基于宏基因组学探讨东平湖水库的菌群结构、耐药基因谱及其公共健康风险

水库中长时间滞留的水体会成为耐药基因的存储库,因此研究其菌群结构、耐药基因谱及其潜在的健康风险具有重要的公共卫生学意义.本研究以东平湖水库的水体和沉积物为研究对象,采用宏基因组测序技术,系统分析其在旱季和雨季中的菌群结构、耐药基因谱、耐药基因水平转移以及携带耐药基因的致病菌类型.结果表明,与水库沉积物相比,水体中的菌群结构和耐药基因谱受季节影响较大,且旱季的总耐药基因丰度明显高于雨季;水库沉积物中总耐药基因丰度高于水体,但水体中耐药基因的水平转移高于沉积物.本研究共检测到20个耐药基因型,377个耐药基因亚型;杆菌素和万古霉素耐药基因分别是水和沉积物中主要的耐药基因型,变形杆菌和放线菌分别是水体和沉积物中主要携带耐药基因的菌群.此外,本研究还鉴定出30株携带耐药基因的致病菌,包括大肠杆菌、肺炎克雷伯菌、铜绿假单胞菌、肠炎沙门氏菌以及鲍曼不动杆菌,其中有2株大肠杆菌携带毒力因子.综上所述,东平湖水库中的耐药基因不仅种类繁多,而且还存在潜在的公共健康风险;因此定期检测水体环境的菌群结构及其耐药基因谱具有重要的科学价值和现实意义.     

不同施肥措施对水稻土壤微生物镉抗性的影响

镉（Cd）是最常见的土壤重金属污染物之一,对植物和人体具有毒害作用,研究不同施肥条件下的土壤微生物Cd抗性水平,可以为有机肥改良重金属污染提供一定的理论依据.为了探讨施无机肥和有机肥对于微生物Cd抗性水平的影响,在中国江苏常州、江西上高和福建福州水稻种植区采集了施有机肥（猪粪）和无机肥的土壤样品及猪粪样品,利用功能基因芯片（GeoChip 5.0）技术,研究了土壤微生物Cd抗性功能基因的响应及影响Cd抗性功能微生物群落互作的因素.结果表明,施有机肥土壤中有效态Cd含量[（1.08±0.70）mg·kg^-1]显著低于施无机肥土壤[（3.75±1.22）mg·kg^-1]（P<0.05）.土壤及猪粪样品中共检测到639个Cd抗性功能基因,施有机肥土壤中微生物Cd抗性基因丰度高于施无机肥土壤;有效态Cd含量、含水率、pH和铵态氮含量是影响Cd抗性微生物分布的重要环境因素.对Cd抗性微生物分子生态网络分析表明,施无机肥条件下,pH、含水率和有效态Cd含量是影响功能微生物潜在互作关系的主要因素;施有机肥条件下,主要影响因素是全钾和含水率.相比较无机肥,施用有机肥提高了土壤中微生物的Cd抗性水平及耐Cd微生物分子生态网络中的正相互作用.     

潮汐-复合流人工湿地系统优化及对抗生素抗性基因的去除效果

抗生素抗性基因随废水排放传播扩散,对环境生物和民众健康构成严重威胁.针对废水中抗性基因的深度去除值得重点关注.在前期研究中发现潮汐流人工湿地能有效去除废水中多种氮素.本研究通过增加隔板和种植植物等方式进一步优化潮汐流人工湿地系统,并考察了工艺优化对抗性基因去除和脱氮功能微生物的影响机制.结果表明,同时增加隔板和种植植物后的潮汐-复合流人工湿地系统能有效提高抗性基因的去除效率,在增加隔板和种植植物组对7类21种抗性基因去除率最高达到83.82%~100.0%,显著高于单一增加隔板或种植植物组.从湿地基质和植物中的抗性基因绝对丰度对比可以看出,增加隔板能促进湿地基质中抗性基因量积累,而植物对抗性基因吸附也是其去除途径之一.同时,结合氮循环功能基因测序结果显示,湿地系统优化能提高基质中硝化和反硝化功能微生物物种多样性和丰富度,这与优化系统对废水中硝化量、反硝化量和总氮的去除率相对更高结果一致.     

宁夏养牛场粪污和周边土壤中抗生素及抗生素抗性基因分布特征

畜禽粪便是抗生素和抗生素抗性基因（antibiotic resistance genes,ARGs）重要的储存库.为揭示宁夏肉牛养殖场牛粪和养殖场周边土壤中抗生素及ARGs的分布特征,采用超高效液相色谱-串联质谱法和高通量荧光定量法对不同规模的养牛场进行调查研究.结果表明：①牛粪中优势抗生素为四环素类、喹诺酮类和磺胺类.不同养殖期牛粪中抗生素含量差异显著;育肥前期和哺乳期抗生素含量较高,犊牛期相反.②土壤中以喹诺酮类和四环素类抗生素为主,喹诺酮类抗生素检出率和含量都最高.养殖场对周边土壤中抗生素的分布无影响;施用牛粪的土壤中喹诺酮类和四环素类抗生素含量显著增加.③牛粪中共检测到ARGs亚型79~142种,其中氨基糖苷类ARGs数目最多.育成期ARGs数目最多且相对丰度最高,犊牛期相反.所有养殖期牛粪中共存ARGs是tetQ、ermF和tetO-01.④土壤中ARGs亚型有35~79种,主要是多药类和氨基糖苷类ARGs.养殖场对周边土壤中ARGs的数目和相对丰度无影响;施用牛粪的土壤中ARGs数目和相对丰度都显著高于其他土壤.⑤磺胺类和氯霉素类ARGs在牛粪和土壤中都有水平移动的风险.牛粪中氨基糖苷类和四环素类ARGs相对丰度与其对应抗生素含量呈极显著相关.⑥对牛粪和土壤中各类ARGs相对丰度而言,氨基糖苷类与磺胺类和氯霉素类、可移动遗传元件（mobile genetic elements,MGEs）与氨基糖苷类和磺胺类均呈显著或极显著正相关,而大环内酯类与万古霉素类则呈显著负相关.本研究结果可为肉牛不同养殖期抗生素的使用和牛粪施用等提供理论依据.     

Characterization of the genes involved in nitrogen cycling in wastewater treatment plants using DNA microarray and most probable number-PCR

To improve nitrogen removal performance of wastewater treatment plants (WWTPs), it is essential to understand the behavior of nitrogen cycling communities, which comprise various microorganisms. This study characterized the quantity and diversity of nitrogen cycling genes in various processes of municipal WWTPs by employing two molecular-based methods:most probable number-polymerase chain reaction (MPN-PCR) and DNA microarray. MPN-PCR analysis revealed that gene quantities were not statistically different among processes, suggesting that conventional activated sludge processes (CAS) are similar to nitrogen removal processes in their ability to retain an adequate population of nitrogen cycling microorganisms. Furthermore, most processes in the WWTPs that were researched shared a pattern:the nirS and the bacterial amoA genes were more abundant than the nirK and archaeal amoA genes, respectively. DNA microarray analysis revealed that several kinds of nitrification and denitrification genes were detected in both CAS and anaerobic-oxic processes (AO), whereas limited genes were detected in nitrogen removal processes. Results of this study suggest that CAS maintains a diverse community of nitrogen cycling microorganisms; moreover, the microbial communities in nitrogen removal processes may be specific.

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Effect of salinity on community structure and naphthalene dioxygenase gene diversity of a halophilic bacterial consortium

The aim of this study is to analyze the effect of salinity on polycyclic aromatic hydrocarbons (PAHs) biodegradation, community structure and naphthalene dioxygenase gene (ndo) diversity of a halophilic bacterial consortium with the denaturing gradient gel electrophoresis (DGGE) approach. The consortium was developed from oil-contaminated saline soil after enrichment for six times, using phenanthrene as the substrate. The prominent species in the bacterial consortium at all salinities were identified as halophilic bacteria Halomonas, Alcanivorax, Marinobacter, Idiomarina, Martelella and uncultured bacteria. The predominant microbes gradually changed associating with the saline concentration fluctuations ranging from 0.1% to 25% (w/v). Two ndo alpha subunits were dominant at salinities ranging from 0.1% to 20%, while not been clearly detected at 25% salinity. Consistently, the biodegradation occurred at salinities ranging from 0.1% to 20%, while no at 25% salinity, suggesting the two ndo genes played an important role in the degradation. The phylogenetic analysis revealed that both of the two ndo alpha subunits were related to the classic nah-like gene from Pseudomonas stutzeri AN10 and Pseudomonas aeruginosa PaK1, while one with identity of about 82% and the other one with identity of 90% at amino acid sequence level. We concluded that salinity greatly affected halophilic bacterial community structure and also the functional genes which were more related to biodegradation.

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城市河流异养菌耐药状况及微生物群落结构特征

为深入了解城市河流中细菌的耐药状况和微生物群落结构特征,在西安市浐河和灞河河段8个采样点以及某污水处理厂二级出水排放口采集水样,利用细菌培养技术、PCR检测和DGGE(变性梯度凝胶电泳)分析技术,对河流中异养菌耐药率、四环素抗性基因以及微生物群落结构进行研究. 结果表明:浐河和灞河水样中异养菌对SMZ(磺胺甲恶唑)、TET(四环素)、CIP(环丙沙星)和CTX(头孢噻肟)的平均耐药率分别为18.3%、6.2%、2.7%和1.3%,耐药率高低顺序与污水厂排放水中的异养菌相同. 城市河流中异养菌浓度与其耐药率并不存在显著的相关性. 与上游河段相比,污水厂排放口下游河段的异养菌浓度没有明显增加,但对多种抗生素的耐药率却有所升高. 浐河和灞河中四环素抗性基因主要是tetA和tetB,检出率分别为100%和75%,这意味着城市河流中异养菌对TET的耐药性可能主要通过编码外输泵蛋白的抗性机制来实现. 污水厂排放水进入城市河流,并未显著影响河流中的微生物群落结构,但却使某些原有的敏感物种消失,而病原菌或条件致病菌在邻近污水厂排放口的下游河段则有所增加.      

Degradation of extracellular genomic,plasmid DNA and specific antibiotic resistance genes by chlorination

Extracellular DNA structure damaged by chlorination was characterized. Integrity of extracellular ARG genetic information after chlorination was determined. Typical chlorine doses will likely effectively diminish extracellular DNA and ARGs. Plasmid DNA/ARGs were less readily broken down than genomic DNA. The Bioanalyzer methodology effectively documented damage incurred to DNA. There is a need to improve understanding of the effect of chlorine disinfection on antibiotic resistance genes (ARGs) in order to advance relevant drinking water, wastewater, and reuse treatments. However, few studies have explicitly assessed the physical effects on the DNA. Here we examined the effects of free chlorine (1–20 mg Cl₂/L) on extracellular genomic, plasmid DNA and select ARGs. Chlorination was found to decrease the fluorometric signal of extracellular genomic and plasmid DNA (ranging from 0.005 to 0.05 mg/mL) by 70%, relative to a no-chlorine control. Resulting DNA was further subject to a fragment analysis using a Bioanalyzer, indicating that chlorination resulted in fragmentation. Moreover, chlorine also effectively deactivated both chromosomal- and plasmid-borne ARGs, mecA and tetA, respectively. For concentrations >2 mg Cl₂//L × 30 min, chlorine efficiently reduced the qPCR signal when the initial concentration of ARGs was 10⁵ copies/mL or less. Notably, genomic DNA and mecA gene signals were more readily reduced by chlorine than the plasmid-borne tetA gene (by ~2 fold). Based on the results of qPCR with short (~200 bps) and long amplicons (~1200 bps), chlorination could destroy the integrity of ARGs, which likely reduces the possibility of natural transformation. Overall, our findings strongly illustrate that chlorination could be an effective method for inactivating extracellular chromosomal- and plasmid-borne DNA and ARGs.     

南京地区污水厂、自来水厂及长江水域MCR-1和NDM-1携带菌耐药特征

为了研究以多粘菌素耐药基因(MCR-1)和新德里金属β-内酰胺酶-1基因(NDM-1)携带菌为代表的"超级细菌"在地表水中的环境行为,以长江南京段典型水域及邻近城市污水厂和自来水厂为研究对象,考察了MCR-1和NDM-1携带菌的耐药特性及其变化特征。结果发现,污水厂进水和生物处理单元2种细菌耐药能力均处于较高水平,随处理流程呈降低趋势,至消毒出水中不再被检出。长江中MCR-1携带菌耐粘菌素的半抑制浓度(EC50)较高,平均达37.89 mg·L-1,顺水流方向无显著降低。仅在下游朱家山河汇入口和长江大桥附近滨江公园检出较高耐药水平的NDM-1携带菌。2种细菌耐药水平受水体污染程度和人类活动影响较明显。自来水厂MCR-1携带菌数目较少,但其EC50平均可达32.75 mg·L~(-1),常规处理单元中未检出NDM-1携带菌。水处理工艺显著降低了细菌耐药水平,但排泥水中2种细菌均检出,且耐药水平相比进水显著提高。本研究可为评价水介质中超级抗性基因及其携带菌的环境风险提供参考。     

乙草胺对蛋白核小球藻的毒性效应研究

乙草胺是我国使用量最大的除草剂之一,在水体中广泛存在。已有研究证明,乙草胺对人类、老鼠和鱼类具有毒害效应,而关于其对浮游植物影响的研究较少。以蛋白核小球藻(Chlorella pyrenoidosa)为模型,使用1～10 000μg·L~(-1)的乙草胺对其进行7 d的暴露实验,考察小球藻生长性能、叶绿素含量、光合作用产氧量以及光合作用相关基因(pbsA、rbcL和rbcS)表达的变化。结果表明,较低浓度的乙草胺可刺激蛋白核小球藻生长,而较高浓度乙草胺则会抑制其生长;并且乙草胺会通过影响小球藻叶绿素的含量而影响光合作用产氧量;小球藻光合作用相关基因pbsA、rbcL和rbcS表达大多显著上升,这可能是对乙草胺胁迫响应的反馈调节。研究表明,乙草胺会对蛋白核小球藻的生长及光合作用产生影响。