Dissertations / Theses on the topic 'Ureaplasmas'

Dissertação para obtenção do grau de Mestre em Biologia Molecular em Saúde
Neste estudo foram analisados os dados referentes às bactérias Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum e Ureaplasma parvum, duma amostra constituída por 16 020 utentes de um laboratório de análises clínicas. Constatou-se que o maior número de pedidos de análises recaía sobre a Chlamydia trachomatis, dado esta ser uma das doenças sexualmente transmissíveis com maior prevalência atualmente, e que a faixa etária com maior pedido de análises se situa entre os 27 e os 34 anos, correspondendo a adultos sexualmente ativos e em idade reprodutiva. De salientar também a elevada incidência de infertilidade em Portugal e a ligação entre este facto e as doenças sexualmente transmissíveis. Verificou-se que do total das análises requeridas apenas 4,6% foram amostras do género masculino. No entanto, são os homens que apresentam globalmente uma maior taxa de positivos, estando este facto associado à existência de sintomatologia aquando do diagnóstico. Da análise da amostra em estudo conclui-se que as colheitas de urina e exsudado uretral são os produtos que apresentam maior positividade para ambos os sexos, no entanto, a colheita com maior número de pedidos é a de exsudado vaginal. Foi possível ainda verificar que os métodos moleculares que permitem a distinção entre ureaplasmas contribuem para o uso racional de antibióticos.

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.

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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

Os Mollicutes causam doenças em várias espécies animais de importância econômica, inclusive em bovinos. Neste estudo, foi avaliada por concentração inibitória mínima (CIM) e citometria de fluxo, a atividade de oito agentes antibacterianos (enrofloxacina, ciprofloxacina, gentamicina, claritromicina, cloranfenicol, oxitetraclina, tiamulina e tilosina) contra Ureaplasma diversum. Foram analisadas 24 amostras de isolados de campo oriundas da mucosa genital de fêmeas bovinas. As amostras foram confirmadas por crescimento em caldo, placa e por PCR. Os inóculos foram submetidos à analise de suscetibilidade aos antibióticos pelo método da microdiluição em microplaca e posteriormente analisados pelo citômetro de fluxo a fim de avaliar a atividade antimicrobiana nas células. A claritromicina apresentou os maiores índices de inibição in vitro, sendo a gentamicina considerada o antibiótico de menor espectro de ação nesse estudo. De acordo com as análises do citômetro, a gentamicina apresentou o menor número de células viáveis enquanto a tiamulina apresentou o maior número. Embora haja resultados destoantes entre as técnicas utilizadas, o citômetro de fluxo pode ser utilizado como uma boa ferramenta para auxiliar a avaliação da suscetibilidade desses microrganismos a antibióticos.
The Mollicutes cause disease in several economically important species, including cattle. In this study, was evaluated by minimum inhibitory concentration (MIC) and flow cytometry, the activity of eight antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, clarithromycin, chloramphenicol, oxitetraclina, tiamulin and tylosin) against Ureaplasma diversum. We analyzed 24 samples of field isolates originating from the genital mucosa of cows. The samples were confirmed by growth in broth, plate, and PCR. The inoculations were subjected to analysis of susceptibility to antibiotics by the method of micro-dilution plate and then analyzed by flow cytometry to assess the antimicrobial activity in cells. Clarithromycin showed the highest levels of inhibition in vitro, the antibiotic gentamicin considered lower spectrum of action in this study. According to the analysis of the flow cytometer, gentamicin showed the lowest number of viable cells as tiamulin showed the greatest number. Although there are divergent results between the techniques used, flow cytometry can be used as a good tool even help assess the susceptibility of microorganisms to antibiotics.

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-β, IL-6 e TNF-α. 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-α 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-β, IL-6 and TNF-α. 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-α 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.

Monoclonal antibodies (Mabs) raised against Ureaplasma urealyticum (serotype 8) revealed the presence of three membrane antigens. One major surface antigen of apparent molecular mass 96 kDa, shown to express four distinct epitopes, was found to be serotype-8 specific. Thus, Mabs raised against this polypeptide will unequivocally differentiate serotype 8 from the other serotypes of human origin. The binding of antibodies to this polypeptide partially suppressed the growth of the organisms. Membrane expressed antigenic polypeptides of apparent molecular masses 16 kDa and 17 kDa were expressed by those serotypes belonging to the large serocluster (A), whereas the 17 kDa polypeptide only was expressed in the smaller serocluster (B). Using this Mab probe serotype 13 was placed in the larger serocluster. Thus Mabs, which recognise one or both of these polypeptides, will unequivocally differentiate the two seroclusters of this organism. The cytosolic urease from U. urealyticum, serotype 8, was purified by immuno-affinity chromatography. Two active forms of the enzyme were demonstrated by non-denaturing electrophoretic analysis and a single peak with urease activity of apparent molecular mass 190 KDa was shown by FPLC. Freezing and thawing of the purified enzyme caused a partial breakdown to inactive sub-units whereas total inactivation of the enzyme and denaturation, achieved by boiling for two minutes in the absence of any added denaturing agents, revealed three subunit polypeptides of apparent molecular masses 72, 14 and 11 KDa. Densitometry suggested that the active enzyme contains equimolar ratios of the three subunits and hence is a hexamer. The active enzyme displayed two pH optima of 6.9 and 6.15. Mabs raised against purified urease bound to both the active enzyme and to the inactive 72 kDa subunit. No evidence of antigenicity was found for the 14 and 11 KDa sub-units. These Mabs cross-reacted with ureases from all the other human serotypes. Competition assays revealed a minimum of four and possibly five distinct epitopes on the enzyme, all distinct from its active site. Ureaplasmas from 5 animal hosts were studied using the various Mabs. The 96 kDa antigen was not found in any of the non-human strains. Variations in the available epitopes on the ureases and the presence or absence of the 16/17 KDa antigens in the non-human strains allowed a putative identification of the source of the non-human ureaplasmas. Such investigations also showed that with the exception of the 96 KDa serotype 8-specific antigen, chimpanzee isolates could not be differentiated from the human ureaplasma serotypes belonging to the large serocluster. These Mabs were also used to develop fluorescent probes and other diagnostic assays which included a slide agglutination system and a sensitive urease catch assay which was also converted into a 'dip-stick' assay.

Com a crescente demanda por produtos ovinos, tem crescido o número de empresários dispostos a investir nessas atividades. Estudos sobre a importância das micoplasmoses e suas diferentes espécies já conhecidas ainda devem ser realizadas no Brasil. Foram estudadas 60 fêmeas da espécie ovina. O material clínico selecionado para analisar foi muco vulvovaginal obtido através de colheita por zaragatoa. Foi obtido o isolamento através de cultivo e a detecção pela técnica de reação em cadeia da polimerase de Mycoplasma spp., Ureaplasma spp. e Acholeplasma laidlawii em fêmeas de ovinos pela primeira vez no Brasil na região vulvovaginal. A porcentagem de micoplasma genérico detectada nas amostras foi de 75%, de Ureaplasma spp. foi de 66,7% e de Acholeplasma laidwaii foi de 1,7%. Houve isolamento em placa de 11,7% de Ureaplasma spp. do total de amostras processadas e de Micoplasma spp. em 8,3%. Não foi possível a identificação da espécie envolvida. Não houve associação da presença de micoplasmas com alterações no trato reprodutivo de fêmeas ovinas. Foi comprovada a infecção de uma fêmea ovina através de monta natural por um macho infectado, provando que uma das principais fontes de infecção dos animais é através do coito.
Sheep industry is growing rapidly in Brazil. However many diseases are the principal bottleneck to the increasing these activities. Among the reproductive diseases the vulvo-vaginites caused by Mycoplasma play an important role. Although no studies has been carried out in brazilian sheep herd so far. For that 60 ewes were ramdomly from different flocks selected at the county of Piedade state of São Paulo, Brazil. The mucus from vulvovagina was obtained by swab for isolation and detectetion of Mycoplasma spp., Ureaplasma spp. and Acholeplasma laidlawii. Generic Mycoplasma was detected in 75% of the samples, Ureaplasma spp. in 66,7% and Acholeplasma laidwaii in 1,7%. Mycoplasma spp. was isolated 8,3% and Ureaplasma spp. in 11,7%. It was not possible the identification of the species found. There was no association of mycoplasmas in the muco on the presence of reproductive problems in the herds. At least in one case, ram with presence of Mycoplasma in the semen infected a negative ewe, suggesting the importance of the ram in spreading the bacteria in the heard.

Introdução: Há tempos Micoplasmas Genitais como o Ureaplasma vêm sendo implicados na patogênese de trabalho de parto prematuro e morbidade neonatal, mas seu real papel permanece obscuro e sua prevalência no sangue de recém-nascidos de muito baixo peso ainda não foi estudada em nosso meio. Objetivo: Determinar a prevalência da infecção por Ureaplasma urealyticum (Uu) e Ureaplasma parvum (Up) em uma amostra de recém-nascidos de muito baixo peso (RNMBP) e avaliar os fatores associados. Pacientes e métodos: Foi realizada extração de DNA de amostras de sangue de RNMBP coletadas nas primeiras 72 horas de vida e a presença de Uu e/ou Up foi identificada por técnica de Reação em Cadeia de Polimerase (PCR). Os recém-nascidos foram acompanhados até a alta hospitalar. Resultados: Noventa e cinco recém-nascidos de muito baixo peso foram incluídos no estudo. A detecção de Uu e/ou Up ocorreu em 12 recém-nascidos (12,63%). Em 5,26% foi detectado somente Uu, em 5,26% somente Up e em 2,11% ambos. Na análise univariada a presença de Ureaplasma foi associada à infecção ovular e a trabalho de parto prematuro. Pré-eclâmpsia e ser PIG foram associados a menor ocorrência de Ureaplasma. Quando analisados apenas os nascimentos decorrentes de trabalho de parto prematuro, a prevalência da infecção por Ureaplasma foi de 25%. Pela regressão logística passo a passo, somente trabalho de parto prematuro manteve-se estatisticamente significante aumentando em 9 vezes a chance de positividade para Ureaplasma. Conclusão: A infecção por Ureaplasma é comum em recém-nascidos de muito baixo peso, principalmente entre os nascidos de trabalho de parto prematuro, reforçando a hipótese de associação entre prematuridade e infecção por Ureaplasma.
Introduction: Ureaplasma has long been implicated in the pathogenesis of both preterm labor and neonatal morbidity, but its actual role remains unclear, and it’s prevalence in the blood of very low birth weight (VLBW) infants has not been studied in our country. Objective: To determine the prevalence of Ureaplasma urealyticum (Uu) and Ureaplasma parvum (Up) bacteremia in a sample of very low birth weight infants and evaluate the associated factors. Patients and methods: DNA was extracted from blood samples collected during the first 72 hours of life of VLBW infants and the presence of Uu and/or Up was identified by the technique of Polymerase Chain Reaction (PCR). The newborns were followed up until hospital discharge. Results: Ninety-five very low birth weight newborns were included in the study. Detection of Uu and / or Up occurred in 12 infants (12.6%). We detected Uu in 5.2%, Up in 5.2% and both in 2.1%. In univariate analysis the presence of Ureaplasma was associated with clinical chorioamnionitis and preterm labor. Pre-eclampsia and SGA were associated with lower incidence of Ureaplasma. When analyzing only the births due to preterm labor, the prevalence of Ureaplasma bacteremia was 25%. Only preterm labor remained statistically significant after step by step logistic regression analysis increasing by 9 times the chance of Ureaplasma occurrence. Conclusion: Ureaplasma bacteremia is common in very low birth weight infants, especially among those born of premature labor, reinforcing the hypothesis of an association between prematurity and Ureaplasma infection.

It was confirmed that U. urealyticum produces an IgAl protease, which cleaves human IgAl only into intact Fab and Fcalpha fragments. By N-terminal amino acid sequencing of Fcalpha fragments, the site of digestion was identified as a Pro235-Thr236 peptide bond within the a chain hinge-region of IgAl. A number of assay systems were examined for their ability to detect and estimate IgAl protease activity. A reliable and reproducible immunoblotting method was developed, in conjunction with a quantifiable assay utilising [125I] IgAl. By these methods, IgAl protease activity was identified in fourteen serotypes of U. urealyticum, all of which appeared to digest IgAl at the same Pro235-Thr236 peptide bond. The enzyme was active over a broad range of pH (pH 3-10) and was inhibited by the serine-protease inhibitors 3,4-DCI and DFP. The IgAl protease was not located in 'spent' ureaplasma cultivation medium but appeared to be cell-associated. The activity was solubilised by a number of non-ionic detergents which were required in purification buffers to maintain enzyme stability, further suggesting a membrane-bound location. Although the enzyme was not purified to homogeneity, a number of protocols were established which provide a basis for future work. A genomic library of U. urealyticum DNA was produced and a variety of strategies adopted for identification of the iga gene. Radiolabelled DNA probes were generated from a plasmid containing the iga gene for N. gonorrhoeae (pIP503). By Southern blot hybridisation, no significant homology was identified between the heterologous probes and ureaplasma genomic DNA. Based on regions of high nucleotide conservation between the iga genes from N. gonorrhoeae and H. influenzae, degenerate PCR primers were designed. While amplification products did not appear to contain regions of the iga, such an approach may be adapted and extended for use in future studies.

Os efeitos clastogênicos causados por diferentes cepas Ureaplasma urealyticum e por cepas do gênero Mycoplasma foram avaliados \"in vitro\", utilizando-se culturas temporárias de linfócitos humanos. Inibições, total ou parcial da mitose foram produzidos pelos sorotipos II, III e X de Ureaplasma urealyticum, independentemente das concentrações dos inóculos de micoplasmas utilizados. Já os sorotipos I, VII e XII do Ureaplasma urealyticum, a cepa Mycoplasma hominis ATCC 23114 e a cepa Mycoplasma pneumoniae foram influenciadas pela concentração dos micoplasmas. Mitoses alteradas foram observadas nas cepas Ureaplasma urealyticum VIII, IX e X. A maior frequência de alterações foram de gaps cromatídicos e quebras cromatídicas. Os resultados obtidos neste modelo experimental \"in vitro\" revelaram haver comportamentos diferentes entre os vários sorotipos de Ureaplasma urealyticum e entre espécies do gênero Mycoplasma. Essas diferenças observadas, \"in vitro\", poderão de alguma forma contribuir para melhor compreensão dos efeitos da colonização desses microorganismos em humanos.
Clastogenic effect caused by different strains of Ureaplasma urealyticum and by strains of Mycoplasma sp were evaluated in vitro by using temporary cultures of human linphocytes. Mitotic inhibitions, either total or partial were produced by serotypes lI, III and X of Ureaplasma urealyticum, indenpendently of the concentration of microorganisms used. Otherwise, the kind of clastogenic effect of Ureaplasma urealyticum of serotypes I, VII and XII, Mycoplasma hominis strain 23144 and Mycoplasma pneumoniae varied with the different concentrations of these mycoplasmas used in the test. Mitotic alterations were observed in Ureaplasma urealyticum of sorotypes VII, IX and X. Cromatidic gaps and cromatidic rupture were the most frequent kind of alterations evidenced. The results obtained in the present in vitro experiment revealed that the occurence of different kind of clastogenic effect depends on the serotypes of Ureaplasma urealyticum and on the strains of Mycoplasma sp. The variability of the effect obtained will contribute to the better comprehension of the effects of colonization of these microorganisms in humans and stimulate future in vivo investigation.

Micoplasmas e ureaplasmas têm sido isolados de pequenos ruminantes, mas existem poucos estudos. O Mycoplasma ovine/caprine sorotipo 11, conhecido como cepa 2D, ainda não classificado como espécie, vem sendo relacionado a casos de vulvovaginite e problemas reprodutivos em ovinos e caprinos. Os ureaplasmas apresentam a habilidade em induzirem doenças no trato genital, confirmada por inoculações experimentais de isolados obtidos de animais doentes em animais saudáveis, resultando em moderada granularidade e hiperemia na vulva. Estes ureaplasmas não receberam ainda a designação de espécie, sendo apenas conhecidas suas características sorológicas, nas quais é fundamentada a classificação em nove sorotipos, sendo o IX relacionado a infertilidade e aborto em ovelhas. Este trabalho teve como objetivo o isolamento de micoplasmas e ureaplasmas do trato reprodutivo de fêmeas e machos das espécies caprina e ovina e tipificação genotípica dos isolados por meio de PFGE e sequenciamento do gene 16S rRNA. Foram obtidos 20 isolamentos de ureaplasma no trato reprodutivo de fêmeas e machos da espécie ovina, um isolamento de micoplasma e sete com crescimento misto de micoplasma e ureaplasma. Nas fêmeas da espécie caprina foram obtidos cinco isolamentos, sendo quatro de ureaplasma e um de micoplasma. Dos isolados submetidos a PFGE , 11 de origem ovina resultaram em oito diferentes perfis confirmando a capacidade da técnica em tipificar ureaplasma de origem ovina. O sequenciamento do gene 16S rRNA, analisado pelo UPGMA, permitiu agrupar seis isolados ao Ureaplasma diversum ATCC 49782, e quatro não foram agrupados com nenhuma outra espécies de ureaplasma, quando utilizado o ponto de corte 97%. Em nosso estudo, os isolados de ovino e caprino agrupados às referências de U. diversum não permite concluir que sejam da mesma espécie. A utilização do gene 16S rRNA gera grande quantidade de informações úteis para inferências filogenéticas e pode ser a primeira escolha na investigação de novas espécies. Técnicas filogenéticas como sequenciamento do espaço intergênico 16S-23S rRNA, e gene da urease podem auxiliaram futuramente na classificação dos isolados obtidos de ovino e caprino.
Mycoplasmas and or ureaplasmas have been isolated from small ruminants but are few studied. Mycoplasma ovine/caprine serotype 11, known as 2D strain, has not been classified yet as specie. However, it has been associated to vulvovaginitis and reproductive disorder in caprine and ovine. Ureaplasmas may cause genital tract diseases. Experimental infections with isolates recovered from sick animals resulted in granularity and hyperemia of the vulva. These have not been designated as specie but are divide in nine serological characteristics types. The serotype IX is associated to infertility and abortion in sheep. The present study aims the isolation of mycoplasmas and ureaplasmas from reproductive tract of ovine and caprine, genotyping of isolates by PFGE, and sequencing of their 16S rRNA. Ureaplasma isolates were recovered from the reproductive tract of 20 ovine, being them, male and female. Mycoplasma was isolated alone in one sample. A mixture of mycoplasma and ureaplasma was obtained in seven samples. On the material obtained from female caprine, five isolations were performed. Four of them were ureaplasma and one was a mycoplasma. Eleven isolates from ovine showed eight distinct profiles at PFGE, confirm that the method can typify ovine origin ureaplasmas. Six isolates were grouped to the Ureaplasma diversum ATCC 49782, through the sequencing of their 16S rRNA using the UPGMA. However, when using a 97% cutoff, four isolates could not be grouped to none of the ureaplasma specie. The isolates from ovine and caprine grouped to U. diversum, do not allow conclude that they are all the same specie. The use of 16S rRNA sequencing showed many useful information to phylogenetic inference, and can be the first choice for when investigating new species. Phylogenetic techniques, as sequencing of the intergenic space 16S-23S rRNA and urease gene, can be used to help the classification of new ureaplasma isolates from ovine and caprine.

Bacterial and fungal culturing was conducted on samples taken from the vaginal fornix of 106 cows, of which 42 had vaginitis and 64 had normal vaginas. The diagnosis of vaginitis and non-vaginitis samples was determined by histologic examination. Aerobic, anaerobic, and microaerophilic cultures were done. In addition, cultures were performed for Campylobacter sp., Ureaplasma sp., Mycoplasma sp., Tritrichomonas foetus, and fungi. All 106 samples contained mixed aerobic bacterial cultures. The more frequent aerobic isolates included Acinetobacter lwoffii, Arcanobacterium pyogenes, Escherichia coli, Corynebacterium spp., and Streptococcus spp. These organisms were isolated from both groups of cows, but more frequently from the vaginitis group. Anaerobic isolates included Peptostreptococcus spp., Prevotella spp., and Fusobacterium spp. The fungal isolates included Aspergillus sp., Mucor sp., and Penicillium sp.

Pesquisou-se a freqüência de Mollicutes na orofaringe, conjuntiva e trato urogenital de 58 macacos. Na orofaringe detectou-se Mollicutes em 55,17 % e Ureaplasma spp em 43,10%. As espécies identificadas nesta região foram: M. arginini (43,10%), M salivarium (41,37%), e M. pneumoniae (18,96%). No trato genital detectou-se Mollicutes em 27,58% sendo identificado M. arginini em 8,62%, A. laidlawii em 1,72 % e Ureaplasma spp em 32,75%. Na conjuntiva detectou-se Mollicutes em 29,31 % e A. laidlawii em 1,72 %. Os cultivos apresentaram limitações pelo alto índice de contaminação e obtiveram-se apenas dois isolamentos da conjuntiva. As espécies detectadas constituem-se o achado inicial destas bactérias em macacos no Brasil.
The frequency of Mollicutes in oropharynx, conjunctiva, and urogenital tract were accessed in 58 monkeys. In oropharynx, Mollicutes and Ureaplasma spp were detected in 55.17% and 43.01% of the samples, respectively. The identified species in this site included: M. arginini (43.10%), M. salivarium (41.37%), and M. pneumoniae (18.96%). In the urogenital tract, Mollicutes were detected in 27.58% of the samples; including M. arginini (8.62%), A. laidlawii (1.72%) and Ureaplasma spp (32.75%). In the conjunctiva, Mollicutes were detected in 29.31% and A. laidlawii in 1.72% of the animals. Mollicutes culture showed technical limitations because of the high level of contamination and only two isolates were obtained; both from conjunctival sites. The Mollicute specie surveillance of this study provided initial and new insights about these bacteria in Brazilian monkeys.

O presente estudo teve como objetivo a caracterização genômica das espécies: Mycoplasma hominis, Ureaplasma urealyticum e Ureaplasma parvum em amostras cervicais de gestantes. Cepas de Mycoplasma hominis e de Ureaplasma spp., foram identificadas através do cultivo e por PCR utilizando-se primers genéricos e específicos. Os achados na literatura são contraditórios quanto a participação dessas bactérias nas doenças humanas, especialmente quanto a participação destas em casos de infertilidade e alterações perigestacionais. Considerando a marcante heterogeneidade das cepas de Mycoplasma hominis, utilizamos a reação de RAPD com a finalidade de definir os perfis genômicos das cepas isoladas. A caracterização dos biótipos 1 e 2 do gênero Ureaplasma, respectivamente, Ureaplasma urealyticum e Ureaplasma parvum, através da análise da MBA (Múltipla Banda Antigênica) por PCR, teve como finalidade determinar a predominância de cada uma dessas espécies. Foram estudadas amostras de material cervical de 163 gestantes em várias idades gestacionais. Os resultados obtidos neste trabalho foram: M. hominis foi detectado em 14,7% (24/163) das amostras, sendo a RAPO realizada em 7 das amostras positivas e puras de M. hominis. A reação RAPD demonstrou similaridade do perfil gênico entre as amostras 3 e 4 e entre as amostras 6 e 8, enquanto que as amostras 2, 5 e 7 demonstraram uma grande heterogeneidade. O gênero Ureaplasma foi isolado em 54,6% (89/163) das amostras, sendo 31,5% (28/89) cepas de U. urealyticum e 68,5% (61/89) U. parvum. Foram detectados 10,4% (17/163) casos de co-infecções M. hominis e Ureaplasma spp. O estudo da suscetibilidade das cepas isoladas frente a tetraciclina foi realizado com o intuito de se determinar a freqüência de cepas resistentes a essa droga. Considerando ser este antibiótico o de eleição no tratamento das micoplasmoses humanas e devido aos altos índices de resistência apresentados, resistência esta conferida pela presença do plasmídeo tetM, submetemos nossas amostras à pesquisa deste plasmídeo através da PCR. A presença do plasmídeo tetM foi detectada em 29,5% (7/24) das amostras positivas para Mycoplasma hominis e em 60,7% (54/89) das amostras positivas para Ureaplasma spp., sendo que dentre estas, 57,4% (31/54) foram detectados na espécie U. parvum e 42,6% (23/54) na espécie Ureaplasma urealyticum. O estudo da heterogeneidade intra-espécie das cepas de Mycoplasma hominis pela técnica de RAPD é inédito no Brasil. Da mesma maneira a caracterização das espécies do gênero Ureaplasma em U. parvum e U. urealyticum, através da PCR pela análise da MBA, foi pioneiramente realizada pelo nosso grupo de pesquisa. Esperamos com esse trabalho poder contribuir para um conhecimento melhor da freqüência dessas espécies na nossa população e tentar explicar as divergências das avaliações nas interações entre os micoplasmas e o hospedeiro.
The objective of this study was to characterize the species Mycoplasma hominis, Ureaplasma urealyticum and Ureaplasma parvum in cervical samples from pregnant women. Strains of Mycoplasma hominis and Ureaplasma spp., were identified by means of culture and by PCR using generic and specific primers. The findings in the literature with regard to the involvement of these bacteria in human diseases are contradictory, especially regarding their involvement in cases of infertility and perigestational changes. In view of the strong heterogeneity among Mycoplasma hominis strains, we used RAPD to define the profiles of the strains isolated. Biotypes 1 and 2 of the genus Ureaplasma - Ureaplasma urealyticum and Ureaplasma parvum respectively - were characterized by means of MBA (Multiple Banded Antigenic) analysis using PCR to determine the predominance of each of these species. Samples of cervical material from 163 pregnant women at various stages of pregnancy were studied. The results obtained in this study were: M. hominis was detected in 14.7% (24/163) of the samples, and RAPD analyses carried out in 7 M. hominis strains. The RAPD similarly gene profile was showed: between strains 3 and 4, and between strains 6 and 8. The strains 2, 5 and 7 showed a pronounced heterogeneity in the gene profile. The genus Ureaplasma was isolated in 54.6% (89/163) of the samples, of which 31.5% (28/89) were U. urealyticum and 68.5% (61/89) U. parvum. Mycoplasma hominis and Ureaplasma spp., coinfection were detected in 10.4% (17/163) of the samples. The study of the susceptibility of the isolated strains to tetracycline was carried out with a view to determining the frequency of strains resistant to this drug. As this is the antibiotic of choice in the treatment of human mycoplasmoses, and in view of the high resistance indices observed, which are conferred by the presence of the \"tetM\" plasmid, we looked for this plasmid in our samples using PCR. The presence of the tetM plasmid, was detected 57,1% (4/7) of the Mycoplasma hominis - positive samples and in 40,2% (29/72) of the Ureaplasma spp - positive samples. Of the latter, 41,6% (25/72) were detected in the species U. parvum and 33,4% (4/12) were detected in the species Ureaplasma urealyticum. This is the first time that a study of the intraspecies heterogeneity of Mycoplasma hominis strains using RAPD has been carried out in Brazil. Characterization of the genus Ureaplasma species into U. parvum and U. urealyticum by means of PCR using MBA analysis was likewise pioneering effort by our research group. We hope that our work will contribute to a greater knowledge of the frequency of these species in our population and that it will help to explain differences in the assessment of the interaction between mycoplasma and the host.

The bacteria Ureaplasma has long been associated with a wide range of adverse health implications, including preterm birth, preterm premature rupture of the membrane and lung disorders, such as bronchopulmonary dysplasia in neonatal infants, but still, little is known about the pathogenic properties of Ureaplasma and possible direct association with adverse health complications. Estimated prevalence of Ureaplasma colonisation in sexually active adults is between 40 – 80%, therefore further understanding of its pathogenic properties and its ability to initiate an immune response is crucial. Specifically selected human cell-lines were examined in vitro to determine whether an innate immune response could be activated by Ureaplasma infection. If inflammatory immune responses were detected in human cell-lines, pathogenic properties of Ureaplasma would be confirmed, and its role in pregnancy and neonatal complications could be supported. Using a range of techniques, activation of immune response pathways were examined, as too were the production of detrimental pro-inflammatory cytokines that would strengthen the suggested associations of Ureaplasma infection with the above-mentioned complications. Myeloid-derived leukocytic monocytes, human bronchial epithelial cells and human amniotic epithelial cells were examined, as these would be the most relevant cell lines to determine if Ureaplasma could induce preterm birth, preterm premature rupture of the membrane and bronchopulmonary dysplasia. All cell lines studied showed immune response and inflammatory cytokine production after stimulation with Ureaplasma. This supports that Ureaplasma is capable of causing tissue damage in neonatal respiratory tracts that may lead to bronchopulmonary dysplasia and damage to the amniotic and chorion membranes that may lead to preterm premature rupture of the membrane. Ureaplasma was detected at the cell surface of human amniotic epithelial cells (HAECs) by TLR2 and TLR2/6 heterodimers. Results suggest that Ureaplamsma multiple banded antigen (MBA) is the strong ligand for TLR2 and TLR6 and stimulation of HAECs with MBA alone caused an immune response. TLR9 was responsible for the detection of internalised Ureaplasma, which is also able to initiate an immune response and inflammatory cytokine production. v Ureaplasma stimulation results in the production of the inflammatory cytokines TNF-α, IL-8 IL-6 via the NF-κB signaling pathway. Production of the potent inflammatory cytokine IL-1β was also observed, which would suggest the formation of inflammasome complexes. NLRs were investigated to find which NLR inflammasome were activated. It was shown that genetically knocking down NLRP7 significantly reduced the amount of IL-1β that was produced after Ureaplasma stimulation, suggesting that NLRP7 inflammsones are activated by Ureaplasma. Reduction in IL-1β was also observed, but to a lesser extent, when NLRP3 was knocked down. We decided to investigate the role of NLRP7 further and found a novel immune pathway, where NH3 causes activation and formation of the NRLP7 inflammasone. NH3 is produced as a bi-product of urease activity, which an essential process for Ureaplasma. The addition of a potent urease inhibitor to HAECs being stimulated with Ureaplasma significantly reduced the production of IL-1β, strongly supporting that NH3 plays a significant role in the detection of Ureaplasma infection and is responsible for causing the tissue damage that contributes to preterm premature rupture of the membrane leading to preterm birth. This investigation strongly supports that Ureaplasma is responsible for causing preterm birth and health complications in neonates, and that more robust treatment and monitoring of Ureaplasma is required, especially in pregnant women. These undertakings will hopefully reduce the rates of preterm birth and the associated health implications, in addition to reducing rates of bronchopulmonary dysplasia in neonates.

Ureaplasma parvum is one of the smallest (genome <0.8 Mb), free living, self-replicating organisms identified. There are seven species of Ureaplasma but only two infect humans: Ureaplasma parvum (serovars 1, 3, 6 and 14) and Ureaplasma urealyticum (serovars 2, 4, 5, 7–13). While frequently thought to be a commensal, Ureaplasma spp. has been found to be the most frequently identified infectious organism associated with preterm delivery. The mechanisms it uses to escape recognition by the host immune system and establish long term chronic infections in the host are unknown. However, association of infection with pregnancy and preterm birth, which have lower immune response are well known and its infection of and induction of preterm birth leads to short and long term sequel. Objectives of the study Using a model of experimental infection, this thesis sought to investigate differences be-tween systemic Ureaplasma infection in mid-second trimester equivalent and early third trimester equivalent, and relate these differences to the stage of development of the com-plement system in preterm lambs. These studies required the development of a new assay to measure complement function in sheep. The ability of the infecting strain of Ureaplas-ma to adapt to the sheep immune system was also investigated, examined at varying lengths of infection. These studies required the use of a new in vivo catheterisation method to establish the kinetics of early experimental intrauterine Ureaplasma infection and to re-cover Ureaplasma from the amniotic fluid by amniocentesis at various times during chron-ic infection. Emerging resistance to sheep complement was investigated in these strains and related to coincident alterations in the major surface antigen (multiple banded antigen; MBA) and alterations in the whole genome sequence. These studies required the charac-terisation of new monoclonal antibodies that recognise the MBA and establishment of a synthetic MBA expressed in E.coli to aid in reagent characterisation. Materials and Methods: Different erythrocyte sources and sensitising antibodies were utilised to establish the guin-ea pig erythrocyte as the best target for measuring the 50% haemolytic activity in fetal and adult sheep sera. Flow cytometry methods were also used to establish endogenous anti-erythrocyte antibodies in adult, but not fetal sheep, revealing a failure of maternal IgG transfer across the placenta in contrast to humans. A bactericidal assay for the decrease in surviving Ureaplasma following incubation with sheep sera was utilised and modified conditions revealed the rate of killing and calcium-dependence of Ureaplasma killing for both fetal and adult sera. Experimental infection of singleton pregnant ewes by catheter or ultrasound guided needle, followed by caesarean delivery at either 94 or 125 days gestation (term=150 days) was central to all of the studies. Over 3 years, different infection duration (from 5 days to 6 weeks) and gestational age at infection (from 70 days to 116 days gesta-tional age) were investigated in this model. Western blot analysis was also a central meth-od for both examining the antibody response of foetus and ewe to Ureaplasma as well as examination of the size and number of MBA isoforms in evolving strains during chronic infection. The use of genomic analysis software Geneious (Biomatters ltd., New Zealand) was also utilised to compare the genomic sequences of Ureaplasma strains recovered by amniocentesis during chronic infection relative to the original infecting strain to determine number and site of genetic alterations with evolving serum resistance. Only the repeat re-gion of the MBA gene required investigation by PCR and traditional Sanger sequencing, due to limitations of Illumina sequencing methods and repeats greater than 150 bp. Results: The 50% complement haemolytic activity of preterm sheep foetuses was very poor at 95 day gestation and improved by 125 day gestation, but was still much lower than adult sera. This was reflected in both in vivo bacteraemia during experimental infection and in vitro examining Ureaplasma-cidal activity of sera. A direct correlation (R2=0.30; p<0.05) was found between haemolytic activity of sera and capacity to kill Ureaplasma in vitro. The strain utilised for experimental infection (HPA5) has a single low mass isoform of MBA consisting of 17 PAGKEQ repeats in the C-terminus. Following 5-7 days infection two of six of the infected animals began to show emergence of a second larger MBA isoform. The number and size of MBA isoforms continued to increase at 3 and 5 weeks, until recov-ered strains appeared to solely consist of strains with much larger MBA isoforms (72->120 k Da). The underlying mechanisms of increased MBA mass was found to be increasing number of PAGKEQ repeats from 17 to >200 repeats. Genomic analysis of recovered strains identified conserved single nucleotide polymorphisms in the two genes encoding the ammonium ion transport, but otherwise very few alterations were observed and no ob-vious candidate to explain increased serum resistance (other than increased MBA mass) were observed. For the most part, alterations occurred in polyA, polyT and polyAT inter-genic regions. Despite the apparent correlation between increasing complement resistance of recovered strains and increasing MBA number of repeats, transposon delivery and ex-pression of the MBA gene from a resistant recovered strain and the serum-sensitive origi-nal HPA5 strain did not transfer serum resistance in one experiment. Conclusions: Ureaplasma can change its surface epitopes continuously however, the ex-pected phase variation loss of MBA expression with development of anti-MBA antibodies was not observed. A strong selection for increased MBA mass was apparent with longer infections and this was coincident with evolution of serum resistance (which increased steadily after 1 week infection to high serum resistance after 5 weeks infection). The de-velopment of the complement system was directly related to the detection of Ureaplasma in the circulation of infected lambs, but even 6 weeks of chronic persistent Ureaplasma infection was found to induce an inconsistent and low antibody response, and only in the later gestational age foetuses. While increased MBA size appears to be coincident with evolved serum resistance, it must require either loss of the original MBA isoform (domi-nant serum susceptibility determinant) or other less obvious alterations in the genome of Ureaplasma.

Ureaplasma spp colonize the lower genital tract in many healthy men, yet can also cause urethritis and infertility. Since Ureaplasma spp are frequently isolated from the normal urogenital tract, it has been suggested that only certain serotypes are associated with diseases. The genus Ureaplasma consists of 14 serotypes that can be divided into two biotypes, biotype 1 (U. parvum) and biotype 2 (U. urealyticum). Biotype 1 includes four serotypes (1, 3, 6 and 14) and biotype 2 includes ten serotypes (2, 4, 5 and 7 to 13). Identification and characterization of Ureaplasma spp are mainly performed by traditional culture methods that are difficult to perform and take two to seven days. Commercial kits have been an alternative method for the quick identification of Ureaplasma spp (24 to 48 hrs) but these kits cannot speciate the bacteria. However, polymerase chain reaction (PCR) assays have been developed for characterization and speciation of Ureaplasma spp. In this study, 200 first-void urine specimens were collected, 100 from symptomatic men (discharge/dysuria) and 100 from asymptomatic (without discharge/dysuria) men. The specimens were all cultured on U9 broth then subcultured on A2 agar for confirmation of growth. Antibiotic susceptibility determination was performed on the positive isolates using the Mycofast evolution 3 kit (ELITech Microbiology, France). Molecular detection of U. urealyticum was performed using a commercial kit, the Ureaplasma urealyticum real-time PCR kit. Detection and characterization of U. parvum was performed using a multiplex Taqman real-time PCR assay targeting the multiple banded antigen genes (MBA). Culture results of all specimens showed 40% (79/200) urease production on Shepard’s U9 broth and of these specimens 35% (28/79) were positive when subcultured on A2 medium, 25% (20/79) were contaminated with either yeast or Mycoplasma hominis or both and 39% (31/79) of the U9 positive specimens did not grow when subcultured on Shepard’s A2 agar medium. When determining the susceptibility profile of Ureaplasma spp, 32% (9/28) of the isolates did not grow with the kit and four were contaminated. None of the isolates was resistant to all of the antibiotics and all isolates were sensitive to doxycycline, pristinamycin, roxycycline and azithromycin. One isolate was resistant to ciprofloxacin and josamycin but intermediately resistant to ofloxacin and another isolate was resistant to ofloxacin, while a second isolate was only resistant to ciprofloxacin. Ureaplasma urealyticum was detected in 16% (31/100) of the symptomatic men and 15% (15/100) of the asymptomatic men whereas Ureaplasma parvum was detected in 11% (11/100) of the symptomatic men and 18% (18/100) of the asymptomatic men. There was no significant difference between the two groups (p= 01598). The distribution of serotypes did not differ significantly between the two groups (p= 0.309). The predominant serotype was serotype 6 followed by 1, 3 and 14. In conclusion, the real-time PCR assay was rapid, not prone to contamination and implementation of the assay in diagnostic laboratories will help in the rapid detection and administering of treatment to patients (especially neonates) infected with Ureaplasma species. The determination of the susceptibility profiles of Ureaplasma spp will assist in the monitoring of the antibiotic resistant profile trends in Pretoria, so that the correct antibiotic regimens can be administered to people with Ureaplasma infections.
Dissertation (MSc)--University of Pretoria, 2012.
Medical Microbiology
MSc
Unrestricted

The human Ureaplasma species are the most frequently isolated bacteria from the upper genital tract of pregnant women and can cause clinically asymptomatic, intra-uterine infections, which are difficult to treat with antimicrobials. Ureaplasma infection of the upper genital tract during pregnancy has been associated with numerous adverse outcomes including preterm birth, chorioamnionitis and neonatal respiratory diseases. The mechanisms by which ureaplasmas are able to chronically colonise the amniotic fluid and avoid eradication by (i) the host immune response and (ii) maternally-administered antimicrobials, remain virtually unexplored. To address this gap within the literature, this study investigated potential mechanisms by which ureaplasmas are able to cause chronic, intra-amniotic infections in an established ovine model. In this PhD program of research the effectiveness of standard, maternal erythromycin for the treatment of chronic, intra-amniotic ureaplasma infections was evaluated. At 55 days of gestation pregnant ewes received an intra-amniotic injection of either: a clinical Ureaplasma parvum serovar 3 isolate that was sensitive to macrolide antibiotics (n = 16); or 10B medium (n = 16). At 100 days of gestation, ewes were then randomised to receive either maternal erythromycin treatment (30 mg/kg/day for four days) or no treatment. Ureaplasmas were isolated from amniotic fluid, chorioamnion, umbilical cord and fetal lung specimens, which were collected at the time of preterm delivery of the fetus (125 days of gestation). Surprisingly, the numbers of ureaplasmas colonising the amniotic fluid and fetal tissues were not different between experimentally-infected animals that received erythromycin treatment or infected animals that did not receive treatment (p > 0.05), nor were there any differences in fetal inflammation and histological chorioamnionitis between these groups (p > 0.05). These data demonstrate the inability of maternal erythromycin to eradicate intra-uterine ureaplasma infections. Erythromycin was detected in the amniotic fluid of animals that received antimicrobial treatment (but not in those that did not receive treatment) by liquid chromatography-mass spectrometry; however, the concentrations were below therapeutic levels (<10 – 76 ng/mL). These findings indicate that the ineffectiveness of standard, maternal erythromycin treatment of intra-amniotic ureaplasma infections may be due to the poor placental transfer of this drug. Subsequently, the phenotypic and genotypic characteristics of ureaplasmas isolated from the amniotic fluid and chorioamnion of pregnant sheep after chronic, intra-amniotic infection and low-level exposure to erythromycin were investigated. At 55 days of gestation twelve pregnant ewes received an intra-amniotic injection of a clinical U. parvum serovar 3 isolate, which was sensitive to macrolide antibiotics. At 100 days of gestation, ewes received standard maternal erythromycin treatment (30 mg/kg/day for four days, n = 6) or saline (n = 6). Preterm fetuses were surgically delivered at 125 days of gestation and ureaplasmas were cultured from the amniotic fluid and the chorioamnion. The minimum inhibitory concentrations (MICs) of erythromycin, azithromycin and roxithromycin were determined for cultured ureaplasma isolates, and antimicrobial susceptibilities were different between ureaplasmas isolated from the amniotic fluid (MIC range = 0.08 – 1.0 mg/L) and chorioamnion (MIC range = 0.06 – 5.33 mg/L). However, the increased resistance to macrolide antibiotics observed in chorioamnion ureaplasma isolates occurred independently of exposure to erythromycin in vivo. Remarkably, domain V of the 23S ribosomal RNA gene (which is the target site of macrolide antimicrobials) of chorioamnion ureaplasmas demonstrated significant variability (125 polymorphisms out of 422 sequenced nucleotides, 29.6%) when compared to the amniotic fluid ureaplasma isolates and the inoculum strain. This sequence variability did not occur as a consequence of exposure to erythromycin, as the nucleotide substitutions were identical between chorioamnion ureaplasmas isolated from different animals, including those that did not receive erythromycin treatment. We propose that these mosaic-like 23S ribosomal RNA gene sequences may represent gene fragments transferred via horizontal gene transfer. The significant differences observed in (i) susceptibility to macrolide antimicrobials and (ii) 23S ribosomal RNA sequences of ureaplasmas isolated from the amniotic fluid and chorioamnion suggests that the anatomical site from which they were isolated may exert selective pressures that alter the socio-microbiological structure of the bacterial population, by selecting for genetic changes and altered antimicrobial susceptibility profiles. The final experiment for this PhD examined antigenic size variation of the multiple banded antigen (MBA, a surface-exposed lipoprotein and predicted ureaplasmal virulence factor) in chronic, intra-amniotic ureaplasma infections. Previously defined ‘virulent-derived’ and ‘avirulent-derived’ clonal U. parvum serovar 6 isolates (each expressing a single MBA protein) were injected into the amniotic fluid of pregnant ewes (n = 20) at 55 days of gestation, and amniotic fluid was collected by amniocentesis every two weeks until the time of near-term delivery of the fetus (at 140 days of gestation). Both the avirulent and virulent clonal ureaplasma strains generated MBA size variants (ranging in size from 32 – 170 kDa) within the amniotic fluid of pregnant ewes. The mean number of MBA size variants produced within the amniotic fluid was not different between the virulent (mean = 4.2 MBA variants) and avirulent (mean = 4.6 MBA variants) ureaplasma strains (p = 0.87). Intra-amniotic infection with the virulent strain was significantly associated with the presence of meconium-stained amniotic fluid (p = 0.01), which is an indicator of fetal distress in utero. However, the severity of histological chorioamnionitis was not different between the avirulent and virulent groups. We demonstrated that ureaplasmas were able to persist within the amniotic fluid of pregnant sheep for 85 days, despite the host mounting an innate and adaptive immune response. Pro-inflammatory cytokines (interleukin (IL)-1â, IL-6 and IL-8) were elevated within the chorioamnion tissue of pregnant sheep from both the avirulent and virulent treatment groups, and this was significantly associated with the production of anti-ureaplasma IgG antibodies within maternal sera (p < 0.05). These findings suggested that the inability of the host immune response to eradicate ureaplasmas from the amniotic cavity may be due to continual size variation of MBA surface-exposed epitopes. Taken together, these data confirm that ureaplasmas are able to cause long-term in utero infections in a sheep model, despite standard antimicrobial treatment and the development of a host immune response. The overall findings of this PhD project suggest that ureaplasmas are able to cause chronic, intra-amniotic infections due to (i) the limited placental transfer of erythromycin, which prevents the accumulation of therapeutic concentrations within the amniotic fluid; (ii) the ability of ureaplasmas to undergo rapid selection and genetic variation in vivo, resulting in ureaplasma isolates with variable MICs to macrolide antimicrobials colonising the amniotic fluid and chorioamnion; and (iii) antigenic size variation of the MBA, which may prevent eradication of ureaplasmas by the host immune response and account for differences in neonatal outcomes. The outcomes of this program of study have improved our understanding of the biology and pathogenesis of this highly adapted microorganism.

The human Ureaplasma species are among the smallest and simplest self-replicating bacteria known to date. These microbes cause infection in humans, particularly in the upper genital tract during pregnancy, leading to several adverse outcomes including preterm birth, chorioamnionitis, and respiratory diseases of neonates. Little is known about the pathogenesis of Ureaplasma and mechanisms by which they avoid recognition and killing by the complement system. In this thesis, some mechanisms underlying serum resistance of Ureaplasma spp. were investigated. This goal was achieved by creating serum-resistant models of serum-sensitive laboratory Ureaplasma strains and developing and using some proteomic and molecular biology methods to study the role of potential factors, which mediate serum resistance and play a role in pathogenesis of Ureaplasma. My original contribution to the knowledge in this work was the development of transposon mutagenesis method that can now be used to study virulence genes of Ureaplasma. This method will also allow genetic manipulation of Ureaplasma for future studies. Monitoring and investigating induced serum-resistant strains using immunoblot analysis and proteomics revealed significant changes in two candidate proteins coincident with serum resistance. The first was the elongation factor Tu protein that found to be immunogenic and had altered pI isoforms. The observed change in this protein was consistent in all serum-resistant strains, which suggests a possible role in mechanism of serum resistance, possibly as a mediator for binding complement regulators, such as factor H and C4BP, at the cell surface of Ureaplasma. The second candidate protein was a novel 41 kDa protein that was uniquely expressed in all induced serum-resistant strains. Expression of this protein in all resistant strains strongly indicates its involvement in mechanism(s) of serum resistance of Ureaplasma. The possible gene that encodes for this 41 kDa protein has putatively been identified as UUR10_0137 in the genome of U. urealyticum serovar 10 (strain ATCC 33699) using the transposon mutagenesis method developed in this study. Although the gene product of UUR10_0137 gene is not known (hypothetical protein), this protein is now identified and proposed to have a role in serum resistance of Ureaplasma. The product of the UUR10_0137 gene could function as a complement regulator or inhibitor that prevents the activation of complement system, protecting Ureaplasma from the complement attack. The contribution of the multiple- banded antigen, MBA, was proven to be unimportant to serum resistance. Sole antigenic variations in this major surface antigen of Ureaplasma did not play any role in mediating serum resistance. Confirmation of a gene that mediates complement resistance would dramatically increase our understanding of Ureaplasma pathogenicity and provide a target for future human studies with preterm birth and Ureaplasma infection.

O presente trabalho teve como objetivo o estudo da variabilidade genética e dos fatores de virulência de isolados de U. diversum. As cepas foram submetidas a sequenciamento dos genes da urease e 16S rRNA e a testes para verificar os fatores de virulência: cápsula, fosfolipase C, IgAse e adesão e invasão. A análise do sequênciamento parcial do gene 16S rRNA resultou na presença de polimorfismos em 44 posições da seqüência, que diferenciou as amostras em sete grupos. Em relação aos fatores de virulência, os dados mostraram que as cepas estudadas apresentaram uma camada densa ao redor da membrana celular dos microrganismos e atividade de fosfolipase C. No entanto, não foi observado a atividade de IgAse nas cepas. Em relação a atividade de invasão, observou-se que os ureaplasma estudados puderam ser visualizados no interior de células Hep-2 com apenas um minutos de infecção, sendo observados em uma região perinuclear, mas não no interior do núcleo. Além disto, pode verificar que entre 1% a 10% dos ureaplasmas estudos penetraram na célula pelo teste da gentamicina.
The aim of the present study was the study of genetic variability and virulence factors of U. diversum clinical isolates. The strains were submitted to sequencing for 16S rRNA and urease genes. Moreover, the strains were analyzed to the virulence factors: capsule, phospholipase C, IgA protease and adhesion and invasion into Hep-2 cells. The sequencing of parcial 16S rRNA gene showed polymorphic patterns into 44 positions. These polymorphisms clustered the strains in seven groups. For the virulence factors, ureaplasma cells showed a dense-stained external capsule-like structure surrounding the cell membrane. A high level of phospholipase C activity was also detected in 31 studied ureaplasma. However, no strains showed IgA protease activity. For the invasion assay, the isolates and strains used were detected inside the cells after infection of one minute. The invasions of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% to 10% of studied ureaplasmas were inside the infected cells.

This thesis investigated, for the first time, the prevalence of Ureaplasma species infection within the placentae of women who delivered in the late preterm stages of pregnancy. The presence of these microorganisms was associated with either severe inflammation within the placenta or, for some women, there were no pregnancy complications and these women delivered at term. Ureaplasmas are able to vary their surface exposed lipoproteins and we demonstrated that different host immune responses were generated in vivo to different sized surface lipoproteins. This may explain why ureaplasma infections do not always result in adverse pregnancy and neonatal outcomes.

Foram analisadas, para fins de diagnóstico das micoplasmoses da reprodução, 105 amostras de muco prepucial e 80 amostras de sêmen in natura, provenientes de touros de quatro propriedades rurais e 70 amostras de muco prepucial e 63 amostras de sêmen in natura, provenientes de touros de uma central de inseminação artificial, localizadas no Estado de São Paulo. As amostras foram submetidas ao isolamento bacteriano e PCR, para demonstração de presença de Mycoplasma bovis, M. bovigenitalium e Ureaplasma diversum. Colheitas foram realizadas no período de 1999 à 2002, antes e após a implantação de dois métodos de antibioticoterapia, utilizando-se para tal, oxitetraciclina di-hidratada ou fumarato de hidrogênio de tiamulin. O Sistema MGSO/GPO-1 de detecção de Mycoplasmatales pela PCR foi testado frente ao isolamento bacteriano. A associação entre a idade e forma de manejo reprodutivo, além da qualidade seminal e a presença dos microrganismos foi analisada através da odds ratio. A eficiência dos métodos de tratamento sobre a qualidade seminal dos touros foi analisada estatisticamente pelos testes de Mann Whitney e Wilcoxon. As freqüências de detecção de Mycoplasma spp pelo isolamento e pela PCR nas amostras de muco prepucial, foram 45,1 e 63,7%, respectivamente. Nas amostras de sêmen, as freqüências foram 22,5% (isolamento) e 24,1 (PCR). O Sistema MGSO/GPO-1 mostrou-se pouco eficiente na detecção dos agentes. Análise estatística revelou que animais com menos de quatro anos têm de 1,39 (M. bovigenitalium) até 7,54 (U. diversum) vezes mais chance de serem portadores dos microrganismos. A odds ratio de 4,01 até 4,68 (muco prepucial) e de 1,71 até 18,29 (sêmen) mostrou a importância da higiene no manejo reprodutivo. Os animais da Central de Inseminação Artificial apresentaram resposta significativa sobre a motilidade espermática no tratamento com oxitetraciclina (p=0,033). As estimativas de risco, pós-tratamento, foram menores para os parâmetros motilidade e defeitos menores, associados à Mycoplasma spp e, motilidade e defeitos maiores, associados à U. diversum .
Prepucial mucuses and fresh semen were collected from bulls under two kinds of reproductive management: a) natural breeding with cows in four farms, b) an Artificial Insemination Center. All of them located in São Paulo State, Brazil. The samples were collected from 1999 to 2002. In the farms it were collected 105 samples of prepucial mucuses and 80 samples of fresh semen and in the Artificial Insemination Center, 70 samples of prepucial mucuses and 63 of fresh semen. The laboratory tests applied for demonstration of Mycoplasma bovis, M. bovigenitalium and Ureaplasma diversum were classical cultivation and polymerase chain reaction (PCR). The therapeutic assay were performed with oxytetracicline, 20 mg./kg. of body weight, IM, or tiamulin hydrogen fumarate, 15 mg./kg. of body weight, IM. Mycoplasma spp was demonstrated in 45,1% and 63,7% of prepucial mucuses samples, respectively by cultivation and PCR and in 22,5% and 24,1% of fresh semen samples, respectively by cultivation and PCR. Ureaplasma diversum was demonstrated in 66,5% and 72,9% of prepucial mucuses samples, respectively by cultivation and PCR and in 51,7% and 56,6% of fresh semen samples, respectively by cultivation and PCR. The MGSO/GPO-1 PCR System, was able for demonstration of Mycoplasma spp DNA in bovine semen and U. diversum in prepucial mucuses, with results in agreement with cultivation. Animals younger than four years of age presented a higher infection rate than older: odds ratio 1.39 (M. bovigenitalium) and up to 7.54 (U. diversum). Odds ratio from 4.01 to 4.68 (prepucial mucuses) and from 1.71 to 18.29 (semen) showed the importance of the hygienic status of the reproduction management. The animals from Insemination Center, showed better motility after the treatment with oxytetracicline (p=0,033). After therapeutic assay, the odds ratio was small for the motility and morphologic aspects of the semen associated to Mycoplasma spp and U. diversum contamination.

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The reduction of reproductive rates is measured by the reduction in the number of calves born that immediately reflected in the reduction of milk production and profitability of commercial property. The effect is the increased administrative costs of maintenance with dry cows, higher rates of rejection and a higher number of doses of semen for conception. Parameters related to the level of hygiene of the udder and the milking system and environmental criteria as the overall control on the suction pressure of the milking, the ventilation system for livestock and hygiene in the milking parlor, contribute to the satisfaction of the control of pathogens transmissible among livestock. Study on monitoring the herd showed that the time of calving interval increases significantly in the first lactation cows with vaginitis, but the injury was not associated with cases of voluntary discharge. Research on the likelihood of introduction of infectious agents into herds free of the disease found that the significant risk factors for this spread were related to the return of animals that were removed for marketing, proximity between pastures of different properties and especially the clothing of a veterinarian or visitors. When analyzing the risk factors for the occurrence of a disease the causative factors are considered. Regression analysis to identify risk factors usually consider the multifactorial effects and outcome. The identification of the risk factor is to compare the pattern of distribution of the dependent variables between the levels of occurrence of the disease. Infections with mycoplasma (Mollicutes) in farm animals, have historical importance and continuing today interfering with livestock productivity. The M. Canadense is still little known in the pathological processes of reproduction. The M. bovis is highly adapted to cattle, however has been isolated in samples of buffalo, small ruminants and humans. The M. bovigenitalium was related to diseases of the reproductive system for more than three decades and was considered as the primary agent of vaginitis. The granular vulvitis sponsored by the U. diversum was demonstrated both in animals vaccinated as in unvaccinated. The agent has to interact with cellular factors, the ability to bind to the surface membrane of the host and even biological mechanisms to escape immune response submitted by host.Young animals and adults are susceptible. Transmission is by the introduction of carrier animals, semen and contact with infected genital secretions of infected animals
A queda dos índices reprodutivos é medida pela redução no número de bezerros nascidos que reflete imediatamente na redução da produção de leite e na rentabilidade comercial da propriedade. A repercussão administrativa é o incremento das despesas de manutenção com vacas secas, maiores taxas de descarte e maior número de doses de sêmen por concepção. Parâmetros relacionados ao nível de higiene do úbere e do sistema de ordenha e os critérios ambientais globais como o controle na pressão de sucção da ordenhadeira, o sistema de ventilação para os animais assim como a higiene na sala de ordenha, contribuem de forma satisfatória para o controle de patógenos transmissíveis entre os animais de produção. Estudo sobre monitoramento de rebanho demonstrou que o tempo do intervalo de partos aumenta significativamente em vacas de primeira lactação com vulvovaginite, mas a ocorrência da lesão não apresentou associação com os casos de descarte voluntário. Pesquisas sobre a probabilidade de introdução de agentes infecciosos em rebanhos livres da doença identificaram que os fatores de risco significativos para esta disseminação estiveram relacionados com a devolução de animais que foram retirados para comercialização, proximidade entre pastos de diferentes propriedades e principalmente pela vestimenta do veterinário ou de visitantes. Ao analisar os fatores de risco para a ocorrência de determinada doença as causas multifatoriais são consideradas. As análises de regressão para identificar fatores de risco normalmente consideram os efeitos multifatoriais e o desfecho. Assim, a identificação do fator de risco consiste em comparar o padrão de distribuição das variáveis dependentes entre os níveis de ocorrência da doença. O M. canadense ainda é pouco conhecido nos processos patológicos da reprodução. O M. bovis é altamente adaptado aos bovinos, porém já foi isolado em amostras de búfalos, pequenos ruminantes e humanos. O M. bovigenitalium foi relacionado com patologias do sistema reprodutivo há mais de três décadas e foi considerado como o agente primário da vulvovaginite. A vulvite granular promovida pelo U. diversum foi demonstrada tanto em animais vacinados quanto nos não vacinados. O agente apresenta fatores de interação celular, a habilidade de se ligar à superfície de membrana do hospedeiro e ainda mecanismos biológicos de escape à resposta imunológica apresentada pelo hospedeiro.Tanto animais jovens quanto adultos são susceptíveis. A transmissão ocorre pela introdução de animais portadores, sêmen contaminado e contato com as secreções genitais de animais infectados

A vulvovaginite granular (VVG) é uma infecção caracterizada pela presença de secreção, granulações e vesículas nas mucosas vaginal e vulvar descrita em fêmeas bovinas de várias partes do mundo. Bactérias dos gêneros Mycoplasma spp. e Ureaplasma diversum, que se caracterizam por serem fastidiosos e de difícil diagnóstico, são frequentemente descritos como os principais agentes etiológicos envolvidos nessa infecção. O objetivo deste estudo foi desenvolver um sistema de diagnóstico molecular, constituído por uma PCR genérica seguida de uma nested-PCR, para a identificação de espécies bacterianas da classe Mollicutes em material biológico proveniente de animais com VVG. Para a PCR genérica foram desenhados primers consensuais degenerados (MolliFw1 / MolliRv1) tendo como alvo uma região conservada denominada espaço intergênico (ITS) e parte dos genes ribossomais 16S e 23S do genoma dos Mollicutes. Posteriormente, foram desenhados primers internos para reações individuais de nested-PCR específica para as espécies de M. bovis (MybovisFw2 / MybovisRv2), M. bovigenitalium (MygenitFw3 / MygenitRv3) e U. diversum (UdivFw4 / UdivRv4). Para determinar a especificidade dos primers os produtos amplificados por meio de nested-PCR foram submetidos ao sequenciamento e análise da sequência de nucleotídeos. O sistema de diagnóstico desenvolvido foi ainda avaliado frente a 31 amostras de swabs vaginais colhidos de vacas com vulvovaginite clínica, provenientes de três rebanhos sendo dois de corte dos estados de Minas Gerais (n=12) e Mato Grosso do Sul (n=11) e um leiteiro do estado do Paraná (n=8). Todas as 31 amostras foram previamente avaliadas quanto a presença de herpesvírus bovino - 1 por meio de semi nested-PCR e foram negativas. Em 87,1% (27/31) das amostras avaliadas foram identificados pelo menos um dos micro-organismos investigados. Infecções singulares e mistas ocorreram em 54,8% (17/31) e 32,2% (10/31) das amostras, respectivamente. U. diversum foi identificado em 83,8% (26/31) e o M. bovigenitalium em 35,4% (11/31) das amostras analisadas. Nenhum dos três rebanhos bovinos incluídos no estudo apresentou infecção singular por M. bovis. Os resultados obtidos nesse estudo sugerem a importância de U. diversum e do M. bovigenitalium na etiologia da VVG. As taxas de positividade para Mycoplasma spp. e U. diversum obtidas foram semelhantes e/ou superiores aos resultados apresentados em estudos que incluíram como métodos de diagnóstico técnicas de cultivo bacteriológico e também reações moleculares como PCR e nested-PCR. O sistema molecular de diagnóstico de infecções genitais em fêmeas bovinas ocasionadas por bactérias da classe Mollicutes revelou-se como uma alternativa viável, rápida, sensível e específica para o diagnóstico de M. bovis, M. bovigenitalium e U. diversum a partir de material biológico. O diagnóstico precoce dos três principais patógenos bacterianos envolvidos em infecções genitais em vacas com VVG é de grande importância, pois possibilita a implantação de medidas de tratamento, controle e profilaxia mais rápidas e efetivas das infecções.
Granular vulvovaginitis (GVV) is an infection characterized by the presence of vaginal granular discharge, and vesicles in vaginal and vulvar mucosa described in cows worldwide. Bacteria of the genera Mycoplasma spp. and Ureaplasma diversum are characterized by being fastidious and difficult to diagnose are frequently described as the main etiological agents involved in this infection. The objective of this study was to develop a molecular diagnostic method, consisting of a generic PCR followed by a nested-PCR for identification of bacterial species of the Mollicutes class in biological material obtained from animals with GVV. Degenerate primers were designed for generic consensus PCR (MolliFw1 / MolliRv1) targeting the conserved intergenic spacer region denominated ITS and part of the 16S and 23S ribosomal genes of Mollicutes. Subsequently, internal primers were designed for individual nested-PCR reactions specific for the M. bovis (MybovisFw2 / MybovisRv2), M. bovigenitalium (MygenitFw3 / MygenitRv3) and U. diversum (UdivFw4 / UdivRv4) species. To determine the specificity of the primers, the nested-PCR amplified products were subjected to sequencing and nucleotides sequence analysis. The diagnostic system developed was used to screening of 31 vaginal swabs collected from cows with clinical vulvovaginitis from three herds; two beef herds in Minas Gerais (n=12) and Mato Grosso do Sul (n=11) and one dairy herd of Paraná (n=8) state. All 31 samples were negative for the presence of bovine herpesvirus - 1 by semi nested-PCR. At least one of the microorganisms investigated was present in 87.1% (27/31) samples. Single and mixed infections occurred in 54.8% (17/31) and 32.2% (10/31) of sample, respectively. U. diversum was identified in 83,8% (26/31) and M. bovigenitalium in 35.4% (11/31) of samples. None of the three herds included in the study showed M. bovis single infection. The results of this study suggest the importance of U. diversum and M. bovigenitalium in the etiology of GVV. The positivity rates for Mycoplasma spp. and U. diversum reported here were similar or higher than the results of previous studies usin diagnostic methods such as bacterial culture and molecular techniques including PCR and nested-PCR. The molecular tools for diagnosis of genital infections in cows caused by bacteria of the class Mollicutes revealed itself as a reliable, rapid, sensitive and specific alternative for the diagnosis of M. bovis, M. bovigenitalium and U. diversum from biological material. Early diagnosis of the three major bacterial pathogens involved in genital infections in cows with GVV is of major importance, because it enables the implementation of more effective measures of treatment, control and prophylaxis of infections.

Na área da reprodução, a importância das infecções genitais surge pela sua influência evitável no desenvolvimento de infertilidade. Infecções genitais têm potencial para alterar a cérvice e o muco cervical dificultando a passagem dos espermatozóides, e para causar doença inflamatória pélvica e consequente oclusão tubária, sendo esta um dos principais factores de infertilidade. A eficácia de técnicas de Procriação Medicamente Assistida é alterada na presença de infecções genitais. Adicionalmente, a sua propagação ascendente para o útero gravídico provoca infecção e inflamação da decídua e dos anexos embrionários sendo a causa de abortos espontâneos recorrentes. O maior foco de interesse científico incide sobre infecções de etiologia sexualmente transmissível (como Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis) e as espécies Mycoplasma hominis e Ureaplasma urealyticum. Bactérias associadas a Vaginose Bacteriana, Streptococcus agalactiae, e espécies Candida foram também identificadas em mulheres com infertilidade. Este estudo retrospectivo visa calcular a prevalência de agentes infecciosos genitais em mulheres com tempo de infertilidade igual ou superior a doze meses (n=190) seguidas na Unidade de Medicina da Reprodução do Centro Hospitalar Cova da Beira, no período de Agosto de 2009 a Agosto de 2011. Como rotina da Unidade, realizou-se a colheita de amostras vaginais e endocervicais para pesquisar Trichomonas vaginalis, espécies Ureaplasma e Mycoplasma hominis via teste “MycoView”®, Chlamydia trachomatis via teste “QuickVue”®, e outras espécies via cultura em meios específicos. Das mulheres estudadas, 42,63% apresentam infecção genital. O agente mais prevalente é o Ureaplasma urealyticum, numa percentagem (29,47%) superior à detectada noutros estudos. Candida spp. foi o segundo agente com maior prevalência(15,26%), e a pesquisa de Chlamydia trachomatis foi positiva em apenas uma mulher. Nenhuma das mulheres estudadas apresentava infecção por Neisseria gonorrhoeae nem Trichomonas vaginalis. É recomendável a aplicação dos métodos mais sensíveis e específicos disponíveis para rastreio de infecções genitais, promovendo uma prevenção eficaz de problemas de subfertilidade e de complicações após procedimentos invasivos e contribuindo para o sucesso da actuação da Unidade de Medicina da Reprodução.
In the reproduction area, the importance of genital infections emerges by their avoidable influence in the development of infertility. Genital infections have the potential to modify cervix and cervical mucus obstructing the passage of sperm, and to cause a pelvic inflammatory disease and subsequent tubal occlusion, being this one of the main factors of infertility. The effectiveness of techniques of Medically Assisted Procreation is altered in the presence of genital infections. In addition, his ascending spreading to the pregnant uterus causes infection and inflammation of the deciduous membrane and embryonic annexes being the cause of recurrent miscarriages. The major focus of scientific interest relates on infections of sexually transmitted etiology (like Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis) and Mycoplasma hominis and Ureaplasma urealyticum species. Bacteria associated to Bacterial Vaginosis, Streptococcus agalactiae, and Candida spp. were also identified in women with infertility problems. This retrospective study aims to estimate the prevalence of genital infectious agents in women with a infertility period equal or more than twelve months (n=190) observed in the Reproductive Medicine Unit of the Centro Hospitalar Cova da Beira, since August 2009 to August 2011. As routine of Unit, was performed vaginal and endocervical sampling to search Trichomonas vaginalis, Ureaplasma and Mycoplasma hominis species using “MycoView”® test, Chlamydia trachomatis testing via “QuickVue”® and other species from culture in specific environment. Of the studied women, 42,63% have genital infection, being the most prevalent agent the Ureaplasma urealyticum, in a percentage (29,47%) higher than that found in others studies. Candida spp. was the second most frequent agent (15,26%) and Chlamydia trachomatis was positive in only one woman. None of the women studied were infected with Neisseria gonorrhoeae e Trichomonas vaginalis. It is recommended the application of the most sensitive and specific screening methods available, promoting an effective prevention of subfertility problems and complications after invasive procedures and contributing to a successful performance of the Reproductive Medicine Unit.

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This study’s objective was to conduct a Mycoplasma bovigenitalium and Ureaplasma diversum infections epidemiological investigation in cattle in the Ipanema Valley microregion, Pernambuco state. Vaginal swabs were collected from 355 reproductive age cows and submitted to polymerase chain reaction (PCR) and microbiological isolation. An investigative questionnaire was used to risk factors analysis associated with Mollicutes infection. According to PCR, 9.29% (33/355) of the samples were positive for Mycoplasma bovigenitalium and 21.69% (77/355) for Ureaplasma diversum. Coinfection was observed in 2.81% (10/355) of the samples. According to isolation, there was growth on Hayflick medium of 81.81% (27/33) from the samples classified as Mycoplasma spp. and 24.67% (19/77) growth on UB medium, being classified as Ureaplasma diversum. Risk factors associated to Mollicutes infection were considered: semi-intensive breeding system (OR = 4.6), pasture rent (OR = 3.6), reproductive disorders nonisolated animals (OR = 3.2), and natural mating plus artificial insemination (OR = 3.5). This is the first record of Mycoplasma bovigenitalium and Ureaplasma diversum infection in semiarid region cattle herds of Pernambuco state, Brazil. From this, preventive measures directed to the identified risk factors can contribute to Mollicutes’ occurrence decrease in the cattle herds studied.
Objetivou-se, com este estudo, realizar uma investigação epidemiológica da infecção por Mycoplasma bovigenitalium e Ureaplasma diversum em bovinos na microrregião do Vale do Ipanema, estado de Pernambuco. Foram coletados suabes vaginais de 355 vacas em idade reprodutiva que foram submetidos à Reação em Cadeia da Polimerase (PCR) e isolamento microbiológico. Um questionário investigativo foi utilizado para a análise dos fatores de risco associados à infecção por Mollicutes. Pela PCR, 9,29% (33/355) amostras foram positivas para Mycoplasma bovigenitalium e 21,69% (77/355) para Ureaplasma diversum. Observou-se coinfecção em 2,81% (10/355) das amostras. A partir do isolamento microbiológico das amostras positivas na PCR, houve crescimento no meio Hayflick de 81,81% (27/33) das amostras, sendo classificadas como Mycoplasma spp. e 24,67% (19/77) cresceram no meio “UB”, sendo classificadas como U. diversum. Foram considerados fatores de risco associados à infecção por Mollicutes: sistema de criação semi-intensivo (OR=4,6); aluguel de pastagens (OR=3,6); não isolamento dos animais com distúrbios reprodutivos (OR=3,2) e realização de monta natural mais inseminação artificial (OR=3,5). Este é o primeiro registro da infecção por Mycoplasma bovigenitalium e Ureaplasma diversum em rebanhos bovinos na região semiárida do estado de Pernambuco, Brasil. A partir dos resultados obtidos, medidas de prevenção direcionadas aos fatores de riscos identificados podem contribuir com a diminuição da ocorrência de Mollicutes nos rebanhos bovinos estudados.

Les activites dna polymerases de differents membres des mollicutes ont ete etudiees. Une seule dna polymerase a ete trouvee chez mycoplasma mycoides et ureaplasma urealyticum, especes types des genres mycoplasma et ureaplasma. Ces deux enzymes ont ete comparees a celle de mycoplasma orale. La plupart des proprietes catalytiques et physiques sont semblables; une relation immunologique a ete demontree par immunoblotting entre l'enzyme de m. Orale et celle de m. Mycoides. Par contre, trois dna polymerases ont ete purifiees chez acholeplasma laidlawii, espece type du genre acholeplasma qui de ce point de vue ressemble au genre spiroplasma. Une activite 3-5 exonuclease a pu etre mise en evidence dans les preparations purifiees, excepte pour l'enzyme de u. Urealyticum. Celle-ci semble ne pas associee a la dna polymerase. De plus, une activite 5-3 exonuclease a ete egalement mise en evidence dans ces preparations. En resume, la presence d'une seule dna polymerase semble etre une caracteristique des mycoplasmataceae (genre mycoplasma et ureaplasma) par opposition a la presence de trois enzymes chez les acholeplasmataceae et les spiroplasmataceae. Ces resultats sont compatibles avec l'arbre phylogenique etabli par comparaison des sequences des rna ribosomaux 5s et 16s, dans lequel l'evolution des branches acholeplasma et spiroplasma conduit, par reductions genomiques, aux especes mycoplasma et ureaplasma

Ureaplasmas are some of the smallest and simplest free-living organisms known. Little is understood regarding the effects of the complement system upon clearance of these pathogens, but when the immune system fails to control Ureaplasma colonization disease, such as chronic lung disease of prematurity (CLD), can occur. Treatment of such Ureaplasma infections, especially in neonates, is limited by pathogen and host factors. Treatment can be further compromised by antibiotic resistance. Firstly this thesis examines an in vitro system for determining the bactericidal capacity of a selection of human sera against four representative serovars of Ureaplasma parvum. Results showed that the classical activation pathway was essential for the killing of all U. parvum serovars, with little effect being attributed to the alternative or lectin pathways. Additionally serovar 3 was shown to be the most serum sensitive isolate. Secondly the association between presence of Ureaplasma and 16S rRNA with development of CLD was examined in a prospective cohort of 192 neonates. Data suggested that presence of Ureaplasma as well as 16S rRNA was significantly associated with development of CLD as well as increased levels of inflammatory mediators IL-6 and IL-8. Finally a retrospective cohort of 61 Ureaplasma isolates was examined for resistance to various antibiotics. High level macrolide resistance in isolate UHWO10 resulted from a two amino acid deletion within the L4 ribosomal protein (R66Q67). Tetracycline resistance in isolate HPA23 resulted from the presence of the tetM gene while a ciprofloxacin resistance in isolate HPA18 resulted from a D82N substitution within the ParC protein. No differences were found in the GyrA, GyrB or ParE proteins. Comparison of type II topoisomerase genes from all Ureaplasma serovars revealed that mutations previously associated with resistance where wrongly identified and were a result of species or serovar specific polymorphisms.

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The objective of this study was to detect the DNA of Ureaplasma spp. and Mycoplasma agalactiae in sheep semen by the technique of Polymerase Chain Reaction (PCR). Were analyzed 240 samples, 120 frozen semen obtained from central artificial insemination (AI) and 120 fresh semen of breeding sheep from the states of Alagoas, Ceará, Paraíba, Pernambuco and Rio Grande do Norte collected during the Northeastern Exposure animals in Recife. After collection of the samples were carried out DNA extraction and detection of genomic DNA of the agents studied. For the first time in Brazil, was detected the presence of agents Ureaplasma spp. and Mycoplasma agalactiae in samples of frozen semen of sheep. Samples of frozen semen, DNA was detected Ureaplasma spp. in 2.5% (3/120) and Mycoplasma agalactiae of 4.2% (5/120). For fresh semen was detected DNA of Ureaplasma spp. in 8.3% (10/120) of the samples and 6.7% (8/120) of Mycoplasma agalactiae When evaluating the association between the type of semen and DNA detection for Ureaplasma spp. and Mycoplasma agalactiae, a significant association was observed only for Ureaplasma spp. (p = 0.046), being more common detection of this micro-organism in fresh semen samples. We also observed positive samples Ureaplasma spp. and Mycoplasma agalactiae from the states of Paraíba, Pernambuco and Rio Grande do Norte. All samples from the states of Alagoas and Ceará results were negative. Results obtained in this study, there is the presence of DNA of these microorganisms in semen of the breedings sheep. Thus, it is suggested that techniques for detecting these agents must be used in insemination centers and breeding sheep with high genetic potential to maximize the efficiency of the sheep reproduction by preventing the spread of these pathogens.
Objetivou-se com este estudo detectar o DNA de Ureaplasma spp. e Mycoplasma agalactiae em sêmen de ovinos pela técnica da Reação em Cadeia da Polimerase (PCR). Foram analisadas 240 amostras, sendo 120 de sêmen congelado obtidas de centrais de inseminação artificial (IA) e 120 de sêmen fresco de reprodutores ovinos provenientes dos estados de Alagoas, Ceará, Paraíba, Pernambuco e Rio Grande do Norte coletados durante a realização da Exposição nordestina de animais em Recife,PE. Após a colheita das amostras, foram realizadas a extração de DNA e detecção de DNA genômico dos agentes estudados. Pela primeira vez no Brasil, foi detectada a presença dos agentes Ureaplasma spp. e Mycoplasma agalactiae em amostras de sêmen congelado da espécie ovina. Nas amostras de sêmen congelado, detectou-se DNA de Ureaplasma spp. em 2,5% (3/120) e de Mycoplasma agalactiae em 4,2% (5/120). Para o sêmen fresco, detectou-se DNA de Ureaplasma spp. em 8,3% (10/120) das amostras analisadas e 6,7% (8/120) de Mycoplasma agalactiae Ao avaliar a associação entre o tipo de sêmen e a detecção de DNA para Ureaplasma spp. e Mycoplasma agalactiae, observou-se associação significativa somente para Ureaplasma spp. (p= 0,046), sendo mais comum a detecção deste micro-organismo em amostras de sêmen fresco. Foram observadas ainda, amostras positivas para Ureaplasma spp. e Mycoplasma agalactiae provenientes dos estados da Paraíba, Pernambuco e Rio Grande do Norte. Todas as amostras oriundas dos estados de Alagoas e do Ceará obtiveram resultados negativos. Diante dos resultados obtidos neste estudo, constata-se a presença de DNA destes micro-organismos em sêmen de reprodutores ovinos. Desta forma, sugere-se que técnicas para detecção desses agentes sejam utilizadas em centrais de inseminação e em reprodutores ovinos com alto potencial genético, maximizando a eficiência da prática na reprodução de ovinos, evitando a disseminação destes patógenos.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Microbial invasion of the amniotic cavity has been described in term deliveries and its role on the immune modulation is of interest to the better understanding of the underlying labor processes. The aim of this study was to determine the prevalence of Mycoplasma hominis and Ureaplasma urealyticum in the amniotic fluid of term pregnancies and to evaluate its influence on cytokines production at the end of pregnancy. A cross sectional study was conducted with fifty five pregnant women out of labor with intact membranes and gestational age between 37 and 41 weeks seen at the Bom Jesus hospital in Ariquemes, Rondônia, between June 2009 and May 2010. Amniotic fluid samples and fragments of chorioamniotic membranes were collected at cesarean section. M. hominis and U. urealyticum detection was performed by PCR and Interleukin (IL)-1β, IL-6, IL-8, IL-10 and Tumor Necrosis Factor (TNF)- levels were determined by ELISA. Chorioamniotic membranes were submitted to histopatological analyses. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not shown detectable concentrations of TNF- and IL-1β. The median concentration of IL-6 and IL-8 were 107.9 pg/mL (0-517.1) and 208.1 pg/mL (0-1897.4), respectively. Interleukin-1, IL-6, IL-8 and TNF- concentrations were not associated with the presence of M. hominis in amniotic fluid, regardless the gestational age. No sample had detectable IL-10 levels. The histopatological analyses have shown no chorioamnionitis in any of the membranes, only a discreet mononuclear infiltration in the decidua could be observed in 40.4% of the samples. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not... (Complete abstact click electronic access below)

Orientador: Márcia Guimarães da Silva
Banca: Rodrigo Paupéro de Camargo Soares
Banca: Vera Lúcia Mores Rall
Não disponível
Abstract: Microbial invasion of the amniotic cavity has been described in term deliveries and its role on the immune modulation is of interest to the better understanding of the underlying labor processes. The aim of this study was to determine the prevalence of Mycoplasma hominis and Ureaplasma urealyticum in the amniotic fluid of term pregnancies and to evaluate its influence on cytokines production at the end of pregnancy. A cross sectional study was conducted with fifty five pregnant women out of labor with intact membranes and gestational age between 37 and 41 weeks seen at the Bom Jesus hospital in Ariquemes, Rondônia, between June 2009 and May 2010. Amniotic fluid samples and fragments of chorioamniotic membranes were collected at cesarean section. M. hominis and U. urealyticum detection was performed by PCR and Interleukin (IL)-1β, IL-6, IL-8, IL-10 and Tumor Necrosis Factor (TNF)- levels were determined by ELISA. Chorioamniotic membranes were submitted to histopatological analyses. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not shown detectable concentrations of TNF- and IL-1β. The median concentration of IL-6 and IL-8 were 107.9 pg/mL (0-517.1) and 208.1 pg/mL (0-1897.4), respectively. Interleukin-1, IL-6, IL-8 and TNF- concentrations were not associated with the presence of M. hominis in amniotic fluid, regardless the gestational age. No sample had detectable IL-10 levels. The histopatological analyses have shown no chorioamnionitis in any of the membranes, only a discreet mononuclear infiltration in the decidua could be observed in 40.4% of the samples. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not... (Complete abstact click electronic access below)
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Ureaplasmas são comumente isolados do trato urogenital humano. O objetivo do estudo foi detectar ureaplasmas em mulheres sexualmente ativas, relacionar aos aspectos de saúde sexual, e com polimorfismo das citocinas IL-6 e IL-1b. Foram incluídas amostras de swab vaginal e sangue periférico de 302 mulheres. A frequência de detecção por PCR foi de 76,2% para Mollicutes, 7,0% para U. urealyticum e 52,0% para U. parvum. Na qPCR foi encontrada frequência de 16,6% para U. urealyticum e 60,6% U. parvum. As amostras de U. parvum foram subtipadas e os sorotipos 6 e 3/14 foram os mais freqüentes, seguidos do sorotipo 1. A frequência encontrada para Trichomonas vaginalis, Neisseria gonorrhoeae, Gardnerella vaginalis e Chlamydia trachomatis foi de 3,0%, 21,5%, 42,4% e 1,7%, respectivamente. No polimorfismo para IL-6, o genótipo GG foi o mais frequente e para o polimorfismo de IL-1b, o genótipo CC apresentou maior prevalência. Não foi observado diferenças entre os níveis das citocinas no plasma sanguíneo entre mulheres do grupo caso e controle.
Ureaplasmas are commonly isolated from the human urogenital tract. The purpose of this study was to detect the presence of ureaplasmas in sexually active women and relate to sexual health, and polymorphism of cytokines IL-6 and IL-1b. It was included samples of vaginal swab and peripheral blood of 302 women. The frequency of detection by PCR was 76.2% for Mollicutes, 7.0% to U. urealyticum and 52.0% for U. parvum. In the qPCR the frequency found was of 16.6% to U. urealyticum and 60.6% U. parvum. Samples of U. parvum were subtyped and serotypes 6 and 3/14 were the most frequent, followed by serotype 1. The frequency found for Trichomonas vaginalis, Neisseria gonorrhoeae, Gardnerella vaginalis and Chlamydia trachomatis was 3.0%, 21.5%, 42.4% and 1.7%, respectively. In the polymorphism for IL-6, GG genotype was the most frequent and the polymorphism of IL-1b, the CC genotype presented higher prevalence. No differences were observed between the levels of cytokines in the blood plasma of women in the case group and control.