Effect Of Atmospheric Pressure Nonequilibrium Plasmas On Neisseria Gonorrhoeae

Objective To investigate preliminarily the sterilizing effect of atmospheric pressure nonequilibrium plasmas(APNPs) on Neisseria gonorrhoeae and further exploit the possible mechanism.Methods N. gonorrhoeaeFA1090,FA19 and MS11 were treated by APNPs. Their survival rate was analyzed and studied under laser scanning confocal microscope. The morphological changes of bacterial cellmembrane and wall were studied under TEM.Results APNPs had strong sterilization effect on N. gonorrhoeae. The survival rate of MS 11 in Gc liquid medium was 60.65% after disinfection with the APNPs for 5min, whereas, the survival rate of FA 19 was 92.60% and the survival rate of FA 1090 was 96.40%. The survival rate of MS 11 was 21.13% after disinfection with the APNPs for 6min, whereas, the survival rate of FA19 was 31.60% and the survival rate of FA1090 was 91.00%. Under laser scanning confocal microscopy, for t*= 6 min plasma treatment, we get 30% dead bacteria in MS11. However, for the same time plasma treatment, we get 20% dead bacteria in FA1090,FA19. The gross morphological damage of N. gonorrhoeae cell was observed.Conclusion APNPs is able to kill the N. gonorrhoeae effectively and rapidly, it is related to the cell membrane damage. Our results suggested that APNPs be widely used in disinfection and sterilization, prevention and cure of some local infection of N. gonorrhoeae prospectively. Objective To compare the decontamination performances of the atmospheric pressure nonequilibrium plasmas (APNP) on planktonic Neisseria gonorrhoeae and biofilm-resident Neisseria gonorrhoeae of medical interest.Methods The decontamination efficiency of the process was evaluated against bacteria embedded within a bio film, as well as planktonic cells placed on a glass surface. Bacterial survival was assessed using a combination of Colony Forming Unit (CFU) ability and vital staining with a combination of SYTO9 plus Propidium Iodide. Transmission electron microscopic obserbations revealed the morphological changes of N. gonorrhoeae following plasma treatment.Results CFU ability and vital staining with a combination of SYTO9 plus Propidium Iodide revealed an increased resistance of biofilm-resident bacteria, when compared to planktonic cells. The logioN of survival planktonic N. gonorrhoeae was 3.42 after disinfection with the APNPs for 6min, whereas, the log10N of survival N. gonorrhoeae in biofilm was 3.71. The log10N of survival planktonic N. gonorrhoeae was 0 after disinfection with the APNPs for 12min, whereas, the logioN of survival N. gonorrhoeae in biofilm was 2.36. Under laser scanning confocal microscopy, for t*= 8-min plasma treatment, we get 92% dead bacteria and 8% of green fluorescent living cells for planktonic cell. The same experiments realized with biofilm generate 90% of dead bacteria. For 8< t*< 12 min, the number of green fluorescent cells falls close to zero for planktonic bacterial cultures. The number of red fluorescent (dead) bacteria increases with t* for biofilm cells. The ultrastructural morphological damage of planktonic N. gonorrhoeae cell was more serious than N. gonorrhoeae in biofilm.Conclusion APNPs is more efficient for planktonic N. gonorrhoeae than for N. gonorrhoeae in biofilm. Our results suggested that N. gonorrhoeae in bioflm should take into consideration when use APNPs in disinfection and sterilization of N. gonorrhoeae, prevention of gonorrhea prospectively. Objective To examine gene expression during the biofilm formation in Neisseria gonorrhoeae and explore differential gene expression in Neisseria gonorrhoeae within biofilms following exposure to atmospheric pressure nonequilibrium plasmas.Methods Neisseria gonorrhoeae biofilm model was prepared.The levels of pilC1,pilE,mntC mRNA were detected on the first, second, fourth, sixth day during the culture using fluorescent quantitative PCR technology.The samples cultured for two days were exposed to atmospheric pressure nonequilibrium plasmas for 2,4,8, 10min and then cultured for 6h. The levels of pilC1,pilE mRNA were detected using fluorescent quantitative PCR. The samples cultured for four days were also exposed to atmospheric pressure nonequilibrium plasmas for 2,4,8, 10min and then cultured for 6h. The levels of mntC mRNA were detected using fluorescent quantitative PCR.Results The levels of pilCl,pilE mRNA increased gradually from day 1 to day 2. From the fourth to the sixth day, they decreased gradually. The levels of mntC mRNA increased gradually from the first day to the fourth day. But on the sixth day, the level of mntC mRNA decreased dramatically. The samples cultured for two days were exposed to atmospheric pressure nonequilibrium plasmas and then measured the levels of pilC1,pilE mRNA. The levels of pilCl,pilE mRNA decreased gradually with the prolonged exposure. The samples cultured for four days were exposed to atmospheric pressure nonequilibrium plasmas and then measured mntC mRNA levels. The mntC mRNA levels increased gradually with the prolonged exposure when the exposure time was less than 4min. However, the levels of mntC mRNA decreased gradually with the prolonged exposure when the exposure time was more than 8min.Conclusion The gene pilC1,pilE play role in the initial period of biofilm formation of Neisseria gonorrhoeae. The gene mntC play role in Neisseria gonorrhoeae biofilm maturation. Atmospheric pressure non-thermal plasma exposure was found to significantly down-regulate the expression of genes pilC1,pilE. The expression of mntC was up-regulated after exposure to plasma for some time, and then down-regulated. Objective To determine the effect of atmospheric pressure non-thermal plasma on the susceptibilities of Neisseria gonorrhoeae to antibiotics.Methods Cultures of N. gonorrhoeae MS 11 and FA 1090 taken in stationary phase were exposed to the atmospheric pressure non-thermal plasma. Following plasma exposure for various time, the in vitro activities of five antimicrobial agents against N. gonorrhoeae were investigated by MIC assays and killing curve studies. Scanning electron microscopic obserbations revealed the morphological changes of N. gonorrhoeae following plasma treatment.Results The MICs of a variety of antibiotics (penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone) following 8min plasma treatment were reduced by 4-to 8-fold. In the killing curve studies, combinations of 8min plasma treatment with penicillin, tetracycline, ciprofloxacin, spectinomycin or ceftriaxone resulted in 4-to 6-log10FU/ml reductions in bacterial counts when 25% of the MIC of each antibiotic was used.Conclusion These observations are consistent with our killing curve studies. These results indicate that atmospheric pressure non-thermal plasma could increase the susceptibilities of Neisseria gonorrhoeae to antibiotics.