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Pneumococcus is one of the most important human pathogens that causes life-threatening invasive diseases, especially at the extremities of age. Capsular polysaccharides (CPSs) are known to induce protective antibodies; however, it is not feasible to develop CPS-based vaccines that cover all of the 90 disease-causing serotypes. We applied a genomic approach and described the antibody repertoire for pneumococcal proteins using display libraries expressing 15-150 amino acid fragments of the pathogen's proteome. Serum antibodies of exposed, but not infected, individuals and convalescing patients identified the ANTIGENome of pneumococcus consisting of approximately 140 antigens, many of them surface exposed. Based on several in vitro assays, 18 novel candidates were preselected for animal studies, and 4 of them showed significant protection against lethal sepsis. Two lead vaccine candidates, protein required for cell wall separation of group B streptococcus (PcsB) and serine/threonine protein kinase (StkP), were found to be exceptionally conserved among clinical isolates (>99.5% identity) and cross-protective against four different serotypes in lethal sepsis and pneumonia models, and have important nonredundant functions in bacterial multiplication based on gene deletion studies. We describe for the first time opsonophagocytic killing activity for pneumococcal protein antigens. A vaccine containing PcsB and StkP is intended for the prevention of infections caused by all serotypes of pneumococcus in the elderly and in children.

Paneth cell-derived enteric antimicrobial peptides provide protection from intestinal infection and maintenance of enteric homeostasis. Paneth cells, however, evolve only after the neonatal period, and the antimicrobial mechanisms that protect the newborn intestine are ill defined. Using quantitative reverse transcription-polymerase chain reaction, immunohistology, reverse-phase high-performance liquid chromatography, and mass spectrometry, we analyzed the antimicrobial repertoire in intestinal epithelial cells during postnatal development. Surprisingly, constitutive expression of the cathelin-related antimicrobial peptide (CRAMP) was observed, and the processed, antimicrobially active form was identified in neonatal epithelium. Peptide synthesis was limited to the first two weeks after birth and gradually disappeared with the onset of increased stem cell proliferation and epithelial cell migration along the crypt-villus axis. CRAMP conferred significant protection from intestinal bacterial growth of the newborn enteric pathogen Listeria monocytogenes. Thus, we describe the first example of a complete developmental switch in innate immune effector expression and anatomical distribution. Epithelial CRAMP expression might contribute to bacterial colonization and the establishment of gut homeostasis, and provide protection from enteric infection during the postnatal period.

A group A Streptococcus (GAS) isolate, serotype M12, recovered from a patient with streptococcal toxic shock syndrome was analyzed for superantigen-carrying prophages, revealing phi149, which encodes superantigen SSA. Sequence analysis of the att-L proximal region of phi149 showed that the phage had a mosaic nature. Remarkably, we successfully obtained lysogenic conversion of GAS clinical isolates of various M serotypes (M1, M3, M5, M12, M19, M28, and M94), as well as of group C Streptococcus equisimilis (GCSE) clinical isolates, via transfer of a recombinant phage phi149::Km(r). Phage phi149::Km(r) from selected lysogenized GAS and GCSE strains could be transferred back to M12 GAS strains. Our data indicate that horizontal transfer of lysogenic phages among GAS can occur across the M-type barrier; these data also provide further support for the hypothesis that toxigenic conversion can occur via lysogeny between species. Streptococci might employ this mechanism specifically to allow more efficient adaptation to changing host challenges, potentially leading to fitter and more virulent clones.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Pneumococci can counteract the action of neutrophils with an antiphagocytic capsule and through electrochemical repulsion of antimicrobial peptides via addition of positive charge to the surface. Pneumococci are captured, but not killed in neutrophil extracellular traps (NETs). Here, we study the role of the polysaccharide capsule and lipoteichoic acid (LTA) modification on pneumococcal interaction with NETs. Expression of capsule (serotypes 1, 2, 4 and 9V) significantly reduced trapping by NETs, but was not required for resistance to NET-mediated killing. Pneumococci contain a dlt operon that mediates the incorporation of d-alanine residues into LTAs, thereby introducing positive charge. Genetic inactivation of dltA in non-encapsulated pneumococci rendered the organism sensitive to killing by antimicrobial components present in NETs. However, the encapsulated dltA mutant remained resistant to NET-mediated killing in vitro. Nevertheless, in a murine model of pneumococcal pneumonia, the encapsulated dltA-mutant strain was outcompeted by the wild-type upon invasion into the lungs and bloodstream. This suggests a non-redundant role for LTA alanylation in pneumococcal virulence at the early stage of invasive disease when capsule expression has been shown to be low.

Surveillance of group A streptococcal (GAS) infections was undertaken as a major component of the European Commission-funded project on severe GAS disease in Europe (strep-EURO). One aim of strep-EURO was to improve the quality of GAS characterization by standardization of methods. An external quality assurance study (EQA) was therefore carried out to evaluate current global performance. Eleven strep-EURO and seven other streptococcal reference centers received a panel of 20 coded GAS isolates for typing. Conventional phenotypic typing (based on cell surface T and M protein antigens and opacity factor [OF] production) and molecular methods (emm gene typing) were used either as single or combined approaches to GAS typing. T typing was performed by 16 centers; 12 centers found one or more of the 20 strains nontypeable (typeability, 89%), and 11 centers reported at least one incorrect result (concordance, 93%). The 10 centers that tested for OF production achieved 96% concordance. Limited availability of antisera resulted in poor typeability values from the four centers that performed phenotypic M typing (41%), three of which also performed anti-OF typing (typeability, 63%); however, concordance was high for both M (100%) and anti-OF (94%) typing. In contrast, the 15 centers that performed emm gene sequencing achieved excellent typeability (97%) and concordance (98%), although comparison of the performance between centers yielded typeability rates from 65 to 100% and concordance values from 83 to 100%. With the rapid expansion and use of molecular genotypic methods to characterize GAS, continuation of EQA is essential in order to achieve international standardization and comparison of type distributions.

Toll-like receptor 9 (TLR9) induces an inflammatory response by recognition of unmethylated CpG dinucleotides, mainly present in prokaryotic DNA. So far, TLR9-deficient mice have been shown to be more sensitive than wild-type mice to viral, but not to bacterial infections. Here, we show that mice deficient in TLR9 but not in TLR1, TLR2, TLR4 and TLR6 or IL-1R/IL-18R are more susceptible to a respiratory tract bacterial infection caused by Streptococcus pneumoniae. Intranasal challenge studies revealed that TLR9 plays a protective role in the lungs at an early stage of infection prior to the entry of circulating inflammatory cells. Alveolar as well as bone marrow-derived macrophages deficient in either TLR9 or the myeloid adaptor differentiation protein MyD88 were impaired in pneumococcal uptake and in pneumococcal killing. Our data suggest that in the airways, pneumococcal infection triggers a TLR9 and MyD88-dependent activation of phagocytic activity from resident macrophages leading to an early clearance of bacteria from the lower respiratory tract.

Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re-differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti-chlamydial drugs.

Neutrophil extracellular traps (NETs) are considered to be part of the human innate immunity because they trap and kill pathogens. NETs are formed by activated neutrophils and consist of a DNA backbone with embedded antimicrobial peptides and enzymes. They are involved in host defense during pneumococcal pneumonia, streptococcal necrotizing fasciitis, appendicitis and insemination. Recently, bacterial virulence factors that counteract NETs have been identified. These include the degradation of the NET-backbone by DNases enabling the liberation of bacteria from NETs, as well as capsule formation, which reduces bacterial trapping. Furthermore, pathogens can resist NET-mediated killing by adding positive charge to their cell surface.

While newly developed technologies have revolutionized the classical approaches to combating infectious diseases, the difficulties associated with developing novel antimicrobials mean that these technologies have not yet been used to introduce new compounds into the market. The new technologies, including genomics and structural biology, open up exciting possibilities for the discovery of antibiotics. However, a substantial effort to pursue research, and moreover to incorporate the results into the production chain, is required in order to bring new antimicrobials to the final user. In the current scenario of emerging diseases and the rapid spread of antibiotic resistance, an active policy to support these requirements is vital. Otherwise, many valuable programmes may never be fully developed for lack of "interest" and funds (private and public). Will we react in time to avoid potential disaster?

The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3beta to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity.

Streptococcus pneumoniae (pneumococcus) is the most common cause of community-acquired pneumonia, with high morbidity and mortality worldwide. A major feature of pneumococcal pneumonia is an abundant neutrophil infiltration . It was recently shown that activated neutrophils release neutrophil extracellular traps (NETs), which contain antimicrobial proteins bound to a DNA scaffold. NETs provide a high local concentration of antimicrobial components and bind, disarm, and kill microbes extracellularly. Here, we show that pneumococci are trapped but, unlike many other pathogens, not killed by NETs. NET trapping in the lungs, however, may allow the host to confine the infection, reducing the likelihood for the pathogen to spread into the bloodstream. DNases are expressed by many Gram-positive bacterial pathogens, but their role in virulence is not clear. Expression of a surface endonuclease encoded by endA is a common feature of many pneumococcal strains. We show that EndA allows pneumococci to degrade the DNA scaffold of NETs and escape. Furthermore, we demonstrate that escaping NETs promotes spreading of pneumococci from the upper airways to the lungs and from the lungs into the bloodstream during pneumonia.

Streptococcus pneumoniae isolates of serotypes 1, 4, 6B, 7F, 14, and 19F belonging to clonal types with known invasive disease potential in humans were used to infect C57BL/6 and BALB/c mice. Most isolates were able to colonize the nasopharynx for 7 days. One serotype 19F isolate of the clonal type ST162 had higher bacterial numbers than other isolates and clonal types of the same serotype. Serotype 4 clones caused the most-severe invasive disease, whereas serotype 1 clones caused low-level bacteremia without disease symptoms. BALB/c mice were more likely than C57BL/6 mice to develop meningitis. Disease kinetics varied significantly between clonal types. Although most induced a robust tumor necrosis factor response, some isolates of serotype 1 and 7F did not, suggesting that invasive disease caused by different clonal types may result in different degrees of host response. Capsular serotype, other clonal properties, and host factors are important for the development of pneumococcal disease.

The present study compares the molecular epidemiology of Streptococcus pneumoniae causing invasive disease and carriage, respectively, in one geographic area (Stockholm, Sweden) during a specific point in time (the year 1997). A total of 273 invasive isolates (257 from adults and 16 from children) obtained from the 2 major hospitals in Stockholm, as well as 246 nasopharyngeal isolates recovered from children attending 16 day-care centers in the Stockholm area, were analyzed by serotyping, molecular typing (by pulsed-field gel electrophoresis and multilocus sequence typing), and antibiotic susceptibility testing. Of the 34 different serotypes plus nontypeable strains identified in the present study, 12 were never found among the 246 colonizing isolates, whereas only 3 were never found among the 273 invasive isolates. The isolates formed 2 major classes: 1 class that was found mainly among invasive isolates (type 1, 4, 7F, and 9V isolates) and was clonally highly related and 1 class that caused invasive disease but was also common in carriage (including type 6A, 6B, 14, and 19F isolates) and was genetically more diverse. Clones were found that belonged to the same serotype but had different abilities to cause invasive disease. Also, isolates belonging to the same clone were found, although they had different capsules because of serotype switch, and were found to have the same disease potential. Hence, properties associated with a particular clonal type, in addition to capsular serotype, are likely to be important for the potential of pneumococci to cause invasive disease.

Policies for handling cases of penicillin-non-susceptible Streptococcus pneumoniae (PNSP) in day-care groups vary between different counties in Sweden. The aim of this study was to evaluate the epidemiological effect of excluding PNSP-carriers from children's day-care centres (DCC). We followed the incidence in 14 DCC groups with ongoing PNSP-spread, by repeated group screens until no further cases could be identified. All identified carriers were excluded from DCC attendance in study area A (Skåne region) while they remained in the group in study area B (Göteborg and örebro), according to local policies. The intervention effect was evaluated by comparing the number of additional cases after the baseline screen (start of the intervention period) between the 2 study areas. All PNSP-isolates were characterized by resistance pattern, serotype and pulse-field gel electrophoresis. The relative risk for children in DCCs without active intervention was 6.4 (95% CI: 2.0-20.7). Each prevented case in area A can be estimated to have demanded the exclusion of 2 other children from day care for approximately 4 weeks each. The total cost-benefit outcome of this action has to be seen in the light of the local situation with regard to the population prevalence and the distribution of other risk factors.

To investigate the serotype distribution and antibiotic susceptibility patterns 204 isolates of Streptococcus pneumoniae obtained from blood or cerebrospinal fluid (CSF) of children < or = 18 y of age were collected from 19 clinical microbiological laboratories in Sweden during the years 1998-2001. 166 isolates were from blood only, and 38 isolates were from CSF. The most common serotypes found were 6B, 1, 7F, 14, 18C, 19F, 6A, 4, 23F, 9V and 19A, in descending order of frequency. During the study period serotype 6B increased in frequency from 14.3% in 1998 to 28.3% in 2001 and serotype 1 decreased simultaneously from 20.4% to 9.4%. Serotype 1 was the most common serotype among children > or = 2 y of age or older, but was not found among children < 2 y of age. The potential coverage rate for the heptavalent pneumococcal conjugate vaccine varied between 53 and 68% during the studied years, and was higher for children < 2 y of age (74%) than for older children (51%). The majority of isolates were susceptible to penicillin and other antibiotics tested.

When bacteria such as Staphylococcus aureus and Streptococcus pneumoniae are exposed to lytic antibiotics such as penicillin and vancomycin, a self-induced killing process is initiated in the organism. This killing occurs via both non-lytic and lytic processes. Recent data suggest that the non-lytic killing system, which might affect the cytoplasmic membrane, secondarily activates murein hydrolases that eventually lyse the cell. Disturbances in this suicide pathway can lead to antibiotic tolerance, a process whereby the antibiotic still exerts its bacteriostatic effects but the self-induced killing system is impaired. In mutants obtained in vitro, signaling pathways have been affected that show either increased or decreased antibiotic-induced killing. Among clinical isolates of S. pneumoniae that are tolerant to penicillin and/or vancomycin, we do not yet know whether these signaling pathways are affected. We could, however, demonstrate that the activity of murein hydrolases is negatively controlled by the production of capsular polysaccharides in one vancomycin-tolerant isolate. Hence, type and level of capsular expression might constitute one factor that determines the degree of lysis, once the killing signal has been elicited by the antibiotic.

The ability of Streptococcus pneumoniae to escape lysis and killing by vancomycin, a property termed "tolerance," has recently been noted in a laboratory strain of the species. Vancomycin tolerance in clinical isolates represents a potential new health risk. We determined the prevalence of vancomycin and penicillin tolerance among 116 clinical isolates of pneumococci by monitoring lysis and viability after exposure to the respective antibiotic for 4 hours. Eight percent of the strains were tolerant to penicillin and 3% were tolerant to vancomycin. The 3 vancomycin-tolerant isolates also had a high ratio of minimum bactericidal concentration to minimum inhibitory concentration, in contrast to nontolerant strains. They were of serotype 9V and had reduced susceptibility to penicillin. Only 1 was also tolerant to penicillin. Growth rate and ability to divide were not affected in the 3 vancomycin-tolerant strains, and they all lysed with deoxycholate, which indicates autolysin production. Vancomycin tolerance among clinical isolates of pneumococci will necessitate tracking to determine the magnitude of the evolving health risk, since tolerance may contribute to treatment failure (in particular, cases of meningitis, in which bactericidal activity is critical for eradication) and since it may also be a favored background for acquisition of resistance of vancomycin.