The Effect Of Density On The Persistence Of Staphylococcus Aureus And Its Molecular Mechanisms

Staphylococcus aureus is a common clinical pathogen that causes suppurative infections.The development of drug resistance and the formation of persisters are the challenges faced by clinical treatment of S.aureus infections.At present,the formation mechanism of S.aureus persister is still unclear.Therefore,exploring the mechanism of persister formation is of great significance for targeted treatment.This subject is based on the phenotypic phenomenon that the density of S.aureus in different growth phases and the degree of persistence is also different.The effect of bacterial density on the formation of persister was discussed.Transcriptome sequencing and related gene knockout were used to explore related molecular mechanisms.Objective:To explore the effect and molecular mechanisms of the density in the bacterial solution on the persister formation of S.aureus.Methods:1.After washed by PBS two times,the exponential phase of S.aureus Newman strain was concentrated 100-fold,and the stationary phase was diluted 1000-fold to prepare low-density bacterial suspension（including exponential phase and the diluted stationary phase bacterial suspension）and high-density bacterial suspension（including stationary phase and the concentrated exponential phase bacterial suspension）.After placed at 37℃ for 6 hours,ampicillin（10μg/mL）,norfloxacin（200μg/mL）,gentamicin（200μg/mL）and vancomycin（100 μg/mL）were used to perform antibiotics exposure tests to determine the formation of persister in S.aureus at different densities and different growth phase.At the same time,the antibiotics exposure tests were carried out immediately after the low-and high-density bacterial suspension were prepared,and the formation of persister was also measured.2.The 1:10-fold,1:1000-fold,and 1:100,000-fold diluted S.aureus stationary phase suspension by PBS were prepared respectively,and ampicillin,norfloxacin,gentamicin and vancomycin were used as above to perform antibiotic exposure tests to determine the formation of persister at different dilutions.3.Starvation test in PBS on low-and high-density bacteria was used to determine the effect of density on persistence in the absence of nutrients.According to the dilution of the bacterial suspension,hydrogen peroxide was added to carry out the oxidative stress test to determine the effect of the density change of the persistence on the antioxidant capacity of S.aureus.4.The growth curve of S.aureus in 1:10,1:100,1:1000,1:10000,1:100,000 and 1:1000000-fold diluted with TSB were measured.Based on the quantity of S.aureus in cultures which had been cultured 5 hours after diluted 1:1000-fold with TSB,according to the growth curve,the time required for growing to the similar amount of bacteria after they were diluted 1:100,1:10000,1:100,000,and 1:1000000-fold in TSB were determined.The samples selected at the determined time point and one time point before and after it were used to perform ampicillin exposure test,and the forming ability of persistence at each time point was determined.5.The samples of the S.aureus in exponential phase and its concentrated suspension,the stationary phase and its diluted suspension were collected.The total RNA was extracted using the Trizol method,and the transcriptome sequencing was used to determine the transcript level of genomic transcription in bacterial suspension at different growth phase and different densities.QPCR validated the results and used bioinformatics analysis to explore the molecular mechanisms by which the bacteria density affects the formation of S.aureus persister.6.Using homologous recombination technology,the plasmid pMX10 was selected to construct the S.aureus gene knockout strain ΔNWMN₂288.The growth features on TSA and the ability to ferment mannitol were detected.Through the exposure test of wild type and ΔNWMN₂288 against ampicillin and norfloxacin,the effects of NWMN₂288 on the persistence of S.aureus in different growth phases and different density bacterial suspension were observed.Results:1.For the high-density and low-density bacterial suspension which were placed for 6 hours after concentrated or diluted,could,the bacteria in low-density suspension（the diluted stationary phase/exponential phase）can be eradicated completely after they were exposure to lethal dose of ampicillin,norfloxacin,gentamicin,and vancomycin for 5/3,1/2,3/1 and 4/2 days,while the bacteria in high-density bacterial suspension（the concentrated exponential phase/stationary phase）persisted to the drugs,and displayed that ampicillin and vancomycin failed to completely eradicated them after exposed for 10 days,and norfloxacin and gentamicin also required at least 6/7 and 6/7 days to completely eliminate them.The status of persister formation in the different suspension added the antibiotics immediately after they were prepared showed similar results.2.Different diluted suspension of S.aureus in stationary phase showed different persistence to antibiotics.The bacteria in 1:10-fold suspension were eliminated completely after exposed to ampicillin and vancomycin for 8 days,and to norfloxacin and gentamicin for 5-6 days.The bacteria in 1:1000-fold suspension were eliminated completely after exposed to ampicillin and vancomycin for 2-4 days,and to norfloxacin and gentamicin for 1-2 days.The bacteria in 1:100000-fold suspension were eliminated completely after they were exposed to ampicillin 2 days,and it could be completely eliminated within 1 day under the action of vancomycin,norfloxacin and gentamicin.3.The starvation test results showed that the most bacteria in the exponential phase and its concentrated suspension and the stationary phase suspension could survive after they were placed in PBS for 20 days,while the bacteria in the diluted stationary phase suspension all died after lacked with nutrition 14 days.The bacteria in the low density suspension were susceptible to hydrogen peroxide than that of high-density suspension.4.The 1:10,1:100,1:1000,1:10000,1:100,000 and 1:1000000-fold diluted inoculum of S.aureus could reach the stationary phase after cultured 2,4,6,8,10 and 12 hours respectively.The quantity of the bacteria（logarithmic value）in 5 hours the culture which was inoculated at 1:1000-fold with TSB initial was about 8.3,and the suspension which inoculated at 1:100,1:10000,1:100,000 and 1:1000000-fold needed to culture 3,7,9 and 10 hours respectively to reach the same quantity which was the key number of the bacteria in the liquid to form persisters.5.Transcriptome sequencing results showed 92 up-regulaed genes in exponential phase,52 up-regulated genes in stationary phase samples,and 8 of these genes（pflB,NWMN₀163,coa,NWMN₀175,ldh1,pyrB,NWMN₂288,nrdG）were up-regul-ated together.134 genes were down-regulated in the exponential phase,and 30 genes were down-regulated in the stationary phase samples,and 3 of these genes（sarA,NWMN₁252,trpG）were down-regulated together.The main functions of the differential genes were distributed in cell metabolism and biosynthesis processes,transport and binding,metabolic process regulation,cell membrane and cell components.The significant enrichment KEGG pathways shared by the high-density and low-density suspension were included two-component system,nitrogen metabolism,pyruvate metabolism,propanoate metabolism,amino acid metabolism,oxidative phosphorylation,chloroalkane and chloroolefin degradation,pyrimidine metabolism,fatty acids degradation,riboflavin metabolism,biological synthesis of secondary metabolites,microbial metabolism in different environments,carbon metabolism pathways.6.Compared with the wild type,ΔNWMN₂288 had no significant changes in the colony shape,size,and pigment on TSA,but the ability to ferment mannitol was somewhat reduced.After 1:1000-fold in oculation and incubated for 5 and 9 hours,the persistence of ΔNWMN₂288 to ampicillin and norfloxacin decreased.The capacity for persister formation of ΔNWMN₂288 in the high-density suspension was also decreased,while there was shown significant difference in the low-density suspensionConclusions:1.The density of bacteria is an important factor affecting the formation of persister in S.aureus,and plays a "switching" role.When the number of bacteria（logarithmic value）reaches a threshold of about 8.3,a large number of persisters can be formed.2.Bacterial density can affect and regulate the formation of persister in S.aureus by regulated the two-component system,nitrogen metabolism,pyruvate metabolism,propionic acid metabolism,alanine,aspartate and glutamate metabolism,oxidative phosphorylation,chloroalkane and chloroalkene degradation,pyrimidine metabolism,fatty acid degradation,riboflavin metabolism,biological synthesis of secondary metabolites,microbial metabolism in different environments,carbon metabolism pathways.3.NWMN₂288,which encodes and regulate nitrate transport in two-component system,participates in the regulation of density affecting the formation of persister in S.aureus.