No / Metal ion acquisition and homeostasis are essential for bacterial survival, growth and physiology. A family of metal ion, ABC-type import systems have been identified in Gram-positive bacteria, in which the solute-binding proteins are predicted to be membrane-anchored lipoproteins. The prediction that the MtsA protein of Streptococcus agalactiae A909 is a lipoprotein was confirmed. The expression of MtsA was co-ordinately regulated by the presence of both manganese and ferrous ions suggesting that MtsA may be involved in the uptake of both these ions. MtsA was shown to be expressed at levels of ferrous ions known to be present in amniotic fluid, a growth medium for S. agalactiae during neonatal infection.

No / Streptococcus agalactiae is a significant pathogen causing invasive disease in neonates and thus an understanding of the molecular basis of the pathogenicity of this organism is of importance. N-terminal lipidation is a major mechanism by which bacteria can tether proteins to membranes. Lipidation is directed by the presence of a cysteine-containing 'lipobox' within specific signal peptides and this feature has greatly facilitated the bioinformatic identification of putative lipoproteins. We have designed previously a taxon-specific pattern (G+LPP) for the identification of Gram-positive bacterial lipoproteins, based on the signal peptides of experimentally verified lipoproteins (Sutcliffe I.C. and Harrington D.J. Microbiology 148: 2065-2077). Patterns searches with this pattern and other bioinformatic methods have been used to identify putative lipoproteins in the recently published genomes of S. agalactiae strains 2603/V and NEM316. A core of 39 common putative lipoproteins was identified, along with 5 putative lipoproteins unique to strain 2603/V and 2 putative lipoproteins unique to strain NEM316. Thus putative lipoproteins represent ca. 2% of the S. agalactiae proteome. As in other Gram-positive bacteria, the largest functional category of S. agalactiae lipoproteins is that predicted to comprise of substrate binding proteins of ABC transport systems. Other roles include lipoproteins that appear to participate in adhesion (including the previously characterised Lmb protein), protein export and folding, enzymes and several species-specific proteins of unknown function. These data suggest lipoproteins may have significant roles that influence the virulence of this important pathogen.

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Streptococcus agalactiae, ou Estreptococo do Grupo B, é um microrganismo que encontrado na microbiota intestinal, vaginal e/ou geniturinária de 10-30% de mulheres saudáveis. A principal infecção causada por S. agalactiae é a sepse neonatal. O bebê pode adquirir o microrganismo durante o parto ao passar pelo canal vaginal, ou até mesmo durante a gestação, caso haja ascensão de S. agalactiae para o útero. Existem diversos fatores associados à infecção do feto por S. agalactiae quando a mãe é colonizada, tais como fator CAMP, cápsula de polissacarídeos, hialuronidase, β-citolisina/hemolisina e pili. Não existe consenso ou recomendação técnica sobre o tema no Brasil. Segundo o Caderno de Atenção Básica ao Pré-Natal, não existem estudos que levem à recomendação da antibioticoterapia intraparto. É necessário elucidar as características genotípicas de cepas de S. agalactiae isoladas no Brasil para alinhar as práticas clínicas às características fenotípicas do microrganismo. Desta forma, os objetivos deste projeto são: i) classificar cepas de S. agalactiae isoladas de gestantes e não gestantes quanto ao sorotipo capsular, por PCR Multiplex, ii) avaliar a presença e distribuição de fatores de virulência, por PCR e iii) avaliar o perfil de resistência antimicrobiana, pelo método de disco difusão e teste D. Os achados de virulência e resistência a antimicrobianos foram comparados com os sorotipos, gestação, localização geográfica e sítio de isolamento. Foram analisadas 292 cepas isoladas de gestantes e não gestantes em São Paulo, São José dos Campos e Rio de Janeiro. O sorotipo Ia foi o mais prevalente entre as cepas. Na cidade de São José dos Campos não houve diferença significativa entre a prevalência dos sorotipos Ia e V, sendo que o sorotipo V foi mais abundante do que nas cidades de São Paulo e Rio de Janeiro. O sorotipo II foi mais abundante em mulheres não gestantes do que gestantes. Não foram encontradas cepas resistentes à Penicilina e vancomicina; contudo a resistência a Cefepima, Eritromicina e Clindamicina ficou em torno de 22%. Foram encontradas diferenças entre os sorotipos quanto à resistência, genes de virulência e sítio de isolamento das cepas. Portanto essas diferenças podem se refletir no perfil epidemiológico da infecção por S. agalactiae quanto à localização geográfica também quanto à gestação. A incidência de sepse causada por S. agalactiae diminuiu muito nas últimas décadas, contudo o monitoramento constante é necessário para alinhar as práticas clínicas às características fenotípicas do microrganismo. / Streptococcus agalactiae, or Group B Streptococcus, is a microorganism found in intestinal, vaginal and/or genitourinary microbiota from about 10-30% of all healthy women. The main infection caused by S. agalactiae is neonatal sepsis. The baby can contract the infection during labor when passing through the vaginal canal, or even during pregnancy, if S. agalactiae ascends from the vaginal canal to the uterus. There are several factors associated to the infection of the fetus by S. agalactiae when the mother is colonized, such as the CAMP factor, polysaccharide capsule, hyaluronidase, β-cytolysin/hemolysin and pili. There is no consensus or technical recommendation regarding this theme in Brazil. According to the Brazilian guidelines to prenatal care, there is no research that justifies the implementation of intrapartum antibiotic therapy. There is a need to clarify genotype characteristics of S. agalactiae strains isolated in Brazil in order to align clinical practices to phenotypical characteristics of this microorganism. This way, the goals of this project are: i) to classify S. agalactiae strains isolated from pregnant and nonpregnant women according to their capsular serotype, using PCR Multiplex, ii) to evaluate the presence and distribution of virulence factors, using PCR and iii) to evaluate their antibiotic resistance profile, using disk-diffusion and D-zone tests. The findings regarding virulence and resistance were compared to serotypes, pregnancy, geographic localization and the site where the sample was isolated. A total of 292 strains from pregnant and nonpregnant women from the cities of São Paulo, São José dos Campos and Rio de Janeiro were analyzed. Serotype Ia was the most prevalent among the strains. In São José dos Campos there was no significate difference in the prevalence of serotypes Ia and V. Serotype V was the most abundant in São Paulo and Rio de Janeiro. Serotype II was most prevalent in nonpregnant women when compared to pregnant women. No resistance to Penicillin nor Vancomycin was found. However, resistance to Cefepime, Erythromycin or Clindamycin was found in around 22% of strains. There were differences among serotypes regarding resistance, virulence genes and site where the strain was isolated. Therefore these differences can reflect into the epidemiologic profile of S. agalactiae infection in regards to geographic localization and pregnancy. The incidence of sepsis caused by S. agalactiae has decrease in the last few decades, however constant monitoring is necessary in order to align clinical practice to the microorganisms phenotypical characteristics.

Objetivos: Determinar a taxa de prevalência de colonização materna por Estreptococo do grupo B na população de gestantes com idade gestacional de 35 semanas ou mais frequentadoras do pré-natal em dois hospitais (Hospital Universitário Júlio Muller - HUJM e Hospital Beneficente Santa Helena) na cidade de Cuiabá, Mato Grosso. Materiais e Métodos: Entre outubro de 2011 e 2013 foram avaliadas 258 gestantes no HUJM e do Hospital Santa Helena. Após concordarem e assinarem o TCLE, as gestantes de 35 semanas ou mais que não tinham sido submetidas ao exame ginecológico e não estavam em uso de antibióticos e atendiam aos critérios de inclusão, foram submetidas a coleta de swab vaginal e retal conforme protocolo estabelecido. Acondicionado em meio de transporte Stuart e no laboratório cultivado em caldo Granada bifásico IGBL. Após 24 horas, amostras com coloração laranja ou avermelhada foram consideradas positivas, caso contrário, nova leitura com 48 horas de cultivo. Os dados foram submetidos a análise estatística utilizando o EPI-Info da OMS. Resultados: Das 258 amostras 13,95% foram positivas para o EGB com IC (95%) de 9.70% a 18.21%. A avaliação estratificada pela idade gestacional predominou nas gestantes de 36 semanas com 35% de positividade, 10.87% para 37 semanas. E 5.88% para 35 semanas. No trabalho de parto prematuro 33.33% e na amniorrexe prematura 28,57% dos casos eram positivos para o EGB. Os demais parâmetros analisados não mostraram significância estatística. Conclusões: A taxa de prevalência da colonização pelo EGB de uma forma global foi estimada em 13.95%, sendo mais elevada na idade gestacional de 36 semanas com taxa de 35%. Este trabalho deve mudar as políticas públicas de saúde na cidade de Cuiabá / Purpose: To determine the prevalence rate of maternal colonization by Group B Streptococcus in the population of pregnant women in the gestational age of 35 or more weeks, attending prenatal care in two hospitals (HUJM - Hospital Universitário Júlio Muller and Hospital Santa Helena), in the city of Cuiabá, Mato Grosso. Materials and methods: Between October 2011 and October 2013, 258 pregnant women were assessed in HUJM and Hospital Santa Helena. After agreeing and signing the FCCT (Free and clarified Consent Term), those pregnant women of 35 weeks or more, who had not undergone gynecological examination, who were not on antibiotics and who also met the inclusion criteria, were subjected to vaginal and rectal swab collection, according to the established protocol. Stowed in Stuart transport medium and cultivated in IGBL biphasic Granada Broth in the laboratory. After 24 hours, samples with orange or reddish colors were considered positive, otherwise, new evaluation with a 48-hour culture was done. Data were submitted to statistical analysis, using OMS\' EPI-Info. The results: From the 258 given samples, 13.21% were positive for EGB, CI (95%) from 9.70% to 18.21%. Evaluation stratified by gestational age was predominant in pregnant women of 36 weeks with 35% positivity rate, 10.87% of pregnant women of 37 weeks and 5.88% of women of 35 weeks. During preterm labor 33.33% and in premature rupture of membranes, 28.57% cases were positive for GBS. Other analyzed parameters showed no significant statistically. Conclusion: The overall prevalence rate of GBS colonization was estimated at 13.95%, being higher in the gestational age of 36 weeks, with a rate of 35%. The present work should change public health policies in the city of Cuiabá

Objetivos: Determinar a taxa de prevalência de colonização materna por Estreptococo do grupo B na população de gestantes com idade gestacional de 35 semanas ou mais frequentadoras do pré-natal em dois hospitais (Hospital Universitário Júlio Muller - HUJM e Hospital Beneficente Santa Helena) na cidade de Cuiabá, Mato Grosso. Materiais e Métodos: Entre outubro de 2011 e 2013 foram avaliadas 258 gestantes no HUJM e do Hospital Santa Helena. Após concordarem e assinarem o TCLE, as gestantes de 35 semanas ou mais que não tinham sido submetidas ao exame ginecológico e não estavam em uso de antibióticos e atendiam aos critérios de inclusão, foram submetidas a coleta de swab vaginal e retal conforme protocolo estabelecido. Acondicionado em meio de transporte Stuart e no laboratório cultivado em caldo Granada bifásico IGBL. Após 24 horas, amostras com coloração laranja ou avermelhada foram consideradas positivas, caso contrário, nova leitura com 48 horas de cultivo. Os dados foram submetidos a análise estatística utilizando o EPI-Info da OMS. Resultados: Das 258 amostras 13,95% foram positivas para o EGB com IC (95%) de 9.70% a 18.21%. A avaliação estratificada pela idade gestacional predominou nas gestantes de 36 semanas com 35% de positividade, 10.87% para 37 semanas. E 5.88% para 35 semanas. No trabalho de parto prematuro 33.33% e na amniorrexe prematura 28,57% dos casos eram positivos para o EGB. Os demais parâmetros analisados não mostraram significância estatística. Conclusões: A taxa de prevalência da colonização pelo EGB de uma forma global foi estimada em 13.95%, sendo mais elevada na idade gestacional de 36 semanas com taxa de 35%. Este trabalho deve mudar as políticas públicas de saúde na cidade de Cuiabá / Purpose: To determine the prevalence rate of maternal colonization by Group B Streptococcus in the population of pregnant women in the gestational age of 35 or more weeks, attending prenatal care in two hospitals (HUJM - Hospital Universitário Júlio Muller and Hospital Santa Helena), in the city of Cuiabá, Mato Grosso. Materials and methods: Between October 2011 and October 2013, 258 pregnant women were assessed in HUJM and Hospital Santa Helena. After agreeing and signing the FCCT (Free and clarified Consent Term), those pregnant women of 35 weeks or more, who had not undergone gynecological examination, who were not on antibiotics and who also met the inclusion criteria, were subjected to vaginal and rectal swab collection, according to the established protocol. Stowed in Stuart transport medium and cultivated in IGBL biphasic Granada Broth in the laboratory. After 24 hours, samples with orange or reddish colors were considered positive, otherwise, new evaluation with a 48-hour culture was done. Data were submitted to statistical analysis, using OMS\' EPI-Info. The results: From the 258 given samples, 13.21% were positive for EGB, CI (95%) from 9.70% to 18.21%. Evaluation stratified by gestational age was predominant in pregnant women of 36 weeks with 35% positivity rate, 10.87% of pregnant women of 37 weeks and 5.88% of women of 35 weeks. During preterm labor 33.33% and in premature rupture of membranes, 28.57% cases were positive for GBS. Other analyzed parameters showed no significant statistically. Conclusion: The overall prevalence rate of GBS colonization was estimated at 13.95%, being higher in the gestational age of 36 weeks, with a rate of 35%. The present work should change public health policies in the city of Cuiabá

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Lietuvos žaliavinio pieno užterštumo B grupės teršalais analizė 2005-2010 metais. Tyrimo metu gautų rodiklių palyginimas su Europos Sąjungos šalių narių rodiklių palyginimas. / Lithuania milk contamination Group B contamination analysis in 2005-2010 years. The study of performance comparison with the European Union member states are shown.

The neonate is still at risk of acquiring Group B Streptococcus (GBS) infection upon delivery even with the implementation of early onset GBS neonatal disease preventative protocols. GBS was reported as the most prevalent organism causing neonatal morbidity and mortality in the USA and Australia in the 1990s. GBS is also known to cause disease in children, women, the immunocompromised adult and the elderly, but it is the preterm neonates who are at greatest risk of GBS neonatal disease. The aim of this study was to determine the prevalence of lower genital tract (LGT) colonisation with GBS in Brisbane women of child bearing age. We also aimed: (i) to compare the GBS LGT prevalence rate of Indigenous and non Indigenous women; (ii) to determine whether previously reported risk factors for LGT colonisation with GBS were also risk factors associated with GBS colonisation of women in this study; (iii) to further develop and optimise a rapid PCR assay that could detect maternal LGT GBS colonisation; and (iv) to serotype the GBS strains that were isolated from pregnant and non pregnant women who participated in this study. This study recruited 374 women of childbearing age attending public medical providers and found an overall GBS prevalence of 98/374 (26.2%) for these Brisbane women, a rate higher than previously reported in Australia. When the GBS prevalence for pregnant women (25.6%) was compared to non pregnant women (27.2%) they were similar. We also compared the GBS LGT colonisation rate of women attending different medical providers. The GBS LGT prevalence rate for pregnant women attending the Mater was 36/118 (30.5%), whilst those women attending the Redlands Hospital antenatal clinic had a LGT GBS prevalence rate of only 7/53 (13.2%). By comparison, the LGT GBS prevalence rate for non pregnant women attending Biala Sexual Health clinic was 21/69 (30.4%) and 34/127 (26.8%) of women attending the Brisbane Family Planning Queensland were also GBS positive. The seven women recruited from Inala community centre tested negative for GBS LGT colonisation. The LGT GBS prevalence of Australian Aboriginal women was 5/22 (22.7%), a rate which was not significantly different from non-Aboriginal women 78/288 (27.1%). Established early onset GBS neonatal disease preventative policies have been recently revised. The CDC now recommends that all pregnant women are screened for LGT GBS colonisation during late gestation, and that any GBS isolates be tested for resistance to antibiotics if the GBS positive women have an allergy to penicillin. Queensland's Department of Health recommend that Queensland medical agencies implement a non screening based preventative protocol, where clinicians monitor: women prior to labour for reported risk factors associated with maternal GBS colonisation: women in labour for 'obstetric risk factors'. A number of risk factors have previously been reported in association with GBS LGT colonisation. However, in this current study we found that only one risk factor was significantly associated with current GBS: previous reported LGT GBS colonisation was significantly associated with maternal LGT GBS colonisation reported in this study. Women who previously tested positive for GBS were significantly more likely to be GBS positive in subsequent tests (OR 4.7; 95%CI, 1.8-12.5) compared to women with no previous history of GBS colonisation. An assessment of adverse pregnancy outcomes, preterm deliveries, and GBS colonisation data was made. It was established that 30 women had previously given birth to one or more preterm neonates and of these 30 women, nine (30%) of them tested positive for GBS in this current study. Of the 71 women who had given birth to neonates and who had suffered an adverse pregnancy outcome 25.3% also tested positive for GBS in this current study. GBS was identified in up to 30% of all mothers who had delivered their neonate preterm, 27.4% of women who had previously suffered miscarriages and 16.7% of women who had previously had stillbirths. In this study we found that Australian Aboriginal women also had a greater risk of delivering neonates who suffered from an adverse pregnancy outcome in comparison to all other women. Twenty one of the 22 Aboriginal women had previously been pregnant at least once, and nine (42.9%) of these women had at least one prior adverse pregnancy outcome while seven (33.3%) of these women had previously delivered at least one neonate preterm. Of the 21 Aboriginal women who had a previous pregnancy more than half the total number of Aboriginal women (11/21) had either delivered one or more neonates preterm or had suffered from one or more adverse pregnancy outcomes. When the incidence of adverse pregnancy outcomes was compared for Aboriginal and all other women the results were surprising. Overall, this study found 216 women including Aboriginal women had previously been pregnant and of these women 71 (32.8%) of them suffered an adverse pregnancy outcome. By comparison, only 62 of 195 (31.8%) non Aboriginal women but nine out of 21 (41.9%) Australian Aboriginal women suffered from a previous adverse pregnancy outcome. The clinical LGT GBS isolates found in this study of Brisbane women were typed and all nine GBS serotypes plus non typeable GBS serotypes were detected. Seventy women tested GBS culture positive and vaginal and/or perianal samples obtained from these women were evaluated. GBS serotype III was the serotype most frequently isolated from this total population, from 47.4% of pregnant women and 51.7% of non pregnant women. From some women only a single GBS serotype was isolated: in these women we found that GBS serotype III (50%) was the predominant isolate, followed by GBS serotype Ia isolated from 16.7% women. In addition 4.2% of women were colonised with GBS serotypes; Ib, II and V, whilst GBS serotypes IV and VII were isolated from 2.1% women. Non typeable GBS strains confirmed by latex agglutination tests accounted for 11.9% of all strains isolated from these Brisbane women. This study identified multiple serovars in 15 clinical samples and found that 22 (31.4%) women were colonised with mixed GBS serotypes in samples collected from both vaginal and perianal regions. In five women the combination of serotypes III/Ia were identified and in other women combinations of serotypes III/II, III/IV, III/V, III/VIII, Ia/IV and Ib/NT were also detected. In two instances three serotype combinations were detected in samples from one woman and these included serotypes III/Ib/II and III/Ia/Ib. Isolates were also typed for women who were colonised in both vaginal and perianal regions and it was found that only 10 participants had identical isolates in both regions. GBS serotype III was the predominant serotype detected in women tested in this study and this is the serotype that has previously been associated with invasive infections in neonates. GBS neonatal disease is a world wide economic, health and social burden affecting different ethnic groups and is preventable. Currently no vaccine technology is available for the prevention of GBS neonatal disease and the most effective EOGND preventative protocol would be to test for maternal GBS colonisation during labour, or screen women for GBS at &gt36 weeks' gestation and administer intrapartum antibiotic prophylaxis (IAP) to all women who tested positive for GBS. In this current study we utilised a rapid bsp PCR assay to detect LGT GBS colonisation in women of child bearing age. The PCR assay identified 62.5% of all vaginal and perianal positive culture GBS samples. The specificity of the PCR assay was 89% while the positive and negative predictive values were 56.8% and 91.1% respectively. This PCR assay using the current parameters is not an effective GBS detection assay but could be further optimised in the near future. This PCR assay could be an effective test in the future with the development of an alternative DNA extraction method to InstaGene (BioRad). However, this PCR assay if used in conjunction with the current culture method is able to detect a further 8.9% of women colonised with asymptomatic GBS. Brisbane women aged between 26 to 35 years who are pregnant and who are attending public health care agencies are at greatest risk of being colonised with GBS. No disparity was identified when ethnicity or social standing were assessed. The overall results of this study demonstrate that the LGT GBS prevalence rate in Brisbane women is 26.2% but this rate was higher at 30.5% for women attending a Brisbane sexual health clinic and for pregnant women attending the Mater Mothers' antenatal clinic. GBS serovar III has been identified as the dominant serovar in our population group and this strain has been reported as the major cause of GBS disease in neonates and infants aged to three months. Disparity (11.1%) was reported when the incidence of adverse pregnancy outcomes amongst Aboriginal women was compared to non Aboriginal women. From the outcomes of this study it has been suggested that Queensland adopt a screening based GBS preventative protocol. It has also been suggested that an Australian wide GBS prevention strategy may further reduce the incidence of neonatal disease.

Group B Sox genes play a critical developmental role in both vertebrates and insects. Within the model species Drosophila melanogaster, two SoxB genes, Dichaete and SoxNeuro, have been shown to act as ‘master regulators’ in the early development of the central nervous system. SoxB genes have only been characterised in a handful of arthropod species thus far, with most work to date focusing on drosophilids. The purpose of this investigation was twofold. First, I set out to resolve the phylogenetic origins of arthropod SoxB genes, as mutually exclusive models explaining their emergence are still contested. I have identified and annotated the SoxB of several invertebrate taxa. In total, my investigation includes 24 different metazoan taxa, and represents the largest investigation of arthropod SoxB phylogeny to date. In light of this research, I have proposed a new model of SoxB evolution which resolves the conflicting elements of the two primary competing models. Second, to study the evolution of SoxB in terms of functional conservation/divergence, I selected the emerging model organism Tribolium castaneum to draw a comparative analysis with Drosophila melanogaster. I first began by characterising the spatiotemporal expression patterns of SoxNeuro mRNA in early Tribolium embryos using whole mount in situ hybridisation, and examined published Dichaete expression patterns in the context of central nervous system development in T. castaneum. Using these data, I draw a comparison to the expression profiles of Dichaete and SoxNeuro orthologues in Drosophila melanogaster and other species. I have found that both Dichaete and SoxNeuro expression patterns in the developing central nervous system are remarkably well-conserved across species. I also attempted to characterise genome-wide binding for both Dichaete and SoxNeuro proteins in Tribolium in what would have represented the first genomic investigation of its kind in this emerging species. Using a tethered DNA adenine methyltransferase (Dam) enzyme for both SoxNeuro and Dichaete, I hoped to characterise the genomic loci with which each protein interacts within the beetle genome (a technique known as DamID). Unfortunately, these last set of experiments have proved unsuccessful, despite several attempts which have made use of different promoters, different DNA enrichment methodologies, and tackling unforeseen DNA contamination issues. Nevertheless, the troubleshooting experiments that I have carried out will pave the way for further genomic experiments in Tribolium, easing the establishment of genomic research in this emerging organism.