Confocal raman microspectroscopy : a novel diagnostic tool in medical microbiology

The aim of the research described in this thesis was to develop confocal Raman microspectroscopy techniques for the rapid identification and characterisation of clinically relevant microorganisms. Chapter 2 describes a study in which the accuracy of the identification of Enterococcus spp., based on Raman spectroscopy, is exemplified by a comparison with identification results based on phenotypic and genotypic methods. This chapter clearly shows the high accuracy that can be obtained when applying vibrational spectroscopies for microbial identifications. Chapter 3 reports on the unique method that was developed to analyse microbial microcolonies, directly on the solid culture medium. With this approach it is possible to "record" Raman spectra from very young cultures, where the colonies are typically between 10 and 100 microns in diameter. Culturing times therefore can be decreased to several hours, allowing rapid identification schemes to be developed. Chapter 4 describes the possibilities of this technique to probe the heterogeneity of microbial colonies directly on the solid culture medium. It was shown that microbial colonies after 6 hours incubation were most homogeneous as compared to older (12 and 24 hours) colonies, and consequently these 6 hour old colonies are better suited for the composition of a database. From the results obtained in this study, standardized protocols were derived, based on which reproducible Raman spectra can be obtained. With the methodology for rapidly performing measurements now developed, and the culturing conditions optimized for obtaining reproducible Raman spectra. the potential ofthe technique to identifY microbes by their Raman spectrum was evaluated in chapters. A collection of yeast strains from the genus Candida was used to arrive at the most appropriate manner of analysing large amounts of data. Based on a library of Raman spectra from known Candida strains, an identification method was developed to identify these strains based on their Raman spectrum. Finally, to test the methodology developed in all previous studies, a prospective study of clinical isolates is described in chapter 6. Parallel to the routine analysis of positive blood samples from patients in intensive care units and a random selection of other wards, Raman identification was performed of these samples. A summary of all results is provided in chapter 7

The use of Raman spectroscopy in the epidemiology of methicillin-resistant Staphylococcus aureus of human- and animal-related clonal lineages

AbstractIn order to perform a cost-effective search and destroy policy for methicillin-resistant Staphylococcus aureus (MRSA), a quick and reliable typing method is essential. In an area with a high level of animal-related MRSA ST398, pulsed field gel electrophoresis (PFGE) typing and spa-typing are not sufficient to discriminate between co-incidental findings and true transmission of MRSA. This study is the first to retrospectively show the performance of Raman spectroscopy in 16 well-documented outbreaks. We analysed 525 isolates, 286 MRSA ST398 and 239 from other PFGE clusters with Raman spectroscopy. When epidemiologically linked isolates from the outbreaks were analysed with PFGE as the reference standard, Raman spectroscopy correctly identified 97% of cases that were indistinguishable from the index case. With Raman cluster analysis, the most dominant distinction was between MRSA ST398 and other MRSA of human clonal lineages. Within MRSA ST398, 22 different Raman clusters were identified. Raman typing correctly identified an ST398 (spa type t567) outbreak in a hospital setting. No direct correlation was observed between Raman clusters and spa types. We conclude that Raman spectroscopy is a quick and reliable method of MRSA typing, which can be used in outbreak settings and it is comparable to PFGE, with the added advantage that PFGE non-typeable isolates can also be readily typed using the same sample preparation protocol

Structural properties and Raman spectroscopy of lipid Langmuir monolayers at the air-water interface

Spectra of octadecylamine (ODA) Langmuir monolayers and egg phosphatidylcholine (PC)/ODA-mixed monolayers at the air-water interface have been acquired. The organization of the monolayers has been characterized by surface pressure-area isotherms. Application of polarized optical microscopy provides further insight in the domain structures and interactions of the film components. Surface-enhanced Raman scattering (SERS) data indicate that enhancement in Raman spectra can be obtained by strong interaction between headgroups of the surfactants and silver particles in subphase. By mixing ODA with phospholipid molecules and spreading the mixture at the air-water interface, we acquired vibrational information of phospholipid molecules with surfactant-aided SERS effect.Comment: 8 pages, 9 figure

Raman spectroscopy-based identification of nosocomial outbreaks of the clonal bacterium Escherichia coli

DNA-based techniques are frequently used to confirm the relatedness of putative outbreak isolates. These techniques often lack the discriminatory power when analyzing closely related microbes such as E. coli. Here the value of Raman spectroscopy as a typing tool for E. coli in a clinical setting was retrospectively evaluated

TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts.

The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R-dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons

Unsuitability of MALDI-TOF MS to discriminate Acinetobacter baumannii clones under our routine experimental conditions

MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) is now in the forefront for routine bacterial species identification methodologies, being its value for clonality assessment controversial. In this work we evaluated the potential of MALDI-TOF MS for assisting infection control by depicting Acinetobacter baumannii clones. Mass spectra of 58 A. baumannii clinical isolates belonging to the worldwide spread lineages (ST98, ST103, ST208 and ST218) isolated in our country, were obtained and analysed with several chemometric tools (pseudo gel views, peakfind function and partial least squares discriminant analysis). The clonal lineages were obtained using the Oxford scheme, belonging ST98, ST208 and ST218 to the international clone II and ST103 to an epidemic clonal lineage (SG5). Additionally, mass spectra of a highly diverse international collection of 38 isolates belonging to 22 STs were obtained for further comparisons. Pseudo gel views and direct peak pattern analysis didnt allow the discrimination of A. baumannii isolates belonging to ST98, ST103, ST208 or ST218. Moreover, a partial least square discriminant analysis of the mass spectra considering two spectral ranges (2-20kDa and 4-10kDa) revealed a poor degree of discrimination with only 64.6% and 65.8% of correct ST assignments, respectively. Also, mass spectra of the international isolates (n=38, 22STs) revealed a very congruent peak pattern among them as well as among the four lineages included in this work. Despite the increasing interest of MALDI-TOF MS for bacterial typing at different taxonomical levels, we demonstrated, using routine experimental conditions, the unsuitability of this methodology for A. baumannii clonal discrimination.This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE and by National Funds through FCT - Foundation for Science and Technology under the project UID/MULTI/04378/2013. LS was supported by doctoral grant (SFRH/BD/88028/2012) and FG by a post doc grant (SFRH/BPD/95556/2013) from Fundacao para a Ciencia e a Tecnologia. CS was funded by a CIENCIA2008 contract from FCT

Variability in the composition of extracellular polymeric substances from a full-scale aerobic granular sludge reactor treating urban wastewater

Within the framework of the circular economy, there is a need for waste management alternatives that promote the reuse of materials produced in wastewater treatment plants (WWTP). An interesting option is the recovery of extracellular substances from sludge. The variability of characteristics of potential recovered bioproducts has to be assessed in full scale operational settings. In this study, aerobic granular sludge (AGS) from a full-scale WWTP treating urban wastewater was regularly collected for 4 months to assess variability in extracellular polymeric substances (EPS) composition and in granular morphology. Variations in the EPS composition occurred with time. Proteins and humic substances were the main EPS components (329-494 and 259-316?mg/g VSS of AGS, respectively), with polysaccharides and DNA representing minor components. The application of an extra purification step after extraction to obtain a purer EPS led to a decrease in the yield of each EPS component, particularly pronounced for the polysaccharides. The final product had a rather constant composition for the monthly samples. The granules showed morphological stability throughout the sampling period and the yield of EPS was correlated to the size of the granules, higher when there was a higher content of small granules (Deq<150?µm) comparing to intermediate (150???Deq<1500?µm) or large granules (Deq?1500?µm). This is the first time that a potential valorization strategy for surplus AGS biomass is studied in a full-scale environment. Knowledge on yield and product homogeneity is important as these features are essential for downstream application of the recovered EPS.The authors wish to thank the company SIMTEJO for supplying the granules and influent and effluent characterization data. This work was financed by FCT under the project AGeNT - PTDC/BTA-BTA/31264/2017 (POCI-01-0145-FEDER-031264). We would like to thank the scientific collaboration of CBQF under the FCT project UID/Multi/50016/2019 and NORTE-08-5369-FSE-000007 and CEB under the FCT project UID/BIO/044697/2019 and BioTecNorte operation (NORTE-01-0145-FEDER-000004).info:eu-repo/semantics/publishedVersio

Potential mechanisms underlying the acute lung dysfunction and bacterial extrapulmonary dissemination during Burkholderia cenocepacia respiratory infection

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia cenocepacia</it>, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity of <it>B. cenocepacia </it>from the ET-12 lineage, that has been linked to the cepacia syndrome, and four clinical isolates recovered from CF patients with mild clinical courses were analysed in both <it>in vitro </it>and <it>in vivo </it>assays.</p> <p>Methods</p> <p><it>B. cenocepacia-</it>infected BEAS-2B epithelial respiratory cells were used to investigate the bacterial cytotoxicity assessed by the flow cytometric detection of cell staining with propidium iodide. Bacteria-induced procoagulant activity in cell cultures was assessed by a colorimetric assay and by the flow cytometric detection of tissue factor (TF)-bearing microparticles in cell culture supernatants. Bronchoalveolar lavage fluids (BALF) from intratracheally infected mice were assessed for bacterial proinflammatory and procoagulant activities as well as for bacterial cytotoxicity, by the detection of released lactate dehydrogenase.</p> <p>Results</p> <p>ET-12 was significantly more cytotoxic to cell cultures but clinical isolates Cl-2, Cl-3 and Cl-4 exhibited also a cytotoxic profile. ET-12 and CI-2 were similarly able to generate a TF-dependent procoagulant environment in cell culture supernatant and to enhance the release of TF-bearing microparticles from infected cells. In the <it>in vivo </it>assay, all bacterial isolates disseminated from the mice lungs, but Cl-2 and Cl-4 exhibited the highest rates of recovery from mice livers. Interestingly, Cl-2 and Cl-4, together with ET-12, exhibited the highest cytotoxicity. All bacteria were similarly capable of generating a procoagulant and inflammatory environment in animal lungs.</p> <p>Conclusion</p> <p><it>B. cenocepacia </it>were shown to exhibit cytotoxic and procoagulant activities potentially implicated in bacterial dissemination into the circulation and acute pulmonary decline detected in susceptible CF patients. Improved understanding of the mechanisms accounting for <it>B. cenocepacia</it>-induced clinical decline has the potential to indicate novel therapeutic strategies to be included in the care <it>B. cenocepacia</it>-infected patients.</p