| Wastewater from the mariculture and municipal sewage are discharged into coastal zone, causing residues of antibiotics in the coastal environments including the mangrove wetland. These compounds may trigger the increases of antibiotic-resistant bacteria and antibiotic resistance gene (ARGs), and disturb the structure and function of microbial community, eventually leading to unforeseen influences on the flora, fauna and humans. Therefore, the potted experiment in indoor simulative way was conducted to investigate these effects, the ciprofloxacin (CIP) as the pollutant agent was added into the potted sediments with the series concentrations, namely pollution level I (7mg/kg), pollution level II (14mg/kg), pollution level III (21mg/kg), pollution level IV(28mg/kg), and the controlled group without ciprofloxacin was simultaneously set up. The plant and sediment samples were collected on 0,9,18 and 27 days to measure the impact of the mangrove plant Avicennia marina on the residues of ciprofloxacin, changes of community structure and antibiotic resistance (genotypes and phenotypes) changes in bacteria caused by antibiotic residues on the basis of the experiment method from the modern microbiology, molecular biology and instrumental analysis. The results revealed as following:1. Concentrations of CIP in sediment and plant samples (root, stem and leaf) were determined with ultra-high performance liquid chromatography-tandem mass spectrometry. The results showed that the fast trend with declining CIP level was observed in sediments at beginning (9d) and then slowed down gradually along with potted time in planted (containing Avicennia marina) and implanted (no plant as the controlled) systems. Concentrations of CIP in planted sediments were somewhat less than that in unplanted sediment, however, no significant difference was found between them (Two-way ANOVA, p>0.05), indicating that the biennial Avicennia marina was potentially conducive to CIP degradation in sediment to a certain extent. The level of CIP in Avicennia marina planted in sediment with CIP treatment was higher than in that without CIP treatment. The different levels of CIP were present among root, stem and leaf, of which the CIP level was higher in root at early growth of young Avicennia marina in all pollution levels except the pollution level of 14mg/kg and the CIP levels were higher in stem and leaf than in root at later growth of young Avicennia marina. These results suggested that the young Avicennia marina had the capabilities to absorb and transport CIP.2. Antibiotic resistance of bacteria was determined by the single disc diffusion method. No significant differences in antibiotic resistances of bacteria between planted and unplanted systems were found among different CIP treatments (Two-way ANOVA, p>0.05), whereas the bacterial resistance to TMP, AMP and CEF were decreased in some degree in planted systems. Furthermore, the results also exhibited that the bacterial resistance to AMP, TMP and CEF was significantly stronger than to others in different periods (Wilcoxon text,p<0.05).3. ARGs in sediment were surveyed by real-time fluorescent quantitative PCR. TEM was the only detected P-lactam resistance gene and present in all the sediment samples. At 9 days of the pot experiment, the levels of TEM in sediments with CIP treatments were higher 1-3 order of magnitudes than that without CEP treatment, and the levels of TEM at 9 days was higher 1-2 order of magnitudes than at 0 day. The level of TEM was dramatically reduced in later growth of young Avicennia marina. No significant differences in antibiotic resistance of bacteria isolated from sediment were discovered between planted and unplanted systems in different periods (Two-way ANOVA,p>0.05).4. Densities of total bacteria in sediments were enumerated by acridine orange direct count in different periods. In early growth of young Avicennia marina, the bacteria densities increased firstly, and then decreased with the increasing CIP levels. For different CIP treatments, the bacteria densities varied from 106 ind./g.dw to 107 ind./g.dw in later growth of young Avicennia marina. Densities of total bacteria isolated from planted sediments were slightly higher than those isolated from unplanted sediments and no significant deference was found (Two-way ANOVA, p>0.05), implying that the biennial Avicennia marina can maintain bacteria densities to some extent.5. High throughput sequencing was utilized to analyze the bacteria communities in sediments. The slight alteration for the relative abundance in most bacteria was obviously present in different periods, especially the relative abundance of Campylobacterales increased initially, subsequently decreased with the progressive process. In addition, the relative abundance of Thiotrichales also exhibited the increasing trend along with the potted time. Bacterial diversities in sediments decreased initially with the progressive time, and then restored to level same as the controlled group. Moreover, bacterial diversities in sediments increased firstly, and then decreased with the rising levels of CIP treatment. No significant differences in the relative abundance, community composition and diversity of bacteria were observed along with time and between planted and unplanted systems (Two-way ANOVA, p>0.05). In a word, the biennial Avicennia marina was in some degree able to maintain the bacterial diversity and community composition. |
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