Improved MALDI-TOF MS identification of Mycobacterium tuberculosis by use of an enhanced cell disruption protocol.

ABSTRACT - Mycobacterium tuberculosis is the microorganism that causes tuberculosis, a disease affecting millions of people worldwide. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable, and cost-effective method for microorganism identification which has been used for the identification of Mycobacterium spp. isolates. However, the mycobacteria cell wall is rich in lipids, which makes it difficult to obtain proteins for MALDI-TOF MS analysis. In this study, two cell preparation protocols were compared: the MycoEx, recommended by MALDI-TOF instrument manufacturer Bruker Daltonics, and the MycoLyser protocol described herein, which used the MagNA Lyser instrument to enhance cell disruption with ethanol. Cell disruption and protein extraction steps with the two protocols were performed using the Mycobacterium tuberculosis H37Rv strain, and the MALDI-TOF MS results were compared. The MycoLyser protocol allowed for improved Biotyper identification of M. tuberculosis since the log(score) values obtained with this protocol were mostly > 1.800 and significantly higher than that underwent MycoEx processing. The identification reliability was increased as well, considering the Bruker criteria. In view of these results, it is concluded that the MycoLyser protocol for mycobacterial cell disruption and protein extraction improves the MALDI-TOF MS method's efficacy for M. tuberculosis identification.Communication

Identificação por espectrometria de massa MALDI-TOF de Salmonella spp. e Escherichia coli isolados de carcaças bovinas.

The aim of this study was to introduce matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry to improve the traditional microbiological method for the detection of Salmonella spp. and Escherichia coli in beef carcasses. Two hundred seventy samples from 90 beef carcasses were evaluated. The methodologies described in ISO 6579:2002 and in the Compendium of Methods for the Microbiological Examination of Foods were used for Salmonella spp. and E. coli isolation, respectively. MALDI-TOF analysis were performed on tryptone soya broth suspension isolates or directly from nutrient agar colonies, from the positive, inconclusive or negative biochemically tested samples for Salmonella and E. coli. Mass profiles were acquired on an Autoflex III SmartBeam MALDI-TOF mass spectrometer and the raw spectra were processed using the MALDI Biotyper software (Bruker Daltonics). According to the preliminary identification based on colony morphology and the biochemical reactions, seven isolates were positive for Salmonella spp. Through MALDI Biotyper these seven isolates were also classified as belonging to the genus Salmonella and further identified as S. enterica. Four isolates showing unusual phenotypic characteristics and inconclusive results in biochemical tests for Salmonella were identified as belonging to Citrobacter and Proteus genera after MALDI analysis. Regarding Escherichia coli, 37 were positive for species biochemical testing which MALDI Biotyper confirmed. MALDI-TOF methodology allowed rapid Salmonella spp. and E. coli identity confirmation and may be used to detect these microrganisms within bacterial isolates from beef carcasses.Título em inglês: MALDI-TOF mass spectrometry identification of Salmonella spp. and Escherichia coli isolated from bovine carcasses

Identification and classification of Mycobacterium by MALDI-TOF mass spectrometry.

Bovine tuberculosis is a zoonotic disease caused by the Mycobacterium bovis bacillum, which accounts for significant economic losses in cattle production besides being a public health threat. Our purpose is to establish MALDI-TOF (Matrix Assisted Laser Desorption Ionization –Time-of-Flight) mass spectrometry method to identify and classify mycobacteria species, by means of building a specific biomarker library after solid media culture for these microrganims. We have identified 26 different protocols employing MALDI-TOF to identify and classify mycobacteria from solid media culture within 58 articles published since 2010. Four consensus protocols were designed and our results lead to the following cell treatment conditions before MALDI-TOF analysis: heat inactivation at 95ºC for 45 minutes, disruption with zirconia beads and MagNA lyser (Roche) and formic acid 70% and acetonitrile protein extraction

Sequenciamento de novo de peptídeos por espectrometria de massas MALDI-TOF.

Neste trabalho apresentam-se os resultados do estabelecimento dessa técnica no Laboratório de Proteômica e Espectrometria de Massas da Sanidade Animal/Embrapa Gado de Corte (PEMSA). No espectrômetro obtêm-se espectros com séries de massas, que por sua vez correspondem aos produtos da quebra ordenada de determinadas ligações presentes em cada molécula ionizada

Identificação de Mycobacterium bovis por espectrometria de massas MALDI-TOF.

A tuberculose bovina é uma doença zoonótica, causada pela bactéria Mycobacterium bovis . Diversas espécies de mamíferos, inclusive o homem, podem infectar-se por esse bacilo. Além do risco à saúde pública, ocorrem perdas econômicas devido à menor produtividade nos rebanhos acometidos. Objetivou-se definir e implementar metodologia de espectrometria de massas MALDI-TOF (Matrix Assisted Laser Desorption Ionization ? Time-of-Flight) para classificar gênero e espécies de Mycobacterium, por meio da construção de uma biblioteca de biomarcadores específicos desses microrganismos após cultivo microbiológico

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDITOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future

Application of an endo-xylanase from Aspergillus japonicus in the fruit juice clarification and fruit peel waste hydrolysis.

The endo-xylanase from Aspergillus japonicus (UFMS 48.136) was purified in a single step using carboximethylcellulose chromatographic column and applied in fruit juice clarification process and fruit peel waste hydrolysis. This purification procedure resulted in 38.9-fold purification of endo-xylanase with 83.3% final yield. MALDITOF analysis confirmed the molecular mass of 32 kDa. The optimal purified endo-xylanase activity was at a range of pH from 5.0 to 6.0 and from 50 to 60 +-C, retaining more than 70% of its activity at all pH studied (3.0?8.0) for 24 h at room temperature. The A. japonicus endo-xylanolytic activity stimulation curve was assayed in the presence of different birchwood xylan concentrations (ranging from 0.02 to 0.5% w/v) and the endoxylanase activity presented a Vmax of 467.4 +- 30.38 μmol/min/mg, with a km of 2.59 +- 0.17 mg/mL, a kcat of 253.95 +- 16.51 s -1 and a kcat/km value of 98.05 +- 4.41 mL s -1 mg -1. The endo-xylanase was activated by Mn2þ (34.5%) and inhibited by Cu2þ (56.9%). The endo-xylanase was activated by β-mercaptoethanol, Triton X-100, Tween-20, Tween-80 and ferulic acid. In the clarification assay, endo-xylanase successfully clarified the juices of mango (51.11%), banana (9.99%) and tangerine (8.54%). Furthermore, the enzyme also hydrolysed all fruit peel wastes that were tested. In summary, A. japonicus endo-xylanase showed potential for applications in fruit juice clarification and in the treatment of fruit peel wastes, and it is a good candidate for the food industry due to its wide pH stability under acidic conditions

Staphylococcus aureus and Methicillin-resistant coagulase-negative staphylococci in nostrils and buccal mucosa of healthy camels used for recreational purposes

Several different species of animals host staphylococci as normal microbiota. These animals can be a source of staphylococci zoonotic infections. People with routine or occupational exposure to infected/colonized animals are at risk of a potential transmission. Therefore, we aimed to investigate the presence of S. aureus and other staphylococci in camels used for recreational purposes as well as their antimicrobial resistance, virulence factors and genetic lineages. A total of 172 samples were collected from 86 healthy camels (nose and mouth) from different farms located in the Canary Islands, Spain. Antimicrobial susceptibility testing was performed against 14 antimicrobial agents. The presence of virulence genes was studied by PCR. Multilocus sequence typing, spa typing and agr typing were performed in all S. aureus isolates. From the 86 camels tested, 42 staphylococci were isolated, of which there were 11 S. aureus, 13 S. lentus, 12 S. sciuri, 3 S. xylosus, S. epidermidis, S. hominis and S. chromogenes. Staphylococci isolates were resistant to penicillin, ciprofloxacin, clindamycin and fusidic acid. All S. aureus isolates harbored the hla, hlb and hld virulence genes. S. aureus isolates were ascribed to three sequence types (STs) and three spa types. All S. aureus isolates belonged to agr type III. Camels from Gran Canaria used in recreational purposes have a moderate prevalence of S. aureus and other coagulase-negative staphylococci. Nevertheless, S. aureus isolates are susceptible to almost all antibiotics tested.Na publicação: Newton Verbisck