Community-acquired pneumonia in adult patients with special reference to rapid methods for etiological diagnosis

Aikuispotilaan kotisyntyisen keuhkokuumeen etiologinen diagnostiikka mikrobiologisilla pikamenetelmillä Tausta. Keuhkokuume on vakava sairaus, johon sairastuu Suomessa vuosittain n. 60 000 aikuista. Huolimatta siitä, että taudin hoito on kehittynyt, siihen liittyy yhä merkittävä, 6-15%:n kuolleisuus. Alahengitystieinfektion aiheuttajamikrobien tunnistaminen on myös edelleen haasteellista. Tavoitteet. Tämän työn tavoitteena oli tutkia Turun yliopistollisessa keskussairaalassa hoidettujen aikuispotilaiden keuhkokuumeen etiologiaa sekä selvittää uusien mikrobiologisten pikamenetelmi¬en hyödyllisyyttä taudinaiheuttajan toteamisessa. Aineisto. Osatöiden I ja III aineisto koostui 384 Turun yliopistollisen keskussairaalaan infektio-osastolla hoidetusta keuhkokuumepotilaasta. Osatyössä I tutkittiin keuhkokuumeen aiheuttaja¬mikrobeja käyttämällä perinteisten menetelmien lisäksi antigeeniosoitukseen ja PCR-tekniikkaan perustuvia pikamenetelmiä. Osatyö II käsitti 231 potilaasta koostuvan alaryhmän, jossa tutkittiin potilaiden nielun limanäytteestä rinovirusten ja enterovirusten esiintyvyyttä. Osatyössä III potilailta tutkittiin plasman C-reaktiivisen proteiinin (CRP) pitoisuus ensimmäisten viiden sairaalahoitopäi¬vän aikana. Laajoja tilastotieteellisiä analyysejä käyttämällä selvitettiin CRP:n käyttökelpoisuutta sairauden vaikeusasteen arvioinnissa ja komplikaatioiden kehittymisen ennustamisessa. Osatyössä IV 68 keuhkokuumepotilaan sairaalaan tulovaiheessa otetuista näytteistä määritettiin neutrofiilien pintareseptorien ekspressio. Osatyössä V analysoitiin sisätautien vuodeosastoilla vuosina 1996-2000 keuhkokuumepotilaille tehtyjen keuhkohuuhtelunäytteiden laboratoriotutkimustulokset. Tulokset. Keuhkokuumeen aiheuttaja löytyi 209 potilaalta, aiheuttajamikrobeja löydettiin kaikkiaan 230. Näistä aiheuttajista 135 (58.7%) löydettiin antigeenin osoituksella tai PCR-menetelmillä. Suu¬rin osa, 95 (70.4%), todettiin pelkästään kyseisillä pikamenetelmillä. Respiratorinen virus todettiin antigeeniosoituksella 11.1% keuhkokuumepotilaalla. Eniten respiratorisia viruksia löytyi vakavaa keuhkokuumetta sairastavilta potilailta (20.3%). 231 keuhkokuumepotilaan alaryhmässä todettiin PCR-menetelmällä picornavirus 19 (8.2%) potilaalla. Respiratorinen virus löytyi tässä potilasryh¬mässä kaiken kaikkiaan 47 (20%) potilaalta. Näistä 17:llä (36%) löytyi samanaikaisesti bakteerin aiheuttama infektio. CRP-tasot olivat sairaalaan tulovaiheessa merkitsevästi korkeammat vakavaa keuhkokuumetta (PSI-luokat III-V) sairastavilla potilailla kuin lievää keuhkokuumetta (PSI-luokat I-II) sairastavilla potilailla (p <0.001). Yli 100 mg/l oleva CRP-taso neljän päivän kuluttua sairaa¬laan tulosta ennusti keuhkokuumeen komplikaatiota tai huonoa hoitovastetta. Neutrofiilien komple¬menttireseptorin ekspressio oli pneumokokin aiheuttamaa keuhkokuumetta sairastavilla merkitse¬västi korkeampi kuin influenssan aiheuttamaa keuhkokuumetta sairastavilla. BAL-näytteistä vain yhdessä 71:stä (1.3%) todettiin diagnostinen bakteerikasvu kvantitatiivisessa viljelyssä. Uusilla menetelmilläkin keuhkokuumeen aiheuttaja löytyi vain 9.8% BAL-näytteistä. Päätelmät. Uusilla antigeeniosoitus- ja PCR-menetelmillä keuhkokuumeen etiologia voidaan saada selvitettyä nopeasti. Lisäksi näitä menetelmiä käyttämällä taudin aiheuttajamikrobi löytyi huomattavasti suuremmalta osalta potilaista kuin pelkästään tavanomaisia menetelmiä käyttämällä. Pikamenetelmien hyödyllisyys vaihteli taudin vaikeusasteen mukaan. Respiratorinen virus löytyi huomattavan usein keuhkokuumetta sairastavilta potilailta, ja näiden potilaiden taudinkuva oli usein vaikea. Tulovaiheen korkeaa CRP-tasoa voidaan käyttää lisäkeinona arvioitaessa keuhkokuumeen vaikeutta. CRP on erityisen hyödyllinen arvioitaessa hoitovastetta ja riskiä komplikaatioiden ke¬hittymiseen. Neutrofiilien komplementtireseptorin ekspression tutkiminen näyttää lupaavalta pi¬kamenetelmältä erottamaan bakteerien ja virusten aiheuttamat taudit toisistaan. Antimikrobihoitoa saavilla potilailla BAL-tutkimuksen löydökset olivat vähäiset ja vaikuttivat hoitoon vain harvoin.

Improvements in the Assessment of Bacterial Viability and Killing

It is axiomatic that our planet is extensively inhabited by diverse micro-organisms such as bacteria, yet the absolute diversity of different bacterial species is widely held to be unknown. Different bacteria can be found from the depths of the oceans to the top of the mountains; even the air is more or less colonized by bacteria. Most bacteria are either harmless or even advantageous to human beings but there are also bacteria, which can cause severe infectious diseases or spoil the supplies intended for human consumption. Therefore, it is vitally important not only to be able to detect and enumerate bacteria but also to assess their viability and possible harmfulness. Whilst the growth of bacteria is remarkably fast under optimum conditions and easy to detect by cultural methods, most bacteria are believed to lie in stationary phase of growth in which the actual growth is ceased and thus bacteria may simply be undetectable by cultural techniques. Additionally, several injurious factors such as low and high temperature or deficiency of nutrients can turn bacteria into a viable but non-culturable state (VBNC) that cannot be detected by cultural methods. Thereby, various noncultural techniques developed for the assessment of bacterial viability and killing have widely been exploited in modern microbiology. However, only a few methods are suitable for kinetic measurements, which enable the real-time detection of bacterial growth and viability. The present study describes alternative methods for measuring bacterial viability and killing as well as detecting the effects of various antimicrobial agents on bacteria on a real-time basis. The suitability of bacterial (lux) and beetle (luc) luciferases as well as green fluorescent protein (GFP) to act as a marker of bacterial viability and cell growth was tested. In particular, a multiparameter microplate assay based on GFP-luciferase combination as well as a flow cytometric measurement based on GFP-PI combination were developed to perform divergent viability analyses. The results obtained suggest that the antimicrobial activities of various drugs against bacteria could be successfully measured using both of these methods. Specifically, the data reliability of flow cytometric viability analysis was notably improved as GFP was utilized in the assay. A fluoro-luminometric microplate assay enabled kinetic measurements, which significantly improved and accelerated the assessment of bacterial viability compared to more conventional viability assays such as plate counting. Moreover, the multiparameter assay made simultaneous detection of GFP fluorescence and luciferase bioluminescence possible and provided extensive information about multiple cellular parameters in single assay, thereby increasing the accuracy of the assessment of the kinetics of antimicrobial activities on target bacteria.

Childhood community-acquired pneumonia

Background: Community-acquired pneumonia is a leading cause of morbidity and mortality in children worldwide. New, rapid methods are needed to improve the microbiologic diagnosis of pneumonia in clinical practice. The increasing incidence of parapneumonic empyema in children accentuates the importance of the identification of the causative agent and clinical predictors of empyema. Aims and methods: Two prospective studies were conducted to find feasible diagnostic methods for the detection of causative agents of pneumonia. The usefulness of pneumolysin-targeted real-time PCR in the diagnosis of pneumococcal disease was studied in children with pneumonia and empyema, and the clinical utility of induced sputum analysis in the microbiologic diagnosis of pneumonia was investigated in children with pneumonia. In addition, two retrospective clinical studies were performed to describe the frequency and clinical profile of influenza pneumonia in children and the frequency, clinical profile and clinical predictors of empyema in children. Results: Pneumolysin-PCR in pleural fluid significantly improved the microbiologic diagnosis of empyema by increasing the detection rate of pneumococcus almost tenfold to that of pleural fluid culture (75 % vs. 8 %). In whole blood samples, PCR detected pneumococcus in only one child with pneumonia and one child with pneumococcal empyema. Sputum induction provided good-quality sputum specimens with high microbiologic yield. Streptococcus pneumoniae (46 %) and rhinovirus (29 %) were the most common microbes detected. The quantification results of the paired sputum and nasopharyngeal aspirate specimens provided support that the majority of the bacteria (79 %) and viruses (55 %) found in sputum originated from the lower airways. Pneumonia was detected in 14 % of children with influenza infection. A history of prolonged duration of fever, tachypnea, and pain on abdominal palpation were found to be independently significant predictors of empyema. Conclusions: Pneumolysin-targeted real-time PCR is a useful and rapid method for the diagnosis of pneumococcal empyema in children. Induced sputum analysis with paired nasopharyngeal aspirate analysis can be of clinical value in the microbiologic diagnosis of pneumonia. Influenza pneumonia is an infrequent and generally benign disease in children with rare fatalities. Repeat chest radiograph and ultrasound imaging are recommended in children with pneumonia presenting with clinical predictors of empyema and in children with persistent fever and high CRP levels during hospitalization.

Inactive endosialidase-based detection of bacterial and oncofetal polysialic acid

Polysialic acid is a carbohydrate polymer which consist of N-acetylneuraminic acid units joined by alpha2,8-linkages. It is developmentally regulated and has an important role during normal neuronal development. In adults, it participates in complex neurological processes, such as memory, neural plasticity, tumor cell growth and metastasis. Polysialic acid also constitutes the capsule of some meningitis and sepsis-causing bacteria, such as Escherichia coli K1, group B meningococci, Mannheimia haemolytica A2 and Moraxella nonliquefaciens. Polysialic acid is poorly immunogenic; therefore high affinity antibodies against it are difficult to prepare, thus specific and fast detection methods are needed. Endosialidase is an enzyme derived from the E. coli K1 bacteriophage, which specifically recognizes and degrades polysialic acid. In this study, a novel detection method for polysialic acid was developed based on a fusion protein of inactive endosialidase and the green fluorescent protein. It utilizes the ability of the mutant, inactive endosialidase to bind but not cleave polysialic acid. Sequencing of the endosialidase gene revealed that amino acid substitutions near the active site of the enzyme differentiate the active and inactive forms of the enzyme. The fusion protein was applied for the detection of polysialic acid in bacteria and neuroblastoma. The results indicate that the fusion protein is a fast, sensitive and specific reagent for the detection of polysialic acid. The use of an inactive enzyme as a specific molecular tool for the detection of its substrate represents an approach which could potentially find wide applicability in the specific detection of diverse macromolecules.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

Nasopharyngeal colonization by pathogenic bacteria: effect of polymorphisms in innate immune genes of young children

Nasopharyngeal bacteria can asymptomatically colonize the nasopharynx of infants and young children but are also associated with the development of respiratory infections and diseases. Such nasopharyngeal bacteria include Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae and Staphylococcus aureus. The host defense against invading pathogens is largely relies germline-encoded pattern recognition receptors (PRR), which are expressed on the cells of innate immunity, and different cytokines. These include toll-like receptors (TLR), mannose-binding lectin (MBL) and different cytokines such as IL-17A. Single nucleotide polymorphisms (SNP) in these receptors and cytokines have been reported. The aim of this study was to investigate genetic polymorphisms in the genes for TLR2, 3 and 4, MBL as well as for IL-17A and their associations with nasopharyngeal pathogenic bacterial colonization during a two-year follow-up. The study revealed that polymorphisms in TLRs, MBL2 and IL17A are associated with the nasopharyngeal bacterial colonization in young children. Healthy young (2.6 months of age) children with variant types of MBL2, TLR2 R753Q or TLR4 D299G had an increased risk to be colonized by S. pneumonia, S. aureus or M. catarrhalis, respectively. Moreover, variant types of MBL2 in healthy children with might facilitate human rhinovirus (HRV)-induced S. pneumoniae colonization at 2.6 months of age. The polymorphism of TLR4 D299G was shown to be associated with M. catarrhalis colonization throughout the whole two-year follow-up (2.6, 13 and 24 months of age) and also with the bacterial load of this pathogen. Also, the polymorphism of IL17A G152A was shown to be associated with increased risk to be colonized by S. pneumoniae at 13 and 24 months of age. Furthermore, the results suggest that IL17A G152A has an effect on production of serum IL-17A already at young age. In conclusion, the results of this study indicate that polymorphisms in the key PRRs and IL17A seem to play an important role to colonization of S. pneumoniae, M. catarrhalis, and S. aureus in healthy young Finnish children. The nasopharyngeal colonization by these pathogenic bacteria may further promote the development of respiratory infections and may be related to development of asthma and allergy in the later life of children. These findings offer a possible explanation why some children have more respiratory infections than other children and provide a rational basis for future studies in this field.

Bacterial community structure and petroleum hydrocarbon degradation in the Baltic Sea

The Baltic Sea is unique by its biological, geochemical and physical features. The number of species of larger organisms is small and the species composition is distinctive. On the contrary microbial communities are diverse. Because of the low salinity levels, bacterial communities differ from the ones in the oceans. Knowing the structure of these communities better and how they response to different environmental conditions helps us to estimate how different factors affect the balance and function of the Baltic Sea ecosystem. Bacteria are the key players when it comes to natural biogeochemical processes and human-induced phenomena like eutrophication, oil spills or disposal of other harmful substances to the sea ecosystem. In this thesis, bacterial community structure in the sea surface microlayer and subsurface water of the Archipelago Sea were compared. In addition, the effect of diatom derived polyunsaturated aldehydes on bacterial community structure was studied by a mesocosm experiment. Diesel, crude oil and polycyclic aromatic hydrocarbon degradation capacity of the Baltic Sea bacteria was studied in smaller scale microcosm experiments. In diesel oil experiments bacteria from water phase of the Archipelago Sea was studied. Sediment and iron manganese concretions collected from the Gulf of Finland were used in the crude oil and polycyclic aromatic hydrocarbon experiments. The amount of polycyclic aromatic hydrocarbon degradation genes was measured in all of the oil degradation experiments. The results show how differences in bacterial community structure can be seen in the sea surface when compared to the subsurface waters. The mesocosm experiment demonstrated how diatom-bacteria interactions depend on other factors than diatom derived polyunsaturated aldehydes, which do not seem to have an effect on the bacterial community structure as has been suggested in earlier studies. The dominant bacterial groups in the diesel microcosms differed in samples taken from a pristine site when compared to a site with previous oil exposure in the Archipelago Sea area. Results of the study with sediment and iron-manganese concretions indicate that there are diverse bacterial communities, typical to each bottom type, inhabiting the bottoms of the Gulf of Finland capable to degrade oil and polycyclic aromatic hydrocarbon compounds.  

Bacterial adhesion and biofilm formation on monolithic zirconia

Tutkimuksen tavoitteena oli selvittää bakteerien kiinnittymistä ja bakteeribiofilmin muodostumista implanttimateriaalien pinnalla. Monoliittisen zirkonian ja lasikeramien käyttö implanttikruunujen materiaaleina kasvaa jatkuvasti. Zirkoniaa käytetään myös abutmenttien materiaalina esteettisillä alueilla. Tällä hetkellä on vain vähän tutkimustietoa näiden implanttikruunumateriaalien sekä implanttikruunujen sementoimiseen käytetyn sementin pinnalla tapahtuvasta bakteeriadheesiosta ja biofilmin muodostumisesta. Bakteerien adheesiota ja biofilmin muodistumista tutkittiin neljän eri materiaalin pinnalla. Tutkimuksessa käytetyt materiaalit olivat: (1) Litiumdisilikaatti (LDS; IPS e.max CAD, Ivoclar Vivadent,kontrolli), (2) Kokonaan stabiloitu zirkonia (FSZ; Prettau Anterior, Zirkonzahn), (3) Osittain stabiloitu zirkonia (PSZ; Katana, Noritake), ja (4) Kaksoiskovetteinen sementti (DCC; Multilink hybrid abutment cement, Ivoclar Vivadent). Kaikki tutkimuksessa käytetyt materiaalit valmisteltiin ja kiillotettiin valmistajien ohjeiden mukaisesti Tutkittavat pinnat inkuboitiin Streptococcus mutans-suspensiossa +37°C:ssä asteessa. Bakteeriadheesiotestissä inkubointiaika oli 30 minuuttia ja biofilmitestissä vastaava aika oli 24 tuntia. Materiaalien pintoja tarkasteltiin myös elektronimikroskooppia käyttäen. Tutkimuksessa todettiin, että bakteeriadheesiossa oli eroja eri materiaalien välillä. Biofilmin. muodostumisessa ei todettu tilastollisesti merkittäviä eroja tutkittavien materiaalien välillä.