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Background: Streptococcus pneumoniae serotype 1 has a high likelihood of causing invasive disease. Serotype 1 isolates belonging to CC228 are associated with low mortality, while CC217 isolates exhibit high mortality in patients., Methods: Clinical pneumococcal isolates and mutants were evaluated in wild-type C57BL/6 mice, macrophage-depleted mice, neutrophil-depleted mice, and SIGN-R1 knockout mice. In vitro models included binding and phagocytosis by THP-1 cells, capsule measurements, hydrogen peroxide production, and viability assays., Results: During early systemic infection in mice with serotype 1, large-colony variants appeared in blood. Similar large colonies were found in blood specimens from patients with invasive disease. Large morphotypes contained higher numbers of viable bacteria, grew faster, produced no or little hydrogen peroxide, and contained mutations in the spxB gene. spxB mutants were considerably more virulent in wild-type mice, less susceptible to early host clearance than wild-type strains after intravenous infection, but impaired in colonization. spxB mutants were less efficiently phagocytosed by macrophages than wild-type bacteria, which, in contrast to spxB mutants, caused more-severe disease when macrophages or SIGN-R1 were depleted., Conclusions: Hypervirulent spxB mutants are selected in both mice and patients and are resistant to early macrophage-mediated clearance.

Background: The invasive disease potential (IDP) of Streptococcus pneumoniae differs between serotypes, but the reason for this is unknown., Methods: A total of 47 pneumococcal isolates from 13 serotypes with different IDPs in humans that belonged to 37 multilocus sequence types were compared by whole genome microarrays and mutant analyses., Results: Approximately 34% of the genes were variable, including 95 genes previously shown by signature-tagged mutagenesis (STM) to be required for invasive disease in mice. Many variable genes were localized to 41 accessory regions (ARs), of which 24 contained genes previously identified by STM as required for invasive disease. Only AR6 and AR34 were preferentially found in isolates of serotypes with high IDPs. Neither AR6, which carries a gene previously identified bySTMas required for invasive disease and encodes a 6-phospho-betaglucosidase, nor the putative adhesin expressed by AR34 was required for mouse virulence in TIGR4., Conclusions: Pneumococci possess a repertoire of ARs that differ between clones and even between isolates of the same clone. The ARs required for invasive disease in humans may be redundant, as no unique pattern distinguished the most invasive clones from others. The ARs that contained genes previously identified by STM as required for virulence in mice were frequently absent from invasive human isolates. Only 1 AR (AR6) was present in almost all isolates from the serotypes with the highest IDP (1, 4, and 7F), whereas it was missing from many others.

Background: Streptococcus pneumoniae is a genetically diverse major human pathogen, yet a common colonizer of the nasopharynx. Here we analyzed the influence of defects affecting in vitro growth rate, on the ability of S. pneumoniae to colonize and to cause invasive disease in vivo., Results: Of eleven different clinical isolates one serotype 14 carrier isolate showed a significantly longer generation time as compared to other isolates, and was severely attenuated in mice. To directly investigate the impact of growth rate on virulence, a panel of mutants in five non-essential housekeeping genes was constructed in the virulent TIGR4 background by insertion-deletion mutagenesis. Three of these mutants (ychF, hemK and yebC) were, to different degrees, growth defective, and showed a reduced invasiveness in an intranasal murine challenge model that correlated to their in vitro growth rate, but remained capable of colonizing the upper airways. The growth defect, as well as virulence defect of the hemK insertion-deletion mutant, was mediated by polarity effects on the downstream yrdC gene, encoding a probable chaperone in ribosome assembly., Conclusion: We conclude that large fitness defects are needed to completely prevent pneumococci from causing invasive disease after intranasal challenge. However, even severe growth defects still allow pneumococci to persistently colonize the upper airways.

Antibiotic resistance in pneumococci is due to the spread of strains belonging to a limited number of clones. The Spain(9V)-3 clone of sequence type (ST)156 is one of the most successful clones with reduced susceptibility to penicillin [pneumococci nonsusceptible to penicillin (PNSP)]. In Sweden during 2000-2003, a dramatic increase in the number of PNSP isolates was observed. Molecular characterization of these isolates showed that a single clone of sequence type ST156 increased from 40% to 80% of all serotype 14, thus causing the serotype expansion. Additionally, during the same time period, we examined the clonal composition of two serotypes 9V and 19F: all 9V and 20% of 19F isolates belonged to the clonal cluster of ST156, and overall approximately 50% of all PNSP belonged to the ST156 clonal cluster. Moreover, microarray and PCR analysis showed that all ST156 isolates, irrespective of capsular type, carried the rlrA pilus islet. This islet was also found to be present in the penicillin-sensitive ST162 clone, which is believed to be the drug-susceptible ancestor of ST156. Competitive experiments between related ST156 serotype 19F strains confirmed that those containing the rlrA pilus islet were more successful in an animal model of carriage. We conclude that the pilus island is an important biological factor common to ST156 isolates and other successful PNSP clones. In Sweden, a country where the low antibiotic usage does not explain the spread of resistant strains, at least 70% of all PNSP isolates collected during year 2003 carried the pilus islet.

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.

Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality world-wide. The initial event in invasive pneumococcal disease is the attachment of encapsulated pneumococci to epithelial cells in the upper respiratory tract. This work provides evidence that initial bacterial adhesion and subsequent ability to cause invasive disease is enhanced by pili, long organelles able to extend beyond the polysaccharide capsule, previously unknown to exist in pneumococci. These adhesive pili-like appendages are encoded by the pneumococcal rlrA islet, present in some, but not all, clinical isolates. Introduction of the rlrA islet into an encapsulated rlrA-negative isolate allowed pilus expression, enhanced adherence to lung epithelial cells, and provided a competitive advantage upon mixed intranasal challenge of mice. Furthermore, a pilus-expressing rlrA islet-positive clinical isolate was more virulent than a nonpiliated deletion mutant, and it out-competed the mutant in murine models of colonization, pneumonia, and bacteremia. Additionally, piliated pneumococci evoked a higher TNF response during systemic infection, compared with nonpiliated derivatives, suggesting that pneumococcal pili not only contribute to adherence and virulence but also stimulate the host inflammatory response.

Penicillin and vancomycin induce a lytic response in Streptococcus pneumoniae that requires the N-acetylmuramyl-l-alanine amidase LytA. We show that clinical isolates of pneumococci of capsular serotypes 1, 4, 6B, and 23F were generally less lytic to penicillin than pneumococci of serotypes 14 and 3. In addition, most 9V isolates were less lytic to vancomycin, compared with isolates of other serotypes. Parent-mutant pairs expressing and not expressing capsular serotypes 2, 4, and 9V were compared for antibiotic-induced lysis. The nonencapsulated variants were considerably more lytic after beta-lactam and/or vancomycin treatment, and antibiotic tolerance was seen only in the context of capsule expression. Conversion from a nonlytic to a lytic phenotype, after loss of capsule expression, required an intact lytA autolysin gene. Exogenous addition of purified LytA gave a lower lytic response in capsulated strains, compared with that in nonencapsulated mutants. Spontaneous autolysis in stationary phase also was negatively affected by capsule expression in an autolysin-dependent manner. Long-term starvation in the stationary phase of the vancomycin- and penicillin-tolerant isolate I95 yielded nonencapsulated mutants that had lost antibiotic tolerance and were lytic to penicillin and vancomycin. The 9V capsular locus of I95 and one of these stationary phase-selected mutants were completely sequenced. The only difference found was a 1-bp frameshift deletion in the cps9vE gene of the lytic mutant, encoding a uridine diphosphate-glucosyl-1-phosphate transferase. Two additional independently isolated lytic mutants of I95 from the stationary phase also contained mutations in the same region of cps9vE, which identified it as a mutational hot spot. This report demonstrates that capsular polysaccharides negatively influence the lytic process and contribute to antibiotic tolerance in clinical isolates of pneumococci.