Isolation And Degrading Characteristics Of Highly Efficient Sulfamonomethoxine(SMMX) Bacteria In Mangrove Sediment

Sulfamonomethoxine(SMMX), one of the new generation of long-term sulfonamides antibiotics, has been widely used in medicine, livestock and aquaculture to treat diseases caused by bacteria, protozoa or fungi infection, with the characteristics of high efficiency, broad-spectrum and selective. Antibiotic residues in the coastal environments including the mangrove area, caused by municipal wastewater effluent and aquaculture, may weaken the microbial decomposition and thus reduce ecosystem functions. The aim of the study is to screen highly effective SMMX degrading bacteria from the sediment samples collected from mangrove area located from Sea-View gallery, Zhanjiang and investigate the bacterial characteristics with the capacity to degrade SMMX.1 Six preliminary screening strains were isolated from sediment of mangrove through flask domesticated enrichment culture method, named HA-J1, HA-J4, HA-J5, HA-J7, HA-J8, HA-J9 with veterinary medicine SMMS as a sole carbon source. The bacteria count increased at first and then decreased with the increasing concentrations of veterinary drugs and grow best in 800mg/L. Through the determination of growth curve, bacteria thrived during 15~24h which can be used as a suitable sampling time to determine biodegradable content.2 Based on the morphological appearances, physiological biochemical characters and 16 S r DNA sequence analyse, strains HA-J1, HA-J4, HA-J5 and HA-J8 were identified as Aeromonas, while strains HA-J7 and HA-J9 identified as Klebsiella. Using housekeeping genes gyr B and gyr A gene to further identification, the result showed that strains HA-J1, HA-J4, HA-J5 and HA-J8 were 100% homologous with Aeromonas caviae, while strains HA-J7 and HA-J9 were 100% homologous with Klebsiella pneumoniae.3 An ultra high-performance liquid chromatography(UPLC) method was developed for the determination of Sulfamonomethoxine(SMMX). The SMMX degradation percentages of the six preliminary screening strains HA-J1, HA-J4, HA-J5, HA-J7, HA-J8, HA-J9 were 24.13%, 6.8%, 1.4%, 4%, 13%, 3.75% respectively after cultured 24 h in liquid mineral media containing 100mg/L SMMX at 30℃. So the strain HA-J1 was identified as highly efficient degradation bacteria. Pearson correlation analysis revealed that there was no significant correlation between degradation percentage and the bacteria increment(p>0.05).4 To study the degradation characteristics of HA-J1, we designed orthogonal experiment of four factors including temperature, p H, salinity and antibiotic concentration respectively with five levels. The strain HA-J1 in temperature 25℃, p H5.0, and when the antibiotic concentration was the 200mg/L can maintain a higher degradation percentage(14.47%). However there was no significant differences between the four factors(ANOVA, p>0.05). The degradation percentage and the bacteria increment had significant positive correlation(r=0.735,p<0.05). The best bacterial inoculum was 16% under the optimum combination of orthogonal detection and the degradation percentage was 19.29%.5 To investigate the degradation characteristics of strains to other sulfonamides, the biological degradability of the 6 preliminary screening strains was studied in liquid mineral media containing 100mg/L sulfamonomethoxine(SMMX) and sulfamethoxazole(SMX) as the sole carbon source respectively. The degradation percentage of SMX(14.11%,1.55%, 1.47%, 4.87%, 8.49%, 3.33%, respectively)was significantly lower than that of SMMX(0.64%, 0.32%, 0.47%, 0.45%, 0.14%)(T-test, p<0.05).6 Adding carbon resources enhanced the strain HA-J1 to degrate SMMX in 100mg/L medium. By adding glucose, alcohol and sucrose, the degeneration percentages of the strain HA-J1 reached 23.02%, 18.36%, 14.61% respectively. However, there was no significant differences among the adding carbon and the control with SMMX as the sole carbon resource(ANOVA,p>0.05).This study has isolated a pure culture bacteria with highly efficient degradation abilities identified as Aeromonas caviae. However the functional genes and degradation pathway is not clear which need for further research.