Whole Genome Sequencing To Study On Antibiotic Resistance Genes In Ceftriaxone~low Sensitivity Neisseria Gonorrhoeae Isolates In Guangzhou

[Background]Gonorrhea is the suppurative inflammation of the genitourinary tract following infection with Neisseria gonorrhoeae.With highly prevalence,there are about 106 million new cases every year of gonorrhea reported by WHO.The main infections of gonorrhea are Young and middle^aged,which not only seriously affects the quality of life and physical and mental health of patients,but also brings enormous health,medical and economic burdens to society.Gonorrhea has become an increasingly serious problem of public health.The continuous emergence of antibiotic resistance to Neisseria gonorrhoeae and the difficulty of transmission control make these problems more and more serious.Neisseria gonorrhoeae have a remarkable evolutionary capacity for drug resistance and have eliminated antimicrobial agents once used as first^line treatments for gonorrhea.At present,ceftriaxone is used alone or in combination with azithromycin as a first^line treatment in most countries or regions,and is also threatened by drug resistance.Since 2011,Japan has reported the first case of failed clinical treatment of ceftriaxone caused by Extensively drug^resistant（XDR）Neisseria gonorrhoeae H041 strain.Many countries or regions have reported the failure of clinical treatment of ceftriaxone.From 2000 to 2016,we have found that the sensitivity of Neisseria Gonorgonococcus was significantly decreased.A method of genome analysis that uses known reference genomes to sequence individuals or populations is called whole genome sequencing,which is used to analyze genetic variation at the individual or population level.At present,the drug resistance of ceftriaxone is mainly concerned with the penA gene,in addition,there is penB、mtrR、ponA、pilQ、gyrA、parC and so on.However,it could not explain the drug resistance of Neisseria gonorrhoeae ceftriaxone by combination of penA/23 S rRNA/mtrR or themselves alone.The whole genome sequencing is helpful to identify known or unknown resistance sites of Neisseria gonorrhoeae to ceftriaxone.[Objective]In this study,the drug resistance of 7 antibiotics of Neisseria gonorrhoeae in Guangzhou in 2017 was monitored and in comparison and analysis of its trend with previous results.At the same time,from January 1999 to December 2017,about 1700 strains of Neisseria gonorrhoeae were collected from Guangzhou medical university institute of dermatology.Experimental strains screening from 1700 strains of Neisseria gonorrhoeae were identify the associated candidate known / unknown genes by the whole genome sequencing and confirm their association with the resistance of Neisseria gonorrhoeae to ceftriaxone in further.[Methods]A total of 101 strains of Neisseria gonorrhoeae isolated from Guangzhou in 2017 were collected.The minimum inhibitory concentration（MIC）of several antibiotics was determined by agar dilution method,and the sensitivity was determined according to the standard of the World Health Organization（WHO）.From January 1999 to December 2017,about 1700 strains of Neisseria gonorrhoeae were resuscitated,isolated and identified,and other antibiotics with similar MIC values were screened out for whole genome sequencing of hypersensitive strains and sensitive strains of ceftriaxone.[Results]1.In 2017,the number of penicillin producing gonococcal gonococci（PPNG）in Guangzhou area was 21.42%（21 / 98）,the TRNG of highly resistant Neisseria gonorrhoeae was 24.49%,the resistance rate of ciprofloxacin was 100 %（98 / 98）,the resistance rate of azithromycin was 6.12%（6 / 98）,no drug^resistant strain was found to be resistant to spectinomycin,and the hypersensitivity rate of ceftriaxone was 6.93%（7 / 101）.2.Whole genome sequencing showed that ceftriaxone hypersensitive strain and ceftriaxone sensitive strain had significant differences in 9 gene mutation sites,including NC₀11035₂141888、NC₀11035₅4696、NC₀11035₁852095、NC₀11035₅4846、NC₀11035₁756014、NC₀11035₂148154、NC₀11035₂148180、NC₀11035₁385558、NC₀11035₂182987.Their gene products were transcriptase PilC2,hydrogenase expression protein,α / β hydrolase,respectively.Enteromycin receptor FetA,periplasmic transporter.[Conclusion]1.In 2017,the minimal inhibitory concentration（MIC）of gonorrhoeae to 7 antibiotics in Guangzhou area showed that the detection rate of PPNG and TRNG was still high,ciprofloxacin was not sensitive,spectinomycin was not resistant,and the sensitivity of ceftriaxone was decreased.2.Whole genome sequencing showed that ceftriaxone hypersensitive strain and ceftriaxone sensitive strain had significant differences in 9 gene mutation sites.The gene products were transcriptase PilC2,hydrolase expression protein,α / β hydrolase,enterocectin receptor FetA,periplasmic transporter,which may be related to the resistance mechanism of Neisseria gonorrhoeae ceftriaxone.