Studies On The Genes And Proteins Related To The Antibiotic Resistance In Edwardsiella Tarda Biofilm

The discovery of antibiotics has played a major role in the control of many bacterial infections, but the widespread use of antibiotics, especially the abuse and misuse, has led to the emergence of a large number of resistant strains, and even the emergence of multidrug resistant bacteria and super-resistant bacteria. Moreover, biofilm formation has aggravated the severity of antibiotic resistance in clinical or aquaculture industry. Therefore, elucidating the mechanisms of antibiotics resistance in biofilm is a great significance for solving the problems of antibiotic resistance. Edwardsiella tarda has been an important pathogeny of fish and the formation of its biofilm results in chronic infection of fish and huge economic losses. However, the long-term use of antibiotics has leaded to very serious antibiotic resistance and it is urgent to deal with and resolve the problems caused by the antibiotic resistance.In this study, the quantitative proteomics was used to identify the alteration of the whole-extracted proteins in adaptive resistance and genetic acquired resistance of E. tarda ATCC15947 biofilm, and the genomes were compared between oxytetracycline(OXY)-susceptible E.tarda (E.tarda-OXY-O) and its resistant strain (E.tarda-OXY-R) by genome re-sequencing method to investigate the proteins and genes correlated to the antibiotic resistance in E.tarda biofilm.Using iTRAQ labeling based quantitative proteomics technology, we firstly quantified the altered proteins among OXY-resistant E.tarda (genetic acquired resistance) and OXY-susceptible E.tarda (control) without or with OXY (adaptive resistance) in biofilm. Results showed 281 and 70 altered proteins in adaptive and genetic acquired resistance, respectively. The former included 193 down-regulated and 88 up-regulated proteins, and the latter included 20 down-regulated and 50 up-regulated proteins. Then, the following bioinformatics analysis showed that ribosome related proteins were down-regulated in adaptive resistance biofilm whereas some outer membrane proteins such as OppA, MalE and GlnH, and proteins related with energy generation pathways such as glycolysis, pentose phosphate pathway and TCA cycle were decreased. Besides these, some ATP dependent transport proteins such as NikA, OppA and MalE were down-regulated in genetic acquired resistance biofilm, while proteins related to RNA polymerase were up-regulated. The KEGG pathways analysis found that the bacterial chemotaxis decreased and the translation was significantly increased.We used the q-PCR and Western blotting methods to further verify the differential expression of selected altered proteins. As results, most of the transcription levels of these selected genes were consistent with their proteins levels, which were relevant to ribosome, the enzymes of metabolic pathways and ABC transporter. Western blotting analysis showed that PflB and PspB were indeed altered in biofilm resistance, and they may be the antibiotics resistance functional proteins in biofilm status. Besides these, based on the facts that the energy metabolic pathways were decreased in both antibiotics resistance behaviors, we measured the enzyme activates of the two important enzymes in the TCA cycle. Results showed that the activities of succinate dehydrogenase (SDH) and a-ketoglutarate dehydrogenase a-KGDH) were significantly decreased in biofilm resistance, which suggest that the reduction of energy metabolism may be helpful for the biofilm resistance.In addition, in order to explore the effect of genetic mutations of the resistant strain, the genomes of OXY-resistant E.tarda and control strain were sequenced by the whole-genome sequencing technology. The results of analysis and comparison demonstrated that some important genes in resistant strain were mutated, such as pgm, ppsA, gplK and tolC, and the mutational sites of these genes may be involved in the process of bacterial resistance. Furthermore, the protein levels of some mutated genes in the biofilm were also changed, indicating that the mutations of these genes may have a certain relationship with the acquired resistance.In summary, the resistance of E. tarda in biofilm may be mediated by the following aspects:(1) down-regulate the expressions of some outer membrane proteins such as OppA、MalE and GlnH to decrease the permeability of cell membrane; (2) reducing energy generation metabolisms; (3) increasing translation process; (4) down-regulated bacterial chemotaxis; (5) mutate some genes which associated with some enzymes and outer membrane proteins. In summary, the discovery of these antibiotic resistance mechanisms provides new ideas and effective candidate target sites for solving the problems of biofilm resistance and has important significance to the treatment of biofilm infection diseases.