Characteristics of two transferable aminoglycoside resistance plasmids in Escherichia coli isolated from pig and chicken manure

pRKZ3 is a non-conjugative IncQ plasmid, while pKANJ7 is a conjugative IncX plasmid. The optimal mating time of pKANJ7 varied under different conditions. Both of the two transferable ARPs had little impact on the growth of their hosts. A relatively high level of fitness cost was observed for pKANJ7. The fitness cost of ARPs depended on their hosts. Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.     

Effect of culture time on the growth curve and power performance in a microbial fuel cell at a fixed amount of liquid culture

The microbial fuel cell uses the microorganism biochemistry to carry on the energy conversion. Concerning the experimental precision, the colony culture would be replaced by a fixed amount of liquid culture for Microbial fuel cell of Escherichia coli. The anode and cathode chambers whose each volume is 100 mL were utilized, the effective surface area of proton exchange membrane Nafion-117 is about 9 cm². In addition, the electrode area of carbon cloth is 20 cm². Three kinds of Escherichia coli, named as BCRC No. 10322, 10675 and 51534, respectively, would be selected. Results show that the electricity performance of Escherichia coli of BCRC No.51534 is better than the other microorganism studied because of having a larger open circuit voltage of 1.01 V and limiting current 22 mA, the maximum power density of 1342 mW/m^2, and average working power density of 295 mW/m² would be produced. These results would be useful to improve the performance of microbial fuel cell.     

Antimicrobial resistance in Escherichia coli and Salmonella spp. isolates from fresh produce and the impact to food safety

Foodborne diseases associated with fresh produce consumption have escalated worldwide, causing microbial safety of produce of critical importance. Bacteria that have increasingly been detected in fresh produce are Escherichia coli and Salmonella spp., both of which have been shown to progressively display antimicrobial resistance. The study focused on the assessment of antimicrobial resistance of these enteric bacteria from different kinds of fresh produce from various open air markets and supermarkets in the Philippines. Using the disk diffusion assay on a total of 50 bacterial isolates obtained from 410 fresh produce surveyed, monoresistance to tetracycline was observed to be the most prevalent (38%), followed by multidrug resistance to tetracycline, chloramphenicol, ciprofloxacin, and nalidixic acid (4%), and lastly by dual resistance to tetracycline and chloramphenicol (2%). Using multiplex and simplex polymerase chain reaction (PCR) assays, tetA (75%) and tetB (9%) were found in tetracycline resistant isolates, whereas catI (67%) and catIII (33%) were detected in chloramphenicol resistant isolates. Sequence analysis of gyr and par genes from the ciprofloxacin and nalidixic acid resistant isolates revealed different mutations. Based on the results, fresh produce act as a reservoir of these antibiotic resistant bacteria which may pose health threat to consumers.     

群体感应抑制剂与磺胺甲恶唑、盐酸强力霉素对大肠杆菌的联合毒性及其机制初探

群体感应抑制剂(QSIs)具有不产生抗药性的特点,从而被作为抗生素的可能替代品,具有广阔的应用前景,因此其存在着与传统抗生素环境联合暴露的可能,但是目前尚缺乏相关联合效应的研究。本文以大肠杆菌(Escherichia coli)为受试生物,测定了7种QSIs(DL-焦谷氨酸、N-乙烯基吡咯烷酮、呋喃酮乙酸酯、2-甲基四氢呋喃-3-酮、3,4-二溴-2(5H)-呋喃酮、(R)-3-吡咯烷醇、D-脯氨醇)分别与磺胺甲恶唑(SMX)和盐酸强力霉素(DH)的二元联合毒性,并初步探讨了它们的联合作用机制。结果表明,前5种QSIs作用于AI-2类信号分子介导的群体感应系统,与AI-2类信号分子竞争结合LsrB蛋白,此通路与SMX、DH的作用通路互不影响,因此联合效应为相加;后2种QSIs作用于AI-1类信号分子介导的群体感应系统,与AI-1类信号分子竞争结合SdiA蛋白,而SMX、DH的作用可能刺激SdiA蛋白的表达,从而需要消耗更多的QSIs与SdiA结合,因而联合效应为拮抗。本实验研究可为传统抗生素与QSIs联合暴露的生态风险评价提供一定理论基础。     

水环境中大肠杆菌多特征抗原及抗体的制备研究

为了建立用于水环境中大肠杆菌的酶联免疫快速检测方法,针对水中大肠杆菌属多种血清型的特征,制备了水环境样品中大肠杆菌的多特征抗原,包括全菌体抗原、破碎全菌体抗原、菌体抗原、鞭毛抗原和菌毛抗原;采用这5种大肠杆菌抗原分别免疫新西兰大白兔,获得了5种大肠杆菌多克隆抗体,抗体效价高、纯度好,具有较强且稳定的特异性结合抗原的功能;间接ELISA方法表明,全菌体抗体和破碎全菌体抗体的效价均大于1×10⁵,性能优良.基于上述抗体,建立了水中大肠杆菌间接ELISA检测方法,实际水样的检测结果表明,检测限可达10⁴个/L.     

避光处理对污水紫外线消毒后大肠杆菌光复活的影响研究

以大肠杆菌为受试菌种,研究了避光处理对紫外线消毒后三级出水中细菌光复活的影响.将紫外线消毒后的水样避光放置一定时间后再置于光复活条件下,研究了大肠杆菌的复活特性.结果表明,避光放置6h以内,见光后大肠杆菌的光复活能力即刻恢复,并最终达到与非避光处理基本相同的最大复活值;5 mJ/cm2和20mJ/cm2紫外线剂量照射后,最大复活百分比分别为31.1%和0.45%,复活速率分别为1.67×104CFU·(mL·h)-1和125 CFU·(mL·h)-1.延长避光时间至24 h,5 mJ/cm2和20mJ/cm2紫外线剂量照射后的大肠杆菌见光后,最大复活百分比分别减小到10.0%和0.3%,复活速率分别减小到830CFU·(mL·h)-1和20CFU·(mL·h)-1.以上结果表明,延迟消毒后出水见光时间,只能在一定程度上推迟或削弱大肠杆菌的光复活,并不能有效保证复活受到抑制,即紫外线消毒后出水中的微生物仍然存在高的复活风险.     

MTT法测定大肠杆菌活菌数实验研究

实验分析MTT比色法中的工作波长、反应时间、MTT用量、菌龄、二甲基亚砜（DMSO）等因素对大肠杆菌活细菌计数的影响.同时,通过与平板计数对比,确定了MTT细菌计数的可行性.结果表明,当细菌数在107～109cell·mL-1范围内时,双波长MTT法可用于检测大肠杆菌的活菌数量,最佳反应时间为60min,MTT溶液用量...     

木聚糖酶基因xynB在不同大肠杆菌表达系统中的表达比较

从黑曲霉(Aspergillus niger)nl-1中克隆获得木聚糖酶基因xynB.该基因全长745 bp,含有67 bp内含子,与公布的黑曲霉xynB基因有较高的同源性.将PCR扩增的木聚糖酶成熟肽基因和含有信号肽的基因分别连接到表达载体肠杆菌Top10 F’、DH10B和两种BL21宿主中获得重组菌株.通过IPTG诱导,xynB基因在重组菌株中获得特异性表达.表达产物以胞内可溶性蛋白和不溶性包涵体形式存在.诱导4 h,重组菌株pTrc-99a-xynB[BL21 condon plus(DE3)]表达量最高,胞内酶活达到299 IU/L.重组质粒pTrc-99a-xyn B(S)在不同宿主胞内和胞外均能分泌目的蛋白,诱导10 h,胞外酶活pTrc-99a-xynB(S)[BL21 condon plus(DE3)]达到347 IU/L.而pET-20b-xynB(S)在两种BL21宿主中均不表达.经SDS-PAGE分析,以pTrc-99a和pET-20b作为表达载体时,重组蛋白相对分子质量分别约为45×103和30×103.与pET-20b相比,pTrc-99a以及自身信号肽的引导更有利于重组木聚糖酶的表达.     

大肠杆菌对赤子爱胜蚓体表超微结构的影响

以赤子爱胜蚓(Eisenia fetida)为实验动物,研究了细菌诱导(浸浴诱导和注射诱导)对蚯蚓体表超微结构的影响.结果表明:(1)用细菌浸浴4h的实验组、用细菌注射4h的实验组及对照组蚯蚓的角质层厚度分别为(0.51±0.03)μm、(0.56±0.02)μm、(0.50±0.01)μm;上角质层突起的高度分别为(0.16±0.02)μm、(0.12±0.01)μm、(0.11±0.01)μm;上角质层游离面中微绒毛的高度分别为(0.18±0.02)μm、(0.11±0.01)μm、(0.11±0.01)μm.统计分析表明,蚯蚓在抵御微生物侵袭过程中,体表角质层中的上角质层突起及上角质层游离面中的微绒毛发挥着重要作用.(2)细菌浸浴4h的实验组、用细菌注射4h实验组及对照组蚯蚓体表的表皮厚度分别为(2.63±0.15)μm、(2.23±0.06)μm、(1.97±0.15)μm;表皮中小颗粒蛋白细胞的颗粒直径分别为(0.38±0.04)μm、(0.26±0.02)μm、(0.24±0.02)μm;大颗粒粘液细胞的颗粒直径分别为(0.50±0.04)μm、(0.26±0.03)μm、(0.21±0.02)μm;网状粘液细胞的直径分别为(0.93±0.06)μm、(0.63±0.06)μm、(0.28±0.03)μm.统计分析表明,蚯蚓在抵御微生物侵袭的过程中,表皮中的腺细胞(小颗粒蛋白细胞、大颗粒粘液细胞及网状粘液细胞)发挥着重要作用.图4表2参17     

Effects of lead，cadmium，mercury chlorides and aquatic environmental samples on the growth of Escherichia coli

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