Host-pneumococcal interactions - from the lung to the brain

Mechanically ventilated patients are at risk of ventilator-associated pneumonia, a serious infection of the lungs. Not every ventilated patient develops pneumonia due to a combination of the protective layer of mucus in the airways, the immune system and prophylactic antibiotic therapy. To date, only little was known about the antimicrobial factors produced by humans that protect the lungs against infection. Research described in this thesis was therefore aimed at investigating to what extent the lungs of ventilated patients can inhibit the growth of bacteria, the major causative agent of pneumonia Streptococcus pneumoniae in particular. To this end, the accumulated mucus in the patients’ lungs, sputum, was investigated. The most important conclusion was that sputum can indeed possess antimicrobial activity, explained either by a combination of antibiotics and S. pneumoniae-specific antibodies, or by the innate immune defenses. Thus, sputum may serve as a valuable source of information to unravel the complex interactions between the human host, antimicrobial factors and the microbiome of the lower respiratory tract. A possible consequence of pneumonia is the dissemination of bacteria from the lungs to the bloodstream and the brain, which may lead to meningitis. This thesis describes how this process takes place, and how the so-called choline-binding protein CbpL contributes to invasive pneumococcal infections. In addition, possible future approaches to prevent meningitis caused by this bacterium are proposed.Bei künstlich beatmeten Patienten besteht das Risiko einer beatmungsassoziierten Lungenentzündung, d.h. einer schweren Infektion der Lunge. Aufgrund der schützenden Schleimschicht in den Atemwegen, dem Immunsystem und der prophylaktischen Antibiotikatherapie entwickelt nicht jeder künstlich beatmete Patient eine Lungenentzündung. Bisher war nur wenig über die vom Menschen produzierten antimikrobiellen Faktoren bekannt, die die Lunge vor Infektionen schützen. Die in dieser Arbeit beschriebenen Studien hatten das Ziel aufzuzeigen, inwieweit die Lunge beatmeter Patienten das Wachstum von Bakterien hemmen kann. Ein Hauptaugenmerk galt dabei insbesondere dem Hauptverursacher von Lungenentzündungen, Streptococcus pneumoniae. Der angesammelte Schleim, das Sputum, aus der Lunge von künstlich beatmeten und hospitalisierten Patienten, wurde zu diesem Zweck genauer untersucht. Eines der wichtigsten Ergebnisse war, dass Sputum eine antimikrobielle Aktivität aufweisen kann. Diese Aktivität konnte durch eine Kombination von Antibiotika und S. pneumoniae-spezifischen Antikörpern bzw. durch Faktoren der angeborenen Immunabwehr erklärt werden. Das Sputum kann daher als wertvolles Material in infizierten Patienten genutzt werden, um die komplexen Wechselwirkungen zwischen dem menschlichen Wirt, antimikrobiellen Faktoren und dem Mikrobiom der unteren Atemwege aufzuzeigen. Eine mögliche Folge einer schweren Lungenentzündung ist die Verbreitung von Bakterien aus der Lunge in den Blutkreislauf und ins Gehirn, was zu einer Hirnhautentzündung (Meningitis) führen kann. Diese Arbeit beschreibt auch den Prozess der Pathogenese unter besonderer Berücksichtigung des Cholin-Bindungsproteins CbpL für invasive Pneumokokkeninfektionen. Darüber hinaus werden mögliche zukünftige Ansätze zur Verhinderung der Pneumokokken-induzierten Meningitis vorgeschlagen

Heterogeneous antimicrobial activity in broncho-alveolar aspirates from mechanically ventilated intensive care unit patients

Pneumonia is an infection of the lungs, where the alveoli in the affected area are filled with pus and fluid. Although ventilated patients are at risk, not all ventilated patients develop pneumonia. This suggests that the sputum environment may possess antimicrobial activities. Despite the generally acknowledged importance of antimicrobial activity in protecting the human lung against infections, this has not been systematically assessed to date. Therefore, the objective of the present study was to measure antimicrobial activity in broncho-alveolar aspirate ('sputum") samples from patients in an intensive care unit (ICU) and to correlate the detected antimicrobial activity with antibiotic levels, the sputum microbiome, and the respective patients' characteristics. To this end, clinical metadata and sputum were collected from 53 mechanically ventilated ICU patients. The antimicrobial activity of sputum samples was tested against Streptococcus pneumoniae, Staphylococcus aureus and Streptococcus anginosus. Here we show that sputa collected from different patients presented a high degree of variation in antimicrobial activity, which can be partially attributed to antibiotic therapy. The sputum microbiome, although potentially capable of producing antimicrobial agents, seemed to contribute in a minor way, if any, to the antimicrobial activity of sputum. Remarkably, despite its potentially protective effect, the level of antimicrobial activity in the investigated sputa correlated inversely with patient outcome, most likely because disease severity outweighed the beneficial antimicrobial activities.</p

Host-pneumococcal interactions - from the lung to the brain

Mechanically ventilated patients are at risk of ventilator-associated pneumonia, a serious infection of the lungs. Not every ventilated patient develops pneumonia due to a combination of the protective layer of mucus in the airways, the immune system and prophylactic antibiotic therapy. To date, only little was known about the antimicrobial factors produced by humans that protect the lungs against infection. Research described in this thesis was therefore aimed at investigating to what extent the lungs of ventilated patients can inhibit the growth of bacteria, the major causative agent of pneumonia Streptococcus pneumoniae in particular. To this end, the accumulated mucus in the patients’ lungs, sputum, was investigated. The most important conclusion was that sputum can indeed possess antimicrobial activity, explained either by a combination of antibiotics and S. pneumoniae-specific antibodies, or by the innate immune defenses. Thus, sputum may serve as a valuable source of information to unravel the complex interactions between the human host, antimicrobial factors and the microbiome of the lower respiratory tract. A possible consequence of pneumonia is the dissemination of bacteria from the lungs to the bloodstream and the brain, which may lead to meningitis. This thesis describes how this process takes place, and how the so-called choline-binding protein CbpL contributes to invasive pneumococcal infections. In addition, possible future approaches to prevent meningitis caused by this bacterium are proposed.Bei künstlich beatmeten Patienten besteht das Risiko einer beatmungsassoziierten Lungenentzündung, d.h. einer schweren Infektion der Lunge. Aufgrund der schützenden Schleimschicht in den Atemwegen, dem Immunsystem und der prophylaktischen Antibiotikatherapie entwickelt nicht jeder künstlich beatmete Patient eine Lungenentzündung. Bisher war nur wenig über die vom Menschen produzierten antimikrobiellen Faktoren bekannt, die die Lunge vor Infektionen schützen. Die in dieser Arbeit beschriebenen Studien hatten das Ziel aufzuzeigen, inwieweit die Lunge beatmeter Patienten das Wachstum von Bakterien hemmen kann. Ein Hauptaugenmerk galt dabei insbesondere dem Hauptverursacher von Lungenentzündungen, Streptococcus pneumoniae. Der angesammelte Schleim, das Sputum, aus der Lunge von künstlich beatmeten und hospitalisierten Patienten, wurde zu diesem Zweck genauer untersucht. Eines der wichtigsten Ergebnisse war, dass Sputum eine antimikrobielle Aktivität aufweisen kann. Diese Aktivität konnte durch eine Kombination von Antibiotika und S. pneumoniae-spezifischen Antikörpern bzw. durch Faktoren der angeborenen Immunabwehr erklärt werden. Das Sputum kann daher als wertvolles Material in infizierten Patienten genutzt werden, um die komplexen Wechselwirkungen zwischen dem menschlichen Wirt, antimikrobiellen Faktoren und dem Mikrobiom der unteren Atemwege aufzuzeigen. Eine mögliche Folge einer schweren Lungenentzündung ist die Verbreitung von Bakterien aus der Lunge in den Blutkreislauf und ins Gehirn, was zu einer Hirnhautentzündung (Meningitis) führen kann. Diese Arbeit beschreibt auch den Prozess der Pathogenese unter besonderer Berücksichtigung des Cholin-Bindungsproteins CbpL für invasive Pneumokokkeninfektionen. Darüber hinaus werden mögliche zukünftige Ansätze zur Verhinderung der Pneumokokken-induzierten Meningitis vorgeschlagen

How Does Streptococcus pneumoniae Invade the Brain?

Streptococcus pneumoniae (the pneumococcus) is the major cause of bacterial meningitis. The mechanisms by which pneumococci from the bloodstream penetrate the blood-brain barrier to reach the brain are not fully understood. Receptor-mediated adhesion of the bacteria to the brain endothelium is considered a key event leading to meningitis development. The aim of this review is to discuss recent advances and perspectives related to the interactions of S. pneumoniae with the blood-brain barrier during the events leading to meningitis. Altogether, the available data suggest that, by precisely defining the pathways and ligands by which S. pneumoniae adheres to specific receptors, it may be possible to interfere with the respective mechanisms and develop strategies to prevent or even cure pneumococcal meningitis

The sortase A substrates FnbpA, FnbpB, ClfA and ClfB antagonize colony spreading of staphylococcus aureus

Staphylococcus aureus is an important human pathogen that is renowned both for its rapid transmission within hospitals and the community, and for the formation of antibiotic resistant biofilms on medical implants. Recently, it was shown that S. aureus is able to spread over wet surfaces. This motility phenomenon is promoted by the surfactant properties of secreted phenol-soluble modulins (PSMs), which are also known to inhibit biofilm formation. The aim of the present studies was to determine whether any cell surface-associated S. aureus proteins have an impact on colony spreading. To this end, we analyzed the spreading capabilities of strains lacking non-essential components of the protein export and sorting machinery. Interestingly, our analyses reveal that the absence of sortase A (SrtA) causes a hyper-spreading phenotype. SrtA is responsible for covalent anchoring of various proteins to the staphylococcal cell wall. Accordingly, we show that the hyper-spreading phenotype of srtA mutant cells is an indirect effect that relates to the sortase substrates FnbpA, FnbpB, ClfA and ClfB. These surface-exposed staphylococcal proteins are known to promote biofilm formation, and cell-cell interactions. The hyper-spreading phenotype of srtA mutant staphylococcal cells was subsequently validated in Staphylococcus epidermidis. We conclude that cell wall-associated factors that promote a sessile lifestyle of S. aureus and S. epidermidis antagonize the colony spreading motility of these bacteria

Specific associations between fungi and bacteria in broncho-alveolar aspirates from mechanically ventilated intensive care unit patients

The detection of fungi in the human respiratory tract may represent contamination, colonization or a respiratory infection. To develop effective management strategies, a more accurate and comprehensive understanding of the lung fungal microbiome is required. Therefore, the objective of the present study was to define the “mycobiome” of mechanically ventilated patients admitted to an intensive care unit (ICU) using broncho-alveolar aspirate (“sputum”) samples and correlate this with clinical parameters and the bacterial microbiota. To this end, the mycobiome of 33 sputum samples was analyzed by Internal Transcribed Spacer2 (ITS2) amplicon sequencing of the ribosomal operons. The results show that in the investigated sputa of mechanically ventilated patients Candida spp. were most frequently detected, independent of pneumonia or antimicrobial therapy. The presence of Candida excluded in most cases the presence of Malassezia, which was the second most-frequently encountered fungus. Moreover, a hierarchical clustering of the sequence data indicated a patient-specific mycobiome. Fungi detected by culturing (Candida and Aspergillus) were also detected through ITS2 sequencing, but other yeasts and fungi were only detectable by sequencing. While Candida showed no correlations with identified bacterial groups, the presence of Malassezia and Rhodotorula correlated with oral bacteria associated with periodontal disease. Likewise, Cladosporium correlated with other oral bacteria, whereas Saccharomyces correlated more specifically with dental plaque bacteria and Alternaria with the nasal-throat-resident bacteria Neisseria, Haemophilus and Moraxella. In conclusion, ITS2 sequencing of sputum samples uncovered patient-specific lung mycobiomes, which were only partially detectable by culturing, and which could be correlated to specific nasal-oral-pharyngeal niches

Sputum Proteome Signatures of Mechanically Ventilated Intensive Care Unit Patients Distinguish Samples with or without Anti-pneumococcal Activity

Respiratory pathogens like Streptococcus pneumoniae can cause severe pneumonia. Nonetheless, mechanically ventilated intensive care patients, who have a high risk of contracting pneumonia, rarely develop pneumococcal pneumonia. Mechanically ventilated patients are at risk of contracting pneumonia. Therefore, these patients often receive prophylactic systemic antimicrobial therapy. Intriguingly however, a previous study showed that antimicrobial activity in bronchoalveolar aspirates (here referred to as “sputa”) from ventilated patients was only partially explained by antibiotic therapy. Here we report that sputa from these patients presented distinct proteome signatures depending on the presence or absence of antimicrobial activity. Moreover, we show that the same distinction applied to antibodies against Streptococcus pneumoniae , which is a major causative agent of pneumonia. Specifically, the investigated sputa that inhibited growth of S. pneumoniae , while containing subinhibitory levels of the antibiotic cefotaxime, presented elevated levels of proteins implicated in innate immune defenses, including complement and apolipoprotein-associated proteins. In contrast, S. pneumoniae -inhibiting sputa with relatively high cefotaxime concentrations or noninhibiting sputa contained higher levels of proteins involved in inflammatory responses, such as neutrophil elastase-associated proteins. In an immunoproteomics analysis, 18 out of 55 S. pneumoniae antigens tested showed significantly increased levels of IgGs in inhibiting sputa. Hence, proteomics and immunoproteomics revealed elevated levels of antimicrobial host proteins or S. pneumoniae antigen-specific IgGs in pneumococcal growth-inhibiting sputa, thus explaining their anti-pneumococcal activity. IMPORTANCE Respiratory pathogens like Streptococcus pneumoniae can cause severe pneumonia. Nonetheless, mechanically ventilated intensive care patients, who have a high risk of contracting pneumonia, rarely develop pneumococcal pneumonia. This suggests the presence of potentially protective antimicrobial agents in their lung environment. Our present study shows for the first time that bronchoalveolar aspirates, “sputa,” of ventilated patients in a Dutch intensive care unit were characterized by three distinct groups of proteome abundance signatures that can explain their anti-pneumococcal activity. Importantly, this anti-pneumococcal sputum activity was related either to elevated levels of antimicrobial host proteins or to antibiotics and S. pneumoniae -specific antibodies. Further, the sputum composition of some patients changed over time. Therefore, we conclude that our study may provide a novel tool to measure changes that are indicative of infection-related conditions in the lungs of mechanically ventilated patients

Sputum Proteome Signatures of Mechanically Ventilated Intensive Care Unit Patients Distinguish Samples with or without Anti-pneumococcal Activity

Mechanically ventilated patients are at risk of contracting pneumonia. Therefore, these patients often receive prophylactic systemic antimicrobial therapy. Intriguingly however, a previous study showed that antimicrobial activity in bronchoalveolar aspirates (here referred to as "sputa") from ventilated patients was only partially explained by antibiotic therapy. Here we report that sputa from these patients presented distinct proteome signatures depending on the presence or absence of antimicrobial activity. Moreover, we show that the same distinction applied to antibodies against Streptococcus pneumoniae, which is a major causative agent of pneumonia. Specifically, the investigated sputa that inhibited growth of S. pneumoniae, while containing subinhibitory levels of the antibiotic cefotaxime, presented elevated levels of proteins implicated in innate immune defenses, including complement and apolipoprotein-associated proteins. In contrast, S. pneumoniae-inhibiting sputa with relatively high cefotaxime concentrations or noninhibiting sputa contained higher levels of proteins involved in inflammatory responses, such as neutrophil elastase-associated proteins. In an immunoproteomics analysis, 18 out of 55 S. pneumoniae antigens tested showed significantly increased levels of IgGs in inhibiting sputa. Hence, proteomics and immunoproteomics revealed elevated levels of antimicrobial host proteins or S. pneumoniae antigen-specific IgGs in pneumococcal growth-inhibiting sputa, thus explaining their anti-pneumococcal activity. IMPORTANCE Respiratory pathogens like Streptococcus pneumoniae can cause severe pneumonia. Nonetheless, mechanically ventilated intensive care patients, who have a high risk of contracting pneumonia, rarely develop pneumococcal pneumonia. This suggests the presence of potentially protective antimicrobial agents in their lung environment. Our present study shows for the first time that bronchoalveolar aspirates, "sputa," of ventilated patients in a Dutch intensive care unit were characterized by three distinct groups of proteome abundance signatures that can explain their anti-pneumococcal activity. Importantly, this anti-pneumococcal sputum activity was related either to elevated levels of antimicrobial host proteins or to antibiotics and S. pneumoniae-specific antibodies. Further, the sputum composition of some patients changed over time. Therefore, we conclude that our study may provide a novel tool to measure changes that are indicative of infection-related conditions in the lungs of mechanically ventilated patients

Hyper-spreading phenotype of a <i>srtA</i> mutant of <i>S. epidermidis</i> 1457.

<p>Spreading motility of <i>S. epidermidis</i> 1457 (WT), a <i>srtA</i> mutant derivative of this strain (<i>srtA</i>), and a complemented derivative of the <i>srtA</i> mutant (<i>srtA^Se</i>-pCN51) was assayed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044646#pone-0044646-g001" target="_blank">Figure 1</a>.</p