Pretreatment of urine samples with SDS improves direct identification of urinary tract pathogens with matrix-assisted laser desorption ionization-time of flight mass spectrometry.

We pretreated with SDS 71 urine samples with bacterial counts of >10(5) CFU/ml and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) identification scores of <2, in order to minimize failure rates. Identification improved in 46.5% of samples, remained unchanged in 49.3%, and worsened in 4.2%. The improvement was more evident for Gram-negative (54.3%) than for Gram-positive (32%) bacteria.

Rapid method for direct identification of bacteria in urine and blood culture samples by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: intact cell vs. extraction method.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable technology for the identification of microorganisms with proteomics approaches. Here, we compare an intact cell method and a protein extraction method before application on the MALDI plate for the direct identification of microorganisms in both urine and blood culture samples from clinical microbiology laboratories. The results show that the intact cell method provides excellent results for urine and is a good initial method for blood cultures. The extraction method complements the intact cell method, improving microorganism identification from blood culture. Thus, we consider that MALDI-TOF MS performed directly on urine and blood culture samples, with the protocols that we propose, is a suitable technique for microorganism identification, as compared with the routine methods used in the clinical microbiology laboratory.

Microorganisms direct identification from blood culture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows a fast and reliable bacterial identification from culture plates. Direct analysis of clinical samples may increase its usefulness in samples in which a fast identification of microorganisms can guide empirical treatment, such as blood cultures (BC). Three hundred and thirty BC, reported as positive by the automated BC incubation device, were processed by conventional methods for BC processing, and by a fast method based on direct MALDI-TOF MS. Three hundred and eighteen of them yield growth on culture plates, and 12 were false positive. The MALDI-TOF MS-based method reported that no peaks were found, or the absence of a reliable identification profile, in all these false positive BC. No mixed cultures were found. Among these 318 BC, we isolated 61 Gram-negatives (GN), 239 Gram-positives (GP) and 18 fungi. Microorganism identifications in GN were coincident with conventional identification, at the species level, in 83.3% of BC and, at the genus level, in 96.6%. In GP, identifications were coincident with conventional identification in 31.8% of BC at the species level, and in 64.8% at the genus level. Fungaemia was not reliably detected by MALDI-TOF. In 18 BC positive for Candida species (eight C. albicans, nine C. parapsilosis and one C. tropicalis), no microorganisms were identified at the species level, and only one (5.6%) was detected at the genus level. The results of the present study show that this fast, MALDI-TOF MS-based method allows bacterial identification directly from presumptively positive BC in a short time (<30 min), with a high accuracy, especially when GN bacteria are involved.

Neutral glycosphingolipid content of ovine milk.

Milk glycosphingolipids (GSL) have been reported to participate in the newborn's defense against pathogens. Taking this into account, in this study we determined the neutral GSL content of ovine milk, including its fatty acid profile. Its role in bacterial adhesion was also addressed by immunodetection of separate GSL in a high-performance thin-layer chromatography overlay assay. Ovine milk has a neutral GSL pattern similar to human milk and includes lactosylceramide (LacCer; 45.7%), monohexosylceramide (glucosylceramide and galactosylceramide, 31.2%), globotriaosylceramide (Gb3; 19.1%), and globotetraosylceramide (Gb4; 3.5%). Globotriaosylceramide and Gb4 are present in human but not bovine milk. Neutral GSL contained C23:0 and C24:0 as the most abundant fatty acids, a finding consistent with its high content of very long chain fatty acids (longer than C20). Most fatty acids were saturated and had a low content of polyunsaturated fatty acids. Bovine enterotoxigenic Escherichia coli strains bound strongly to LacCer and showed a weak binding to monohexosylceramide. The K99 strain also bound strongly to Gb3, and F41 to Gb4. Lactosylceramide, monohexosylceramide, and Gb3 were also observed to bind to human uropathogenic E. coli strains. The results reported here show the ability of neutral GSL in ovine milk to bind to E. coli strains. These compounds could be used as an alternative and available source to supplement infant or bovine formulas with a view to preventing bacterial infections.