Antimicrobial Susceptibility Survefflance From 2005 To 2013 And Mechanism Of Fluoroquinolone Resistance In Ureaplasma Species

Ureaplasma urealyticum（Uu）commonly colonizes in human urogenital tract.However it is also associated with diverse types of infection.Therefore,it seriously influences human reproductive health and quality of life.In recent years,Due to the extensive use of antibiotics,unreasonable use of antimicrobial agents,acquired resistance rates to all antimicrobials,especially fluoroquinolones,have been documented in clinical isolates and are going up rapidly.The existing mechanisms of drug resistance cannot fully explain the clinical isolates of fluoroquinolones resistance mechanism.Therefore,it is necessary to continue to monitor the resistance of Ureaplasma species fluoroquinolones and to further explore the resistant mechanism.The first part aims to analyze the prevalence and antibiotic resistance rate of Ureaplasma Species from Urogenital outpatient clinic in Hangzhou,China during 2005-2013.In this part,we analyzed the mycoplasma culture,identification and in vitro antibiotic susceptibility monitoring results of 2689 women and 2336 male patients from Urogenital outpatient clinic in Hangzhou during 2005 to 2013.In the 5025 urogenital tracts specimens tested,1942（38.6%）were positive for Ureaplasma Species.1513（30.1%）and 429（8.5%）cultures,respectively,were positive for single Ureaplasma Species identification and simultaneous identification of both M.hominis（Mh）and Ureaplasma Species.Among the female,1434 were positive for Ureaplasma Species,the total positive rate was 53.3%,of which 1073 strains of Ureaplasma Species single positive（39.9%）,361 strains of Ureaplasma Species and M.hominis co-positive（13.4%）.Among the male,508 were positive for Ureaplasma Species,the total positive rate was 21.7%,of which 440 strains of Ureaplasma Species single positive（18.8%）,68 strains of Ureaplasma Species and M.hominis co-positive（2.9%）.Among the nine antibiotic agents asessed,josamycin,doxycycline,tetracycline,and pristinamycin were found to be the most effective antibiotics in the treatment of Ureaplasma Species infection,and the resistance rates did not change significantly from 2005 to 2013.The resistance rates of Ureaplasma Species to fluoroquinolones（ofloxacin and ciprofloxacin）were much higher（>50%）and increased significantly during the study period.Our rsults demonstrate that the prevalence of Ureaplasma Species is higher in outpatients of urogenital tract during 2005-2013 in Hangzhou,China.The prevalence of Ureaplasma Species in female is significantly higher than that in male.The resistance rates of Ureaplasma Species to fluoroquinolones were the highest and increased significantly.The second part aims to understand the molecular epidemiological characteristics and the mechanism of fluoroquinolone resistance in Ureaplasma Species.A total of 114 Ureaplasma strains were screened from female patients with a clear diagnosis of infection（such as cervicitis,vaginitis,salpingemphraxis,etc.）in Sir Run Run Shaw Hospital,Hangzhou,China.Commercially available Mycoplasma IST2 was used for primary isolation of Ureaplasma Species.The accurate MIC values of four fluoroquinolones including ciprofloxacin,ofloxacin,levofloxacin and moxifloxacin were tested by a broth microdilution method with 96-well microtitre plates.DNA extraction and 114 clinical isolates were distinguished based on the difference of Ureaplasma parvum（UPA）specific genes（UU295）and Ureplasma parvum（UUR）specific genes（UUR10₀888）.The Expanded Multilocus Sequence Typing（eMLST）and bioinformatics analysis were performed on 93 clinical strains and 14 standard strains.Moreover,the QRDRs of four genes including gyrA,gyrB,parC and parE in 114 clinical strains were amplified by PCR and then sequenced.We analysed these conserved QRDRs and compared them with reference strains of Ureaplasma to identify mutations that associated with antimicrobial resistance.Our results showed that MICs of three commonly used fluoroquinolone antibiotics were relatively high.Moxifloxacin was the most effective fluoroquinolone for treatment of Ureaplasma Species.（MIC range:0.125-32 μg/ml）.However,ciprofloxacin（MIC range:1-256 μg/ml）and ofloxacin（MIC range:0.5-128 μg/ml）had extremely high MICs,followed by levofloxacin whose MIC range is from 0.5 μg/ml to 64 μg/ml.Of all the 114 clinical isolates,87（76.32%）were UP A,14（12.28%）belonged to UUR and 13（11.4%）including UPA and UUR.The resistant rate of levofloxacin of UPA was 75.9%.UUR was 71.4%.UPA and UUR were 76.9%.The resistance rate of moxifloxacin of UPA was 25.3%.UUR was 50%.UPA and UUR were 23.1%.The 93 clinical isolates were divided into seven subgroups and one singleton by eMLST.There was no significant difference in resistance rates among subgroups.Seven types of point mutations were found in 114 strains of clinical isolates,involving gyrB,parC and parE genes.Among them,89 strains carrying site mutation in parC gene,and the site mutation S83L（C248T）is the most common.In addition,we also found two novel substitutions that have not been reported in parC or parE gene:C248G（S83W）and G1343A（R448K）.In conclusion,there was no significant difference in Ureaplasma Species fluoroquinolones resistance among subgroups based on eMLST typing.Amino acid mutation in topoisomerase IV seems to be the main cause of fluoroquinolone resistance,especially the substitution of Ser-83→Leu（C248T）in ParC.Moxifloxacin had the best activity against strains with Ser-83→Leu mutation.