Physicochemical quality of and Escherichia coli resistance profiles in urban surface waters / Qualidade físico-química e perfis de resistência de Escherichia coli em águas superficiais urbanas

Abstract Aquatic ecosystems of urban rivers are contaminated through waste disposal, which poses a public health problem. The objective of this research was to evaluate the quality of water used for recreation and public supply of six rivers in the city of Cascavel - Paraná, including Cascavel, Quati, Bezerra, Antas, Clarito and Amambay. Samples were collected every 4 months in 2017, and their physicochemical and microbiological parameters, as well as resistance profiles of strains of Escherichia coli to antimicrobials distributed by pharmacies of the primary healthcare network, were evaluated. Parameters such as water temperature, turbidity, total nitrogen, total phosphorus, total coliforms and thermotolerant coliforms showed significant differences. The allowed limit for thermotolerant coliforms, which was set by National Environment Council, Resolution 357/2005, was exceeded in all of the six analyzed rivers. It was determined that 48.1% of E. coli strains showed resistance to nine antimicrobial tested. The highest levels of resistance were found for ampicillin (27.7%), tetracycline (27.7%) and amoxicillin (24.0%). The results of this study contribute to the understanding of the hazards associated with the contamination of springs in urban centers with wastewater containing resistant bacteria. Therefore, recovery work is necessary in these areas because of the importance of these water sources for the entire western region of Paraná state.

Resumo Os ecossistemas aquáticos dos rios urbanos são contaminados pela disposição de resíduos, o que representa um problema de saúde pública. Esta pesquisa teve por objetivo avaliar a qualidade das águas utilizadas para recreação e abastecimento público de seis rios da cidade de Cascavel - Paraná, sendo eles: Cascavel, Quati, Bezerra, Antas, Clarito e Amambay. Amostras foram coletadas a cada 4 meses em 2017, e seus parâmetros físico-químicos e microbiológicos, bem como os perfis de resistência das cepas de Escherichia coli aos antimicrobianos distribuídos pelas farmácias da rede básica de saúde, foram avaliados. Parâmetros como temperatura da água, turbidez, nitrogênio total, fósforo total, coliformes totais e coliformes termotolerantes apresentaram diferenças significativas. O limite permitido para coliformes termotolerantes, estabelecido pelo Conselho Nacional do Meio Ambiente, Resolução 357/2005, foi excedido em todos os seis rios analisados. Foi determinado que 48,1% das cepas de E. coli apresentaram resistência aos nove antimicrobianos testados. Os maiores níveis de resistência foram encontrados para ampicilina (27,7%), tetraciclina (27,7%) e amoxicilina (24,0%). Os resultados deste estudo contribuem para a compreensão dos riscos associados à contaminação de nascentes em centros urbanos com efluentes contendo bactérias resistentes. Portanto, o trabalho de recuperação é necessário nessas áreas, devido à importância dessas fontes de água para toda a região oeste do estado do Paraná.

Crotoxin stimulates an M1 activation profile in murine macrophages during Leishmania amazonensis infection.

American tegumentary leishmaniasis is caused by different species of Leishmania. This protozoan employs several mechanisms to subvert the microbicidal activity of macrophages and, given the limited efficacy of current therapies, the development of alternative treatments is essential. Animal venoms are known to exhibit a variety of pharmacological activities, including antiparasitic effects. Crotoxin (CTX) is the main component of Crotalus durissus terrificus venom, and it has several biological effects. Nevertheless, there is no report of CTX activity during macrophage - Leishmania interactions. Thus, the main objective of this study was to evaluate whether CTX has a role in macrophage M1 polarization during Leishmania infection murine macrophages, Leishmania amazonensis promastigotes and L. amazonensis-infected macrophages were challenged with CTX. MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] toxicity assays were performed on murine macrophages, and no damage was observed in these cells. Promastigotes, however, were affected by treatment with CTX (IC50 = 22·86 µg mL-1) as were intracellular amastigotes. Macrophages treated with CTX also demonstrated increased reactive oxygen species production. After they were infected with Leishmania, macrophages exhibited an increase in nitric oxide production that converged into an M1 activation profile, as suggested by their elevated production of the cytokines interleukin-6 and tumour necrosis factor-α and changes in their morphology. CTX was able to reverse the L. amazonensis-mediated inhibition of macrophage immune responses and is capable of polarizing macrophages to the M1 profile, which is associated with a better prognosis for cutaneous leishmaniasis treatment.

Physicochemical quality of and Escherichia coli resistance profiles in urban surface waters

Abstract Aquatic ecosystems of urban rivers are contaminated through waste disposal, which poses a public health problem. The objective of this research was to evaluate the quality of water used for recreation and public supply of six rivers in the city of Cascavel - Paraná, including Cascavel, Quati, Bezerra, Antas, Clarito and Amambay. Samples were collected every 4 months in 2017, and their physicochemical and microbiological parameters, as well as resistance profiles of strains of Escherichia coli to antimicrobials distributed by pharmacies of the primary healthcare network, were evaluated. Parameters such as water temperature, turbidity, total nitrogen, total phosphorus, total coliforms and thermotolerant coliforms showed significant differences. The allowed limit for thermotolerant coliforms, which was set by National Environment Council, Resolution 357/2005, was exceeded in all of the six analyzed rivers. It was determined that 48.1% of E. coli strains showed resistance to nine antimicrobial tested. The highest levels of resistance were found for ampicillin (27.7%), tetracycline (27.7%) and amoxicillin (24.0%). The results of this study contribute to the understanding of the hazards associated with the contamination of springs in urban centers with wastewater containing resistant bacteria. Therefore, recovery work is necessary in these areas because of the importance of these water sources for the entire western region of Paraná state.

Resumo Os ecossistemas aquáticos dos rios urbanos são contaminados pela disposição de resíduos, o que representa um problema de saúde pública. Esta pesquisa teve por objetivo avaliar a qualidade das águas utilizadas para recreação e abastecimento público de seis rios da cidade de Cascavel Paraná, sendo eles: Cascavel, Quati, Bezerra, Antas, Clarito e Amambay. Amostras foram coletadas a cada 4 meses em 2017, e seus parâmetros físico-químicos e microbiológicos, bem como os perfis de resistência das cepas de Escherichia coli aos antimicrobianos distribuídos pelas farmácias da rede básica de saúde, foram avaliados. Parâmetros como temperatura da água, turbidez, nitrogênio total, fósforo total, coliformes totais e coliformes termotolerantes apresentaram diferenças significativas. O limite permitido para coliformes termotolerantes, estabelecido pelo Conselho Nacional do Meio Ambiente, Resolução 357/2005, foi excedido em todos os seis rios analisados. Foi determinado que 48,1% das cepas de E. coli apresentaram resistência aos nove antimicrobianos testados. Os maiores níveis de resistência foram encontrados para ampicilina (27,7%), tetraciclina (27,7%) e amoxicilina (24,0%). Os resultados deste estudo contribuem para a compreensão dos riscos associados à contaminação de nascentes em centros urbanos com efluentes contendo bactérias resistentes. Portanto, o trabalho de recuperação é necessário nessas áreas, devido à importância dessas fontes de água para toda a região oeste do estado do Paraná.

Involvement of formyl peptide receptors in the stimulatory effect of crotoxin on macrophages co-cultivated with tumour cells.

Crotoxin (CTX) is the main neurotoxic component of Crotalus durissus terrificus snake venom. It inhibits tumour growth and modulates the function of macrophages, which are essential cells in the tumour microenvironment. The present study investigated the effect of CTX on the secretory activity of monocultured macrophages and macrophages co-cultivated with LLC-WRC 256 cells. The effect of the macrophage secretory activities on tumour cell proliferation was also evaluated. Macrophages pre-treated with CTX (0.3 µg/mL) for 2 h were co-cultivated with LLC-WRC 256 cells, and the secretory activity of the macrophages was determined after 12, 24 and 48 h. The co-cultivation of CTX-treated macrophages with the tumour cells caused a 20% reduction in tumour cell proliferation. The production of both H2O2 and NO was increased by 41% and 29% after 24 or 48 h of co-cultivation, respectively, compared to the values for the co-cultures of macrophages of control. The level of secreted IL-1ß increased by 3.7- and 3.2-fold after 12 h and 24 h of co-cultivation, respectively. Moreover, an increased level of LXA4 (25%) was observed after 24 h of co-cultivation, and a 2.3- and 2.1-fold increased level of 15-epi-LXA4 was observed after 24 h and 48 h, respectively. Boc-2, a selective antagonist of formyl peptide receptors, blocked both the stimulatory effect of CTX on the macrophage secretory activity and the inhibitory effect of these cells on tumour cell proliferation. Taken together, these results indicate that CTX enhanced the secretory activity of macrophages, which may contribute to the antitumour activity of these cells, and that activation of formyl peptide receptors appears to play a major role in this effect.

Role of distinct immune components in the radiation-induced abrogation of systemic lupus erythematosus development in mice.

The New Zealand Black x New Zealand White F1 [(NZB/NZW) F1] mouse develops an autoimmune condition resembling aspects of human systemic lupus erythematosus (SLE). We investigated the effects of a novel prophylactic thoraco-abdominal gamma irradiation protocol on the onset and evolution of lupus in these animals. Survival of irradiated mice was higher when compared with nonirradiated mice. Kidney lesions were milder and autoantibody levels were lower in irradiated mice. To identify possible mechanisms involved in the radiation-induced improvement of disease, distinct components of humoral and cellular immune responses were evaluated. Because B-1 cells are known to be involved in various autoimmune diseases, we investigated the participation of these cells in SLE progression. Unexpectedly, B-1 cells were not depleted in (NZB/NZW) F1, even after several rounds of irradiation. No alterations were found in viability and physiology of B-1 cells in SLE animals with the exception of constitutive overexpression of the anti-apoptotic molecule Bcl-2, which may account for the observed radioresistance. Thus, a role for B-1 cells in murine SLE cannot be excluded, since the irradiation protocol did not effectively eliminate these cells. Additionally, we demonstrate a marked delay in the ability of splenocytes to repopulate the spleen after irradiation in (NZB/NZW) F1, in contrast to leucocytes in other cellular compartments. The implications of these findings for the fate of SLE in this model are discussed.

The C-terminus of murine S100A9 inhibits spreading and phagocytic activity of adherent peritoneal cells.

OBJECTIVE AND DESIGN: In the present study, the effect of a synthetic peptide (H(92)-G(102)) identical to the C-terminus of murine S100A9 (mS100A9p) was investigated on adherent peritoneal cell function. MATERIALS AND METHODS: For in vitro assays, peritoneal cells were obtained from the abdominal cavity of mice and incubated, with the different concentrations of mS100A9p, for 1 h, and then their spreading and phagocytosis activities were evaluated. For ex-vivo assays, cells obtained from animals treated for 1 h with the peptide were submitted to the mannose-receptor phagocytosis assay. Shorter homologue peptides to the C-terminus of mS100A9p were also evaluated on in vitro phagocytosis assays of Candida albicans particles. RESULTS: mS100A9p reduced both the spreading index and phagocytic activity, in vitro and ex-vivo, independent of the receptor evaluated. The homologue peptide corresponding to the H(92)-E(97) region of mS100A9p, the zinc-binding motif, was responsible for such an effect. CONCLUSION: These results suggest a modulator effect of the C-terminus of S100A9 protein on the function of adherent peritoneal cells.

Inhibitory effect of phospholipase A(2) isolated from Crotalus durissus terrificus venom on macrophage function.

Recent work demonstrated that crotoxin, the main toxin of Crotalus durissus terrificus venom, inhibits macrophage spreading and phagocytic activities. The crotoxin molecule is composed of two subunits, an acidic non-toxic and non-enzymatic polypeptide named crotapotin and a weakly toxic basic phospholipase A(2) (PLA(2)). In the present work, the active subunit responsible for the inhibitory effect of crotoxin on macrophage function was investigated. Peritoneal macrophages harvested from naive rats were used. Crotapotin (2.12, 3.75, or 8.37nM/ml), added for 2h to the medium of peritoneal cell incubation, did not modify the spreading and phagocytic activities of these cells. On the other hand, the PLA(2) (1.43, 2.86, or 6.43nM/ml) subunit caused a significant reduction (30, 33, and 35%, respectively) of the spreading activity. The PLA(2) also inhibited the phagocytosis of opsonised zymosan, opsonised sheep erythrocytes, and Candida albicans, indicating that this inhibitory effect is not dependent on the type of receptor involved in the phagocytosis process. The inhibitory effect of PLA(2) was not due to loss of cell membrane integrity, since macrophage viability was higher than 95%. These findings indicate that the inhibitory effect of crotoxin on macrophage spreading and phagocytic activities is caused by the phospholipase A(2) subunit.

Transfer of arachidonic acid from lymphocytes to macrophages.

The incorporation and oxidation of arachidonic acid (AA) by rat lymphocytes (LY), the transfer of AA from LY to rat macrophages (Mphi) in co-culture, and the subsequent functional impact on Mphi phagocytosis were investigated. The rate of incorporation of [1-14C]AA by untreated-LY and TG (thioglycolate treated)-LY (TG-LY) was 158 +/- 8 nmol/10(10) LY per h for both untreated-LY and TG-LY. The oxidation of AA was 3.4-fold higher in TG-LY as compared with untreated cells. LY from TG-injected rats had a 2.5-fold increase in the oxidation of palmitic (PA), oleic (OA), and linoleic (LA) acids. After 6 h of incubation, [14C] from AA was distributed mainly into phospholipids. The rate of incorporation into total lipids was 1071 nmol/10(10) cells in untreated-LY and 636 nmol/10(10) cells in TG-LY. [14C]AA was transferred from LY to co-cultured Mphi in substantial amounts (8.7 nmol for untreated and 15 nmol per 10(10) for TG cells). Exogenously added AA, PA, OA, and LA caused a significant reduction of phagocytosis by resident cells. Mphi co-cultured with AA-preloaded LY showed a significant reduction of the phagocytic capacity (about 40% at 35 microM). LY preloaded with PA, LA, and OA also induced a reduction in phagocytic capacity of co-cultured Mphi. TG treatment abolished the AA-induced inhibition of phagocytosis in Mphi co-cultured with TG-LY. Therefore, the transfer of AA between leukocytes is a modulated process and may play an important role in controlling inflammatory and immune response.