Evaluation of Raman spectroscopy as identification tool for bacteria

Ramanspectroscopie is een snelle en eenvoudig te gebruiken analytische techniek, die recent werd geïntroduceerd voor de snelle identificatie van bacteriën. Het bekomen Ramanspectrum is zeer specifiek en dient als een vingerafdruk die uniek is voor de moleculen die worden geëxciteerd. In het geval van micro-organismen geeft dit spectrum dus de volledige moleculaire samenstelling weer, die ten dele wordt bepaald door taxonomische merkers (vb. vetzuren, eiwitten, polyamines, enz.), ten dele door moleculen die niet speciesspecifiek zijn (e.g. carotenoïden) en ten dele door groeiparameters. Rekening houdend met de recente publicaties over de identificatie van micro-organismen met Ramanspectroscopie, evalueert deze scriptie de toepasbaarheid van deze techniek binnen het genus Bacillus. Om een realistisch beeld te verkrijgen van de mogelijkheden van Ramanspectroscopie, als een toekomstige techniek binnen de taxonomie en/of de identificatie van bacteriën, ging speciale aandacht uit naar de standaardisatie van de benadering en de keuze van goed gekozen species (modelsystemen) voor de evaluatie. Wanneer Ramanspectra sterk gelijkend zijn, zoals in het geval van nauw verwante bacteriën, wordt de instrumentele stabiliteit een belangrijk punt. Om de lange termijn stabiliteit van de spectrale databank te garanderen wordt een methode beschreven voor (1) de volledige kalibratie van de Raman set-up en (2) de standaardisatie van de bekomen Ramanspectra waardoor voor spectrale verschuivingen wordt gecorrigeerd. Het effect van de groeiomstandigheden op de Ramanspectra werd onderzocht voor drie, fenotypisch verschillende species B. cereus, B. licheniformis en B. pumilus. Een brede waaier aan groeiomstandigheden werd getest, wat uiteindelijk leidde tot de ontwikkeling van een gestandaardiseerd protocol. De aldus ontwikkelde methodologie werd grondig gevalideerd d.m.v. acht nauw verwante Bacillus species die vertegenwoordigd zijn door 69 stammen. Gebaseerd op kleine, maar reproduceerbare, spectrale verschillen was het mogelijk de verschillende species te identificeren. Er kon besloten worden dat Raman-spectroscopie een algemeen toepasbare techniek is voor de identificatie van bacteriën. De volgende stap betreft een situatie waarin geen betrouwbare a priori kennis over de identiteit van stammen beschikbaar is. Hier wordt het potentieel van Ramanspectroscopie binnen de taxonomie verkend a.d.h.v. de fylogenetisch homogene ‘Bacillus cereus’-groep. Er werd onderzocht of de clusters die konden afgelijnd worden d.m.v. Ramanspectroscopie overeenkwamen met deze bekomen met FAFLP. Verder werd in een correlatiestudie een sterk significante correlatie vastgesteld tussen Raman spectroscopie en de genomische techniek FAFLP. Als besluit kan men stellen dat Ramanspectroscopie een eenvoudige, snelle (< 8u, inclusief groei) en accurate identificatie techniek is die geen (schadelijke) reagentia of tijdrovende monstervoorbereiding vereist en dus relatief goedkoop is in gebruik. Bijgevolg onderscheidt deze techniek zich duidelijk van alternatieve fenotypische en genotypische methoden. Deze scriptie heeft zich geconcentreerd op een groep van fylogenetisch nauw verwante en fenotypisch sterk gelijkende species (e.g. ‘B. subtilis’ en ‘B. cereus’ groep) om de identificatie-mogelijkheden van Raman spectroscopie te evalueren. Binnen de meeste toepassingen (vb. voedings-, grond- en waterisolaten; plantpathogenen; klinisch relevante organismen, etc.) komen zulke nauw verwante species echter zelden voor en er kan dus verwacht worden dat deze techniek gemakkelijk kan uitgebreid worden naar dergelijke toepassingen

Evaluation of an accurate calibration and spectral standardization procedure for Raman spectroscopy

Due to modern developments Raman spectroscopy has evolved into a fast vibrational technique. Detailed fingerprints in combination with non-destructivity and minimal sample preparation has allowed the construction of reference libraries in a variety of research fields. Long-term stability and comparability are important characteristics when developing reference libraries. In addition, small shifts in highly similar spectra of different samples may limit the full potential of Raman spectroscopy. Since libraries often contain a large number of different and/or highly similar spectra, it is important that each data point in all the spectra corresponds to the exact Raman wavenumber. This is often not the case, due to shifts in optical pathway and/or shifts in laser wavelength. This paper describes a complete calibration protocol ( wavelength and intensity) and evaluates the procedure for both short and long term stability, by means of 60 randomly selected measurement sessions spread over a period of nine months. A two-step standardization procedure is proposed to deal with spectral shifts

Psychiatric stigma in treatment-seeking adults with personality problems: evidence from a sample of 214 patients

Stigmatization is a major burden in adult psychiatric patients with Axis-I diagnoses, as shown consistently in most studies. Significantly fewer studies on the emergence of psychiatric stigma in adult patients with personality disorders (PDs) exist, although the resulting evidence is conclusive. Some authors consider patients with PDs at risk for severe stigmatization because of intense difficulties during interpersonal contact, even in a psychotherapeutic relationship. The aim of this study was primarily the assessment of pre-existing stigma in patients referred for intensive treatment for PDs. The study enrolled 214 patients admitted to the adult department of a highly specialized mental health care institute offering psychotherapy for patients with severe and complex personality pathology. All patients underwent a standard assessment with self-report questionnaires and a semi-structured interview to measure Axis II PDs. The stigma consciousness questionnaire and the perceived devaluation-discrimination questionnaire, both validated instruments, were used to measure perceived and actual experiences of stigma. Independent sample t-tests were used to investigate differences in the mean total stigma scores for patients both with and without a PD. One-way ANOVAs were performed to assess the differences between having a borderline PD, another PD, or no PD diagnosis. Multiple regression main effect analyses were conducted in order to explore the impact of the different PD diagnosis on the level of stigma. The mean scores across all patient groups were consistent with rather low stigma. No differences were found for patients with or without a PD diagnosis. Level of stigma in general was not associated with an accumulating number of PDs. Given the remarkable results, we would strongly recommend further investigations in the field to better understand the phenomenon of stigma in all its aspects

Adolescents with personality disorders suffer from severe psychiatric stigma : evidence from a sample of 131 patients

BACKGROUND: The aim of the study is to assess the severity of psychiatric stigma in a sample of personality disordered adolescents in order to evaluate whether differences in stigma can be found in adolescents with different types and severity of personality disorders (PDs). Not only adults but children and adolescents with mental health problems suffer from psychiatric stigma. In contrast to the abundance of research in adult psychiatric samples, stigma in children and adolescents has hardly been investigated. Personality disordered adolescents with fragile identities and self-esteem might be especially prone to feeling stigmatized, an experience which might further shape their identity throughout this critical developmental phase. MATERIALS AND METHODS: One hundred thirty-one adolescent patients underwent a standard assessment with Axis I and Axis II diagnostic interviews and two stigma instruments, Stigma Consciousness Questionnaire (SCQ) and Perceived Devaluation–Discrimination Questionnaire (PDDQ). Independent sample t-tests were used to investigate differences in the mean SCQ and PDDQ total scores for patients with and without a PD. Multiple regression main effect analyses were conducted to explore the impact of the different PDs on level of stigma, as well as comorbid Axis I disorders. Age and sex were also entered in the regression models. RESULTS AND CONCLUSIONS: Adolescents with severe mental health problems experience a burden of stigma. Personality disordered patients experience more stigma than adolescents with other severe psychiatric Axis I disorders. Borderline PD is the strongest predictor of experiences of stigma. More severely personality disordered adolescents tend to experience the highest level of stigma

Possibilities of laser ablation-inductively coupled plasma-mass spectrometry for diamond fingerprinting

In this paper the performance of laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) for the fingerprinting of diamonds of different deposits has been evaluated. The results show that, after optimization of the experimental conditions, the LA unit used (a homogenized 193 nm excimer laser with a flat-top beam profile) is capable of controlled ablation of the diamonds and the sensitivity of the ICP-MS device suffices for the detection of more than ten elements. 31 diamonds originating from four different mines-Premier (South Africa), Orapa (Botswana), Udachnaya (Russia) and Panda (Canada)-were the subject of the study. Every diamond was ablated on 8 different spots for 30 s (crater diameter: 120 mm; energy output: 200 mJ; repetition rate: 20 Hz) and the median of the eight integrated signals was considered as the representative value for subsequent statistical analysis. Under these conditions, the total mass of material removed from a single diamond was similar to16 mug. Finally, 9 elements were selected for fingerprinting purposes (Al, Hg, Na, Ni, Pb, Sb, Sn, Ti and Zn) and different pattern recognition techniques (ternary plots, cluster analysis and partial least squares analysis) were used in order to classify the data. The results obtained can be considered as promising, especially for the partial least squares approach, providing that appropriate data standardization is carried out

Preliminary study of associative stigma among trainee psychiatrists in Flanders, Belgium

AIM: To study the degree of stigmatization among trainee psychiatrists, individual characteristics potentially leading to higher associative stigma, and coping mechanisms. METHODS: Two hundred and seven trainee psychiatrists in Flanders (Belgium), all member of the Flemish Association of Trainee Psychiatrists, were approached to participate in the survey. A non-demanding questionnaire that was specifically designed for the purpose of the study was sent by mail. The questionnaire consisted of three parts, each emphasizing a different aspect of associative stigma: devaluing and humiliating interactions, the focus on stigma during medical training, and identification with negative stereotypes in the media. Answers were scored on a Likert scale ranging from 0 to 3. The results were analyzed using SPSS Version 18.0. RESULTS: The response rate of the study was 75.1%. The internal consistency of the questionnaire was good, with a Cronbach’s α of 0.71. Seventy-five percent of all trainee psychiatrists confirmed hearing denigrating or humiliating remarks about the psychiatric profession more than once. Additionally, more than half of them had had remarks about the incompetence of psychiatrists directed at them. Only 1.3% remembered having stigma as a topic during their psychiatric training. Trainees who had been in training for a longer period of time had experienced a significantly higher level of stigmatization than trainees with fewer years of experience (mean total stigma scores of 16.93 ± SD 7.8 vs 14.45 ± SD 6.1, t = -2.179 and P < 0.05). In addition, senior trainees effectively kept quiet about their profession significantly more often than their junior colleagues (mean item score 0.44 ± SD 0.82 vs 0.13 ± SD 0.48, t = 2.874, P < 0.01). Comparable results were found in trainees working in adult psychiatry as were found in those working in child or youth psychiatry (mean item score 0.38 ± SD 0.77 vs 0.15 ± SD 0.53, t = -2.153, P < 0.05). Biologically oriented trainees were more inclined to give preventive explanations about their profession, which can be seen as a coping mechanism used to deal with this stigma (mean item score 2.05 ± SD 1.05 vs 1.34 ± SD 1.1, t = -3.403, P < 0.01). CONCLUSION: Associative stigma in trainee psychiatrists is underestimated. More attention should be paid to this potentially harmful phenomenon in training

Effect of culture conditions on the achievable taxonomic resolution of Raman spectroscopy disclosed by three Bacillus species

Confocal micro-Raman spectroscopy requires a minimum of sample handling and no reagents and allows fast identification of microorganisms. Since it reflects the overall molecular composition of the cells, it provides much more information than classical, microbial analyses. However, since the molecular makeup of a cell depends on culture conditions, it can be argued that this will affect the reproducibility and discrimination ability of Raman spectroscopy. We used Bacillus cereus, Bacillus pumilus, and Bacillus licheniformis, which are known to be clearly distinct from each other and each displaying important phenotypic heterogeneity, in a wide variety of culture conditions to analyze this. It is illustrated that the influence of culture conditions on the identification accuracy and taxonomic resolution of Raman spectroscopy is important though the effect on the final identification is limited within the set of stains studied. Furthermore, some conditions even allow for better discrimination than others. From a practical point of view, it is especially important that differences in culturing time (and culturing temperature) can be accommodated

Univariate en multivariate kalibratiemodellen voor de kwantitatieve determinatie van styreen m.b.v. Ramanspectroscopie /

Scriptie voor het behalen van de graad Licentiaat in de wetenschappen, groep Scheikund