蛋鸡场中大肠杆菌对抗生素及消毒剂的耐药性

为了解蛋鸡场中抗生素、消毒剂的耐药情况,从蛋鸡场的环境(粪便、土壤、水体等)和鸡蛋中分离大肠杆菌,测定7种抗生素和2种季铵盐消毒剂对大肠杆菌的最低抑制浓度(Minimum inhibitory concentration,MIC),检测18种抗生素耐药基因和5种季铵盐消毒剂耐药基因,并对大肠杆菌进行脉冲场凝胶电泳(Pulsed-field gel electrophoresis,PFGE)分析.共得到44株大肠杆菌;大肠杆菌对抗生素耐药率为6.82%-90.91%,对土霉素(OTC)耐药率最高,对庆大霉素(CN)耐药率最低,所有大肠杆菌对头孢噻呋钠(CFS)表现敏感,一共产生14种不同的耐药谱型,其中,AMLTMP-OTC是最主要的谱型(N=10,P=23.81%);抗生素耐药基因检出率为2.27%-84.09%,tet C检出率最高,qnr A检出率最低.消毒剂苯扎氯铵(Benzalkonium chloride,BC)、氯化十六烷基吡啶(Cetylpyridinium chloride,CPC)对大肠杆菌的MIC范围分别为8-64μg/m L、4-256μg/m L,仅检测出qac EΔ1耐药基因(N=18,P=40.91%),qac EΔ1阳性大肠杆菌与甲氧苄啶(TMP)耐药表型存在显著相关性(P0.05);44株大肠杆菌PFGE基因型相似性在32.11%-96.62%之间.本研究表明蛋鸡场中大肠杆菌存在交叉传播的情况,对部分抗生素耐药性严重,同时对消毒剂出现耐药性,应引起足够重视.     

链霉菌S227抗耐药性物质的分离及其活性研究

在抗耐药性活性筛选过程中,发现分离自四川峨嵋山森林土壤的一株链霉菌S227(Streptomyces sp.)的发酵液具有抗细菌抗生素耐药性生物活性.利用已建立的抗耐药性的活性检测方法(专利号:ZL01128969.4)[1]为跟踪手段,采用有机溶剂萃取、硅胶柱层析以及薄层层析等方法,对该菌发酵液中抗耐药性活性物质进行分离纯化,得到了具有抗耐药性的活性单体S227-4,初步鉴定为四聚糖.利用MIC法对该样品的抗耐药活性进行研究:在证明该样品本身不具有抗菌活性的基础上,以临床分离的耐药菌株为指示菌,考察了该样品与抗生素联合使用时对耐药菌抗生素MIC(最小抑菌浓度)值的影响,结果表明,S227-4在不影响耐药菌生长的浓度下与不同的抗生素联合使用,可以明显提高不同耐药菌对不同抗生素的敏感性,如S227-4(200μg/mL)可以使S.aureus12334对红霉素的敏感性提高128倍.图2表2参8     

不同补给水源的城市人工湖中异养菌耐药性及耐药菌种属分布研究

为了阐明不同补给水源的城市人工湖中异养菌耐药状况和耐药菌种属分布特征,选取分别以地表水和再生水为补水来源的XQ湖和FQ湖为代表进行研究。从2018年4—11月逐月采集水样,考察了各水样中对氨苄西林(AMP)、磺胺甲恶唑(SMZ)和四环素(TET)这3种不同种类抗生素具有耐药性的异养菌含量,并对分离菌株的耐药表型、耐药菌株的种属分布以及水质理化指标进行了分析。结果表明,2处人工湖中的AMP耐药菌和SMZ耐药菌含量约为10~2～10~4CFU·(100 m L)~(-1),而TET耐药菌含量则约10~1～10~3CFU·(100 m L)~(-1)。分离出的84株耐药菌归属于19个种,其中蜡状芽孢杆菌、大肠埃希氏菌、维氏气单胞菌、豚鼠气单胞菌和鸟氨酸拉乌尔菌为2个湖共有耐药菌。71.4%的耐药菌都是对AMP单一耐药,以蜡状芽孢杆菌为主。由于具有固有耐药性的细菌在分离出的耐药菌中占比很低,获得性耐药很可能在城市人工湖中异养菌耐药性的发展上发挥了主要贡献作用。地表水补水的XQ湖和再生水补水的FQ湖在总异养菌含量、耐药菌含量和检出率上均无显著差异。分离来源对16株耐药性气单胞菌的聚类无明显影响。     

畜禽养殖场附近污水中多粘菌素E耐药性肠杆菌的分离分析及内源质粒检测

畜禽养殖业大量使用抗生素引起了广泛的微生物耐药性问题,研究抗生素耐药菌株可以了解环境中细菌的耐药性情况和抗性基因的传播机制。采集畜禽养殖场周边的污水,分析了污水中多粘菌素E耐药性细菌和耐药性肠杆菌的丰度;同时以一株具有较高多粘菌素E耐药水平的肠杆菌R11为例,首先进行16S r DNA测序和抗生素耐药谱分析,然后通过质粒提取、酶切电泳以及脉冲场凝胶电泳(PFGE),分析其内源性质粒等情况。结果表明:在畜禽养殖场周边污水环境中,多粘菌素E耐药性细菌占细菌总数的0.14%~0.35%,多粘菌素E耐药性肠杆菌占肠杆菌总数的1.50%~7.40%,而肠杆菌占细菌总数的1.13%~2.45%,说明肠杆菌可能是携带多粘菌素E抗性基因的主要细菌种类,同时也是养殖业污水中细菌的主要组成部分。16S r DNA序列分析确定R11是一株Enterobacter cloacae,抗生素耐药谱分析R11具有多重耐药性,除对林可霉素敏感外,阿米卡星、四环素和环丙沙星的最小抑菌浓度(MICs)为10μg?m L~(-1),阿莫西林的MICs为25μg?m L~(-1),磺胺甲恶唑的MICs为32μg?m L~(-1),氨苄西林的MICs为40μg?m L~(-1),链霉素的MICs为90μg?m L~(-1),而多粘菌素E的MICs最高,达到96μg?m L~(-1)。质粒提取酶切和PFGE分离鉴定结果显示,R11菌株可能有多个内源性质粒。综上所述,R11菌株具有较高水平的多粘菌素E耐药性,并且具有多重耐药性和多个内源性质粒,对于研究多粘菌素E耐药性机制具有一定的参考价值。     

养殖场空气中E.coli磺胺类抗生素的抗性

本文从养殖场空气中分离出360株E.coli(大肠杆菌,Escherichia coli),应用肉汤微量稀释法和PCR方法,分离磺胺甲唑敏感菌株,检测抗生素抗性和抗性基因.在分离的E.coli中,对磺胺甲唑敏感菌株为95株(26.4%),有48株含有青霉素、氯霉素、四环素、环丙沙星、庆大霉素和利福平的抗性,而47株未含有抗性.其中,7株菌株含有1种抗生素抗性、11株菌株含有2种抗生素抗性、17株菌株含有3种抗生素抗性、6株菌株含有4种抗生素抗性、4株菌株含有5种抗生素抗性、3株菌株含有6种抗生素抗性.对抗生素的耐受能力依次为:氯霉素、青霉素、四环素、环丙沙星、庆大霉素、利福平.磺胺甲唑敏感菌株共检出163个抗性基因,sul1、int1、sul2、Int2、sul3检出数量依次为49、44、29、20和19;含一种、二种、三种、四种、五种抗性基因菌株分别为45、22、10、7、2;但有6株未检测出抗性基因.结果表明养殖场建场时间、抗生素使用、养殖规模等与抗生素抗性菌的抗性呈正相关;养殖场空气中分离的E.coli抗生素抗性较高,且具有多重抗性;抗生素抗性的表现型与其基因型之间出现不完全吻合现象.     

养殖虾塘常见耐药菌的分离鉴定与耐药基因检测

了解水产养殖区中抗生素耐药菌的分布及抗生素耐药基因的污染现状,有助于科学、合理地使用抗生素。采用诺氟沙星抗性平板和红霉素抗性平板统计虾塘泥样和水样中耐药微生物数量,从中筛选得到60株耐药菌株。基于16Sr DNA序列分析完成部分菌株的鉴定,同时,对耐药菌株携带的喹诺酮类(诺氟沙星)抗性基因gyr A及大环内酯类(红霉素)抗性基因erm B进行了扩增及测序分析。结果表明,虾塘水样细菌在诺氟沙星抗性平板中均未生长,虾塘泥样细菌对诺氟沙星的耐药率分别为0.36%、0.82%;1号虾塘泥样和水样中细菌对红霉素的耐药率分别为23.33%、18.50%,2号虾塘泥样和水样中细菌对红霉素的耐药率分别为20.00%、12.50%。耐药菌16S rDNA鉴定结果如下:南极适冷菌(Rheinheimera sp.)7株、不动杆菌(Acinetobacter sp.)6株、微杆菌(Microbacterium sp.)3株、厦门希瓦氏菌(Shewanella xiamenensis)2株、嗜水气单胞菌(Aeromonashydrophila)2株、豚鼠气单胞菌(Aeromonascaviae)1株。1号虾塘gyr A检出率为43.33%,erm B检出率为30.00%;2号虾塘gyr A检出率为10.00%,erm B检出率为70.00%。耐药基因序列比对结果显示,所测菌株序列与数据库中相应耐药基因序列相似性均≥97%。     

紫云英根瘤菌共生基因分布及其共生效应的多样性

从同一植株不同根瘤分离40株紫云英根瘤菌，所有菌株对10种抗生素的抗药性测定表明，该群体分为22个抗药类群，质粒检测显示所有公离株都含有质粒，质粒数1～4条，用快生型大豆根瘤菌USDA205质粒作参考，估测质粒Mr分布范围为83～226MU．根据图谱分析表明，该菌群可分为6个不同质粒型．各类型质粒通过与Dig-nodABC和Dig-nifHDK杂交，结果显示带有1条质粒的菌株其共生基因定位在染色体上．带有2条或2条以上质粒的菌株各拥有1条共生质粒，共生质粒Mr范围有差异，大约为117～220MU．研究结果也显示，不同质粒型的菌株其共生效应存在明显差异，其中第6质粒型的菌株共生固氮率最强，第1质粒型菌株共生固氮率较低．共生固氮能力最强的第6质粒型菌株，只占总菌数7．5％．     

畜禽养殖场排放物病原微生物危险性调查

以粪大肠菌群和沙门菌为指标,采集江苏省内10家不同养殖类型畜禽养殖场的排放物及其周边水、土样共37个进行观察与计数,同时分析排放污水中大肠杆菌的耐药性以及禽畜排泄物对土壤和水体耐药细菌数的影响.结果表明:调查的10家养殖场中有9家粪便未经无害化处理直接排放到水体或施于农田,且排放物的粪大肠菌群数全部严重超标,沙门菌检出率达19%,施新鲜粪肥的土壤中粪大肠菌群数(MPN,最大可能数)在105 g-1以上,水体中分离出的大肠杆菌表现出多重耐药性,抗生素抗性细菌总数也远高于未施新鲜粪肥的土壤及水体.     

亚抑菌浓度氟苯尼考作用下质粒pSD11对大肠杆菌适应性的影响

通过分析cfr阳性质粒pSD11对大肠杆菌的适应性影响,并测定氟苯尼考对携带质粒pSD11的大肠杆菌MG1655(Escherichia col MG1655)的最低筛选浓度(MSC),评价亚抑菌浓度氟苯尼考作用下多重耐药基因cfr在大肠杆菌中的持续存在和扩散传播的风险.通过电转化的方法将质粒pSD11导入模式菌E.coli MG1655中,获得耐药菌株E.coli MG1655/pSD11.通过测定生长曲线,比较无抗生素作用下耐药菌株E.coli MG1655/pSD11与同源敏感菌株E.coli MG1655的生长动力学参数,评价质粒pSD11对宿主菌适应性的影响.并通过比较分析不同亚抑菌浓度(0.031 25,0.062 5,0.125,0.25,0.5,1.0和2.0μg/mL)氟苯尼考作用下的生长速率,测定氟苯尼考对携带质粒pSD11的宿主菌最小筛选浓度(MSC).携带质粒pSD11的耐药菌E.coli MG1655/pSD11的延滞时间(2.853 3±0.031 8 h/λ)相对于同源敏感菌E.coli MG1655极显著延长(2.825 3±0.024 4 h/λ),且世代时间也显著延长(分别为:1.705 7±0.006 4 h/G和1.526 9±0.007 8 h/G).氟苯尼考对耐药菌E.coli MG1655/pSD11的MSC为0.042 5μg/mL约为相应敏感菌的MICsus的1/100.本研究表明携带cfr基因的质粒pSD11在无抗生素选择下对宿主菌有明显的适应性影响,但是在极低的氟苯尼考浓度的作用下存在选择优势,能够平衡适应性成本使宿主菌得到富集与传播,以期为对该质粒在环境中的传播风险评估及氟苯尼考的临床应用提供的参考.(图2表2参18)     

无苯酚培养基分离到的降酚菌多样性的分子分析

用3种不含苯酚的培养基(YPG、10%YPG和LB)从上海焦化厂废水处理系统(A2/O法)好氧池的悬浮污泥分离到24株降酚菌株.通过16SrDNAPCR扩增产物的限制性酶切分析(amplifiedribosomalDNArestrictionanalysis,ARDRA)、ERICPCR指纹图谱分析、多组分苯酚羟化酶大亚基(thelargestsubunitofthemulticomponentphenolhydroxylase,LmPH)基因的PCR扩增及16SrRNA基因测序的方法对这些降酚菌株进行表征.通过ARDRA分型,将这24株降酚菌株分为8个类型;利用ERICPCR可以将这24个菌株分为17种类型,说明同一ARDRA类型内菌株具有多样性.对17个ERIC类型代表菌株的16SrDNA扩增产物进行克隆并测序,测序结果在GenBank和RDP中进行比对.结果表明,与这17个代表菌株同源性最高的菌中,有6株是未见报道具有降酚功能的菌株.在这24株降酚菌中,有19株在苯酚含量为200mg/L的MP培养基中培养5d,苯酚降解率为20%左右,其余5株苯酚降解率达到100%,且均属于Rhodococcus属.本研究在降酚菌生物多样性上作了有益的探索.图2表1参20     

Plasmid mediated antibiotic resistance in marine bacteria

This research work was conducted in Uppanar estuary to ascertain the role of plasmids in the antibiotic resistance of bacteria. Water and sediment samples were collected for a period of three months. When tested against 20 antibiotics 22 MAR strains were isolated from the samples, which were found resistant to 5-13 antibiotics. They belong to 7 genera and 10 species. Gram-negative bacteria namely Neisseria mucosa, N. sicca, Branhamella catarrhalis, Klebsiella ozaenae, Citrobacterintermedius, Pseudomonas fluorescens and Enterobacter aerogenes were isolated. Gram-positive bacteria were of Bacillus subtilis, B. megaterium and Micrococcus luteus. When plasmid curing was done using acredine orange, the resistance against penicillin-G, ampicillin, tetracycline, amoxycillin, kanamycin, and chloramphenicol were totally lost in all strains, which confirmed the role of plasmid in these strains against antibiotics. Ten strains belong to different species were selected for the plasmid isolation and electrophoresis was done. Presence of plasmids in all strains was confirmed and the molecular weight was in the range of 2850 to 3170 bp. The study revealed that MAR strains are common in Uppanar estuary and they are plasmid mediated. This environment is seemed to be deteriorating at an alarming rate.     

Multiple antibiotic resistance patterns of rhizospheric bacteria isolated from Phragmites australis growing in constructed wetland for distillery effluent treatment

Susceptibility patterns of 12 different antibiotics were investigated against rhizospheric bacteria isolated from Phragmites australis from three different zones i.e. upper (0-5 cm), middle (5-10 cm), lower (10-15 cm) in constructed wetland system with and without distillery effluent. The major pollutants of distillery effluent were phenols, sulphide, heavy metals, and higher levels of biological oxygen demand (BOD), chemical oxygen demand (COD) etc. The antibiotic resistance properties of bacteria were correlated with the heavy metal tolerance (one of distillery pollutant). Twenty-two species from contaminated and seventeen species from non-contaminated site were tested by agar disc-diffusion method. The results revealed that more than 63% of total isolates were resistance towards one or more antibiotics tested from all the three different zones of contaminated sites. The multiple-drug resistance property was shown by total 8 isolates from effluent contaminated region out of which 3 isolates were from upper zone, 3 isolates from middle zone and 2 isolates were from lower zone. Results indicated that isolates from contaminated rhizosphere were found more resistant to antibiotics than isolates from non-contaminated rhizosphere. Further this study produces evidence suggesting that tolerance to antibiotics was acquired by isolates for the adaptation and detoxification of all the pollutants present in the effluent at contaminated site. This consequently facilitated the phytoremediation of effluent, which emerges the tolerance and increases resistance to antibiotics.     

Influence of lignin, pentachlorophenol and heavy metal on antibiotic resistance of pathogenic bacteria isolated from pulp paper mill effluent contaminated river water

Pulp paper mill pollutants are the major source of aquatic contamination having metals, lignin and chlorophenols. Study was conducted to see the effect of these contaminants on antibiotic resistance pattern of isolated bacteria. Pulp paper effluents were evaluated for its physico-chemical properties i.e, BOD 72143 +/- 164.81 to 22.32 +/- 2.48, COD 213136 +/- 583.59 to 60.40 +/- 6.34, total phenol 386 +/- 71.24 to 0.43 +/- 0.0, lignin 26312 +/- 258.59 to 73.67 +/- 31.81and microbial quality i.e. K. pneumonae, S. typhi, S. faecalis, P. aeruginosa, E. coli, Ent. faecalis, A. hydrophila, B. subtilis, S. aureus, Y enterolitica and V vulrificus. Antibiotic sensitivity (10-30 microg), heavy metal resistance (100-1000 microg ml(-1)), lignin (1000-10,000 ppm) and pentachlorophenol (100-1000 ppm) tolerance of bacterial strains were assessed by seven classes of antibiotics. Eleven bacterial isolates were found multidrug resistant towards antibiotics, heavy metal, lignin and PCP. Out of 11 isolates, 90.9% were found resistant against eleven antibiotics which acquired 100% resistant in presence of heavy metal, lignin and chlorophenols. Results also revealed that concentration of lignin (50-350 ppm) and PCP (5-30 ppm) induced maximum growth (273-8050 cfu ml(-1)) of pathogenic bacteria in river water.     

驯化菌株降解油制气废水能力的比较

从3种污泥中驯化筛选出10种菌株,研究了各菌株对油制气废水不同污染指标的处理能力差别,以及各菌株对废水中芳烃化合物的降解能力.结果表明,各菌株可在初始阶段提高废水的BOD值,在高低废水浓度条件的降解能力基本一致;酚的去除率可达93.9%,但对废水中氨氮的去除率小于27.3%;实验采用的3种菌株对废水中的芳烃化合物都能降解,但其对芳环数≤3的芳烃化合物的降解能力强于对芳环数为4~6的芳烃化合物的降解能力.图4表2参10     

Antibiotic resistance patterns of Salmonella and Escherichia coli in the groundwater of Cyprus

In addition to diet-based vectors of disease, the contribution of water-borne zoonotic agents to gastrointestinal illnesses may be significant, but this has yet to be investigated for Cyprus. Our main objective was to evaluate antibiotic resistance patterns of Salmonella and Escherichia coli in groundwater samples collected at confined animal feeding operations. This is the first report on the occurrence of antibiotic-resistant Salmonella and E. coli strains in the groundwater of Cyprus. Most of Salmonella isolates belonged to the subgroup enterica, whereas none of the E. coli isolates expressed the verotoxin-encoding gene. Out of 27 isolated Salmonella strains, nearly half of them were resistant to at least one or more antibiotic, whereas the highest resistance was exhibited by sulphamethoxazole (85%), followed by streptomycin (39%), and tetracycline (31%). For the E. coli isolates, nearly a third of them showed resistance to at least one antibiotic, whereas the selection of antibiotic resistance was equal among sulphamethoxazole, tetracycline and streptomycin (20%). This study demonstrated that Salmonella and E. coli in groundwater could pose a public health risk via oral ingestion of contaminated water. Best management practices are needed for overexploited groundwater supplies of rural areas, minimizing human exposure to antibiotic-resistant pathogens.     

莱州湾近岸海域中典型抗生素与抗性细菌分布特征及其内在相关性

为了揭示海陆衔接区环境中抗生素与抗性细菌分布特征及其内在相关性,以莱州湾及其主要入海河流为研究区域,利用HPLC-MS/MS分析样品中15种磺胺类抗生素(SAs)和6种喹诺酮类抗生素(QNs)的浓度,并通过改良的Method 1604(US EPA)评估海水与沉积物中2种典型水传病原微生物大肠杆菌(E.coli)与金黄色葡萄球菌(S.aureus)的抗生素抗性水平,进而探讨该区域水体中抗性菌株的分布特点以及微生物抗性率与相应抗生素浓度的相关性。结果显示,莱州湾水体与沉积物中普遍存在磺胺与喹诺酮类抗生素残留及抗性污染问题。两大类抗生素在水体中平均残留浓度分别为3.89 ng·L~(-1)(SAs)和234.68ng·L~(-1)(QNs),在沉积物中分别为0.91 ng·g~(-1)(SAs)和49.37 ng·g~(-1)(QNs),且分布特征基本呈现自河流向海洋逐渐递减的趋势,说明河流输入是莱州湾抗生素污染的主要来源。在水体中,具有磺胺类抗性的E.coli和S.aureus平均检出量分别达到2 018和4 683 CFU·L~(-1),抗性率范围分别在0%~37.3%和10.6%~45.8%之间;而2种喹诺酮类抗性病原微生物的平均检出量则相对较低,分别为1 315 CFU·L~(-1)(E.coli)和1 461 CFU·L~(-1)(S.aureus),抗性率分别为0%~50.0%和0%~20.8%;此外,相比于E.coli,S.aureus为沉积物中的主要抗性病原微生物,磺胺与喹诺酮类抗性S.aureus检出率均高于80%,平均检出量分别为24CFU·g~(-1)和18 CFU·g~(-1)。相关性分析表明,莱州湾近岸海域水体中磺胺类抗生素浓度与磺胺类抗性微生物总量之间具有良好的线性关系,然而其与微生物抗性率之间并未表现出相似的规律,说明近岸海洋环境中抗生素的残留量不是影响抗性菌株丰度的唯一因素。     

应用全细胞蛋白SDS-PAGE分子标记技术验证含羞草根瘤菌的结瘤能力

对云南省热带及亚热带地区的含羞草根瘤菌进行了分离,选择其中40株菌为接种菌株,通过结瘤试验并采用全细胞蛋白SDS-PAGE分子标记方法研究了其结瘤能力.经过结瘤试验,发现除菌株SWF66075和SWF66093没有结瘤外,其它38株菌株均与含羞草植物结瘤.结瘤率为95%.从结瘤试验所获根瘤中,分离得到结瘤菌株,采用全细胞蛋白SDS-PAGE分子标记对结瘤菌株与接种菌株进行了比较研究.蛋白图谱及聚类分析显示,26株接种菌株与其结瘤菌株的全细胞蛋白分子图谱完全相同,在100%的相似水平上与其结瘤菌株聚在一起,说明宿主植物所形成的根瘤确系接种菌株侵入所致,因而可将这些菌株确认为根瘤菌菌株;而SWF66012、SWF66029、SWF66044和SWF66058等12株菌株的结瘤菌株与其各自接种菌株的全细胞蛋白图谱存在较大差异,推测这12株接种菌株与其结瘤菌株可能不是同一菌株,尚不能确定它们与含羞草植物的结瘤能力,这些菌株是否为根瘤菌菌株仍需进一步验证.研究结果表明,全细胞蛋向SDS-PAGE分子标记技术是一种快速、准确地验证根瘤菌结瘤能力的方法.该方法进一步完善了结瘤试验,并初步揭示了根瘤菌的竞争结瘤能力,适用于对大量根瘤内分离菌株进行根瘤菌的证实研究.图2参28