土壤中以抗生素为单一碳源的抗性细菌

Resistant bacteria subsisting on antibiotic as sole carbon source in soil

通过单一碳源选择性培养基(sole carbon source,SCS),从4种不同利用类型的土壤中分离得到2株以青霉素为碳源的菌株(p_4,p_5)以及3株以四环素为碳源的菌株(t_1,t_5,t_9),5种菌株均为革兰阴性菌。研究结果表明抗生素能刺激相应抗性菌株的生长。分别将p_4,t_5,t_9菌株接种到100mg/L相应抗生素SCS培养液中,30d后培养液中抗生素质量浓度与不接种菌株的培养液相比分别下降19.5%,29.1%,24.9%。通过系统发育分析,5种菌株分别属于溶杆菌属(Lysobacter)、贪噬菌属(Variovorax)、假单胞菌属(Pseudomona)、噬几丁质菌属(Chitinophaga)和慢生根瘤菌属(Bradyrhizobium),说明土壤中以抗生素为单一碳源分离的抗性细菌具有群落结构多样性,甚至有菌株与机会致病菌相关,应引起重视。

Resistance genes and strains were increased significantly with antibiotic accumulation in soil.Some resistant strains showed multiple drug resistance.Resistance genes can not only transfer between different species of bacteria,but also to humans by edible crops.Thus,the harm of resistant strains to the environment and human health is alarming.Studies showed that many microorganisms grew in an extreme environment and could degrade toxic substances,therefore,there must be some bacteria living with antibiotics as carbon sources for growth in the environment.Although many studies reported antibiotic resistant strains,little effort had been found in the literature to isolate strains with antibiotics for carbon sources. With the aim to promote domestic antibiotic resistance genes related research development of new antimicrobial agent,we studied growth characteristics and phylogenetic positions for strains with penicillin or tetracycline as the sole carbon source.We collected four kinds of soil（forest soil,tea garden soil,farm soil,and hospital nearby soil）with a potential antibiotic accumulation for the experiment.Sole carbon source medium with penicillin or tetracycline was used to isolate strains.The purity of each reagent in the medium was more than 99.5%.Morphological observation and gram staining were carried out after inoculating culture.Growth curves of strains and antibiotic concentrations in themedium were analyzed to understand the characteristics of the strains using antibiotics.Primers 27 Fand 1492Rwere used for strains 16 SrRNA genes amplification to determine phylogenetic positions of strains.We isolated five soil bacterial strains（p4,p5,t1,t5,t9）from soil with the capacity to grow on antibiotic as sole carbon source.The strains of p4,p5 are resistant to penicillin and the other three are to tetracycline.The strain of t1 was isolated from the tea garden and other four strains were isolated from the forest soil.Five strains were all gram-negative bacteria.The results showed that antibiotics were limiting factors for these bacteria and antibiotic could stimulate the strains growth.The concentration of penicillin and tetracycline degraded 22.6% and 16.5%,respectively,after inoculation compared with the control in 100mg/L sole carbon source（SCS）antibiotic medium.It showed that the isolates could use antibiotics as carbon sources to support their own growth.The bacterial isolates were identified based on analysis of the 16 SrRNA gene sequences.They were placed into a phylogenetic tree and were considered to be surprisingly diverse.They were classified into five distinct genera,Lysobacter genus,Variovorax genus,Pseudomona genus,Chitinophaga genus and Bradyrhizobium genus,respectively.It is concluded that there are a considerable number of microbes in soil that can grow with antibiotics as the sole carbon source,unlike previous resistant bacteria,and the increase of concentration of antibiotics can stimulate the growth of strains.In theory,the strain can degrade antibiotics in soil,but as a kind of environmental pollutants,the ecological risk of isolated strains still needs further study.Although the mechanism of strains resistance is not obvious,we are sure that these strains are diverse in phylogenetic positions.