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1.
Braz. j. biol ; 82: e233523, 2022. tab
Article in English | LILACS | ID: biblio-1153470

ABSTRACT

Abstract Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.


Resumo Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.


Subject(s)
Humans , Animals , Psittaciformes , Gram-Negative Bacteria , Proteus , Providencia , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Enterobacteriaceae
2.
Pesqui. vet. bras ; 42: e06958, 2022. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1360626

ABSTRACT

Bovine mastitis is the most common disease in dairy cattle and responsible for economic losses in the milk industry. The present study aimed to identify the main species and to evaluate the antimicrobial susceptibility of bacterial isolates from cow herds with mastitis in dairy farms from southern Brazil. A total of 107 milk samples were collected from different cow herds in one important dairy producing region in southern Brazil, including farms located in ten cities from the Northeast region in the Rio Grande do Sul state. Bacterial strains were isolated and submitted to presumptive identification by classical bacteriological methods. Bacterial species were also identified by MALDI-TOF MS and antimicrobial susceptibility testing was performed with 12 antimicrobials commonly used in dairy farms. Fifty-one bacterial strains were isolated and the presumptive identification demonstrated the occurrence of Staphylococcus spp. (82.3%), Bacillus spp. (3.9%), Klebsiella spp. (3.9%), Streptococcus spp. (3.9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) and Serratia sp. (2%). Forty-one isolates were successfully identified in the MALDI-TOF analysis, including 35 isolates from eleven different bacterial species. Importantly, there were eight different Staphylococcus species, with a high frequency of Staphylococcus chromogenes (48.6%) and Staphylococcus aureus (20%). Overall, bacterial isolates demonstrated resistance to penicillin (46.3%), tetracycline (39%), amoxicillin (36.6%), ampicillin (34.1%) and sulfamethoxazole/trimethoprim (31.7%). Enrofloxacin was the unique antimicrobial that all isolates were susceptible. In addition, there were six multidrug resistant isolates (five S. chromogenes and one S. aureus). This study highlights that bacterial pathogens with resistance to several antimicrobials were identified in cows from dairy farms in a very important milk producing region located in southern Brazil. Microbial identification of the bovine mastitis pathogens and determination of the antimicrobial profile is necessary for the rational use of the medicines.(AU)


A mastite bovina é a doença mais comum em gado leiteiro e responsável por perdas econômicas na indústria de laticínios. O presente estudo teve como objetivo identificar as principais espécies e avaliar a suscetibilidade antimicrobiana de isolados bacterianos de rebanhos bovinos com mastite em fazendas leiteiras no sul do Brasil. Um total de 107 amostras de leite foram coletadas em diferentes rebanhos bovinos em uma importante região produtora de leite do sul do Brasil, incluindo fazendas localizadas em 10 cidades da região Nordeste do estado do Rio Grande do Sul. As cepas bacterianas foram isoladas e submetidas à identificação presuntiva por métodos bacteriológicos clássicos. A identificação bacteriana foi confirmada por MALDI-TOF MS e o teste de sensibilidade antimicrobiana foi realizado com antimicrobianos comumente usados em fazendas leiteiras. Cinquenta e uma cepas bacterianas foram isoladas e a identificação presuntiva demonstrou a ocorrência de Staphylococcus spp. (82,3%), Bacillus spp. (3,9%), Klebsiella spp. (3,9%), Streptococcus spp. (3,9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) e Serratia sp. (2%). Os 41 isolados foram identificados com sucesso na análise MALDI-TOF, incluindo 35 isolados de onze espécies bacterianas diferentes. É importante ressaltar que houve a ocorrência de oito espécies diferentes de Staphylococcus, com alta frequência de Staphylococcus chromogenes (48,6%) e Staphylococcus aureus (20%). No geral, os isolados bacterianos tiveram alta resistência à penicilina (46,3%), tetraciclina (39%), amoxicilina (36,6%), ampicilina (34,1%) e sulfametoxazol/trimetoprima (31,7%). A enrofloxacina foi o único antimicrobiano que todos os isolados foram suscetíveis. Além disso, havia seis isolados multirresistentes (cinco S. chromogenes e um S. aureus). Este estudo destaca que os patógenos bacterianos com resistência aos antimicrobianos estão presentes em fazendas leiteiras de subsistência em uma importante região produtora no sul do Brasil. É necessário o monitoramento constante dos patógenos da mastite bovina e a determinação de seu perfil antimicrobiano para o uso racional dos medicamentos.(AU)


Subject(s)
Animals , Female , Cattle , Staphylococcus/isolation & purification , Drug Resistance, Microbial , Milk/microbiology , Mastitis, Bovine , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
3.
J. venom. anim. toxins incl. trop. dis ; 28: e20210017, 2022. graf
Article in English | LILACS, VETINDEX | ID: biblio-1365075

ABSTRACT

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.(AU)


Subject(s)
Animals , Spider Venoms/toxicity , Staphylococcus aureus , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Escherichia coli , Anti-Infective Agents
4.
Cienc. tecnol. salud ; 8(1): 93-103, 2021. il 27 c
Article in Spanish | LILACS, LIGCSA, DIGIUSAC | ID: biblio-1352961

ABSTRACT

Las enfermedades infecciosas son un problema de salud que a pesar de los avances médicos siguen cobrando vi-das en todo el mundo; como las septicemias. La presente investigación tuvo por objetivo diseñar, estandarizar e implementar un protocolo inexistente en Guatemala, para el diagnóstico rutinario de hemocultivos positivos dentro de las instalaciones del Laboratorio Clínico del Hospital General San Juan de Dios, lugar en donde se encuentra el único espectrómetro de masas de tipo Maldi-tof (Matrix Assisted Laser Desorption Ionization-Time of flight-mass spectrometry).Se utilizaron 240 muestras de pacientes de los diferentes servicios. El diagnóstico se realizó compa-rando las identificaciones obtenidas a partir de cultivos microbiológicos puros con muestras directas de botellas con caldo BHI(Brain Heart Infusion).Los resultados de las dos metodologías fueron evaluados con el diseño estadístico "apareado o emparejado en grupo". La comparación no evidenció discordancia en las identificaciones; pero sí en los tiempos de respuesta. La reducción de tiempo fue de 42.9 h para bacterias Gram positivo, 45.0 h para bacterias Gram negativo y 126.2 h para levaduras, todos a favor de identificaciones a partir de muestras directas. Con esta investigación se pretende ofrecer una nueva alternativa que permitirá brindar un diagnóstico rápido, confiable y certero a la población guatemalteca. También permitirá reducir la morbimortalidad de los pacientes con septicemias, promover el ahorro de insumos hospitalarios, disminuir el tiempo de estancia hospitalaria, ahorrar el consumo de antibióticos innecesarios y contribuir indirectamente a combatir la resistencia antimicrobiana; un problema actual de gran importancia a nivel mundial.


Infectious diseases are a health problem that despite medical advances in terms of diagnosis continue to take lives worldwide, such is the case of sepsis. The purpose of this research was to design, standardize and implement a non-existent protocol in Guatemala, for the routine diagnosis of positive blood cultures, within the facilities of the clinical laboratory of the San Juan de Dios General Hospital; where the only Maldi-tof (Matrix Assisted Laser Desorp-tion Ionization-Time of flight-mass spectrometry) type mass spectrometer is located. For this, 240 samples of positive blood cultures were used, coming from patients of the different services. The microbiological diagnosis was made by comparing the identification data obtained from pure microbiological cultures and direct samples of BHI broth (Brain Heart Infusion) bottles. The results of the two methodologies were evaluated based on "paired or matched in groups" statistical design. The Maldi-tof technique did not show disagreement regarding identification between the two types of samples; but it did in the response time. The time reduction was 42.9 h for Gram positive bacteria, 45.0 h for Gram negative and 126.2 h for yeasts, supporting identification from direct samples. This research aims to provide a new diagnostic alternative that will allow access to fast, reliable, and accurate results for the Guatemalan population. It will also help to reduce e morbidity and mortality rates of patients with sepsis, to promote hospital supplies savings, decrease the patient length of stay, save unnecessary antibiotics and indirectly contribute to combating antimicrobial resistance; a critical problem faced by the world today.


Subject(s)
Humans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation , Blood Culture/methods , Gram-Positive Bacteria , Drug Resistance, Microbial/drug effects , Sepsis/blood
5.
J. venom. anim. toxins incl. trop. dis ; 27: e20200171, 2021. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1279405

ABSTRACT

Background Solitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated. Methods A reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides. Results Online mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and β-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, β-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, β-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine. Conclusion Comprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. β-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.(AU)


Subject(s)
Animals , Peptides/classification , Wasp Venoms , Wasps/metabolism , Neuroprotection , Oxidative Stress , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
6.
Infectio ; 24(4): 224-228, oct.-dic. 2020. tab, graf
Article in Spanish | LILACS, COLNAL | ID: biblio-1114873

ABSTRACT

Resumen Objetivo: Comparar los resultados obtenidos de diferentes sistemas de identificación de C. auris. Métodos: Análisis descriptivo con datos recopilados durante 2016-19 mediante la vigilancia nacional. Se evaluaron los resultados generados por los sistemas MicroScan, Phoenix BD, VITEK 2 y MALDI-TOF MS de instituciones hospitalarias de 843 aislamientos clínicos sospechosos de C. auris remitidos al INS y se compararon con los resultados generados de confirmación a través de MALDI- TOF MS (Bruker Daltonics) o PCR. Resultados: De los 843 aislamientos clínicos remitidos al INS, el 81,7% fueron confirmados como C. auris mediante MALDI- TOF MS o PCR en el INS y el resto, 18,3%, fueron identificados como otras especies de Candida spp. Las identificaciones correctas enviadas por los laboratorios representaron el 42,4%. MicroScan identificó C. auris principalmente como C. haemulonii, C. guilliermondii, C. albicans y C. famata; Phoenix BD, VITEK 2 y MALDI-TOF MS identificó C. auris como C. haemulonii. Discusión: Estudios señalan que C. auris exhibe una estrecha relación filogenética con C. haemulonii. Las identificaciones discrepantes pueden darse debido a que las bases de datos de los sistemas de diagnóstico son limitadas para este patógeno. Las deficiencias de los sistemas comerciales para la identificación de C. auris deben ser complementados con otros sistemas como MALDI-TOF MS o pruebas moleculares.


Abstract Objective: To compare the identification results obtained by different identification systems of C. auris isolates. Methods: A descriptive study with data collected during the years 2016-19 through surveillance. The results generated by the MicroScan, Phoenix BD, VITEK 2 and MALDI-TOF MS systems of 843 clinical isolates of C. auris submitted to the INS were evaluated and compared with the results generated from confirmation through MALDI-TOF MS (Bruker Daltonics) or PCR. Results: Out of 843 clinical isolates submitted to the INS, 81.7% were confirmed as C. auris by MALDITOF MS or PCR in the INS and the rest, 18.3%, were identified as other species of Candida spp. The correct identifications sent by the laboratories was 42.4%. MicroScan identified C. auris as C. haemulonii, C. guilliermondii, C. albicans and C. famata; Phoenix BD, VITEK 2 and MALDI-TOF MS identified C. auris as C. haemulonii. Discussion: Studies indicate that C. auris exhibits a close phylogenetic relationship with C. haemulonii. In addition, discrepant identifications may occur because the databases of diagnostic systems are limited with reference to this pathogen. The deficiencies of commercial systems for the identification of C. auris must be complemented with other systems such as MALDI-TOF MS or molecular tests.


Subject(s)
Humans , Female , Candida , Surveillance , Diagnosis , Laboratories , Polymerase Chain Reaction , Epidemiology, Descriptive , Colombia , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Molecular Diagnostic Techniques , Alkalies
7.
Rev. chil. infectol ; 37(3): 252-256, jun. 2020. tab, graf
Article in Spanish | LILACS | ID: biblio-1126117

ABSTRACT

Resumen Introducción: Las enfermedades producidas por micobacterias son de gran importancia clínica y epidemiológica presentando el complejo Mycobacterium tuberculosis (MTBc) una morbi-mortalidad mayor que la producida por micobacterias no tuberculosas (MNTB). La identificación tradicional está basada en sus características fenotípicas mediante procesos laboriosos e incapaces en algunos casos de distinguir entre especies. Actualmente, la mayoría de las técnicas utilizadas se basan en métodos moleculares que tienen alta veracidad, pero son complejas y de alto costo. La espectrometría de masas con desorción/ionización láser asistida por una matriz asociada a tiempo de vuelo (MALDI-TOF MS) se basa en la comparación del espectro proteico producido con respecto al de una base de datos de referencia. Objetivo: Evaluar el rendimiento de MALDI-TOF MS en la identificación de micobacterias comparado con métodos moleculares: Material y Métodos: Se analizaron 28 aislados de nueve especies distintas mediante MALDI-TOF MS. Resultados: Se identificó correctamente 78,5% de las aislados (22/28), concordante en 100% (9/9) de MNTB de crecimiento rápido, 60% (9/15) en las MNTB de crecimiento lento y 100% (4/4) de MTBc. Todas las especies no identificadas (6/6) pertenecen al complejo M. avium/intracellulare. Conclusión: MALDI-TOD MS es una metodología rápida, fácil y de bajo costo, con adecuada veracidad respecto a los métodos moleculares.


Abstract Background: Mycobacterial diseases are very important both clinically and epidemiologically. Mycobacterium tuberculosis complex (MTBc) infections confer higher morbidity and mortality rate than non-tuberculous mycobacteria (NTM) infections. Traditional species identification techniques are based on phenotypic characteristics which take a long time by laborious processes and in occasions are no conclusive. Currently, most used techniques are based on molecular methods, which are accurate but are expensive and complex. Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) is a simple, cheap and fast identification method based on comparing protein spectra with a reference database. Aim: To assess the performance of MALDI-TOF MS in the identification of MTBc and NTM, compared with molecular methods. Methods: For that purpose, 28 isolates of 9 different species were analyzed through MALDI-TOF MS. Results: 78.5% (22/28) of isolates were correctly identified, 100% (9/9) of rapidly growers NTM, 60% (9/15) of slow growing NTM and 100% (4/4) of MTBc. Every unidentified isolate (6/6) corresponded to M. avium/intracellulare complex. Conclusion: MALDI-TOF MS is fast, simple and cheaper than molecular methods and also has adequate accuracy.


Subject(s)
Humans , Mycobacterium , Tuberculosis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
8.
Rev. Ateneo Argent. Odontol ; 62(1): 52-56, jun. 2020.
Article in Spanish | LILACS | ID: biblio-1148211

ABSTRACT

Si partimos de que la microbiología es una ciencia fundante, podemos estar de acuerdo también en la necesidad de la continua actualización de sus contenidos y su vinculación con la odontología. Nuevas técnicas de diagnóstico permiten, no solo poder identificar características especiales de cada microorganismo y su reubicación en la taxonomía general, sino también habilitan a reconocer a aquellos ­hasta el momento­ desconocidos en la cotidianeidad de la práctica profesional y que revisten importancia por sus afecciones sistémicas ya que pueden transformar, en algunos casos, a que el paciente sea considerado de riesgo. En este trabajo, se abordan tres ejemplares bacterianos seleccionados por su complejidad en la identificación y por la magnitud de las lesiones que producen. Granulicatella spp., Kingela kingae y Bilophila wadsworthia afectan no solo adultos sino también pacientes pediátricos, siendo afectados por patologías severas. Se describen cuadros clínicos que afectan tejido óseo, corazón, cerebro, hígado, bazo, riñón y las manifestaciones orales a las cuales pueden asociarse grupos microbianos que agravan el pronóstico. Aplicar la tecnología adecuadamente, no solo a procedimientos odontológicos, sino también para diagnóstico (PCR ­ MALDI ­ TOF) facilita la detección e identificación con mayor celeridad de estos agentes microbianos, evitando la rotación farmacológica, la resistencia microbiana y la automedicación (AU)


Considering microbiology as a key science in the approach of infectious processes, we understand the need for a continuous update of its contents and its link with dentistry. The incorporation of new technological approaches, such as molecular methods or mass spectrometry, allow us not only to identify special characteristics of the microorganism and its relocation in taxonomy, but also to know those microorganisms until now unknown in professional´s life everyday practice and that are important for their systemic implications, modifying in some cases, the risk assessment of the patient. Three bacterial specimens are developed in this work, due to their complexity in the identification and the magnitude of the lesions they produce, Granulicatella spp., Kingela kingae and Bilophila wadsworthia. These affects both adult and paediatric patients, describing several clinical conditions that affect bone tissue, heart, brain, liver, spleen, kidney and oral manifestations to which these microbial groups can be associated, aggravating the prognosis. Applying new technology, not only to dental procedures but also to diagnosis, facilitates the detection and identification with greater speed of these microbial agents, avoiding pharmacological rotation, microbial resistance and self-medication (AU)


Subject(s)
Microbiology , Mouth Diseases/microbiology , Drug Resistance, Microbial , Polymerase Chain Reaction , Kingella kingae , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Bilophila
9.
An. bras. dermatol ; 95(3): 298-306, May-June 2020. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS | ID: biblio-1130892

ABSTRACT

Abstract Background: The increasingly frequent use of dermoscopy makes us think about the possibility of transfer of microorganisms, through the dermatoscope, between doctor and patients. Objectives: To identify the most frequent gram-positive cocci in dermatoscopes and smartphone adapters, as well as the resistance profile, and to evaluate the factors associated with a higher risk of bacterial contamination of the dermatoscopes. Methods: A cross-sectional study was carried out with 118 dermatologists from Porto Alegre/Brazil between September 2017 and July 2018. Gram-positive cocci were identified by MALDI-TOF MS and habits of use of the dermatoscope were evaluated through an anonymous questionnaire. Results: Of the dermatoscopes analysed, 46.6% had growth of gram-positive cocci on the lens and 37.3% on the on/off button. The microorganisms most frequently found were S. epidermidis, S. hominis and S. warneri. Attending a hospital, using the dermatoscope at the hospital, with inpatients and in the intensive care unit were significantly associated with colonisation by gram-positive cocci. The highest resistance rates were observed for penicillin, erythromycin and sulfamethoxazole-trimethoprim. Study limitations: The non-search of gram-negative bacilli, fungi and viruses. Moreover, the small number of adapters did not make it possible to better define if the frequency differences were statistically significant. Conclusion: Coagulase-negative staphylococci were frequently identified. S. aureus was detected only on the lens.


Subject(s)
Humans , Male , Female , Adult , Gram-Positive Bacterial Infections/epidemiology , Gram-Positive Cocci/isolation & purification , Dermoscopy/instrumentation , Smartphone , Dermatologists/statistics & numerical data , Brazil/epidemiology , Microbial Sensitivity Tests , Cross-Sectional Studies , Surveys and Questionnaires , Risk Factors , Gram-Positive Bacterial Infections/microbiology , Gram-Positive Cocci/growth & development , Gram-Positive Cocci/drug effects , Sex Distribution , Age Distribution , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Drug Resistance, Bacterial , Middle Aged , Anti-Bacterial Agents/pharmacology
10.
Infectio ; 23(4): 364-370, Dec. 2019. tab, graf
Article in Spanish | LILACS, COLNAL | ID: biblio-1040007

ABSTRACT

Resumen Objetivo: evaluar la utilidad de la identificación directa de microorganismos en muestras de orina y hemocultivos empleando la tecnología MALDI-TOF MS, mediante el análisis de concordancia en la identificación, tiempo necesario para la obtención de un resultado y costos asociados a cada método de identificación. Materiales y métodos: estudio descriptivo de febrero de 2017 a octubre de 2017. Se seleccionaron a conveniencia 180 muestras de orinas y 129 hemocultivos de pacientes de la Clínica El Rosario, Medellín, se realizó identificación del microorganismo directamente de la muestra y a partir del cultivo por MALDI-TOF (Vitek® MS‚ bioMérieux). Se analizaron los costos y tiempo, para determinar la utilidad de esta tecnología en los procesos del laboratorio de microbiología. Resultados: En el 79,6% de las orinas positivas y en el 76% de los hemocultivos se obtuvo una identificación de microorganismos directamente por MALDI-TOF MS. El tiempo de identificación directa tuvo una media de 6 horas y por cultivo una media de 29 horas. El costo total por aislamiento identificado de forma directa (sin incluir el valor del equipo) fue de $8.200 (2,58 USD) en muestras de orina y de $9.720 (3,06 USD) en hemocultivos positivos. El equipo introduce un costo variable en cada identificación de acuerdo con el número de identificaciones que se realicen en el laboratorio. Conclusiones: Estos resultados confirman la utilidad del MALDI-TOF MS para generar identificaciones más rápidas cuando se utiliza directamente en muestras clínicas, sin embargo, tiene un bajo desempeño en la identificación directa de bacterias gram positivas, siendo necesario evaluar otros protocolos que mejoren la identificación directa. El costo de los consumibles es bajo, pero la adquisición de esta tecnología introduce un costo variable que depende del volumen de muestras identificadas en el laboratorio.


Abstract Objective: To evaluate the utility of the direct identification of microorganisms in urine and blood cultures samples, using MALDI-TOF by evaluating concordance for identification, time to obtain an identification result and associated costs. Materials and Methods: A descriptive study from February to October 2017 in 180 urine samples and 129 positive blood cultures samples of patients from the El Rosario Clinic in Medellin- Colombia. The clinical samples were processed directly for microorganisms identification by using MALDI-TOF (Vitek® MS‚ bioMérieux). This result was compared with the result obtained with Maldi tof -MS done for the cultured microorganism. An analysis of cost and time to achieve an identification result was made to determinate the utility of this technology in the laboratory procedures. Results: 79,6 % of positive urines and the 76 % of blood cultures were identified directly from the sample by MALDI-TOF. MALDI-TOF applied directly had a mean time for obtaining an identification of 6 hours compared to 29 hours to obtain an identification from cultures. The cost of direct identification was $8.200 (2,58 USD) in urine samples and $9.720 (3,06 USD) in blood cultures (without including the equipment cost). This cost is variable depending of the number of identifications that the laboratory performs. Conclusions: These results support the usefulness of MALDI-TOF for getting rapid identification results using the direct methodology in clinical samples. However, the capability to identify gram positive bacteria needs to be improved. The incorporation of this methodology in microbiology laboratories may improve the opportunity in the etiological diagnosis and should have a positive impact on patient care.


Subject(s)
Humans , Bacteria , Urine , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Blood Culture , Mass Spectrometry , Cost Control
11.
Rev. chil. infectol ; 36(6): 790-793, dic. 2019. tab
Article in Spanish | LILACS | ID: biblio-1058113

ABSTRACT

Resumen La espectrometría de masas MALDI-TOF MS es una técnica rápida y sencilla para identificar microorganismos por análisis proteico. Se estudiaron 304 aislados de levaduras procedentes de micosis superficiales y profundas, con el objetivo de comparar tres métodos: convencional (bioquímico y morfológico), MALDI-TOF MS, y reacción en cadena de la polimerasa (RPC, método de referencia). Se estudiaron 24 especies con predominio de Candida spp y Cryptococcus spp. La identificación por método convencional fue de 258/304 cepas, mientras que por MALDI-TOF MS fue de: 277/304 cepas (84,8 versus 91,2%, p = no significativo). El coeficiente Kappa entre el MALDI-TOF MS y la RPC reportó una excelente concordancia (0,99). La sensibilidad y la especificidad de MALDI-TOF MS para la identificación de levaduras patógenas oportunistas de muestras clínicas fueron de 94,6% y 99%; respectivamente. MALDI-TOF MS demostró ser una herramienta de alta precisión para la identificación de levaduras patógenas.


MALDI-TOF MS mass spectrometry is a rapid and straightforward technique to identify microorganisms by protein analysis. The study was performed in 304 yeast isolates from superficial and deep mycoses, in order to compare three methods: conventional (biochemical and morphological), MALDI-TOF MS, and polymerase chain reaction (PCR, reference). We included 24 species with predominance of Candida spp and Cryptococcus spp. The identification by conventional methods was 258/304 strains, while by MALDI-TOF MS was: 277/304 strains (84.8% versus 91.2%, P = not significant). The Kappa coefficient comparing MALDI-TOF-MS with PCR reported excellent concordance (0.99). The sensitivity and specificity of MALDI-TOF MS for the diagnosis of opportunistic pathogenic yeasts of clinical samples were 94.6% and 99% respectively. MALDI-TOF MS is a simple, fast and reliable tool for pathogenic yeasts.


Subject(s)
Humans , Mycoses , Yeasts , Candida/genetics , Sensitivity and Specificity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
12.
Braz. j. infect. dis ; 23(3): 164-172, May-June 2019. tab, graf
Article in English | LILACS | ID: biblio-1019558

ABSTRACT

ABSTRACT Bloodstream infections (BSIs) are serious infections associated with high rates of morbidity and mortality. Every hour delay in initiation of an effective antibiotic increases mortality due to sepsis by 7%. Turnaround time (TAT) for conventional blood cultures takes 48 h, forcing physicians to streamline therapy by exposing patients to broad-spectrum antimicrobials. Our objective was (1) to evaluate the accuracy and TAT of an optimized workflow combining direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in-house real-time polymerase chain reaction (PCR) for bacterial identification and antimicrobial resistance profiling directly from positive blood bottles for diagnosing bloodstream infections and (2) to verify the effect of reporting results to medical staff. A total of 103 BSI episodes from 91 patients admitted to three hospitals in São Paulo, Brazil were included. TAT from molecular versus conventional methods was measured and compared. Our protocol showed an overall agreement of 93.5% for genus and 78.5% for species identification; 74.2% for methicillin resistance detection, 89.2% for extended-spectrum β-lactamase profiling, 77.8% for metallo-β-lactamase profiling, and 100% for carbapenemase profile and vancomycin-resistance detection when compared with conventional testing. TAT of molecular sample processing according to our protocol was 38 h shorter than conventional methods. Antimicrobial interventions were possible in 27 BSI episodes. Antimicrobial discontinuation was achieved in 12 BSI episodes while escalation of therapy occurred in 15 episodes. Antimicrobial therapy was inadequate in three (12%) BSI episodes diagnosed using results of molecular testing. Our in-house rapid protocol for identifying both bacteria and antimicrobial resistance provided rapid and accurate results, having good agreement with conventional testing results. These results could contribute to faster antimicrobial therapy interventions in BSI episodes.


Subject(s)
Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , Bacteremia/diagnosis , Gram-Negative Bacteria/classification , Gram-Positive Bacteria/classification , Time Factors , Prospective Studies , Bacteremia/microbiology , Bacteremia/drug therapy , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Real-Time Polymerase Chain Reaction , Gram-Negative Bacteria/genetics , Gram-Positive Bacteria/genetics , Anti-Bacterial Agents/administration & dosage
13.
Rev. chil. infectol ; 36(3): 392-395, jun. 2019. tab, graf
Article in Spanish | LILACS | ID: biblio-1013799

ABSTRACT

Resumen Presentamos un caso de bacteriemia por Vibrio cholerae no-O1/ no-O139 en una mujer de 81 años con un cuadro de dolor abdominal, fiebre, vómitos, diarrea, coluria e ictericia, mientras visitaba una zona rural sin acceso a agua potable. La identificación se realizó por la técnica de espectrometría de masa MALDI-TOF, confirmándose una cepa no toxigénica no-O1/no-139. La caracterización molecular del aislado demostró la ausencia del gen de la toxina del cólera (CTX), y pilus TCP; sin embargo, presentó cinco de los seis genes de virulencia presentes en la isla de patogenicidad homóloga denominada VPaI-7 del V. parahaemolyticus (vcs N2+, vcs C2+, vcs V2+,toxR-, vspD+, T vopF+). Además, el aislado presentó los genes de virulencia hylA y rtxA. Este es el primer caso reportado en Chile de una cepa clínica de V. cholerae no-O1, no-O139 aislada de hemocultivos portador de un segmento homólogo de la isla de patogenicidad denominada VPaI-7 de V. parahaemolyticus, el cual codifica para un sistema de secreción tipo III (TTSS), que probablemente contribuye a su virulencia.


We report a case of V. cholerae non-O1 / non-O139 bacteremia in an 81-year-old woman with abdominal pain, fever, vomiting, liquid stools, choluria and jaundice, while visiting a rural area without access to potable water. The identification was made by the MALDI-TOF mass spectrometry technique and subsequently the non-toxigenic non-O1 / non-139 strain was confirmed in the national reference laboratory. The molecular characterization demonstrated the absence of the cholera toxin gene (CTX), and the TCP pilus, however, presented 5 of 6 virulence genes present in an island of homologous pathogenicity named VPaI-7 of V. parahaemolyticus (vcs N2 +, vcs C2 +, vcs V2 +, toxR-, vspD +, T vopF +) and in addition it was positive for hylAy rtxA virulence genes recognized outside the island. This is the first case reported in Chile of a clinical strain of V. cholerae non-O1, non-O139 isolated from blood culture that carries in its genome a homologous segment of the pathogenicity island named VPaI-7 of V. parahaemolyticus, which codifies for a type III secretion system (TTSS) that probably contributes to his virulence.


Subject(s)
Humans , Female , Aged, 80 and over , Bacterial Proteins/chemistry , Vibrio cholerae/chemistry , Bacteremia/etiology , Vibrio cholerae non-O1/chemistry , Bacterial Proteins/isolation & purification , Vibrio cholerae/isolation & purification , Vibrio cholerae/pathogenicity , Virulence , Cholera/complications , Cholera/microbiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Vibrio cholerae non-O1/isolation & purification , Vibrio cholerae non-O1/pathogenicity , Genomic Islands
14.
Clin. biomed. res ; 39(2): 128-135, 2019.
Article in English | LILACS | ID: biblio-1022788

ABSTRACT

Introduction: Identification of yeast species has clinical and epidemiological value. Different methods can be used, such as chromogenic media, microculture on corn meal agar with Tween 80, as well as conventional biochemical and automated methods. Recently, proteomic studies employing matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry have been a major advance in diagnosis due to speed of execution and accuracy of results. Methods: For this study, 79 yeast samples were submitted to identification using chromogenic medium, microculture on corn meal-Tween 80 agar, VITEK 2 Compact identification, and MALDI-TOF mass spectrometry. Results: Most of the 79 samples were identified, with differences in the performance of the methods used. Colonial morphology and microscopy were compatible with the genus Candida. MALDI-TOF mass spectrometry had the best performance, with 78 strains identified (98.7%), compared to VITEK 2 Compact (92.4%) and microculture on corn meal agar (70.9%). Conclusions: MALDI-TOF mass spectrometry using the VITEK MS instrument performed best and has proven to be a revolutionary method in clinical microbiology laboratories. Regarding the identification of C. albicans and C. tropicalis, the chromogenic medium had excellent performance, thus being a good option to optimize the process. (AU)


Subject(s)
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Candida/isolation & purification , Candidiasis/diagnosis
15.
Clin. biomed. res ; 39(2): 171-174, 2019.
Article in English | LILACS | ID: biblio-1023235

ABSTRACT

(MALDI-TOF MS) has been used in clinical diagnostic laboratories for the identification of microorganisms. It has a relevant advantage compared to other methods in terms of speed to provide results, being an alternative for addressing restrictions in clinical diagnosis as it may replace or complement existing identification techniques. This is especially important because some rare microorganisms would be identified only by higher cost techniques which are not widely available, such as genetic sequencing. Thus, the present paper reports two cases in which uncommon microorganisms were identified effectively and quickly. (AU)


Subject(s)
Humans , Female , Middle Aged , Bacterial Infections , Actinomycetaceae/pathogenicity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Mycobacterium abscessus , Nontuberculous Mycobacteria
16.
J. venom. anim. toxins incl. trop. dis ; 25: e147018, 2019. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1002495

ABSTRACT

Lachesis muta rhombeata is one of the venomous snakes of medical importance in Brazil whose envenoming is characterized by local and systemic effects which may produce even shock and death. Its venom is mainly comprised of serine and metalloproteinases, phospholipases A2 and bradykinin-potentiating peptides. Based on a previously reported fractionation of L. m. rhombeata venom (LmrV), we decided to perform a subproteome analysis of its major fraction and investigated a novel component present in this venom. Methods: LmrV was fractionated through molecular exclusion chromatography and the main fraction (S5) was submitted to fibrinogenolytic activity assay and fractionated by reversed-phase chromatography. The N-terminal sequences of the subfractions eluted from reversed-phase chromatography were determined by automated Edman degradation. Enzyme activity of LmrSP-4 was evaluated upon chromogenic substrates for thrombin (S-2238), plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) and upon fibrinogen. All assays were carried out in the presence or absence of possible inhibitors. The fluorescence resonance energy transfer substrate Abz-KLRSSKQ-EDDnp was used to determine the optimal conditions for LmrSP-4 activity. Molecular mass of LmrSP-4 was determined by MALDI-TOF and digested peptides after trypsin and Glu-C treatments were analyzed by high resolution MS/MS using different fragmentation modes. Results: Fraction S5 showed strong proteolytic activity upon fibrinogen. Its fractionation by reversed-phase chromatography gave rise to 6 main fractions (S5C1-S5C6). S5C1-S5C5 fractions correspond to serine proteinases whereas S5C6 represents a C-type lectin. S5C4 (named LmrSP-4) had its N-terminal determined by Edman degradation up to the 53rd amino acid residue and was chosen for characterization studies. LmrSP-4 is a fibrinogenolytic serine proteinase with high activity against S-2302, being inhibited by PMSF and benzamidine, but not by 1,10-phenantroline. In addition, this enzyme exhibited maximum activity within the pH range from neutral to basic and between 40 and 50 °C. About 68% of the LmrSP-4 primary structure was covered, and its molecular mass is 28,190 Da. Conclusions: Novel serine proteinase isoforms and a lectin were identified in LmrV. Additionally, a kallikrein-like serine proteinase that might be useful as molecular tool for investigating bradykinin-involving process was isolated and partially characterized.(AU)


Subject(s)
Plasminogen , Snake Venoms , Lachesis muta , Serine Proteases , Kallikreins , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Phospholipases A2
17.
Article in Chinese | WPRIM | ID: wpr-813290

ABSTRACT

To establish a two-dimensional gel electrophoresis (2-DE) map for comparative proteomic analysis of rat spinal cord with chronic morphine tolerance, and to detect differentially expression proteins that are associated with chronic morphine tolerance.
 Methods: Sixteen male SD rats received the intrathecal catheterization operation and they were randomly divided into a morphine tolerance group (MT group, n=8) and a saline group (NS group, n=8). The lumbar enlargement segments of the MT group and the NS group spinal cord were harvested and proteins were separated by 2-DE. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The 2-DE maps were visualized after coomassie blue staining and analyzed using PDQuest analysis software. Identification of differential protein spots was conducted by MALDI-TOF-MS, and the Mascot query software was used to search Swiss-Prot database for bioinformatics analysis. Western blotting was used to verify the expression of some differentially expressed proteins.
 Results: A total of 1 000 spots were identified in 2-DE maps of rat spinal cord tissues from the MT group and the NS group, and 36 proteins were significantly differentially expressed in the MT group compared with the NS group. Identification was conducted by MALDI-TOF-MS and Swiss-Prot database through Mascot query software, and a total of 14 proteins were obtained. Among them, 2 protein spots were down-regulated in the MT group compared with that in the NS group, and 12 protein spots were up-regulated in the MT group compared with that in the NS group. Two kinds of proteins (NUDAA, ENOG) were verified by Western blotting and the results were consistent with proteomics data.
 Conclusion: The optimized 2-DE profiles for the proteome of spinal cord tissue in rats with chronic morphine tolerance is established preliminarily, which showed that morphine tolerance can cause changes in the expression of various proteins in the spinal cord.


Subject(s)
Animals , Electrophoresis, Gel, Two-Dimensional , Male , Morphine , Proteome , Proteomics , Rats , Rats, Sprague-Dawley , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spinal Cord
18.
Braz. j. microbiol ; 49(4): 900-908, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-974290

ABSTRACT

ABSTRACT Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.


Subject(s)
Humans , Bacterial Typing Techniques/methods , Tandem Mass Spectrometry/methods , Leptospira/isolation & purification , Leptospirosis/microbiology , Brazil , DNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Leptospira/classification , Leptospira/genetics , Leptospira/chemistry
19.
Braz. j. microbiol ; 49(4): 801-807, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-974302

ABSTRACT

ABSTRACT The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 ºC for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.


Subject(s)
Animals , Female , Infant , Cattle , Staphylococcus/isolation & purification , Streptococcus/isolation & purification , Bacterial Typing Techniques/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Milk/microbiology , Mastitis, Bovine/microbiology , Staphylococcus/genetics , Staphylococcus/chemistry , Streptococcus/genetics , Streptococcus/chemistry , Milk/chemistry , Mastitis, Bovine/physiopathology
20.
Braz. j. infect. dis ; 22(3): 239-242, May-June 2018.
Article in English | LILACS | ID: biblio-974204

ABSTRACT

ABSTRACT Febrile Neutropenia represents a medical emergency and the use of appropriate antimicrobial therapy is essential for a better outcome. Although being time-consuming, conventional cultures and antimicrobial susceptibility tests remain the golden standard practices for microbiology identification. Final reports are typically available within several days. Faster diagnostic tools, such as species identification trough Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) and molecular techniques might help to shorten time to diagnostic and also guide definitive therapy in this scenario. Here we present a case in which the use of a diagnostic molecular workflow combining MALDI-TOF and real-time PCR for relevant genes codifying antibiotic resistant integrated with instant communication report, led to a tailored and more appropriate treatment in a patient presenting with febrile neutropenia.


Subject(s)
Humans , Male , Middle Aged , Ceftazidime/administration & dosage , Azabicyclo Compounds/administration & dosage , Febrile Neutropenia/microbiology , Febrile Neutropenia/drug therapy , beta-Lactamase Inhibitors/administration & dosage , Klebsiella pneumoniae/drug effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Reverse Transcriptase Polymerase Chain Reaction , Drug Resistance, Multiple, Bacterial , Drug Combinations , Precursor Cell Lymphoblastic Leukemia-Lymphoma/complications , Klebsiella pneumoniae/isolation & purification
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