The Molecular Epidemiological Characteristics And Comparative Genomics Of Ureaplasma Species Isolated From Infertile Patients

Background and objective: Ureaplasma spp.as commensal bacteria are present on the mucosal surfaces of the human genitourinary tract and can cause a variety of infectious diseases.The expanded multilocus sequence typing(e MLST)scheme for Ureaplasma spp.was developed recently,only a few studies have reported the population distribution of this species with the new typing method.Moreover,a difference in the relationships between Ureaplasma subgroups and disease has been revealed.The aim of this study was to use e MLST to explore the distribution of Ureaplasma spp.in infertile females and males and to understand the genetic differences between Ureaplasma strains in different subgroups,which may provide insight into the pathogenicity of Ureaplasma spp.Methods: 1.A total of 226 vaginal swab samples were obtained from infertile females at Sir Run Run Shaw Hospital of Zhejiang University and 480 semen samples were obtained from infertile males at Shanghai Corps Hospital of Chinese People's Armed Police.Ureaplasma spp.were identified in all samples using the commercially available Mycoplasma Kit.U.urealyticum and U.parvum were identified in all broth cultures that were positive for Ureaplasma spp.The e MLST was performed for single infections of U.urealyticum or U.parvum.The distributions of Ureaplasma spp.between infertile females and males were analyzed.Semen characteristics,including sperm concentration,total motility,progressive motility,non-progressive motility and immotile sperm,were compared between negative group and Ureaplasma sub-groups.2.Three Ureaplasma strains UPA106(belonging to sub-group A),UUR132(belonging to sub-group 2)and UUR315(belonging to sub-group 1)were chosen.Wholegenome sequencing was performed using combined Hi Seq X10(Illumina)and Min ION(Nanopore)platforms.Strain ATCC 27815 was regarded as a reference strain for U.parvum and strain ATCC 33699 was regarded as a reference strain for U.urealyticum.Multiple genome alignment and whole-genome orthologous clusters of three clinical strains and two reference strains were performed.The single nucleotide polymorphisms between UPA106 and known sequenced U.parvum strains in NCBI was performed.The single nucleotide polymorphisms between UUR132,UUR315 and known sequenced U.urealyticum strains in NCBI was performed.The fifteen virulence genes were analyzed between each of the three isolates and the corresponding reference strain.Comparisons of multiple banded antigen(MBA)loci between UPA106 and ATCC 27818,UUR132 and ATCC 27618,UUR315 and ATCC 33696 were analyzed,respectively.Next,the MBA proteins of three strains were detected using monoclonal antibody 6522 and were identified by mass spectrum.Three strains were subjected to antimicrobial susceptibility testing for levofloxacin,ciprofloxacin,tetracycline,erythromycin,azithromycin,chloromycetin,and gentamicin using the broth microdilution method.The mechanisms underlying quinolone and macrolide resistance were determined.Results: 1.The clonal diversity of Ureaplasma species in infertile females and males(1)A total of 101 Ureaplasma strains were detected in infertile females,85 isolates were U.parvum,11 isolates were U.urealyticum,the remaining five samples were mixed species.Except for the five mixed samples,e MLST revealed 24 expanded sequence types(e STs)in U.parvum strains and 10 e STs in U.urealyticum strains.Among them,e ST16 and e ST41 were the most frequent clones in U.parvum and e ST82 was the most prevalent clones in U.urealyticum.(2)A total of 104 Ureaplasma strains were detected in infertile males,78 isolates were U.parvum,24 isolates were U.urealyticum,the remaining two samples were mixed species.Except for the two mixed samples,e MLST revealed 32 e STs in U.parvum strains and 16 e STs in U.urealyticum strains.Among them,e ST16 and e ST41 were the most frequent clones in U.parvum and e ST82 was the most prevalent clones in U.urealyticum.(3)The distribution of U.parvum was higher than U.urealyticum in infertile females and males.Compared with infertile males,the distribution of U.urealyticum was lower in infertile females(P<0.05)and no strains were found in sub-group 2.(4)The sperm concentration,total motility,and progressive motility in Ureaplasma sub-groups tended to be lower or higher than in the negative group,but the differences were without statistical significance(P>0.05).Non-progressive motility in sub-group 2 was statistically significantly lower than in the negative group(P<0.05).2.Genome features and comparative genomics of three Ureaplasma species from different Ureaplasma sub-groups(1)The genomes of UPA106,UUR132 and UUR315 each consisted of a single,circular chromosome with a low GC content of approximately 25.5%.The genome sizes of the three strains ranged from 741,809 to 853,955 bp.The genes in the three strains ranged from 627 to 699.(2)The three clinical strains had significant genomic synteny with the two reference strains.Ortho Venn analysis revealed 652 clusters among them,with 541 clusters(82.98%)shared among the five strains.Strains UPA106,UUR132 and UUR315 shared the most similarity with strains ATCC 27818,ATCC 27618 and 1000,respectively. (3)There was no nonsynonymous mutation in genes encoding glutathione peroxidase,thioredoxin and urease families of the three strains.UPA106 and UUR132 showed one nonsynonymous variant site of virulence genes encoding O-sialoglycoprotein endopeptidase,A661G(I221V)and G709A(A237T),respectively.UPA106 showed one nonsynonymous variation in the gene encoding thioredoxin-disulfide reductase,A425G(Y142C).UUR132 showed one nonsynonymous variation in the gene encoding serine/threonine kinase,A148G(S50G).Three nonsynonymous mutations,A55G(I19V),A981T(K327N)and A981T(K327N),were observed in genes coding for hemolysin in UPA106,UUR132 and UUR315,respectively.(4)The comparisons of MBA loci between UPA106 and ATCC 27818,UUR132 and ATCC 27618 revealed DNA inversion events,which occurred between the Nterminal conserved domain of the mba gene and one flanking intergenic region.The inverted N-terminal domain was fused with the adjacent gene(A0412),forming an mba(01920)without the C-terminal repeat region in UPA106.The inverted N-terminal domain was fused with the insertion gene(0418)forming an mba(02045)without tandem repeating units in UUR132.The N-terminal sequence of mba mutated in UUR315,resulting in a stop codon in the 21 st amino acid(MKLLKKKSFFFQNTKKQLFS*).Relative to strain ATCC 33696,UUR315 exhibited an inversion event in the C-terminal variable domain of mba.Gene(02080)of UUR315 contained the complete C-terminal domain of mba(A0418)of strain ATCC 33696.Two sequences with identical tandem repeating unit ‘QAMVQQAQKN' inserted into a gene(02090)were observed in UUR315.Moreover,alterations of the number of repeat units in the downstream region were observed in the flanking genes of mba in the three strains.Monoclonal antibody 6522 recognized three,three and six bands for UPA106,UUR132 and UUR315,respectively.The upper bands of UPA106 and UPA132 were corresponded MBA,which was encoded by the gene(01920)in UPA106 and by the gene(02045)in UUR132.(5)UPA106 and UUR132 were resistant to levofloxacin,and UUR132 was also resistant to erythromycin.Three stains were all sensitive to tetracycline.Two substitutions,R448 K in par E and S83 L in par C,were observed in UPA106 and UUR132,respectively.However,no mutations were found in L4,L22 and 23 r RNA of UUR132.Conclusions: 1.The distribution of Ureaplasma spp.is high diversity in infertile females and males,and the main Ureaplasma clones were e ST16,e ST41 and e ST82.2.The virulence factors of Ureaplasma spp.are conserved,especially for ureaseencoding genes.However,MBA loci show a great variation among different strains.3.The mechanisms of MBA loci variation are flexible and complex.The conserved N-terminal domain of the MBA can mutate,resulting in the absence of the typical MBA sequence.DNA inversion events can occur not only in the N-terminal domain of the MBA,but also happen in the C-terminal domain of the MBA.Variation in the number of repeat unit occurs in any genes which containing tandem repeating units in their the C-terminal domain.Insertion mutation also plays a role in MBA variation.