| Since penicillin was invented in the 1940 s,antibiotics have been widely used in the prevention and treatment of bacterial diseases in human medicine,animal husbandry and aquaculture worldwide.With the development of intensive livestock and aquaculture,consumption of veterinary antibiotics has far exceeded that of medical antibiotics.The continuous overuse of antibiotics not only threatens the safety of cultured organisms as food sources and causes ecological environment pollution,but also induces the generation of a large number of antibiotic-resistant bacteria under the action of this continuous selective pressure.In this paper,the screening of intestinal drug-resistant bacteria and the investigation of drug-resistant genes of sea cucumber at seedling stage were carried out in three main feeding areas of sea cucumber in Shandong Province,and 16 s rDNA analysis were applied to the screening of strains,preliminary reveals the trepang intestinal bacteria resistant and the present situation of the existence of the resistance gene.Finally,in vitro artificial induction technology was used to induce the drug resistance of Vibrio harveyi and Photobacterium damselae to verify the inducibility of vibrio to antibiotic resistance.The changes of the minimum inhibitory concentration(MIC),the spectrum of drug-resistant bacteria and the characteristics of bacterial reaction after bacteriostasis of the artificially induced drug-resistant strains were monitored and analyzed,to explore the mechanism of biological characteristics change.1.Distribution Characteristics of Antibiotic Resistant Bacteria and Antimicrobial Resistant Genes in Intestinal of Cultured Sea Cucumber(Apostichopus japonicus)Seedlings in Shandong provinceIn order to understand the current status of drug-resistant bacteria and drug-resistant genes in sea cucumber,seedlings were collected from 6 aquaculture farms in Shandong province.The number,proportion and species of antibiotic-resistant bacteria in the intestinal were tested.Meanwhile,the distribution of seven drug-resistant genes of four classes of antibiotics was analyzed by RT-PCR.The results were listed as follows: all of the six kinds of antibiotic-resistant bacteria are detected at all six sampling sites.According to the proportion of drug-resistant bacteria to total cultured bacteria,the top three highest proportion are mequindox,nalidixic acid and tetracycline-resistant bacteria,with the proportions are 0.05%-40.06%,2.16%-39.94%,0.06%-23.15%,respectively.The proportion of florfenicol,gentamicin and streptomycin-resistant bacteria range from 0.01% to 4.15%.The 98 antibiotic-resistant bacteria strains are identified 4 phyla,5 classes and 30 genera based on 16 S rDNA sequence.The top 3 genera are Vibrio,Bacillus and Psychrobacter with the detection rates are15.30%,13.27% and 12.25%,respectively.The components of drug-resistant bacteria species varied at different sampling sites,and there are compound antibiotic-resistant strains in Vibrio,Bacillus and Psychrobacter genera.The abundances of seven antibiotic resistance genes in six samples show that the contents of different antibiotic resistance genes against the same antibiotic are significantly different.There is significant correlation between the relative copy number ratio of aminoglycoside resistance gene(aadA)and streptomycin and gentamicinresistant bacteria.The correlation between the abundance of other the six genes and the proportion of drug-resistant bacteria is not significant.The results indicate that there is a certain risk of drug-resistant bacteria and drug-resistant genes in the seedling stage of sea cumber.2.Artificial induction of drug resistance of Photobacterium damsela and analysis of its related biological characteristicsThe drug resistance of lab-isolated Photobacterium damsela was induced,and the sensitive florfenicol and ofloxacin were selected as the inducing antibiotics according to the drug sensitivity results.The initial inhibitory circle diameters of the sensitive strains to florfenicol and ofloxacin were 25 mm and 21 mm,respectively.The minimum inhibitory concentrations to florfenicol and ofloxacin were 0.125 ug/mL and 0.25 ug/mL,respectively.Photobacterium damsela was induced artificially by MIC step-by-step induction method.It was counted as one generation every 20 hours and induced continuously for 30 generations.During the induction period,the minimum inhibitory concentration(MIC)was measured once every six generations,and the concentration of induced drugs was adjusted according to the results,so that the MIC level was always maintained.The drug sensitivity spectra(38 kinds of antibiotics)were measured every six generations,and the changes of the drug sensitivity spectra were monitored.After induction,the growth curve of artificially induced strains and the growth curve after bacteriostasis were measured,compared with that of sensitive strains.The results showed that the MIC of florfenicol resistant strain(M-FF)increased to 8 ?g/mL(64 times increased),and that of ofloxacin resistant strain(M-OF)increased to 1 ?g/mL(4 times increased),respectively.The results of the drug sensitivity spectrum showed that the diameter of the bacteriostasis circle of M-FF against florfenicol decreased from 25 mm to 19 mm,and the drug resistance changed from "sensitive" to "intermediary".While the diameter of the bacteriostasis circle of M-OF against ofloxacin decreased from 21 mm to 12 mm,and the drug resistance changed from "sensitive" to "resistant".The results of growth curve before and after induction showed that the time to enter the plateauing stage of the induced strain M-OF was delayed by 2h compared with the sensitive strain,but there was no difference in the number of viable bacteria after entering the plateauing stage.There was no significant difference between M-FF and sensitive strains in the number of live bacteria and the time to enter the plateau period.The growth curve of the bacteria after bacteriostasis showed that the sensitivity of the induced strain M-OF against drugs was reduced.After the drug was removed for 2h after the bacteriostasis treatment,the growth rate was the fastest and the intensity was the highest,which was significantly higher than that of the sensitive strain.Even though the growth intensity of the induced strain M-FF was not significantly different from that of the sensitive strain,but the growth rate was still higher than that of the sensitive strain.This indicated that the Photobacterium damsela could be induced to develop drug resistance after continuous stimulation of antibiotics in vitro,and the sensitivity of drug-resistant bacteria to drugs was significantly reduced,and the growth rate after contact with drugs was faster than that of antibiotic-sensitive bacteria.3.Artificial induction of drug resistance and analysis of related biological characteristics of Vibrio harveyiInduction of drug resistance of Vibrio harveyi isolated from fish in laboratory,according to the drug sensitivity results,florfenicol and ofloxacin were selected as induced antibiotics,which were sensitive to vibrio harvevi.The initial inhibitory circle diameters of the sensitive strains to florfenicol and ofloxacin were 30 mm and 20 mm,respectively.The minimum inhibitory concentrations to florfenicol and ofloxacin were 0.5 ug/mL and 2.0 ug/mL,respectively.Vibrio harveyi was induced artificially by MIC step-by-step induction method.It was counted as one generation every 20 hours and induced continuously for 30 generations.During induction period,the minimum inhibitory concentration(MIC)was determined once every six generations,and the concentration of induced drugs was adjusted according to the results,so that the concentration of induced drugs remained at the MIC level.The drug sensitivity spectrum(a total of 38 antibiotics)was determined once every six generations to monitor the change of the drug sensitivity spectrum.After induction,the growth curve of artificially induced strains and the growth curve after bacteriostasis were measured,compared with that of sensitive strains.The results showed that the MIC of flubenicol resistant strain(H-FF)increased to 32 ?g/mL(64 times increased),and that of ofloxacin resistant strain(HOF)increased to 8 ?g/mL(4 times increased),respectively.The results of drug sensitivity spectrum showed that the diameter of inhibition zone of H-FF to florfenicol decreased from 30 mm to 17 mm,the drug resistance changed from "sensitive" to "intermediary",the diameter of inhibition zone of H-OF to ofloxacin decreased from 20 mm to 0 mm,and the drug resistance changed from "sensitive" to "drug resistance".The results of growth curve before and after induction showed that the time of the induced strain H-FF entering the plateau phase was delayed by 2 hours compared with the sensitive strains,but there was no difference in the number of viable bacteria after entering the plateau phase.There was no significant difference between the induced strain H-OF and the sensitive strains in the number of viable bacteria and the time of entering the plateau phase.After bacteriostasis treatment,the growth curve of the induced strain H-FF showed that the sensitivity of the induced strain to drugs decreased,and the growth rate and intensity of the induced strain H-FF were higher than those of the sensitive strain.The growth intensity of the induced strain H-OF was not significantly different from that of the sensitive strain,but the growth rate was still higher than that of the sensitive strain.It is concluded that Vibrio harveyi can be induced to develop drug resistance by sustained stimulation of antibiotics in vitro,and the sensitivity of drug-resistant bacteria to drugs is significantly reduced,and the growth rate after contact with drugs is faster than that of antibiotic-sensitive bacteria.4.Transcriptome analysis of vibrio harveyi sensitive bacteria and ofloxacinresistant bacteriaWith the continuous development of high-throughput sequencing technology,RNA sequencing(RNA-seq)has become a powerful tool for replacing microarrays for transcriptome analysis,in which the expression levels of thousands of genes can be detected at the same time,and the functional pathways and regulatory approaches in biological processes can be further analyzed.In this study,we used the second-generation transcriptome sequencing technology to simultaneously sequence the sensitive strains of vibrio harveyi and the drug-resistant strains of ofloxacin,and studied the changes of vibrio harveyi at the transcriptome level before and after induction,and finally obtained 602 differentially expressed genes.Among them,compared with the sensitive strain h-n,the drug-resistant strain h-of had 314 up-regulated genes and 288 down-regulated genes,among which 4 genes were only expressed in the drug-resistant strain.GO annotation,differentially expressed genes in the biological process in main concentration in the mobile(locomotion),biological adhesion(in adhesion)and carbohydrate utilization(carbohydrate utilization).The COG classification focuses on amino acid transport and metabolism,carbohydrate transport and metabolism,inorganic ion transport and metabolism,transcription,post-translation modification and protein conversion,cell wall/membrane biosynthesis and cell movement.The enrichment results of KEGG pathway showed that bicomponent system(ko02020),ABC transport system(ko02010),flagellum assembly(ko02040),purine metabolism(ko00230)and oxidative phosphorylation(ko00190)had the highest number of differentially expressed genes annotated and significantly enriched. |
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