| Vibrio parahaemolyticus,a Gram-negative food-borne pathogen,widely exists in aquatic products such as fish,shrimp,crab and shellfish.Accidental ingestion of food contaminated with Vibrio parahaemolyticus can cause abdominal pain,diarrhea,vomiting and other poisoning phenomena.With the abuse of antibiotics,the resistance level of V.parahaemolyticus continues to increase,which brings difficulties for antibiotics to treat infected patients.However,the mechanism of drug resistance in V.parahaemolyticus is still not fully understood.Analyzing the drug resistance mechanism of V.parahaemolyticus is of great significance for the development of new antibacterial drugs.In this paper,V.parahaemolyticus ATCC33846 was taken as the research object,this paper first compared its tolerance to 19 antibiotics,and then the genes related to its tolerance to polymyxin and ampicillin were analyzed by transcriptomics.and finally focused on the high tolerance mechanism of V.parahaemolyticus to polymyxin.The main research results of this paper are as follows:(1)Preliminary determination of halophilicity and drug resistance of V.parahaemolyticus ATCC33846.V.parahaemolyticus showed high resistance to polymyxin B,ampicillin,amoxicillin and lincomycin by antibiotic susceptibility test strip analysis.V.parahaemolyticus was cultured in LB solid medium containing 1.5%,2.5% and 3.5% Na Cl,respectively,and the bacterial growth,cell morphology and inhibition zone size were observed.The formation ability is enhanced,and the resistance to antibiotics is more significant.The MIC values of V.parahaemolyticus and E.coli against 19 antibiotics were determined.V.parahaemolyticus was found to be highly resistant to cationic antimicrobial peptides(polymyxin B and polymyxin E)and β-lactams(ampicillin and amoxicillin)compared with E.coli.The MIC values for polymyxin B,polymyxin E and ampicillin were 64,130 and 6000μg/m L for V.parahaemolyticus and 2,0.5 and 15 μg/m L for E.coli,respectively.(2)The mechanism of V.parahaemolyticus tolerance to AMP was investigated based on transcriptomic analysis.By analyzing the transcriptome of V.parahaemolyticus ATCC33846 under AMP stress,670 genes were found to be significantly up-regulated.The most upregulated bla A,VPA0510,VPA0252,VPA0699,VPA0768,VPA0320,VP0636,VPA1096,VPA0947 and VP1775 genes were selected and overexpressed in V.parahaemolyticus ATCC33846,respectively.It was found that under AMP pressure,the growth rate of these genes overexpressed strains was significant improvement.The most notable of these were the VP0636 and VPA0947 genes,which overexpressed strains with the same level of AMP tolerance as bla A(known AMP tolerance gene)overexpressing strain.The two-component system Vbr K/Vbr R in V.parahaemolyticus was found to regulate β-lactamase expression,and its deletion strains showed extreme sensitivity to AMP.Transcriptomic analysis also revealed significant changes in the expression levels of genes related to the outer membrane protein Omp A,cellular antioxidant system,energy metabolism,and amino acid synthesis.(3)The resistance mechanism of V.parahaemolyticus to polymyxin B was investigated by transcriptomic analysis.Transcript levels of genes associated with synthesis of LPS are upregulated in V.parahaemolyticus under polymyxin B stress.LC-MS analysis showed that the number of lipid A fatty acid chains synthesized by V.parahaemolyticus increased under polymyxin B pressure,and the lipid A structure was modified by phosphoethanolamine;at the same time,the composition and molecular structure of phospholipids also changed,and PG accounted for ratio increased,and a three-chain acyl-PG was synthesized.Transcriptome analysis also found that under polymyxin B pressure,the transcription levels of two-component system,thiometabolism,ribosome,cationic antimicrobial peptide resistance,citric acid cycle,pyruvate metabolism,metabolism in different environments,ABC transporter,antibiotic biosynthesis,bacterial secretion system and aminoacyl t RNA biosynthesis in V.parahaemolyticus also changed significantly.(4)The effects of two-component regulatory systems of Pmr A/Pmr B,Pho P/Pho Q and Cpx A/Cpx R on polymyxin resistance in V.parahaemolyticus were found.The mutant strains ATCC33846Δpmr A,ATCC33846Δpmr B,ATCC33846Δpho P,ATCC33846Δpho Q,ATCC33846Δpmr F and ATCC33846Δpmr K were constructed by knocking out genes related to the Pmr A/Pmr B and Pho P/Pho Q two-component regulatory system.LC-MS analysis found that the molecular weight of phospholipids in strains ATCC33846Δpmr A,ATCC33846Δpmr B,ATCC33846Δpho P and ATCC33846Δpho Q decreased,indicating that they contained shorter fatty acid chains and reduced the resistance to polymyxin B;Lipid A was modified by adding a C16:0 secondary fatty acid chain and a phosphoethanolamine group in ATCC33846Δpmr F strain,which increased the resistance to polymyxin B;Loss of phosphoethanolamine modification on lipid A in ATCC33846Δpmr K strain reduces resistance to polymyxin B.The genes related to the Cpx A/Cpx R two-component system were deleted,and the transcription levels of the genes VP＿RS01045 and VP＿RS08405 encoding lipid A secondary fatty acid chain transferase were significantly increased in ATCC33846Δcpx A,ATCC33846Δcpx R and ATCC33846Δcpx AR mutant strains,and lipid A was added 1 C14:0 secondary fatty acid chains that increase resistance to polymyxin B.(5)The VP＿RS21300 gene was found to play a key role in the high polymyxin tolerance and pathogenicity of V.parahaemolyticus.It was found by BLAST that the VP＿RS21300 gene of V.parahaemolyticus had high homology with E.coli ept A gene.After knocking out ept A gene in E.coli and overexpressing VP＿RS21300 gene,lipid A was extracted and analyzed by mass spectrometry.It was found that there was an additional phosphoethanolamine group on the 1-or 4’-phosphate of lipid A,indicating that V.parahaemolyticus VP＿RS21300 gene encodes lipid A phosphoethanolamine transferase.Knockout of the VP＿RS21300 gene in V.parahaemolyticus resulted in the disappearance of phosphoethanolamine on its lipid A structure,again proving that the VP＿RS21300 gene of V.parahaemolyticus encodes lipid A phosphoethanolamine transferase.The MIC values of wild-type V.parahaemolyticus ATCC33846 for polymyxin B and polymyxin E were 45.7 and 42.2 times higher than those of the VP＿RS21300 gene-deleted strain ATCC33846ΔRS21300,respectively,indicating that the VP＿RS21300 gene is highly tolerant to polymyxin in V.parahaemolyticus plays a key role.The survival rate of RAW264.7 cells stimulated by strain ATCC33846ΔRS21300 was 2.8 times higher than that stimulated by strain ATCC33846,indicating that the VP＿RS21300 gene also plays a key role in the pathogenicity of V.parahaemolyticus. |
PDF Full Text Request