| Food-borne pathogens are an important biological factor leading to food safety problems,which seriously endanger human health.The emergence of drug-resistant food-borne pathogens not only increases the difficulty and burden of medical treatment,but also can spread in the food chain,leading to aggravation of bacterial drug resistance.In the food industry,most pathogenic microorganisms mainly adhere to the food surface in the form of biofilms.These biofilms not only affect food safety,but are also considered to be closely related to the formation of bacterial resistance and can help bacteria resist.The invasion of external antibiotics increases the difficulty of disease treatment and causes serious economic losses and social burdens.However,at this stage,the exploration of bacterial biofilm drug resistance is mostly focused on the field of clinical research,and there are relatively few studies on the biofilm drug resistance mechanism of food-borne pathogens.Clarifying the law of drug resistance of foodborne pathogens in biofilms will help to better control the harm of food-borne pathogens.Therefore,this study aimed at the two key and difficult issues of food safety:biofilm and drug resistance of food-borne pathogens.First,the Meta-analysis technology was used to explore the prevalence and relationship between biofilm and drug resistance of food-borne pathogens in the world.distribution,and further selected Vibrio parahaemolyticus as the research object to analyze the drug resistance differences between planktonic and enveloped Vibrio parahaemolyticus.Finally,confocal laser microscopy,extracellular polymer lyase treatment,and transcriptomic techniques were used.comprehensively and systematically revealed the resistance mechanism of Vibrio parahaemolyticus biofilms against aminoglycoside antibiotics.The main research contents and results of this study are as follows:1.Meta-analysis reveals global foodborne pathogen resistance and biofilm prevalenceIn order to better understand the formation and distribution of drug resistance and biofilms of food-borne pathogens around the world,and to clarify the common problems of the formation rules and mechanisms of drug resistance of food-borne pathogens.In this study,332 groups of research data from 36 countries were obtained through literature collection in the electronic resource database,and statistical methods such as meta-analysis were used to summarize and analyze the related researches on the drug resistance and biofilm of food-borne pathogens in the world.Multivariate analysis.The results showed that the resistance levels of foodborne pathogens isolated from Africa and South America were significantly higher than those of the other three continents.Globally,the resistance rate of food isolates is higher than 10%,of which the resistance to β-lactamase antibiotics is the strongest,and the resistance of Bacillus cereus is as high as 94%.The resistance rates of all clinical isolates were greater than19%,and were more resistant to various antibiotics than food isolates.The overall biofilm formation rate for foodborne pathogens was 90%(95% CI,68%-96%),and there was no direct linear relationship between biofilm formation and antibiotic resistance(free state).This study is the first to use meta-analysis to reveal the global prevalence of food-borne pathogen resistance and biofilms,laying a solid data foundation for revealing the drug resistance mechanism of food-borne pathogens in biofilms.2.Correlation between drug resistance of Vibrio parahaemolyticus and biofilm formation abilityIn this study,Vibrio parahaemolyticus was further selected as the research object,and the correlation between its drug resistance and biofilm formation ability was analyzed.The resistance of 32 free and enveloped V.parahaemolyticus strains to 8common antibiotics was determined by the micro-broth dilution method and the biofilm resistance assay method constructed for the first time in this study.Combined with crystal violet staining,the biofilm formation ability of Vibrio parahaemolyticus was revealed.Finally,the correlation between its drug resistance and biofilm was studied.Through Spearman correlation analysis,it was found that there was no correlation between the drug resistance(MIC)of free Vibrio parahaemolyticus and its biofilm formation ability(p-value>0.05),which verified the relevant results of the Meta analysis in Chapter 2 of this paper.The amikacin(AK)resistance(BIC)of enveloped Vibrio parahaemolyticus to aminoglycosides was positively correlated with the biofilm formation ability(p-value<0.01).Further comparative analysis found that the drug resistance of the enveloped strains was significantly higher than that of the planktonic strains,and the difference in resistance to aminoglycoside antibiotics was the most obvious.3.Study on the resistance mechanism of Vibrio parahaemolyticus biofilms to aminoglycoside antibioticsIn order to explore the resistance mechanism of Vibrio parahaemolyticus biofilms to aminoglycoside antibiotics,this paper firstly analyzed the microstructural characteristics of Vibrio parahaemolyticus biofilms with the help of laser confocal microscopy,and correlated them with drug resistance.Correlation analysis.The main components of biofilm,e DNA and extracellular proteins,were treated with key enzymes(DNase I and proteinase K)to explore the changes in drug resistance before and after treatment.Finally,the transcriptomes of free and enveloped V.parahaemolyticus were sequenced to analyze the differences in gene expression regulation.The results showed that the biological volume(BV)and average thickness(AT)of the biofilm of Vibrio parahaemolyticus were significantly positively correlated with the amikacin resistance(BIC)of its envelope state,and the Spearman correlation coefficients were 0.813 and 0.642,respectively.There is a certain correlation between BV and the resistance of Vibrio parahaemolyticus in enveloped state to gentamicin(pvalue<0.05).After treatment with key enzymes,the resistance of the enveloped Vibrio parahaemolyticus to aminoglycoside antibiotics was significantly reduced,and the DNase I treatment was more significant.Transcriptome sequencing analysis showed that after Vibrio parahaemolyticus formed a biofilm,a total of 724 genes were significantly up-regulated and 260 genes were down-regulated,among which the differential expression of anion transport and ABC transport system functional pathway genes was obvious.After comprehensive analysis,this paper preliminarily revealed the formation mechanism of aminoglycoside resistance of Vibrio parahaemolyticus in biofilms:(1)Resistance mechanism: Vibrio parahaemolyticus produces aminoglycosides by increasing the biomass,average thickness and other parameters of biofilms.Glycoside antibiotic resistance;(2)Reduction mechanism: Anionic extracellular polymers in Vibrio parahaemolyticus biofilm can combine with cationic antibiotics to reduce the invasion of antibiotics,resulting in drug resistance.(3)Regulatory mechanism: After Vibrio parahaemolyticus forms a biofilm,the expression of its ABC transport system and anion transport is significantly up-regulated,which helps to promote the formation of drug resistance. |
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