Method For Identification Of Microorganisms Research Articles

A new culture-based method for rapid identification of microorganisms in polymicrobial blood cultures by MALDI-TOF MS

BackgroundThe application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) to microbial identification has allowed the development of rapid methods for identification of microorganisms directly in positive, blood cultures (BCs). These methods can yield accurate results for monomicrobial BCs, but often fail to identify multiple microorganisms in polymicrobial BCs. The present study was aimed at establishing a rapid and simple method for identification of bacteria and yeast in polymicrobial BCs from patients with bloodstream infection.ResultsThe rapid method herein proposed is based on short-term culture in liquid media allowing selective growth of microorganisms recovered from polymicrobial BCs, followed by rapid identification by MALDI-TOF MS. To evaluate the accuracy of this method, 56 polymicrobial BCs were comparatively analyzed with the rapid and routine methods. The results showed concordant identification for both microbial species in 43/50 (86%) BCs containing two different microorganisms, and for two microbial species in six BCs containing more than two different species. Overall, 102/119 (85.7%) microorganisms were concordantly identified by the rapid and routine methods using a cut-off value of 1.700 for valid identification. The mean time to identification after BC positivity was about 4.2 h for streptococci/enterococci, 8.7 h for staphylococci, 11.1 h for Gram-negative bacteria, and 14.4 h for yeast, allowing a significant time saving compared to the routine method.ConclusionsThe proposed method allowed rapid and reliable microbial identification in polymicrobial BCs, and could provide clinicians with timely, useful information to streamline empirical antimicrobial therapy in critically ill patients.

An improved in-house lysis-filtration protocol for bacterial identification from positive blood culture bottles with high identification rates by MALDI-TOF MS

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is now a well-established method for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial proteins are a prerequisite for successful identification, and a variety of protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We developed a new, in-house lysis-filtration protocol and prospectively evaluated its performance compared to the Sepsityper kit. The in-house protocol consists of three simple steps: lysis by ammonium chloride, aspiration with a syringe fitted with a 0.45-μm membrane, and centrifugation to collect microbes. The novel protocol requires only 20 min.Performance of the in-house protocol was evaluated using a total of 117 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the in-house protocol or the commercial kit, and isolated cells were subjected to direct identification by mass spectrometry fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with score > 1.7 and > 2.0 obtained using the in-house protocol were 99.2% and 85.5%, respectively, whereas those obtained using the Sepsityper Kit were 85.4% and 61.5%, respectively. For Gram-positive cases, the in-house protocol yielded scores >1.7 and > 2.0 at 98.5% and 76.1%, respectively, whereas the commercial kit yielded these scores at 76.1% and 43.3%, respectively. Although these are preliminary results, these values suggest that this easy lysis-filtration protocol deserves assessment in a larger-scale test.

Identification of rod-shaped Gram-negative bacilli of Enterobacteriaceae family by MALDI-TOF mass spectrometry

Summary Introduction. Matrix assisted laser desorption ionization-time of flight mass spectrometry is a new rapid method for identification of microorganisms isolated from clinical specimens. Aim. The aim of the study was to compare the results of identification Enterobacte- riaceae strains obtained by using MALDI Biotyper and Vitek 2. Material and methods. Identification of 313 Enterobacteriaceae isolates obtained from clinical specimens was performed simultaneously by using MALDI Biotyper and Vitek 2. Results. In the case of 84.98% of the strains (n = 266) the same effectiveness of iden- tification to the species level was obtained using both analyzers. 13.42% strains (n = 42) have been equally identified only to the genus level by both analyzers. Only in the case of 4 strains (1.28%) the results were divergent. Conclusions. MALDI TOF-MS technology provides an excellent tool for identification of bacteria from clinical specimens. It allows obtaining reliable results within time unattain- able for biochemical methods of microorganisms identification.

Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies.

A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspecies-specific biomarker peptides for three Salmonella subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level.

Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory

Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.

A rapid molecular taxonomy technique on the bacteria genus and species by 16S rDNA and 16S-23S rDNA intergenic spacer regions sequences

目的 研究细菌rDNA快速鉴定方法.方法以细菌16S rDNA、16S-23S rDNA基因间隔区(ISR)和常见细菌耐药基因为对象,通过改进标本中细菌DNA提取方法,指纹分析、直接测序与多重聚合酶链反应(PCR)技术,建立快速细菌分子鉴定方法.结果细菌属种不同表现出独特的16S-23S rDNA ISR指纹,在分析软件指导下进行初步细菌鉴定和亲缘关系研究;16S rDNA和16S-23SrDNA ISR序列可确定细菌种与型;序列分析与生化鉴定一致.多重PCR可解决葡萄球菌16S rDNA与mecA基因、产超广谱β内酰胺酶菌16S rDNA与TEM和SHV基因同步检测.结论细菌rDNA分类方法,提高了非培养细菌的鉴定能力。

The electrooptical parameters of suspensions of Escherichia coli XL-1 cells interacting with helper phage M13K07

The electrophysical properties of Escherichia coli XL-1 cells interacting with helper phage M13K07 were studied as a function of the phage-to-cell ratio and the contact time. The electro-optical signal of bacterial cells changed considerably as soon as 10 min after the onset of their incubation with phage particles, presumably due to phage adsorption on the cell surface. The maximum changes in the orientational spectra of cell suspensions were observed when the phage-to-cell ratio was 20. Selectivity studies showed that E. coli XL-1 cells interacting with the helper phage M13K07 in the presence of foreign microflora, such as E. coli K-12 or Azospirillum brasilense Sp7, can be identified by using their electrophysical properties. Changes in the orientational spectra of cell suspensions are interpreted with the stage of phage-bacterium interaction taken into account. The results obtained can probably be used to devise a new rapid method for identification of microorganisms and to study the particular stages of cell infection by bacteriophages.

Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization--RING-FISH.

Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 10(4)-10(5) copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (10(1)-10(3) copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method--a halo-like, ring-shaped concentration of fluorescence in the cell periphery--we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH.