Dominant chloramphenicol-resistant bacteria and resistance genes in coastal marine waters of Jiaozhou Bay, China

Studies of abundance, diversity and distribution of antibiotic-resistant bacteria and their resistance determinants are necessary for effective prevention and control of antibiotic resistance and its dissemination, critically important for public health and environment management. In order to gain an understanding of the persistence of resistance in the absence of a specific antibiotic selective pressure, microbiological surveys were carried out to investigate chloramphenicol-resistant bacteria and the chloramphenicol acetyltransferase resistance genes in Jiaozhou Bay after chloramphenicol was banned since 1999 in China. About 0.15-6.70% cultivable bacteria were chloramphenicol resistant, and the highest abundances occurred mainly in the areas near river mouths or sewage processing plants. For the dominant resistant isolates, 14 genera and 25 species were identified, mostly being indigenous estuarine or marine bacteria. Antibiotic-resistant potential human or marine animal pathogens, such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Shewanella algae, were also identified. For the molecular resistance determinants, the cat I and cat III genes could be detected in some of the resistant strains, and they might have the same origins as those from clinical strains as determined via gene sequence analysis. Further investigation about the biological, environmental and anthropogenic mechanisms and their interactions that may contribute to the persistence of antibiotic-resistance in coastal marine waters in the absence of specific antibiotic selective pressure is necessary for tackling this complicated environmental issue.

Molecular characterizations of chloramphenicol- and oxytetracycline-resistant bacteria and resistance genes in mariculture waters of China

In order to gain an understanding of the diversity and distribution of antimicrobial-resistant bacteria and their resistance genes in maricultural environments, multidrug-resistant bacteria were screened for the rearing waters from a mariculture farm of China. Both abalone Haliotis discus hannai and turbot Scophthalmus maximus rearing waters were populated with abundant chloramphenicol-resistant bacteria. These bacteria were also multidrug resistant, with Vibrio splendidus and Vibrio tasmaniensis being the most predominant species. The chloramphenicol-resistance gene cat II, cat IV or floR could be detected in most of the multidrug-resistant isolates, and the oxytetracycline-resistance gene tet(B), tet(D), tet(E) or tet(M) could also be detected for most of the isolates. Coexistence of chloramphenicol- and oxytetracycline-resistance genes partially explains the molecular mechanism of multidrug resistance in the studied maricultural environments. Comparative studies with different antimicrobial agents as the starting isolation reagents may help detect a wider diversity of the antimicrobial-resistant bacteria and their resistance genes. (C) 2009 Elsevier Ltd. All rights reserved.

羊草细胞差异表达基因分析与遗传转化方法研究

羊草（Leymus chinensis (Trin.) Tzvel），隶属禾本科赖草属，是欧亚大陆草原区东部重要建群种之一。羊草是牧草之王，是我国比较有优势的战略性生物资源，对我国北方畜牧业的发展以及生态环境的保育均具有重要意义。近年来，由于缺乏科学管理、过度放牧等不利影响，加之羊草本身固有的“三低”问题（即抽穗率低、结实率低、发芽率低）已对羊草生物多样性维持构成了严重的威胁，限制了我国人工草地建设和天然草地的改良及沙化治理的步伐。因此，如何通过细胞、分子生物学以及生物技术手段改良羊草、快速评价和创造新的种质;如何加快育种进程便成为当前亟待解决的问题。本文围绕这些问题开展了系统的研究并取得如下结果： 1. 建立了羊草离体培养再生体系，并研究了影响愈伤组织诱导和植株再生的因素，影响植株再生的主要因素为激素配比和基因型。将3～5mm的幼穗接种到含有2,4-D 0～5.0mg/L的N6基本培养基上，随着2,4-D浓度的变化，愈伤组织诱导率不同，最高诱导率为93.21%（基因型C6）、最低为33.35%（吉生1号）;愈伤组织在N6(大)＋B5(微)＋KT1.0mg/L+BA1.0mg/L的培养基上可以分化出芽，并在1/2MS培养基上生根。羊草基因型W4不同幼穗诱导的愈伤组织在继代培养过程中其生长、褐化死亡等方面存在着差异;在分化培养过程中，不同幼穗的愈伤组织最高分化率为9.24%，最低分化率为5.26%。 2. 对来自同一基因型不同幼穗的愈伤组织中差异表达的基因进行了研究。采用DDRT-PCR技术对其差异表达的基因进行了分离，通过银染技术显示差异片段。将得到的差异片段进行回收、克隆测序，得到两个差异片段序列，经过序列分析表明，其中一个片段是与水稻翻译延伸因子eEF-1基因高度同源;另一差异片段与水稻谷胱甘肽转移酶GST基因高度同源。 3. 建立了羊草遗传转化方法。在获得羊草离体培养再生体系的基础上，采用基因枪法对羊草两个基因型进行转抗除草剂基因（PAT）的研究。对分别来自基因型W4和C3的愈伤组织各1430和1850块进行转化。在附加1.0mg/L PPT的培养基上进行一系列的筛选培养，共获得了23株再生苗，经过生根筛选培养，得到5株抗性苗，3株来自基因型W4，2株来自基因型C3。对5株植株进行PCR和Southern 检测，得到2株阳性苗，均来自基因型W4，对阳性植株经过无性繁殖得到的无性系进行PCR检测及Basta耐受性鉴定，外源基因可以在其无性系稳定遗传并表达，无性系除对Basta具有抗性外，其表型特征与对照无明显区别。

耐酸产甲烷菌的分离及其复合菌剂的研究

本文从成都龙泉垃圾填埋场和宜宾造纸厂分离到耐酸性能优良的高温产甲烷菌RY3和中温产甲烷菌SH4，并将其与实验室现有的利用不同底物的产甲烷菌配伍组合成了复合菌剂。采用活性污泥作为固体附着物，研制出了固体产甲烷菌复合菌剂。 菌株RY3的pH耐受范围为5.5～10.5，最适生长pH 6.0～8.0。菌株RY3为革兰氏阳性，长杆状，多数单生，不运动；菌落浅黄色，形状近圆形；利用H2+CO2或甲酸盐作为唯一碳源生长，不利用乙酸盐，对氯霉素非常敏感。该菌最适生长温度为55℃～65℃，最适NaCl浓度为0～2%。根据形态和生理生化特性及16S rDNA序列分析将其初步定为热自养甲烷热杆菌（Methanothermobacter thermautotrophicus）。添加RY3菌液与仅添加厌氧污泥作为接种物相比一周内可使达到最大产甲烷速率所需时间缩短三分之二，甲烷总产量提高约1.8倍。菌株SH4的生长pH范围5.5～9.5，其对酸碱具有良好的适应性，培养3天后，在初始pH值为6.0～8.0的培养基中甲烷产量相差不大，且基本达到最大产量。SH4革兰氏染色阳性，短杆状，多数单生，不运动；菌落近圆形，微黄；利用H2+CO2或甲酸盐作为唯一碳源生长，不利用乙酸盐，对氯霉素非常敏感。SH4最适生长pH 为7.0，最适生长温度为35℃，最适NaCl浓度为0～1.5%。实验表明，添加SH4菌液与仅添加厌氧污泥作为接种物相比可使产甲烷启动时间缩短三分之一，甲烷总产量亦有大幅提高。从形态和生理生化特征以及16S rDNA序列分析表明SH4为嗜树木甲烷短杆菌（Methanobrevibacter arboriphilus）。 以活性污泥为附着物，与培养基和菌种经搅拌后厌氧发酵可得产甲烷菌固体复合菌剂。固体复合菌剂的pH耐受范围为5.5～9.5，温度耐受范围为15℃～65℃，表明其对环境的适应性较强。以猪粪为底物进行厌氧发酵，接种复合菌剂进行试验，以接种实验室长期富集的产甲烷厌氧污泥作为对照，在20℃时，发酵甲烷浓度与对照基本一致，但每日产气量优于对照，第15天时接种复合菌剂的发酵瓶每日产气量是对照的1.59倍；50℃时达到最大甲烷含量所需时间比对照缩短三分之二，三周内总产气量约为对照的2.7倍，甲烷总产量约为2.8倍。以不加接种物为对照，接种复合菌剂20℃时发酵甲烷含量达到50%约需2周，对照2周内甲烷含量最高仅为4.3%；50℃时接种复合菌剂发酵仅需约1周甲烷含量便可达50%，对照则至少需要2周。 In this paper, high-temperature Methanogen RY3 and middle-temperature SH4 were isolated from Chengdu Longquan refuse landfill and Yibin paper mill. They could be used to make compound inoculum that producing methane with the existing Methanogens utilized different substrate. With using anaerobic activated sludge be solid fixture, the process had been designed to produce solid compound inoculum. Strain RY3 possessed excellent capacity of acid and alkali-tolerant. The pH-tolerant scale of RY3 was 5.5～10.5 and its optimum pH value for growth was 6.0～8.0. RY3 was G+, long-rod shape, monothetic and nonmotile, the colony was pale yellow with suborbicular-shape. Formate or H2+CO2 but not acetate was utilized by RY3 as sole C-source, and it was very sensitive to chloramphenicol. Besides, strain RY3 grew fastest at 55℃～65 and 0℃～2% NaCl. Characteristics of modality and physiology with sequence analysis of the 16s rDNA gene of strain RY3 preliminarily showed that it was Methanothermobacter thermautotrophicus. The experiments indicated that the time which began to produce methane with the highest velocity could be shortened two third by adding RY3 in one week, and the total methane production also was 1.8 times than before. Strain SH4 possessed wide scale of growing pH（5.5～9.5）and excellent ability of acclimatizing itself to acid-alkali. The methane production had no apparent difference among those cultivated in different initial pH（6.0～8.0）after three days and equaled to the maximum production basically. Cells of SH4 were G+, short-rod sharp, monothetic and nonmotile. The colony was pale yellow with suborbicular-shape. Formate or H2+CO2 but not acetate was utilized by SH4 as sole C-source, and it was very sensitive to chloramphenicol. Besides, it grew fastest at pH 7.0，55 ℃～65 and 0℃～2% NaCl concentration. The experiment indicated the time that began to produce methane could be shortening one third by adding SH4. And the total methane production also rose apparently. Characteristic of modality and physiology with sequence analysis of the 16S rDNA gene of strain SH4 demonstrated it was Methanobrevibacter arboriphilus. The activated sludge was utilized as fixture, mixed with culture medium and inocolum, that the solid compound inoculum could be produced by anaerobic fermentation. The compound inoculum could grow between pH 5.5～9.5, 15℃～65. It demonstrated the compound inoculum ha℃ve great ability of adapting to circumstance. In the experiment that making pig manure be substrate and taking the anaerobic sludge producing methane that cultured in long term in laboratory to be comparison, the concentration of methane in fermentation added compound inoculum almost equal to the comparison at 20℃, but the volume of gas production could be a little higher. The gas production everyday inoculated compound inoculum was 1.59 times to comparison. The time that the concentration of methane to maximum could be shortening by two third by adding compound inoculum, and the total gas production was 2.7 times to comprison while the total methane production was 2.8 times. If take the no inoculum be the comprasion, anaerobic fermentation added compound inoculum made the concentration of methane to 50% in 2 weeks but the comparison only to 4.3% at 20℃. The time that the concentration of methane to 50% by adding compound inoculum only need 1 week, but the comparison need 2 weeks at 50℃.

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

Genetic mechanisms of multi-antimicrobial resistance in a pathogenic Edwardsiella tarda strain

TX01, a pathogenic Edwardsiella tarda strain isolated from diseased fish at an epidemic-inflicted fish farm in China, exhibits resistance to multiple classes of antimicrobial agents. The genes (kn(R). catA3, and tet(A), respectively) encoding resistance to kanamycin, chloramphenicol, and tetracycline were cloned and found to be 99-100% identical to the corresponding genes carried by known plasmids and transposons of human, animal, and environmental isolates. Further study demonstrated that TX01 harbors a plasmid, pETX, which proved to be (i) the carrier of the tet and cut operons; (ii) a mobile genetic element that is capable of transferring between bacteria of different genera. These results, which, to our knowledge, documented for the first time the co-existence of chloramphenicol and tetracycline resistance determinants on a conjugative plasmid in a pathogenic E tarda strain, indicated that gene acquisition via horizontal transferring of pETX-like mobile genetic entities may have played an important part in the dissemination of antimicrobial resistance and that there have existed for some time widespread genetic exchanges between bacteria of human, animal/fish, and environmental origins. (C) 2008 Elsevier B.V. All rights reserved.

Establishment of a micro-particle bombardment transformation system for Dunaliella saliva

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. saliva with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 mu g/ ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

Review of genetic engineering of Laminaria japonica (Laminariales, Phaeophyta) in China

Progress has been made in establishing a genetic transformation model for Laminaria japonica (Phaeophyta, Laminariales). The model includes introduction of foreign genes by biolistic bombardment, use of promoter SV40 to drive gene expression, algal regeneration by parthenogenesis and selection by chloramphenicol or hygromycin.

Molecular characterizations of oxytetracycline resistant bacteria and their resistance genes from mariculture waters of China

Oxytetracycline-resistant bacteria were isolated from a mariculture farm in China, and accounted for 32.23% and 5.63% of the total culturable microbes of the sea cucumber and the sea urchin rearing waters respectively. Marine vibrios, especially strains related to Vibrio splendidus or V. tasmaniensis, were the most abundant resistant isolates. For oxytetracycline resistance, tet(A), tet(B) and tet(D) genes were detected in both sea cucumber and sea urchin rearing ponds. The dominant resistance type for V. tasmaniensis-like strains was the combination of both tet(A) and tet(B) genes, while the major resistance type for V. splendidus-like strains was a single tet(D) gene. Most of the sea cucumber tet-positive isolates harbored a chloramphenicol-resistance gene, either cat IV or cat II, while only a few sea urchin tet-positive isolates harbored a cat gene, actually cat IV. The coexistence of tet and cat genes in the strains isolated from the mariculture farm studied was helpful in explaining some of the multi-resistance mechanisms. (c) 2006 Elsevier Ltd. All rights reserved.

Concurrence of cat and tet genes in multiple antibiotic-resistant bacteria isolated from a sea cucumber and sea urchin mariculture farm in China

A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for finding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-five percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.