The fate of recombinant plasmids during composting of organic wastes

Composting was investigated as a means for safe disposal of organic waste containing bacteria that carry transgenes in recombinant plasmids. To generate model recombinant plasmids, a mobile IncQ plasmid, RSF1010, and a non-mobile plasmid, pGFP, were genetically modified to carry a DNA segment encoding both green fluorescent protein and kanamycin resistance and were designated as RSF1010-GFPK and pGFPK. Escherichia coli (E. coli) C600 harboring these plasmids were inoculated into chicken manure specimens that were placed in compost at 20 and 60 cm from the bottom of a 1.0-m high compost bin. Control specimens were held at ambient temperature. By day 10, compost temperatures at the lower and upper levels of the bin had reached 45.3 and 61.5°C, respectively, and at both levels the target E. coli had been inactivated and the plasmids had lost their capacity to be transformed or mobilized. Furthermore, based on real time Polymerase chain reaction (PCR), the transgene fragments along with the host chromosomal DNA fragment from specimens at the upper level had been degraded beyond the detection limit. However, at the lower level where temperatures remained below 48°C these fragment persisted to day 21. At ambient temperatures (0–8°C), the E. coli, plasmids and the transgene fragments persisted in manure specimens throughout the 21 day test period. The study showed the potential for composting as a safe procedure for disposal of bacteria carrying transgenes in recombinant plasmids.     

Molecular characterization of conjugative plasmids in pesticide tolerant and multi-resistant bacterial isolates from contaminated alluvial soil

A total of 35 bacteria from contaminated soil (cultivated fields) near pesticide industry from Chinhat, Lucknow, (India) were isolated and tested for their tolerance/resistance to pesticides, heavy metals and antibiotics. Bacterial isolates were identified by 16S rDNA sequencing. Gas Chromatography analysis of the soil samples revealed the presence of lindane at a concentration of 547 ng g⁻¹ and α-endosulfan and β-endosulfan of 422 ng g⁻¹ and 421 ng g⁻¹ respectively. Atomic Absorption Spectrophotometry analysis of the test sample was done and Cr, Zn, Ni, Fe, Cu and Cd were detected at concentrations of 36.2, 42.5, 43.2, 241, 13.3 and 11.20 mg kg⁻¹ respectively. Minimum inhibitory concentrations of all the isolates were determined for pesticides and heavy metals. All the multi-resistant/tolerant bacterial isolates were also tested for the presence of incompatibility (Inc) group IncP, IncN, IncW, IncQ plasmids and for rolling circle plasmids of the pMV158-family by PCR. Total community DNA was extracted from pesticide contaminated soil. PCR amplification of the bacterial isolates and soil DNA revealed the presence of IncP-specific sequences (trfA2 and oriT) which was confirmed by dot blot hybridization with RP4-derived DIG-labelled probes. Plasmids belonging to IncN, IncW and IncQ group were neither detected in the bacterial isolates nor in total soil DNA. The presence of conjugative or mobilizable IncP plasmids in the isolates indicate that these bacteria have gene transfer capacity with implications for dissemination of heavy metal and antibiotic resistance genes. We propose that IncP plasmids are mainly responsible for the spread of multi-resistant bacteria in the contaminated soils.     

Large plasmids governing multiple resistances to heavy metals: A genetic approach∗

Gram negative bacteria classified as Alcaligenes eutrophus and carrying large resistance plasmids (generally two) were found in various industrial sites highly contaminated by heavy metals (Zn⁺⁺, Cu⁺⁺, Co⁺⁺,...). These strains were detected by DNA hybridization with a probe made with a 9kb fragment (ccz⁺ fragment) encoding for resistances to Cd⁺⁺, Co⁺⁺ and Zn⁺⁺, and cloned from plasmid pMOL30. This plasmid was isolated from the representative strain A. eutrophus CH34 which harbours the plasmids pMOL30 (240 kb) and pMOL28 (165 kb). Phenotypes related to pMOL28 and pMOL30 include the tolerance to Cd⁺⁺, Co⁺⁺, Cr0₄ ⁼, Cu⁺⁺, Hg⁺⁺, Ni⁺⁺, Pb⁺⁺ and Zn⁺⁺. The described genetic properties of these plasmids refer to some cloned or mapped functions and to some plasmid rearrangements. Plasmid pMOL85 (250 kb) which is related to pMOL30 was also described. Its host (A. eutrophus DS185) was isolated from a zinc desert. pMOL85 can efficiently self transfer in plasmidfree derivatives.     

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.     

贫营养和痕量抗生素对质粒抗生素抗性适应度代价的影响

本文针对饮用水系统中营养和抗生素水平对质粒抗生素抗性的适应度代价进行了研究.选取了p ACYC184、RP4和PBR322这3种不同的抗性质粒,发现在不同的营养水平下,抗性质粒的适应度代价具有普遍性.而且,营养水平越低,其适应度代价表现得越明显.另外,痕量浓度的抗生素也会影响抗性质粒的适应度代价.本研究中,小于50%MIC的抗生素浓度对野生菌的损伤或抑制作用不足以抗衡抗性质粒的适应度代价,质粒难以维持.因而,推断在饮用水系统(贫营养和ng·L-1水平的抗生素)中,质粒抗生素抗性的适应度代价较大,质粒不易维持,在饮用水系统中传播和扩散的风险较小.     

环境中抗药基因水平转移研究进展

抗药基因是一种新型环境污染物,一旦被致病菌获得,将导致抗生素的临床使用失效,从而危害公共健康。抗药基因可通过转移因子在细菌间传播,加剧抗药基因的扩散效应,提升了环境健康风险性,因此抗药基因水平转移受到了越来越多的关注。本文介绍了抗药基因水平转移的分子机制,总结了不同环境中各种抗药基因水平转移途径的研究现状,并对该领域将来的研究方向提出了展望,以期为环境中抗药基因的调查评估、污染控制及合理管理提供理论参考。