RESUMEN
Patients with orofacial clefts are more likely to develop oral fungal diseases due to anatomo-physiological changes and surgical rehabilitation treatment. This case-series study evaluated the genetic diversity and dynamics of oral colonization and spread of C. albicans and C. tropicalis in four patients with orofacial clefts, from the time of hospital admission, perioperative and outpatient follow-up, with specialized physician. Candida biotypes previously identified by CHROMagar Candida and PCR methods were studied by MALDI-TOF MS assays and clustering analyses. Possible correlations with pathogenicity characteristics were observed, including production of hydrolytic exoenzymes and the antifungal sensitivity profiles. Amphotericin B-sensitive and fluconazole-resistant (low frequency) C. tropicalis and C. albicans, including clinically compatible MIC of nystatin, were found in the oral cavity of these patients. Clusters of isolates revealed phenomena of (i) elimination in the operative phase, (ii) maintenance or (iii) acquisition of oral C. tropicalis in the perioperative period and specialized outpatient and medical follow-up. For C. albicans, these phenomena included (i) elimination in the operative phase, (ii) acquisition in the operative phase and propagation from the hospital environment, and (iii) maintenance during hospitalization and operative phase. Amphotericin B and nystatin were shown to be effective in cases of clinical treatment and/or prophylaxis, especially considering the pre-existence of fluconazole-resistant strains. This study confirmed the phenomena of septic maintenance, septic neocolonization and septic elimination involving the opportunistic pathogens. MALDI-TOF MS associated with clustering analysis may assist the monitoring of clinical isolates or groups of epidemiologically important microbial strains in the hospital setting.
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Anfotericina B , Antifúngicos , Candida albicans , Candida tropicalis , Farmacorresistencia Fúngica , Genotipo , Pruebas de Sensibilidad Microbiana , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Humanos , Candida tropicalis/efectos de los fármacos , Candida tropicalis/aislamiento & purificación , Candida tropicalis/genética , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Candida albicans/genética , Candida albicans/efectos de los fármacos , Candida albicans/aislamiento & purificación , Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Masculino , Femenino , Boca/microbiología , Niño , Fluconazol/farmacología , Fluconazol/uso terapéutico , Candidiasis Bucal/microbiología , Nistatina/farmacología , Nistatina/uso terapéutico , Técnicas de Tipificación Micológica , Fisura del Paladar/cirugía , Labio Leporino/cirugía , Adolescente , Análisis por Conglomerados , PreescolarAsunto(s)
Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/clasificación , Enterobacteriaceae/genética , Técnicas de Tipificación Bacteriana/métodosRESUMEN
The intricate composition of microalgal pigments plays a crucial role in various biological processes, from photosynthesis to biomarker identification. Traditional pigment analysis methods involve complex extraction techniques, posing challenges in maintaining analyte integrity. In this study, we employ Electron Transfer Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (ET-MALDI-MS) to compare the pigmentary profiles of Chlorella vulgaris intact cells, chloroplasts, and solvent extracts. We aim to obtain comprehensive extracts rich in polar and non-polar compounds using ultrasound-assisted and supercritical fluid extraction methods. Additionally, intact chloroplasts are isolated using a lysis buffer and sucrose density gradient centrifugation. Our ET-MALDI-MS analysis reveals distinct compositional differences, highlighting the impact of extraction protocols on microalgal pigment identification. We observe prominent signals corresponding to radical cations of key pigments, including chlorophylls and carotenoids, which are crucial for C. vulgaris identification. Furthermore, ET-MALDI-MS facilitates the identification of specific lipids within chloroplast membranes and other organelles. This study underscores the rapid and precise nature of ET-MALDI-MS in microalgal biomarker analysis, providing valuable insights into phytoplankton dynamics, trophic levels, and environmental processes. C. vulgaris emerges as a promising model for studying pigment composition and membrane lipid diversity, enhancing our understanding of microalgal ecosystems.
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Chlorella vulgaris , Cloroplastos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Chlorella vulgaris/química , Chlorella vulgaris/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Cloroplastos/química , Cloroplastos/metabolismo , Carotenoides/análisis , Carotenoides/química , Clorofila/análisis , Clorofila/química , Pigmentos Biológicos/análisis , Pigmentos Biológicos/químicaRESUMEN
BACKGROUND: Throughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples. RESULTS: The following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus ( 2.7%), E. durans (1.2%), E. galinarum (0.8%). CONCLUSIONS: The difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented.
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Enterococcus , Mastitis Bovina , Leche , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Mastitis Bovina/microbiología , Mastitis Bovina/diagnóstico , Animales , Leche/microbiología , Leche/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/veterinaria , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Femenino , Enterococcus/aislamiento & purificación , Bovinos , Brasil , Infecciones por Bacterias Grampositivas/veterinaria , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/diagnósticoRESUMEN
Forensic chemistry literature has grown exponentially, with many analytical techniques being used to provide valuable information to help solve criminal cases. Among them, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), particularly MALDI MS imaging (MALDI MSI), has shown much potential in forensic applications. Due to its high specificity, MALDI MSI can analyze a wide variety of compounds in complex samples without extensive sample preparation, providing chemical profiles and spatial distributions of given analyte(s). This review introduces MALDI MS(I) to forensic scientists with a focus on its basic principles and the applications of MALDI MS(I) to the analysis of fingerprints, drugs of abuse, and their metabolites in hair, medicine samples, animal tissues, and inks in documents.
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Ciencias Forenses , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Ciencias Forenses/métodos , Humanos , Animales , Cabello/química , Dermatoglifia , Detección de Abuso de Sustancias/métodosRESUMEN
RATIONALE: Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable. METHODS: Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30. RESULTS: A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker. CONCLUSIONS: Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates.
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Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Succinimidas , Espectrometría de Masas en Tándem , Vacunas Conjugadas , Succinimidas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Vacunas Conjugadas/química , Péptidos/química , HidrólisisRESUMEN
Gelatin, a versatile protein derived from collagen, is widely used in the food, pharmaceutical and medical sectors. However, bacterial contamination by spore-forming bacteria during gelatin processing represents a significant concern for product safety and quality. In this study, an investigation was carried out to explore the heat and chemical resistance, as well as the identification and characterization of spore-forming bacteria isolated from gelatin processing. The methodologies involved chemical resistance tests with drastic pH in microplates and thermal resistance tests in capillary tubes of various isolates obtained at different processing stages. In addition, phenotypic and genotypic analyses were carried out to characterize the most resistant isolates of spore-forming bacteria. The findings of this study revealed the presence of several species, including Bacillus cereus, Bacillus licheniformis, Bacillus sonorensis, Bacillus subtilis, Geobacillus stearothermophilus, and Clostridium sporogenes, with some isolates exhibiting remarkable chemical and heat resistances. In addition, a significant proportion of the most resistant isolates showed gelatinase activity (n = 19/21; 90.5 %) and the presence of heat resistance (n = 5/21; 23.8 %), and virulence genes (n = 11/21; 52.4 %). The results of this study suggest that interventions should be done in quality control practices and that process parameter adjustments and effective contamination reduction strategies should be implemented through gelatin processing.
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Gelatina , Calor , ARN Ribosómico 16S , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Esporas Bacterianas , Esporas Bacterianas/genética , ARN Ribosómico 16S/genética , Virulencia/genética , Microbiología de Alimentos , Bacillus/genética , Bacillus/aislamiento & purificaciónRESUMEN
Nocardia pyomyositis in immunocompetent patients is a rare occurrence. The diagnosis may be missed or delayed with the risk of progressive infection and suboptimal or inappropriate treatment. We present the case of a 48-year-old immunocompetent firefighter diagnosed with pyomyositis caused by Nocardia brasiliensis acquired by direct skin inoculation from gardening activity. The patient developed a painful swelling on his right forearm that rapidly progressed proximally and deeper into the underlying muscle layer. Ultrasound imaging of his right forearm showed a 7-mm subcutaneous fluid collection with surrounding edema. Microbiologic analysis of the draining pus was confirmed to be N brasiliensis by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry. After incision and drainage deep to the muscle layer to evacuate the abscess and a few ineffective antibiotic options, the patient was treated with intravenous ceftriaxone and oral linezolid for 6 weeks. He was then de-escalated to oral moxifloxacin for an additional 4 months to complete a total antibiotic treatment duration of 6 months. The wound healed satisfactorily and was completely closed by the fourth month of antibiotic therapy. Six months after discontinuation of antibiotics, the patient continued to do well with complete resolution of the infection. In this article, we discussed the risk factors for Nocardia in immunocompetent settings, the occupational risks for Nocardia in our index patient, and the challenges encountered with diagnosis and treatment. Nocardia should be included in the differential diagnosis of cutaneous infections, particularly if there is no improvement of "cellulitis" with traditional antimicrobial regimens and the infection extends into the deeper muscle tissues.
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Antibacterianos , Jardinería , Inmunocompetencia , Nocardiosis , Nocardia , Piomiositis , Humanos , Masculino , Persona de Mediana Edad , Nocardiosis/diagnóstico , Nocardiosis/tratamiento farmacológico , Nocardia/aislamiento & purificación , Antibacterianos/uso terapéutico , Piomiositis/tratamiento farmacológico , Piomiositis/diagnóstico , Piomiositis/microbiología , Ceftriaxona/uso terapéutico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Drenaje , Moxifloxacino/uso terapéutico , Moxifloxacino/administración & dosificación , Linezolid/uso terapéuticoRESUMEN
In this study, we report the first isolation of Hanseniaspora opuntiae obtained from four pregnant women in Brazil. Clinical isolates were obtained from four samples taken between 35 and 37 gestational weeks, as part of the routine antenatal care for maternal colonization screening for Streptococcus agalactiae group B. The patients were immunocompetent, with two of them diagnosed with gestational diabetes mellitus. Species identification was performed by MALDI-TOF MS and rDNA sequencing. While Hanseniaspora species have not traditionally been considered a typical opportunist pathogen, our findings emphasize the importance of investigating and screening for Hanseniaspora in pregnant populations, highlighting H. opuntiae as a potential agent of human infections.
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Complicaciones Infecciosas del Embarazo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto , Femenino , Humanos , Embarazo , Adulto Joven , Brasil , Diabetes Gestacional/microbiología , Diabetes Gestacional/diagnóstico , ADN Ribosómico/genética , Complicaciones Infecciosas del Embarazo/microbiología , Complicaciones Infecciosas del Embarazo/diagnóstico , Análisis de Secuencia de ADN , Streptococcus agalactiae/aislamiento & purificación , Streptococcus agalactiae/genética , Streptococcus agalactiae/clasificación , Vagina/microbiologíaRESUMEN
A group of Gram-negative plant-associated diazotrophic bacteria belonging to the genus Nitrospirillum was investigated, including both previously characterized and newly isolated strains from diverse regions and biomes, predominantly in Brazil. Phylogenetic analysis of 16S rRNA and recA genes revealed the formation of a distinct clade consisting of thirteen strains, separate from the formally recognized species N. amazonense (the closest species) and N. iridis. Comprehensive taxonomic analyses using the whole genomes of four strains (BR 11140T = AM 18T = Y-2T = DSM 2788T = ATCC 35120T, BR 11142T = AM 14T = Y-1T = DSM 2787T = ATCC 35119T, BR 11145 = CBAmC, and BR 12005) supported the division of these strains into two species: N. amazonense (BR 11142 T and BR 12005) and a newly proposed species (BR 11140 T and BR 11145), distinct from N. iridis. The phylogenomic analysis further confirmed the presence of the new Nitrospirillum species. Additionally, MALDI-TOF MS analysis of whole-cell mass spectra provided further evidence for the differentiation of the proposed Nitrospirillum species, separate from N. amazonense. Analysis of chemotaxonomy markers (i.e., genes involved in fatty acid synthesis, metabolism and elongation, phospholipid synthesis, and quinone synthesis) revealed that the new species highlights high similarity and evolutionary convergence with other Nitrospirillum species. This new species exhibited nitrogen fixation ability in vitro, it has similar NifHDK protein phylogeny position with the closest species, lacked denitrification capability, but possessed the nosZ gene, enabling N2O reduction, distinguishing it from the closest species. Despite being isolated from diverse geographic regions, soil types, and ecological niches, no significant phenotypic or physiological differences were observed between the proposed new species and N. amazonense. Based on these findings, a new species, Nitrospirillum viridazoti sp. nov., was classified, with the strain BR 11140T (DSM 2788T, ATCC 35120T) designated as the type strain.
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Nitrógeno , Poaceae , Filogenia , ARN Ribosómico 16S/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The aim of this study was to compare the performance of two MALDI-TOF MS systems in the identification of clinically relevant strict anaerobic bacteria. The 16S rRNA gene sequencing was the gold standard method when discrepancies or inconsistencies were observed between platforms. A total of 333 isolates were recovered from clinical samples of different centers in Buenos Aires City between 2016 and 2021. The isolates were identified in duplicate using two MALDI-TOF MS systems, BD Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMèrieux, Marcy-l'Etoile, France). Using the Vitek MS system, the identification of anaerobic isolates yielded the following percentages: 65.5% (n: 218) at the species or species-complex level, 71.2% (n: 237) at the genus level, 29.4% (n: 98) with no identification and 5.1% (n: 17) with misidentification. Using the Bruker Biotyper system, the identification rates were as follows: 85.3% (n: 284) at the species or species-complex level, 89.7% (n: 299) at the genus level, 14.1% (n: 47) with no identification and 0.6% (n: 2) with misidentification. Differences in the performance of both methods were statistically significant (p-values <0.0001). In conclusion, MALDI-TOF MS systems speed up microbial identification and are particularly effective for slow-growing microorganisms, such as anaerobic bacteria, which are difficult to identify by traditional methods. In this study, the Bruker system showed greater accuracy than the Vitek system. In order to be truly effective, it is essential to update the databases of both systems by increasing the number of each main spectrum profile within the platforms.
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Bacterias Anaerobias , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bacterias Anaerobias/genética , ARN Ribosómico 16S/genética , ArgentinaRESUMEN
Bartonella spp. are bacteria responsible for neglected diseases worldwide. Bartonella henselae is the species most associated with human infections. It is associated with a large spectrum of clinical manifestations and is potentially fatal. The identification of Bartonella spp. is considered a challenge in clinical routine. These bacteria are fastidious, and the time required to isolate them varies from one to six weeks. MALDI-TOF mass spectrometry has emerged as an application for research on Bartonella spp. , and has still been little explored. We investigated whether three different B. henselae strains with different growth times-14 and 28 days-could be correctly identified by MALDI-TOF mass spectra fingerprint comparison and matching. We found that the spectra from strains with different growth times do not match each other, leading to misidentification. We suggest creating database entries with multiple spectra from strains with different growth times to increase the chances of accurate identification of Bartonella spp. by MALD-TOF MS.
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Bartonella henselae , Bartonella , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Mass spectrometry (MS) is a powerful analytical technique that plays a central role in modern protein analysis and the study of proteostasis. In the field of advanced molecular technologies, MS-based proteomics has become a cornerstone that is making a significant impact in the post-genomic era and as precision medicine moves from the research laboratory to clinical practice. The global dissemination of COVID-19 has spurred collective efforts to develop effective diagnostics, vaccines, and therapeutic interventions. This chapter highlights how MS seamlessly integrates with established methods such as RT-PCR and ELISA to improve viral identification and disease progression assessment. In particular, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) takes the center stage, unraveling intricate details of SARS-CoV-2 proteins, revealing modifications such as glycosylation, and providing insights critical to formulating therapies and assessing prognosis. However, high-throughput analysis of MALDI data presents challenges in manual interpretation, which has driven the development of programmatic pipelines and specialized packages such as MALDIquant. As we move forward, it becomes clear that integrating proteomic data with various omic findings is an effective strategy to gain a comprehensive understanding of the intricate biology of COVID-19 and ultimately develop targeted therapeutic paradigms.
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COVID-19 , Proteómica , Humanos , Proteómica/métodos , COVID-19/diagnóstico , SARS-CoV-2 , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteínas , Prueba de COVID-19RESUMEN
CONTEXT.: Carbapenem-resistant Enterobacterales are disseminated worldwide and associated with infections with high rates of morbidity and mortality. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a useful tool for identification of pathogens directly from blood cultures in clinical microbiology laboratories. Furthermore, it has been applied for the detection of carbapenemase production, by evaluating carbapenem hydrolysis. OBJECTIVE.: To determine meropenem hydrolysis to detect carbapenemase production directly from positive blood cultures, using logRQ to establish a quantitative measure of hydrolysis. DESIGN.: We evaluated 100 Enterobacterales from positive blood cultures, with 81 carrying a carbapenemase gene (blaKPC, blaGES, blaNDM-1, blaIMP, blaVIM, and blaOXA-48-like), as determined by real-time multiplex polymerase chain reaction with high-resolution melting (HRM-qPCR). Bacterial proteins extracted from positive blood culture bottles were incubated in a meropenem solution (2-4 hours) followed by centrifugation for MALDI-TOF MS analysis. The intensity of peaks of the hydrolyzed and nonhydrolyzed forms were used to calculate the logRQ value. RESULTS.: Overall, sensitivity was 86.8% and specificity, 89.5%. Of note, sensitivity varied depending on enzyme type. For blaKPC-positive isolates, sensitivity was 97.9%, while it reduced significantly for blaNDM-1 and blaOXA-48-like isolates: 62.5% (10 of 16) and 66.7% (6 of 9), respectively. Indeed, logRQ was higher in blaKPC-positive isolates (0.37-1.97) than in blaNDM-1 (-1.37 to 0.83) and blaOXA-48-like isolates (-1.08 to 1.79). CONCLUSIONS.: This is an inexpensive and rapid test to identify carbapenemase activity directly from blood culture bottles, which contributes to early adequate antimicrobial therapy and implementation of infection control measures.
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Antibacterianos , Proteínas Bacterianas , Cultivo de Sangre , Carbapenémicos , Meropenem , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , beta-Lactamasas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Cultivo de Sangre/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Carbapenémicos/farmacología , Meropenem/farmacología , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/diagnóstico , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Hidrólisis , Sensibilidad y EspecificidadRESUMEN
Artisanal cheese from Serra Geral, Minas Gerais, Brazil, stands out for its cultural asset and socio-economic relevance. However, standards of identity and quality and the peculiar terroir associated with the edaphoclimatic conditions have not been established. Therefore, the production flow diagram and the physico-chemical and microbiological quality of the raw milk, pingo (natural starter culture), production benches, water and fresh cheese were investigated for the first time. In addition, lactic acid bacteria (LAB) from cheese and its production environment were identified by MALDI-TOF. For that, 12 cheese making facilities were selected. The raw milk and pingo showed adequate physico-chemical characteristics for cheesemaking; however, high microbial counts were found. In the water, total and thermotolerant coliforms were also identified. The fresh cheeses were classified as 'high moisture and fat' and 'soft mass'. Most physico-chemical parameters were satisfactory; however, there were high counts of total coliforms, Staphylococcus spp. and coagulase-positive staphylococci. There were high counts of LAB in the raw milk, pingo, bench surface and fresh cheese. A total of 84 microbial biotypes from MRS agar were isolated. Lactococcus lactis was the predominant LAB, followed by Lactococcus garvieae. Leuconostoc mesenteroides (benches), Leuconostoc pseudomesenteroides (fresh cheese), and Enterococcus faecium (pingo) were identified sporadically. These results indicate the risks to public health associated with the consumption of the fresh cheese, and measures to improve its safety are needed.
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Queso , Lactobacillales , Lactococcus lactis , Animales , Queso/análisis , Leche/microbiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Brasil , Microbiología de Alimentos , AguaRESUMEN
The purpose of this study was to evaluate the MBT-ASTRA to determine susceptibility to ceftazidime/avibactam (CZA) and meropenem (MEM) of Enterobacterales directly from positive blood cultures (BC). Bacterial suspension was incubated with antibiotic and analyzed by MALDI-TOF MS. The relative growth was calculated and cutoff values were determined to categorize isolates as "S," "I," and "R." Klebsiella spp. with CZA 20/8 mg/L and 1.5-h incubation presented 1 (5.9%) major discrepancy and 96.3% category agreement; other species required 2.5 h for 100% category agreement. For MEM, 4 mg/L and 1.5h were necessary, demonstrating 2 (6.67%) minor discrepancies and 93.3% categorical agreement.
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Cultivo de Sangre , Ceftazidima , Humanos , Ceftazidima/farmacología , Meropenem/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Antibacterianos/farmacología , Combinación de Medicamentos , Pruebas de Sensibilidad Microbiana , beta-LactamasasRESUMEN
Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) based on micro/nanostructured materials with different natures has received increasing attention for the analysis of a wide variety of analytes. However, up to now, only a few studies have shown the application of simple platforms in MALDI-MS for the identification of intact proteins. The present work reports on the application of copper oxide particles (Cu2O PS), obtained by a greener route, in combination with low amounts of 2,5-dihydroxybenzoic acid (DHB) as a novel hybrid platform. The combined Cu2O PS@DHB matrix, containing only 2.5 mg mL-1 of particles and 10 mg mL-1 of DHB, was easily applicable in MALDI-MS without surface modification of target plates. Under optimal conditions, the analysis of intact proteins up to 150,000 Da was possible, including immunoglobulin G, bovine serum albumin, and cytochrome C with adequate spot-to-spot signal reproducibility (RSD < 10%). In addition, the analysis of glycopeptides from IgG digests was carried out to prove the multipurpose application of the Cu2O PS@DHB platform in the low m/z range (2500-3000 Da). From the obtained results, it can be concluded that the optical and surface properties of as-synthesized Cu2O PS are likely to be responsible for the superior performance of Cu2O PS@DHB in comparison with conventional matrices. In this sense, the proposed user-friendly methodology opens up the prospect for possible implementation in bioanalysis and diagnostic research.
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Cobre , Glicopéptidos , Hidroxibenzoatos , Reproducibilidad de los Resultados , Gentisatos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteínas/análisis , Rayos Láser , ÓxidosRESUMEN
RATIONALE: The thiosuccinimide linker is widely used in the synthesis of bioconjugates. However, it is susceptible to hydrolysis and is transformed into its hydrolyzed and/or the isobaric thiazine forms, the latter of which is a fairly common product in a conjugate that contains a cysteinyl peptide. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS) are useful for differentiating these isobaric species. METHODS: Four cross-linked peptides with thiosuccinimide linkers were synthesized. Analogs with linkers that were transformed into thiazine and/or the hydrolyzed thiosuccinimide linkers were then synthesized by incubating the samples at neutral or basic pH. All the cross-linked peptides were purified using RP-HPLC (reversed-phase high-performance liquid chromatography) and differentiated using MALDI-MS, MALDI-MS/MS, and ultraviolet photodissociation. RESULTS: A cysteinyl peptide-containing conjugate, the thiosuccinimide form, was largely transformed into the hydrolyzed or thiazine forms after incubation at neutral or basic pH. MALDI-MS allowed the three forms to be differentiated: the thiosuccinimide and its hydrolysis product yielded two constituent peptides after reductive cleavage between the Cys and succinimide moieties; no fragment ions were produced from the thiazine form. In addition, MALDI-MS/MS of the thiosuccinimide form yielded two pairs of complementary fragment ions via 1,4-elimination: Cys-SH and maleimide, and dehydro-alanine and thiosuccinimide, which are different from those produced via reductive cleavage in MALDI-MS. The thiazine form yielded fragment ions resulting from the cleavage of the newly formed amide bond in the linker that resulted from thiazine formation. CONCLUSIONS: The thiosuccinimide (but not thiazine) form of the cross-linked peptide yielded individual constituent peptides using MALDI-MS and MALDI-MS/MS, showing specific 1,4-elimination for the thiosuccinimide form and cleavage at the newly formed peptide bond via transcyclization for the thiazine form.
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Espectrometría de Masas en Tándem , Tiazinas , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Péptidos/química , Iones , MaleimidasRESUMEN
AIMS: This study aimed to evaluate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial subtyping for the rapid detection of biomarkers in Staphylococcus aureus from subclinical bovine mastitis. METHODS AND RESULTS: A total of 229 S. aureus isolates were obtained from milk samples collected from cows with subclinical mastitis using microbiological culture. Staphylococcus aureus isolates were also submitted to PCR analysis targeting the mecA and mecC genes, which are indicative of methicillin resistance. Confirmation of the species was achieved through MALDI-TOF MS analysis. To analyze antimicrobial resistance patterns, the MALDI BioTyper Compass Explorer and ClinProTools Bruker software were employed, and dendrograms were generated using Bionumerics software. CONCLUSIONS: MALDI-TOF MS successfully identified S. aureus at the species level, but no methicillin resistance was observed. Moreover, spectral typing displayed limited similarity when compared to pulsed-field gel electrophoresis (PFGE).
Asunto(s)
Mastitis Bovina , Infecciones Estafilocócicas , Animales , Bovinos , Femenino , Staphylococcus aureus/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Mastitis Bovina/diagnóstico , Mastitis Bovina/microbiología , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/veterinaria , Infecciones Estafilocócicas/microbiología , BiomarcadoresRESUMEN
MALDI-TOF mass spectrometry (MS) has proven to be a fast and reliable method for the identification of a large number of taxonomic groups. It offers the advantage of being able to incorporate protein spectra of microorganisms that are absent or poorly represented in commercial databases, such as the genus Brucella. The aim of the study was to build the first database of protein spectra of local biological variants of Brucella in Argentina and of standard strains. First, the identification performance of a panel of 135 strains was evaluated with the Swedish database ¨Folkhälsomyndigheten¨ (containing protein spectra of several international standards of the genus Brucella) imported from the open access site https://spectra.folkhalsomyndigheten.se/spectra/. With this library 100 % of the strains were correctly identified by mass spectrometry to genus level, but not to species level. Due to the limitation found, an in-house database was designed with local Brucella isolates from Argentina and standard strains used in routine bacteriological diagnosis. For its validation, a panel of strains, different from those used to develop the extended local database (n: 177), was used to, simultaneously, challenge both libraries. The samples were processed by triplicate and the results obtained were: 177 strains correctly identified to genus and species level compared to the gold standard method (phenotypic typing), meeting the criteria accepted by the literature and the manufacturer as reliable identification. Only 2 of these isolates had score values lower than 2 (1.862) and were therefore not included in the calculation of results. According to these results, MALDI-TOF MS is a fast and reliable method for the routine identification of the different Brucella species, and even has the advantage of reducing the time of exposure to pathogenic microorganisms for laboratorians. It could be considered a valuable technique to replace, in the near future, the current conventional techniques due to the ease of transferring protein spectra, avoiding the use of reference strains that are difficult to find commercially available and commonly used in phenotypic typing.