温室内土壤-气溶胶中四环素类抗生素抗性基因的分布特征

目的研究温室内四环素类抗生素抗性基因(antibiotic resistance genes,ARGs)在土壤和气溶胶中的分布特征。方法以日光温室为研究对象,通过对温室内土壤、气溶胶样品中的17种四环素类ARGs进行实时荧光定量PCR测定,分析ARGs在土壤样品、不同切割粒径下的气溶胶样品中的分配分布特征。结果研究显示土壤及气溶胶中Tet(32s)检出率为100%,其次是Tet(A)、Tet(W)。随着气溶胶采样的切割粒径变小,检出的四环素类ARGs种类增加,丰度提高。PM2.5中的Tet(32s)平均丰度分别是土壤、TSP、PM10中的571.8倍、4.66倍和0.80倍。细胞保护是温室内四环素类ARGs的主要抗性机制。结论气溶胶和土壤均为ARGs的储库,不同切割粒径的气溶胶样品中ARGs的富集程度差别较大。鉴于气溶胶ARGs流动扩散能力强,比土壤ARGs的潜在风险更大,因此气溶胶ARGs亟待深入研究。     

商品灭菌乳中常见抗生素抗性基因的筛查与定性分析

探究了市售常见商品灭菌乳中四环素类、氨基糖苷类、β-内酰胺类等抗生素抗性基因(Antibiotic resistance genes,ARGs)的分布与带染特征。以12种市售常见商品灭菌乳为研究对象,用CTAB法提取预处理后样品中的总DNA;超微量分光光度计测量DNA纯度与浓度;PCR扩增ARGs;PCR产物测序后提交GenBank进行序列比对,分析ARGs的种类;使用SPSS软件对ARGs检出率、样品品牌、包装类型、产品类型等进行相关性分析。结果显示,灭菌乳中可同时检出多种ARGs,检出率较高的基因分别为str B(93.75%)、aad B (92.71%)、sul1(85.42%)、bla_TEM82.29%)和tet C(58.33%),未在35种ARGs中检出bla_VEB、bla_PER、bla_DHA、bla_CMY2和bla_GIM。商品灭菌乳的品牌、包装、产地和保质期与aad A、aad B、str A、str B等抗性基因检出率显著相关。市售商品灭...     

4种市售水产品抗生素耐药基因和intⅠ1实时荧光定量PCR检测

为掌握水产品中抗生素耐药基因(ARGs)和Ⅰ类整合子intⅠ1含量及分布,本研究建立了水产品常见抗生素耐药基因实时荧光定量PCR(qPCR)检测体系。以含目的基因tetA、sul2、blaPSE、cmlA、qnrS、aac(6')-Ib、intⅠ1的重组质粒为标准品,建立qPCR标准曲线,按照已建立的qPCR体系对市售沼虾、鲫鱼、黄颡鱼、大黄鱼中ARGs和intⅠ1进行定量。结果表明,6种ARGs的最低检出限在11.5～202拷贝/μL之间,blaPSE的最低检出限为6.6×10~4拷贝/μL。沼虾、鲫鱼、黄颡鱼、大黄鱼4种样品都存在高丰度的blaPSE,相对丰度范围为1.17×10~(-4)～5.96×10~(-3),aac(6')-Ib的相对丰度在4种样品中都较低,在2.38×10~(-8)～6.71×10~(-7)之间。该检测体系以样品中细菌总基因组为模板,可对水产品中6种常见ARGs同时定量检测,能够较客观地揭示市售水产样品中ARGs的种类和数量分布信息,对控制水产品中ARGs污染并保障水产食品安全意义深远。     

基于SmartChip HT-qPCR分析不同食品中抗生素抗性基因的多样性及丰度

抗生素抗性基因（Antibiotics Resistance Genes,ARGs）广泛存在于各种环境中,被认为是一种新型环境污染物,对环境及人类健康具有潜在威胁。但是目前对于不同食品中多种ARGs赋存状况的研究仍然有限。该研究采用SmartChip高通量实时定量PCR方法（High-Throughput Quantitative Real-Time PCR,HT-qPCR）对水产品、发酵食品、肉类、蔬菜四大类样品中的ARGs及可移动遗传元件（Mobile Genetic Elements,MGEs）进行了定量分析。结果显示,被分析的15个样品中共检出9个ARGs大类,涉及234个ARGs亚类。所有样品中共有的ARGs亚类为79个,特有的为51个;水产品和肉类样品富集多种ARGs,这些基因表现出较高的丰度（最高可达3.6 copies/16S rRNA gene）。MGEs的检出数量达10个,其中所有样均检出整合子基因intI-1(clinic)和转座酶基因tnpA-01。ARGs抵抗机制分析结果表明,抗生素灭活机制、外排泵机制和细胞保护机制为主要的ARGs抵抗机制,抵抗机制贡献大小依次为：抗生素灭活机制>外排泵机制>细胞保护机制。该研究进一步丰富了对不同食品中ARGs赋存情况的认识,可为食品中ARGs的风险分析提供依据。     

水产品中6类抗生素抗性基因和Ⅰ类整合子的定量检测

论文选取广州市天河区两家菜市场、一家海鲜市场和三家超市的48份水产品为研究对象,提取了三种不同水产品:虾、鱼和贝类的总DNA,采用实时荧光定量PCR(Quantitative Real-time PCR)法,对市售水产品中6类抗生素抗性基因(Antibiotic Resistance Genes,ARGs):tet A、tet B、tet M、sulⅡ、flo R、aph A-1、aad A、erm B、cml A和Ⅰ类整合子5’端整合酶基因int I1、3’端磺胺耐药基因sulⅠ、季铵盐化合物及溴化乙锭的耐药基因qac EΔ1进行定量。研究显示:6类ARGs及Ⅰ类整合子基因在水产品中均有检出,基因拷贝数大小从10~2~10~8跨越七个数量级,最小值为1.86×10~2 copies/g,最大值为8.98×10~8 copies/g。三种水产品虾、鱼、贝类中,虾类的ARGs含量最高,贝类最低;三类采样地点中,菜市场来源的水产品中ARGs含量最高,海鲜市场来源的次之,超市来源的最低。本研究为进一步探索ARGs和可移动耐药元件整合子介导的细菌耐药性传播而引起的食源性疾病机理奠定了基础。     

水产品批发市场中抗生素抗性基因的污染分析

为了探究待售水产品中抗生素抗性基因(Antibiotic resistance genes,ARGs)在水产品批发市场中的扩散污染情况,选取上海市3个大型水产品批发市场作为研究对象,采用PCR的方法,对市场内部和市场附近的排污沟表层淤泥中5大类(4种磺胺类、8种四环素类、6种β-内酰胺类、5种氯霉素类、4种链霉素类)共27种ARGs进行了检测,并利用PCR-DGGE技术分析了细菌种群多样性。结果显示共有17种ARGs在批发市场淤泥中被检出,其中磺胺类、四环素类和链霉素类ARGs的检出率较高且检出种类较多,表明水产品批发市场含有种类丰富的ARGs,水产品以磺胺类、四环素类和链霉素类ARGs污染为主。对市场外围的检测显示市场外围区域ARGs的种类分布略低于市场内部,表明水产品批发市场中ARGs的存在会通过污水排放途径扩散到周边环境。DGGE图谱的分析结果显示,3个批发市场污泥样品细菌种类差异性较大,表明环境中ARGs的种类分布与细菌的种类并无直接关联。本研究表明水产品携带的ARGs会扩散到水产品批发市场中,导致水产品批发市场成为ARGs的重要储存库,以此造成的污染会进一步传播到周边环境,此外,这些种类丰富的ARGs重新扩散到待售水产品中的风险很大,需引起人们足够的重视。     

猪肉生产链四环素抗性基因污染分布研究进展

四环素作为药物和促生长剂,是农业生产和畜牧业中最常用的抗生素之一。然而,不规范的抗生素滥用造成了大量抗生素抗性基因(Antibiotic resistance genes,ARGs)的形成,使得药物治疗效益显著下降,对人类健康也产生了巨大威胁。目前已发现的四环素类抗性基因(Tet-R)的种类有40种以上,在各地猪肉生产链的各个环节中均有不同程度的检出,Tet-R的分布存在一定的空间分布特征和时间分布特征。目前分子生物学方法因在检测抗生素耐药性(antimicrobial resistance,AMR)的潜在遗传机制方面具有较高的速度和准确性而被广泛应用。文中结合国内外文献,主要介绍了猪肉生产链中猪肉及生产废弃物中ARGs的分布特征、研究方法等研究进展,为相关政策的制定提供科学理论依据,为养殖业兽医用药抗生素耐药性风险评估提供一定支持。     

四川泡豇豆原料表皮附生乳酸菌抗生素抗性及抗性基因污染分析

以鲜豇豆为研究对象,利用生理生化特征、16S r RNA、抗生素抗性和抗性基因检测分析其表皮附生乳酸菌对氯霉素(CHL)、四环素(TET)和氨苄青霉素(AMP)的抗性与抗性基因。结果表明:从鲜豇豆表皮共分离得到182株乳酸菌,分别属于Weissella confuse(50.0%)、Weissella cibaria(15.4%)、Enterococcus sulfurous(7.1%)和Lactococcus lactis subsp.lactis(27.5%)。32株(17.6%)乳酸菌对CHL和TET有抗性,其中,6株W.confuse(3.3%)和4株L.lactis subsp.Lactis(2.1%)对CHL单一抗性,4株W.confuse(2.1%)和5株L.lactis subsp.Lactis(2.7%)对TET单一抗性,6株W.confuse(3.2%)、2株W.cibaria(1.1%)、1株E.sulfurous(0.5%)和4株L.lactis subsp.Lactis(2.1%)对CHL和TET二重抗性;编码外排泵基因efr A、efr B、nor B、nor C、nor E和sug E中,efr B的检出率最高,达31.3%,efr B和sug E的检出率最低,为3.1%。TET被检抗性基因tet(A)、tet(B)、tet(C)、tet(D)、tet(H)和tet(L)中,tet(C)的检出率最高,达90.9%,tet(B)的检出率最低,为4.5%。     

南美白对虾肠道内乳酸菌的分离鉴定及其对抗生素的耐药性

以南美白对虾为研究对象,采用平板分离法从其肠道内筛得26株乳酸菌,通过16S rRNA测序鉴定可归为4种:乳酸乳球菌乳亚种L. lactis subsp. lactis、台湾乳球菌L. taiwanensis、格式乳球菌L. garvieae、乳酸乳球菌L. lactis。选取7株具有代表性的乳酸菌,用PCR的方法检测对虾中检出率较高的6类18种抗性基因(ARGs)在乳酸菌中的分布,辅以平板涂布的方法用抗生素选择性培养基研究乳酸菌对抗生素的耐药性。研究发现:7株乳酸菌具有相似的耐药谱,对四环素、磺胺吡啶、乙酰螺旋霉素表现出耐药性(抑菌率50.00%),对盐酸金霉、土霉素、硫酸庆大霉素、氯霉素、红霉素敏感(抑菌率100%);含有多重抗性基因,ARGs的检出率:磺胺类(92.86%)四环素类(53.06%)喹诺酮类(23.81%)氨基糖苷类(4.76%)氯霉素类=大环内酯类(0.00%),其ARGs基因型与菌株的抗生素表型并不能完全吻合。     

市售鸡肉中四环素耐药菌污染特征初探

为描述鸡肉产品中四环素耐药菌的污染状况,初步评价四环素耐药基因通过市售鸡肉产品进行迁移的潜在风险。在上海13个区采集55个冷冻鸡肉样品、50个冷鲜鸡肉样品和3个现宰鸡样品,分离对四环素或强力霉素不敏感的细菌(Tet~(is)或Dox~(is)),研究其耐药特征,并筛查7种四环素耐药基因和一类整合酶基因int I。结果表明,所有样品均分离出Tet~(is)菌,菌落数为1.00×103~1.51×108CFU/g;从96.19%的样品中分离出Dox~(is)菌,菌落数为5.01×10~7.76×106CFU/g;两类抗生素不敏感菌的数量在现宰样中最高,冷鲜样次之,冷冻样最低。738株Tet~(is)和Dox~(is)菌普遍对多种抗生素不敏感,其中165株菌中耐药基因tet A,tet B,tet C,tet E,tet G,tet L,tet M和int I的检出率分别为9.70%,20.00%,4.24%,9.70%,15.80%,29.70%,61.80%和46.10%。耐药基因的寄主菌包括埃希氏菌、葡萄球菌、肠球菌、奇异变形杆菌、不动杆菌、鞘氨醇杆菌和土地杆菌等,大多数菌的四环素最小抑菌质量浓度大于64μg/m L。综上,市售鸡肉产品中四环素耐药菌污染较为严重,可能包含巨大的耐药基因池,存在潜在的迁移风险。     

北京市售生鲜猪肉和牛肉中有害细菌的鉴定及耐药性分析

采用选择性培养基初步筛选,结合16S rRNA同源性分析的方法,对北京市农贸市场及超市中所销售的生鲜猪、牛肉中含有的有害细菌的分布情况进行考察。对分离得到的细菌针对7种常见抗生素进行耐药表型检测。同时,利用特异性PCR扩增法检测了2个常见红霉素耐药基因和7个常见四环素耐药基因在分离细菌中的分布情况。结果表明:市场中购买的生鲜肉中分离得到的细菌耐药情况较之超市中的更为严重;猪肉和牛肉中分离的细菌耐药情况均十分严重;分离得到的不同细菌中包含某些条件致病菌的Escherichia/Shigella以及Aeromonas属细菌耐药情况更加严重。同时,研究还发现在北京市售鲜肉中分离得到的细菌中,绝大多数细菌对不止1种抗生素表现出非敏感(耐药及中介),且一些菌株携带多种不同的耐药基因。因此,市售生鲜肉中分离得到的细菌耐药情况较为严重以及普遍,应得到监管部门以及普通消费者的足够重视。     

Effect of various sludge digestion conditions on sulfonamide, macrolide, and tetracycline resistance genes and class I integrons

Wastewater treatment processes are of growing interest as a potential means to limit the dissemination of antibiotic resistance. This study examines the response of nine representative antibiotic resistance genes (ARGs) encoding resistance to sulfonamide (sulI, sulII), erythromycin (erm(B), erm(F)), and tetracycline (tet(O), tet(W), tet(C), tet(G), tet(X)) to various laboratory-scale sludge digestion processes. The class I integron gene (intI1) was also monitored as an indicator of horizontal gene transfer potential and multiple antibiotic resistance. Mesophilic anaerobic digestion at both 10 and 20 day solids retention times (SRTs) significantly reduced sulI, suII, tet(C), tet(G), and tet(X) with longer SRT exhibiting a greater extent of removal; however, tet(W), erm(B) and erm(F) genes increased relative to the feed. Thermophilic anaerobic digesters operating at 47 °C, 52 °C, and 59 °C performed similarly to each other and provided more effective reduction of erm(B), erm(F), tet(O), and tet(W) compared to mesophilic digestion. However, thermophilic digestion resulted in similar or poorer removal of all other ARGs and intI1. Thermal hydrolysis pretreatment drastically reduced all ARGs, but they generally rebounded during subsequent anaerobic and aerobic digestion treatments. To gain insight into potential mechanisms driving ARG behavior in the digesters, the dominant bacterial communities were compared by denaturing gradient gel electrophoresis. The overall results suggest that bacterial community composition of the sludge digestion process, as controlled by the physical operating characteristics, drives the distribution of ARGs present in the produced biosolids, more so than the influent ARG composition.     

Antibiotic resistance genes as emerging contaminants: studies in northern Colorado

This study explores antibiotic resistance genes (ARGs) as emerging environmental contaminants. The purpose of this study was to investigate the occurrence of ARGs in various environmental compartments in northern Colorado, including Cache La Poudre (Poudre) River sediments, irrigation ditches, dairy lagoons, and the effluents of wastewater recycling and drinking water treatment plants. Additionally, ARG concentrations in the Poudre River sediments were analyzed at three time points at five sites with varying levels of urban/agricultural impact and compared with two previously published time points. It was expected that ARG concentrations would be significantly higher in environments directly impacted by urban/agricultural activity than in pristine and lesser-impacted environments. Polymerase chain reaction (PCR) detection assays were applied to detect the presence/absence of several tetracycline and sulfonamide ARGs. Quantitative real-time PCR was used to further quantify two tetracycline ARGs (tet(W) and tet(O)) and two sulfonamide ARGs (sul(I) and sul(II)). The following trend was observed with respect to ARG concentrations (normalized to eubacterial 16S rRNA genes): dairy lagoon water > irrigation ditch water > urban/agriculturally impacted river sediments (p < 0.0001), except for sul(II), which was absent in ditch water. It was noted that tet(W) and tet(O) were also present in treated drinking water and recycled wastewater, suggesting that these are potential pathways for the spread of ARGs to and from humans. On the basis of this study, there is a need for environmental scientists and engineers to help address the issue of the spread of ARGs in the environment.     

Prevalence of clinically relevant antibiotic resistance genes in surface water samples collected from Germany and australia

The prevalence and proliferation of antibiotic resistant bacteria is profoundly important to human health, but the extent to which aquatic environments contribute toward the dissemination of antibiotic resistant genes (ARGs) is poorly understood. The prevalence of 24 ARGs active against eight antibiotic classes (β-lactams, aminoglycosides, glycopeptides, chloramphenicols, tetracycline, macrolides, trimethoprim, and sulfonamides) was evaluated in surface water samples collected from Germany and Australia with culture independent methods. The ARGs most frequently detected both in Germany and Australia were sulI, sulII (77-100%), and dfrA1 (43-55%) which code for resistance to sulfonamide and trimethoprim. Macrolides resistance gene ermB was relatively more prevalent in the surface water from Germany (68%) than Australia (18%). In contrast, the chloramphenicol resistance gene catII was more frequently detected in Australia (64%) than Germany (9%). Similarly, β-lactams resistance gene ampC was more prevalent in the samples from Australia (36%) than Germany (19%). This study highlights wide distribution of ARGs for sulfonamide, trimethoprim, macroline, β-lactams and chloramphenicol in the aquatic ecosystems. Aquatic ecosystems can therefore be reservoirs of ARGs genes which could potentially be transferred from commensal microorganisms to human pathogens.     

A new strategy for the analysis of tetracycline residues in foodstuffs by a surface plasmon resonance biosensor

A new strategy was developed for the establishment of an indirect tetracycline assay using surface plasmon resonance (SPR). The principle for the assay is based on the most important resistance mechanism against tetracycline in gram-negative bacteria. Tetracyclines release the 46.6 kDa Tet repressor protein (TetR) from the tet operator (tetO), a biotinylated short double strand DNA sequence bound to a streptavidin biosensor chip. Tetracyclines present in a sample solution bind to the repressor protein, inducing a conformational change accompanied with a reduction of the affinity constant of TetR to tetO. We were able to quantify tetracycline residues in spiked raw milk samples in concentrations corresponding to the maximum residue limit (MRL) set by the European Union. Honey samples could also be analysed but with lower sensitivity. The assay detects seven of the most commonly used tetracyclines in veterinary medicine. Nine antibiotics of five other antibiotic classes were tested and no interference with the SPR assay was found. Thus, the described principle forms the basis for screening assays to routinely test for tetracycline residues in foodstuffs.     

婴幼儿食品中金黄色葡萄球菌污染情况及其耐药基因和新型肠毒素基因的检测

本文调查了婴幼儿奶粉和米粉中金黄色葡萄球菌的污染状况,并且检测其耐药基因和新型肠毒素基因。采集陕西省2010年和2012年间不同品牌的婴幼儿奶粉和米粉692份,进行金黄色葡萄球菌污染检验,采用PCR对分离菌株进行31种耐药基因和9种新型肠毒素基因的检测。692份样品中金黄色葡萄球菌的检出率为7.80%,其中2010年所采样品的检出率为8.17%,2012年所采样品的检出率为7.38%。分离的75株菌最终检测出12种耐药基因和2种新型肠毒素基因,检出率最高的耐药基因是tet K(72.00%),其次是bla Z(36.00%),erm C(29.33%),tet(K)F(21.33%),lin A/lin A’(12.00%),dfr G(8.00%),tet L(6.67%)和acc(6')(5.33%),最少的为erm B,msr A、msr B和drf K(均为1.33%)。新型肠毒素基因仅检测出sen(44.00%)和ser(4.00%)。不同采样年份、生产季节和货架期的样品中金黄色葡萄球菌的污染率不存在显著差异。并且这些菌株主要携带四环素、青霉素和大环内酯类抗生素的耐药基因,以及新型毒素基因sen。     

Fate of tetracycline resistance genes in aquatic systems: migration from the water column to peripheral biofilms

Antibiotic resistance genes (ARGs) are emerging contaminants that are being found at elevated levels in sediments and other aquatic compartments in areas of intensive agricultural and urban activity. However, little quantitative data exist on the migration and attenuation of ARGs in natural ecosystems, which is central to predicting their fate after release into receiving waters. Here we examined the fate of tetracycline-resistance genes in bacterial hosts released in cattle feedlot wastewater using field-scale mesocosms to quantify ARG attenuation rate in the water column and also the migration of ARGs into peripheral biofilms. Feedlot wastewater was added to fifteen cylindrical 11.3-m3 mesocosms (some of which had artificial substrates) simulating five different receiving water conditions (in triplicate), and the abundance of six resistance genes (tet(O), tet(W), tet(M), tet(Q), tet(B), and tet(L)) and 16S-rRNA genes was monitored for 14 days. Mesocosm treatments were varied according to light supply, microbial supplements (via river water additions), and oxytetracycline (OTC) level. First-order water column disappearance coefficients (kd) for the sum of the six genes (tetR) were always higher in sunlight than in the dark (-0.72 d(-1) and -0.51 d(-1), respectively). However, water column kd varied among genes (tet(O) < tet(W) < tet(M) < tet(Q); tet(B) and tet(L) were below detection) and some genes, particularly tet(W), readily migrated into biofilms, suggesting that different genes be considered separately and peripheral compartments be included in future fate models. This work provides the first quantitative field data for modeling ARG fate in aquatic systems.     

Enterococci from Appenzeller and Schabziger raw milk cheese: antibiotic resistance, virulence factors, and persistence of particular strains in the products

Enterococci are natural residents of human and animal intestinal tracts, and grow to high numbers in a variety of cheeses. The aim of this study was to determine the diversity of enterococci in two types of artisanal raw milk cheese (Schabziger and Appenzeller) and to investigate whether particular strains with triple resistance against chloramphenicol (Chl), tetracycline (Tet), and erythromycin (Ery) persist in the production system. Of 46 cheese samples, a total of 312 Enterococcus strains were isolated over a 5-month period on selective agar plates containing Chl, Tet, or, Ery. Enterococcus faecalis was the predominant species (80.7%), followed by Enterococcus faecium (5.1%), and Enterococcus durans (11.7%). According to the phenotypic resistance patterns, a selection of 150 strains was analyzed with PCR for the presence of genes encoding resistance to Ery (ereA, ereB, mphA, ermA, ermB, ermC, mrsA/mrsB, mefA/mefE), and Tet (tetM, tetL). Because virulence factors have been linked to the pathogenicity of enterococci, the strain selection was also tested for the presence of the following virulence factors: Agg, GelE, Cyl, Esp, EfaAfs, EfaAfm, Cpd, Cob, and Ccf. All tested strains contained at least two of the nine virulence genes taken into analysis. Pulsed-field gel electrophoresis patterns of the isolates showed a limited persistence of several strains over a period of 1 to 2 months in Schabziger, and more than 2 months in Appenzeller. Finally, the enterococcal flora in the two types of cheeses seems to be rather unrelated. Within 150 strains from 25 different cheese samples (11 Appenzeller and 14 Schabziger), 41 pulsed-field gel electrophoresis patterns could be identified, and only 1 of these was found in enterococci from both types of cheese.