Laura Ferreira

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.

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.

MALDI-TOF mass spectrometry is a fast and reliable platform for identification and ecological studies of species from family Rhizobiaceae.

Family Rhizobiaceae includes fast growing bacteria currently arranged into three genera, Rhizobium, Ensifer and Shinella, that contain pathogenic, symbiotic and saprophytic species. The identification of these species is not possible on the basis of physiological or biochemical traits and should be based on sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of members from family Rhizobiaceae. In this work we evaluated the suitability of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for this purpose. Firstly, we evaluated the capability of this methodology to differentiate among species of family Rhizobiaceae including those closely related and then we extended the database of MALDI Biotyper 2.0 including the type strains of 56 species from genera Rhizobium, Ensifer and Shinella. Secondly, we evaluated the identification potential of this methodology by using several strains isolated from different sources previously identified on the basis of their rrs, recA and atpD gene sequences. The 100% of these strains were correctly identified showing that MALDI-TOF MS is an excellent tool for identification of fast growing rhizobia applicable to large populations of isolates in ecological and taxonomic studies.

Identification of Brucella by MALDI-TOF Mass Spectrometry. Fast and Reliable Identification from Agar Plates and Blood Cultures

Background MALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures. Methodology/Principal Findings We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct. Conclusions/Significance MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles.

Synergic antitumoral effect of an IGF-IR inhibitor and trastuzumab on HER2-overexpressing breast cancer cells

BACKGROUND Receptor tyrosine kinases play an important role in breast cancer. One of them, the type I insulin-like growth factor, has been linked to resistance to trastuzumab (Herceptin), an agent that targets human epidermal growth factor receptor 2. Here, we show that the insulin-like growth factor-I receptor (IGF-IR) antagonist NVP-AEW541 inhibits proliferation of breast cancer cells and synergizes with trastuzumab. PATIENTS AND METHODS Patient samples and breast cancer cell lines were evaluated for IGF-IR expression or activation by western blotting. 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) uptake assays and Annexin V staining were used for the analyses of cell proliferation/apoptosis. Biochemical and genomic studies were carried out to gain insights into the mechanism of action of NVP-AEW541. RESULTS The IGF-IR was expressed above normal levels in a number of breast cancer samples. Activation of this receptor was inhibited by NVP-AEW541 that also decreased cell proliferation and increased apoptosis. NVP-AEW541 decreased the amount of pAkt and increased the level of p27. Combination studies with several drugs used in the breast cancer clinic showed that NVP-AEW541 synergistically increased the action of trastuzumab. CONCLUSIONS Our results show the anti-breast cancer action of NVP-AEW541 and support the clinical development of anti-IGF-IR agents, especially in combination with trastuzumab.

Nitric oxide protects against pancreatic subcellular damage in acute pancreatitis.

Objectives Oxidative stress involvement in damage to the pancreas in acute pancreatitis (AP) is well documented. However, little is known about oxidative damage occurring in the different subcellular fractions of pancreatic cells. The aim of this study was to ascertain the main targets of oxidative damage inside cells after AP and the role of endogenous nitric oxide (NO) in it. Methods A model of cerulein-induced AP in rats was used and N-nitro-l-arginine methyl ester (l-NAME) was administered as an NO production inhibitor. After pancreatitis induction, indicative parameters of lipid peroxidation and protein oxidation together with some enzymatic and nonenzymatic endogenous free radical scavengers were assessed in serum and pancreatic subcellular fractions. Conclusions In pancreatitic rats, malondialdehyde and protein carbonyl group concentrations were significantly increased (P < 0.05) in serum and some fractions. The increases were higher in l-NAME-treated rats (P < 0.05). Superoxide dismutase and catalase activities were also increased (P < 0.05) but were decreased (P < 0.05) with l-NAME. The &agr;-tocopherol concentration diminished (P < 0.05) in serum and all the studied subcellular fractions and the decrease was stronger in l-NAME-treated rats. Our data suggest that microsomes followed by lysosomal + mitochondrial are the fractions most susceptible to oxidative damage in AP. Endogenous NO plays a protective role against oxidative damage to subcellular fractions.

Urinary levels of regenerating islet-derived protein III β and gelsolin differentiate gentamicin from cisplatin-induced acute kidney injury in rats

A key aspect for the clinical handling of acute kidney injury is an early diagnosis, for which a new generation of urine biomarkers is currently under development including kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. A further diagnostic refinement is needed where one specific cause among several potentially nephrotoxic insults can be identified during the administration of multidrug therapies. In this study we identified increases in regenerating islet-derived protein III beta (reg IIIb) and gelsolin as potential differential urinary markers of gentamicins nephrotoxicity. Indeed, urinary levels of both reg IIIb and gelsolin distinguish between the nephrotoxicity caused by gentamicin from that caused by cisplatin where these markers were not increased by the latter. Reg IIIb was found to be overexpressed in the kidneys of gentamicin-treated rats and excreted into the urine, whereas urinary gelsolin originated from the blood by glomerular filtration. Our results illustrate an etiological diagnosis of acute kidney injury through analysis of urine. Thus, our results raise the possibility of identifying the actual nephrotoxin in critically ill patients who are often treated with several nephrotoxic agents at the same time, thereby providing the potential for tailoring therapy to an individual patient, which is the aim of personalized medicine.

Sub-nephrotoxic doses of gentamicin predispose animals to developing acute kidney injury and to excrete ganglioside M2 activator protein

We studied whether nephrotoxic drug administration sensitizes to acute renal failure (ARF) by administering a sub-nephrotoxic dose of gentamicin. This pre-treatment sensitized animals with no sign of renal injury to develop ARF when exposed to a second potential nephrotoxic drug, also given at sub-nephrotoxic doses that would be otherwise harmless to non-sensitized animals. We identified urinary ganglioside M2 activator protein (GM2AP) as a biomarker of an enhanced sensitivity to suffer ARF following sub-nephrotoxic treatment with gentamicin. Sub-nephrotoxic gentamicin did not alter renal GM2AP gene expression or protein levels, determined by reverse transcriptase-PCR, western blot, and immunostaining, nor was its serum level modified. The origin of increased GM2AP in the urine is thought to be a defective tubular handling of this protein as a consequence of gentamicin action. Hence, markers of acquired sensitivity may improve the prevention of ARF by enhancing our capacity to monitor for this condition, in a preemptive manner.

MALDI-TOF mass spectrometry as a tool for differentiation of Bradyrhizobium species: Application to the identification of Lupinus nodulating strains

Genus Bradyrhizobium includes slow growing bacteria able to nodulate different legumes as well as species isolated from plant tumours. The slow growth presented by the members of this genus and the phylogenetic closeness of most of its species difficults their identification. In the present work we applied for the first time Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to the analysis of Bradyrhizobium species after the extension of MALDI Biotyper 2.0 database with the currently valid species of this genus. With this methodology it was possible to identify strains belonging to phylogenetically closely related species of genus Bradyrhizobium allowing the discrimination among species with rrs gene identities higher than 99%. The application of MALDI-TOF MS to strains isolated from nodules of different Lupinus species in diverse geographical locations allowed their correct identification when comparing with the results of rrs gene and ITS analyses. The nodulation of Lupinus gredensis, an endemic species of the west of Spain, by B. canariense supports the European origin of this species.

Increased urinary excretion of albumin, hemopexin, transferrin and VDBP correlates with chronic sensitization to gentamicin nephrotoxicity in rats.

Drug nephrotoxicity is a serious health and economic problem worldwide. Rats can be acutely sensitized to acute kidney injury (AKI) by subnephrotoxic treatments with potentially nephrotoxic drugs. Acquired sensitization to AKI poses a silent risk impossible to diagnose pre-emptively with the technology available at the clinical level. Herein, we hypothesized whether a chronic, subnephrotoxic insult to the kidneys might result in chronically acquired sensitization to AKI, and whether chronic sensitization might be detected through specific urinary markers. To this end, rats were treated with a subtoxic dosage of the experimental nephrotoxin uranyl nitrate (UN) in the drinking water for 21 weeks, or plain water (as control), and then with low-dose gentamicin for 7 days. Renal function and renal tissue damage were evaluated through the experiment. The mild renal damage caused by gentamicin was markedly magnified in rats having received UN chronically, which was evident both at the functional and histological level. Four proteins, namely albumin, hemopexin, transferrin and vitamin D binding protein were increased in the urine in temporal association with the appearance of chronic predisposition. Although further studies are necessary, our results suggest that these proteins might be potentially used as markers of hidden, chronic predisposition to gentamicin nephrotoxicity, in order to appropriately and pre-emptively stratify and handle individuals according to their specific risk in the long term, and to conveniently optimize their life conditions or additional clinical procedures or treatments that might trigger the disease. This might reduce AKI incidence and severity and the associated costs.