O Streptococcus do Grupo B (EGB) é um dos principais agentes de infecção no período neonatal, sendo responsável por altos índices de morbimortalidade materno-fetal. Este estudo tem por objetivo avaliar a passagem transplacentária de anticorpos anti-Streptococcus B e imunoglobulina G em recém-nascidos de termo e pré-termo, bem como comparar seus níveis séricos. Foi realizado estudo transversal incluindo 44 recém-nascidos (18 pré-termo e 26 de termo) do Berçário Anexo à Maternidade do Hospital das Clínicas da FMUSP no período de dezembro de 2006 a julho de 2007. Após consentimento esclarecido, foram obtidas amostras de sangue das mães e do cordão umbilical de seus respectivos recém-nascidos, realizadas dosagens de IgG total por nefelometria e de anticorpos anti-EGB através do ensaio imunoenzimático (ELISA). Observou-se que nos dois grupos de mães da casuística, compostos por 16 mães de RN pré-termo e 26 mães de RN de termo, não houve diferença significativa em relação aos níveis séricos de anticorpos anti-EGB. O nível sérico médio de anticorpos maternos anti-EGB foi de 1697,98, com variação de 456 a 5200 (em títulos). O nível sérico médio de anticorpos anti-EGB das mães de RNPT foi de 1570,72, com variação de 588 a 3829 (em títulos), enquanto nas mães de RNT o nível médio foi de 1786,08, com variação de 456 a 5200. Os níveis séricos de anticorpos anti-Streptococcus do grupo B dos recém-nascidos foram significantemente mais baixos nos RN pré-termo em relação aos RN de termo. O nível sérico médio de anticorpos anti-EGB dos RNPT foi de 1059,22, com variação de 416 a 3924 (em títulos), enquanto nos RNT foi 2025,50, com variação de 542 a 5476. Houve correlação positiva entre os níveis de imunoglobulina G e de anticorpos anti-Streptococcus B com a idade gestacional, demonstrando correlação entre prematuridade e baixos níveis séricos de anticorpos anti-EGB. A associação entre idade gestacional inferior a 37 semanas e diminuição dos níveis de anticorpos anti-EGB concorda com a maior vulnerabilidade dos neonatos pré-termo à infecção por esta bactéria. Os autores concluem que houve passagem transplacentária de imunoglobulina G e anticorpos anti-Streptococcus B nos RN de termo e pré-termo, sendo, porém, os níveis séricos significantemente mais baixos nos RNPT, tanto em relação às suas mães quanto em relação aos níveis observados nos RN de termo. Os recém-nascidos de termo apresentaram níveis séricos de anticorpos anti-Streptococcus B semelhantes aos maternos, devido ao incremento da transferência transplacentária no final da gestação. Houve correlação positiva entre os níveis de imunoglobulina G e de anticorpos anti-Streptococcus B com a idade gestacional, enfatizando a importância da prematuridade como fator determinante das baixas concentrações séricas destes componentes imunológicos. Não houve diferença significante entre as mães dos recém-nascidos de termo e pré-termo em relação aos níveis séricos de anticorpos anti-Streptococcus B. / Group B Streptococcus (GBS) is one of the leading causes of infections in mothers and newborn babies, and it is responsible for high mortality rates. The purpose of this study was to evaluate the transplacental transfer of anti-Streptococcus B antibodies and immunoglobulin G in term and preterm newborns and compare the serum levels between these two groups. A transversal study was conducted with 44 newborns (18 preterm and 26 term infants) admitted to the Nursery next to FMUSP Hospital das Clínicas Maternity in the period of December 2006 to July 2007. After they gave their informed consent, blood and umbilical cord samples were collected from the mothers and from their respective newborn babies. Total IgG measurements were performed using nephelometry and the presence of antibodies anti-GBS was evaluated by the immunoenzimatic test (ELISA). In both groups of mothers (16 preterm`s mothers and 26 term`s mothers) the serum levels of anti-Streptococcus B antibodies were similar and there was no statistical significance between them. The mean serum levels of anti-GBS antibodies in mothers was 1697,98, ranging from 456 to 5200 (in titles). The mean serum levels of anti-GBS antibodies in mothers of preterm babies was 1570,72, ranging from 588 to 3829 (in titles), while in term`s mothers the mean level was 1786,08, ranging from 456 to 5200. Anti-Streptococcus B antibodies in the newborns demonstrated significantly lower levels in preterm newborn compared to the levels of the term newborns. The mean serum levels of anti-GBS antibodies in preterm newborns was 1059,22, ranging from 416 to 3924 (in titles), while in term newborns the mean level was 2025,50, ranging from 542 to 5476. There was a positive correlation between the levels of immunoglobulin G and anti-Streptococcus B antibodies and the gestational age which shows the correlation between prematurity and low levels of anti-Streptococcus B antibodies. The association between gestational age less than 37 weeks and reduction of anti-GBS antibody levels corroborates with the fact that preterm neonates are more vulnerable to the infection by this bacteria. The authors have come to the conclusion that transplacental transfer of immunoglobulin G and anti-Streptococcus B antibodies have been proved in term and preterm newborns. The transfer of immunoglobulin and antibodies was less effective in preterm newborns, whose serum levels were significantly lower compared to the levels of their mothers and the term newborns. Term newborns showed levels of anti-Streptococcus B antibodies similar to their mothers due to the increase of transplacental transfer of antibodies during the end of gestation. The positive correlation between the levels of immunoglobulin G and anti-Streptococcus B antibodies with gestational age, proves the importance of prematurity as a determining factor of the low serum concentrations in the components of this newborn\'s immunological repertoire. There was no significant difference between the levels of anti-Streptococcus B antibodies in mothers of term and preterm newborns.

Há poucos dados na literatura sobre o transporte transplacentário de imunoglobulinas em gestações múltiplas. O objetivo deste estudo foi observar fatores que influenciam a concentração de imunoglobulina G (IgG) no cordão umbilical dos neonatos e a transferência transplacentária de IgG total e de IgG contra o Streptococcus grupo B (EGB), e lipopolissacarídeos (LPS) de Klebsiella spp. e Pseudomonas spp.. Métodos: estudo prospectivo realizado no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo no período de 2012 a 2013. Foram coletadas amostras de sangue materno e de cordão umbilical no momento do parto. Os critérios de inclusão foram gestações gemelares com ausência de sinais de infecção por HIV, citomegalovírus, Hepatites B e C, toxoplasmose e rubéola e ausência de doenças autoimunes, malformação fetal e síndromes genéticas. A análise multivariada foi realizada para avaliar a associação entre os níveis de IgG em cordão umbilical e as taxas de transferência de anticorpos com a concentração materna de IgG, a corionicidade da gestação, a presença de insuficiência placentária, a restrição de crescimento intrauterino, a idade gestacional de nascimento, o peso de nascimento, o tabagismo, a doença materna e a via de parto. Resultados: a concentração de IgG total em cordão umbilical apresentou correlação positiva com os níveis maternos séricos de IgG total e a idade gestacional do parto. Os níveis de IgG total em cordão umbilical foram significativamente menores em gestações monocoriônicas quando comparadas às dicoriônicas. A taxa de transferência de IgG total apresentou correlação positiva com a idade gestacional do parto, mas negativa com as concentrações maternas de IgG total. As concentrações de IgG contra EGB e LPS de Klebsiella spp. e Pseudomonas spp. apresentaram associação com os níveis maternos de IgG específicos contra esses antígenos e com o diabetes. Os níveis de IgG contra LPS de Klebsiella spp. também foram associados com o peso de nascimento e com hipertensão materna. As taxas de transferência de IgG contra EGB e LPS de Pseudomonas spp. apresentaram correlação com os níveis maternos de IgG específicos contra os antígenos referidos. A taxa de transferência de IgG contra EGB também esteve associada com a idade gestacional do parto, enquanto a taxa de transferência de IgG contra LPS de Pseudomonas spp. apresentou correlação com diabetes. Não houve correlação entre a taxa de transferência de IgG contra a LPS de Klebsiella spp. com nenhum fator analisado. Conclusão: em gestações gemelares, a concentração total de IgG em cordão umbilical foi influenciada pela concentração materna de IgG total, pela idade gestacional do parto e pela corionicidade placentária. As concentrações de IgG total foram significativamente menores em gestações monocoriônicas que em dicoriônicas. As concentrações séricas de IgG contra EGB e LPS de Klebsiella spp. e Pseudomonas spp. em cordão umbilical apresentaram associação com os níveis maternos de IgG específicos contra esses antígenos e com a presença de diabetes. Todos os outros parâmetros estudados apresentaram diferentes associações com as concentrações de IgG e com as taxas de transferências de IgG específicas contra cada antígeno investigado / There is a lack of data in the literature regarding the placental transport of immunoglobulins in twin pregnancies. The objective of this study was to examine factors that influence the concentration of immunoglobulin G (IgG) in cord serum and the placental transfer of total IgG and of IgGs against Klebsiella spp. LPS and Pseudomonas spp. LPS, and Group B Streptococcus (GBS). Methods: A prospective study was conducted at the Hospital das Clinicas in the São Paulo University Medical School between 2012 and 2013. Maternal and umbilical cord samples were collected at birth. The inclusion criteria were twin pregnancies with no evidence of infection with HIV, cytomegalovirus, hepatitis B or C, toxoplasmosis, or rubella. Twin pregnancies with evidence of autoimmune disease, fetal malformations or genetic syndromes were also excluded. Stepwise multivariate regression analysis was used to evaluate the association between cord serum concentrations of IgG and IgG transfer ratios as well as the associations between cord serum concentrations of IgG and maternal serum concentrations of IgG, pregnancy chorionicity, the presence of an abnormal umbilical artery pulsatility index, intrauterine growth restriction, gestational age at delivery (GAD), birth weight, placental weight, smoking during pregnancy, maternal disease, and mode of delivery. Results: Total IgG concentrations in cord sera were positively correlated with total IgG concentrations in maternal sera. Cord serum concentrations of IgG were also positively correlated with GAD. Cord serum concentrations of total IgG were significantly lower in monochorionic versus dichorionic pregnancies. The total IgG transfer ratio was positively correlated with GAD but was inversely correlated with total IgG concentration in maternal serum. Cord serum concentrations of IgGs against GBS, Klebsiella spp. LPS and Pseudomonas spp. LPS were significantly associated with maternal concentrations of specific IgGs and the presence of maternal diabetes. Cord serum concentrations of anti-Klebsiella spp. LPS IgG were also correlated with birth weight and the presence of maternal hypertension. The transfer ratios of IgGs against GBS and Pseudomonas spp. LPS were related to maternal concentrations of specific IgGs. The transfer ratios of IgGs against GBS and Pseudomonas spp. LPS were also associated with GAD and the presence of diabetes, respectively. None of the examined parameters were found to be correlated with the transfer ratio of IgG against Klebsiella spp. LPS. Conclusions: In twin pregnancies, in addition to the influences of maternal serum concentrations of total IgG and of GAD, chorionicity was also found to influence cord serum concentrations of total IgG. Compared with dichorionic twins, monochorionic twins were found to have lower concentrations of total IgG in cord sera. Umbilical cord serum concentrations of IgGs against GBS, Klebsiella spp. LPS and Pseudomonas spp. LPS were associated with maternal serum concentrations of specific IgGs and with maternal diabetes. All of the remaining parameters that were investigated had varying associations with concentrations of specific IgGs in cord serum and with placental transfer and were dependent on the antigen being studied

O Streptococcus do Grupo B (EGB) é um dos principais agentes de infecção no período neonatal, sendo responsável por altos índices de morbimortalidade materno-fetal. Este estudo tem por objetivo avaliar a passagem transplacentária de anticorpos anti-Streptococcus B e imunoglobulina G em recém-nascidos de termo e pré-termo, bem como comparar seus níveis séricos. Foi realizado estudo transversal incluindo 44 recém-nascidos (18 pré-termo e 26 de termo) do Berçário Anexo à Maternidade do Hospital das Clínicas da FMUSP no período de dezembro de 2006 a julho de 2007. Após consentimento esclarecido, foram obtidas amostras de sangue das mães e do cordão umbilical de seus respectivos recém-nascidos, realizadas dosagens de IgG total por nefelometria e de anticorpos anti-EGB através do ensaio imunoenzimático (ELISA). Observou-se que nos dois grupos de mães da casuística, compostos por 16 mães de RN pré-termo e 26 mães de RN de termo, não houve diferença significativa em relação aos níveis séricos de anticorpos anti-EGB. O nível sérico médio de anticorpos maternos anti-EGB foi de 1697,98, com variação de 456 a 5200 (em títulos). O nível sérico médio de anticorpos anti-EGB das mães de RNPT foi de 1570,72, com variação de 588 a 3829 (em títulos), enquanto nas mães de RNT o nível médio foi de 1786,08, com variação de 456 a 5200. Os níveis séricos de anticorpos anti-Streptococcus do grupo B dos recém-nascidos foram significantemente mais baixos nos RN pré-termo em relação aos RN de termo. O nível sérico médio de anticorpos anti-EGB dos RNPT foi de 1059,22, com variação de 416 a 3924 (em títulos), enquanto nos RNT foi 2025,50, com variação de 542 a 5476. Houve correlação positiva entre os níveis de imunoglobulina G e de anticorpos anti-Streptococcus B com a idade gestacional, demonstrando correlação entre prematuridade e baixos níveis séricos de anticorpos anti-EGB. A associação entre idade gestacional inferior a 37 semanas e diminuição dos níveis de anticorpos anti-EGB concorda com a maior vulnerabilidade dos neonatos pré-termo à infecção por esta bactéria. Os autores concluem que houve passagem transplacentária de imunoglobulina G e anticorpos anti-Streptococcus B nos RN de termo e pré-termo, sendo, porém, os níveis séricos significantemente mais baixos nos RNPT, tanto em relação às suas mães quanto em relação aos níveis observados nos RN de termo. Os recém-nascidos de termo apresentaram níveis séricos de anticorpos anti-Streptococcus B semelhantes aos maternos, devido ao incremento da transferência transplacentária no final da gestação. Houve correlação positiva entre os níveis de imunoglobulina G e de anticorpos anti-Streptococcus B com a idade gestacional, enfatizando a importância da prematuridade como fator determinante das baixas concentrações séricas destes componentes imunológicos. Não houve diferença significante entre as mães dos recém-nascidos de termo e pré-termo em relação aos níveis séricos de anticorpos anti-Streptococcus B. / Group B Streptococcus (GBS) is one of the leading causes of infections in mothers and newborn babies, and it is responsible for high mortality rates. The purpose of this study was to evaluate the transplacental transfer of anti-Streptococcus B antibodies and immunoglobulin G in term and preterm newborns and compare the serum levels between these two groups. A transversal study was conducted with 44 newborns (18 preterm and 26 term infants) admitted to the Nursery next to FMUSP Hospital das Clínicas Maternity in the period of December 2006 to July 2007. After they gave their informed consent, blood and umbilical cord samples were collected from the mothers and from their respective newborn babies. Total IgG measurements were performed using nephelometry and the presence of antibodies anti-GBS was evaluated by the immunoenzimatic test (ELISA). In both groups of mothers (16 preterm`s mothers and 26 term`s mothers) the serum levels of anti-Streptococcus B antibodies were similar and there was no statistical significance between them. The mean serum levels of anti-GBS antibodies in mothers was 1697,98, ranging from 456 to 5200 (in titles). The mean serum levels of anti-GBS antibodies in mothers of preterm babies was 1570,72, ranging from 588 to 3829 (in titles), while in term`s mothers the mean level was 1786,08, ranging from 456 to 5200. Anti-Streptococcus B antibodies in the newborns demonstrated significantly lower levels in preterm newborn compared to the levels of the term newborns. The mean serum levels of anti-GBS antibodies in preterm newborns was 1059,22, ranging from 416 to 3924 (in titles), while in term newborns the mean level was 2025,50, ranging from 542 to 5476. There was a positive correlation between the levels of immunoglobulin G and anti-Streptococcus B antibodies and the gestational age which shows the correlation between prematurity and low levels of anti-Streptococcus B antibodies. The association between gestational age less than 37 weeks and reduction of anti-GBS antibody levels corroborates with the fact that preterm neonates are more vulnerable to the infection by this bacteria. The authors have come to the conclusion that transplacental transfer of immunoglobulin G and anti-Streptococcus B antibodies have been proved in term and preterm newborns. The transfer of immunoglobulin and antibodies was less effective in preterm newborns, whose serum levels were significantly lower compared to the levels of their mothers and the term newborns. Term newborns showed levels of anti-Streptococcus B antibodies similar to their mothers due to the increase of transplacental transfer of antibodies during the end of gestation. The positive correlation between the levels of immunoglobulin G and anti-Streptococcus B antibodies with gestational age, proves the importance of prematurity as a determining factor of the low serum concentrations in the components of this newborn\'s immunological repertoire. There was no significant difference between the levels of anti-Streptococcus B antibodies in mothers of term and preterm newborns.

O presente trabalho foi organizado em dois estudos. O objetivo do primeiro estudo foi determinar o efeito da infecção intramamária (IIM) causada por S. aureus e S. agalactiae na contagem de células somáticas (CCS) e na composição do leite (gordura, proteína, lactose, sólidos totais e extrato seco desengordurado), o efeito da contagem de S. aureus e S. agalactiae sobre a composição do leite e a estimativa da frequência de vacas e de quartos infectados com S. aureus e S. agalactiae por meio da contagem do tanque. O objetivo do segundo estudo foi avaliar a reação em cadeia da polimerase em Tempo Real (qPCR) como metodologia alternativa à cultura microbiológica, por meio da sensibilidade diagnóstica, da concordância e da equivalência entre as metodologias para identificação e contagem de S. aureus e S. agalactiae de origem das vacas com IIM. O experimento foi realizado em quatro rebanhos comerciais com histórico de mastite causada por S. aureus e S. agalactiae. Foram coletadas 785 amostras compostas de leite, 3049 amostras de leite de quartos mamários de todas as vacas em lactação e 12 amostras de leite de tanque dos rebanhos selecionados, durante 3 meses consecutivos, totalizando 3 coletas por rebanho. As amostras de leite foram submetidas à detecção e contagem de S. aureus e S. agalactiae por meio de cultura microbiológica e de qPCR, e à análise de composição e CCS. No primeiro estudo, foi observado que amostras compostas de leite com S. agalactiae apresentaram menores teores de lactose e maiores teores de gordura, proteína, sólidos totais e CCS, e com S. aureus apresentaram menores teores de lactose e maiores teores de proteína e de CCS do leite. O aumento da contagem de S. aureus e S. agalactiae em amostras compostas de leite pode ocasionar aumento linear nos teores de lactose e de extrato seco desengordurado. Foi observado que as amostras compostas de leite e de quartos mamários apresentaram relação positiva referente às contagens de S. aureus e S. agalactiae e ao percentual (%) de amostras de leite com S. aureus e S. agalactiae. A partir de amostras compostas de leite pode-se estimar o percentual de quartos mamários com S. aureus e S. agalactiae. A contagem de S. aureus e de S. agalactiae de amostras de leite de tanque pode estimar o percentual de vacas com S. aureus e o percentual de quartos mamários com S. agalactiae. No segundo estudo, foi observado equivalência e concordância de resultados de identificação e contagem de S. agalactiae entre as metodologias de qPCR e cultura microbiológica somente em amostras de leite de tanque e de quartos mamários. Em amostras de leite com S. aureus e S. agalactiae, a qPCR apresentou sensibilidade diagnóstica de 71,54% e 90.20% em amostras de leite de quartos mamários, 71,79% e 87,72% em amostras compostas de leite e 50,00% e 90,91% em amostras de leite de tanque, respectivamente. Portanto, o leite de tanque e a qPCR podem se utilizadas para monitoramento da mastite bovina no rebanho leiteiro / This work was organized in two studies. The purpose of the first study was to determine the effect of intramammary infection (IMI) caused by S. aureus and S. agalactiae in somatic cell count (SCC) and composition of milk (fat, protein, lactose, total solids and nonfat dry), the effect of S. aureus count and S. agalactiae on milk composition and an estimated frequency of cows and quarters infected with S. aureus and S. agalactiae by counting the tank. The purpose of the second study was to evaluate the polymerase chain reaction in real time (qPCR) as an alternative methodology to microbiological culture, through the diagnostic sensitivity, for consistency and equivalence between the methodologies for identification and enumeration of S. aureus and S. agalactiae source of cows with IMI. The experiment was conducted in four commercial herds with mastitis history caused by S. aureus and S. agalactiae. We collected 785 samples composed of milk, 3049 milk samples from mammary glands of all lactating cows and 12 from the selected herds tank milk samples for 3 consecutive months, totaling 3 samples per herd. The milk samples were subjected to the detection and counting of S. aureus and S. agalactiae through microbial culture and qPCR, and the composition analysis and SCC. In the first study, it was observed that composite milk samples with S. agalactiae had lower lactose content and higher fat content, protein, total solids and SCC, and S. aureus showed lower lactose content and higher protein content and SCC milk. Increased count of S. aureus and S. agalactiae in composite samples of milk can cause a linear increase in the levels of lactose and nonfat dry. It was observed that the composite milk sample and mammary glands were closely related to the counts of S. aureus and S. agalactiae and the percentage (%) of samples of milk with S. aureus and S. agalactiae. From composite samples of milk can estimate the percentage of mammary quarters with S. aureus and S. agalactiae. The count of S. aureus and S. agalactiae of tank milk samples can estimate the percentage of cows with S. aureus and the percentage of mammary quarters with S. agalactiae. In the second study, it was observed equivalence agreement and identification results and S. agalactiae count between the methodologies of qPCR and microbiological culture only in tank milk samples and mammary glands. In milk samples with S. aureus and S. agalactiae, qPCR showed diagnostic sensitivity of 71.54% and 90.20% in milk samples from mammary quarters, 71.79% and 87.72% on composite samples of milk and 50.00% to 90.91% in tank milk samples, respectively. Therefore, the tank and the qPCR milk can be used for monitoring of bovine mastitis in dairy cattle

Há poucos dados na literatura sobre o transporte transplacentário de imunoglobulinas em gestações múltiplas. O objetivo deste estudo foi observar fatores que influenciam a concentração de imunoglobulina G (IgG) no cordão umbilical dos neonatos e a transferência transplacentária de IgG total e de IgG contra o Streptococcus grupo B (EGB), e lipopolissacarídeos (LPS) de Klebsiella spp. e Pseudomonas spp.. Métodos: estudo prospectivo realizado no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo no período de 2012 a 2013. Foram coletadas amostras de sangue materno e de cordão umbilical no momento do parto. Os critérios de inclusão foram gestações gemelares com ausência de sinais de infecção por HIV, citomegalovírus, Hepatites B e C, toxoplasmose e rubéola e ausência de doenças autoimunes, malformação fetal e síndromes genéticas. A análise multivariada foi realizada para avaliar a associação entre os níveis de IgG em cordão umbilical e as taxas de transferência de anticorpos com a concentração materna de IgG, a corionicidade da gestação, a presença de insuficiência placentária, a restrição de crescimento intrauterino, a idade gestacional de nascimento, o peso de nascimento, o tabagismo, a doença materna e a via de parto. Resultados: a concentração de IgG total em cordão umbilical apresentou correlação positiva com os níveis maternos séricos de IgG total e a idade gestacional do parto. Os níveis de IgG total em cordão umbilical foram significativamente menores em gestações monocoriônicas quando comparadas às dicoriônicas. A taxa de transferência de IgG total apresentou correlação positiva com a idade gestacional do parto, mas negativa com as concentrações maternas de IgG total. As concentrações de IgG contra EGB e LPS de Klebsiella spp. e Pseudomonas spp. apresentaram associação com os níveis maternos de IgG específicos contra esses antígenos e com o diabetes. Os níveis de IgG contra LPS de Klebsiella spp. também foram associados com o peso de nascimento e com hipertensão materna. As taxas de transferência de IgG contra EGB e LPS de Pseudomonas spp. apresentaram correlação com os níveis maternos de IgG específicos contra os antígenos referidos. A taxa de transferência de IgG contra EGB também esteve associada com a idade gestacional do parto, enquanto a taxa de transferência de IgG contra LPS de Pseudomonas spp. apresentou correlação com diabetes. Não houve correlação entre a taxa de transferência de IgG contra a LPS de Klebsiella spp. com nenhum fator analisado. Conclusão: em gestações gemelares, a concentração total de IgG em cordão umbilical foi influenciada pela concentração materna de IgG total, pela idade gestacional do parto e pela corionicidade placentária. As concentrações de IgG total foram significativamente menores em gestações monocoriônicas que em dicoriônicas. As concentrações séricas de IgG contra EGB e LPS de Klebsiella spp. e Pseudomonas spp. em cordão umbilical apresentaram associação com os níveis maternos de IgG específicos contra esses antígenos e com a presença de diabetes. Todos os outros parâmetros estudados apresentaram diferentes associações com as concentrações de IgG e com as taxas de transferências de IgG específicas contra cada antígeno investigado / There is a lack of data in the literature regarding the placental transport of immunoglobulins in twin pregnancies. The objective of this study was to examine factors that influence the concentration of immunoglobulin G (IgG) in cord serum and the placental transfer of total IgG and of IgGs against Klebsiella spp. LPS and Pseudomonas spp. LPS, and Group B Streptococcus (GBS). Methods: A prospective study was conducted at the Hospital das Clinicas in the São Paulo University Medical School between 2012 and 2013. Maternal and umbilical cord samples were collected at birth. The inclusion criteria were twin pregnancies with no evidence of infection with HIV, cytomegalovirus, hepatitis B or C, toxoplasmosis, or rubella. Twin pregnancies with evidence of autoimmune disease, fetal malformations or genetic syndromes were also excluded. Stepwise multivariate regression analysis was used to evaluate the association between cord serum concentrations of IgG and IgG transfer ratios as well as the associations between cord serum concentrations of IgG and maternal serum concentrations of IgG, pregnancy chorionicity, the presence of an abnormal umbilical artery pulsatility index, intrauterine growth restriction, gestational age at delivery (GAD), birth weight, placental weight, smoking during pregnancy, maternal disease, and mode of delivery. Results: Total IgG concentrations in cord sera were positively correlated with total IgG concentrations in maternal sera. Cord serum concentrations of IgG were also positively correlated with GAD. Cord serum concentrations of total IgG were significantly lower in monochorionic versus dichorionic pregnancies. The total IgG transfer ratio was positively correlated with GAD but was inversely correlated with total IgG concentration in maternal serum. Cord serum concentrations of IgGs against GBS, Klebsiella spp. LPS and Pseudomonas spp. LPS were significantly associated with maternal concentrations of specific IgGs and the presence of maternal diabetes. Cord serum concentrations of anti-Klebsiella spp. LPS IgG were also correlated with birth weight and the presence of maternal hypertension. The transfer ratios of IgGs against GBS and Pseudomonas spp. LPS were related to maternal concentrations of specific IgGs. The transfer ratios of IgGs against GBS and Pseudomonas spp. LPS were also associated with GAD and the presence of diabetes, respectively. None of the examined parameters were found to be correlated with the transfer ratio of IgG against Klebsiella spp. LPS. Conclusions: In twin pregnancies, in addition to the influences of maternal serum concentrations of total IgG and of GAD, chorionicity was also found to influence cord serum concentrations of total IgG. Compared with dichorionic twins, monochorionic twins were found to have lower concentrations of total IgG in cord sera. Umbilical cord serum concentrations of IgGs against GBS, Klebsiella spp. LPS and Pseudomonas spp. LPS were associated with maternal serum concentrations of specific IgGs and with maternal diabetes. All of the remaining parameters that were investigated had varying associations with concentrations of specific IgGs in cord serum and with placental transfer and were dependent on the antigen being studied

Introdução: O estreptococo do grupo B (EGB) tem sido motivo de preocupação para obstetras e neonatologistas pela possibilidade de sepse neonatal, que apresenta um risco importante de mortalidade. O rastreamento do EGB entre 35-37 semanas associado com a profilaxia intra-parto com penicilina cristalina tem propiciado uma diminuição de sepse neonatal precoce por EGB. O índice de colonização por EGB é muito variável de acordo com região e condição social das gestantes. A freqüência de clones virulentos é desconhecida em nosso meio. Objetivos: 1 - Determinar a prevalência de EGB no trato genital-anal em gestantes que internam no Centro Obstétrico de um Hospital Universitário na Cidade de Porto Alegre, verificando também os fatores associados a esta colonização. 2- Verificar a presença de cepas invasivas do EGB nos casos positivos Métodos: O estudo envolveu 319 gestantes, com idade gestacional que variou entre24 e 41 semanas, que internaram na Maternidade do Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul. Nestas pacientes foi feito um swab vaginal-anal e semeado em meio de cultura Todd-Hewitt. As amostras positivas para EGB foram enviadas para realização de reação em cadeia da polimerase (PCR) para extração do DNA e tipagem de cepas altamente virulentas do EGB. Resultados: A prevalência de EGB encontrada foi de 23,4% (IC 95%: 17,8- 27,2). Em relação aos possíveis fatores associados a colonização a variável idade mostrou uma tendência de maior colonização em pacientes com mais de 30 anos, quando comparada com idades mais jovens. Isso também aconteceu em relação a comparação com a cor da paciente onde apareceu uma tendência de maior colonização em pacientes de cor preta ou mista. As pacientes com idade gestacional abaixo de 35 semanas apresentaram um índice de colonização inferior às gestantes entre 35-37 semanas e também entre 37-39 semanas. A presença de ruptura prematura das membranas ovulares não apareceu como fator associado a índice maior de colonização pelo EGB. Em relação a freqüência de cepas virulentas a análise de 60 casos revelou 10 casos positivos (16,6%) para clone "sequence typing" - 17 (ST-17). Conclusões: A prevalência de EGB encontrado em nosso estudo é similar aos índices de países desenvolvidos, que realizam o rastreamento rotineiro no prénatal e fazem a intervenção com profilaxia intra-parto nos casos positivos. Houve um maior índice de colonização em gestações à termo comparadas com gestações de pré-termo. A freqüência de cepas "altamente virulentas" encontrada foi 16,6%. / Background: Group B Streptococcus (GBS) has been of concern to obstetrician and neonatologists to the possibility of neonatal sepsis, which presents a significant risk of mortality. Screening for GBS between 35-37 weeks associated with intrapartum prophylaxis with crystalline penicillin has resulted in reduction of neonatal sepsis by GBS. The rate of GBS colonization varies widely according to region and social status of women. The frequency of virulent clones is unknown in our contry. Objectives: 1. To determine the prevalence of GBS in the genital-anal tract of pregnant patients that are hospitalized at the obstetric center of a University Hospital of Porto Alegre, also check the factors associated with this colonization. 2. Verify the presence of invasive strains of GBS positive cases Methods: The study involved 319 pregnant women with gestational ages ranging from 24 and 41 weeks, who were hospitalized at the São Lucas Hospital of Catholic University of Rio Grande do Sul. These patients was done a vaginalanal swab and seeded un culture medium Todd-Hewitt. Samples positive for GBS were sent to carry out polymerase chain reaction (PCR) for DNA extraction and typing of higly virulent strains of GBS. Results: GBS prevalence found was 23.4% (CI 95%: 17.8-27.2). Regarding the possible factors associated with colonization of the age variable showed a trend of increased colonization in patients over 30 years compared with younger ages. This also happened for comparison with the color of the patient where it appeared a tend of increased colonization in patients of gestacional age below 35 weeks among pregnant women at 35-37 weeks and between 37-39 weeks. The presence of premature rupture of ovular membranes did not appear as a factor associated with higer rate of GBS colonization. Regarding the frequency of virulent strains analysis of 60 cases revealed 10 positive cases (16.6%) to clone sequence typing -17 (ST-17). Conclusions: GBS prevalence found in our study is similar to rates in developed countries, carrying out routine screening during prenatal care and make a intervention with intrapartum prophylaxis in positive cases. There was a higer rate of colonization in term pregnacies compared with preterm. The frequency of strains "highly virulent" found was 16.6%.

Introdução: O estreptococo do grupo B (EGB) tem sido motivo de preocupação para obstetras e neonatologistas pela possibilidade de sepse neonatal, que apresenta um risco importante de mortalidade. O rastreamento do EGB entre 35-37 semanas associado com a profilaxia intra-parto com penicilina cristalina tem propiciado uma diminuição de sepse neonatal precoce por EGB. O índice de colonização por EGB é muito variável de acordo com região e condição social das gestantes. A freqüência de clones virulentos é desconhecida em nosso meio. Objetivos: 1 - Determinar a prevalência de EGB no trato genital-anal em gestantes que internam no Centro Obstétrico de um Hospital Universitário na Cidade de Porto Alegre, verificando também os fatores associados a esta colonização. 2- Verificar a presença de cepas invasivas do EGB nos casos positivos Métodos: O estudo envolveu 319 gestantes, com idade gestacional que variou entre24 e 41 semanas, que internaram na Maternidade do Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul. Nestas pacientes foi feito um swab vaginal-anal e semeado em meio de cultura Todd-Hewitt. As amostras positivas para EGB foram enviadas para realização de reação em cadeia da polimerase (PCR) para extração do DNA e tipagem de cepas altamente virulentas do EGB. Resultados: A prevalência de EGB encontrada foi de 23,4% (IC 95%: 17,8- 27,2). Em relação aos possíveis fatores associados a colonização a variável idade mostrou uma tendência de maior colonização em pacientes com mais de 30 anos, quando comparada com idades mais jovens. Isso também aconteceu em relação a comparação com a cor da paciente onde apareceu uma tendência de maior colonização em pacientes de cor preta ou mista. As pacientes com idade gestacional abaixo de 35 semanas apresentaram um índice de colonização inferior às gestantes entre 35-37 semanas e também entre 37-39 semanas. A presença de ruptura prematura das membranas ovulares não apareceu como fator associado a índice maior de colonização pelo EGB. Em relação a freqüência de cepas virulentas a análise de 60 casos revelou 10 casos positivos (16,6%) para clone "sequence typing" - 17 (ST-17). Conclusões: A prevalência de EGB encontrado em nosso estudo é similar aos índices de países desenvolvidos, que realizam o rastreamento rotineiro no prénatal e fazem a intervenção com profilaxia intra-parto nos casos positivos. Houve um maior índice de colonização em gestações à termo comparadas com gestações de pré-termo. A freqüência de cepas "altamente virulentas" encontrada foi 16,6%. / Background: Group B Streptococcus (GBS) has been of concern to obstetrician and neonatologists to the possibility of neonatal sepsis, which presents a significant risk of mortality. Screening for GBS between 35-37 weeks associated with intrapartum prophylaxis with crystalline penicillin has resulted in reduction of neonatal sepsis by GBS. The rate of GBS colonization varies widely according to region and social status of women. The frequency of virulent clones is unknown in our contry. Objectives: 1. To determine the prevalence of GBS in the genital-anal tract of pregnant patients that are hospitalized at the obstetric center of a University Hospital of Porto Alegre, also check the factors associated with this colonization. 2. Verify the presence of invasive strains of GBS positive cases Methods: The study involved 319 pregnant women with gestational ages ranging from 24 and 41 weeks, who were hospitalized at the São Lucas Hospital of Catholic University of Rio Grande do Sul. These patients was done a vaginalanal swab and seeded un culture medium Todd-Hewitt. Samples positive for GBS were sent to carry out polymerase chain reaction (PCR) for DNA extraction and typing of higly virulent strains of GBS. Results: GBS prevalence found was 23.4% (CI 95%: 17.8-27.2). Regarding the possible factors associated with colonization of the age variable showed a trend of increased colonization in patients over 30 years compared with younger ages. This also happened for comparison with the color of the patient where it appeared a tend of increased colonization in patients of gestacional age below 35 weeks among pregnant women at 35-37 weeks and between 37-39 weeks. The presence of premature rupture of ovular membranes did not appear as a factor associated with higer rate of GBS colonization. Regarding the frequency of virulent strains analysis of 60 cases revealed 10 positive cases (16.6%) to clone sequence typing -17 (ST-17). Conclusions: GBS prevalence found in our study is similar to rates in developed countries, carrying out routine screening during prenatal care and make a intervention with intrapartum prophylaxis in positive cases. There was a higer rate of colonization in term pregnacies compared with preterm. The frequency of strains "highly virulent" found was 16.6%.

Introdução: O estreptococo do grupo B (EGB) tem sido motivo de preocupação para obstetras e neonatologistas pela possibilidade de sepse neonatal, que apresenta um risco importante de mortalidade. O rastreamento do EGB entre 35-37 semanas associado com a profilaxia intra-parto com penicilina cristalina tem propiciado uma diminuição de sepse neonatal precoce por EGB. O índice de colonização por EGB é muito variável de acordo com região e condição social das gestantes. A freqüência de clones virulentos é desconhecida em nosso meio. Objetivos: 1 - Determinar a prevalência de EGB no trato genital-anal em gestantes que internam no Centro Obstétrico de um Hospital Universitário na Cidade de Porto Alegre, verificando também os fatores associados a esta colonização. 2- Verificar a presença de cepas invasivas do EGB nos casos positivos Métodos: O estudo envolveu 319 gestantes, com idade gestacional que variou entre24 e 41 semanas, que internaram na Maternidade do Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul. Nestas pacientes foi feito um swab vaginal-anal e semeado em meio de cultura Todd-Hewitt. As amostras positivas para EGB foram enviadas para realização de reação em cadeia da polimerase (PCR) para extração do DNA e tipagem de cepas altamente virulentas do EGB. Resultados: A prevalência de EGB encontrada foi de 23,4% (IC 95%: 17,8- 27,2). Em relação aos possíveis fatores associados a colonização a variável idade mostrou uma tendência de maior colonização em pacientes com mais de 30 anos, quando comparada com idades mais jovens. Isso também aconteceu em relação a comparação com a cor da paciente onde apareceu uma tendência de maior colonização em pacientes de cor preta ou mista. As pacientes com idade gestacional abaixo de 35 semanas apresentaram um índice de colonização inferior às gestantes entre 35-37 semanas e também entre 37-39 semanas. A presença de ruptura prematura das membranas ovulares não apareceu como fator associado a índice maior de colonização pelo EGB. Em relação a freqüência de cepas virulentas a análise de 60 casos revelou 10 casos positivos (16,6%) para clone "sequence typing" - 17 (ST-17). Conclusões: A prevalência de EGB encontrado em nosso estudo é similar aos índices de países desenvolvidos, que realizam o rastreamento rotineiro no prénatal e fazem a intervenção com profilaxia intra-parto nos casos positivos. Houve um maior índice de colonização em gestações à termo comparadas com gestações de pré-termo. A freqüência de cepas "altamente virulentas" encontrada foi 16,6%. / Background: Group B Streptococcus (GBS) has been of concern to obstetrician and neonatologists to the possibility of neonatal sepsis, which presents a significant risk of mortality. Screening for GBS between 35-37 weeks associated with intrapartum prophylaxis with crystalline penicillin has resulted in reduction of neonatal sepsis by GBS. The rate of GBS colonization varies widely according to region and social status of women. The frequency of virulent clones is unknown in our contry. Objectives: 1. To determine the prevalence of GBS in the genital-anal tract of pregnant patients that are hospitalized at the obstetric center of a University Hospital of Porto Alegre, also check the factors associated with this colonization. 2. Verify the presence of invasive strains of GBS positive cases Methods: The study involved 319 pregnant women with gestational ages ranging from 24 and 41 weeks, who were hospitalized at the São Lucas Hospital of Catholic University of Rio Grande do Sul. These patients was done a vaginalanal swab and seeded un culture medium Todd-Hewitt. Samples positive for GBS were sent to carry out polymerase chain reaction (PCR) for DNA extraction and typing of higly virulent strains of GBS. Results: GBS prevalence found was 23.4% (CI 95%: 17.8-27.2). Regarding the possible factors associated with colonization of the age variable showed a trend of increased colonization in patients over 30 years compared with younger ages. This also happened for comparison with the color of the patient where it appeared a tend of increased colonization in patients of gestacional age below 35 weeks among pregnant women at 35-37 weeks and between 37-39 weeks. The presence of premature rupture of ovular membranes did not appear as a factor associated with higer rate of GBS colonization. Regarding the frequency of virulent strains analysis of 60 cases revealed 10 positive cases (16.6%) to clone sequence typing -17 (ST-17). Conclusions: GBS prevalence found in our study is similar to rates in developed countries, carrying out routine screening during prenatal care and make a intervention with intrapartum prophylaxis in positive cases. There was a higer rate of colonization in term pregnacies compared with preterm. The frequency of strains "highly virulent" found was 16.6%.

O presente trabalho foi organizado em dois estudos. O objetivo do primeiro estudo foi determinar o efeito da infecção intramamária (IIM) causada por S. aureus e S. agalactiae na contagem de células somáticas (CCS) e na composição do leite (gordura, proteína, lactose, sólidos totais e extrato seco desengordurado), o efeito da contagem de S. aureus e S. agalactiae sobre a composição do leite e a estimativa da frequência de vacas e de quartos infectados com S. aureus e S. agalactiae por meio da contagem do tanque. O objetivo do segundo estudo foi avaliar a reação em cadeia da polimerase em Tempo Real (qPCR) como metodologia alternativa à cultura microbiológica, por meio da sensibilidade diagnóstica, da concordância e da equivalência entre as metodologias para identificação e contagem de S. aureus e S. agalactiae de origem das vacas com IIM. O experimento foi realizado em quatro rebanhos comerciais com histórico de mastite causada por S. aureus e S. agalactiae. Foram coletadas 785 amostras compostas de leite, 3049 amostras de leite de quartos mamários de todas as vacas em lactação e 12 amostras de leite de tanque dos rebanhos selecionados, durante 3 meses consecutivos, totalizando 3 coletas por rebanho. As amostras de leite foram submetidas à detecção e contagem de S. aureus e S. agalactiae por meio de cultura microbiológica e de qPCR, e à análise de composição e CCS. No primeiro estudo, foi observado que amostras compostas de leite com S. agalactiae apresentaram menores teores de lactose e maiores teores de gordura, proteína, sólidos totais e CCS, e com S. aureus apresentaram menores teores de lactose e maiores teores de proteína e de CCS do leite. O aumento da contagem de S. aureus e S. agalactiae em amostras compostas de leite pode ocasionar aumento linear nos teores de lactose e de extrato seco desengordurado. Foi observado que as amostras compostas de leite e de quartos mamários apresentaram relação positiva referente às contagens de S. aureus e S. agalactiae e ao percentual (%) de amostras de leite com S. aureus e S. agalactiae. A partir de amostras compostas de leite pode-se estimar o percentual de quartos mamários com S. aureus e S. agalactiae. A contagem de S. aureus e de S. agalactiae de amostras de leite de tanque pode estimar o percentual de vacas com S. aureus e o percentual de quartos mamários com S. agalactiae. No segundo estudo, foi observado equivalência e concordância de resultados de identificação e contagem de S. agalactiae entre as metodologias de qPCR e cultura microbiológica somente em amostras de leite de tanque e de quartos mamários. Em amostras de leite com S. aureus e S. agalactiae, a qPCR apresentou sensibilidade diagnóstica de 71,54% e 90.20% em amostras de leite de quartos mamários, 71,79% e 87,72% em amostras compostas de leite e 50,00% e 90,91% em amostras de leite de tanque, respectivamente. Portanto, o leite de tanque e a qPCR podem se utilizadas para monitoramento da mastite bovina no rebanho leiteiro / This work was organized in two studies. The purpose of the first study was to determine the effect of intramammary infection (IMI) caused by S. aureus and S. agalactiae in somatic cell count (SCC) and composition of milk (fat, protein, lactose, total solids and nonfat dry), the effect of S. aureus count and S. agalactiae on milk composition and an estimated frequency of cows and quarters infected with S. aureus and S. agalactiae by counting the tank. The purpose of the second study was to evaluate the polymerase chain reaction in real time (qPCR) as an alternative methodology to microbiological culture, through the diagnostic sensitivity, for consistency and equivalence between the methodologies for identification and enumeration of S. aureus and S. agalactiae source of cows with IMI. The experiment was conducted in four commercial herds with mastitis history caused by S. aureus and S. agalactiae. We collected 785 samples composed of milk, 3049 milk samples from mammary glands of all lactating cows and 12 from the selected herds tank milk samples for 3 consecutive months, totaling 3 samples per herd. The milk samples were subjected to the detection and counting of S. aureus and S. agalactiae through microbial culture and qPCR, and the composition analysis and SCC. In the first study, it was observed that composite milk samples with S. agalactiae had lower lactose content and higher fat content, protein, total solids and SCC, and S. aureus showed lower lactose content and higher protein content and SCC milk. Increased count of S. aureus and S. agalactiae in composite samples of milk can cause a linear increase in the levels of lactose and nonfat dry. It was observed that the composite milk sample and mammary glands were closely related to the counts of S. aureus and S. agalactiae and the percentage (%) of samples of milk with S. aureus and S. agalactiae. From composite samples of milk can estimate the percentage of mammary quarters with S. aureus and S. agalactiae. The count of S. aureus and S. agalactiae of tank milk samples can estimate the percentage of cows with S. aureus and the percentage of mammary quarters with S. agalactiae. In the second study, it was observed equivalence agreement and identification results and S. agalactiae count between the methodologies of qPCR and microbiological culture only in tank milk samples and mammary glands. In milk samples with S. aureus and S. agalactiae, qPCR showed diagnostic sensitivity of 71.54% and 90.20% in milk samples from mammary quarters, 71.79% and 87.72% on composite samples of milk and 50.00% to 90.91% in tank milk samples, respectively. Therefore, the tank and the qPCR milk can be used for monitoring of bovine mastitis in dairy cattle

Streptococcus (S.) agalactiae sind grampositive, in Ketten gelagerte Kokken, die auf Blutagar eine Hämolyse zeigen. Aufgrund ihrer Zugehörigkeit zur Lancefield-Gruppe B werden sie auch als Gruppe B Streptokokken (GBS) bezeichnet (Hof, 2005). GBS sind die Hauptursache von Sepsis, Meningitis und Pneumonie bei Neugeborenen (Schrag, et al., 2000). Die Arbeit beschäftigte sich mit der Genotypisierung von GBS. Darüber hinaus konnten auch Einblicke in die Phylogenese sowie die Populationsstruktur von GBS gewonnen werden. Ziel war es, einen DNA-Microarray zu entwickeln und zur Genotypisierung von GBS einzusetzen. Während der Evaluierung des DNA Microarray konnten stammspezifische Muster beobachtet werden, diese wurden durch bereits etablierte Typisierungmethoden (MLST) bekannten Genotypen zugeordnet. Die Ergebnisse wurden in einer Datenbank zusammengefasst. Mithilfe der Datenbank konnte die Software zur Auswertung entwickelt werden. (siehe http://alere-technologies.com/en/products/lab-solutions.html). Der DNA-Microarray trägt Sonden für GBS-spezifische Virulenzfaktoren und Oberflächenmarker. Für die auf dem Microarray basierende Typisierung wurden 11 über das ganze Genom verteilte Gene bzw. Gencluster (bac, alp, pil1 locus, pepS8, fBsB, capsule locus, hylB, abiG-I/-II plus Q8DZ34, pil2 locus, nss plus srr plus rogB2 und rgfC/A/D/B) ausgewählt. Ubiquitär vorkommende, konservierte Gene (z. B. cylD/cylE) eignen sich nicht als Marker für eine Typisierung, wurden aber als Kontrollen und zur Normierung eingeschlossen. Zur vollständigen Charakterisierung wurden außerdem Sonden für hochmobile plasmidgebundene Resistenzgene wie z. B. erm(A), erm(B), erm(C), tet(M), emrB/qacA aufgetragen (Aracil, et al., 2002; Betriu, et al., 2003; Uh, et al., 2001). Diese Gene sind nicht für GBS spezifisch. Sie eignen sich z. B. für eine Unterscheidung einzelner Isolate, nicht jedoch für die Unterteilung der GBS Population in verschiedene Stämme. Für die vorliegende Arbeit wurden insgesamt 448 klinische Isolate von GBS ausgewählt und untersucht. Darunter waren Isolate, die schwerwiegende Erkrankungen wie Sepsis und Meningitis verursacht haben, Isolate aus lokalen Infektionen sowie Isolate von asymptomatischen/gesunden Trägern. Zu Vergleichszwecken wurden außerdem Isolate aus der Veterinärmedizin (von bovinen Mastitisfällen) und humane Isolate aus einer geographisch weit entfernten Region (Trinidad und Tobago) genotypisiert. Für 36 ausgewählte Isolate mit repräsentativen Hybridisierungsmustern wurde zusätzlich eine Typisierung mittels Multilocus-Sequenztypisierung (MLST) (Jones, et al., 2003) durchgeführt. Die Hybridisierungsmuster vom Microarray wurden mit Daten aus diesem bereits etablierten Typisierungssystem verbunden. Durch die Verknüpfung beider Methoden konnte eine Einteilung der GBS Isolate in verschiedene Stämme vorgenommen werden. Mit Hilfe des eBURST-Algorithmus wurde gezeigt, dass einige Hybridisierungsmuster sich zu Gruppen zusammenfügen. Dieses Verfahren veranschaulicht die Populationsstruktur und beschreibt die genetische Vielfalt. Mit den 11 definierten Markern konnten die untersuchten Isolate 76 verschiedenen Stämmen bzw. „hybridization profiles“ (HP) zugeordnet werden. Die Einteilung beruht auf dem Fehlen bzw. Vorhandensein einzelner Gene/Gencluster bzw. deren allelischen Varianten. Diese Stämme korrelieren mit den durch MLST definierten klonalen Komplexen (CC). Isolate mit identischen oder ähnlichen Hybridisierungsprofilen gehören zum selben CC. Dagegen können Isolate mit einem ähnlichen MLST-Profil verschiedene Hybridisierungsmuster zeigen. Es konnte außerdem häufig beobachtet werden, dass ansonsten ähnliche Stämme sich in einzelnen Merkmalen, z. B. Kapsel-Genen, alp- oder pili-Genen, voneinander unterscheiden, und dass diese Gene unabhängig voneinander variieren. Zusätzlich zeigten einige ubiquitäre Gene/Gencluster, die sich in den publizierten Genomsequenzen immer an derselben Position befinden, zahlreiche verschiedene Allele. Welches Allel in einem gegebenen Stamm gerade vorliegt, scheint dabei eher zufällig zu sein. Eine Erklärung dieses Phänomens könnte in vergangenen Rekombinationsereignissen liegen. Auch eine konvergente Evolution könnte diskutiert werden. Ähnliche Stämme/ „hybridization profiles“ wurden in Analogie zu den MLST-definierten klonalen Komplexen zu Gruppen zusammengefügt. Das bedeutet jedoch nicht notwendigerweise eine direkte Verwandtschaft der Isolate im Sinne des Vorhandenseins eines unmittelbaren gemeinsamen Vorfahren. Die Typisierung sowohl über den DNA-Microarray als auch über die MLST kann nicht die „wahre“ Phylogenese im Rahmen der Evolutionsgeschichte und Herkunft widerspiegeln. Sie stellt lediglich ein zufälliges Modell dar, ein Ordnungssystem im Sinne eines genetischen Fingerabdrucks, das einen Vergleich von Isolaten, aber keine Rückschlüsse über deren Abstammung und Herkunft erlaubt.Die untersuchten GBS Isolate konnten in fünf klonale Komplexe (CC19, CC23, CC26, CC103, CC130) eingeteilt werden, deren Häufigkeit unterschiedlich war. Deutsche humanmedizinische Isolate konnten vorwiegend CC19 zugeordnet werden. Karibische humanmedizinische Isolate sind zumeist CC19 und CC23 zugehörig. Bovine Isolate gehören meist zu CC19 und CC103. Unter humanen Isolaten ist CC103 rar. Vermutlich basierend auf der geografischen und wirtsspezifischen Herkunft der untersuchten GBS Isolate gibt es Unterschiede in der Populationsstruktur. In der vorliegenden Arbeit war CC19 der am häufigsten gefundene und außerdem ein genetisch besonders inhomogener CC. Er besteht aus mehreren unterschiedlichen, bisher als eigenständig angesehenen CCs (darunter CC1, CC17, CC19 und CC22). Diese werden von dem zur MLST-Verwandtschaftsanalysen verwendeten eBURST-Algorithmus zu CC19 zusammengefasst, seit die MLST-Profile von 'missing links' zwischen den CCs identifiziert wurden, da eBURST „gemeinsame Vorfahren“ nicht von durch horizontalem Gentransfer bzw. durch Hybridisierungen entstandenen „Chimären“ unterscheiden kann. Da diese Komplexe klar unterscheidbare Hybridisierungsmuster aufweisen, wurden sie hier als CC19/01, CC19/17, CC19/19 und CC19/22 bezeichnet. Einzelne Gene traten in Gruppen von Isolaten aus verschiedener Herkunft unterschiedlich häufig auf. So fand sich der Virulenzfaktor scpB in 412 von 418 humanen Isolaten (98,6 %), aber nur in 10 von 21 Rinderisolaten (48 %). Ferner ließ sich beobachten, dass invasive Isolate weniger wahrscheinlich abiGI-/II und Q8DZ34 tragen, jedoch häufiger pil1 locus, fbsB (515) und Kapseltyp III sowie pil2b, nss/srr und rgf (COH1 like) aufweisen. Einige dieser Marker erscheinen zusammen in CC19/17-Stämmen, welche häufig bei invasiven Krankheitsverläufen beobachtet werden. CC19 (incl. ST01, ST17, ST19) konnte bei neonatalen Sepsis-Fällen in verschiedenen geografischen Regionen isoliert werden (Brzychczy-Wloch, et al., 2012; Ryu, et al., 2014; Sorensen, et al., 2010; Strakova, et al., 2010; Tien, et al., 2011). Zusätzlich wurden andere Virulenzfaktoren wie speM (Exotoxin M) und das cyl-Operon (beta-Hämolysin) untersucht. In lediglich sieben Isolaten wurde speM nachgewiesen. Das cyl-Operon konnte in allen Isolaten gefunden werden, sein Nachweis ist daher für eine Vorhersage der Virulenz eines gegebenen Isolates nicht hilfreich. Es konnte kein einzelner Faktor zur definitiven Unterscheidung zwischen invasiven Isolaten und Trägerisolaten bestimmt werden. Für die Virulenz eines Isolates ist wahrscheinlich nicht das bloße Vorhandensein oder Fehlen eines bestimmten Genes ausschlaggebend, sondern dessen Expression in vivo. Wichtig wäre in diesem Zusammenhang auch die genaue Betrachtung der Sequenz eines als Virulenzfaktor angesehenen Genes sowie die Untersuchung der zugehörigen regulatorischen Gene. Über den Nachweis der Gene erm(A), erm(B) und erm(C) konnte eine Aussage über die Macrolid-/Clindamycinresistenz eines GBS Isolates getroffen werden. Bei keinem der karibischen Isolate wurden erm Gene nachgewiesen. Innerhalb der deutschen GBS Population wurde erm(B) am häufigsten beobachtet. Die Gene erm(A) und erm(C) waren in humanen Isolaten selten und wurden in bovinen Isolaten überhaupt nicht gefunden. Das Tetracyclinresistenzgen tet(M) wurde häufig in humanen Isolaten und sehr selten in veterinärmedizinischen Isolaten gefunden. Für weiterführende Untersuchungen könnte die beschriebene Typisierungsmethode verfeinert werden. So lassen sich z. B. die oben beschriebenen 11 ausgewählten Typisierungsmarker des Microarrays mit denen der MLST zu einem 18 Marker-System verknüpfen. Daneben können auch erm-, cad-, mer- oder tet-Gene zur Feststellung oder zum Ausschluss der Identität verwandter Isolate in vitro oder in silico verwendet werden. Mit der nun einsatzbereiten Genotypisierungsmethode können in Zukunft weitere Studien zur Untersuchung regionaler und wirtsspezifischer Unterschiede der GBS Population durchgeführt werden. In dieser Arbeit konnte gezeigt werden, dass der DNA-Microarray stabile und reproduzierbare Resultate erbringt. Es kann ein detaillierter Befund erstellt werden, die Ergebnisse sind mit denen anderer Typisierungsmethoden und der Genomsequenzierung vergleichbar. Jedoch steht mit dem DNA-Microarray ein wesentlich unkomplizierteres und schnelleres Procedere zur Verfügung, welches zudem geringere Kosten verursacht.:1. Einleitung 5 1.1 Gegenstand der Untersuchung 5 1.2 Entdeckungsgeschichte 6 1.3 Klinische Bedeutung 7 1.4 Veterinärmedizinische Bedeutung 9 1.5 Stand der Forschung mit Hinblick auf Typisierung von GBS 9 1.6 Ziele der vorliegenden Untersuchung 10 1.7 Vorgehensweise 11 1.7.1 Untersuchungsmaterial 11 1.7.2 Untersuchungsmethode 11 1.8 Zur Typisierung ausgewählte Marker 12 2. DNA Microarray-Based Typing of Streptococcus agalactiae Isolates 15 2.1 Abstract 16 2.2 Introduction 17 2.3 Materials and methods 18 2.3.1 Bacterial isolates 18 2.3.2 Ethics statement 18 2.3.3 Preparation of genomic DNA 19 2.3.4 MLST 19 2.3.5 Microarray design and protocol optimization 19 2.3.6 Microarray procedures 20 2.3.7 eBURST 21 2.4 Results 22 2.4.1 Typing GBS by MLST 22 2.4.2 Genotyping GBS by microarray hybridization 22 2.4.3 Population structure 25 2.4.4 Detection of antibiotic resistance markers 25 2.4.5 Detection of heavy metal resistance markers 26 2.5 Discussion 27 2.6 Acknowledgments 30 2.7 References 31 2.8 Tables and figures 33 3. Zusammenfassung 45 3.1 Zusammenfassung 45 3.2 Summary 49 4. Korrespondenz mit dem Editor 53 4.1 Hinweise des Editors und der Gutachter 53 4.2 Antworten an den Editor 56 4.3 Endgültige Annahme 58 Anhang 61 / Streptococcus (S.) agalactiae are Gram-positive, chain-forming cocci, which show hemolysis on blood agar. They are also referred to group B streptococci (GBS) because of their affiliation to Lancefield-group B (Hof, 2005). GBS are the main cause of sepsis, meningitis and pneumonia in newborns (Schrag, et al., 2000). The work focused on genotyping of GBS. The aim was to develop a DNA microarray and to use it for epidemiological typing of GBS. During the evaluation of the microarray, strain-specific patterns could be observed and these patterns assigned to genotypes as defined by other typing methods (MLST). The results were summarized in a database that subsequently was developed into software for automated analysis of experiments (http://alere-technologies.com/en/products/lab-solutions.html). The DNA microarray carries probes for GBS specific virulence factors and surface markers. For the microarray-based typing, 11 genes or gene clusters were selected that are distributed across the entire genome (bac, alp, pil1 locus, pepS8, fBsB, capsule locus, hylB, abiG-I/-II plus Q8DZ34, pil2 locus, nss plus srr plus rogB2 and rgfC/A/D/B). Ubiquitous, conserved genes (e.g. cylD/cylE) were included to be used as species markers and controls. Furthermore, probes for highly mobile plasmid-borne resistance genes such as erm(A), erm(B), erm(C), tet(M), emrB/qacA were also included (Aracil, et al., 2002; Betriu, et al., 2003; Uh, et al., 2001). These genes are not specific to GBS, but they are found in some isolates. They can be used to distinguish individual, related isolates, rather than for a definition of distinct strains. A total of 448 isolates of GBS was selected and examined for the present work. Among them were isolates from severe diseases, such as sepsis and meningitis, isolates from local infections as well as isolates from asymptomatic/healthy carriers. For comparison, isolates from veterinary medicine (from cases of bovine mastitis) and human isolates from a geographically distant region (Trinidad and Tobago) were genotyped. For 36 selected isolates with representative hybridization patterns, parallel typing was performed using a second method, multilocus sequence typing (MLST) (Jones, et al., 2003). Hybridization patterns on the Microarray could thus be linked to this already established typing system. With the array based GBS typing isolates could be divided into 76 different strains or 'hybridization profiles', HP. The classification with both methods is based on the absence or presence of individual genes or gene clusters or their allelic variants. Similar isolates were lumped together. The eBURST algorithm was used to group strain-specific patterns into groups of related strains illustrating the population structure and describing the genetic diversity. Groups of similar hybridization patterns largely correlate with the clonal complexes (CC) defined by MLST. While isolates with identical or similar hybridization profiles belong to the same CC, isolates with a similar MLST profile can show different hybridization patterns. It has also often been observed that otherwise similar strains differ from each other in individual traits, e.g. capsule genes, alp or pili genes, and that these genes vary independently of one another. In addition, some ubiquitous genes/gene clusters, which are always localized at the same position in the published genomic sequences, show numerous different alleles and related strains (that belong to one clonal complex) might differ in the presence of one allele. Alleles are thus not linked to clonal complexes, but rather randomly distributed. An explanation of this phenomenon could be a frequent occurrence of recombination events or horizontal gene transfers. A convergent evolution could also be discussed as an alternative explanation. A similarity of hybridization profiles does not necessarily mean a direct phylogenetic relationship between the isolates in the sense of being derived from a direct common ancestor. Typing both the DNA microarray and the MLST cannot reflect the 'true' phylogenesis, evolutionary history and origin. Assuming frequent recombination i.e., random events, the MLST profiles as well as the hybridization patterns can be used as genetic fingerprints, allowing a comparison of isolates, but no conclusions about their phylogeny and origin. The investigated GBS isolates were classified into five clonal complexes (CC19, CC23, CC26, CC103, CC130) with very different relative abundances indicating differences in the population structure with regard to geographic origin and host organisms. German medical isolates were mainly assigned CC19. Caribbean medical isolates mostly were assigned to CC19 and CC23. Bovine isolates usually belonged to CC19 and CC103. Among human isolates, CC103 was rare. In the present work, CC19 was the most abundant and the genetically most inhomogeneous CC. Several different clusters that previously been regarded as CCs (CC1, CC17, CC19 and CC22) have recently been merged to CC19 by the eBURST algorithm since MLST profiles of missing links between the CCs have been identified. Unfortunately, eBURST cannot distinguish whether two MLST types are linked by true common ancestors or by hybrid or chimera strains originating from horizontal gene transfers or hybridization events. Since these complexes within CC19 have clearly distinguishable hybridization patterns, they have been referred to herein as CC19/01, CC19/17, CC19/19 and CC19/22, and we assume that they are linked by hybridizations or gene transfers rather than by shared ancestry. Few differences were found between isolates from different origins. The virulence factor scpB was found in 412 of 418 human isolates (98.6%), but only in 10 of 21 bovine isolates (48%). Furthermore, it was observed that invasive isolates are less likely to carry abiGI-/II and Q8DZ34, but are more likely to have pil1 locus, fbsB (515) and capsule type III as well as pil2b, nss/srr and rgf (COH1 like). Some of these markers appear together in CC19/17 strains, which are often observed in invasive disease. speM (exotoxin M) was also investigated. It was detected only in seven isolates. Contrarily, the cyl (beta-hemolysin) operon was found in all isolates. Thus, it detection is not helpful for a prediction of the virulence of a given isolate. No single factor could be identified that allowed a definitive distinction between invasive isolates and carrier isolates. Probably, the virulence of an isolate does not depend on the presence or absence of one particular gene. In this context, it would be important to investigate the expression in vivo of the various putative virulence factors as well as the allelic variants of the factors and of their associated regulatory genes. Macrolide-/clindamycin resistance genes erm(A), erm(B) and erm(C) can also be detected by the microarray. None of these genes was identified in any of the Caribbean isolates. Within the German GBS population, erm(B) was most frequently observed. The genes erm(A) and erm(C) were rare in human isolates, and they were not found in bovine isolates. The tetracycline resistance gene tet(M) was observed frequently in human isolates but only very rarely in veterinary isolates. With the genotyping method that was developed during the present work, further studies can be carried out to study regional and host-specific differences in the GBS population. For future investigations, the described typing method could further be refined. For example, the 11 selected typing markers on the microarray can be combined with those from MLST to one comprehensive marker system. In addition, it is also possible to use genes on mobile genetic elements such as resistance genes (erm, cad, mer or tet) to prove or to rule out the identity of related isolates in vitro or in silico. In our study, it was shown that the DNA microarray provides stable and reproducible results that are comparable to those of other typing methods and genome sequencing. However, since the DNA microarray offers a much more uncomplicated and faster procedure, which also results in lower costs, it is more suitable to a routine setting.:1. Einleitung 5 1.1 Gegenstand der Untersuchung 5 1.2 Entdeckungsgeschichte 6 1.3 Klinische Bedeutung 7 1.4 Veterinärmedizinische Bedeutung 9 1.5 Stand der Forschung mit Hinblick auf Typisierung von GBS 9 1.6 Ziele der vorliegenden Untersuchung 10 1.7 Vorgehensweise 11 1.7.1 Untersuchungsmaterial 11 1.7.2 Untersuchungsmethode 11 1.8 Zur Typisierung ausgewählte Marker 12 2. DNA Microarray-Based Typing of Streptococcus agalactiae Isolates 15 2.1 Abstract 16 2.2 Introduction 17 2.3 Materials and methods 18 2.3.1 Bacterial isolates 18 2.3.2 Ethics statement 18 2.3.3 Preparation of genomic DNA 19 2.3.4 MLST 19 2.3.5 Microarray design and protocol optimization 19 2.3.6 Microarray procedures 20 2.3.7 eBURST 21 2.4 Results 22 2.4.1 Typing GBS by MLST 22 2.4.2 Genotyping GBS by microarray hybridization 22 2.4.3 Population structure 25 2.4.4 Detection of antibiotic resistance markers 25 2.4.5 Detection of heavy metal resistance markers 26 2.5 Discussion 27 2.6 Acknowledgments 30 2.7 References 31 2.8 Tables and figures 33 3. Zusammenfassung 45 3.1 Zusammenfassung 45 3.2 Summary 49 4. Korrespondenz mit dem Editor 53 4.1 Hinweise des Editors und der Gutachter 53 4.2 Antworten an den Editor 56 4.3 Endgültige Annahme 58 Anhang 61