Introduction
According to a proposal of Yassin et al. (2011) genus Arcanobacterium consists of four species namely Arcanobacterium haemolyticum, Arcanobacterium phocae, Arcanobacterium pluranimalium and Arcanobacterium hippocoleae. More recently Arcanobacterium canis and Arcanobacterium phocisimile were described as novel species of genus Arcanobacterium (Hijazin et al., 2012a, Hijazin et al., 2013). The closely related species Arcanobacterium pyogenes, Arcanobacterium abortisuis, Arcanobacterium bernardiae, Arcanobacterium bialowiezense and Arcanobacterium bonasi were reclassified to the newly described genus Trueperella as Trueperella pyogenes, Trueperella abortisuis, Trueperella bernardiae, Trueperella bialowiezensis and Trueperella bonasi (Yassin et al., 2011). However, both genera belong to the family Actinomycetaceae (http://www.ncbi.nlm.nih.gov/taxonomy).
A. pluranimalium was initially described based on two isolates which were recovered from a harbour porpoise and a fallow deer, respectively (Lawson et al., 2001). In the following years this bacterial species was isolated in a mixed culture from a dog with pyoderma (Ülbegi-Mohyla et al., 2010a) and from abortion material, semen, abscesses, viscera, navel ill and peritonitis of sheep and from a milk sample of a cow with mastitis (Foster and Hunt, 2011). The identification of the hitherto described A. pluranimalium was performed with phenotypic methods (Foster and Hunt, 2011) and by sequencing 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR) and 23S rDNA (Lawson et al., 2001, Ülbegi-Mohyla et al., 2010a).
The present study was designed to investigate the usefulness of MALDI-TOF MS and the pluranimaliumlysin encoding target gene pla for identification of novel and previously described A. pluranimalium strains of various origins.
Section snippets
Materials and methods
The strains used in the present study included reference strain A. pluranimalium DSM 13483, other strains of genus Arcanobacterium (Sammra et al., 2014), A. pluranimalium 1128 isolated from a dog with pyoderma (Ülbegi-Mohyla et al., 2010a) and 12 hitherto not characterized A. pluranimalium strains isolated in Scotland, England and Germany. Further data about the origin of the 13 A. pluranimalium strains are summarized in Table 1.
The strains were investigated phenotypically (Ülbegi-Mohyla et
Results and discussion
All 12 A. pluranimalium strains newly investigated in the present study showed the typical phenotypical properties of this species and could be classified as A. pluranimalium by 16S rDNA sequencing. A typical dendrogram of the 16S rDNA sequencing results is shown in Fig. 1. The phenotypical properties included a moderate hemolysis on sheep blood agar, a slightly enhanced hemolysis on rabbit blood agar, positive CAMP-like reactions with Staphylococcus aureus β-hemolysin, Rhodococcus equi and
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It was of interest that despite some sequence variation of gene phl, all 12 A. phocae strains in the present study and the reference strains A. phocae DSM 10002T and A. phocae DSM 10003 uniformly displayed the undecapeptide sequence EATGLAWDPWW which differs from the undecapeptide sequence of ALN of A. haemolyticum, PLO of T. pyogenes, undecapeptides of other CDCs and the consensus sequence (Fig. 2B). Comparable to gene plo of T. pyogenes, which appeared to be a constant characteristic of all investigated T. pyogenes (Billington et al., 1997; Ülbegi-Mohyla et al., 2010; Hijazin et al., 2011) and gene pla as typical characteristic of all investigated A. pluranimalium (Moser et al., 2013; Balbutskaya et al., 2014, Risse et al., 2014; Wickhorst et al., 2016; Ningrum et al., 2017), phl of A. phocae seems to be also constantly present in all strains of this species and could be used for molecular identification of A. phocae. Further studies will focus on providing more information on the consistent presence and the pathogenic importance of this hitherto unknown virulence factor of A. phocae.
In the present study 12 Arcanobacterium phocae strains isolated from fur animals in Finland, including foxes, minks and Finnraccoons, could successfully be identified phenotypically, by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and phocaelysin (PHL) encoding gene phl. The PHL of all 12 A. phocae strains in the present study and reference strains A. phocae DSM 10002T and A. phocae DSM 10003 displayed, as typical members of the cholesterol dependent cytolysin-group of toxins, the variant undecapeptide sequence EATGLAWDPWW which appeared to be most closely related to arcanolysin of Arcanobacterium haemolyticum and pyolysin of Trueperella pyogenes. In addition, gene phl could be determined with a newly designed loop-mediated isothermal amplification (LAMP) assay. The detection of mass spectra by MALDI-TOF MS and the LAMP assay based on gene phl might help to reliably identify A. phocae in future and also elucidate the role this species plays in infections of fur animals.
Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium
2015, Molecular and Cellular Probes
Citation Excerpt :
The present study was designed to investigate a newly developed LAMP assay targeting the pluranimaliumlysin encoding gene pla for identification of novel and previously described A. pluranimalium strains of various origins. The strains used in the present study included the reference strain A. pluranimalium DSM 13483, other presently available type strains and other strains of genus Arcanobacterium (n = 8) and Trueperella (n = 10) obtained from the strain collection of the Institute of Pharmacology and Toxicology at Justus Liebig University Giessen, Germany, and field strains of A. pluranimalium strains from ovine (n = 23), bovine (n = 3) [7,10] origin and from a juvenile giraffe (n = 1) [9], (Table 2). The bacterial DNA was isolated using the DNeasy tissue isolation kit (Qiagen, Germany) according to the manufacturer's instructions.
In the present study 28 Arcanobacterium pluranimalium strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene pla encoding pluranimaliumlysin. No comparable reaction could be observed with control strains representing five species of genus Arcanobacterium and five species of genus Trueperella. The presented pla LAMP assay might allow a reliable and low cost identification of this bacterial pathogen also in laboratories with less specified equipment.
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FAQs
What is matrix assisted laser desorption ionization time of flight mass spectrometry? ›
In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation.
What is matrix assisted laser desorption ionization time of flight mass spectrometry in clinical microbiology? ›A new method, Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), has been recently developed to profile bacterial proteins from whole cell extracts and obtain a bacterial fingerprint able to discriminate microorganisms from different genera and species.
What are the 4 steps in MALDI-TOF? ›MALDI-TOF MS-based yeast identification requires a pretreatment step for which four are described in the literature, i.e., direct smear, fast formic acid and two complete formic acid/acetonitrile extractions.
What are the 4 stages of TOF mass spectrometry? ›The four stages of mass spectrometry are – ionization, acceleration, deflection, and detection. The sample is vaporized before being passed into an ionization chamber where it is bombarded by a stream of electrons emitted by an electrically heated metal coil.
What is matrix assisted laser desorption ionization used for? ›Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that enables the simultaneous detection and identification of biomolecules in analytes.
Which is the most common laser used in Maldi-TOF MS? ›In the case of MALDI-TOF MS, which uses a UV laser, the matrix molecule must also have a strong chromophore as part of its composition to help absorb energy, thus preserving the protein fragmentation.
What is matrix for Maldi imaging? ›MALDI-TOF MS is based on the use of a matrix that absorbs the energy of the laser irradiation and is, thus, crucial for the successful generation of ions. There are basically three different classes of matrices available: Classical organic matrices, such as benzoic acid or cinnamic acid derivatives.
What are the three key major components of a Maldi-TOF MS instrument? ›MALDI-TOF MS's ability to quantify the presence of different analytes depends on many factors. These factors are based on three MS components: the ion source, mass analyser and detector.
What is MALDI-TOF and how does it work? ›Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) is a diagnostic tool of microbial identification and characterization based on the detection of the mass of molecules.
What is MALDI-TOF method for bacterial identification? ›Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) is replacing traditional methods for identifying microorganisms in the clinical laboratory. This relatively simple technique overcomes many of the challenges of identifying bacteria and fungi.
How does a TOF detector work? ›
Time-of-Flight (TOF) is a mass analyser that utilises an electric field to accelerate generated ions through the same electrical potential, and then measures the time each ion takes to reach the detector.
What is the major advantage of TOF? ›Advantages of using TOF for broad spectrum analysis includes increased mass accuracy and mass resolution, greater sensitivity, rapid acquisition, and increased dynamic range when profiling over a broad molecular weight range.
What is the difference between quad and TOF? ›A quadrupole and a TOF each measure ten mass-to-charge ratios. The quadrupole can spend no more than 1 second accumulating signal for each mass-to-charge ratio, while the TOF accumulates the full ten seconds of signal for each m/Q.
How does MALDI-TOF identify proteins? ›Proteins and peptides have been characterized by high pressure liquid chromatography (HPLC) or SDS PAGE by generating peptide maps. These peptide maps have been used as fingerprints of protein or as a tool to know the purity of a known protein in a known sample.
What is the purpose of the laser in MALDI-TOF? ›MALDI MS units have been increasingly used in imaging applications where the highly localized nature of laser ablation allows the determination of the concentrations and spatial locations of compounds of interest in a wide range of sample types, especially biological tissues.
What are the limitations of MALDI-TOF? ›The most significant limitation of MALDI-TOF is its low analytical sensitivity without prior cultivation and the discrimination of phyletically related microorganisms such as Shigella and Escherichia coli [41] Consequently, MALDI-TOF is unsuitable for detecting the small number of bacteria in sterile samples.
How accurate is MALDI-TOF mass spectrometry? ›With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100-500).
What organism can be identified using MALDI-TOF MS? ›A number of published studies have demonstrated the accuracy of MALDI-TOF MS for the identification of a broad spectrum of bacteria,12,14–16,28–33 including gram-positive cocci and rods, fermentative and nonfermentative gram-negative rods, and anaerobes.
What is the difference between mass spectrometry and MALDI-TOF? ›Mass spectrometry is an analytical technique in which samples are ionized into charged molecules and ratio of their mass-to-charge (m/z) can be measured. In MALDI-TOF mass spectrometry, the ion source is matrix-assisted laser desorption/ionization (MALDI), and the mass analyzer is time-of-flight (TOF) analyzer.
Why are these common features important for a MALDI matrix? ›Why are these common features important for a MALDI matrix? A. They have aromatic rings so they have strong absorption in the ultraviolet region of the spectrum. The have a carboxylic acid group so they can transfer a proton to the biomolecule giving it a positive charge.
What is typically MALDI technique used for? ›
Matrix Assisted Laser Desorption/Ionization (MALDI) is used to analyze extremely large molecules. This technique directly ionizes and vaporizes the analyte from the condensed phase. MALDI is often used for the analysis of synthetic and natural polymers, proteins, and peptides.
What are the advantages of Maldi imaging? ›MALDI-MS is a valuable analytical tool in pathology and laboratory medicine. Advantages of MALDI-MS include ease of sample preparation and analysis. Uses include ID of pathogens, monoclonal proteins, variants and diseased tissue.
What is ionization time of flight mass spectrometry? ›Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined by a time of flight measurement. Ions are accelerated by an electric field of known strength.
What is the purpose of the matrix in matrix assisted laser desorption ionization? ›The matrix performs two important functions: (1) it absorbs photon energy from the laser beam and transfers it into excitation energy of the solid system, and (2) it serves as a solvent for the analyte, so that the intermolecular forces are reduced and aggregation of the analyte molecules is held to a minimum.
What is desorption ionization mass spectrometry? ›Laser desorption mass spectrometry uses a laser to ionize the analyte. In some cases, the laser can be applied directly to the sample. It will remove molecules from the surface of the material and then ionize them. Very little decomposition of the parent ion takes place so molecular ions are always prominent.
How does matrix assisted laser desorption ionization work? ›Matrix Assisted Laser Desorption/Ionization (MALDI) is used to analyze extremely large molecules. This technique directly ionizes and vaporizes the analyte from the condensed phase. MALDI is often used for the analysis of synthetic and natural polymers, proteins, and peptides.
What is the purpose of time of flight mass spectrometry? ›Time of flight mass spectrometry (TOF–MS) is one approach to capturing a broad molecular weight range of signals associated with polar and nonpolar compounds in a single sample. This method uses an electric field to accelerate ions to the same potential.
What are the two ionization methods for mass spectrometry? ›- Chemical ionization (CI) ...
- Plasma and glow discharge. ...
- Electron impact (EI) ...
- Electrospray ionization (ESI) ...
- Fast-atom bombardment (FAB) ...
- Field ionization. ...
- Laser ionization (LIMS) ...
- Matrix-assisted laser desorption ionization (MALDI)
Time-of-Flight (TOF) is a mass analyser that utilises an electric field to accelerate generated ions through the same electrical potential, and then measures the time each ion takes to reach the detector.
What are the disadvantages of matrix-assisted laser desorption ionization? ›There are limitations to matrix-assisted laser desorption ionization mass spectrometry. No susceptibility information is provided, and the technology is not generally useful for direct testing of clinical specimens. Some organisms require repeat analyses and additional processing.
Why is the matrix method used? ›
Conveniently, matrix methods are ideally suited for expressing structural theory and expressing that theory in a form suitable for numerical solution by computer. A structural problem may be formulated in either of the two ways.
What makes a good Maldi Matrix? ›Your matrix should have a strong optical absorption in either the ultraviolet or infrared range, in order to rapidly and efficiently absorb the laser irradiation. The matrix needs to contain polar groups, which allow them to be used in aqueous solutions.
What is the difference between laser ablation and desorption? ›There are two representative methods for the gasification of the condensed samples, i.e., ablation and desorption. While ablation is based on the explosion induced by the energy accumulated in the condensed matrix, desorption is a single molecular process taking place on the surface.
What does MALDI-TOF detect? ›Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a widely used technique for the rapid and accurate identification of bacteria, mycobacteria and certain fungal pathogens in the clinical microbiology laboratory.
What is the difference between MALDI and ESI? ›For MALDI, an analyte is embedded into a typically acidic matrix which heavily absorbs UV light. Excited by a short laser pulse, parts of the matrix heat rapidly and are vaporized/ionized together with the analyte. (3) In ESI, an electric field is applied to an analyte solution flowing through a capillary.
What is the purpose of MALDI? ›MALDI MS units have been increasingly used in imaging applications where the highly localized nature of laser ablation allows the determination of the concentrations and spatial locations of compounds of interest in a wide range of sample types, especially biological tissues.
What is the principle of MALDI? ›MALDI – Principle and Methodology. The sample for analysis by MALDI MS is prepared by mixing or coating with solution of an energy-absorbent, organic compound called matrix. When the matrix crystallizes on drying, the sample entrapped within the matrix also co-crystallizes.
How does MALDI imaging work? ›After tissue sectioning and transfer onto a conductive and transparent sample plate, the MALDI matrix is deposited, and data are acquired by recording mass spectra according to a raster of points covering the surface to be analyzed.