| Aeromonas hydrophila is an important bacterial pathogen of aquatic animals and human. It is one of the most common aquatic pathogens that cause a significant harm to the normal cultured fishes. It infects a variety of aquatic and terrestrial animals resulting in huge economic losses in the aquaculture industry each year. Currently, people use antibiotics to prevent and treat fish diseases, leading to the emergence of A. hydrophila antibiotics resistance strains. Furthermore, this pathogen could live in biofilm formation in the natural environment, which is another status different to planktonic cells. It was reported that biofilm plays an important role in the bacterial pathogenesis and resistance. Thus, it is of great significance to study the antibiotic resistance related proteins and their functions in the state of planktonic and biofilm.In this study, a TMT labeling quantitative proteomics technology combined with LC-MS/MS mass spectrometry was used to compare the differential expression of proteins between the fitness and genetic acquired resistance to chlortetracycline (CTC) in A. hydrophila biofilm firstly. Results showed that a total 66 differential proteins including 24 down-regulated and 42 up-regulated proteins were quantified in fitness behavior (Ah-CTC-O+CTC/Ah-CTC-O). Meanwhile, a total of 141 proteins, including 66 down-regulated and 75 up-regulated proteins were quantified in genetic acquired resistance behavior (Ah-CTC-R/Ah-CTC-O) in biofilm. The correlation assay showed a moderate correlation between both behaviors, suggesting an intrinsic evolutionary correlation between them. The further bioinformatics analysis showed that translation related ribosomal proteins increased in both factors, fatty acid biosynthesis related proteins increased in fitness, while some metabolic pathways including propanoate metabolism, microbial metabolism in diverse environments and biosynthesis of secondary metabolites decreased abundance in acquired resistance. Selected proteins involved in fatty acid biosynthesis and propanoate metabolism were further validated by q-PCR assay. Results showed that all of the transcription levels of the fatty acid biosynthesis related genes in fitness were in accordance with the proteomics results, while most of the mRNA levels in acquired resistance showed higher abundance than the protein levels. Meanwhile, compared to the proteomics analysis, the transcription behaviors of the propanoate metabolism related genes showed greater fluctuation with about 50-60% related mRNA levels consisted with protein levels.Based on the fact that fatty acid biosynthesis may play an important role in antibiotics resistance, a fatty acid biosynthesis inhibitor, triclosan (TCS), was used to compare the differential behaviors of CTC sensitive and resistant strains when fatty acid biosynthesis was inhibited. The survival rate of CTC susceptible and resistance strains showed related strains were significantly killed with a low concentration cocktail of CTC plus TCS. Furthermore, the expression of FabD which involved in fatty acid biosynthesis in CTC and TCS treatment in A.h-CTC-O and A.h-CTC-R was validated by western blotting method. Results showed that fatty acid biosynthesis should be a protective antibiotics resistance mechanism and a cocktail of CTC and TCS exhibited more efficient antimicrobial capability than each antibiotic individually on biofilm, specifically for CTC sensitive biofilm. We therefore demonstrate that the up-regulation of fatty acid biosynthesis may play an important role in antibiotic resistance and suggest that a cocktail of CTC and TCS may be a potential cocktail therapy for pathogenic infections in biofilm.Secondly, the iTRAQ labeling based quantitative proteomics method was used to analysis the differential expression of proteins between the fitness and acquired resistance to chlortetracycline in A. hydrophila planktonic status. Results showed a total of 310 proteins, including 116 up-regulated and 194 down-regulated proteins were quantified in fitness; and a total 252 differential proteins including 93 up-regulated and 159 down-regulated proteins were quantified in genetic resistance behavior. We found a total of 92 proteins overlapped between fitness and acquired resistance, indicating a common antibiotic resistance mechanisms to deal with antibiotic stress in fitness and acquired resistance. Bioinformatics analysis showed that the ribosomal subunit protein was significantly up-regulated in both resistances, which was consistent with the results in above biofilm assay. Selected proteins involved in translation pathway and bacterial chemotaxis were further validated by q-PCR assay. Results showed that all of the transcription levels of the translation and bacterial chemotaxis related genes were in accordance with the proteomics results in fitness and acquired resistance. Further, RplI and GlmS which involved in translation in fitness and acquired resistance were validated using western blotting method, suggesting both proteins may play an important role in bacterial resistance in planktonic status.In general, our findings provide a theoretical basis for the study of A.hydrophila resistant mechanisms in both planktonic and biofilm status, and may provide candidate targets for future novel vaccines screening. |
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