Study On The Characteristics Of Joint Toxicity Interaction And Mechanism Within Some Antibiotics To Vibrio Qinghaiensis Sp.-Q67

Antibiotics are widely present in our environment,and even in drinking water.No substance in nature can exist alone,contaminants coexist in the form of a mixture,and contaminants of the same composition can exist in the environment in thousands of forms and concentrations.Therefore,it is necessary to carry out researches on the combined toxicity characteristics and mechanisms of different types of antibiotics,and provide data reference for the antibiotics'environmental and ecotoxicological risk assessment.Here are five?-lactam antibiotics:penicillin G potassium?BLP?,carbenicillin sodium?CBD?,cloxacillin sodium?CLS?,piperacillin sodium?PSS?and sulbactam sodium?SBS?,4kinds of aminoglycoside antibiotics:spectinomycin hydrochloride?SPC?,succinomycin sulfate?MCR?,amikacin sulfate?AMK?,tobramycin?TOB?and chloramphenicol?CHL?for the test substance.Vibrio-qinghaiensis sp.-Q67?Q67?was selected as an indicator organism,a binary mixture was designed by the equivalence method?EquRay?,and ternary,quaternary,five-component and ten-component mixtures were designed by the uniform design ray method?UD-Ray?.The inhibitory toxicity of substances and their mixtures to Q67 was determined by the time microplate toxicity analysis?t-MTA?.The effect concentration and other parameters were calculated according to the S-type and J-type concentration-effect curve?CRC?,and their time-dependent toxicity,component and concentration ratio-dependent toxicity were analyzed.The concentration addition?CA?model was used as a reference model for toxicity interaction analysis.At the same time,representive mixture rays was selected.The sample was collected by exposure to the erlenmeyer flask method,and the Q67 at special exposure concentrations and time(two characteristic concentrations:half effect concentration(EC₅₀),minimum effect concentration(EC_min),and one exposure time node?12 h?)was subjected to electron microscope scanning and result analysis,and then the content of the luminescent substance?flavin mononucleotide?FMN?,nicotinamide adenine dinucleotide reduced state?NADH?,superoxide dismutase?SOD?,catalase?CAT?,and total protease?TP??was measured by using a microbial ELISA kit.Finally,combined with toxicity experiments,electron microscopy experiments,and experimental results of luminescent related substances,toxicity characteristics and mechanism of the tested substances were discussed preliminarily.The main conclusions are as follows:?1?The ten tested substances show significant time-dependent toxicity to Q67,both in unit and in mixture.The difference is that the five kinds of?-lactam antibiotics and their mixtures have“hormesis”effect in 8 h and 12 h,and the concentration effect curves are“J”type.The ten-component mixture rays have the same regularity.The concentration effect curves of the four aminoglycoside antibiotics and chloramphenicol and their mixtures are classical"S"type.When the exposure time is 12 h,the negative logarithmic value of half concentration effect(pEC₅₀)was used as toxicity index,the toxicity order of the ten antibiotics is TOB>CHL>MCR>PSS>AMK>SPC>BLP>CBD>SBS>CLS.?2?Except for BLP-CBD-CLS-PSS quaternary mixture,there is no component-concentration dependence in the mixture of five beta-lactam antibiotics.The other mixtures'toxicity regularity depends on the mixing components and concentration ratio.The effects of different antibiotics on cell morphology are also different:under EC₅₀ of BLP,bacterial cells break up from straight rod to scale;under EC₅₀of PSS,bacterial cells change from straight rod to filament;under EC_min of PSS,most bacterial cells grow obviously,and the morphological characteristics of cells under the action of multicomponent mixtures are component-dependent.The content of luminescent related substances of most luminescent bacteria is greater than EC_min under EC₅₀.Combined with the results of electron microscopy,it is concluded that the mechanism of action may be inhibition of cell wall synthesis,resulting in cell wall defects,water infiltration,bacterial swelling and necrosis of luminescent bacteria.?3?The characteristics of combined toxicity of four aminoglycoside antibiotics and mixture of chloramphenicol,generally speaking,most of the mixtures have no obvious toxicity interaction,showing typical addition effect.The cell morphology of most Q67 cells did not change significantly at 12 h after exposure to EC₅₀.The content of luminescence-related substances in most of the luminescent bacteria poisoned by the mixture is lower than that in the blank group under EC₅₀ effect.Combined with the results of electron microscopy,it is concluded that the mechanism of action may be through the interference of protein synthesis in luminescent bacteria,leading to bacterial metabolic disorders and even death.?4?The stimulus effect of ten-component mixture increases significantly,which probably because aminoglycoside antibiotics and chloramphenicol promoted the stimulus effect of beta-lactam antibiotics mixture.The ten-component mixture does not reflect the composition and concentration ratio dependence.Luminescent bacteria have the most complete cell morphology under EC_min-ten-component mixture,which probably because aminoglycoside antibiotics and chloramphenicol weaken the ability of beta-lactam antibiotics to destroy Q67cell wall.The content of luminescence-related substances in Q67 cells is higher than that in EC_min at EC₅₀.Combined with the results of scanning electron microscopy,the mechanism may be that it can inhibit the synthesis of cell wall and interfere with the synthesis of protein in cells,and the inhibition of cell wall synthesis plays a major role.