| Dairy mastitis,one of the critical factors restricting dairy cows’ healthy breeding,has brought huge economic losses to the breeding industry.Antibiotics are the availably essential means of treating dairy mastitis.However,with the widespread application of antibiotics and the increasingly rapid evolution of bacteria,the problem of bacterial resistance has gradually become a major global problem threatening the development of animal husbandry.The resistance of Staphylococcus aureus,one of the important pathogens of dairy mastitis,has plagued the dairy farming industry.Solving this problem relied on both the continuous development of new antibiotics,the implementation of policies to regulate the rational use of antibiotics,and the innovation of strategies to predict and prevent the evolution of antibiotic resistance based on a deep understanding of the mechanisms of adversarial evolution.This experiment revealed the effect of the efflux pump on the evolution of antibiotic resistance in Staphylococcus aureus through in vivo induced evolutionary tests supplemented by molecular and cellular experimental techniques.The main findings are as follows:1.Elevated expression of efflux pumps in tolerant strains facilitates the development from tolerance to resistance.The expression of six efflux pump genes was significantly higher in nine tolerant strains(acquired by early induction in our laboratory)than in the wildtype strains.Further resistant evolutionary tests were performed on two of the tolerated strains,which revealed that the tolerant strains established resistance earlier than the wildtype strains.This implies that the efflux pump played an essential role in the evolution of drug resistance.To determine whether the expression level of the efflux pump results in the resistance evolution,we first successfully constructed overexpression and knockout strains of the efflux pump norA and the efflux pump abcA.An in vitro resistance evolution test showed that the overexpression of the two efflux pumps promoted the resistance evolution,while knockouts delayed resistance evolution.These results indicate that the expression level of the efflux pumps in Staphylococcus aureus positively correlates with the resistance evolution.2.The mechanism underlying the development from tolerance to resistance mediated by efflux pumps.To explore how efflux pumps affect the evolution of resistance,we determined the random mutation rate of efflux pump mutants and their survivability under antibiotic and non-antibiotic conditions.The random mutation rates of norA and abcA efflux pump-overexpressed strains were significantly higher than those of the control strain.Killing-time tests revealed that the norA and abcA overexpression strains had a survival advantage compared to wildtype strains.Meanwhile,the survival rates of norA and abcA knockout mutants significantly reduced this effect,with norA showing the highest significance.Analyzing the growth rate of the exponential growth stage,we found that overexpression of norA and abcA led to an increase in the exponential growth rate.Knockout of norA led to a decrease in the growth rate of bacteria.However,the knockout of abcA showed the opposite result: the growth rate increased instead.These results indicate that during resistance evolution,efflux pumps increase the probability of resistance mutation through its high expression and enhances the adaptability to make it fixed,promoting the evolution of drug resistance.Additionally,the detection of mutation sites of the norA knockout showed no adaptive genetic preference,indicating that the efflux pump mainly affected the resistance evolution by influencing the generation and spread of drug resistance mutations.To illustrate the relationship between multiple efflux pumps in the resistance evolution of the same antibiotic in Staphylococcus aureus,two other efflux pump genes targeting ciprofloxacin,mepA and sav1866,were knocked out.The in vitro resistance evolution test found that the emergence of resistance in the mepA and sav1866 knockout strains was delayed by 4 and 6 cycles,respectively,with the relative resistance significantly reduced compared to the control.By measuring the expression of the other two pump genes in the norA,mepA,and sav1866 knockout strains,we found that the knockout of any efflux pumps would directly cause a compensatory effect on the other two.These results suggest that,rather than the specific effect of the efflux pump norA,other efflux pumps(mepA and sav1866)were also involved in ciprofloxacin’s resistance evolution in Staphylococcus aureus,with a complementary role between these efflux pumps.Through studying the transcriptional regulator mgrA knockout strains,we found that mgrA positively regulated the expression of norA and affected resistance evolution by regulating the expression of other efflux pumps.3.Efflux pump inhibitors delayed the resistance development from tolerance to resistance.The important role of efflux pumps in the resistance evolution of resistance illuminated us to block or delay the evolution of resistance by inhibiting the expression of the efflux pump.To explore the feasibility of this strategy,we studied the effect of the efflux pump’s inhibition on resistant evolution and bacterial infection of cells.The results from the resistant evolutional experiment found that the addition of the efflux pump’s inhibitor,reserpine,delayed resistant emergence by 5 cycles,indicating that reserpine can block the evolution of resistance to some extent.Bacteria adherence and hemolysis experiments showed that inhibiting efflux pumps can reduce the bacteria’s ability to adhere to cells and virulence,thereby preventing bacteria from infecting host cells.Together,these results suggest that an efflux pump inhibitor not only blocked the development of antibiotic resistance but also disrupted the bacterial infection of cells.4.Tolerant mutations protect the fixation of drug-resistant mutations in the population.To explore whether the resistance mutation can be fixed in the population by other means outside efflux pumps,the relationship between tolerant and resistant mutations was studied.Firstly,the tolerant,resistant,and tolerant and resistant mutations of ciprofloxacin and oxacillin were constructed by CRISPR/Cas9 gene-editing technology and molecular technology,respectively.Then,by detecting the viability of the mutant strain under antibiotics and competitiveness tests,it was found that tolerant and resistant mutations synergistically enhanced the survival of bacteria under antibiotic treatment.Moreover,the tolerant background can protect the resistance mutations and is beneficial for their fixation in populations.In summary,the expression level of efflux pumps positively correlated with the evolution of antibiotic resistance in Staphylococcus aureus.The efflux pump improves the probability of resistance mutation generation through its high expression.On the one hand,the generated mutations were fixed in the population by enhancing the survival ability and adaptability of bacteria.On the other hand,the tolerant background protects resistance mutations to promote resistance,which is regulated by mgrA.The inhibition of the efflux pump blocks the evolution of antibiotic resistance and destroys the bacterial infection of cells.This study reveals the role of the efflux pump in the evolution of resistance and its potential mechanisms,provides new evidence for understanding the evolutionary mechanism of antibiotics and proposes a new strategy to inhibit the evolution of drug resistance by blocking the expression of the efflux pump. |
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