两株高效相思根瘤菌的抗药性研究及其质粒组成分析

目的:研究相思根瘤菌质粒与其抗药性之间的关系。方法:研究了相思根瘤菌MZ和AJ018在含不同浓度的抗生素的固体培养基和液体培养基的生长情况,并用碱裂解方法对其质粒组成进行检测。结果:两菌株对链霉素和卡那霉素均无抗性,而对其它抗生素都有不同程度的抗性。MZ菌株对实验中的氯霉素、氨苄青霉素、四环素三种抗生素的耐受范围与最大耐受值都比AJ018强,当平板培养基中氨苄青霉素、四环素、氯霉素的终浓度分别为250μg/ml、75μg/ml、150μg/ml时,AJ018在平板上无菌落生长,当三种抗生素终浓度分别为800μg/ml1、50μg/ml、400μg/ml时无菌落生长。两菌株都含有一个大约50kb的质粒。     

B-animalisV9质粒检测及抗生素敏感性试验

本实验对动物双歧杆菌Vg（B．animalisvg）菌株进行质粒检测及抗生素敏感性测试。结果表明,Baninalis V9菌株无质粒检出。该菌株对针对G＋菌的抗生素,如青霉素类（青霉素G、氨苄青霉素）和大环内酯类（氯霉素、红霉素）表现出高度敏感;对抑制细菌蛋白质合成的广谱（G＋、G-）抗生素（四环素、利福平、痢特灵）表现出中度敏感;对G-菌的抗生素,如氨基糖苷类（庆大霉素、链霉素）、喹诺酮类（诺氟沙星）及内酰胺类（卡那霉素、丁胺卡那霉素）表现出耐药性;而对广谱抗生素磺胺甲基异嗯唑也表现为耐药。     

双歧杆菌模拟胃肠环境中抗性的研究

采用PYG培养基,模拟胃肠环境,即低pH值(1．5—4．5),高胆汁酸盐(9．1％-0．4％)对青春双歧杆菌进行抗性研究。同时对肠道致病性大肠埃希菌、福氏志贺菌和金黄色葡萄球菌拮抗性以及对氨苄青霉素、庆大霉素、卡那霉素和氯霉素的耐药性进行研究。结果表明,青春双歧杆菌在pH4．5时有较强的生存能力,4h活菌效可达10^7CFU／m1以上。且能在含0．1％-0．4％的胆汁盐中存活,在0．1％-0．2％时4h活菌总数可达10^5CFU／m1以上。同时对致病性大肠埃希菌、福氏志贺菌和金黄色葡萄球菌具有明显的抑菌作用,且对庆大霉素中度敏感,对氨苄青霉素、卡那霉素和氯霉素有耐受性。     

发光酶基因lux AB标记硅酸盐细菌NBT菌株的研究

外源基因标记技术为研究土壤引入细菌的生态行为提供了有效的检测手段,通过选择不同的碳源和降低碳氮比筛选获得0．25％麦芽糖作为碳源的菌体制备培养基,对硅酸盐细菌BT菌株进行紫外诱变和抗生素抗性驯化获得—株抗利福平200μg·ml^-1的NBT-R200菌株,含发光酶基因luxAB的质粒pTR102：：luxAB在辅助质粒pRK2013的帮助下转入该菌株中,从而赋予NBT菌株以发光活性和利福平、卡那霉素、四环素三种抗生素抗性．以对数生长期的菌体制备受体细胞,发现对数生长前期的细胞转移频率最高,可达6．70×10^-5,杂交比例以1：1：1适宜．标记菌株RL85的释钾能力没有丧失且有提高,发光特性稳定,连续转接20次后仍具有发光活性和3种抗生素抗性,适用于根际微生态学研究。     

苏芸金杆菌抗药性的研究

本文报道了苏芸金杆菌包括30个亚种标准菌株在内的34个菌株对6种抗菌素的抗性研究结果。结果表明所有被试菌株对氨苄青霉素50—200单位/ml表现出较强的抗性,其中有17个菌株对链霉素12.5ug/ml、18个菌株对四环素25单位/ml显示出弱抗性。全部供试菌株对链霉素25—50ug/ml、四环素50单位/ml、红霉素12.5—50ug/ml、氯霉素12.5—100单位及卡那霉素12.5单位/ml均未显示出任何抗性。显微镜检查了部分菌株在含四环素25单位/ml,链霉素12.5ug/ml培养基上形成的抗性菌落的菌体发育情况,在所检查的11株链霉素抗性菌落中,有6个菌株的抗性菌落可形成正常的芽孢和晶体,在10株四环素抗性菌落中,有8个菌株可形成正常的芽孢和晶体。试验还测定了其中5株苏芸金杆菌对四环素25单位/ml的抗性频率为7.7—13.2％。     

大肠杆菌抗药性质粒pFD3的链霉素抗性表达

Cohen等‘31研究大肠杆菌抗药性质粒R6 （此质粒携带卡那霉素、新霉素、氯霉素、四环 素、链霉素抗性）,发现在用卡那霉素抗性、新 霉素抗性或氯霉素抗性作为选择性标记进行转 化时,极少数转化子失掉链霉素抗性。本文报 道大肠杆菌抗药性质粒pFD 3（此质粒携带四 环素、链霉素、氯霉素、氨苇青霉素、磺胺抗性） 在经结合转移或转化途径传递给大肠杆菌C'₆₀₀ 时,所得的转移子、转化子中四环素、氯霉素、氨 苇青霉素及磺胺抗性都能正确表达,而链霉素 抗性则不能完全表达。     

绿脓杆菌抗药性质粒接合和转化作用的研究

绿脓杆菌是重要的院内感染菌,多数菌株具有多重抗药性,且近年来有不断增多的趋势,因而,该菌的抗药性传播途径越来越受到重视。本文对临床分离的3株绿脓杆菌(PA1、PA2、PA3)进行了抗药性测定,并以E。coli为受体菌,对其抗药性质粒的接合和转化作用进行了研究。结果表明供试菌对多种临床常用的抗生素(氨苄青霉素Amp、氯霉素Chl、红霉素Ery、庆大霉素Gen、青霉素Pen和链霉素Str)都具有抗性,尤其对Gen、Str、Ery等临床常用药物的抗性为强,所能耐受的药物浓度可高达100u/ml左右;不同的菌株所抗的抗生素种类及浓度不完全相同,表明不同的菌株具有不同的抗药谱。     

温泉嗜热菌LY的分离鉴定及质粒的初步研究

目的:从海南温泉中分离鉴定嗜热微生物,并了解其生理生化特征,同时对其质粒进行初步研究.方法:稀释平板法分离嗜热菌;形态学、生理生化和分子生物学方法进行菌种鉴定;氯化铯超速离心法测定菌株(G+ C) mol％含量;利用吖啶橙消除菌株质粒并分析质粒消除前后的生物学特性.结果:获得1株温泉嗜热菌菌株LY,其最适生长温度在80 ～ 85℃之间,最适pH值为6.0,对链霉素、卡那霉素、四环素、氨苄青霉素、氯霉素敏感.结合形态学、生理生化测试和16S rRNA序列分析鉴定菌株为Bacillus sp.LY.菌株的(G+C)mol％含量为66.90％.菌株质粒大于3000 bp,质粒消除前后,其生物学特性无明显差异.结论:温泉嗜热菌LY可作为耐热候选菌株进行后续深入研究.     

嗜热脂肪芽孢杆菌抗药性质粒的转化

从堆肥和污泥中分得8株抗药性高温细菌,经电泳检查有质粒存在,对其中1株具有卡那霉素和链霉素抗性的嗜热脂肪芽孢杆菌T617-8的质粒DNA,用电镜测得分子量为26.6×10~6道尔顿。T617-8(Km~rSm~r)菌株经溴化乙锭处理后,对卡那霉素敏感,同时质粒消失。为此确证,卡那霉素抗性是由质粒所控制。以T617-8的质粒(Km~r)DNA,对消除质粒后的菌株(Sm~r)的原生质体进行转化,获得了抗卡那霉素和链霉素的转化子。     

大肠杆菌抗药性质粒pFD3和pFD11的研究

从人的粪水中分离到大肠杆菌A2和B5,经药物敏感度测定,大肠杆菌A2抗链霉素,四环素、氯霉素、氨苄青霉素和磺胺药;大肠杆菌B5抗链霉素、四环素、磺胺药。通过接触转移实验证明,这些抗性决定因子分别位于质粒pFD3和pFD11上。     

Broad host range cloning vectors for gram-negative bacteria

A series of cloning vectors has been constructed based on the broad-host-range plasmid R300B. One of these vectors, pGSS33, has a size of 13.4 kb and carries four antibiotic resistance genes [ampicillin (Apr), chloramphenicol (Cmr), streptomycin (Smr) and tetracycline (Tcr)], all of which have restriction sites for insertional inactivation. The derivation, structure and uses of the plasmids are described.     

Plasmid vehicles for direct cloning of Escherichia coli promoters.

A multicopy plasmid cloning vehicle, pGA22, which carries genes for ampicillin resistance (Apr), tetracycline resistance (Tcr), chloramphenicol resistance (Cmr), and kanamycin resistance (Kmr) has been constructed. This plasmid has five unique sites for restriction endonucleases EcoRI, PstI, XhoI, SmaI, and SalI within antibiotic resistance genes. pGA22, which is 5.1 megadaltons in size, has a low copy number (probably fewer than 10 per genome), is capable of relaxed replication, and is mobilized by F-factor at a frequency of 10(-5). A series of promoter-cloning vehicles, pGA24, pGA39, and pGA46, has been developed from pGA22. In these plasmids the natural promoter for Tcr has been removed and has been replaced by small deoxyribonucleic acid fragments carrying unique sites for several restriction endonucleases. Cells carrying these vectors are sensitive to tetracycline unless insertional activation of the Tcr occurs by cloning a promoter-carrying deoxyribonucleic acid fragment in one of the unique sites adjacent to the 5' end of Tcr. In this way, promoters carried on a HindIII-generated deoxyribonucleic acid fragment can be inserted at the HindIII site of plasmid pGA24, pGA39, or pGA46. A promoter in fragments generated by digestion with restriction endonuclease XmaI or PstI or by any restriction endonucleases which generate flush ends, such as SmaI, PvuII, HpaI, HincII, or HaeIII, can be clones in plasmid pGA39. Plasmid pGA46 can be used to detect a promoter fragment carried on a BglII, BamHI, MboI, or PstI fragment. We also describe a plasmid, pGA44, with a unique KpnI site in the rifampin resistance gene rpoB.     

Isolation of a new plasmid pIRL19: its use in construction of small vectors and detection of specific sequence in a foreign DNA

A new plasmid, pIRL19, was constructed by ligating a 1875-bp HaeII fragment carrying the ampicillin-resistance (Apr) gene to a 370-bp HaeII fragment containing the replication origin of the plasmid pBR322. The plasmid essentially contains only the basic replicator and the Apr gene. This basic replicator provides a valuable initial building block for in vitro construction of other very small vectors with antibiotic-resistance determinants. To illustrate this potential, we have transferred the chloramphenicol-resistance (Cmr) gene and a part of the Apr gene from the plasmid pBR329 into pIRL19 such that the new plasmid pIRL20 acquired the Cmr gene and maintained the integrity of its Apr structural gene.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA

An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4.5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10(7) to 4 x 10(8) transformants per micrograms DNA. The transformation frequency (transformants per regenerant) was 0.5 to 1.0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2.5 x 10(5) transformants (microgram DNA)-1] and pTHT15 [1.8 x 10(5) transformants (micrograms DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 degrees C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 degrees C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 degrees C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50, 60, and 65 degrees C was 69, 18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 degrees C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 degrees C.     

Copper amendment of agricultural soil selects for bacterial antibiotic resistance in the field

AIMS: The objective of this study was to determine whether Cu-amendment of field plots affects the frequency of Cu resistance, and antibiotic resistance patterns in indigenous soil bacteria. METHODS AND RESULTS: Soil bacteria were isolated from untreated and Cu-amended field plots. Cu-amendment significantly increased the frequency of Cu-resistant isolates. A panel of isolates were characterized by Gram-reaction, amplified ribosomal DNA restriction analysis and resistance profiling against seven antibiotics. More than 95% of the Cu-resistant isolates were Gram-negative. Cu-resistant Gram-negative isolates had significantly higher incidence of resistance to ampicillin, sulphanilamide and multiple (> or =3) antibiotics than Cu-sensitive Gram-negative isolates. Furthermore, Cu-resistant Gram-negative isolates from Cu-contaminated plots had significantly higher incidence of resistance to chloramphenicol and multiple (> or =2) antibiotics than corresponding isolates from control plots. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this field experiment show that introduction of Cu to agricultural soil selects for Cu resistance, but also indirectly selects for antibiotic resistance in the Cu-resistant bacteria. Hence, the widespread accumulation of Cu in agricultural soils worldwide could have a significant effect on the environmental selection of antibiotic resistance.     

Lack of expression of RP4-specified beta-lactamase in Azospirillum brasilense

Plasmid RP4, which normally confers resistance to ampicillin (Apr), tetracycline (Tcr), and kanamycin (Kmr) to its hosts, failed to express enhanced Apr when transferred from Escherichia coli to Azospirillum brasilense which has its own intrinsic beta-lactamase. Even in a beta-lactamase-deficient mutant, A. brasilense RG-D16, no increase in beta-lactamase or significant Apr appeared following transfer of RP4. However, A. brasilense RG (RP4) and A. brasilense RG-D16 (RP4) did exhibit Tcr Kmr. When RP4 was transferred back from A. brasilense to E. coli all three drug resistances and beta-lactamase activity were fully expressed.     

Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils

Abstract: A total of 132 different fluorescent Pseudomonas strains were isolated from several agricultural and industrial soils. The bacteria from the two different soil environments were compared for species and biotype variation, antibiotic and heavy metal resistance profiles, ability to degrade polyaromatic hydrocarbons, and plasmid incidence. Irrespective of the soil type, the isolates belonged to Pseudomonas fluorescens biotypes I–VI and Pseudomonas putida biotype B. Except for a streptomycin resistant isolate from one of the industrial soils, all the strains had the same antibiotic resistance profile. However, there was a higher incidence of heavy metal resistance and polyaromatic hydrocarbon degradation phenotypes in the isolates from industrial soils than from the agricultural soils. Only 2 out of 68 strains from agricultural soil were found to carry plasmids, while 28 out of 64 strains from industrial soil had plasmids. A majority of the plasmids (56%) were estimated to be larger than 50 kb, indicating that they could encode transfer functions. However, transferability as indicated by the ability to mobilize an IncQ plasmid (tra⁻, mob⁺), was observed with only one plasmid. None of the plasmid(s) containing isolates hybridized to a ³²P-labelled repP probe suggesting that none of the indigenous plasmids in the soil fluorescent Pseudomonas strains was related to the IncP group of conjugative plasmids commonly associated with resistance and catabolic genes.