Detection of EPEC and STEC strains isolated from children with diarrhea in Argentina.

Rectal swabs (122) from pediatric patients were analyzed by polymerase chain reaction (PCR) for the detection of EPEC and STEC. STEC isolates were tested for the presence of stx1, stx2, eae, saa and ehxA. All eae-positive samples were tested for the presence of bfpA, and antigen O was determined using the agglutination test. Int1 and Int2 were detected to identify the presence of integrons class 1 and 2, respectively. Escherichia coli was detected in 68% of the samples, of which 18.8% were STEC (2.45%) and EPEC (16.3%). Serogroups STEC O145 and EPEC O130, O113 and O157 were observed, while three strains were non-typable. None of the EPEC strains carrying tbfpA and class 1 and 2 integrons was detected in any of the samples. The results obtained are important considering the virulence profiles found in the isolated EPEC and STEC strains and the serogroups associated with disease in humans.

Superoxide, nitric oxide and peroxynitrite production by macrophages under different physiological oxygen tensions.

Macrophages count on two O2-consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O2•-) and nitric oxide (•NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O2•- and •NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO2) normally ranges within 2-14 %, with a typical average pO2 value for most tissues ca. 5 %. Given that O2 is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O2•- and •NO production. Also, O2 is a modulator of the macrophage adaptative response and may influence iNOS expression in a hypoxia inducible factor 1-α (HIF1α-)-dependent manner. However, most of the reported experiments in cellula, analyzing the formation and effects of O2•- and •NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO2; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O2) which are far from the physiological pO2. Here, the role of O2 as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO2 and O2•- and •NO formation rates were measured, obtaining a KM of 26 and 42 µM O2 for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO2, reaching a maximum at ≥ 10 % O2, but even at levels as low as 2 % O2, a substantial formation rate of this oxidant was detected. Indeed, the cytotoxic capacity of immunostimulated macrophages against the intracellular parasite T. cruzi was significant, even at low pO2 values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions.