Identification of new flagellin-encoding fliC genes in Escherichia coli isolated from domestic animals using RFLP-PCR and sequencing methods

Identification of Escherichia coli requires knowledge regarding the prevalent serotypes and virulence factors profiles allows the classification in pathogenic/non-pathogenic. However, some of these bacteria do not express flagellar antigen invitro. In this case the PCR-restriction fragment length polymorphism (RFLP-PCR) and sequencing of the fliC may be suitable for the identification of antigens by replacing the traditional serology. We studied 17 samples of E. coli isolated from animals and presenting antigen H nontypeable (HNT). The H antigens were characterized by PCR-RFLP and sequencing of fliC gene. Three new flagellin genes were identified, for which specific antisera were obtained. The PCR-RFLP was shown to be faster than the serotyping H antigen in E. coli, provided information on some characteristics of these antigens and indicated the presence of new genes fliC.

In situ differentiation of labile/inert metal species in Brazilian tropical rivers by means of a time-controlled batch-procedure based on TEPHA resin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Simultaneous determination of furanocoumarins in infusions and decoctions from Carapia (Dorstenia species) by high-performance liquid chromatography

The use of furanocoumarins, which are photosensitizing compounds, combined with exposure to UV-A radiation is a common treatment for vitiligo, psoriasis, and a number of other skin diseases. Although furanocoumarins plus UV-A treatment is highly effective, several studies have shown that exposure to high doses increases the risk to development of cutaneus carcinoma. Several Dorstenia species are used in folk medicine, mainly against skin diseases, because of the presence of biologically active compounds. We present here analysis of the chemical composition of furanocoumarins from infusion and decoction of Carapia (Dorstenia species), which is used in Brazil against several diseases. We have employed high-performance liquid chromatography (HPLC) procedures for the quantitative determination of psoralen, bergapten, and isopimpinellin. The contents of furanocoumarins revealed an insignificant difference between infusion and decoction. Dorstenia tubicina and D. asaroides contained psoralen and bergapten only in the rhizomes, whereas D. vitifolia shows solely isopimpinellin in both rhizomes and aerial parts.

Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland

Antimicrobial susceptibility testing was performed on a total of 581 clinical Escherichia coli isolates from diarrhea and edema disease in pigs, from acute mastitis in dairy cattle, from urinary tract infections in dogs and cats, and from septicemia in laying hens collected in Switzerland between 1999 and 2001. Among the 16 antimicrobial agents tested, resistance was most frequent for sulfonamides, tetracycline, and streptomycin. Isolates from swine presented significantly more resistance than those from the other animal species. The distribution of the resistance determinants for sulfonamides, tetracycline, and streptomycin was assessed by hybridization and PCR in resistant isolates. Significant differences in the distribution of resistance determinants for tetracycline (tetA, tetB) and sulfonamides (sulII) were observed between the isolates from swine and those from the other species. Resistance to sulfonamides could not be explained by known resistance mechanisms in more than a quarter of the sulfonamide-resistant and sulfonamide-intermediate isolates from swine, dogs and cats. This finding suggests that one or several new resistance mechanisms for sulfonamides may be widespread among E. coli isolates from these animal species. The integrase gene (intI) from class I integrons was detected in a large proportion of resistant isolates in association with the sulI and aadA genes, thus demonstrating the importance of integrons in the epidemiology of resistance in clinical E. coli isolates from animals.

In situ differentiation of labile/inert metal species in Brazilian tropical rivers by means of a time-controlled batch-procedure based on TEPHA resin

The present study deals with a new analytical procedure based on a cellulose diffusion membrane and immobilised tetraethylene-pentamine-hexaacetate chelator (DM-TEPHA) for an in situ differentiation of labile and inert metal species in aquatic systems. The DM-TEPHA system was prepared by placing TEPHA chelator in pre-purified cellulose bags and in situ applied immersing the system in two Brazilian rivers to study the relative lability of metal species (Cu, Pb, Fe, Mn and Ni) as a function of the time and the quantity of exchanger, respectively. The procedure is simple and enables a new perspective for understanding the complexation, transport, stability and lability of metal species in aquatic systems rich in organic matter.

Direct detection of infectious bursal disease virus from clinical samples by in situ reverse transcriptase-linked polymerase chain reaction

The presence of the very virulent (vv) Brazilian strain of infectious bursal disease virus (IBDV) was determined in the bursa of Fabricius, thymus and liver of 2-week-old broilers from a flock with a higher than expected mortality. For this purpose, a direct in situ reverse transcriptase (RT)-linked polymerase chain reaction (PCR) method was developed using specific primers for vvIBDV. Unlabelled forward and reverse biotinylated oligonucleotides were used for RT-PCR in a one-step method and the respective products were revealed by a direct enzymatic reaction. The results were compared with those obtained by standard RT-PCR using general primers for IBDV and virus isolation. The virus isolation, RT-PCR and in situ RT-PCR revealed positive results on the bursa of Fabricius in 86%, 80% and 100%, respectively. The in situ RT-PCR detected vvIBDV in all tested thymus and liver samples, whereas the standard RT-PCR detected virus in 80% and 90% of the samples, respectively. After three consecutive passages on chicken embryonated eggs, IBDV was isolated from 64% of the thymus samples and 30% of the liver samples. In the present study, no classical or antigenic variants of IBDV were detected. The developed in situ RT-PCR assay was able to detect the very virulent strain of IBDV with a higher sensitivity than the conventional RT-PCR and virus isolation.

A comparison of in situ vs. ex situ filtration methods on the assessment of dissolved and particulate metals at hydrothermal vents

The objective of this study was to assess the impact of the filtration method (in situ vs. ex situ) on the dissolved/particulate partitioning of 12 elements in hydrothermal samples collected from the Lucky Strike vent field (Mid-Atlantic Ridge; MAR). To do so, dissolved ( <0.45 mu m) and particulate Mg, Li, Mn, U, V, As, Ba, Fe, Zn, Cd, Pb and Cu were measured using different techniques (HR-ICP-MS, ICP-AES and CCSA). Using in situ filtration as a baseline, we showed that ex situ filtration (on-board and on shore after freezing) resulted in an underestimation of the dissolved pool, which was counterbalanced by an overestimation of the particulate pool for almost all the elements studied. We also showed that on-board filtration was acceptable for the assessment of dissolved and particulate Mn, Mg, Li and U for which the measurement bias for the dissolved fraction did not exceed 3%. However, in situ filtration appeared necessary for the accurate assessment of the dissolved and particulate concentrations of V, As, Fe, Zn, Ba, Cd, Pb and Cu. In the case of Fe, on-board filtration underestimated the dissolved pool by up to 96%. Laboratory filtration (after freezing) resulted in a large bias in the dissolved and particulate concentrations, unambiguously discounting this filtration method for deep-sea chemical speciation studies. We discuss our results in light of the precipitation processes that can potentially affect the accuracy of ex situ filtration methods.