| Both Acinetobacter baumannii(AB)and Neisseria gonorrhoeae(NG)belong to Neisseriaceae bacteria that have become serious global public health problems.A.baumannii often causes pneumonia,bloodstream infections,meningitis and wound infections,etc,and the mortality rate is very high.Gonorrhea caused by N.gonorrhoeae is the second popular sexually transmitted disease in the world that causes serious consequences such as infertility.The situation of drug resistance of the two pathogens is more and more severe,and cases of treatment failure due to drug resistance have been reported continuously.A comprehensive understanding of the resistance related biological characteristics of the two pathogens could provide useful data for the control of the serious drug resistance problems.Bacterial resistances often lead to variations in virulence.A.baumannii resistant strains probably have a stronger ability to form biofilms,however there were also reports of contrary conclusions.And research on the relationship between drug resistance and other virulences has not been reported,so the first part of this study aims to explore the relationship between drug resistance and virulences of A.baumannii,to provide an important theoretical basis for the effective prevention and treatment of infection caused by A.baumannii.There are a large number of small non-coding RNA(sRNA)in bacteria,and their roles in the regulation of drug resistance had been widely demonstrated;however there is no report on the regulation of drug-resistant sRNA in N.gonorrhoeae.The second part of this study aims to discover the sRNAs that regulate ceftriaxone resistance in N.gonorrhoeae to provide a new idea for solving it's drug resistance problem.1.Studies on the relationship between drug resistance and virulence of A.baumanniiIn order to study the relationship between drug resistance and virulence of A.baumannii,the 16S rRNA genes of the 36 clinical isolates collected by our group were identified.Then the antibiotic resistance to 21 drugs covering all the 9 antimicrobial categories was assessed for each A.baumannii clinical isolate by Kirby-Bauer disc diffusion method or the broth microdilution method.Serum resistance was performed by mixing 10%bacterial solution with 90%normal human serum(heat-inactivated human serum as a control)and then incubated in a 37 ?incubator for 1-3 h.The serum resistance of the strain was considered to be weak as the number of surviving bacteria in the normal human serum group was lower than that of the control group with a significant difference.Otherwise the serum resistance was considered to be strong.A549 cells were mixed at MOI of 100 with bacterial solution and then incubated in a 37 ? incubator for 1 h,the number of bacteria adhering to each cell was measured as the adhesion level of the strain.The mouse pneumonia model of was established to detect the mortality rate of mice caused by A.baumannii infection.Five strains with different drug resistance were selected for the virulence test in vivo.Statistical method was performed to analyze the relationship between drug resistances and the 3 kind of virulences of A.baumannii.The results showed that 34 of the 36 clinical isolates were identified as A.baumannii,among which the proportion of multidrug-resistant strains and extensively-resistant strains was as high as 97.06%.The serum resistance tests revealed 26 strong serum resistance strains and 9 week serum resistance strains,and the resistant strains had stronger serum resistance.The ability of drug-resistant strains to adhere to A549 cells was stronger than that of non-resistant strains.The in vivo virulence test showed that the extensively-resistant A.baumannii strains resulted in higher mortality of mice.The above data suggested that the drug resistance problem of A.baumannii was very serious,there was a positive relationship between drug resistances and virulences of A.baumannii.2.Screening of ceftriaxone resistance related sRNAs in N.gonorrhoeaeTo screen the ceftriaxone resistance related sRNAs in N.gonorrhoeae WHO-A strain,the strain was transcriptome sequenced to obtain the information of it's non-coding RNA(sRNA).Then the strain was induced with gradient dose of ceftriaxone to get resistant strains.A combination of bioinformatic methods and Real-time Quantitative PCR were used to screen and verify the ceftriaxone resistance related sRNAs,and reverse transcription PCR was used to vilify the distribution of drug-resistant sRNAs in N.gonorrhoeae clinical isolates.The results showed that the mutant resistant gonococcal strains(MIC=1 ?g/ml)were obtained by drug induction.The wide-type WHO-A strain contained 1051 sRNAs,of which 80 sRNAs were complementary to 8 resistance-related genes,e.g.,penA,porB,mtrR,etc.The expression levels of 10 sRNAs were decreased with the increasing of ceftriaxone resistance,among which 6 sRNAs were transcribed in 3 gonococcal clinical isolates.These results indicated that some sRNAs were involved in the ceftriaxone resistance regulation and widely distributed in N.gonorrhoeae. |
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