人工湿地中抗生素抗性大肠杆菌和抗性基因的去除与分布

抗生素的滥用导致抗生素抗性菌和抗性基因随生活污水和养殖废水的排放在环境中肆意散播,其去除及环境行为越来越受到关注。采用K-B纸片法测定了9套不同工艺构型模拟人工湿地中大肠杆菌对7种抗生素的抗性率,并应用多重PCR检测磺胺类sul1、2、3与四环素tetA、B、C、D抗性基因,探究人工湿地对抗性菌的去除效率及抗性菌、抗性基因的分布规律。结果显示,人工湿地能有效去除污水中70%左右的抗性大肠杆菌,有效降低了细菌抗性的传播风险;共计分离出535株大肠肝菌中有378株对一种以上抗生素有抗性性,以四环素、磺胺类和氨苄西林抗性率最高,达到25%以上,其他4种抗性率较低,不足20%;2种抗性基因的检出率都在70%以上;对不同采样点大肠杆菌的抗性性及抗性基因的比较发现,各部分大肠杆菌的抗性水平、多重抗性指数(MRI)以及抗性基因(sul、tet)检出率和组合数表现出:基质≥出水>进水,推测抗性菌被湿地基质截留,在基质生物膜上发生抗性基因的重组,并释放抗性菌,提高了出水中抗性水平和抗性基因检出率。     

Xenobiotics: Substrates and inhibitors of the plant cytochrome P450

The ability of a plant cytochrome P450 to bind and metabolise plant endogenous molecules and xenobiotics was investigated. The work was performed on the yeast-expressed CYP73A1, a cinnamate 4-hydroxylase isolated fromHelianthus tuberosus. CYP73 controls the general phenylpropanoid pathway and is likely to be one of the most abundant sources of P450 in the biosphere. The enzyme shows a high selectivity toward plant secondary metabolites. Nevertheless, it oxygenates several small and planar xenobiotics with low efficiency, including an herbicide (chlorotoluron). One xenobiotic molecule, 2naphthoic acid, is hydroxylated with an efficiency comparable to that of the physiological substrate. This reaction was used to devise a fluorimetric test for the rapid measurement of enzyme activity. A series of herbicidal molecules (hydroxybenzonitriles) are shown to bind the active site without being metabolised. These molecules behave as strong competitive inhibitors of CYP73 with a K_i in the same micromolar range as the K_m for the physiological substrate. It is proposed that their inhibition of the phenylpropanoid pathway reinforces their other phytotoxic effects at the level of the chloroplasts. All our results indicate a strong reciprocal interaction between plant P450s and xenobiotics.     

Toxicity of cobalt-complexed cyanide to <Emphasis Type="Italic">Oncorhynchus mykiss,Daphnia magna</Emphasis>, and <Emphasis Type="Italic">Ceriodaphnia dubia</Emphasis>

Background Cobalt cyanide complexes often result when ore is treated with cyanide solutions to extract gold and other metals. These have recently been discovered in low but significant concentrations in effluents from gold leach operations. This study was conducted to determine the potential toxicity of cobalt-cyanide complexes to freshwater organisms and the extent to which ultraviolet radiation (UV) potentiates this toxicity. Tests were also conducted to determine if humic acids or if adaptation to UV influenced sensitivity to the cyanide complexes. Methods Rainbow trout (Oncorhynchus mykiss), Daphnia magna, and Ceriodaphnia dubia were exposed to potassium hexacyanocobaltate in the presence and absence of UV radiation, in the presence and absence of humic acids. Cyano-cobalt exposures were also conducted with C. dubia from cultures adapted to elevated UV. Results With an LC50 concentration of 0.38 mg/L, cyanocobalt was over a 1000 times more toxic to rainbow trout in the presence of UV at a low, environmentally relevant irradiance level (4 μW/cm² as UVB) than exposure to this compound in the absence of UV with an LC50 of 112.9 mg/L. Toxicity was immediately apparent, with mortality occurring within an hour of the onset of exposure at the highest concentration. Fish were unaffected by exposure to UV alone. Weak-acid dissociable cyanide concentrations were observed in irradiated aqueous solutions of cyanocobaltate within hours of UV exposure and persisted in the presence of UV for at least 96 hours, whereas negligible concentrations were observed in the absence of UV. The presence of humic acids significantly diminished cyanocobalt toxicity to D. magna and reduced mortality from UV exposure. Humic acids did not significantly influence survival among C. dubia. C. dubia from UV-adapted populations were less sensitive to metallocyanide compounds than organisms from unadapted populations. Conclusions The results indicate that metallocyanide complexes may pose a hazard to aquatic life through photochemically induced processes. Factors that decrease UV exposure such as dissolved organic carbon or increased pigmentation would diminish toxicity.     

Detecting points as developmental delay based on the life-history development and urosome deformity of the harpacticoid copepod, Tigriopus japonicus sensu lato, following exposure to benzo(a)pyrene

To identify ecotoxicological responses to an endocrine disrupter, benzo(a)pyrene, we examined the life-history of the harpacticoid copepod, Tigriopus japonicus sensu lato. Based on the life-history of copepods, survival rate of nauplii (NSR) and copepodites (CSR), copepodite emergence day (CED) and adult male emergence day (AMED), sex ratio (MER), brooding success rate (BSR), and first brooding day of adult females (FBD) were measured. Significant differences were observed in the survival and development of nauplii (NSR and CED) and sex ratio (MER) of exposed and non-exposed copepods. Moreover, high concentration of BaP can be lethal to copepodite and exhibited a delay of growth. In this study, the CED and AMED among ecotoxicological response based on life-history developments were delayed and the body characteristics decreased in response to exposure to benzo(a)pyrene. The dwarfism and urosome deformity of the T. japonicus s.l. was exhibited in response to chemical exposure. Specifically, the body characteristics and biomass of dwarf copepods that had been exposed to benzo(a)pyrene were 30% and 50% lower than the control group, respectively. The incidence of abnormal urosomes was divided into two types. The first deformity type was signs of shrinkage in the middle of the urosome or the entire urosome was narrower than those of the control organisms. In the second type, the anal somite and the distal side of the urosome had abnormally swelled. Taken together, the nauplii and copepodid development of T. japonicus s.l. can be used as a useful biomaker for detecting developmental delay based on their entire life-history. In addition, the urosome deformity was used a good potential monitoring tool invading various chemicals and environmental contamination into water system.     

Research Articles

The metabolism of phenanthrene was studied both in cell suspension cultures of wheat (Triticum aestivum) and soybean (Glycine max), and in intact plants of the water mossFontinalis antipyretica. Metabolism in cell suspension cultures strongly differed between the monocotyle and the dicotyle plant. Only small amounts oftrans-phenanthrene-9,10-dihydrodiole and phenanthrene-9,10-dione were detectable in the wheat culture. Soybean cultures, in contrast demonstrated a strong turnover resulting in a 75% reduction of the initial phenanthrene concentration. Metabolites were phenanthrene-9,10-dione, not further characterized polar metabolites and bound residues. Intact plants ofFontinalis antipyretica metabolized only small amounts of phenanthrene. Data obtained from cell cultures did not provide information for the metabolic potential in intact plants. Therefore standardized tests with model systems like suspension cultures lead to inadequate assessment of the ecological risk of certain xenobiotics.     

Racemic,R-, and S-tebuconazole altered chitinase and chitobiase activity of Daphnia magna

Tebuconazole is a chiral trizole fungicide and widely used in many crops for controlling disease. Tebuconazole is potential toxic to some aquatic organisms but relative information of its isomers is scarce. To detect the endocrine disrupting effects and difference of rac-, R-, and S-tebuconazole, the chitinase activity in Daphnia magna and chitobiase activity in each test medium were used as biomonitors after a 14-day exposure. Results showed that chitinase activity was significantly reduced by rac-, R-, and S-tebuconazole. The chitobiase activity in the test medium was reduced by rac- and R-tebuconazole before day 10, and only one peak was observed at day 10 or day 12 compared with two obvious peaks in the control group (days 6 and 12). S-tebuconazole delayed and reduced the reproduction of D. magna, but did not delay the first chitobiase activity peak, whereas the second peak could not be characterized as the exposure concentration and time increased. Compared with chitinase activity, chitobiase activity can still be used as a rudimentary model for identifying molt-interfering xenobiotics, and further studies should focus on the analysis of correlations between these parameters.     

Bioindicators for toxicity assesment of effluents from a wastewater treatment plant

The discharge of effluents and toxic compounds into aquatic systems represents a growing environmental problem involving an impact on water ecology and potential effect on human health. Most municipal wastewaters are complex mixtures; their complexity led as to carry out a hazard assessment using chemical analyses and biological tests.This study investigates biochemical alteratiobns in two sentinel organisms, the Anodonta cygnea mussel and the Xenopus laevis frog exposed for different lengths of time to various concentrartion of wastewater of the S. Antonino Ticino treatment plant. The results point out the long-life of toxic compounds.     

全氟辛烷磺酸盐(PFOS)在藻渣/小球藻上的吸附行为及机理

为了开发新型廉价生物吸附剂,以高效吸附去除水体中全氟辛烷磺酸盐(PFOS),对小球藻提取生物柴油后的藻渣吸附酸性水体中的PFOS进行了吸附行为及机理的研究。小球藻提取生物质柴油后,比表面积、孔容、孔径几乎没有变化;等电点由3.3降低至2.7;蛋白质含量由51.45%提高到57.35%。在酸性条件下(pH≤3),小球藻和藻渣对PFOS的吸附率均达到99%以上;随着pH值增加至7,二者的吸附去除率迅速降低,但仍保持在22%~26%。小球藻和藻渣对PFOS的最大吸附容量分别为353.69 mg/g和444.83 mg/g。Freundlich模型能较好地拟合二者对PFOS的吸附数据,表明为多层吸附,即小球藻以静电吸引的形式吸附PFOS阴离子,并疏水分配至所含蛋白质中;而藻渣中含量较高的蛋白质对PFOS的疏水性分配作用是导致藻渣吸附量增高的主要原因。     

Localization of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) in poplar and switchgrass plants using phosphor imager autoradiography

Phosphor imager autoradiography is a technique for rapid, sensitive analysis of the localization of xenobiotics in plant tissues. Use of this technique is relatively new to research in the field of plant science, and the potential for enhancing visualization and understanding of plant uptake and transport of xenobiotics remains largely untapped. Phosphor imager autoradiography is used to investigate the uptake and translocation of the explosives 1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene within Populus deltoides × nigra DN34 (poplar) and Panicum vigratum Alamo (switchgrass). In both plant types, TNT and/or TNT-metabolites remain predominantly in root tissues while RDX and/or RDX-metabolites are readily translocated to leaf tissues. Phosphor imager autoradiography is further investigated for use in semi-quantitative analysis of uptake of TNT by switchgrass.     

Proteomic analysis of chromate-induced modifications in Pseudokirchneriella subcapitata

In this work, we have analyzed the changes in the protein expression profile elicited by chromium (Cr) exposure in the freshwater green alga Pseudokirchneriella subcapitata, a well known bio-indicator of water pollution. We tested two experimental conditions, namely 0.2 and 1 ppm of potassium dichromate; this concentration range includes the environmentally-relevant concentrations. Results show that neither concentration of potassium dichromate tested inhibited algal growth. However, the proteomic approach allowed the identification of relevant modifications in protein expression. In fact, among 800 protein spots detected by two-dimensional electrophoresis, 16 Cr-regulated proteins, including predicted and novel ones, were identified using tandem mass spectromic protein analysis.The results demonstrate a Cr-specific action in altering several photosynthetic proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), RuBisCO activase, Light Harvesting Chla/b protein complex, and stress related Chla/b binding protein1. Although Cr toxicity with respect to photosynthesis has been already documented, here we have identified, for the first time, the target proteins of this toxicity. Cr also induced a modulation of some proteins involved in the metabolism of the amino acids glutamine, arginine and methionine. These data are supported by changes in cellular polyamine (PA) accumulation. Present findings provide new insight into the molecular mechanisms underlying Cr toxicity in P. subcapitata.     

改进钒基SCR脱硝催化剂的抗碱金属中毒性能

以锐钛矿型二氧化钛和钛钨粉(5%WO3-TiO2)为载体,制备了系列钒和钨负载量不同的钒钛催化剂,考察碱金属和碱土金属(钾、钠和钙)对催化剂在氨选择性催化还原(NH3-SCR)氮氧化物反应中催化活性的影响。钾、钠和钙对钒钛催化剂的中毒影响大小顺序为钾钠钙。提高钒钛催化剂中钒的含量可显著提高催化剂的SCR活性和抗碱金属中毒性能,但高钒负载量(4.5%V2O5)造成催化剂氮气选择性明显下降,氧化亚氮生成显著增加。钨的添加有利于提高钒钛催化剂的低温活性和抗碱金属中毒性能,对氮气选择性无明显影响。     

Physical-chemical and ecotoxicological evaluation of water based drilling fluids used in Italian off-shore

In order to evaluate the effects on the marine ecosystem caused by an eventual discharge into sea of water based drilling fluids, as current legislation allows, chemical and ecotoxicological analyses were performed on the most common drilling muds and products used in Italian off-shore activities. The chemical analysis on drilling fluids involved the leaching test and the measurement of total content of heavy metals, whereas biodegradation tests were performed on the products used in mud's formulations. As for ecotoxicological evaluation, two marine organisms, the crustacean Artemia salina and the diatom Phaeodactylum tricornutum, were selected to determine the LC₅₀ and the EC₅₀ respectively.     

Chronic copper exposure and fatty acid composition of the amphipod Dikerogammarus villosus: results from a field study

Field study allows assessment of long-term effects on fatty acid (FA) composition of organisms under chronic exposure to metals. One expected effect of copper is peroxidation of lipids and essentially polyunsaturated fatty acids (PUFA). FA analysis was established for the amphipod Dikerogammarus villosus subjected to different degrees of copper exposure (4–40 μg Cu L⁻¹). A previous study in our team showed that this species regulates its body Cu concentration (106–135 mg Cu kg⁻¹ dry weight). Despite the high capacity of bioaccumulation, the absence of a correlation between copper concentration in D. villosus and water prevents its use as bioindicator of copper pollution. Both sexes from the most polluted site showed the lowest total FA content, but the highest PUFA percent, mainly of the long-chained variety (C20–C22). Mechanisms leading to the prevention of lipid peroxidation in this species were discussed (metallothioneins and intracellular granules) and proposed with support from literature data.     

Detection of Class I and II integrons for the assessment of antibiotic and multidrug resistance among Escherichia coli isolates from agricultural irrigation waters in Bulacan,Philippines

Contaminated irrigation water may greatly affect not only the quality of produce but also the people exposed to it. In this study, agricultural irrigation waters in Bulacan, Philippines were assessed and found to be contaminated with Escherichia coli (E. coli) ranging from 0.58 to 4.51 log₁₀ CFU/mL. A total of 79 isolates of E. coli were confirmed through polymerase chain reaction (PCR) amplifying the uidA gene and were tested for phenotypic resistance using 10 antimicrobials through the Kirby–Bauer disc diffusion method. Forty-six isolates (58.22%) were noted to be multidrug resistant (MDR) with high resistance rate to cephalothin, tetracycline, streptomycin, ampicillin, trimethoprim, nalidixic acid, and chloramphenicol. Moreover, this study also examined the prevalence of Class I and II integrons accounting to 67.39% and 17.39%, respectively, of the MDR E. coli strains using multiplex PCR. The results imply that the agricultural water used in Bulacan is contaminated with the fecal material of man or other animals present in the area, and the presence of MDR bacteria, which pose a potential threat to individuals in these areas, is alarming. In addition, detection of integrons could be a good marker for the identification of MDR isolates. Lastly, this study could develop strategies for the proper management of farming sites leading to the detection of food-borne pathogens and prevention of infectious diseases.     

Response of antioxidant enzymes in Nicotiana tabacum clones during phytoextraction of heavy metals

Background, aim, and scope  Tobacco, Nicotiana tabacum, is a widely used model plant for growth on heavy-metal-contaminated sites. Its high biomass and deep rooting system make it interesting for phytoextraction. In the present study, we investigated the antioxidative activities and glutathione-dependent enzymes of different tobacco clones optimized for better Cd and Zn accumulation in order to characterize their performance in the field. Main features  The improved heavy metal resistance also makes the investigated tobacco clones interesting for understanding the plant defense enzyme system in general. Freshly harvested plant material (N. tabacum leaves) was used to investigate the antioxidative cascade in plants grown on heavy metal contaminated sites with and without amendments of different ammonium nitrate and ammonium sulfate fertilizers. Materials and methods  Plants were grown on heavily polluted soils in north-east Switzerland. Leaves were harvested at the field site and directly deep frozen in liquid N₂. Studies were concentrated on the antioxidative enzymes of the Halliwell–Asada cycle, and spectrophotometric measurements of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), glutathione S-transferase (GST, EC 2.5.1.18) were performed. Results and discussion  We tried to explain the relationship between fertilizer amendments and the activity of the enzymatic defense systems. When tobacco (N. tabacum) plants originating from different mutants were grown under field conditions with varying fertilizer application, the uptake of cadmium and zinc from soil increased with increasing biomass. Depending on Cd and Zn uptake, several antioxidant enzymes showed significantly different activities. Whereas SOD and CAT were usually elevated, several other enzymes, and isoforms of GST were strongly inhibited. Conclusions  Heavy metal uptake represents severe stress to plants, and specific antioxidative enzymes are induced at the cost of more general reactions of the Halliwell–Asada cycle. In well-supplied plants, the glutathione level remains more or less unchanged. The lack of certain glutathione S-transferases upon exposure to heavy metals might be problematic in cases when organic pollutants coincide with heavy metal pollution. When planning phytoremediation of sites, mixed pollution scenarios have to be foreseen and plants should be selected according to both, their stress resistance and hyperaccumulative capacity.     

活性污泥体系中聚糖菌的富集与鉴定

活性污泥体系中，聚糖菌(GAOs)在厌氧环境下与聚磷菌(PAOs)形成对底物的竞争关系，对聚糖菌的研究对于优化生物除磷工艺有重要意义。以葡萄糖为惟一碳源，在磷限制条件下，利用特殊运行方式对活性污泥进行驯化培养出了稳定的聚糖菌颗粒污泥，厌氧阶段磷释放量与有机物吸收量浓度(mg/L)比从7.4%下降为0.25%。从培养好的活性污泥反应器中分离获得2株聚糖菌，经菌落PCR和16S rRNA序列分析确定了所得聚糖菌菌株G1和菌株G2分别是枯草芽孢杆菌(Bacillus subtilis)和解鸟氨酸克雷伯氏菌(Klebsiella ornithinolytica)。     

Distribution and bioaccumulation of organochlorine pesticides in surface sediments and benthic organisms from Taihu Lake, China

The spatial distribution and bioaccumulation of organochlorine pesticides (OCPs) in surface sediments and benthic organisms from Taihu Lake were studied. OCPs were detected in all sediment samples with total concentrations ranging from 4.22 to 461 ng g⁻¹ dry weight (dw). The ratios of certain metabolites to their parent compounds indicated there are still new inputs of parent DDT (dichlorodiphenyltrichloroethane) to Taihu Lake, while the highest residues of HCHs (hexachlorocyclohexanes) mainly came from earlier usage and fresh γ-HCH (lindane). No positive correlation was found between the distribution of OCPs and organic matter contents in sediments. Concentrations of OCPs and lipids in typical large benthic organisms, Bellamya aeruginosa (B. aeruginosa) and Corbicula fluminea (C. fluminea), increased with body weight. HCHs, DDTs, chlordanes and heptachlors were the dominant compounds detected in organisms and C. fluminea accumulated much more OCP than B. aeruginosa. Higher values of biota-sediment accumulation factor (BSAF) were detected in C. fluminea, which was both affected by biological characteristics of the organisms and physicochemical properties of the compounds.     

Improved xenobiotic-degrading activity of Rhodococcus opacus strain 1cp after dormancy

The goals of the present work were as follows: to obtain the dormant forms of R. opacus 1cp; to study the phenotypic variability during their germination; to compare phenotypic variants during the growth on selective and elective media; and to reveal changes in the ability of the strain to destruct xenobiotics that had not been degradable before dormancy. It was shown that Rhodococcus opacus 1cp (the strain degrading chlorinated phenols) became able to utilize a broader spectrum of xenobiotics after storage in the dormant state. Germination of the dormant forms of R. opacus 1cp on an agarized medium was followed by emergence and development of phenotypic variants that could grow on 4-chlorophenol and 2,4,6-trichlorophenol without adaptation. The cells of R. opacus 1cp phenotypic variants also utilized all of the tested chlorinated phenols: 2,3-, 2,5-, and 2,6-dichloro-, 2,3,4- and 2,4,5-trichloro-, pentachlorophenol, and 1,2,4,5-tetrachlorobenzene in concentrations up to 60 mg/L, though at the lower rates than 4-CP and 2,4,6-TCP. The improved degradation of chlorinated phenols by R. opacus strain 1cp exposed to the growth arrest conditions demonstrates the significance of dormancy for further manifestation of the adaptive potential of populations. A new principle of selection of variants with improved biodegradative properties was proposed. It embraces introduction of the dormancy stage into the cell life cycle with subsequent direct inoculation of morphologically different colonies into the media with different toxicants, including those previously not degraded by the strain.     

Diversity of antibiotic resistance genes and encoding ribosomal protection proteins gene in livestock waste polluted environment

The rapid development and increase of antibiotic resistance are global phenomena resulting from the extensive use of antibiotics in human clinics and animal feeding operations. Antibiotics can promote the occurrence of antibiotic resistance genes (ARGs), which can be transferred horizontally to humans and animals through water and the food chain. In this study, the presence and abundance of ARGs in livestock waste was monitored by quantitative PCR. A diverse set of bacteria and tetracycline resistance genes encoding ribosomal protection proteins (RPPs) from three livestock farms and a river were analyzed through denaturing gradient gel electrophoresis (DGGE). The abundance of sul(I) was 10³ to 10⁵ orders of magnitude higher than that of sul(II). Among 11 tet-ARGs, the most abundant was tet(O). The results regarding bacterial diversity indicated that the presence of antibiotics might have an evident impact on bacterial diversity at every site, particularly at the investigated swine producer. The effect of livestock waste on the bacterial diversity of soil was stronger than that of water. Furthermore, a sequencing analysis showed that tet(M) exhibited two genotypes, while the other RPPs-encoding genes exhibited at least three genotypes. This study showed that various ARGs and RPPs-encoding genes are particularly widespread among livestock.