Group B streptococcus (GBS, S. agalactiae) and human ureaplasmas (U. parvumand U. urealyticum) are two clinically and phylogenetically related, potential perinatal pathogens. Their relationships between genotypes and pathogenesis of GBS and ureaplasma infection were still not well understood, one of the reason is that both of them are still short of a very practical genotyping system. In the study, to solve the above problem we developed genotyping systems for the organisms (the second section). For human ureaplasmas, based on four genes/gene clusters (rRNAgene clusters, the elongation factor Tu genes, urease gene complexes and multiplebanded antigen genes), we designed many primer pairs suitable for developing species identification assays for the two newly established human ureaplasma species (U. parvum and U. urealyticum). Further, based on the heterogeneity of ureaplasma multiple banded antigen gene (which contains species- and serovar-specific regions), we developed genotyping methods for each ureaplasma species.For GBS, based on three sets of molecular markers (capsular polysaccharidesynthesis gene clusters, surface protein antigen genes and mobile genetic elements),we developed a genotyping system. The primary evaluation of the genotyping systems showed that the genotyping systems were practical alternative assays for the conventional serotyping and they will be useful to further explore the relationships between genotypes and pathogenesis of GBS and ureaplasma infection. In the study, we introduced novel data and tools into GBS and ureaplasma studies especially from genomic- and bioinformatics-based molecular microbiology(the third section). For two newly established human ureaplasma species, based on the U. parvum serovar-3 genome, and using the above four important genes/geneclusters, we exposed some interesting problems in the understanding of newureaplasma taxonomy especially in the post genomic era. For GBS, we studied the two published full genomes and exposed some new problems or possible future new research fields. In particular we found the two finished and one ongoing GBS genomes were all non-typical and suggest that future genomic project had better have genetic population structure viewpoint. Finally, we suggested that integrated studies of the two potential or conditional perinatal pathogens, from the viewpoint of evolution, would provide a new understanding angle of the pathogenesis of the two organisms. Studies suggested that during coevolution, human ureaplasmas(especially U. parvum) became friendlier than their ancestors to their human host (by losing most of its virulence genes); however, GBS tried to increase its invasive abilities (by getting more virulence genes) to fight against the human host attack.

Group B streptococcus (GBS, S. agalactiae) and human ureaplasmas (U. parvumand U. urealyticum) are two clinically and phylogenetically related, potential perinatal pathogens. Their relationships between genotypes and pathogenesis of GBS and ureaplasma infection were still not well understood, one of the reason is that both of them are still short of a very practical genotyping system. In the study, to solve the above problem we developed genotyping systems for the organisms (the second section). For human ureaplasmas, based on four genes/gene clusters (rRNAgene clusters, the elongation factor Tu genes, urease gene complexes and multiplebanded antigen genes), we designed many primer pairs suitable for developing species identification assays for the two newly established human ureaplasma species (U. parvum and U. urealyticum). Further, based on the heterogeneity of ureaplasma multiple banded antigen gene (which contains species- and serovar-specific regions), we developed genotyping methods for each ureaplasma species.For GBS, based on three sets of molecular markers (capsular polysaccharidesynthesis gene clusters, surface protein antigen genes and mobile genetic elements),we developed a genotyping system. The primary evaluation of the genotyping systems showed that the genotyping systems were practical alternative assays for the conventional serotyping and they will be useful to further explore the relationships between genotypes and pathogenesis of GBS and ureaplasma infection. In the study, we introduced novel data and tools into GBS and ureaplasma studies especially from genomic- and bioinformatics-based molecular microbiology(the third section). For two newly established human ureaplasma species, based on the U. parvum serovar-3 genome, and using the above four important genes/geneclusters, we exposed some interesting problems in the understanding of newureaplasma taxonomy especially in the post genomic era. For GBS, we studied the two published full genomes and exposed some new problems or possible future new research fields. In particular we found the two finished and one ongoing GBS genomes were all non-typical and suggest that future genomic project had better have genetic population structure viewpoint. Finally, we suggested that integrated studies of the two potential or conditional perinatal pathogens, from the viewpoint of evolution, would provide a new understanding angle of the pathogenesis of the two organisms. Studies suggested that during coevolution, human ureaplasmas(especially U. parvum) became friendlier than their ancestors to their human host (by losing most of its virulence genes); however, GBS tried to increase its invasive abilities (by getting more virulence genes) to fight against the human host attack.

Group B streptococcus (GBS, S. agalactiae) and human ureaplasmas (U. parvumand U. urealyticum) are two clinically and phylogenetically related, potentialperinatal pathogens. Their relationships between genotypes and pathogenesis ofGBS and ureaplasma infection were still not well understood, one of the reason isthat both of them are still short of a very practical genotyping system. In the study,to solve the above problem we developed genotyping systems for the organisms (thesecond section). For human ureaplasmas, based on four genes/gene clusters (rRNAgene clusters, the elongation factor Tu genes, urease gene complexes and multiplebanded antigen genes), we designed many primer pairs suitable for developing species identification assays for the two newly established human ureaplasmaspecies (U. parvum and U. urealyticum). Further, based on the heterogeneity ofureaplasma multiple banded antigen gene (which contains species- and serovar-specific regions), we developed genotyping methods for each ureaplasma species.For GBS, based on three sets of molecular markers (capsular polysaccharidesynthesis gene clusters, surface protein antigen genes and mobile genetic elements),we developed a genotyping system. The primary evaluation of the genotypingsystems showed that the genotyping systems were practical alternative assays forthe conventional serotyping and they will be useful to further explore therelationships between genotypes and pathogenesis of GBS and ureaplasmainfection. In the study, we introduced novel data and tools into GBS and ureaplasmastudies especially from genomic- and bioinformatics-based molecular microbiology(the third section). For two newly established human ureaplasma species, based onthe U. parvum serovar-3 genome, and using the above four important genes/geneclusters, we exposed some interesting problems in the understanding of newureaplasma taxonomy especially in the post genomic era. For GBS, we studied thetwo published full genomes and exposed some new problems or possible future newresearch fields. In particular we found the two finished and one ongoing GBSgenomes were all non-typical and suggest that future genomic project had better have genetic population structure viewpoint. Finally, we suggested that integratedstudies of the two potential or conditional perinatal pathogens, from the viewpointof evolution, would provide a new understanding angle of the pathogenesis of thetwo organisms. Studies suggested that during coevolution, human ureaplasmas(especially U. parvum) became friendlier than their ancestors to their human host(by losing most of its virulence genes); however, GBS tried to increase its invasiveabilities (by getting more virulence genes) to fight against the human host attack.

Group B streptococcus (GBS, S. agalactiae) and human ureaplasmas (U. parvumand U. urealyticum) are two clinically and phylogenetically related, potentialperinatal pathogens. Their relationships between genotypes and pathogenesis ofGBS and ureaplasma infection were still not well understood, one of the reason isthat both of them are still short of a very practical genotyping system. In the study,to solve the above problem we developed genotyping systems for the organisms (thesecond section). For human ureaplasmas, based on four genes/gene clusters (rRNAgene clusters, the elongation factor Tu genes, urease gene complexes and multiplebanded antigen genes), we designed many primer pairs suitable for developing species identification assays for the two newly established human ureaplasmaspecies (U. parvum and U. urealyticum). Further, based on the heterogeneity ofureaplasma multiple banded antigen gene (which contains species- and serovar-specific regions), we developed genotyping methods for each ureaplasma species.For GBS, based on three sets of molecular markers (capsular polysaccharidesynthesis gene clusters, surface protein antigen genes and mobile genetic elements),we developed a genotyping system. The primary evaluation of the genotypingsystems showed that the genotyping systems were practical alternative assays forthe conventional serotyping and they will be useful to further explore therelationships between genotypes and pathogenesis of GBS and ureaplasmainfection. In the study, we introduced novel data and tools into GBS and ureaplasmastudies especially from genomic- and bioinformatics-based molecular microbiology(the third section). For two newly established human ureaplasma species, based onthe U. parvum serovar-3 genome, and using the above four important genes/geneclusters, we exposed some interesting problems in the understanding of newureaplasma taxonomy especially in the post genomic era. For GBS, we studied thetwo published full genomes and exposed some new problems or possible future newresearch fields. In particular we found the two finished and one ongoing GBSgenomes were all non-typical and suggest that future genomic project had better have genetic population structure viewpoint. Finally, we suggested that integratedstudies of the two potential or conditional perinatal pathogens, from the viewpointof evolution, would provide a new understanding angle of the pathogenesis of thetwo organisms. Studies suggested that during coevolution, human ureaplasmas(especially U. parvum) became friendlier than their ancestors to their human host(by losing most of its virulence genes); however, GBS tried to increase its invasiveabilities (by getting more virulence genes) to fight against the human host attack.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-12-05T13:27:43Z No. of bitstreams: 1 Dissertação - Cecília Da Cunha Camilo.pdf: 1238897 bytes, checksum: e26e3eb86bc9e96ee8e82757d4858f3e (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-12-05T13:28:00Z (GMT) No. of bitstreams: 1 Dissertação - Cecília Da Cunha Camilo.pdf: 1238897 bytes, checksum: e26e3eb86bc9e96ee8e82757d4858f3e (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-12-05T13:28:20Z (GMT) No. of bitstreams: 1 Dissertação - Cecília Da Cunha Camilo.pdf: 1238897 bytes, checksum: e26e3eb86bc9e96ee8e82757d4858f3e (MD5) / Made available in DSpace on 2016-12-05T13:28:20Z (GMT). No. of bitstreams: 1 Dissertação - Cecília Da Cunha Camilo.pdf: 1238897 bytes, checksum: e26e3eb86bc9e96ee8e82757d4858f3e (MD5) Previous issue date: 2012-12-06 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Mycoplasmas are microorganisms lacking cell walls with reduced ability Biosintética, which makes them extremely fastidious growth. The strength of these microorganisms to antimicrobials used in his therapy as tetracyclines, macrolides and quinolones have been reported with increased frequency. The determination of antimicrobial susceptibility is particularly difficult because not shown in broth turbidity which complicates the standardization of the inoculum and are extremely susceptible pH conditions. The method most commonly used for this purpose is based on metabolic inhibition using specific substrates. This study evaluated the susceptibility of isolates of Mycoplasma hominis and Ureaplasma sp. stored in the period from 2011 to 2012. The methodology used for the isolation comprised culture techniques, for storage and freezing of the strains we used selective broth enriched PPLO and BHI, Thawing of isolates was succeeded subculture in broth enriched selective and differential agar A7 for phenotypic characterization. Quantification, identification and antimicrobial susceptibility testing was performed using the the triage system Mycofast Evolution ® Screening (Elitech Group), whose identification is based on bacterial susceptibility by Identibiotiqué system. The susceptibility of the isolates was determined against antimicrobial agents Doxiciclina 8 μg/ml, Roxitromicina 4 μg/ml and Ofloxacina 4 μg/ml, widely used in the empirical treatment of patients with urogenital tract infections. Our study showed high level of resistance of the isolates to doxicycline and ofloxacin. This is the first study involving isolation and susceptibility profile of these agents undertaken in the Amazonia. / Micoplasmas são microrganismos desprovidos de parede celular, com reduzida capacidade Biossintética, o que os torna extremamente fastidiosos ao crescimento. A resistência destes microrganismos aos agentes antimicrobianos utilizados na sua terapia como as tetraciclinas, macrolídeos e quinolonas tem sido relatadas com frequência cada vez maior. A determinação da susceptibilidade a antimicrobianos é particularmente difícil porque não mostram turbidez em caldo o que dificulta a padronização do inóculo e são extremamente susceptíveis as condições de pH. A metodologia utilizada para este fim baseia-se na inibição metabólica utilizando substratos específicos. O presente trabalho avaliou a susceptibilidade de isolados de Mycoplasma hominis e Ureaplasma sp. arrmazenados no período de 2011 a 2012. A metodologia utilizada englobou técnicas de cultura; para a estocagem e congelamento das cepas empregou-se caldo seletivo enriquecido PPLO e BHI, O descongelamento dos isolados foi sucedido de subcultivos em caldos enriquecidos seletivos diferenciais e Agar A7 para caracterização fenotípica. A quantificação, a identificação e o teste de susceptibilidade a antimicrobianos foi realizada através do sistema de triagem Mycofast® Screening Evolution (Elitech Group), cuja identificação se baseia na susceptibilidade bacteriana pelo sistema Identibiotiqué. A susceptibilidade dos isolados foi determinada frente aos antimicrobianos doxiciclina 8 μg/ml, roxitromicina 4 μg/ml e ofloxacina 4 μg/ml, amplamente utilizados no tratamento empírico de pacientes com infecções do trato urogenital. Nosso estudo detectou alto nível de resistência dos isolados armazenados para doxiciclina e ofloxacina. Este é o primeiro estudo envolvendo isolamento e o perfil de susceptibilidade destes agentes realizado na região norte.

Bibliography / Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Overview: Mycoplasmas and ureaplasmas are not routinely diagnosed and are under researched in South Africa. Prevalence, population shifts especially concerning genital flora and implications in infection or other conditions are unknown. Information pertaining to Mycoplasma pneumoniae in respiratory disease is similarly lacking. There is little information on antimicrobial susceptibilities and resistance development against Sexually Transmitted Infections (STI) syndromic management approaches. Aims: a) Elucidate mycoplasmal and ureaplasmal prevalence and contributing factors concerning cervical colonisation or preterm delivery in conjunction with HIV and Chlamydia trachomatis b) Investigate prevalence of M. pneumoniae in respiratory infections in conjunction with HIV, Mycobacterium tuberculosis and Pneumocystis jiroveci. c) Determine antimicrobial susceptibilities of mycoplasmas and ureaplasmas and analyse resistance genes. d) Assess the inter-generic transfer potential of resistance gene (tetM) between Ureaplasma spp. and Neisseria gonorrhea. Genital setting: The prevalence of genital mycoplasmas, ureaplasmas and Chlamydia on women attending their first prenatal visit, in conjunction with preterm labour or HIV status was investigated. For preterm labour (2003), 199 women were monitored for preterm delivery (<37 weeks); for colonisation and HIV (2005), 219 women were screened. Microbial detection was performed on DNA extracted from endocervical swabs employing PCR techniques. Colonisation was seen to be highest in the 14-20 year group from 2003. In women aged ±21 years, co-colonisation was 13% although there was a shift from co-colonisation with Mycoplasma hominis and Ureaplasma spp. in 2003 to other dual/triple combinations in 2005. Overall major trends from both collection periods were that the prevalence of Ureaplasma spp. tended to be higher in women ±26 years, whilst prevalence of C. trachomatis and M. hominis were lower. No association was evident between colonisation with M. hominis, U. urealyticum, Ureaplasma parvum and labour outcome. HIV status had no effect on the prevalence/co-colonisation of M. hominis, Ureaplasma spp. or C. trachomatis. Respiratory setting: Studies were conducted to determine the prevalence of community acquired atypical pneumonias in adults (M. pneumoniae and P. jiroveci) and neonates (mycoplasmas, ureaplasmas and Chlamydia trachomatis) in order to improve treatment management programmes in the Port Elizabeth region. Sputum specimens from 102 adult patients presenting with pneumonia/symptoms of pneumonia admitted to hospitals were assessed by PCR. Details of patient’s gender, age, HIV and Mycobacterium tuberculosis status were provided by the hospitals. Women were seen to be at high risk for community-acquired P. jiroveci colonisation. Overall, prevalence of P. jiroveci was 52.9% (54/102 patients). P. jiroveci was mainly associated with HIV (25/74) (P. jiroveci and HIV positive patients in patient sample for which clinical data and HIV status was available) and co-infection with M. tuberculosis was observed in 12 HIV cases and one HIV negative patient. No DHPS (20) or DHFR (17) resistance associated mutations were found in P. jiroveci. M. pneumoniae was detected in one patient. For prevalence studies (2007-2008) on atypical pneumonia in neonates, 69 endotracheal aspirates were obtained. PCR detection of M. hominis, U. urealyticum and C. trachomatis was performed and U. parvum detected in two specimens. Antibiotic susceptibilities and resistance genes: The following investigations on clinical isolates of U. parvum and U. urealyticum were conducted (i) antibiotic susceptibility profiles, (ii) detection of drug target gene mutations, or gene acquisitions and (iii) inter-generic resistance gene transfer potential to Neisseria gonorrhoeae. Culture techniques applied to 132 endocervical specimens provided 66 Ureaplasma cultures (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIC determinations to ofloxacin, erythromycin, tetracycline, doxycycline, azithromycin and josamycin were performed. Thirty-seven ureaplasma cultures were fully susceptible to all antibiotics tested; 21 showed intermediate resistance to erythromycin, azithromycin and ofloxacin; while seven were resistant to tetracycline, three of which were also resistant to doxycycline and one also resistant to azithromycin. Concerning ofloxacin resistance directed at quinolone resistance determining regions, a substitution of Ser83Leu in ParC was demonstrated in one intermediately-resistant Ureaplasma (MIC 4 µg/ml) while a triple substitution of Asp112Glu in GyrA along with Ala125Thr and Ala136Thr in ParC was found in six further intermediately-resistant strains. No mutations were found in strains with MICs 1 µg/ml. No mutations were detected in 23S rRNA operons, L4 or L22 proteins. TetM and int-Tn genes were found in seven tetracycline-resistant strains. On screening 59 tetracycline-susceptible and -intermediate strains, eleven whilst possessing an int-Tn gene lacked a large region of tetM and 48 only contained small regions of tetM. The tetM genes of the seven tetracycline-resistant strains were sequenced and comparisons performed against GenBank sequences of Neisseria gonorrhoeae, Streptococcus pneumoniae and U. urealyticum. For five strains tetM was seen to be highly mosaic in structure containing regions that were similar to those of the GenBank strains and others that were unique. In the tetM leader region, four hot spot recombination sites were identified that could certainly influence the formation of the mosaic structures, upstream insertion sequences/open reading frames and transposon regions that regulate expression. On characterising the int-Tn genes of the seven tetracycline-resistant strains, three types were present indicating transposons from different origins had integrated into ureaplasma genomes. Reciprocal tetracycline resistance gene transfer between ureaplasmas and N. gonorrhoeae were unsuccessful. However, low-level tetracycline resistance (MICs 4-8 µg/ml) was transferred to a U. parvum recipient from one U. urealyticum and three U. parvum donors that carried tetM with MICs 16-64 µg/ml. On tetM PCR analysis, tetM was not detected in the transformants. Conclusions: The importance of genital mycoplasmas, ureaplasmas and C. trachomatis in long term aetiologies requires further investigations, certainly in relation with syndromic management regimens that fail to reduce colonisation rates. The high prevalence of P. jiroveci, the presence of M. pneumoniae in cases of pneumonia and detection of U. parvum in two cases of neonatal pneumonia investigated emphasises that in the absence of definitive diagnoses, it is crucial to monitor treatment responses carefully, especially when first line antibiotic preferences are ß-lactams, in order to ensure adequate and informed delivery of medical care. The finding of transposon and/or tetM regions in all ureaplasmas investigated with or without full expression of tetracycline resistance, in conjunction with tetM gene diversity, certainly places ureaplasmas strongly in the picture for intra- and inter-generic exchange of antibiotic resistance genes. / AFRIKAANSE OPSOMMING: Oorsig: Mikoplasma en ureaplasma word nie roetineweg gediagnoseer nie en in Suid Afrika is nog min navorsing daaroor gedoen. Prevalensie, populasie verskuiwings, veral in genital flora, en die impliksies van infeksie en ander toestande is onbekend. Inligting rakende Mycoplasma pneumoniae in respiratoriese siekte is ook gebrekkig. Daar is min inligting beskikbaar rakende die antimikrobiale vatbaarheid en die ontwikkeling van weerstandigheid gesien teen die benadering tot sindromiese hantering van seksueel oordraagbare siektes. Doelwitte: a) Om inligting te verskaf oor die prevalensie van mikoplasma en ureaplasma en bydraende faktore betreffende voortydige kraam tesame met MIV en Chlamydia trachomatis. b) Ondersoek van die prevalensie van M. pneumoniae in respiratoriese infeksies tesame met MIV, Mycobacterium tuberculosis en Pneumocystis jiroveci. c) Bepaling van die antimikrobiale vatbaarheid van mikoplasma en ureaplasma en analisevan weerstandigheids gene. d) Bereken die inter-genetiese oordrag potensiaal van weerstandigheids gene (tetM) tussen Ureaplasma spp. en Naisseria gonorrhoeae. Genitale omgewing: Die prevalensie van genitale mikoplasma, ureaplasma en Chlamydia in vroue tydens hul eerste prenatale besoek, tesame met vroegtydige kraam en MIV status is ondersoek. In voortydige kraam (2003), is 199 vroue gemonitor vir voortydige kraam (<37 weke); vir kolonisasie en MIV (2005), is 219 vroue getoets. Mikrobiale toetsing is gedoen deur DNS te win vanaf endoservikale deppers met PKR tegnieke. Kolonisasie was die hoogste in die ouderdomsgroep 14.20 jaar, in 2003. In vroue van ±21 jaar was medekolonisasie 13% alhoewel daar en verskuiwing was van mede-kolonisasie met Mycoplasma hominis en Ureaplasma spp. in 2003 tot ander dubbel/trippel kombinasies in 2005. Die oorkoepelende tendens in altwee die tydperke van waarneming was dat die prevalensie van Ureoplasma spp. geneig was om hoër te wees in vroue ±26 jaar, terwyl prevalensie van C. trachomatis en M. hominis laer was. Geen assosiasie kon getoon word tussen koloniesasie met M. hominis, U. urealyticum, Ureaplasma parvum en uitkoms van kraam nie. MIV status het geen effek gehad op die prevalensie/mede-kolonisasie van M. hominis, Ureaplasma spp. of C. Trachomatis nie. Respiratories: Studies is gedoen om die prevalensie van gemeenskaps verworwe atipiese pneumonie in volwassenes (M. pneumoniae en P. jiroveci) en neonate (mikoplasma, ureaplasma en Chlamydia trachomatis) te bepaal om behandeling en hantering programme in die Port Elizabeth area te verbeter. Sputum monsters van 102 volwasse pasiënte wat presenteer het met pneumonie of simptome van pneumonie en wat tot hospitale toegelaat was, is ontleed. Besonderhede van die pasiënte se geslag, ouderdom, MIV en Mycobacterium tuberculosis status is deur die hospitale verskaf. PKR is gedoen met inleiers gerig teen die volgende gene: P. jiroveci vir die aantoning van mitokondriale groot subeenheid RNS en vir die analise van mutasies vir ko-trimoksasool weerstandigheid dihydropteroaat sintetase (DHPS) en dihydrofolaat reduktase (DHFR); M. pneumoniae vir die aantoning van P1 adhesien en 16S rRNS. Vroue het ‘n hoë risiko vir gemeenskapsverworwe P. jiroveci kolonisasie gehad. In die algemeen was die prevalensie van P. jiroveci 52.9% (54/102 pasiënte). P. jiroveci was hoofsaaklik geassosieerd met MIV (25/74) (P. jiroveci en MIV positiewe pasiënte in die pasiënt monster waarvoor daar kliniese data en MIV status bekend was) en mede-infeksie met M. tuberculosis is gesien in 12 MIV gevalle en een MIV negatiewe pasiënt. Geen DHPS (20) of DHFR (17) weerstandigheids geassosieerde mutasies is gevind in P. Jiroveci nie. M. pneumoniae was aangetoon in een pasiënt. Vir prevalensie studies (2007-2008) op atipiese pneumonie in neonate is 69 endotrageale aspirate verkry. PKR toetsing vir M. hominis, U. urealyticum en C. trachomatis is gedoen met ‘primers’ soos voorheen gepubliseer. Ureaplasma parvum is aangetoon in twee neonate met PKR met negatiewe kultuur resultate. Antibiotika sensitiwiteite en weerstandigheids gene: Die volgende toetse is gedoen op kliniese isolate van U. parvum en U. urealyticum (i) antibiotika sensitiwiteits profiele, (ii) aantoning van teiken geen mutasies, of geen aanwinste en (iii) potensiaal vir inter-generiese weerstandigheids geen oordrag na Neisseria gonorrhoeae. Kultuur tegnieke toegepas op 132 endoservikale monsters het 66 Ureaplasma kulture gelewer (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIK bepaling vir ofloksasien, eritromisien, tetrasiklien, doksisiklien, azitromisien en josamisien is gedoen. Sewe-en-dertig kulture was ten volle sensitief vir alle antibiotika wat getoets is; een-en twintig het intermediere weerstandigheid teenoor eritromisien, azitromisien en ofloksasien getoon, terwyl sewe weerstandig was vir tetrasiklien, drie daarvan was ook weerstandig vir doksisiklien. Wat betref ofloksasien weerstandigheid gemik teen kwinoloon weerstandigheids bepalende gebiede, is vervanging van Ser83Leu in ParC gedemonstreer in een intermedier weerstandige Ureaplasma (MIK 4 µml) terwyl en trippel vervanging van Asp112Glu in GyrA saam met Ala125Thr en Ala136Thr in ParC gevind is in ses ander intermedier weerstandige stamme. Geen mutasies is gevind in stamme met MIKs van MICs 1 µg/ml nie. Geeneen van die ureaplasma was weerstandig vir eritromisien/azitromisien nie en geen mutasies is gevind in 23S rRNA operons , L4 of L22 proteine nie. TetM en int- Tn gene is gevind in sewe tetrasiklien weerstandige stamme. 58 Tetrasiklien sensitiewe en .intermediere stamme is getoets, waarvan elf en int-Tn geen gekort het sowel as en groot deel van tetM, terwyl 48 slegs klein dele van TetM bevat het. Die tetM gene van die sewe tetrasiklein-werstandige stamme se geenvolgorde is bepaal en vergelykings is getref teenoor die GenBank volgordes van Neisseria gonorrhoeae, Streptococcus pneumoniae en U. urealyticum. In vyf stamme is gevind dat die tetM geen hoogs mosaiek in struktuur was met areas wat ooreenstem met die in GenBank stamme, en ander areas wat uniek is. In die tetM leier area, is vier ehot spot f herkombinasie areas geidentifiseer wat sekerlik die vorming van die mosaiiek strukture kon beinvloed, asook transposon areas wat geenuitdrukking bepaal. Met karakterisering van die int-Tn gene van die sewe tetrasikleinweerstandlige stamme, was drie tipes teenwoordig waarin transposons vanaf verskillende oorsprong aangedui was, geintegreerd met die ureaplama genome. Resiprokale tetrasiklien weerstandigheids geen oordrag tussen ureaplasma en n. gonorrhoea was nie suksesvol nie. Lae-vlak tetrasiklien weerstandigheid (MIK fs van 4 . 8 µg/ml) is wel suksesvol oorgedra na en U. parvum ontvanger vanaf een U. urealyticum en drie U. parvum ontvangers wat tetM gedra het met MIKs van 16-64 µg/ml. Met die analise van tetM met PKR, kon tetM nie aangetoon word in die transformante nie. Gevolgtrekkings: Die belang van genitale mykoplasma, ureaplasma en C. trachomatis in langtermyn etologie benodig verdere ondersoek, veral in die lig van die sindromiese behandeling regimes wat nie kolonisasie verminder nie. Die hoe prevalensie van P. jiroveci, die teenwoordigheid van M. pneumoniae in gevalle van pneumonie en die aantoning van U. parvum in twee gevalle van neonatale pneumonie benadruk dat, in die afwesigheid van en definitiewe diagnose, dit noodsaaklik is om respons tot behandeling sorgvuldig te moniteer, veral indien die eerste lyn antibiotika keuse ß-laktam antimikrobiale middels of kefalosporiene is, sodat behoorlike en ingeligde gesondheidsorg gelewer kan word. Die bevinding van transposon en/of tetM gebiede in alle ureaplasma wat ondersoek is met of sonder volle uitdrukking van tetrasiklien weerstandigheid, in samehang met tetM diversiteit, plaas verseker ureaplasma sterk in die prentjie vir intra- en inter-generiese uitruiling van antibiotika weerstandigheids gene. / Nelson Mandela Metropolitan University / National Research Foundation (NRF Thuthuka) / Medical Research Council

O desenvolvimento do câncer cervical depende da exposição ao HPV, fator necessário, mas não suficiente. Outras bactérias, tais como ureaplasmas, têm sido associadas como cofatores. O objetivo deste estudo foi avaliar a presença de ureaplasmas em mulheres com lesão cervical, e observar alterações em PHK causadas pela infecção por ureaplasmas. 140 swabs vaginais foram coletados. O material foi submetido a PCR para a detecção de HPV, Mollicutes, U. urealyticum, U. parvum e seus sorotipos, e outras bactérias de importância ginecológica; e qPCR para U. urealyticum e U. parvum. Também foi realizada a infecção de ureaplasmas em PHK transformados com HPV. As células foram contadas e realizou-se a dosagem das citocinas IL1-&beta;, IL-6 e TNF-&alpha;. HPV, Mollicutes, U. parvum, sorotipos 1 e 6 de U. parvum, T. vaginalis e G. vaginalis, além de alguns fatores socioeconômicos, foram associados com lesão cervical. Verificou-se maior carga de U. parvum entre mulheres com lesão. Houve diminuição do número de células e maior liberação de IL-6 e TNF-&alpha; nos grupos infectados. Com os resultados obtidos neste estudo, foi possível verificar uma associação entre os ureaplasmas e HPV no início das lesões cervicais, contudo mais estudos precisam ser realizados para aprimorar essa hipótese. / The development of cervical cancer depends on the exposure to HPV, necessary factor, but not enough. Other bacteria, such as ureaplasmas, have been associated as cofactors. The aim of this study was to evaluate the presence of ureaplasmas in women with cervical injury, and observe changes in PHK infected by ureaplasmas. 140 vaginal swabs were collected. The material was subjected to PCR for detection of HPV, Mollicutes, Ureaplasma urealyticum, U. parvum (and serotypes) and other bacteria gynecological importance; qPCR for U. urealyticum and U. parvum was made. PHK transformed by HPV was infected by ureaplasma. Cells were counted and it was done titration of IL1-&beta;, IL-6 and TNF-&alpha;. HPV, Mollicutes, U. parvum, serotypes 1 and 6 U. parvum, T. vaginalis and G. vaginalis, and some socioeconomic factors were associated with cervical injury. Besides this, it was detected higher load U. parvum among women with injury. There was decrease in cell number and increased release of IL-6 and TNF-&alpha; in infected groups. With the results of this study, we found an association among HPV and ureaplasmas at the beginning of cervical lesions, but more studies are needed to enhance this hypothesis.

O desenvolvimento do câncer cervical depende da exposição ao HPV, fator necessário, mas não suficiente. Outras bactérias, tais como ureaplasmas, têm sido associadas como cofatores. O objetivo deste estudo foi avaliar a presença de ureaplasmas em mulheres com lesão cervical, e observar alterações em PHK causadas pela infecção por ureaplasmas. 140 swabs vaginais foram coletados. O material foi submetido a PCR para a detecção de HPV, Mollicutes, U. urealyticum, U. parvum e seus sorotipos, e outras bactérias de importância ginecológica; e qPCR para U. urealyticum e U. parvum. Também foi realizada a infecção de ureaplasmas em PHK transformados com HPV. As células foram contadas e realizou-se a dosagem das citocinas IL1-&beta;, IL-6 e TNF-&alpha;. HPV, Mollicutes, U. parvum, sorotipos 1 e 6 de U. parvum, T. vaginalis e G. vaginalis, além de alguns fatores socioeconômicos, foram associados com lesão cervical. Verificou-se maior carga de U. parvum entre mulheres com lesão. Houve diminuição do número de células e maior liberação de IL-6 e TNF-&alpha; nos grupos infectados. Com os resultados obtidos neste estudo, foi possível verificar uma associação entre os ureaplasmas e HPV no início das lesões cervicais, contudo mais estudos precisam ser realizados para aprimorar essa hipótese. / The development of cervical cancer depends on the exposure to HPV, necessary factor, but not enough. Other bacteria, such as ureaplasmas, have been associated as cofactors. The aim of this study was to evaluate the presence of ureaplasmas in women with cervical injury, and observe changes in PHK infected by ureaplasmas. 140 vaginal swabs were collected. The material was subjected to PCR for detection of HPV, Mollicutes, Ureaplasma urealyticum, U. parvum (and serotypes) and other bacteria gynecological importance; qPCR for U. urealyticum and U. parvum was made. PHK transformed by HPV was infected by ureaplasma. Cells were counted and it was done titration of IL1-&beta;, IL-6 and TNF-&alpha;. HPV, Mollicutes, U. parvum, serotypes 1 and 6 U. parvum, T. vaginalis and G. vaginalis, and some socioeconomic factors were associated with cervical injury. Besides this, it was detected higher load U. parvum among women with injury. There was decrease in cell number and increased release of IL-6 and TNF-&alpha; in infected groups. With the results of this study, we found an association among HPV and ureaplasmas at the beginning of cervical lesions, but more studies are needed to enhance this hypothesis.