scholarly journals An Improved Method for Rapid Detection of Mycobacterium abscessus Complex Based on Species-Specific Lipid Fingerprint by Routine MALDI-TOF

2021 ◽  
Vol 9 ◽  
Author(s):  
Min Jia Khor ◽  
Agnieszka Broda ◽  
Markus Kostrzewa ◽  
Francis Drobniewski ◽  
Gerald Larrouy-Maumus
Keyword(s):  
Ribosomal Proteins ◽  
Mycobacterium Abscessus ◽  
Bacterial Suspension ◽  
Rapid Diagnostics ◽  
Maldi Tof ◽  
Appropriate Treatment ◽  
Subspecies Level ◽  
Successful Recovery ◽  
The Matrix ◽  
Species Specific

Rapid diagnostics of bacterial infection is the key to successful recovery and eradication of the disease. Currently, identification of bacteria is based on the detection of highly abundant proteins, mainly ribosomal proteins, by routine MALDI-TOF mass spectrometry. However, relying solely on proteins is limited in subspecies typing for some pathogens. This is the case for, for example, the mycobacteria belonging to the Mycobacterium abscessus (MABS) complex, which is classified into three subspecies, namely, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Being able to detect bacteria accurately and rapidly at the subspecies level could not only reliably identify the pathogen causing the disease but also enable better antibiotic stewardship. For instance, M. abscessus subsp. abscessus and M. abscessus subsp. bolletii possess a functional erm41 (erythromycin ribosomal methylation gene 41) gene, whilst M. abscessus subsp. massiliense does not, resulting in differences in macrolide antibiotic (e.g., clarithromycin and azithromycin) susceptibilities. This presents a challenge for physicians when designing an appropriate treatment regimen. To address this challenge, in addition to proteins, species-specific lipids have now been considered as a game changer in clinical microbiology diagnostics. However, their extraction can be time-consuming, and analysis requires the use of apolar toxic organic solvents (e.g., chloroform). Here, we present a new method to accurately detect species and subspecies, allowing the discrimination of the mycobacteria within the MABS complex and relying on the use of ethanol. We found that a combination of the matrix named super-DHB with 25% ethanol with a bacterial suspension at McFarland 20 gave robust and reproducible data, allowing the discrimination of the bacteria within the MABS complex strains tested in this study (n = 9). Further investigations have to be conducted to validate the method on a larger panel of strains for its use in diagnostic laboratories.

scholarly journals Konya İli Böğürtlen, Ahududu ve Kuşburnu Yetiştiriciliğinde Potansiyel Bir Tehdit: Ateş Yanıklığı Hastalığı

2022 ◽  
Vol 9 (sp) ◽  
pp. 2663-2669
Author(s):  
Aysun Öztürk ◽  
Kubilay Kurtulus Bastas
Keyword(s):  
Bacterial Suspension ◽  
Pathogenicity Test ◽  
Distilled Water ◽  
Bacterial Strains ◽  
Molecular Tests ◽  
Flight Mass Spectrometry ◽  
The Matrix ◽  
Shoot Blight ◽  
Phytosanitary Measures ◽  
Species Specific

In the present study, totally 49 samples, which showed the symptoms of leaf and shoot blight and cankers with brown discoloration of necrotic tissues on mature branches, were collected from 22 districts and areas of Konya Province between 2017 and 2019. Presence rate of E. amylovora in collected samples, showing symptoms of the disease, from the province was determined to be 40% for blackberry and raspberry and 33% rosehip for rosehip in three years. Bacteria consistently isolated from the diseased tissues were identified on the basis of biochemical, physiological, and molecular tests, comparing with a reference strain of E. amylovora, isolated from blackberry (Kbb 371). Twenty seven representative bacterial strains were gram-negative, rod-shaped, mucoid, fermentative, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. All strains induced a hypersensitive response in tobacco (Nicotiana tobacum cv. White Burley) 24 h after inoculation with a 108 CFU ml-1 bacterial suspension in sterile distilled water. The strains were identified as E. amylovora using the species-specific primers set A/B (1), which amplified a 1-kb DNA fragment in PCR, and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. In order to fulfill the Koch postulates, pathogenicity test was confirmed by injecting bacterial suspensions of 108 CFU ml-1 in sterile distilled water into the shoot tips of 3-year-old blackberry R. fruticosus cv. Chester, raspberry R. idaeus cv. Heritage and rosehip R. canina. All tests were repeated three times. The bacterium was re-isolated from inoculated plants and identified as E. amylovora. Phytosanitary measures are needed to prevent any further spread of the bacterium as potential inoculum sources to new blackberry, raspberry and rosehip growing areas.

scholarly journals Identification of Adult Fasciola spp. Using Matrix-Assisted Laser/Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

2020 ◽  
Vol 9 (1) ◽  
pp. 82
Author(s):  
Issa Sy ◽  
Lena Margardt ◽  
Emmanuel O. Ngbede ◽  
Mohammed I. Adah ◽  
Saheed T. Yusuf ◽  
...  
Keyword(s):  
Laser Desorption ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Specific Identification ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Species Specific ◽  
Tof Ms ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

Fascioliasis is a neglected trematode infection caused by Fasciola gigantica and Fasciola hepatica. Routine diagnosis of fascioliasis relies on macroscopic identification of adult worms in liver tissue of slaughtered animals, and microscopic detection of eggs in fecal samples of animals and humans. However, the diagnostic accuracy of morphological techniques and stool microscopy is low. Molecular diagnostics (e.g., polymerase chain reaction (PCR)) are more reliable, but these techniques are not routinely available in clinical microbiology laboratories. Matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a widely-used technique for identification of bacteria and fungi; yet, standardized protocols and databases for parasite detection need to be developed. The purpose of this study was to develop and validate an in-house database for Fasciola species-specific identification. To achieve this goal, the posterior parts of seven adult F. gigantica and one adult F. hepatica were processed and subjected to MALDI-TOF MS to create main spectra profiles (MSPs). Repeatability and reproducibility tests were performed to develop the database. A principal component analysis revealed significant differences between the spectra of F. gigantica and F. hepatica. Subsequently, 78 Fasciola samples were analyzed by MALDI-TOF MS using the previously developed database, out of which 98.7% (n = 74) and 100% (n = 3) were correctly identified as F. gigantica and F. hepatica, respectively. Log score values ranged between 1.73 and 2.23, thus indicating a reliable identification. We conclude that MALDI-TOF MS can provide species-specific identification of medically relevant liver flukes.

scholarly journals Signatures of conserved and unique molecular features in Afrotheria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Arangasamy Yazhini ◽  
Narayanaswamy Srinivasan ◽  
Sankaran Sandhya
Keyword(s):  
Ribosomal Proteins ◽  
Individual Species ◽  
Functional Domain ◽  
Nucleic Acid Binding ◽  
Uncharacterized Protein ◽  
Homologous Proteins ◽  
Functional Roles ◽  
Molecular Features ◽  
Specific Proteins ◽  
Species Specific

AbstractAfrotheria is a clade of African-origin species with striking dissimilarities in appearance and habitat. In this study, we compared whole proteome sequences of six Afrotherian species to obtain a broad viewpoint of their underlying molecular make-up, to recognize potentially unique proteomic signatures. We find that 62% of the proteomes studied here, predominantly involved in metabolism, are orthologous, while the number of homologous proteins between individual species is as high as 99.5%. Further, we find that among Afrotheria, L. africana has several orphan proteins with 112 proteins showing < 30% sequence identity with their homologues. Rigorous sequence searches and complementary approaches were employed to annotate 156 uncharacterized protein sequences and 28 species-specific proteins. For 122 proteins we predicted potential functional roles, 43 of which we associated with protein- and nucleic-acid binding roles. Further, we analysed domain content and variations in their combinations within Afrotheria and identified 141 unique functional domain architectures, highlighting proteins with potential for specialized functions. Finally, we discuss the potential relevance of highly represented protein families such as MAGE-B2, olfactory receptor and ribosomal proteins in L. africana and E. edwardii, respectively. Taken together, our study reports the first comparative study of the Afrotherian proteomes and highlights salient molecular features.

scholarly journals Discrimination of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

2017 ◽  
Vol 29 (5) ◽  
pp. 622-627 ◽  
Author(s):  
Rinosh J. Mani ◽  
Anil J. Thachil ◽  
Akhilesh Ramachandran
Keyword(s):  
Laser Desorption Ionization ◽  
Biochemical System ◽  
Ionization Time ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Subspecies Level ◽  
Streptococcus Equi ◽  
Level Identification ◽  
Tof Ms

Accurate and timely identification of infectious etiologies is of great significance in veterinary microbiology, especially for critical diseases such as strangles, a highly contagious disease of horses caused by Streptococcus equi subsp. equi. We evaluated a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform for use in species- and subspecies-level identification of S. equi isolates from horses and compared it with an automated biochemical system. We used 25 clinical isolates each of S. equi subsp. equi and S. equi subsp. zooepidemicus. Using the MALDI-TOF MS platform, it was possible to correctly identify all 50 isolates to the species level. Unique mass peaks were identified in the bacterial peptide mass spectra generated by MALDI-TOF MS, which can be used for accurate subspecies-level identification of S. equi. Mass peaks (mass/charge, m/ z) 6,751.9 ± 1.4 (mean ± standard deviation) and 5,958.1 ± 1.3 were found to be unique to S. equi subsp. equi and S. equi subsp. zooepidemicus, respectively. The automated biochemical system correctly identified 47 of 50 of the isolates to the species level as S. equi, whereas at the subspecies level, 24 of 25 S. equi subsp. equi isolates and 22 of 25 S. equi subsp. zooepidemicus isolates were correctly identified. Our results indicate that MALDI-TOF MS can be used for accurate species- and subspecies-level identification of S. equi.

scholarly journals Proteomic Study of Ribosomal Proteins from Escherichia coli, Saccharomyces cerevisiae, Bos taurus, Gallus gallus, and Oncorhynchus tshawytscha: Application in a Teaching Laboratory Setting

2014 ◽  
Vol 12 (1) ◽  
Author(s):  
Yoshihiro Miura ◽  
Eric Yeager ◽  
James MacKenzie ◽  
Kestutis Bendinskas
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Oncorhynchus Tshawytscha ◽  
Bos Taurus ◽  
Laboratory Setting ◽  
Maldi Tof Ms ◽  
Teaching Laboratory ◽  
Maldi Tof ◽  
Proteomic Study ◽  
Tof Ms

Ribosomes are central to protein synthesis and our understanding of ribosomes has advanced antibiotics research. The proteomic study of ribosomes presented here utilizes a combination of differential centrifugation and matrix assisted laser desorption/ionization – time of flight mass spectrometry (MALDI-TOF MS) to analyze ribosomes from various species in a teaching laboratory setting. Five biologically varied species were used: Escherichia coli (bacteria), Saccharomyces cerevisiae (yeast), Bos taurus (cow), Gallus gallus (chicken), and Oncorhynchus tshawytscha (Chinook salmon). Samples were lysed, ribosomes were isolated via ultracentrifugation using a discontinuous sucrose gradient and the individual protein subunits were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis. Tryptic digest and MALDI-TOF MS were then conducted on fifteen bands excised from the gel, and the mass spectra of both the whole protein sample and peptides were analyzed. Five out of these fifteen bands were positively identified as various ribosomal proteins, with two uncertain identifications. Additionally, three of the five positively identified proteins that travelled the same distance on the gel were determined to be orthologous. Finally, a class of 14 Biochemistry II students utilized these protocols, identified 3 ribosomal proteins and provided their evaluations of the ultracentrifugation-proteomics teaching laboratory. Key Words: Proteomics, MALDI-TOF MS, ultracentrifugation, ribosomes, teaching laboratory

scholarly journals Characterization of Clostridium tyrobutyricum Strains Using Three Different Typing Techniques

2020 ◽  
Vol 8 (7) ◽  
pp. 1057 ◽  
Author(s):  
Johanna Burtscher ◽  
Franziska Küller ◽  
Matthias Dreier ◽  
Emmanuelle Arias-Roth ◽  
David Drissner ◽  
...  
Keyword(s):  
Species Diversity ◽  
Clostridium Tyrobutyricum ◽  
Maldi Tof Ms ◽  
Pcr Fingerprinting ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Subspecies Level ◽  
Causative Agents ◽  
Different Strains ◽  
Tof Ms

Clostridium tyrobutyricum is well known as one of the main causative agents of severe cheese spoilage. The metabolism of this anaerobic bacterium during ripening leads to textural and sensory defects in cheese and consequential loss of product value. The potential to induce cheese spoilage, however, may vary among different strains of the same species. Therefore, a better understanding of the intra-species diversity of C. tyrobutyricum may be of practical relevance for the dairy industry. In the present study, we compared the ability of three typing techniques to differentiate 95 C. tyrobutyricum strains on the subspecies level: (1) repetitive element palindromic PCR (rep-PCR) fingerprinting combined with conventional agarose gel electrophoresis, (2) hexaplex-PCR followed by an automated capillary electrophoresis and (3) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) typing. MALDI-TOF MS fingerprinting provided only moderate reproducibility and low discriminatory power. Both PCR-based methods were highly reproducible and discriminative, with hexaplex-PCR fingerprinting being slightly more discriminative than rep-PCR typing. Overall, a high intra-species diversity was observed among the tested strains, indicating that further investigations on the strain level may be of interest.

scholarly journals Sand fly fauna of Crete and the description of Phlebotomus (Adlerius) creticus n. sp. (Diptera: Psychodidae)

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Vít Dvořák ◽  
Nikolaos Tsirigotakis ◽  
Christoforos Pavlou ◽  
Emmanouil Dokianakis ◽  
Mohammad Akhoundi ◽  
...  
Keyword(s):  
Field Work ◽  
Abundant Species ◽  
Protein Profiling ◽  
Sand Fly ◽  
Published Data ◽  
Specific Sequence ◽  
Maldi Tof ◽  
Morphological Criteria ◽  
Species Specific ◽  
Protein Spectra

Abstract Background The Greek island of Crete is endemic for both visceral leishmaniasis (VL) and recently increasing cutaneous leishmaniasis (CL). This study summarizes published data on the sand fly fauna of Crete, the results of new sand fly samplings and the description of a new sand fly species. Methods All published and recent samplings were carried out using CDC light traps, sticky traps or mouth aspirators. The specific status of Phlebotomus (Adlerius) creticus n. sp., was assessed by morphological analysis, cytochrome b (cytb) sequencing and MALDI-TOF protein profiling. Results Published data revealed the presence of 10 Phlebotomus spp. and 2 Sergentomyia spp. During presented field work, 608 specimens of 8 species of Phlebotomus and one species of Sergentomyia were collected. Both published data and present samplings revealed that the two most common and abundant species were Phlebotomus neglectus, a proven vector of Leishmania infantum causing VL, and Ph. similis, a suspected vector of L. tropica causing CL. In addition, the field surveys revealed the presence of a new species, Ph. (Adlerius) creticus n. sp. Conclusions The identification of the newly described species is based on both molecular and morphological criteria, showing distinct characters of the male genitalia that differentiate it from related species of the subgenus Adlerius as well as species-specific sequence of cytb and protein spectra generated by MALDI-TOF mass spectrometry.

scholarly journals Clonal Diversification and Changes in Lipid Traits and Colony Morphology in Mycobacterium abscessus Clinical Isolates

2015 ◽  
Vol 53 (11) ◽  
pp. 3438-3447 ◽  
Author(s):  
In Kwon Park ◽  
Amy P. Hsu ◽  
Hervé Tettelin ◽  
Shamira J. Shallom ◽  
Steven K. Drake ◽  
...  
Keyword(s):  
Acid Fast Bacillus ◽  
Mycobacterium Abscessus ◽  
Colony Morphology ◽  
Culture Methods ◽  
Genetic Changes ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms

The smooth-to-rough colony morphology shift inMycobacterium abscessushas been implicated in loss of glycopeptidolipid (GPL), increased pathogenicity, and clinical decline in cystic fibrosis (CF) patients. However, the evolutionary phenotypic and genetic changes remain obscure. Serial isolates from nine non-CF patients with persistentM. abscessusinfection were characterized by colony morphology, lipid profile via thin-layer chromatography and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), sequencing of eight genes in the GPL locus, and expression level offadD23, a key gene involved in the biosynthesis of complex lipids. All 50 isolates were typed asM. abscessussubspeciesabscessusand were clonally related within each patient. Rough isolates, all lacking GPL, predominated at later disease stages, some showing variation within rough morphology. While most (77%) rough isolates harbored detrimental mutations inmps1andmps2, 13% displayed previously unreported mutations inmmpL4aandmmpS4, the latter yielding a putative GPL precursor. Two isolates showed no deleterious mutations in any of the eight genes sequenced. Mixed populations harboring different GPL locus mutations were detected in 5 patients, demonstrating clonal diversification, which was likely overlooked by conventional acid-fast bacillus (AFB) culture methods. Our work highlights applications of MALDI-TOF MS beyond identification, focusing on mycobacterial lipids relevant in virulence and adaptation. Later isolates displayed accumulation of triacylglycerol and reduced expression offadD23, sometimes preceding rough colony onset. Our results indicate that clonal diversification and a shift in lipid metabolism, including the loss of GPL, occur during chronic lung infection withM. abscessus. GPL loss alone may not account for all traits associated with rough morphology.

scholarly journals Comparison of MALDI-TOF mass spectra of [PdCl(dien)]Cl and [Ru(en)2Cl2]Cl acquired with different matrices

2011 ◽  
Vol 76 (12) ◽  
pp. 1687-1701 ◽  
Author(s):  
Bojana Damnjanovic ◽  
Biljana Petrovic ◽  
Jasmina Dimitric-Markovic ◽  
Marijana Petkovic
Keyword(s):  
Transition Metal Complexes ◽  
Mass Spectra ◽  
Hydroxycinnamic Acid ◽  
Dihydroxybenzoic Acid ◽  
Ionization Time ◽  
Maldi Tof ◽  
Ru Complex ◽  
Maldi Tof Mass Spectra ◽  
The Matrix ◽  
Flavonoid Quercetin

In this work, the matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectra of two cationic complexes, i.e., [PdCl(dien)]Cl and [Ru(en)2Cl2]Cl, acquired under different conditions were analyzed. The spectra were recorded with three matrices with or without trifluoroacetic acid (TFA), i.e., two traditional matrices, i.e., 2,5-dihydroxybenzoic acid and ?-cyano-hydroxycinnamic acid, and one flavonoid, quercetin. The spectra acquired with quercetin appeared to be the simplest, whereas in the spectra obtained with other matrices, peaks arising either from the addition of matrix molecules or from the fragmentation products were detectable. Addition of TFA did not complicate the spectra of the Pd(II) and Ru(III) complexes when the traditional matrices were used. On the other hand, the spectra of Pd complex were simpler, whereas the addition of TFA in the case of the Ru complex resulted in a higher number of peaks, some of which could not be identified. Taken together, the results of this study once more emphasize the differences arising in the MALDI-TOF mass spectra of transition metal complexes in dependence on the applied matrix.

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