Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

<p>Abstract</p> <p>Background</p> <p>Accurate identification is necessary to discriminate harmless environmental <it>Yersinia </it>species from the food-borne pathogens <it>Yersinia enterocolitica </it>and <it>Yersinia pseudotuberculosis </it>and from the group A bioterrorism plague agent <it>Yersinia pestis</it>. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of <it>Yersinia </it>species. As a first step, we built a database of 39 different <it>Yersinia </it>strains representing 12 different <it>Yersinia </it>species, including 13 <it>Y. pestis </it>isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the <it>Yersinia </it>species.</p> <p>Results</p> <p>When compared with a 3,025-profile database, every <it>Yersinia </it>species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of <it>Y. pestis </it>(n = 2) and <it>Y. enterocolitica </it>(n = 11) were compared to the database and correctly identified. In particular, <it>Y. pestis </it>was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes.</p> <p>Conclusion</p> <p>These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of <it>Yersinia </it>isolates.</p

Global Analysis of Circulating Immune Cells by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Background: MALDI-TOF mass spectrometry is currently used in microbiological diagnosis to characterize bacterial populations. Our aim was to determine whether this technique could be applied to intact eukaryotic cells, and in particular, to cells involved in the immune response. Methodology/Principal Findings: A comparison of frozen monocytes, T lymphocytes and polymorphonuclear leukocytes revealed specific peak profiles. We also found that twenty cell types had specific profiles, permitting the establishment of a cell database. The circulating immune cells, namely monocytes, T lymphocytes and polymorphonuclear cells, were distinct from tissue immune cells such as monocyte-derived macrophages and dendritic cells. In addition, MALDI-TOF mass spectrometry was valuable to easily identify the signatures of monocytes and T lymphocytes in peripheral mononuclear cells. Conclusions/Significance: This method was rapid and easy to perform, and unlike flow cytometry, it did not require any additional components such as specific antibodies. The MALDI-TOF mass spectrometry approach could be extended t

Rapid Detection of Carbapenem Resistance in Acinetobacter baumannii Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Rapid detection of carbapenem-resistant Acinetobacter baumannii strains is critical and will benefit patient care by optimizing antibiotic therapies and preventing outbreaks. Herein we describe the development and successful application of a mass spectrometry profile generated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) that utilized the imipenem antibiotic for the detection of carbapenem resistance in a large series of A. baumannii clinical isolates from France and Algeria. A total of 106 A. baumannii strains including 63 well-characterized carbapenemase-producing and 43 non-carbapenemase-producing strains, as well as 43 control strains (7 carbapenem-resistant and 36 carbapenem-sensitive strains) were studied. After an incubation of bacteria with imipenem for up to 4 h, the mixture was centrifuged and the supernatant analyzed by MALDI-TOF MS. The presence and absence of peaks representing imipenem and its natural metabolite was analyzed. The result was interpreted as positive for carbapenemase production if the specific peak for imipenem at 300.0 m/z disappeared during the incubation time and if the peak of the natural metabolite at 254.0 m/z increased as measured by the area under the curves leading to a ratio between the peak for imipenem and its metabolite being <0.5. This assay, which was applied to the large series of A. baumannii clinical isolates, showed a sensitivity of 100.0% and a specificity of 100.0%. Our study is the first to demonstrate that this quick and simple assay can be used as a routine tool as a point-of-care method for the identification of A. baumannii carbapenemase-producers in an effort to prevent outbreaks and the spread of uncontrollable superbugs

Classification of Ancient Mammal Individuals Using Dental Pulp MALDI-TOF MS Peptide Profiling

International audienceBackground The classification of ancient animal corpses at the species level remains a challenging task for forensic scientists and anthropologists. Severe damage and mixed, tiny pieces originating from several skeletons may render morphological classification virtually impossible. Standard approaches are based on sequencing mitochondrial and nuclear targets. Methodology/Principal Findings We present a method that can accurately classify mammalian species using dental pulp and mass spectrometry peptide profiling. Our work was organized into three successive steps. First, after extracting proteins from the dental pulp collected from 37 modern individuals representing 13 mammalian species, trypsin-digested peptides were used for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The resulting peptide profiles accurately classified every individual at the species level in agreement with parallel cytochrome b gene sequencing gold standard. Second, using a 279-modern spectrum database, we blindly classified 33 of 37 teeth collected in 37 modern individuals (89.1%). Third, we classified 10 of 18 teeth (56%) collected in 15 ancient individuals representing five mammal species including human, from five burial sites dating back 8,500 years. Further comparison with an upgraded database comprising ancient specimen profiles yielded 100% classification in ancient teeth. Peptide sequencing yield 4 and 16 different non-keratin proteins including collagen (alpha-1 type I and alpha-2 type I) in human ancient and modern dental pulp, respectively. Conclusions/Significance Mass spectrometry peptide profiling of the dental pulp is a new approach that can be added to the arsenal of species classification tools for forensics and anthropology as a complementary method to DNA sequencing. The dental pulp is a new source for collagen and other proteins for the species classification of modern and ancient mammal individuals

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Identification of Mycobacteria in Routine Clinical Practice

Background: Non-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed. Methodology: We set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 10 5 colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25M. avium and 12 non-tuberculosis clinical isolates with identification scores $2 within 2.5 hours. Conclusions: Our data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heatinactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories i

Matrix-assisted laser desorption ionization--time of flight mass spectrometry: an emerging tool for the rapid identification of mosquito vectors.

BACKGROUND: The identification of mosquito vectors is typically based on morphological characteristics using morphological keys of determination, which requires entomological expertise and training. The use of protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is increasingly being used for the routine identification of bacteria, has recently emerged for arthropod identification. METHODS: To investigate the usefulness of MALDI-TOF-MS as a mosquito identification tool, we tested protein extracts made from mosquito legs to create a database of reference spectra. The database included a total of 129 laboratory-reared and field-caught mosquito specimens consisting of 20 species, including 4 Aedes spp., 9 Anopheles spp., 4 Culex spp., Lutzia tigripes, Orthopodomyia reunionensis and Mansonia uniformis. For the validation study, blind tests were performed with 76 specimens consisting of 1 to 4 individuals per species. A cluster analysis was carried out using the MALDI-Biotyper and some spectra from all mosquito species tested. RESULTS: Biomarker mass sets containing 22 and 43 masses have been detected from 100 specimens of the Anopheles, Aedes and Culex species. By carrying out 3 blind tests, we achieved the identification of mosquito vectors at the species level, including the differentiation of An. gambiae complex, which is possible using MALDI-TOF-MS with 1.8 as the cut-off identification score. A cluster analysis performed with all available mosquito species showed that MALDI-Biotyper can distinguish between specimens at the subspecies level, as demonstrated for An gambiae M and S, but this method cannot yet be considered a reliable tool for the phylogenetic study of mosquito species. CONCLUSIONS: We confirmed that even without any specific expertise, MALDI-TOF-MS profiling of mosquito leg protein extracts can be used for the rapid identification of mosquito vectors. Therefore, MALDI-TOF-MS is an alternative, efficient and inexpensive tool that can accurately identify mosquitoes collected in the field during entomological surveys

MALDI-ToF mass spectrometry for the rapid diagnosis of cancerous lung nodules.

Recently, tissue-based methods for proteomic analysis have been used in clinical research and appear reliable for digestive, brain, lymphomatous, and lung cancers classification. However simple, tissue-based methods that couple signal analysis to tissue imaging are time consuming. To assess the reliability of a method involving rapid tissue preparation and analysis to discriminate cancerous from non-cancerous tissues, we tested 141 lung cancer/non-tumor pairs and 8 unique lung cancer samples among the stored frozen samples of 138 patients operated on during 2012. Samples were crushed in water, and 1.5 µl was spotted onto a steel target for analysis with the Microflex LT analyzer (Bruker Daltonics). Spectra were analyzed using ClinProTools software. A set of samples was used to generate a random classification model on the basis of a list of discriminant peaks sorted with the k-nearest neighbor genetic algorithm. The rest of the samples (n = 43 cancerous and n = 41 non-tumoral) was used to verify the classification capability and calculate the diagnostic performance indices relative to the histological diagnosis. The analysis found 53 m/z valid peaks, 40 of which were significantly different between cancerous and non-tumoral samples. The selected genetic algorithm model identified 20 potential peaks from the training set and had 98.81% recognition capability and 89.17% positive predictive value. In the blinded set, this method accurately discriminated the two classes with a sensitivity of 86.7% and a specificity of 95.1% for the cancer tissues and a sensitivity of 87.8% and a specificity of 95.3% for the non-tumor tissues. The second model generated to discriminate primary lung cancer from metastases was of lower quality. The reliability of MALDI-ToF analysis coupled with a very simple lung preparation procedure appears promising and should be tested in the operating room on fresh samples coupled with the pathological examination

Yersinia pestis halotolerance illuminates plague reservoirs

International audienceThe plague agent Yersinia pestis persists for years in the soil. Two millennia after swiping over Europe and North Africa, plague established permanent foci in North Africa but not in neighboring Europe. Mapping human plague foci reported in North Africa for 70 years indicated a significant location at <3 kilometers from the Mediterranean seashore or the edge of salted lakes named chotts. In Algeria, culturing 352 environmental specimens naturally containing 0.5 to 70 g/L NaCl yielded one Y. pestis Orientalis biotype isolate in a 40 g/L NaCl chott soil specimen. Core genome SNP analysis placed this isolate within the Y. pestis branch 1, Orientalis biovar. Culturing Y. pestis in broth steadily enriched in NaCl indicated survival up to 150 g/L NaCl as L-form variants exhibiting a distinctive matrix assisted laser desorption-ionization time-of-flight mass spectrometry peptide profile. Further transcriptomic analyses found the upregulation of several outer-membrane proteins including TolC efflux pump and OmpF porin implied in osmotic pressure regulation. Salt tolerance of Y. pestis L-form may play a role in the maintenance of natural plague foci in North Africa and beyond, as these geographical correlations could be extended to 31 plague foci in the northern hemisphere (from 15°N to 50°N)

In vitro detection of bacterial contamination in platelet concentrates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a preliminary study

International audiencePlatelet concentrates are at risk of transfusion-related sepsis. The microbial detection methods currently available have reached their limits, as they do not completely prevent transfusion-related bacterial contamination.The aim of this study was to develop a new strategy to detect the risk of platelet transfusion-related bacterial contamination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Rapid Diagnosis of Lung Tumors, a Feasability Study Using Maldi-Tof Mass Spectrometry

International audienceObjective Despite recent advances in imaging and core or endoscopic biopsies, a percentage of patients have a major lung resection without diagnosis. We aimed to assess the feasibility of a rapid tissue preparation/analysis to discriminate cancerous from non-cancerous lung tissue. Methods Fresh sample preparations were analyzed with the Microflex LTTM MALDI-TOF analyzer. Each main reference spectra (MSP) was consecutively included in a database. After definitive pathological diagnosis, each MSP was labeled as either cancerous or non-cancerous (normal, inflammatory, infectious nodules). A strategy was constructed based on the number of concordant responses of a mass spectrometry scoring algorithm. A 3-step evaluation included an internal and blind validation of a preliminary database (n = 182 reference spectra from the 100 first patients), followed by validation on a whole cohort database (n = 300 reference spectra from 159 patients). Diagnostic performance indicators were calculated. Results 127 cancerous and 173 non-cancerous samples (144 peripheral biopsies and 29 inflammatory or infectious lesions) were processed within 30 minutes after biopsy sampling. At the most discriminatory level, the samples were correctly classified with a sensitivity, specificity and global accuracy of 92.1%, 97.1% and 95%, respectively. Conclusions The feasibility of rapid MALDI-TOF analysis, coupled with a very simple lung preparation procedure, appears promising and should be tested in several surgical settings where rapid on-site evaluation of abnormal tissue is required. In the operating room, it appears promising in case of tumors with an uncertain preoperative diagnosis and should be tested as a complementary approach to frozen-biopsy analysis