Insights Into The Properties,mechanisms And Pathogenicity Of L-form In Staphylococcus Aureus

Objective: To explore the properties,mechanisms and pathogenicity of S.aureus L-form.Methods: S.aureus unstable L-form model was established followed with our previous study.Atomic force microscopy(AFM)was used to detect the L-form morphology and turgor pressure effects on the bacteria.To detect the interactions between L-form colonies,the cultivation of transformed Newman-pCM29,Newman-PAH9 and Newman wild type were mixed and inoculated on L-form induced medium(LIM)and the L-form colonies were observed under laser scanning confocal microscope.Zeta potential of L-form and normal form(N-form)were measured and used to analyse the aggregation characters of bacteria.Growth and proliferation properties were observed under optical microscope and transmission electron microscope.The susceptibility of S.aureus L-form to antimicrobial agents was measured by disc diffusion method.The hemolytic ability of S.aureus L-form was determined by hemolysis test in vitro.Mouse subcutaneous infection was used to detect the pathogenicity of S.aureus L-form.RNA sequencing was performed to detect the gene expression changes during S.aureus wild type transition into L-form and bioinformatics analysis results were verified by Q-PCR.Laser scanning confocal microscopy was used to evaluate the effections of fluorescent reporters labeled in S.aureus on properties and pathogenicity of L-form and wild type.Results: 1.S.aureus L-form presented pleomorphism,rough surface.The turgor pressure(68.6 ± 7.0 kPa)of L-form was higher than that of N-form(61.4 ± 5.0 kPa)significantly(P < 0.05).2.There exsited obvious boundary between S.aureus L-form colonies from different sources.The core of “fried egg” colonies embedded in soft agar.Not only the bacteria in the thin edge,but also in the dense core,all possessed the ability to proliferate as L-form colonies.The charge of L-form(-7.7m V)were less than N-form(-16.5mV),significantly(P < 0.05).Blebbing,tabulation and vesiculation appeared in S.aureus L-form proliferation.3.After induced 12 h,compared with N-forms,the susceptibility of L-form to antimicrobial agents showed an increasing trend except for ceftriaxone.After induced 24 hours,compared with N-forms,the L-form displayed resistance to ceftriaxone,tetracycline,and erythromycin completely but was susceptible to levofloxacin and linezolid,and.The susceptibility to vancomycin,trimethoprim / sulfamethoxazole,and chloramphenicol also presented as decrease decreasing trend.4.S.aureus L-form could form distinct ?-hemolytic zone on blood LIM after 72 h incubation while N-form did not form hemolytic zone even if they were incubated 10 days on hyperosmotic control medium blood agar.S.aureus L-form could cause mouse subcutaneous infection.N-form and L-form colonies could be formed after the abscess homogenates were plated on TSA and LIM.5.Compared with the N-form,96 genes were down-regulated and 62 genes up-regulated in S.aureus L-form.Differentially expressed genes were involved mainly in energy production,stress response,protein synthesis,RNA metabolism,and virulence.6.The fluorescence reporter in S.aureus contributed to the observation of L-form “fried egg” colony and embedded growth.The distribution and intensity of S.aureus wild type in mouse infected lesions could be effectively used to detect the distribution and the amount of bacteria,which could be used to estimate the virulence of S.aureus.Conclusions: 1.S.aureus unstable L-forms are chacterized by high turgor pressure and high pleomorphism.L-form colonies were embedded in the agar.It is a principle that one wild type bacterium can be induced into one colony.The bacteria in the thin edge and in the dense core all possessed the ability of to proliferate as L-form colonies.The changes of zeta potential are closely related to aggregation properties in S.aureus L-form.Blebbing,tabulation and vesiculation were important modes of L-form proliferation.The antibiotic susceptibility of L-form exhibited difference from N-form.S.aureus L-form become more tolerant than N-form to most antimicrobial agents except for levofloxacin,chloramphenicol and linezolid.2.S.aureus L-form possessed stronger hemolytic capacity than N-form on hyperosmotic environment.S.aureus L-form could still cause subcutaneous infections in mice.3.Pathways and genes including energy production,stress response,proteins synthesis,RNA metabolism,and virulence were involved in S.aureus L-form formation and survival.3.The fluorescence reporter in S.aureus contributes to observation of S.aureus L-forms formation and pathogenicity in S.aureus.