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.     

Abundance and removal of antibiotic resistance genes (ARGs) in the rearing environments of intensive shrimp aquaculture in South China

Although research regarding antibiotic resistance genes (ARGs) in aquaculture environments has gained increasing scientific interest, further studies are required to understand the abundances and removal mechanisms of ARGs during the entire rearing period of shrimp aquaculture. Thus, in this study, abundances, distributions and removal rates of ARGs in different environmental compartments of intensive shrimp farms in South China were investigated during the entire rearing period. The results indicated that sul1 and cmlA were the predominant ARGs in the water and sediment samples. Additionally, the total abundance of ARGs was higher in shrimp pond water than in the source water and farm effluent. Moreover, sediment samples indicated significantly higher ARG abundances than water samples from the shrimp ponds (P?<?0.05). Environmental factors were found to significantly affect the distribution of ARGs in shrimp rearing environments. Furthermore, stable ponds aided the removal of ARGs from shrimp pond water. This study accounted for temporal variations in ARG abundances as well as removal of ARGs in different environmental compartments during the entire shrimp rearing period. However, additional research is required to optimize the water treatment process for removal of ARGs from the aquaculture.     

Molecular characterization and developmental expression patterns of thyroid hormone receptors (TRs) and their responsiveness to TR agonist and antagonist in Rana nigromaculata

Considering some advantages of Rana nigromaculata as an experimental species, we propose that this species, like Xenopus laevis, could be used to assay thyroid hormone（TH） signaling disrupting actions. To validate the utilizability of R. nigromaculata, we investigated the responsiveness of R. nigromaculata to a TH receptor（TR） agonist（T3） and antagonist（amiodarone） by analyzing expression, based on characterizing TR cDNA and developmental expression patterns. With high levels of identity with the corresponding genes in X. laevis, both TRα and TRβ in R. nigromaculata exhibited roughly similar developmental expression patterns to those of X. laevis, in spite of some species-specific differences. Both TRα and TRβ expression had greater changes in the liver and intestine than in the tail and brain during metamorphosis. T3 exposure for 2 days induced more dramatic increases of TRβ expression in stage 27 than in stage34 tadpoles but not in stage 42 tadpoles, showing that the responsiveness of R. nigromaculata to TH decreased with development and disappeared at the onset of metamorphic climax.Corresponding to greater changes of TRβ expression in the liver and intestine than in the tail and brain during metamorphosis, the liver and intestine had higher responsiveness to exogenous T3 than the tail and brain. Amiodarone inhibited T3-induced TRβ expression. Our results show that R. nigromaculata can be used as a model species for assaying TH signaling disrupting actions by analyzing TRβ expression, and intestine tissues at stage 27 are ideal test materials due to high responsiveness and easy accessibility.     

2,2'',4,4''-四溴联苯醚对赤子爱胜蚓的抗氧化酶、代谢酶及其基因表达的影响

多溴联苯醚(PBDEs)是广泛存在于环境中的一种新型持久性有机污染物.四溴联苯醚同分异构体中的BDE-47是多溴联苯醚中最重要的单体之一.试验采用人工土壤培养法,通过亚急性试验,研究了在不同暴露时间阶段下,不同BDE-47剂量对赤子爱胜蚓(Eisenia fetida)抗氧化酶(过氧化氢酶CAT)、代谢酶(谷胱甘肽转移酶GST)以及二者基因表达的影响.结果表明,在暴露14 d和28 d时,CAT活性被诱导上升并且差异显著;GST活性变化差异不显著;CAT基因表达水平在第14 d时呈现抑制效应,在后续的第28 d和第42 d基因表达水平上调;GST基因表达水平整体呈现诱导效应,并且差异显著,随着暴露时间的增加,低毒处理组(10、50 mg·kg-1)的基因表达水平逐渐下调至低于对照组的水平,高毒处理组(100、200 mg·kg-1)的基因表达量仍高于对照组水平;在BDE-47的暴露试验中,CAT与GST活性及其基因表达水平两项指标对低毒处理组较高毒处理组更为敏感.     

初始pH对厌氧环境下污泥中抗生素抗性基因行为特征的影响

污水处理厂产生大量的剩余污泥中含有丰富的抗性基因,给环境带来了潜在风险。以城市污水处理厂的剩余污泥为研究对象,在不同初始p H(对照组、初始p H=3、5、7、9、11)下观察厌氧条件下,8种抗生素浓度以及四环素类抗性基因(tet A、tet G、tet L、tet M、tet O、tet Q、tet W、tet X)、磺胺类抗性基因(sul I、sul II)和Ⅰ类整合子(int I 1)的行为特征。研究结果显示,初始p H对抗生素的降解影响较小,污泥中总抗生素的平均去除率为42%。对照组及初始p H为3、5、7、9、11下的总四环素类抗性基因分别削减0.65 log、0.96 log、0.75 log、0.62 log、0.86 log和0.98 log。不同四环素类抗性基因表现相似,在初始p H=3和初始p H=11下部分抗性基因削减较多,特别是tet A、tet G、tet L、tet O和tet X。2种磺胺类抗性基因均无削减,浓度平均上升0.18log。相关性分析显示,总抗性基因与TN、NH3-N、TP、SCOD(溶解性COD)均存在显著相关性(P0.05)。上述研究结果为污泥厌氧消化中抗生素抗性基因减量条件提供参考依据。     

一株贫营养异养硝化-好氧反硝化菌的筛选及脱氮特性

为了探究并优化菌剂应用于微污染水源水体修复的机制和条件,主要针对水库沉积物内筛选出的贫营养好氧反硝化菌进行了菌种鉴定及脱氮特性研究,考察菌株在不同环境条件下的脱氮效果,明确了该菌株的最适宜生长条件,并基于水库水体中贫营养条件对菌株进行水源水库原水的驯化培养试验研究,以期实现该菌株对微污染水源水库原水中氮源污染物的脱除,为原位投菌技术实际工程应用提供理论依据。从微污染水源水库沉积物中驯化筛分出一株高效异养硝化-好氧反硝化菌A14,通过扫描电镜观察、生理生化特征、16S rRNA基因测序和Biolog GenⅢ鉴定,确定该菌株为革兰氏阴性短杆菌,鉴定为皮特不动杆菌（Acinetobacter pittii）。在好氧条件下,菌株细胞内表达反硝化功能基因napA,以NO3-为唯一氮源进行反硝化作用时,36 h时NO3-去除率为78.89%。以NH4+为唯一氮源时,48 h NH4+去除率为95.25%,TN去除率达80.42%,TOC去除率达98.30%,表明该菌株具有异养硝化-好氧反硝化特性。在改变环境条件过程中,该菌株在以乙酸钠为碳源,温度为30℃,C/N为12,pH为7,接种量为10%时,NO3-去除率最高为86.62%,并且在10℃下脱氮率达到40.18%。在水源水库原水脱氮实验中,接种处理TN去除率为50.95%,NO3-去除率为80.25%。结果表明,菌株A14在微污染水源水体菌剂脱氮修复中具有良好的应用潜力。     

雌二醇短期暴露对非洲爪蟾性腺和输卵管形态及性二态基因表达影响的初步研究

环境雌激素对两栖动物的生殖内分泌干扰作用已引起关注,目前的研究主要集中在长期暴露对性腺分化和发育的影响。以雌二醇(E2,10 nmol·L-1)为模式化合物,以非洲爪蟾为实验动物,6月龄的幼蛙暴露6 d,研究雌激素对性腺和输卵管形态以及性二态性基因表达的影响。结果发现,6 d的E2暴露对精巢和卵巢的形态没有明显影响,但从组织学结构来看对卵和精子的发育有微弱的促进作用。E2能够明显地促进了雌性输卵管的发育,表现为输卵管变粗变弯曲。E2暴露显著抑制精巢和卵巢中性激素合成相关基因cyp11a1、3βHSD、Srd5α、ARO的表达,对cyp17的表达无影响,使脑中LHβ和FSHβ的表达下调;对性别分化和性腺发育相关基因Dax、AR、ERα、TFⅢA、TBPL2的表达均无影响,但能够促进卵巢中Sox3的表达;同时促进脑中Emi2的表达,抑制脑中Dmrt1的表达。因此,输卵管可作为敏感的形态指标,性腺中Srd5α、cyp11a1、3βHSD、ARO、Sox3和脑中LHβ、FSHβ、Dmrt1、Emi2可作为分子指标。以上结果可为后续建立评价化学品对两栖动物雌激素效应的短期试验方法提供参考。     

连续施用发酵猪粪对土壤中四环素抗性基因数量的影响

为了研究连续施用发酵猪粪之后,土壤中抗性基因的数量以及发酵猪粪的施用量对土壤抗性基因的影响,采用实时荧光定量PCR方法,对稻麦轮作模式下连续6年施用4.5t/hm2和9.0t/hm2发酵猪粪的土壤进行了四环素抗性基因(TRGs)的检测和定量分析.在施用发酵猪粪的土壤中能够检测到9种TRGs.施用发酵猪粪显著增加了土壤中tet G、tet L、tet B(P)、tet O、tet W的绝对数量,其中tet B(P)、tet W、tet O的绝对数量受到了施肥用量的影响;而tet Z、tet C和tet S的绝对数量则没有受到施肥的影响.上述8种TRGs在同一施肥处理的0~5cm、5~10cm以及10~20cm土壤中的相对丰度分布均无显著差异.发酵猪粪的施用也显著增加了tet G、tet L、tet B(P)和tet O的相对丰度,但是仅有tet O的相对丰度受到了施肥用量的显著影响.本研究表明,在稻麦轮作模式下,农田土壤中的TRGs数量会受到发酵猪粪中残留TRGs的影响,连续施用该粪肥会显著增加土壤中tet G、tet L、tet B(P)和tet O的绝对数量和相对丰度.因此,需要优化畜禽粪堆肥发酵工艺,以减少抗性基因在粪肥中的残留.     

Piriformospora indica confers cadmium tolerance in Nicotiana tabacum

Piriformospora indica, a root-colonizing endophytic fungus of Sebacinales, promotes plant growth and confers resistance against biotic and abiotic stresses. In order to confirm the influence of P. indica on growth, proline, malondialdehyde (MDA), chlorophyll, and cadmium (Cd) amounts in Nicotiana tabacum under Cd stress, hydroponics, pot and field trials were conducted. The results showed that P. indica can store Cd in plant roots and reduce leaf Cd content, reduce the concentration of MDA, and increase the proline and chlorophyll content and the activities of catalase, peroxidase, and superoxide dismutase under hydroponic Cd stress. RT-PCR analysis showed that the relative expression level of genes Gsh2, TaPCS1, oas1, GPX, and Hsp70 in colonized plants was 4.3, 1.4, 2.9, 1.7, and 6.9 fold higher than in un-colonized plants respectively. Cd exposure significantly reduced un-colonized plants'' agronomic traits compared to P. indica-colonized ones. Our results suggested that P. indica can sequester Cd in roots, so that much less cadmium was transported to leaves, and the increased concentrations of antioxidant enzymes, pigments and proline contents, as well as the higher expression of stress-related phytochelatin biosynthesis genes in P. indica-inoculated plants, may also serve to protect N. tabacum plants against oxidative damage, enhancing Cd tolerance.     

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone (TH) signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3, whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.