Proteins on microbial identification: past achievements, present state-of-the-art, and future challenges

The use of chemical analysis of microbial components, including proteins, became an important achievement in the 80’s of the last century to the microbial identification. This led a more objective microbial identification scheme, called chemotaxonomy, and the analytical tools used in the field are mainly 1D/2D gel electrophoresis, spectrophotometry, high-performance liquid chromatography, gas chromatography, and combined gas chromatography-mass spectrometry. The Edman degradation reaction was also applied to peptides sequence giving important insights to the microbial identification. The rapid development of these techniques, in association with knowledge generated by DNA sequencing and phylogeny based on rRNA gene and housekeeping genes sequences, boosted the microbial identification to an unparalleled scale. The recent results of mass spectrometry (MS), like Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF), for rapid and reliable microbial identification showed considerable promise. In addition, the technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with other biological techniques. At present, MALDI-TOF MS adds an additional step for polyphasic identification which is essential when there is a paucity of characters or high DNA homologies for delimiting very close related species. The full impact of this approach is now being appreciated when more diverse species are studied in detail and successfully identified. However, even with the best polyphasic system, identification of some taxa remains time-consuming and determining what represents a species remains subjective. The possibilities opened with new and even more robust mass spectrometers combined with sound and reliable databases allow not only the microbial identification based on the proteome fingerprinting but also include de novo specific proteins sequencing as additional step. These approaches are pushing the boundaries in the microbial identification field.

The influence of kaolin application on key metabolic pathways associated with grape berry quality: a molecular and biochemical analysis

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Insights into bacterial colony morphology evolution and diversification during infection development in cystic fibrosis

Tese de Doutoramento em Engenharia Biomédica.

Enzymatic hydrophobic modification of jute fibers via grafting to reinforce composites

Horseradish peroxidase (HRP)/H2O2 system catalyzes the free-radical polymerization of aromatic compounds such as lignins and gallate esters. In this work, dodecyl gallate (DG) was grafted onto the surfaces of lignin-rich jute fabrics by HRP-mediated oxidative polymerization with an aim to enhance the hydrophobicity of the fibers. The DG-grafted jute fibers and reaction products of their model compounds were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results clearly indicated the grafting of DG to the jute fiber by HRP. Furthermore, the hydrophobicity of jute fabrics was determined by measuring the wetting time and static contact angle. Compared to the control sample, the wetting time and static contact angle of the grated fabrics changed from ~1 s to 1 h and from ~0° to 123.68°, respectively. This clearly proved that the hydrophobicity of jute fabrics improved considerably. Conditions of the HRP-catalyzed DG-grafting reactions were optimized in terms of the DG content of modified jute fabrics. Moreover, the results of breaking strength and elongation of DG-grafted jute/ polypropylene (PP) composites demonstrated improved reinforcement of the composite due to enzymatic hydrophobic modification of jute fibers.

Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. In the present study, we prospectively compared MALDI-TOF MS to the conventional phenotypic method for the identification of routine isolates. Colonies were analyzed by MALDI-TOF MS either by direct deposition on the target plate or after a formic acid-acetonitrile extraction step if no valid result was initially obtained. Among 1,371 isolates identified by conventional methods, 1,278 (93.2%) were putatively identified to the species level by MALDI-TOF MS and 73 (5.3%) were identified to the genus level, but no reliable identification was obtained for 20 (1.5%). Among the 1,278 isolates identified to the species level by MALDI-TOF MS, 63 (4.9%) discordant results were initially identified. Most discordant results (42/63) were due to systematic database-related taxonomical differences, 14 were explained by poor discrimination of the MALDI-TOF MS spectra obtained, and 7 were due to errors in the initial conventional identification. An extraction step was required to obtain a valid MALDI-TOF MS identification for 25.6% of the 1,278 valid isolates. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional phenotypic identification for most bacterial strains routinely isolated in clinical microbiology laboratories.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues.

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

Untargeted profiling of urinary steroid metabolites after testosterone ingestion: opening new perspectives for antidoping testing.

AIM: Antidoping procedures are expected to greatly benefit from untargeted metabolomic approaches through the discovery of new biomarkers of prohibited substances abuse. RESULTS: Endogenous steroid metabolites were monitored in urine samples from a controlled elimination study of testosterone undecanoate after ingestion. A platform coupling ultra-high pressure LC with high-resolution quadrupole TOF MS was used and high between-subject metabolic variability was successfully handled using a multiblock data analysis strategy. Links between specific subsets of metabolites and influential genetic polymorphisms of the UGT2B17 enzyme were highlighted. CONCLUSION: This exploratory metabolomic strategy constitutes a first step toward a better understanding of the underlying patterns driving the high interindividual variability of steroid metabolism. Promising biomarkers were selected for further targeted study.

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. II: Confirmatory analysis.

For doping control, analyses of samples are generally achieved in two steps: a rapid screening and, in the case of a positive result, a confirmatory analysis. A two-step methodology based on ultra-high-pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was developed to screen and confirm 103 doping agents from various classes (e.g., beta-blockers, stimulants, diuretics, and narcotics). The screening method was presented in a previous article as part I (i.e., Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. Part I: screening analysis). For the confirmatory method, basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction (SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor and characteristic product ions. The mass accuracy and the elemental composition of precursor and product ions were used for compound identification. After validation including matrix effect determination, the method was considered reliable to confirm suspect results without ambiguity according to the positivity criteria established by the World Anti-Doping Agency (WADA). Moreover, an isocratic method was developed to separate ephedrine from its isomer pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their direct quantification in urine.

A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N′-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N′-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

New insights in carbohydrate-deficient transferrin analysis with capillary electrophoresis-mass spectrometry

Capillary zone electrophoresis (CZE) with UV detection has been widely used for the determination of carbohydrate-deficient transferrin (CDT), an indirect marker of the chronic alcohol consumption (≥60-80g/day). A commercially available method (CEofix? CDT kit), containing a bilayer anionic coating, allows for the analysis of CDT with a high resolution between transferrin (Tf) glycoforms with reduced protein adsorption onto the capillary wall. Although widely used in routine analysis, this procedure presents some limitations in terms of selectivity and sensitivity which may be overcome with mass spectrometry (MS). However, the available method is not MS-compatible due to the non-volatile coating as well as the phosphate and borate buffers present in the background electrolyte (BGE). This study firstly consisted in developing MS-compatible separation conditions, i.e., coating and BGE compositions. Numerous cationic, neutral, and anionic coatings were evaluated in combination with BGEs covering a broad range of pH values. A bilayer coating composed of a cationic layer of 10% polybrene (m/v) and an anionic layer of 10% dextran sulfate (m/v) combined with a BGE composed of 20mM ammonium acetate at pH 8.5 provided the best results in terms of glycoforms' resolution, efficiency, adsorption reduction, migration times' repeatability, and coating stability. The method was then transferred to CZE-MS after investigations of the electrospray ionization (ESI) source, equipped with a sheath-flow interface, and the time-of-flight (TOF/MS) parameters. A successful MS detection of tetrasialo-Tf was obtained during infusion, while the experiments highlighted the challenges and issues encountered with intact glycoprotein analysis by CZE-ESI-MS.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.

Effective empirical treatment is of paramount importance to improve the outcome of patients with Staphylococcus aureus bacteraemia. We aimed to evaluate a PCR-based rapid diagnosis of methicillin resistance (GeneXpert MRSA) after early detection of S. aureus bacteraemia using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Patients with a first episode of S. aureus bacteraemia identified using MALDI-TOF MS were randomized in a prospective interventional open study between October 2010 and August 2012. In the control group, antibiotic susceptibility testing was performed after MALDI-TOF MS identification on blood culture pellets. In the intervention group, a GeneXpert MRSA was performed after S. aureus identification. The primary outcome was the performance of GeneXpert MRSA directly on blood cultures. We then assessed the impact of early diagnosis of methicillin resistance on the empirical treatment. In all, 197 episodes of S. aureus bacteraemia were included in the study, of which 106 were included in the intervention group. Median time from MALDI-TOF MS identification to GeneXpert MRSA result was 97 min (range 25-250). Detection of methicillin resistance using GeneXpert MRSA had a sensitivity of 99% and a specificity of 100%. There was less unnecessary coverage of MRSA in the intervention group (17.1% versus 29.2%, p 0.09). GeneXpert MRSA was highly reliable in diagnosing methicillin resistance when performed directly on positive blood cultures. This could help to avoid unnecessary prescriptions of anti-MRSA agents and promote the introduction of earlier adequate coverage in unsuspected cases.

Histology-driven data mining of lipid signatures from multiple imaging mass spectrometry analyses: application to human colorectal cancer liver metastasis biopsies.

Imaging mass spectrometry (IMS) represents an innovative tool in the cancer research pipeline, which is increasingly being used in clinical and pharmaceutical applications. The unique properties of the technique, especially the amount of data generated, make the handling of data from multiple IMS acquisitions challenging. This work presents a histology-driven IMS approach aiming to identify discriminant lipid signatures from the simultaneous mining of IMS data sets from multiple samples. The feasibility of the developed workflow is evaluated on a set of three human colorectal cancer liver metastasis (CRCLM) tissue sections. Lipid IMS on tissue sections was performed using MALDI-TOF/TOF MS in both negative and positive ionization modes after 1,5-diaminonaphthalene matrix deposition by sublimation. The combination of both positive and negative acquisition results was performed during data mining to simplify the process and interrogate a larger lipidome into a single analysis. To reduce the complexity of the IMS data sets, a sub data set was generated by randomly selecting a fixed number of spectra from a histologically defined region of interest, resulting in a 10-fold data reduction. Principal component analysis confirmed that the molecular selectivity of the regions of interest is maintained after data reduction. Partial least-squares and heat map analyses demonstrated a selective signature of the CRCLM, revealing lipids that are significantly up- and down-regulated in the tumor region. This comprehensive approach is thus of interest for defining disease signatures directly from IMS data sets by the use of combinatory data mining, opening novel routes of investigation for addressing the demands of the clinical setting.

Análisis del perfil de péptidos en sangre y orina en pacientes con nefropatía IgA y su asociación con las lesiones histológicas según la clasificación de Oxford

La nefropatía IgA (NIgA) es causa importante de insuficiencia renal crónica. Se propone estudiar el perfil proteómico en sangre/orina de pacientes con NIgA y su asociación con las lesiones histológicas según la clasificación de Oxford. Se incluyeron pacientes con NIgA entre 2006-2009, evaluando las lesiones histológicas según la Clasificación de Oxford. Se realizó análisis proteómico mediante microesferas magnéticas y espectrometría de masas (MALDI-TOF MS). Encontramos una asociación significativa entre péptidos en sangre/orina y las lesiones histológicas. Uromodulina, alfa-1-antitripsina y bradiquinina, mostraron una asociación significativa con la lesión tubulointersticial y glomerulosclerosis. Los péptidos m/z (1769,1898,1913,1945,2378,2491,2977,3004,3389,3406,4752,5337,9289) se asociaban con una peor función renal. Palabras claves: Clasificación de Oxford, Nefropatía IgA, perfil de péptidos.

Identification and antifungal susceptibility of a large collection of yeast strains isolated in Tunisian hospitals.

Abstract In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a rapid method to identify yeasts isolated from patients in Tunisian hospitals. When identification could not be exstablished with this procedure, sequencing of the internal transcribed spacer with 5.8S ribosomal DNA (rDNA) (ITS1-5.8S-ITS2) and D1/D2 domain of large-subunit (LSU rDNA) were employed as a molecular approach for species differentiation. Candida albicans was the dominant species (43.37% of all cases), followed by C. glabrata (16.55%), C. parapsilosis (13.23%), C. tropicalis (11.34%), C. dubliniensis (4.96%), and other species more rarely encountered in human diseases such as C. krusei, C. metapsilosis, C. lusitaniae, C. kefyr, C. palmioleophila, C. guilliermondii, C. intermedia, C. orthopsilosis, and C. utilis. In addition, other yeast species were obtained including Saccharomyces cerevisiae, Debaryomyces hansenii (anamorph known as C. famata), Hanseniaspora opuntiae, Kodamaea ohmeri, Pichia caribbica (anamorph known as C. fermentati), Trichosporon spp. and finally a novel yeast species, C. tunisiensis. The in vitro antifungal activities of fluconazole and voriconazole were determined by the agar disk diffusion test and Etest, while the susceptibility to additional antifungal agents was determined with the Sensititre YeastOne system. Our results showed low incidence of azole resistance in C. albicans (0.54%), C. tropicalis (2.08%) and C. glabrata (4.28%). In addition, caspofungin was active against most isolates of the collection with the exception of two K. ohmeri isolates. This is the first report to describe caspofungin resistant isolates of this yeast.