Detection of Salmonella enterica serovar Enteritidis using real time PCR, immunocapture assay, PNA FISH and standard culture methods in different types of food samples

Several methods for the rapid and specific detection of Salmonella in food samples have been described. Here, we compare 4 of those methods in terms of assay time, procedure complexity, detection limit, sensitivity, specificity and accuracy. Milk, eggs and mayonnaise samples were artificially contaminated with Salmonella enterica serovar Enteritidis cell concentrations ranging from 1 × 10- 2 to 1 × 102 CFU per 25 g or ml of food. Samples were then pre-enriched and analyzed by either: i) real-time PCR, using the iQ-Check Salmonella kit; ii) immunocapture, using the RapidChek SELECT Salmonella; iii) a peptide nucleic acid fluorescence in situ hybridization (PNA FISH) method and iv) the traditional bacteriological method ISO 6579:2002. All methods were able to detect Salmonella in the different types of food matrixes and presented a similar detection level of 1 CFU per 25 g or ml of food sample. The immunocapture and the PNA FISH methods proved to be very reliable, as their results were 100% in agreement with the ISO method. However, real-time PCR presented a significant number of false positives, which resulted in a specificity of 55.6% (CI 95%, 31.3 – 77.6) and an accuracy of 82.2%(CI 95%, 63.2 – 91.4) for this method. Sensitivity was 100% since no false negative results were observed. In conclusion, the implementation of these molecular techniques, mainly the immunocapture and PNA-FISH methods, provides a reliable and less time-consuming alternative for the detection of Salmonella spp. in food samples.This work was supported by the Portuguese Institute Fundacao para a Ciencia e Tecnologia (Project "DNA mimics" PIC/IC/82815/2007; Post-Doc Fellowship SFRH/BPD/74480/2010 and PhD Fellowship SFRH/BD/38124/2007)

Development of a rapid method for Proteus spp. detection in urine by peptide nucleic acid fluorescence in situ hybridisation (PNA)

The need to avoid empirical treatment of patients, and the fact that urine samples are among the most numerous of specimen types sent for microbiology studies, have prompted many researchers to explore methods to limit the time and expense of urine culture processing. The aim of this work was to develop a new peptide nucleic acid fluorescence in situ hybridization (PNA FISH) method for the rapid detection of Proteus spp., a genus related to the emergence of complicated urinary tract infectios, especially for immuno-compromised patients. Methods: The PNA probe was designed, optimized, tested on representative strains of the genus and other related strains, and, finally, a PNA FISH method was developed for application in urine samples. Results: The PNA FISH method was optimized, and laboratory testing on representative strains from the Proteus genus and several related bacterial species, showed experimental specificity and sensitivity both of 100% (sensitivity, 95% CI, 81.5 – 100 and specificity, 95% CI, 91.4 - 100). Then, the PNA FISH method was adapted to the detection of Proteus in urine. Artificial urine samples were contaminated with decreasing pathogen concentrations and the PNA FISH method was able to detect, in approximately 2 hours, as low as 1x10^4 CFU/mL, a concentration considered indicative of infection for catheter associated urinary tract infections (CAUTI’s). Conclusions: PNA FISH is a very sensitive, specific and rapid method for Proteus detection in urine and it could be a reliable alternative to the currently used culture-based techniques as it may avoid the need for empirical antibiotic treatment

Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method

Despite the emergence of non-O157 shiga toxin-producing Escherichia coli (STEC) infections, E. coli O157 serotype is still the most commonly identified STEC in world. It causes high morbidity and mortality, and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain as the gold standard.In here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157.Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. Then, the method was optimized for the detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1×10-2 to 1×102 CFU per 25g or ml of food. Samples were then pre-enriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrixes with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% CI 82.83 - 100), a sensitivity of 97.22% (95% CI, 83.79 - 99.85) and an accuracy of 98,33% (CI 95%, 83.41 - 99.91). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.This work was supported by the Portuguese Institute Fundacao para a Ciencia e Tecnologia (FCT), project PIC/IC/82815/2007. C.A. acknowledges FCT for individual postdoctoral fellowship SFRH/BPD/74480/2010. We also acknowledge Biomode S.A. for providing some supplies for this project

Characterization of magnetron sputtered sub-stoichiometric CrAlSiNx and CrAlSiOyNx coatings

The influence of varying nitrogen and oxygen partial pressures on microstructure, mechanical and optical properties of magnetron sputtered CrAlSiNx and CrAlSiOyNx coatings has been studied. The partial pressure of nitrogen reactive gaswas varied from0.037 Pa to 0.15 Pa for CrAlSiNx films, and the N2/O2 (85%:15%) partial pressure was varied from 0.046 Pa to 0.21 Pa for CrAlSiOyNx layers. Transmittance and reflectance of samples were measured and were modeled to obtain the spectral optical constants, n and k. Chemical state, composition, morphology and microstructure of films were analyzed by XPS, RBS, XRD, Raman Spectroscopy and SEM. Films' hardness was evaluated using nanoindentation method. XRD results revealed that the two samples CrAlSiNx with PN =0.15 Pa and CrAlSiOyNx with PNO = 0.21 Pa are polycrystalline with cubic (fcc-B1) structure. On contrary, all other films prepared with lower reactive gases partial pressures are amorphous. The chemical composition changed with the variation of reactive gases partial pressure, although the Cr: Al: Si composition ratio remained approximately constant, 1.25:1.5:1. All samples showed low hardness, mainly due to lower content of reactive gases and higher content of Si. However, the sample CrAlSiNx with PN = 0.15 Pa has the highest value of 11.1 GPa. Optical constants are seen to be very sensitive to reactive gases partial pressure. The refractive index and extinction coefficient were lower for coatings with higher reactive gases partial pressure. These coatings are good candidates for designing selective solar absorber stacks for different applications.The authors acknowledge the support of FCT in the framework of the Strategic Funding UID/FIS/04650/2013 and the financial support of FCT, FOCI and PORL operational programs through the project POCI-01-0145-FEDER-016907 (PTDC/C11/1-ENE/2882/2014), co-financed by European community fund FEDER.info:eu-repo/semantics/publishedVersio

Fetal stem cells obtained from amniotic fluid and wharton's jelly expanded using platelet lysate for tissue engineering applications

Extra-embryonic tissues, such as amniotic fluid (AF) and Wharton´s Jelly (WJ) of umbilical cord, offer many advantages over both embryonic and adult stem cell sources. These tissues are routinely discarded at parturition and the extracorporeal nature of these cell sources facilitates isolation, as well as the comparatively large volume and ease of physical manipulation theoretically increases the number of stem cells that can be isolated. Autologous approaches to use MSCs, namely from bone marrow, have difficulties regarding the limited availability of large amounts of cells from the patient. Fetal stem cells appear to have even more pronounced immunomodulatory properties than adult MSCs (1, 2). This allogeneic escape mechanism may be of therapeutic value, because transplantation of allogeneic human MSCs in stock would be readily available, as opposed to the culture of autologous cells for subsequent transplantation. Cell expansion protocols are based on the use of media supplemented with fetal bovine serum (FBS) as a source of nutrientes and growth factors. The animal serum is not completely safe, once there is a possibility of contamination by animal viroses, prions or others contaminants and it is described that FBS used systematically in MSCs subcultivation induces more humoral immune response (3). Additionally anti-FBS antibodies could be detected in patients after receiving MSCs expanded in FBS (4). Platelet lysate (PL) has enormous possibilities in cell therapy, namely because of the high concentration of growth factors that promotes higher cell expansion, such as tissue regeneration (5). A recent study showed that proliferation of MSCs was much higher on PL gel compared to tissue culture plastic (6). The immunomodulatory properties of MSCs are maintained when expanded in culture medium supplemented with PL (7) Based on these premises we isolated fetal stem cells from AF obtained from amniocentesis and WJ from umbilical cords. These cells were plated and expanded in low density numbers in basal culture medium with FBS or either supplemented with PL. In each passage cells were counted for proliferation kinetics and prepared for flow cytometry analysis. Expanded populations were analysed both population size and complexity and for the MSCs well-known surface markers (CD34, CD45, CD73, CD44, CD106, CD105, CD29, CD90, CD31) and markers related with immune response (HLADR, 80, 83, 86) and embryonic markers SSEA-4 and TRA-1-60

Observations on parasite fauna of centropomus undecimalis and C. parallelus (perciformes) bred in southern Brazil, and its possible influence on the welfare of fishes.

The metazoan parasite fauna of snooks, Centropomus parallelus and Centropomus undecimalis, cultured in southern Brazil and parasite?s influence on the relative condition factor (Kn), are investigated. Snooks were parasitized by two species of gill monogeneans belonging to Rhabdosynochus (Diplectanidae) genus and by one endoparasitic digenean species Acanthocollaritrema umbilicatum (Acanthocollaritrematidae). Centropomus parallelus and C. undecimalis showed similar prevalence rates of Rhabdosynochus spp., but greater mean intensity and abundance rates were found in C. parallelus. On the other hand, there was no significant difference on prevalence, mean intensity and abundance of A. umbilicatumfor both hosts. The mean abundance of Rhabdosynochusspp. decreased as the hosts´ length and weight increased. Since the most parasitized fish species, C. parallelus, had lower weight than expected (Kn<1.0), the fact suggested that gill monogeneans might alter fish welfare. Current analysis reports a new host and a new locality for A. umbilicatum