长江口近岸地区抗生素抗性基因与微生物群落分布特征

由于抗生素的大量使用,环境中微生物对抗生素的抗性不断增加,抗生素抗性基因(ARGs)问题越来越严重,严重威胁生态安全和人类健康.为研究长江口近岸地区水体和底泥沉积物中的ARGs和微生物群落的分布特征,通过野外调查采集了8个站点的水样和沉积物样本,对2种磺胺类抗性基因(sul1、sul2)、6种四环素类抗性基因(tetM、tetC、tetX、tetA、tetO、tetQ)、1种整合子基因intI1、16S rRNA基因和微生物群落进行检测分析.结果表明,长江口近岸地区10种抗性基因的检出率为100%.其中,整合子基因intI1和水样中多种ARGs呈显著正相关关系.变形菌门(Proteobacteria)和拟杆菌门(Bacteroidota)是长江口近岸地区水环境中的优势菌门;Chloroplast为水体中的主要菌属,Chloroplast和Nitrospira为沉积物中的主要菌属.在水体中,硝化螺旋菌门(Nitrospirota)是4种四环素类抗性基因(tetX、tetA、tetO和tetQ)共同的潜在宿主;在沉积物中,Sva0485是sul1和intI1的共同潜在宿主.微生物群落的分布...     

Combined pollution of arsenic and Polymyxin B enhanced arsenic toxicity and enriched ARG abundance in soil and earthworm gut microbiotas

Polymyxin B (PMB) is considered as the last line of antibiotic defense available to humans. The environmental effects of the combined pollution with PMB and heavy metals and their interaction mechanisms are unclear. We explored the effects of the combined pollution with PMB and arsenic (As) on the microbial composition of the soil and in the earthworm gut, as well as the spread and transmission of antibiotic resistance genes (ARGs). The results showed that, compared with As alone, the combined addition of PMB and As could significantly increase the bioaccumulation factor and toxicity of As in earthworm tissues by 12.1% and 16.0%, respectively. PMB treatment could significantly increase the abundance of Actinobacteria in the earthworm gut (from 35.6% to 45.2%), and As stress could significantly increase the abundance of Proteobacteria (from 19.8% to 56.9%). PMB and As stress both could significantly increase the abundance of ARGs and mobile genetic elements (MGEs), which were positively correlated, indicating that ARGs might be horizontally transferred. The inactivation of antibiotics was the main resistance mechanism that microbes use to resist PMB and As stress. Network analysis showed that PMB and As might have antagonistic effects through competition with multi-drug resistant ARGs. The combined pollution by PMB and As significantly promoted the relative abundance of microbes carrying multi-drug resistant ARGs and MGEs, thereby increasing the risk of transmission of ARGs. This research advances the understanding of the interaction mechanism between antibiotics and heavy metals and provides new theoretical guidance for the environmental risk assessment and combined pollution management.     

菌株WYT降解还原蓝4的关键基因

以印染厂活性污泥中筛选出的WYT菌株为研究对象,探索其对还原蓝4（VB4）染料降解脱色的关键基因.采用全基因组测序分析方法和同源重组法,确定染料代谢的可能关键基因.对该基因进行敲除,构建载体进行基因回补,设计表型验证实验验证基因的脱色效果.结果表明,该可能关键基因属于染料过氧化物酶基因中的B型,命名为DyP.敲除后的菌株对VB4没有脱色效果,基因敲除成功.WYT菌株与敲除载体发生双交换,获得回补株.在降解实验中,回补株对VB4的脱色率为96.04%,野生株的脱色率为96.95%,回补株恢复了对VB4的降解脱色能力,而敲除株几乎失去对VB4的降解能力,DyP基因是VB4降解脱色的关键基因.     

蚯蚓摄食污泥对其肠道功能区微生物种群及耐药基因的影响

以赤子爱胜蚓(Eisenia fetida)为实验蚓种, 研究蚯蚓消化污泥前后, 其肠道各功能区微生物种群结构及耐药基因(ARGs)丰度的变化情况.为表征活体功能性微生物, 所取新鲜样品均采用叠氮溴化丙锭(PMA)预处理后, 再进行DNA相关分析.结果表明: 蚯蚓消化污泥5d后, 细菌16S rDNA和真核生物18S rDNA的丰度在其胃中分别显著增加了28.2倍和42.2倍(P<0.05), 而在砂囊和后肠中均显著性降低(P<0.05).此外, 蚯蚓消化污泥使其砂囊中的优势菌门由变形菌门变为软壁菌门, 胃中的优势菌门由拟杆菌门变为厚壁菌门, 而对后肠微生物种群结构的影响较小.对于ARGs而言, 大环内酯类耐药基因ermF、四环素类耐药基因tetX和磺胺类耐药基因sul2在蚯蚓胃中的丰度分别显著升高了1.9×10²倍、8.4×10⁵倍和25.9倍(P<0.05), 而在其砂囊和后肠中ARGs总丰度分别显著下降了11.0倍和45.2倍(P<0.05).基于网络分析结果显示, 蚯蚓摄食污泥可影响其肠道内ARGs宿主细菌的结构多样性.     

医院空调回风口滤网积尘中抗生素抗性基因污染的比较

为了解医院环境中抗生素抗性基因(ARGs)的污染情况与分布特征,于2018年11月至2021年4月期间分批次采集了武汉市两家医院部分病房空调回风口滤网积尘样本127份,对其进行β-内酰胺类ARGs (mecA、bla_TEM、bla_CTX-M、bla_SHV)和碳青霉烯类ARGs (bla_KPC、bla_NDM-1、bla_IMP、bla_VIM、bla_OXA-51)及一类整合酶基因intI 1的定性与定量检测与评价.结果表明,医院不同科室及ICU空调滤网积尘中可检出以上9种β-内酰胺类,碳青霉烯类ARGs及intI 1,平均检出率分别为55.12%、37.64%和81.89%.ICU的β-内酰胺类与碳青霉烯ARGs的平均检出率显著高于外科与内科(P<0.05);4种β-内酰胺类ARGs中的bla_TEM、mecA检出率及其相对丰度在内科、外科最高(P<0.05);ICU的bla_SHV检出率及其相对丰度高于内科、外科(P<0.05).5种碳青霉烯类ARGs中的bla_NDM-1检出率及其相对丰度在内科、外科显著高于bla_KPC、bla_IMP(P<0.05).intI 1与9种ARGs的相对丰度均呈显著正相关(P<0.05).研究结果表明,两家医院不同科室病房空调滤网积尘中存在β-内酰胺类和碳青霉烯类ARGs以及一类整合酶基因intI 1的污染,内科、外科、ICU均以bla_TEM、mecA、bla_NDM-1为主,由此可认为医院不同科室空气及相关环境中可能存在与此等ARGs相关的耐甲氧西林金黄色葡萄球菌、产ESBLs肠杆菌科细菌、耐碳青霉烯大肠埃希菌与鲍曼不动杆菌等耐药菌的现时与既往污染,并且存在ARGs水平转移的风险;ICU空调滤网积尘中β-内酰胺类与碳青霉烯ARGs的污染较内科、外科严重,其中bla_SHV污染高于内科、外科,由此表明,ICU是ARGs及相关耐药菌污染的高风险科室.     

氯/氯胺和紫外顺序消毒对管网抗性基因的去除

为探究供水系统中氯/氯胺与低压紫外顺序消毒对抗生素抗性基因(ARGs)分布特征的影响,采用生物膜反应器模拟供水管网,对管网出水和生物膜进行60mJ/cm²低压紫外线(254nm)消毒,并利用高通量定量PCR技术检测模拟管网进、出水及生物膜内的典型ARGs和遗传原件(MGEs).结果表明,管网反应器运行150d,氯和氯胺管网出水ARGs总相对丰度分别为0.13和0.137,生物膜ARGs分别为2.45和0.277,表明供水管网中低剂量的氯或氯胺可有效降低水相和生物膜相中ARGs的相对丰度达90%,且氯胺消毒对生物膜中的ARGs控制作用更显著.氯和氯胺消毒后管网出水再经低压紫外线照射后,ARGs相对丰度分别为0.0682和0.0537,管网生物膜中ARGs的相对丰度分别为2.01和0.194.ARGs与MGEs间的相关性发生显著变化,转座子与strB和mepA的相关性增强,与ermX和tetM相关性减弱,而整合子与acrF、cmlA1-01、oprJ及tolC-01的相关性增强.研究表明,将紫外线消毒工艺设置在用水终端可以显著降低氯和氯胺管网水中ARGs丰度,但对管网生物膜中的ARGs影响较小.     

Occurrence and distribution of antibiotic resistance genes in the sediments of the East China Sea bays

The coastal area of the East China Sea has experienced rapid urbanization and industrialization in China since 1980 s, resulting in severe pollution of its environments.Antibiotic resistance genes(ARGs) are regarded as a kind of emerging pollutant with potential high risk. The sediment samples were collected from Hangzhou Bay(HB),Xiangshan Bay(XB), and Taizhou Bay(TB) to investigate the spatial occurrence and distribution of 27 ARGs and class I integron–integrase gene(intI1) in the coastal area of the East China Sea. The PCR results showed the frequent presence of 11 ARGs and intI1 in the sediments of the three bays. The qPCR results further showed that sulfonamide resistance was the most prevalent ARG type and antibiotic target replacement and protection were the most important resistance mechanisms in the sediments. Regarding the subtype of ARGs, sulI, tetW, and dfrA13 were the most abundant ARGs, in which sulI was higher in TB(based on both the absolute and relative abundances) and dfrA13 was higher in HB(based on the relative abundances). The network analysis revealed that intI1 had significant correlations with tetC, sulI, sulII, and blaPSE-1. Oil was the key connected factor, which had positive connections with sulI, sulII, and blaPSE-1. In addition, the joint effect of heavy metals and nutrients organic pollutants might be crucial for the fate of ARGs in the coastal sediments.     

Distribution and relationship between antimicrobial resistance genes and heavy metals in surface sediments of Taihu Lake, China

Heavy metals, pharmaceuticals, and other wastes released into the environment can significantly influence environmental antibiotic resistance. We investigated the occurrence of 22 antimicrobial resistance genes(ARGs) and 10 heavy metal concentrations, and the relationship between ARGs and heavy metals in surface sediment from seven sites of Lake Taihu. The results showed significant correlations(p 0.05) between sediment ARG levels,especially for tetracycline and sulfonamides(e.g., tet(A), tet(D), tet(E), tet(O), sul1, sul2 and int-1) and specific heavy metals(Fe, Mn, Cr, Cu, Zn, among others) in the Lake. In the surface sediments, heavy metals showed an interaction with resistance genes, but the strength of interaction was diminished with increasing depth. For most of the heavy metals, the concentration of elements in the top sediments was higher than that in other depths.Tetracycline resistance genes(tet(A), tet(B), tet(D), tet(E) and tet(O), β-lactam resistance genes(SHV, TEM, CTX, OXA and OXY) and sulfonamide resistance genes(sulA, sul1, sul2, sul3 and int-1) were detected. They showed a trend which inferred a statistically significant increase followed by decreases in the relative abundance of these ARGs(normalized to 16 S rRNA genes) with increasing depth. This study revealed that tet(A), tet(O), TEM, OXY, int-1, sul1 and sul3 were widespread in surface sediments with high abundance, indicating that these genes deserve more attention in future work.     

Vertical response of microbial community and degrading genes to petroleum hydrocarbon contamination in saline alkaline soil

A column microcosm was conducted by amending crude oil into Dagang Oilfield soil to simulate the bioremediation process. The dynamic change of microbial communities and metabolic genes in vertical depth soil from 0 to 80 cm were characterized to evaluate the petroleum degradation potential of indigenous microorganism. The influence of environmental variables on the microbial responds to petroleum contamination were analyzed. Degradation extent of 42.45% of n-alkanes(C8–C40) and 34.61% of 16ΣPAH were reached after 22 weeks. Relative abundance of alkB, nah, and phe gene showed about 10-fold increment in different depth of soil layers. Result of HTS profiles demonstrated that Pseudomonas, Marinobacter and Lactococcus were the major petroleum-degrading bacteria in0–30 and 30–60 cm depth of soils. Fusarium and Aspergillus were the dominant oil-degrading fungi in the 0–60 cm depth of soils. In 60–80 cm deep soil, anaerobic bacteria such as Bacteroidetes, Lactococcus, and Alcanivorax played important roles in petroleum degradation.Redundancy analysis(RDA) and correlation analysis demonstrated that petroleum hydrocarbons(PHs) as well as soil salinity, clay content, and anaerobic conditions were the dominant effect factors on microbial community compositions in 0–30, 30–60, and 60–80 cm depth of soils, respectively.     

Nitrogen-cycling microbial community functional potential and enzyme activities in cultured biofilms with response to inorganic nitrogen availability

Biofilms mediate crucial biochemical processes in aquatic ecosystems. It was hypothesized that eutrophication may promote the growth of biofilms, resulting in larger numbers of functional genes. However, the metabolic activity and the roles of biofilms in N cycling will be affected by ambient inorganic nitrogen availability, not by the abundance of functional genes. Biofilms were cultured either with replete inorganic nitrogen(N-rep) or without exogenous inorganic nitrogen supply(N-def) in a flow incubator, and the N-cycling gene abundances(nifH, N_2 fixation; amoA, ammonia oxidation, archaea and bacteria; nirS and nirK, denitrification) and enzyme activities(nitrogenase and nitrate reductase) were analyzed. The results showed that, comparing the N-def and N-rep biofilms, the former contained lower nifH gene abundance, but higher nitrogenase activity(NA), while the latter contained higher nifH gene abundance, but lower NA. Different patterns of NA diel variations corresponded to the dynamic microbial community composition and different stages of biofilm colonization. Ammonia oxidizing bacteria(AOB), detected only in N-def biofilms, were responsible for nitrification in biofilms. N-rep biofilms contained high nirS and nirK gene abundance and high denitrification enzyme activity, but N-def biofilms contained significantly lower denitrification gene abundance and activity. In general,the strong N_2 fixation in N-def biofilms and strong denitrification in N-rep biofilms assured the balance of aquatic ecosystems. The results suggested that evaluation of the functional processes of N cycling should not only focus on genetic potential, but also on the physiological activity of biofilms.