 6B among carriage isolates declined after vaccine introduction (9.7% to 3.3%; p<0.0001). The major STs circulating in Kenya differed from the major STs of serotype 6 strains circulating elsewhere in the world and there was considerable diversity in these STs. At the capsular locus, serotype 6B grouped into three clusters, with high between-cluster variation. Serotype 6A formed seven clusters but with less between-cluster variation. The most prominent serotype 6B subtype, comprising 75% of isolates, is 6B-III, which is very similar to serotype 6E. Conclusion: Although there is correlation between STs and serotype, we found isolates of unrelated STs had the same subtype. Streptococcus pneumoniae in Kilifi appears to be genetically diverse and distinct from wellcharacterized populations elsewhere. It is unclear whether this will affect the success of vaccination. Background and Aim: Pneumococcal neuraminidase NanA cleaves both α2-3 and α2-6 sialyl linkages, while both NanB and NanC can cleave only α2-3 sialyl linkages. In the present study we aimed to examine the role of the neuraminidases NanB and NanC in the pathogenesis of systemic pneumococcal infection. Methods: Mutants of both nanB and nanC were constructed in Streptococcus pneumoniae serotype 6B by doublecrossover recombination mutagenesis. BALB/c mice were injected with 1 × 10 8 CFU S. pneumoniae in PBS through tail vein intravenously. Thomsen-Friedenrich antigen (T-antigen) exposure on the mice RBC was detected by flow cytometer. Results: To monitor the influence of NanB and NanC on the survival of S. pneumoniae in mice bloodstream, BALB/c mice were intravenously challenged with wild type S. pneumoniae, nanB and nanC mutants. After specific time points day 1-, 2-, 3-, 5-and 7-, the blood was collected by retro-orbital puncture and subjected to bacterial culture. After 2 days infection, nanB mutant had a significantly lower bacteria counts in bloodstream than wild type (p = 0.0207). nanB mutant had a significantly lower survival rate in bloodstream than wild type (p = 0.0340). nanB and nanC mutants had a lower T-antigen exposure on the RBC than wild type. After wild type S. pneumoniae infection, prominent T-antigen exposure on the glomeruli was observed. After infection with nanB and nanC mutants, less T-antigen exposure on the glomeruli was observed. Conclusion: In addition to NanA, NanB and NanC also play a role in pneumococcal infection. Although NanB and NanC can selectively release α2-3 sialyl linkages only, they can provide an additive effect along with NanA to expose Thomsen-Friedenrich antigen on various cells in host.

NanB AND NanC IN THE PATHOGENESIS OF SYSTEMIC PNEUMOCOCCAL INFECTION
No conflict of interest pneumonia 2014 Volume 3 132 ISPPD-9 / pneumonia 2014 Mar 9-13;3:1-286 Background: Pneumococcal infections remain major public health problem worldwide for all age groups, and also for aged population. There is still no molecular epidemiological characteristic of Streptococcus pneumoniae isolates gained from aged people with different forms of pneumococcal infections.Aims: was to perform multilocus sequence typing (MLST) in strains of S. pneumoniae gained in patients elder of 60 years at the territory of Primorsky region as the most southern part of the Russian Far East. Methods: MLST was conducted with housekeeping genes on standard method on recommendations of MC Enright (1998) et al. with previous study of antimicrobial agents resistance and serotyping. Results: There were taken 50 isolates ( group 1, from patients with community-acquired pneumonia), 30 isolates ( group 2, from patients with invasive pneumococcal infections such as bacteremia and meningitis). Isolates form 1st group included 24 serotypes ( 26 of non-typable) and 22 sequence types ( ST) according to MLST of which 7 were novel, 8 were of Taiwanese clones 19,26) of 19F serotype, 3 were of R6, EU38, SP95. In group 2 of 20 strains there was revealed 8 serotypes ( 12 of non-typable) and 6 ST of Taiwanese clones. In strains from patients there were isolated 18% resistant to erythromycin, 16% tetracycline, 4 % chloramphenicol. The pattern of antimicrobial agents resistance from the strains of 2nd group was 35% to erythromycin, 15% to tetracycline, 5% to fluoroquinolones. Conclusion: Multilocus sequence typing allows us to suggest the epidemiological significance of Taiwanese isolates of S. pneumoniae in our region. Aims: The aim of the study is to analyze the serotypes diversity of PMEN clones among pneumococcal invasive isolates before and after the introduction in 2010 of PCV13 vaccine in Catalonia. Methods: Prospective study which included all pneumococcal invasive isolates received in the Catalonia Support Molecular Laboratory for Invasive Pneumococci. All isolates were characterized by serotyping and clonal type. Genetic diversity of isolates was estimated using the Simpson's index of diversity. Periods 2009Periods -2010Periods and 2011Periods -2012 were considered prevaccine and vaccine period respectively. Results: A total of 1831 invasive pneumococci were registered and 1810 (98.8%) were viable for serotype and clonal study. 57.2% were isolated during 2009-2011 and 42.8% during 2011-2012. 20.3% were isolated in patients <5 years old. PCV13 serotypes and PMEN clones were detected in 1131 and 759 isolates respectively. PCV13 serotypes decreased from 69.3% during prevaccine-period to 53.3% during vaccine-period (p < 0.001). PMEN clones declined from 44.1% to 39.1% (p = 0.03). Among PMEN clones non-PCV13 serotypes increased from 10.1% during prevaccine-period to 19.5% during vaccine-period (p < 0.001). A rise of serotype diversity among PMEN clones was observed comparing the two periods: Simpson's index of diversity of 0.821 (95% CI 0.799-0.843) vs. 0.857 (95% CI 0.837-0.877). Conclusion: An increase of serotype diversity among PMEN clones due to an increase of non-PCV13 serotypes has been observed after the introduction of PCV13 vaccine in Catalonia.

IN VIVO TRANSCRIPTOMIC ANALYSIS USING RNA-SEQ OF PNEUMOCOCCAL GENES EXPRESSED IN THE PLEURAL CAVITY
N.D. Ritchie 1 , S.C. Irvine 1 , T.J. Evans 1 1 Infection Immunity & Inflammation, University of Glasgow, Glasgow, United Kingdom Introduction: Specific patterns of gene expression allow the pneumococcus to occupy different niches within the host. We set out to examine what genes were differentially expressed in the pleural cavity following in a murine model of pneumonia Methods: Mice were infected by the intranasal route with strain SRL1 (serotype1) that resulted in pneumonia and pleural infection. Bacteria recovered from the pleural space were treated immediately with Trizol and RNA purified. RNA was similarly purified from SRL1 grown in broth. Following library construction, Paired-end RNA transcripts were sequenced using a Hi-Seq and analysed using CLC Genomics Workbench. Changes in transcript levels between pleura and broth were assessed for statistical significance using Kal's z test; fold-changes > 2 with Bonferroni corrected p < 0.05 were classified as significant. Results: Sequence reads in excess of 15 million were obtained from both samples. 983 genes were significantly up-regulated (2 -40 fold) in pleura compared to broth and 278 were down-regulated (2 -32 fold). Upregulated genes included the blp/pnc operon encoding bacteriocin and related genes, purine biosynthetic genes, the N-acetyl glucosamine specific phosphotransferase system and those controlling competence. Down-regulated genes included many ribosomal proteins, the htrA protease, genes controlling galactose metabolism and the H 2 O 2 producing enzyme spxB. Conclusion: RNA-seq is a powerful tool to identify in vivo patterns of gene expression. Many genes identified as upregulated in the pleura were not previously known to be important in virulence. Further work will define what roles they play in establishing pleural infection.

INFLUENZA A VIRUS-MEDIATED PRIMING OF LUNG INNATE IMMUNITY ENHANCES CYTOKINE SECRETION OF LUNG CELLS AND INHIBITS CLEARANCE OF STREPTOCOCCUS PNEUMONIAE
V. Sender 1 , B. Henriques-Normark 1 1 Microbiology Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden Both, influenza A virus and Streptococcus pneumoniae are a leading cause of morbidity and mortality worldwide. However, most influenza-related mortality is not due to the viral infection alone but rather secondary bacterial infections, mainly caused by S. pneumoniae. The mechanisms driving virulent influenza co-infection are poorly defined, making it difficult to develop effective therapeutic strategies. This study investigates early signaling events evoked by influenza infections affecting the innate immune response and cellular clearance mechanisms in the lung. Using an in vivo model of subsequent infections with influenza A virus and S. pneumonia via the intratracheal infection route we show that sublethal influenza infections clearly predispose for severe pneumococcal infections even at low bacterial doses. Co-infected C57BL/6 mice are more susceptible to pneumococcal infection than single-infected mice, resulting in drastically less survival and earlier development of pneumonia and septicemia. Preliminary flow cytometry data on kinetic changes of cell populations in lavage fluid and post-lavage lung tissue from single and co-infected animals, as well as viable bacterial and viral counts in lungs suggest that virus-induced innate defects impair early bacterial clearance, thereby supporting the development of secondary bacterial pneumonia. Cytokine analyses reveal an up-regulation of IL-6 and TNF-α in bronchoalveolar lavage from co-infected mice compared to single-infected mice. Further investigations of lung cell populations, their possible cross-activation, and reduced clearance potential during influenza-pneumococcal co-infections will increase our understanding of the mechanisms behind influenza-mediated sensitization of the host to pneumococcal infections which will help to find treatment regimens that combat secondary bacterial infections.

NEXT-GENERATION SEQUENCING ANALYSIS OF A PUTATIVE IRON-RESPONSIVE TRANSCRIPTIONAL REGULATOR MUTANT IN STREPTOCOCCUS PNEUMONIAE TIGR4
T. Tower 1 , R. Gupta 2 , E. Swiatlo 2 , B. Nanduri 1 1 College of Veterinary Medicine, Mississippi State University, Starkville, USA; 2 Department of Microbiology, University of Mississippi Medical Center, Jackson, USA Iron is an essential micronutrient for many pathogenic bacteria and plays an important role in gene regulation. Cellular response to environmental iron is critical for pathogens such as Streptococcus pneumoniae that inhabit multiple anatomical sites in the host, since both form and quantity of available iron differ greatly in each location. The genome of serotype 4 pneumococcal strain TIGR4 encodes a putative iron-dependent transcriptional regulator (Idtr). In this study we used the next-generation sequencing (NGS) Illumina MiSeq platform to analyze differential gene expression between TIGR4 and an isogenic mutant deleted at idtr (Δidtr) cultured in vitro in both high and lowiron conditions. Total RNA was collected and rRNA was removed before reverse transcription and sequencing. The RNA-Rocket software suite was used to align the reads and identify significant changes in gene expression. A total of 291 genes were identified as differentially-expressed at ±1.5-fold difference. These genes included choline-binding proteins, heat shock and stress-induced proteins, as well as multiple sugar transport and metabolism genes. These data suggest that intracellular iron levels may affect the regulation of pneumococcal gene expression in different environmental niches in which iron availability varies widely. Background and Aims: The German National Reference Center for Streptococci (GNRCS) has been carrying out surveillance of invasive pneumococcal disease since 1992. Of a total of 37,000 pneumococcal isolates, about 400 were classified as non-typeable (NT). NT isolates are not covered by any of the current vaccines and may have the intrinsic capacity of being replacement serotypes. Additionally, they form the pool in which 'new serotypes' might be detected. Therefore, in this study we (re-)analyzed these isolates using modern molecular techniques. Methods: Identification of the isolates was re-assessed using our current expertise with the Quellung method, PCR of the lytA-gene followed by restriction-analysis, and sequencing of the sodA-gene. MLST, multiplex-PCR, BµG@Sarray and genomic-sequencing were used for identification and characterization of the NT isolates. Results: A total of 404 NT isolates were analysed. Of these, 114 (28.2%) were not Streptococcus pneumoniae -in part identified as S. pseudopneumoniae, 137 isolates (33.9%) were typeable using the Quellung method, most of them with rare serotypes (27,29,31,34,37,38). 123 isolates (30.4%) were really non-typeable, while 16 isolates were not viable (14 isolates have not been assessed so far). MLST of 54 real non-typeable isolates showed that 35 had STs occurring in mlst.net with reported serotypes. Microarray-analyses, genomic-sequencing and electronmicroscopy revealed point-mutations resulting in defective capsular-cassettes. Conclusion: Reassessment of NT-isolates might be useful as they may represent rare serotypes that were previously undetected. Among the remaining real non-typeable isolates some were identified having point-mutations in their capsular-genes. These isolates would have been falsely assigned serotypes using PCR-methods.

EPIDEMIOLOGICAL CHARACTERIZATION OF STREPTOCOCCUS PNEUMONIAE USING MULTI LOCUS SEQUENCE TYPING (MLST) ACROSS INDIA
R. Varghese 1 , R. Jeyaraman 1 , B. Veeraraghavan 1 , K. Thomas 2 1 Microbiology, Christian Medical College, Vellore, India; 2 General Medicine, Christian Medical College, Vellore, India Background and Aim: Streptococcus pneumoniae is a major cause of invasive bacterial disease in both children and adults worldwide especially in India. Geographical diversity of serotypes and serotype exchange (capsular switching) in S.pneumoniae limits the efficacy of new conjugate vaccines produced against limited number of serotypes. So serotyping and molecular typing has to go hand in hand to characterize pneumococcal population. In molecular typing MLST provides unambiguous data that are portable and comparable between the laboratories which in turn help us understand the population and evolution biology of these organisms. In these circumstances we commenced this study with the aim to identify the common invasive sequence types (using MLST) present over different regions of India and to check for correlation between serotypes and sequence types. Materials and Methods: 40 invasive S.pneumoniae isolates from children < 5 years ,collected from different sites within India was serotyped by Co-agglutination and reconfirmed by PCR, followed by MLST. Antimicrobial susceptibility profile was done using Vitek II system. Results: The most frequent serotypes were 6B, 14, 4, 1,19F and 6A. The isolates showed 100 % resistance to Cotrimoxazole followed by erythromycin (32.5%) and penicillin (15%). Serotypes 19F and 6B are common among the Penicillin resistant serotypes. Sequence types by MLST will be discussed during the presentation. Conclusion: Continued surveillance by serotyping and sequence typing combined with antimicrobial susceptibility profile is imperative owing the diverse epidemiology, emergence of multi drug resistance and vaccine escape recombinants of S.pneumoniae.

COINFECTION WITH INFLUENZA A VIRUS EXACERBATES PNEUMOCOCCAL ACUTE OTITIS MEDIA
J.T. Wren 1 , W.E. Swords 1 1 Department of Microbiology and Immunology, Wake Forest University, Winston-Salem, USA Background and Aims: Streptococcus pneumoniae (pneumococcus) is both a nearly ubiquitous nasopharyngeal colonizer of children but also the leading cause of acute otitis media (AOM), accounting for billions of dollars in healthcare expenditures each year. Multiple factors have been proposed to be involved in this transition from colonization to disease; two of which include influenza A virus (IAV) and pneumococcal phase variation. Indeed, IAV is frequently associated with pneumococcal AOM and nasopharyngeal bacterial phase variation may influence otopathogen ascension to the middle ear. Thus, our aim was to investigate the role of pneumococcal-influenza synergism and bacterial phase variation in the pathogenesis of AOM. Methods: Mice were infected intranasally with IAV followed by S. pneumoniae and the magnitude of nasal colonization, middle ear infection, and histopathological inflammation were assessed. Coinfection studies were also performed using isogenic pneumococcal phase variants. Results: Preceding IAV infection enhanced the magnitude of pneumococcal colonization by nearly 9-fold. Inflammation and neutrophilic infiltration was likewise increased in the nasopharynx following IAV infection. Further, IAV established a direct correlation between nasal bacterial load and middle ear ascension, such that the magnitude of infection was increased by nearly 40-fold. The effect of the phase of pneumococci in the nasopharynx on the IAV modulation of bacterial AOM is currently being studied. Conclusion: Based on these studies, we conclude that the preceding IAV infection is inflammatory in nature, disrupting stable pneumococcal colonization and resulting in an enhanced nasal bacterial burden that is then associated with increased bacterial ascension into the middle ear.

STUDIES ON PNEUMOCOCCAL ESTERASES
H. Kahya 1 , P.W. Andrew 1 , H. Yesilkaya 1 1 Infection Immunity & Inflammation, University of Leicester, Leicester, United Kingdom Background and Aims: The pneumococcus has four putative esterase genes (SPD_0534 (estA), SPD_0932, SPD_1239, and SPD_1506). These lipolytic enzymes have been reported to be important for bacterial physiology and virulence in other microorganisms. However, no detailed study has been done on pneumococcal esterases to assess their role in pneumococcal biology. Hence, this study aims to investigate esterases' role in pneumococcal physiology and virulence. Methods: Mariner mutagenesis was used for mutant construction. Total esterase activity was assayed by using chromogenic substrates. Metal affinity column was used to purify his-tagged recombinant proteins. Results: The highest level of esterase activity could be obtained with p-NPA, indicating that the pneumococcus has esterase specific for short acyl chains. All mutants displayed significantly less esterase activity than the parental strain, and the highest reduction in activity was observed in SPD_0534 mutant. The purified EstA showed optimal activity against p-NPA compared to other p-nitrophenyl esters. Michaelis-Menten kinetics showed that Vmax and Km of EstA were 339.2 mU±6.07 and 1.45 mM±0.16, respectively. In addition, EstA activity against Bovine Sub-maxillary Mucin (BSM), highly acetylated substrate, showed that acetate release increased in a time and concentration dependent manner. We also showed that pre-treatment of BSM by EstA increases sialic acid release by neuraminidase. Conclusion: All four genes contribute to total pneumococcal esterase activity though EstA is the major pneumococcal esterase. Pneumococcal esterases are specific for short acyl chains. EstA potentiate neuraminidase activity in vitro. Further work is underway to determine in vivo function of these enzymes. Background and Aims: Streptococcus pneumoniae depends on carbohydrates to produce its metabolic energy. Galactose is the main source of sugar in the respiratory tract, and can be found within the structure of mucin. Fermentative metabolism of galactose leads to generation of mixed acids. The enzyme pyruvate formate lyase (PFL, coded by pfl) is responsible for mixed acid fermentation and was also shown to be important for pneumococcal virulence. However, it is not known how pfl is regulated. Therefore, this study aims to investigate the transcriptional regulation of pfl. Methods: Microarray analysis of a pfl mutant was done. Metal affinity column was used to purify his-tagged recombinant proteins. SOEing-PCR was used for mutant construction. Electrophoretic mobility shift assays (EMSA) were done by using Molecular Probes fluorescence-based EMSA Kit (Invitrogen, UK). Results: Differential expression of seven transcriptional regulators was identified in a pfl mutant relative to the wild type. It was hypothesised that the proteins encoded by these genes are responsible for either direct or indirect regulation of pfl. EMSA results showed that three of these regulatory proteins bound to putative promoter region of pfl. To demonstrate the involvement of these regulators further in pfl regulation, we mutated the transcriptional regulators and grew the mutants in the presence of glucose or galactose with or without oxygen, and analysed the end products. The mutants had different growth patterns, and end-product composition than the wild type strain.

Conclusion:
The results show that some of the transcriptional regulators studied, are involved in the regulation of pfl.