scholarly journals Caracterización fenotípica y genotípica de cepas de Enterococcus spp. aisladas de líquidos obtenidos por punción provenientes de infecciones invasivas humanas

2019 ◽  
Author(s):  
◽  
Celia María Beatriz Schell
Keyword(s):  
Enterococcus Faecalis ◽  
Enterococcus Faecium ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Enterococcus Spp ◽  
America Latina ◽  
Siglo Xx ◽  
Tof Ms

Enterococcus spp. es flora habitual de la microbiota intestinal de humanos y animales, así como importantes patógenos nosocomiales. Desde principios del siglo XX, se conoce que causan infecciones en humanos. Enterococcus faecalis y Enterococcus faecium son las especies más relevantes clínicamente, particularmente en infecciones invasivas humanas. Estas especies se consideran patógenas oportunistas, ya que las infecciones graves se han asociado principalmente a pacientes inmunocomprometidos. Las infecciones invasivas causadas por enterococos pueden tener un origen alóctono o endógeno. Además, los enterococos presentan resistencia intrínseca a varios antibióticos, como los β-lactámicos, trimetoprima-sulfametoxazol y los aminoglucósidos que podrían mejorar su selección en el tracto gastrointestinal humano. E. faecium y E. faecalis han adquirido resistencia a la mayoría de las familias de antibióticos utilizados en terapia humana, en las últimas tres décadas. Esto llevó a la selección de clones enterocócicos resistentes a múltiples fármacos, lo que complica enormemente el tratamiento antimicrobiano, particularmente en el caso de infecciones graves. La adquisición de resistencia a vancomicina, una de las opciones terapéuticas de primera línea para las infecciones por enterococos, es uno de los problemas más preocupantes para la salud humana. El primer enterococo vancomicina resistente (EVR) reportado en América Latina fue una cepa de E. faecium de Argentina en 1998. Posteriormente, se realizaron estudios epidemiológicos para detectar EVR en diferentes hospitales de Argentina. El linaje clonal más frecuente de E. faecium vancomicina resistente (EFMVR) que circula en los hospitales argentinos parece ser el ST17. Se han realizado varios estudios de vigilancia epidemiológica aplicando técnicas moleculares en nuestro país, sin embargo se limitan principalmente a cepas EVR. No hay documentación de los perfiles de resistencia a los antimicrobianos y de la estructura poblacional de E. faecalis y E. faecium aislados de infecciones invasivas humanas en el Hospital Público de Tandil, Argentina hasta la fecha. El objetivo general de este trabajo de tesis fue caracterizar fenotípicamente y genotípicamente Enterococcus spp. aislados de líquidos obtenidos por punción, de infecciones invasivas humanas, de pacientes ingresados en el Hospital Municipal Ramón Santamarina (HMRS) de Tandil. Además, el segundo objetivo general fue detectar la resistencia a los antimicrobianos y evaluar in vitro varias opciones terapéuticas en enterococos resistentes a múltiples fármacos (MDR). Las cepas de Enterococcus spp. se recuperaron de forma retro-prospectiva a través de un estudio transversal a partir de infecciones invasivas de pacientes hospitalizados en el HMRS entre 2010 y 2014. Todas las cepas fueron identificadas con métodos convencionales fenotípicos y confirmadas por MALDI TOF-MS. Cuarenta y cuatro fueron identificadas como E. faecalis (69,8 %) y 19 como E. faecium (30,2 %). La caracterización molecular y la relación clonal se determinaron mediante electroforesis en gel de campo pulsado (PFGE) y tipificación de secuencias multilocus (MLST). La susceptibilidad a 15 antimicrobianos ampicilina, penicilina, ampicilina/sulbactam, cloranfenicol, vancomicina, teicoplanina, estreptomicina (resistencia de alto nivel: RAN), gentamicina (RAN), ciprofloxacina, levofloxacina, quinupristin-dalfopristin, clindamicina, eritromicina, linezolid y tigeciclina fue determinada por el método de difusión de disco y por concentraciones inhibitorias mínimas (CIM). El genotipo de resistencia a glucopéptidos fue determinado por PCR. La actividad antimicrobiana se evaluó in vitro mediante estudios de cinética de muerte utilizando curvas de letalidad. Este trabajo de tesis contribuye a generar conocimiento para continuar avanzando en la temática de la resistencia antimicrobiana y su estrategia de control en Argentina en el contexto de “Una Salud’’. Además, reconoce que, la detección y el control epidemiológico de los enterococos MDR es necesario realizarlo tanto en reservorios humanos como no humanos, para evitar su diseminación e impacto en salud pública.

In Vitro and In Situ Tracking of Choline-Phospholipid Biogenesis by MALDI TOF-MS

2006 ◽  
Vol 78 (4) ◽  
pp. 1174-1180 ◽  
Author(s):  
Rosendo Estrada ◽  
Douglas Borchman ◽  
John Reddan ◽  
Anne Hitt ◽  
M. Cecilia Yappert
Keyword(s):  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Choline Phospholipid ◽  
Tof Ms

scholarly journals Polymorphisms of TLR4: Rapid Genotyping and Reduced Response to Lipopolysaccharide of TLR4 Mutant Alleles

2002 ◽  
Vol 48 (10) ◽  
pp. 1661-1667 ◽  
Author(s):  
Christopher Schmitt ◽  
Andreas Humeny ◽  
Cord-Michael Becker ◽  
Kay Brune ◽  
Andreas Pahl
Keyword(s):  
Rflp Analysis ◽  
Blood Monocytes ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Reverse Transcription Pcr ◽  
Flight Mass Spectrometry ◽  
Pcr Assays ◽  
Factor Α ◽  
Tof Ms

Abstract Background: Pathogen recognition receptors such as Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, lead to the activation of innate immunity. Genetic variations in these receptors may lead to an altered host immune response to pathogens. Methods: We developed homogeneous fluorescence-based PCR assays as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) genotyping assays to detect TLR4 polymorphisms. These assays were compared with restriction fragment length polymorphism (RFLP) analysis. Peripheral blood monocytes from donors with differing genotypes were prepared and exposed to bacterial products in vitro. The abundance of mRNAs of the proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α from these monocytes were monitored by real-time reverse transcription-PCR. Results: By our homogeneous PCR method, the allele frequencies were 5.6% for the TLR4 Asp299Gly and 6.0% for the TLR4 Thr399Ile polymorphism in 116 healthy German Caucasians. Nine incorrect genotype calls were detected in the RFLP analysis and two in the TaqMan genotype analysis. MALDI-TOF-MS allowed clear detection of all TLR4 alleles. Monocytes from donors homozygous for the TLR4 mutant alleles Asp299Gly and Thr399Ile were lipopolysaccharide hyporesponsive and exhibited median effective concentrations (EC50s) approximately fourfold higher than those of monocytes carrying wild-type or heterozygous alleles. In contrast, a TLR2 agonist elicited similar responses in monocytes irrespective of the TLR4 genotype. Conclusions: Homogeneous fluorescence-based PCR assays provide a specific and sensitive method for high-throughput genotyping of TLR4 mutations. The newly developed PCR and MALDI-TOF-MS assays may be useful to evaluate the presence of TLR4 polymorphisms in patients to predict susceptibility to bacterial infection.

scholarly journals Applications of MALDI Mass Spectrometry in Clinical Chemistry

2016 ◽  
Vol 62 (1) ◽  
pp. 134-143 ◽  
Author(s):  
Mark W Duncan ◽  
Dobrin Nedelkov ◽  
Ryan Walsh ◽  
Stephen J Hattan
Keyword(s):  
Mass Spectrometry ◽  
Clinical Chemistry ◽  
Cost Effective ◽  
Maldi Mass Spectrometry ◽  
Ease Of Use ◽  
Mass Detection ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

Abstract BACKGROUND MALDI-TOF mass spectrometry (MS) is set to make inroads into clinical chemistry because it offers advantages over other analytical platforms. These advantages include low acquisition and operating costs, ease of use, ruggedness, and high throughput. When coupled with innovative front-end strategies and applied to important clinical problems, it can deliver rapid, sensitive, and cost-effective assays. CONTENT This review describes the general principles of MALDI-TOF MS, highlights the unique features of the platform, and discusses some practical methods based upon it. There is substantial potential for MALDI-TOF MS to make further inroads into clinical chemistry because of the selectivity of mass detection and its ability to independently quantify proteoforms. SUMMARY MALDI-TOF MS has already transformed the practice of clinical microbiology and this review illustrates how and why it is now set to play an increasingly important role in in vitro diagnostics in particular, and clinical chemistry in general.

scholarly journals High-Throughput Detection and Characterization of Antimicrobial Resistant Enterococcus sp. Isolates from GI Tracts of European Starlings Visiting Concentrated Animal Feeding Operations

Foods ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 890 ◽  
Author(s):  
Jennifer Anders ◽  
Bledar Bisha
Keyword(s):  
High Throughput ◽  
Enteric Bacteria ◽  
European Starlings ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Enterococcus Spp ◽  
Contaminated Food ◽  
High Throughput Detection ◽  
Tof Ms

Antimicrobial resistant enteric bacteria can easily contaminate the environment and other vehicles through the deposition of human and animal feces. In turn, humans can be exposed to these antimicrobial resistant (AMR) bacteria through contaminated food products and/or contaminated drinking water. As wildlife are firmly established as reservoirs of AMR bacteria and serve as potential vectors in the constant spread of AMR, limiting contact between wildlife and livestock and effective tracking of AMR bacteria can help minimize AMR dissemination to humans through contaminated food and water. Enterococcus spp., which are known opportunistic pathogens, constantly found in gastrointestinal tracts of mammalian and avian species, swiftly evolve and cultivate AMR genotypes and phenotypes, which they easily distribute to other bacteria, including several major bacterial pathogens. In this study, we evaluated the use of high throughput detection and characterization of enterococci from wildlife [European starlings (Sturnus vulgaris)] by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) following culture-based isolation. MALDI-TOF MS successfully identified 658 Enterococcus spp. isolates out of 718 presumptive isolates collected from gastrointestinal tracts of European starlings, which were captured near livestock operations in Colorado, Iowa, Kansas, Missouri, and Texas; antimicrobial susceptibility testing was then performed using 13 clinically significant antibiotics.

scholarly journals Human Neutrophil Peptides 1–3 as Gastric Cancer Tissue Markers Measured by MALDI-Imaging Mass Spectrometry: Implications for Infiltrated Neutrophils as a Tumor Target

2012 ◽  
Vol 32 (1) ◽  
pp. 21-31 ◽  
Author(s):  
Chun-Chia Cheng ◽  
Jungshan Chang ◽  
Ling-Yun Chen ◽  
Ai-Sheng Ho ◽  
Ker-Jer Huang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gastric Cancer ◽  
Human Neutrophil ◽  
Binding Capacity ◽  
Tumor Target ◽  
Ags Cells ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

Objective: Human neutrophil peptides (HNPs) -1, -2 and -3 are significantly upregulated and were reported as biomarkers in gastric cancer (GC). However, the tissue location and function of HNPs 1-3 are still unclear in GC, and the spatial distribution of the triad needs to be disclosed. The aims of this study were to investigate the distribution and relationships among HNPs-1, -2 and -3, and assess whether infiltrated neutrophils accumulate in gastric tumor.Methods: In this study, paired samples (n=33) of the GC tissues and adjacent normal tissues from the same patients were obtained from surgery. Expression of HNPs 1-3 were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The distributions of the HNPs 1-3 in GC tissues were investigated. After verification of HNPs-1 by immunohistochemistry, infiltrated neutrophils were also detected. Then, an in vitro assay was used to observe the binding capacity and measure the cytotoxic effect of HNPs-1 against AGS cells.Results: Comparing to neighboring normal tissue, expressional level of HNPs 1-3 were significantly higher and their distributions overlapped in cancerous tissues of GC patients with high abundance in the lamina propria, whereas HNPs-1 was identified as the highest major peak. Moreover, HNPs-1, -2 and -3 correlated with each other. Besides, we also observed that increased infiltrated neutrophils accumulating in GC tissues, indicating that a strong positive correlation between HNPs 1-3 and infiltrated neutrophils. In addition, the further investigated demonstrated that the major peptide, HNPs-1, was statistically increased with the advance of tumor development from the early to advanced stage of GC (p < 0.05). Moreover, we also noticed that HNPs-1 with a great binding capacity to GC AGS cells in vitro can inhibit tumor cell growth.Conclusions: Our results suggest that neutrophil secreted peptides, HNPs 1-3, increased in the GC tissues and could be used as potential biomarkers detected using MALDI-TOF MS, implying that elevated neutrophils may be used as a tumor target for tumor treatment. The binding capacity of HNPs-1 with GC cells implies that tracking molecules conjugated with HNPs-1 could be applied as a specific probe for GC diagnoses.

scholarly journals A Lipidomic Approach to Identify Potential Biomarkers in Exosomes From Melanoma Cells With Different Metastatic Potential

2021 ◽  
Vol 12 ◽  
Author(s):  
Simona Lobasso ◽  
Paola Tanzarella ◽  
Francesco Mannavola ◽  
Marco Tucci ◽  
Francesco Silvestris ◽  
...  
Keyword(s):  
Lipid Analysis ◽  
Lipid Extraction ◽  
Metastatic Potential ◽  
Melanoma Cells ◽  
Lipid Profiles ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms

Melanoma, one of the most lethal cutaneous cancers, is characterized by its ability to metastasize to other distant sites, such as the bone. Melanoma cells revealed a variable in vitro propensity to be attracted toward bone fragments, and melanoma-derived exosomes play a role in regulating the osteotropism of these cells. We have here investigated the lipid profiles of melanoma cell lines (LCP and SK-Mel28) characterized by different metastatic propensities to colonize the bone. We have purified exosomes from cell supernatants by ultracentrifugation, and their lipid composition has been compared to identify potential lipid biomarkers for different migration and invasiveness of melanoma cells. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) lipid analysis has been performed on very small amounts of intact parental cells and exosomes by skipping lipid extraction and separation steps. Statistical analysis has been applied to MALDI mass spectra in order to discover significant differences in lipid profiles. Our results clearly show more saturated and shorter fatty acid tails in poorly metastatic (LCP) cells compared with highly metastatic (SK-Mel28) cells, particularly for some species of phosphatidylinositol. Sphingomyelin, lysophosphatidylcholine, and phosphatidic acid were enriched in exosome membranes compared to parental cells. In addition, we have clearly detected a peculiar phospholipid bis(monoacylglycero)phosphate as a specific lipid marker of exosomes. MALDI-TOF/MS lipid profiles of exosomes derived from the poorly and highly metastatic cells were not significantly different.

scholarly journals Confirmation of fructans biosynthesized in vitro from [1-13C]glucose in asparagus tissues using MALDI–TOF MS and ESI–MS

2013 ◽  
Vol 170 (8) ◽  
pp. 715-722 ◽  
Author(s):  
Takashi Suzuki ◽  
Tomoo Maeda ◽  
Suzanne Grant ◽  
Gordon Grant ◽  
Peter Sporns
Keyword(s):  
Maldi Tof Ms ◽  
Esi Ms ◽  
Maldi Tof ◽  
Tof Ms
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